Objective:Oxaliplatin(OXA)and 5-fluorouracil(5-FU)are 2 commonly used chemotherapeutic agents for colorectal cancer(CRC).MicroRNAs(miRNAs,miRs)play crucial roles in the development of chemoresistance in various cancer...Objective:Oxaliplatin(OXA)and 5-fluorouracil(5-FU)are 2 commonly used chemotherapeutic agents for colorectal cancer(CRC).MicroRNAs(miRNAs,miRs)play crucial roles in the development of chemoresistance in various cancers.However,the role and mechanism of miR-224-5p in regulating CRC chemoresistance remain unclear.This study aims to investigate the function of miR-224-5p in chemoresistant CRC cells and the underlying mechanisms.Methods:CRC datasets GSE28702 and GSE69657 were downloaded from the Gene Expression Omnibus(GEO)database.Differentially expressed miRNAs between drug sensitive and resistant groups(OXA or 5-FU)were analyzed,and miR-224-5p was identified as the target miRNA.Chemoresistant cell lines HCT15-OXR,HCT15-5-FU,SW480-OXR,and SW480-5-FU were established.Transient transfections were performed using miR-224-5p mimics,inhibitors,and their respective negative controls(control mimic,control inhibitor)in these cell lines.Cells were treated with different concentrations of OXA or 5-FU post-transfection,and the half-maximal inhibitory concentration(IC_(50))was determined using the cell counting kit-8(CCK-8)assay.Cell proliferation was assessed by CCK-8 and colony formation assays.The expression levels of miR-224-5p,LC3,and P62 were measured by real-time polymerase chain reaction(real-time PCR)and/or Western blotting.Autophagic flux was assessed using a tandem fluorescent-tagged LC3 reporter assay.TargetScan 8.0,miRTarBase,miRPathDB,and HADb were used to predict B-cell lymphoma-2(Bcl-2)as a potential miR-244-5p target,which was further validated by dual luciferase reporter assays.Results:Chemoresistant CRC cells exhibited down-regulated miR-224-5p expression,whereas up-regulation of miR-224-5p enhanced chemotherapy sensitivity.Exposure to OXA or 5-FU significantly increased autophagic activity in chemoresistant CRC cells,which was reversed by miR-224-5p overexpression.Dual-luciferase assays verified Bcl-2 as a direct target of miR-224-5p.Conclusion:MiR-224-5p regulates chemoresistance in CRC by modulating autophagy through direct targeting of Bcl-2.展开更多
The incidence rate of kidney diseases in China has always remained high.At present,the clinical treatment mainly focuses on symptomatic treatment to delay the progression of the disease,and there is a lack of economic...The incidence rate of kidney diseases in China has always remained high.At present,the clinical treatment mainly focuses on symptomatic treatment to delay the progression of the disease,and there is a lack of economical and effective treatment methods.MicroRNA plays an important regulatory role in the occurrence and development of diseases.This study aims to explore the role and regulatory mechanism of miR⁃142a⁃3p in adriamycin(ADR)⁃induced renal tubular epithelial cell(TCMK⁃1)injury,with a focus on its potential as a therapeutic target for ADR nephropathy.First,cell viability was assessed using the CCK⁃8 kit,and a mouse renal tubular epithelial cell model induced by ADR was established.Subsequently,alterations in miR⁃142a⁃3p and its target gene ATG16L1 mRNA levels were quantified using RT⁃qPCR.Western blotting was used to detect the protein levels of autophagy marker proteins and pyroptosis marker proteins.Monodansylcadaverin(MDC)staining was performed and the autophagy of cells was detected by flow cytometry.The results showed that the relative expression of miR⁃142a⁃3p in TCMK⁃1 cells induced by ADR was increased and the relative expression of its target gene ATG16L1 was decreased(P<0.0001).Western blotting results showed that the levels of p62(P<0.001)and pyroptosis⁃related proteins(P<0.001)were increased,while the protein levels of autophagy⁃related proteins were decreased(P<0.05).The flow cytometry results showed that there was no difference in the mean fluorescence intensity of autoph⁃agosomes between the ADR group and the autophagosome inhibitor group(3⁃MA group)(P>0.05),indicating that after ADR induction,cell autophagy was inhibited and pyroptosis was enhanced.When the expression of miR⁃142a⁃3p was inhibited by transfecting miR⁃142a⁃3p inhibitor,the relative expression level of the target gene ATG16L1 was restored(P<0.001).Western blotting showed that the protein level of p62(P<0.01)and pyroptosis⁃related proteins(P<0.01)were decreased,and the protein level of autophagy⁃related proteins was restored(P<0.001).Flow cytometry results further indicated that cell autophagy was restored(P<0.0001).In conclusion,ADR targets ATG16L1 through miR⁃142a⁃3p to reduce the autophagy level of TCMK⁃1,and simultaneously activates GSDMD⁃mediated pyroptosis.展开更多
Esophageal cancer(EC)is one of the most common malignancies in the world,and there is no specific treatment drug for esophageal cancer yet.Doramectin(DRM)is a broad-spectrum anti-parasitic drug,and it plays an importa...Esophageal cancer(EC)is one of the most common malignancies in the world,and there is no specific treatment drug for esophageal cancer yet.Doramectin(DRM)is a broad-spectrum anti-parasitic drug,and it plays an important role in the treatment of animal diseases,while DRM has not been reported for the treatment of esophageal squamous cell carcinoma(ESCC).The purpose of this study was to investigate the anticancer effects and potential molecular mechanisms of DRM in ESCC.In the present study,the impact of DRM on the viability of ESCC was examined by methylthiazolyldiphenyl-tetrazolium bromide(MTT).Autophagy was measured by transmission electron microscopy(TEM),Western blot and immunohistochemistry.The apoptosis rate was measured by Western blot,flow cytometry and terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL).Meanwhile,autophagy inhibition was achieved by using chloroquine(CQ).After autophagy inhibition,cell proliferation and cloning ability were significantly inhibited,and the expression level of apoptotic protein was significantly changed compared with that of DRM alone.Additionally,Eca109-derived xenografts were established for testing the DRM-induced autophagy in vivo.It was found that DRM significantly inhibited the proliferation of Eca109 and EC9706 cells in vitro and in vivo in a dose-dependent manner by activating autophagy.DRM was able to significantly repress colony formation in Eca109 and EC9706 cells in vitro.At the same time,DRM could induce apoptosis of ESCC in vitro,it was also regulated through mitochondrial pathways.Meanwhile,DRM induced autophagy and inhibited the proliferation of ESCC,and exhibited little toxicity in organs in vivo.Moreover,DRM-induced autophagy could inhibit the apoptosis of EC in vitro and in vivo.Further experiment suggested that DRM might induce autophagy by the Akt/mTOR pathway.In conclusion,the present study was the first to clarify that DRM could inhibit Eca109 and EC9706 cells proliferation through activating autophagy by the Akt/mTOR pathway.DRM might be a potentially effective treatment for EC.展开更多
Aim Breast cancer is one of the lethal gynecological malignancy in the world. Tamoxifen （TAM） and fulvestrant （FUL） are the major drugs for patients with estrogen receptor-positive （ER ＋ ） breast cancers. Howev...Aim Breast cancer is one of the lethal gynecological malignancy in the world. Tamoxifen （TAM） and fulvestrant （FUL） are the major drugs for patients with estrogen receptor-positive （ER ＋ ） breast cancers. Howev- er, the development of endocrine resistance is the impediment for successful treatment. In this study, we explored the mechanisms of endocrine resistance and therapeutic strategy for overcoming resistance against TAM and FUL. Methods The experiments were performed in Ell ＋ and estrogen/TAM-sensitive MCF7 cells and antiestrogen-re- sistant MCF7/LCC9 cells. Western blot and confocal microscopy were used to determine cell autophagy. Cell trans- fection and luciferase activity assay were performed to identify the target gene of miR-214. Results It showed that 4-OHT/FUL treatment induced apoptosis as well as autophagy in breast cancer cells. The increase of autophagy might be the major cause of endocrine resistance to 4-OHT or FUL. Mill-214 increased the sensitivity of breast cancer cells to the 4-OHT/FUL-induced apoptosis through inhibition of autophagy. Importantly, a negative correla- tion was established between miR-214 and UCP2 in human breast cancer tissue specimens by RT-qPCR assay. UCP2 was identified to be a direct target of mill-214. Further study in MCF7/LCC9 cells indicated that endocrine resistance might arise from activation of the PI3 K-Akt-mTOll pathway, thereby inducing autophagy by overexpres- sion of UCP2. Conclusions MiR-214 increased the sensitivity of breast cancer cells to TAM and FUL through in- hibition of autophagy by targeting UCP2. Mill-214 shows potential as a novel therapeutic strategy for overcoming endocrine resistance in ER ＋ breast cancers.展开更多
OBJECTIVE Glycyrrhetinic acid(GA),one of the main bioactive constituents of the famous Chinese medicinal herb Glycyrrhizauralensis Fisch,presents potent cytotoxicity to various cancer cells both in vitro and in vivo.H...OBJECTIVE Glycyrrhetinic acid(GA),one of the main bioactive constituents of the famous Chinese medicinal herb Glycyrrhizauralensis Fisch,presents potent cytotoxicity to various cancer cells both in vitro and in vivo.Herein,we′d like to determine whether GA triggers autophagy in non-small cell lung cancer cells and the mechanisms involved.METHODS Cell proliferation was determined by MTT assay and colony formation.AnnexinⅤ/PI staining,Hoechst33342 staining,and Western blotting were used to detect GA-induced apoptosis.GA-induced autophagy was measured by expression of the lipid modification of light chain-3(LC3)and transfected with GFP-LC3 or GFP-RFP-LC3 plasmid.Pharmacological regulators,siRNA,and plasmid transfection were used to study the mechanisms of GA-triggered autophagy.RESULTS GA inhibited cell proliferation and induced apoptosis in a concentrationdependent manner in non-small cell lung cancer A549 cells.GA induced autophagyas evidenced by up-regulation of LC3-Ⅱexpression when combined treatment with chloroquine and induction of the red punta after GFP-RFP-LC3 plasmid transfection.Knockdown of autophagy related proteins(ATG)7,ATG 5,or beclin 1by siRNA,the expression of LC3-Ⅱand GFP-LC3 punt atriggered by GA were decreased.Furthermore,the c-Jun N-terminal kinase(JNK)pathways were activated after treatment with GA,and pretreatment with JNK inhibitor SP600125 or silence of JNK pathway by siRNA of JNK or c-jun obviously reduced GA-induced LC3-Ⅱexpression and GFP-LC3 punta formation.GA also stimulate dendoplasmic reticulum stress response by triggering inositol-requiring enzyme 1α(IRE1α)pathway,and knockdown of IRE1αinhibited the activation of JNK pathway and autophagy induced by GA.In addition,GA-induced cell proliferative inhibition and apoptosis were both enhanced when silence of autophagy as well as JNK pathway.CONCLUSION Our study demonstrated,for the first time,that GA induced a cytoprotective autophagy in non-small cell lung cancer cells by activating the IRE1α-JNK pathway,which might decreased the anti-cancer effects of GA.展开更多
OBJECTIVE To explore the potential effect and mechanisms of protopanaxadiol deriva.tive 1-(3,4-dimethoxyphenethyl)-3-(3-dehydroxyl-20(s)-protopa-naxadiol-3 b-yl)-urea(DDPU) in the treatment of Alzheimer disease.METHOD...OBJECTIVE To explore the potential effect and mechanisms of protopanaxadiol deriva.tive 1-(3,4-dimethoxyphenethyl)-3-(3-dehydroxyl-20(s)-protopa-naxadiol-3 b-yl)-urea(DDPU) in the treatment of Alzheimer disease.METHODS ELISA assay was performed in both HEK293-APPswe and CHO-APP cells to demonstrate the efficacy of DDPU in reducing Ab level.SH-SY5 Y,primary neurons and astrocyte cellswereused to study the regulation of DDPU against the signaling pathways involved in Aβ/ER-stress pathology.APP/PS1 transgenic mice wereusedto study the regulation of DDPU against ADL and cognitive deficits.APP/PS1 transgenic mice were randomly placed into three groups(n=10):The two 6-month transgenic groups were administrated with 30 mg·kg^(-1) DDPU or vehicle and the 6-month non-transgenic group was administrated with vehicle for 100 days by intraperitonealinjec.tion.After 100-day administration,nest construction assay and Morris water maze(MWM) assay were applied to evaluate the daily living activities and cognitive abilities of the mice with continuous DDPU treatment.Upon completion of behavior assays,mice were euthanized,and the brains were removed and bisected in mid-sagittal plane.The right hemispheres were frozen and stored at-80°C,and the left hemispheres were fixed in 4% paraformaldehyde.RESULTS DDPU effectively improved learning and memory impairments in APP/PS1 transgenic mice,and the underlying mechanisms have been inten.sively investigated.DDPU reduced Ab production by inhibiting the PERK/eIF2 a signaling-mediated BACE1 translation,while promoted Ab clearance as a PI3K inhibitor thus negatively regulating PI3K/AKT/mTOR signaling in promotion of autophagy.Moreover,DDPU also exhibited neuroprotective effect by attenuating ER stress.Therefore,all findings have clearly demonstrated the crosstalk between Ab and ER stress,and confirmed that targeting ER stress should be a potential target for innovative anti-AD drug development,while highlighted the potential of DDPU in the treatment of AD.展开更多
Tau oligomers are the etiologic molecules of Alzheimer disease(AD), and correlate strongly with neuronal loss and exhibit neurotoxicity. Recent evidence indicates that small tau oligomers are the most relevant toxic a...Tau oligomers are the etiologic molecules of Alzheimer disease(AD), and correlate strongly with neuronal loss and exhibit neurotoxicity. Recent evidence indicates that small tau oligomers are the most relevant toxic aggregate species. The aim of the present study was to investigate the mechanisms of cornel iridoid glycoside(CIG) on tau oligomers and cognitive functions. We injected wortmannin and GF-109203 X(WM/GFX, 200 μmol·L-1 each) into the lateral ventricles to induce tau oligomer and memory impairment in rats. When oral y administered with CIG at 60 and 120 mg·kg-1 per day for 14 d, CIG decreased the escape latency in Morris water maze test. We also found that CIG restored the expression of presynaptic p-synapsin, synaptophysin, and postsynaptic density-95(PSD-95) decreased by WM/GFX in rat cortex. CIG reduced the accumulation of tau oligomers in the brain of WM/GFX rats and in cells transfected with wild type glycogen synthase kinase-3β(wt GSK-3β). In addition, CIG up-regulated the levels of ATG7, ATG12, Beclin-1, and LC3 II in vivo and in vitro, suggesting the restoration of autophagy function. These results suggest that CIG could ameliorate memory deficits and regulate memory-associated synaptic proteins through the clearance of tau oligomers accumulation. Moreover, CIG clears tau oligomers by restoring autophagy function.展开更多
OBJECTIVE To explore the protective effects of total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja on STZ-stimulated INS-1 cells through regulating of autophagy and apoptosis.METHODS INS-1cells wer...OBJECTIVE To explore the protective effects of total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja on STZ-stimulated INS-1 cells through regulating of autophagy and apoptosis.METHODS INS-1cells were cultured in media containing 3n M STZ and different doses of total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja.The proliferation of cells was examined by MTT assay,ROS content were detected by fluorescence enzyme label.The levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),hydrogen peroxidase(CAT),malondialdehyde(MDA)were also measured by colorimetry.The activities of caspase-3,9 were also observed by Caspase colorimetric assay kit in each group.The expressions of Bcl-2 m RNA and Bax m RNA were detected by Real time PCR,protein expression of LC3-Ⅱand PARP were detected by Western blotting.RESULTS Compared with the control group,total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja could promote the proliferation of INS-1 cells no matter with STZ or not when its concentration lower than 25μg·m L^(-1);but when its concentration higher than 100μg·m L^(-1)(use individually)or 50μg·m L^(-1)(combined use),total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja might significantly inhibite the growth of the cells whether STZ existed or not.Total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja(6.25,12.5,25μg·m L^(-1))might inhibit INS-1cell apoptosis,decrease intra-cellular ROS contents,improve the s upernatant liquid SOD,GSH-PX,CAT activities,decrease MDA level,promote INS-1 cell secreting insulin,decrease the protein expressions of autophagy protein LC3Ⅱand apoptosis regulating protein cleaved PARP protein,up-regulate the anti-apoptotic protein Bcl-2 m RNA expression,down-regulate the pro-apoptotic protein Bax m RNA expression and Bcl-2and Bax,reduce the activity of caspase-9 and caspase-3(P<0.05 or P<0.01).CONCLUSION Total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja has good protective effect on STZ-stimulated INS-1cells.It can inhibit STZ injured INS-1 cells to overproduce ROS production,enhance endogenous antioxidant enzymes(GSH-Px,SOD,CAT activities),reduce the expression of autophagy protein LC3Ⅱand apoptosisregulating protein cleaved PARP,up-reglute the antiapoptosis protein Bcl-2 expression,down-regulate the pro-apoptotic protein Bax expression,decrease the caspase-9 and caspase-3 activities,and improve the INS-1cell survival rate,and then play a protective effect on damaged INS-1 cells.展开更多
Porcine parvovirus(PPV) is one of the major causes of reproductive failure in pigs, which poses a great threat to the pig breeding industry and results in tremendous economic losses worldwide. Autophagy is the biologi...Porcine parvovirus(PPV) is one of the major causes of reproductive failure in pigs, which poses a great threat to the pig breeding industry and results in tremendous economic losses worldwide. Autophagy is the biological process of cell self-defense and self-protection. Despite many viruses can cause cell autophagy, when they enter cell or copied, the relationship between autophagy and PPV infection has not been reported. In this study, impact of autophagy after swine testicular(ST) cells infected by PPV was studied. Autophagy was demonstrated by the effective replication of PPV through transmission electron microscopy, immunofluorescence and western blot analysis. Moreover, autophagy was confirmed to benefit PPV replication by real-time fluorescence quantitative PCR and determination of median tissue culture infective dose(TCID). For the first time, the complex interaction between PPV infection and autophagy was explored in this study. It indicated that PPV could induce autophagy in ST cells, which in turn facilitated its own replication, which might be one of the mechanisms of the virus infection. These findings could facilitate the study of the pathogenesis of PPV infection and provide new insight into the development of effective therapeutic strategies.展开更多
OBJECTIVE Inhibition of phosphodiesterase 4(PDE4) improves the learning and memory abilities in Alzheimer disease animal models. The cognition-enhancing effects of PDE4 inhibition involve reduced inflammatory response...OBJECTIVE Inhibition of phosphodiesterase 4(PDE4) improves the learning and memory abilities in Alzheimer disease animal models. The cognition-enhancing effects of PDE4 inhibition involve reduced inflammatory responses in the brain. However,the underlying mechanisms are illunderstood. cA MP induces autophagy,and deficiency of autophagy leads to elevated inflammatory factors.In the present study,we aimed to investigate the contribution of autophagy to the anti-inflammatory effect of PDE4 inhibitor ROF. METHODS Acidic vesicles were traced by Lysotracker(LYT) red and acridine orange(AO) staining. Autophagosomes in BV-2 cells was observed by immunofluorescence staining of microtubule-associated protein 1 light chain 3(LC3). Aβ_(25-35) or lipopolysaccharide(LPS) with ATP were used to activate microglial cells and inflammasome. Cytokine levels were measured by ELISA method. The levels of pro-inflammatory factors and essential proteins involved in the formation of autophagosome were detected by Western blotting. RESULTS ROF increased the level of LC3-Ⅱ,while the level of p62 was decreased. Enhanced fluorescent signals were observed in BV-2 cells treated with ROF by AO and LYT red staining. In addition,immunofluorescence indicated a significant increase in punctate LC3. Both LPS plus ATP and Aβ_(25-35) enhanced the conversion of pro-caspase-1 to cleaved-caspase-1 and increased the production of mature IL-1β. Interestingly,these effects were blocked by the treatment of ROF. Moreover,ROF decreased the apoptosis of neuronal N2 a cells in conditioned media from BV-2 microglia. These effects were reversed by inhibition of microglial autophagy.Treatment with ROF also showedenhanced autophagy in mcie treated with LPS. CONCLUSION PDE4 inhibitor ROF inhibits inflammasome activities and reduces the release of IL-1β by inducing autophagy.展开更多
OBJECTIVE To discover a small-molecule activator of ULK1 for Parkinson disease treatment and exploreits potential mechanisms.METHODS Candidate ULK1 activator was found by using structure-based design and high-through ...OBJECTIVE To discover a small-molecule activator of ULK1 for Parkinson disease treatment and exploreits potential mechanisms.METHODS Candidate ULK1 activator was found by using structure-based design and high-through put screening,then modified by chemical synthesis and screened by kinase and autophgic activities.The amino acid residues that key to the activation site of the best candidate ULK1 activator(BL-918) were determined by site-directed mutagenesis,as well as in vitro kinase assay,ADP-Glo kinase assay and surface plasmon resonance(SPR) analysis.The mechanisms of BL-918 induced cytoprotective autophagy were investigated by electron microscopy,fluorescence microscopy,Western blotting,co-immunoprecipitation assay,si RNA and GFP-LC3 plasmid transfections.The therapeutic effect of BL-918 was determined by MPTP-mouse model,including behavioral tests,the levels of dopamine and its derivatives,as well as immunofluorescence and Western blotting.The toxicity of BL-918 was assessed by blood sample analysis and hematoxylin-eosin staining.RESULTS We discovered a small molecule(BL-918) as a potent activator of ULK1 by structure-based drug design.Subsequently,some key amino acid residues(Arg18,Lys50,Asn86 and Tyr89) were found to be crucial to the binding pocket between ULK1 and BL-918,by site-directed mutagenesis.Moreover,we found that BL-918 could induce autophagy via the ULK complex in neuroblastoma SH-SY5Y cells.Intriguingly,this activator displayed a cytoprotective effect on MPP+-treated SH-SY5Y cells,as well as protected against MPTP-induced motor dysfunction and loss of dopaminergic neurons by targeting ULK1-modulated autophagy in mouse models of PD.CONCLUSION We discovered a novel ULK1 activator(BL-918) that potently activated ULK1.This activator could induce cytoprotective autophagy via the ULK1 complex in SH-SY5Y cells,and also exerted its neuroprotective effects by targeting ULK1-modulated autophagy in a MPTP-induced PD mouse model,which may serve as a candidate drug for future PD therapy.展开更多
Objective:IgA nephropathy(IgAN)is the most common primary glomerular disease in China,but its pathogenesis remains unclear.This study aims to explore the regulatory role of the mammalian target of rapamycin(mTOR)signa...Objective:IgA nephropathy(IgAN)is the most common primary glomerular disease in China,but its pathogenesis remains unclear.This study aims to explore the regulatory role of the mammalian target of rapamycin(mTOR)signaling pathway in autophagy and mesangial proliferation during renal injury in IgA.Methods:The activity of mTOR and autophagy was evaluated in kidney samples from IgAN patients and in an IgAN mouse model induced by oral bovine serum albumin and carbon tetrachloride(CCl4)injection.mTOR inhibitors(rapamycin)and activators[bpV(phen)]were administered to the IgAN mouse model to observe the effects of mTOR on autophagy and renal lesions.In human mesangial cells treated with polymeric IgA1(p IgA1)and mTOR modulators,the expression and distribution of cell cycle proteins were assessed,along with the effects of mTOR on mesangial cell proliferation and autophagy.Results:Increased mTOR activity and decreased autophagy were observed in kidney tissues from IgAN patients and the mouse model,as evidenced by elevated phosphorylated mTOR(p-mTOR)levels and reduced LC3 expression.In the IgAN mouse model,rapamycin inhibited mTOR,restored autophagy,reduced mesangial IgA deposition,alleviated mesangial cell proliferation,and decreased proteinuria(all P<0.05).In contrast,bpV(phen)activated mTOR,further suppressed autophagy,exacerbated kidney damage,and increased proteinuria(all P<0.05).In vitro,p-IgA1 induced mesangial cell proliferation and inhibited autophagy,effects that were reversed by rapamycin and aggravated by bpV(phen)(all P<0.05).mTOR regulated mesangial cell proliferation by altering cell cycle distribution,with rapamycin inducing G1 phase arrest and bpV(phen)promoting cell cycle progression.Additionally,cyclinD1 expression in renal cortex was up-regulated in the IgAN mouse model,further increased by bpV(phen),and reduced by rapamycin(all P<0.05).Conclusion:Inhibition of the mTOR signaling pathway enhances renal autophagy,reduces mesangial cell proliferation,and improves renal injury in IgAN.展开更多
OBJECTIVE Reactive oxygen species(ROS)mediated both apoptosis and protective autophagy in response to natural products while the detailed mechanisms remain unclear.Cucurbitacin B(Cuc B),a natural tetracyclic triterpen...OBJECTIVE Reactive oxygen species(ROS)mediated both apoptosis and protective autophagy in response to natural products while the detailed mechanisms remain unclear.Cucurbitacin B(Cuc B),a natural tetracyclic triterpene,induced both protective autophagy and apoptosis mediated by ROS.This study was designed to explore the mechanism of Cuc B-induced apoptosis and autophagy in hepatocellular carcinoma BEL-7402cells.METHODS AND RESULTS Cuc B decreased cell viability in concentration-and time-dependent manners.Cuc B caused long comet tails and increased expression ofγHA2X,phosphorylation of ATM/ATR,and Chk1/Chk2.This DNA damage response was inhibited by KU55933 and caffeine.Cuc B induced autophagy as evidenced by monodansylcadaverine(MDC)staining,increased expression of LC3Ⅱ,phosphorylated ULK1and decreased expression of phosphorylated AKT,mT OR.Cuc B induced apoptosis mediated by Bcl-2 family proteins and caspase activation.Furthermore,Cuc B induced ROS formation,which was inhibited by N-acetylL-cysteine(NAC).NAC pretreatment dramatical y reversed Cuc B-induced DNA damage,autophagy,and apoptosis.Cuc B-induced apoptosis was reversed by NAC but enhanced by 3-methyladenine(3-MA),chloroquine(CQ),and silencing phosphatase and tensin homolog(PTEN).3-MA and CQ showed no effect on Cuc B-induced DNA damage.In addition,Cuc B increased PTEN phosphorylation.Silence PTEN restored Cuc B-induced autophagic protein expressions without affecting DNA damage.CONCLUSION Cuc B induced DNA damage,apoptosis and protective autophagy mediated by ROS.PTEN activation in response to DNA damage bridged apoptosis and pro-survival autophagy.These observations provide insights for better understanding the crosstalk between apoptosis and autophagy.展开更多
Aim This study is aimed to determine whether SPK2 （Sphingosine kinase-2） is involved in isoflurane preconditioning induced autophagy activation in primary cultured neurons. We also examined whether SPK2 pro- tects n...Aim This study is aimed to determine whether SPK2 （Sphingosine kinase-2） is involved in isoflurane preconditioning induced autophagy activation in primary cultured neurons. We also examined whether SPK2 pro- tects neurons from ischemic injury by activating autophagy, and explored the molecular mechanism of SPK2 contrib- uting to the autophagy activation in neurons. Methods Isoflurane preconditioning （ISO） and oxygen glucose dep- rivation （OGD） model was established in primary cultured murine cortical neurons. Neurons were transfected by siRNA to interfere SPK2 and Beclin 1, or lentivirus to overexpress SPK2. Protein expression of SPK2, LC3, and Beclinl were detected with immunofluorescence and Western blot analysis. The neurons were treated with lysosomal inhibitor ammonium chloride （NH4 C1） to test the autophagy flux. The protection of SPK2 on OGD/R induced neu- ronal death was detected with CCK-8 （cell counting kit-8） and LDH cytotoxicity assay kit. Autophagy inhibitor 3- MA （3-Methyladenine） was used to detect the protection of autophagy on SPK2 induced isehemie tolerance. Co-im- munoprecipitation was used to detect the interaction between Beclin 1 and Bel-2. Results In primary cultured neu- ISO enhanced SPK2 and LC3 immunofluorescenee. SPK2 siRNA inhibits LC3II upregulation induced by rons, ISO. Beclin 1 siRNA also inhibits LC3II upregulation induced by ISO. Lentivirus-indueed SPK2 overexpression in- creased LC3II/LC3I ratio and enhanced the autophagy flux in neurons. SPK2 overexpression also exerted neuropro- teetion against OGD model in cortical neurons, as evidenced by improvement of neuronal morphology, increased cellular viability and reduced LDH leakage, while 3-MA partly abolished the SPK2-induced neuroprotection. After SPK2 overexpression, Beclin 1 siRNA inhibited SPK2 induced LC3II upregulation, and the coimmunoprecipitation of Beclin 1 and Bcl-2 was reduced.. Conclusion ISO increases SPK2 and activates autophagy in neurons. SPK2 or Beclin 1 interference cancels ISO induced autophagy activation. SPK2 overexpression activates autophagy, and protects the neurons against ischemic injury. SPK2 may induce autophagy by disrupting Beclin 1/Bcl-2 interaction.展开更多
Aim To investigate the nephroprotective activity of berberine in diabetic nephropathy （DN） mice. Methods Diabetic nephropathy was induced by intraperitoneal injection with 55 mg · kg^-1 streptozotocin（STZ） , ...Aim To investigate the nephroprotective activity of berberine in diabetic nephropathy （DN） mice. Methods Diabetic nephropathy was induced by intraperitoneal injection with 55 mg · kg^-1 streptozotocin（STZ） , Berberine was administered at daily doses of 50, 100 and 200 mg· kg^-1 by gavage for 8 weeks. To detect serum creatinine and blood urea nitrogen （BUN） levels, blood were collected after the last dose of berberine, renal cortex was separated on ice after heart peffusion by precooled normal saline. The specimen was stored in -80℃ for the next experiments, and some of the kidney tissue were immobilized by 4% paraformaldehyde solution and 3% glut- araldehyde solution for the preparation of paraffin tissue slides and electron microscope biopsy respectively. After that, PAS staining and electron microscope were used to observe the glomerular morphology changes; ELISA was used to measure proinflammatory chemokines and cytokines levels in renal cortex. Real time RT-PCR was taken to detect the level of nucleotide binding oligomerzation domain 2 （NOD2） mRNA, Western blot was used to test the ex- pression of NOD2 and autophagy marker light chain 3 （LC3） in renal cortex. Results Histopathological changes and the increase in serum creatinine and BUN in DN mice were significantly ameliorated by berberine in a dose-de- pendent manner. Additionally, The expression of tumor necrosis factor-or （TNF-α）, interleukin-6 （IL-6） and in- tercellular adhesion molecule-1 （ICAM-1） was markedly suppressed by berberine, indicating the inhibition of in- flammatory response. Treatment of DN mice with berberine also significantly reduced the expression of NOD2 and LC3 in the kidneys. Conclusion The current study showed the nephroprotective activity of berberine in DN mice could be attributed to the inhibition of inflammation and展开更多
Aim Endoplasmic reticulum （ER） stress is increasingly recognized as an important contributor to the pathophysiology of many diseases, and therapeutic interventions that target ER stress response emerge as new thera-...Aim Endoplasmic reticulum （ER） stress is increasingly recognized as an important contributor to the pathophysiology of many diseases, and therapeutic interventions that target ER stress response emerge as new thera- peutic modalities to treat cardiovascular diseases driven by prolonged ER stress. Ginkgolides K （GK） is a diterpene lactone constituent isolated from the leaves of Ginkgo biloba and has been found to possess potent neuroprotective properties. This study is aimed to investigate the cytoprotective effect of GK in cultured cardiomyocytes subjected to ER stress injury. Neonatal rat cardiomyocytes （NRCMs） were treated with ER stress inducer tunicamycin to mimic the ER stress injury. We demonstrated that GK pre-treatment mitigated ER stress-induced apoptosis in tunicamycin treated NRCMs. We observed that the activation of ER-associated degradation （ERAD） and autophagy were in- volved in the ER stress inhibition exerted by GK. These beneficial effects of GK were nearly abolished by the addi- tion of specific short interfering RNA （siRNA） for IRElα and XBP-1. Therefore, we conclude that GK might be a promising therapeutic agent for ER stress-mediated cardiovascular diseases, and ER-associated degradation （ERAD） and autophagy play a vital role in GK mediated cytoprotection.展开更多
Autophagy is a highly evolutionarily conserved pathway that depends on lysosome to degrade misfolded proteins and damaged organelles. Besides canonical autophagy, studies have shown some chemicals could bypass sever...Autophagy is a highly evolutionarily conserved pathway that depends on lysosome to degrade misfolded proteins and damaged organelles. Besides canonical autophagy, studies have shown some chemicals could bypass several core genes to induce autophagy, but the targets and regulatory mechanism is still unclear. In this work one novel chemical, G1, was screened out which could trigger both canonical autophagy and non-canonical autophagy by recruiting Atgl6L1 to pre-autophagosomal site and causing LC3 lipidation. The Gl-induced non-canonical auto- phagy was ULK1, and Beclinl-independent but ubiquitin-like conjugation system-dependent, indicating G1 might target the upstream of Atgl6L1. Moreover, inhibition of V-ATPase by specific V-ATPase inhibitiors could suppress the formation of Gl-indueed autophagosomes in FIP200-defieient MEF cells. While other classic lysosomal inhibi- tors could not block the puneta of Atgl2, Atgl6L1 and LC3, in different stages, suggesting V-ATPase activity in- stead of lysosome function is required for Gl-indueed non-canonical autophagy. These studies broaden the under- standing of different working pattern of autophagy and the crucial roles of V-ATPase in the regulation of different au- tophagy.展开更多
Aim p53 up-regulated modulator of apoptosis （PUMA） is a known apoptosis inducer; however its role in microglial survival remains poorly understood. In addition to the classical transcription factor p53, microRNA- ...Aim p53 up-regulated modulator of apoptosis （PUMA） is a known apoptosis inducer; however its role in microglial survival remains poorly understood. In addition to the classical transcription factor p53, microRNA- 143 （miR-143） is involved in PUMA expression at the post-transcriptional level. Furthermore, they identify unique roles of miR-143/PUMA in mediating microglial survival via the regulation of apoptosis and autophagy interplay. Results Blockage of autophagy accelerated methamphetamine-induced apoptosis, whereas the induction of autoph- agy attenuated the decrease in microglial survival. Moreover, anti-miR-143-dependent PUMA up-regulation re- versed the methamphetamine-induced decrease in microglial survival via the regulation of apoptosis and autophagy. The in vivo relevance of these findings was confirmed in mouse models, which demonstrated that the microinjection of anti-miR-143 into the hippocampus ameliorated the methamphetamine-induced decrease in microglia as well as that observed in heterozygous miR-143 ^＋/- mice. Conclusion These findings provided new insight for the specific contributions of miR-143/PUMA to microglial survival in the context of drug abuse.展开更多
Aim Leptin, a 16 ku hormone from the ob gene, has been identified as an important protein involved obesity. As a chronic metabolic disorder, Obesity is associated with a high risk of developing cardiovascular and meta...Aim Leptin, a 16 ku hormone from the ob gene, has been identified as an important protein involved obesity. As a chronic metabolic disorder, Obesity is associated with a high risk of developing cardiovascular and metabolic diseases, such as heart failure. The main aim of this paper is to probe the effects of leptin on contractile function of cardiomyocyte in adult rat. And the mechanism of the action involved of oxidative stress and autophagy were investigated. Methods Isolated adult rat cardiomyocytes were exposed to leptin （1, 10, and 100 nmol · L^-1） for 1 hour. The calcium transients and contraction in adult rat cardiomyocytes were recorded with SoftEdge MyoCam system. Using the corresponding agents such as apocynin, tempol and rapamycin, the underlying mecha- nism of leptin involved of oxidative stress and autophagy was determined. Werstern blot was employed to evaluate the expression of LC3B and Beclin-1. Results While perfusing the cardiomyocytes with leptin, peak shortening （PS） and maximal velocity of shortening/relengthening （ ± dL/dtmax） of cell shortening were markedly decreased, and prolonged time to 50% relengthening （TR50%）. As well as, baseline （bl） , peak and time to 50% baseline （TB50%） of calcium transient were markedly depressed. Leptin attenuated autophagy as evidenced by decreased LC3-Ⅱ and Beclin-1. All of the abnormalities were significantly attenuated by apocynin, tempol or rapamyein. Conclusion Leptin has depressing effects on intracellular free calcium and myocardial systolic function. Apocy-nin, tempol or rapamycin could block the effects of leptin. The mechanism involved oxidative stress and autophagy.展开更多
Aim It has been widely accepted that autophagy plays a key role in some human diseases such as Par- kinson＇ s disease （PD）. UNC-51-1ike kinasesl （ ULK1 ） has been widely reported to initiate autophagy via its com...Aim It has been widely accepted that autophagy plays a key role in some human diseases such as Par- kinson＇ s disease （PD）. UNC-51-1ike kinasesl （ ULK1 ） has been widely reported to initiate autophagy via its com- plex ULKl-mAtg13-FIP200 at the first stage; however, targeting ULK1 as a therapeutic strategy in PD still remains in its infancy. This study aimed at developing a novel ULK1 activator as candidate drugs for PD therapy and valida- ting the possible mechanism and efficacy in vitro and in vivo. Methods Sequence alignment, phylogenetic analy- sis, homology modeling, molecular dockingand structure modificationwere applied forscreening of candidate com- pounds. Surface plasmon resonance （SPR） analysis and molecular dynamics （MD） simulations were carried outto prove the binding betweenULKland BL-UA07. Observations of cell morphology were executed through several methods including MDC staining and GFP-LC3 transfection. Flow cytometric analysis of MDC was used for quantifi- cation of autophagy ratio. Western blot and RNAi transfection were used to explore the detailed mechanisms of BL- UA07-induced autophagy. Furthermore, an in vivo PD mouse model was established for validating the PD treatment efficacy of BL-UA07. Results After a series of screening and structure modification, a novel compound BL-UA07 targeting ULK1 was obtained, which couldeffectivelybind with its target. Then, our results showed that BL-UA07 could induce autophagy via ULK1 complex and decrease damage induced by MPP ~ in SH-SY5Y cells. In addition, in vivomouse model was established to evaluate the protective effect of BL-UA07. The results demonstrated that BL- UA07 has a therapeutic effect on the in vivomouse model without apparent toxicity, which is dependent on the cyto- protective autophagy mediated by ULK1. Conclusion In this study, a novel specific ULK1 activator （BL-UA07） was computationally designed, chemically synthesized and biologically validatedthat could induce cytoprotective au- tophagy in neuroblastoma SH-SYSY cells and in vivo mouse models. Together, these results may uncover this small-molecule compound BL-UA07 as a novel ULK1 activator in autophagy and thus would provide a new clue for exploring more candidate drug targeting ULK1 for future PD therapy.展开更多
基金supported by the National Natural Science Foundation(82072729)Wuhan Municipal Health Commission Medical Research Project(WX19Q30),China。
文摘Objective:Oxaliplatin(OXA)and 5-fluorouracil(5-FU)are 2 commonly used chemotherapeutic agents for colorectal cancer(CRC).MicroRNAs(miRNAs,miRs)play crucial roles in the development of chemoresistance in various cancers.However,the role and mechanism of miR-224-5p in regulating CRC chemoresistance remain unclear.This study aims to investigate the function of miR-224-5p in chemoresistant CRC cells and the underlying mechanisms.Methods:CRC datasets GSE28702 and GSE69657 were downloaded from the Gene Expression Omnibus(GEO)database.Differentially expressed miRNAs between drug sensitive and resistant groups(OXA or 5-FU)were analyzed,and miR-224-5p was identified as the target miRNA.Chemoresistant cell lines HCT15-OXR,HCT15-5-FU,SW480-OXR,and SW480-5-FU were established.Transient transfections were performed using miR-224-5p mimics,inhibitors,and their respective negative controls(control mimic,control inhibitor)in these cell lines.Cells were treated with different concentrations of OXA or 5-FU post-transfection,and the half-maximal inhibitory concentration(IC_(50))was determined using the cell counting kit-8(CCK-8)assay.Cell proliferation was assessed by CCK-8 and colony formation assays.The expression levels of miR-224-5p,LC3,and P62 were measured by real-time polymerase chain reaction(real-time PCR)and/or Western blotting.Autophagic flux was assessed using a tandem fluorescent-tagged LC3 reporter assay.TargetScan 8.0,miRTarBase,miRPathDB,and HADb were used to predict B-cell lymphoma-2(Bcl-2)as a potential miR-244-5p target,which was further validated by dual luciferase reporter assays.Results:Chemoresistant CRC cells exhibited down-regulated miR-224-5p expression,whereas up-regulation of miR-224-5p enhanced chemotherapy sensitivity.Exposure to OXA or 5-FU significantly increased autophagic activity in chemoresistant CRC cells,which was reversed by miR-224-5p overexpression.Dual-luciferase assays verified Bcl-2 as a direct target of miR-224-5p.Conclusion:MiR-224-5p regulates chemoresistance in CRC by modulating autophagy through direct targeting of Bcl-2.
文摘The incidence rate of kidney diseases in China has always remained high.At present,the clinical treatment mainly focuses on symptomatic treatment to delay the progression of the disease,and there is a lack of economical and effective treatment methods.MicroRNA plays an important regulatory role in the occurrence and development of diseases.This study aims to explore the role and regulatory mechanism of miR⁃142a⁃3p in adriamycin(ADR)⁃induced renal tubular epithelial cell(TCMK⁃1)injury,with a focus on its potential as a therapeutic target for ADR nephropathy.First,cell viability was assessed using the CCK⁃8 kit,and a mouse renal tubular epithelial cell model induced by ADR was established.Subsequently,alterations in miR⁃142a⁃3p and its target gene ATG16L1 mRNA levels were quantified using RT⁃qPCR.Western blotting was used to detect the protein levels of autophagy marker proteins and pyroptosis marker proteins.Monodansylcadaverin(MDC)staining was performed and the autophagy of cells was detected by flow cytometry.The results showed that the relative expression of miR⁃142a⁃3p in TCMK⁃1 cells induced by ADR was increased and the relative expression of its target gene ATG16L1 was decreased(P<0.0001).Western blotting results showed that the levels of p62(P<0.001)and pyroptosis⁃related proteins(P<0.001)were increased,while the protein levels of autophagy⁃related proteins were decreased(P<0.05).The flow cytometry results showed that there was no difference in the mean fluorescence intensity of autoph⁃agosomes between the ADR group and the autophagosome inhibitor group(3⁃MA group)(P>0.05),indicating that after ADR induction,cell autophagy was inhibited and pyroptosis was enhanced.When the expression of miR⁃142a⁃3p was inhibited by transfecting miR⁃142a⁃3p inhibitor,the relative expression level of the target gene ATG16L1 was restored(P<0.001).Western blotting showed that the protein level of p62(P<0.01)and pyroptosis⁃related proteins(P<0.01)were decreased,and the protein level of autophagy⁃related proteins was restored(P<0.001).Flow cytometry results further indicated that cell autophagy was restored(P<0.0001).In conclusion,ADR targets ATG16L1 through miR⁃142a⁃3p to reduce the autophagy level of TCMK⁃1,and simultaneously activates GSDMD⁃mediated pyroptosis.
基金Supported by the Academic Backbone Fund of Northeast Agricultural University(19XG20)the Excellent Young Scholars Fund of Harbin Medical University(HYD2020JQ0016)。
文摘Esophageal cancer(EC)is one of the most common malignancies in the world,and there is no specific treatment drug for esophageal cancer yet.Doramectin(DRM)is a broad-spectrum anti-parasitic drug,and it plays an important role in the treatment of animal diseases,while DRM has not been reported for the treatment of esophageal squamous cell carcinoma(ESCC).The purpose of this study was to investigate the anticancer effects and potential molecular mechanisms of DRM in ESCC.In the present study,the impact of DRM on the viability of ESCC was examined by methylthiazolyldiphenyl-tetrazolium bromide(MTT).Autophagy was measured by transmission electron microscopy(TEM),Western blot and immunohistochemistry.The apoptosis rate was measured by Western blot,flow cytometry and terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL).Meanwhile,autophagy inhibition was achieved by using chloroquine(CQ).After autophagy inhibition,cell proliferation and cloning ability were significantly inhibited,and the expression level of apoptotic protein was significantly changed compared with that of DRM alone.Additionally,Eca109-derived xenografts were established for testing the DRM-induced autophagy in vivo.It was found that DRM significantly inhibited the proliferation of Eca109 and EC9706 cells in vitro and in vivo in a dose-dependent manner by activating autophagy.DRM was able to significantly repress colony formation in Eca109 and EC9706 cells in vitro.At the same time,DRM could induce apoptosis of ESCC in vitro,it was also regulated through mitochondrial pathways.Meanwhile,DRM induced autophagy and inhibited the proliferation of ESCC,and exhibited little toxicity in organs in vivo.Moreover,DRM-induced autophagy could inhibit the apoptosis of EC in vitro and in vivo.Further experiment suggested that DRM might induce autophagy by the Akt/mTOR pathway.In conclusion,the present study was the first to clarify that DRM could inhibit Eca109 and EC9706 cells proliferation through activating autophagy by the Akt/mTOR pathway.DRM might be a potentially effective treatment for EC.
文摘Aim Breast cancer is one of the lethal gynecological malignancy in the world. Tamoxifen （TAM） and fulvestrant （FUL） are the major drugs for patients with estrogen receptor-positive （ER ＋ ） breast cancers. Howev- er, the development of endocrine resistance is the impediment for successful treatment. In this study, we explored the mechanisms of endocrine resistance and therapeutic strategy for overcoming resistance against TAM and FUL. Methods The experiments were performed in Ell ＋ and estrogen/TAM-sensitive MCF7 cells and antiestrogen-re- sistant MCF7/LCC9 cells. Western blot and confocal microscopy were used to determine cell autophagy. Cell trans- fection and luciferase activity assay were performed to identify the target gene of miR-214. Results It showed that 4-OHT/FUL treatment induced apoptosis as well as autophagy in breast cancer cells. The increase of autophagy might be the major cause of endocrine resistance to 4-OHT or FUL. Mill-214 increased the sensitivity of breast cancer cells to the 4-OHT/FUL-induced apoptosis through inhibition of autophagy. Importantly, a negative correla- tion was established between miR-214 and UCP2 in human breast cancer tissue specimens by RT-qPCR assay. UCP2 was identified to be a direct target of mill-214. Further study in MCF7/LCC9 cells indicated that endocrine resistance might arise from activation of the PI3 K-Akt-mTOll pathway, thereby inducing autophagy by overexpres- sion of UCP2. Conclusions MiR-214 increased the sensitivity of breast cancer cells to TAM and FUL through in- hibition of autophagy by targeting UCP2. Mill-214 shows potential as a novel therapeutic strategy for overcoming endocrine resistance in ER ＋ breast cancers.
基金The project supported by Science and Technology Development Fund,Macao S.A.R(FDCT)(074/2012/A)the Research Fund of University of Macao(MRG013/WYT/2013/ICMS,MYRG2015-00101-ICMS-QRCMand CPG2014-00012-ICMS)
文摘OBJECTIVE Glycyrrhetinic acid(GA),one of the main bioactive constituents of the famous Chinese medicinal herb Glycyrrhizauralensis Fisch,presents potent cytotoxicity to various cancer cells both in vitro and in vivo.Herein,we′d like to determine whether GA triggers autophagy in non-small cell lung cancer cells and the mechanisms involved.METHODS Cell proliferation was determined by MTT assay and colony formation.AnnexinⅤ/PI staining,Hoechst33342 staining,and Western blotting were used to detect GA-induced apoptosis.GA-induced autophagy was measured by expression of the lipid modification of light chain-3(LC3)and transfected with GFP-LC3 or GFP-RFP-LC3 plasmid.Pharmacological regulators,siRNA,and plasmid transfection were used to study the mechanisms of GA-triggered autophagy.RESULTS GA inhibited cell proliferation and induced apoptosis in a concentrationdependent manner in non-small cell lung cancer A549 cells.GA induced autophagyas evidenced by up-regulation of LC3-Ⅱexpression when combined treatment with chloroquine and induction of the red punta after GFP-RFP-LC3 plasmid transfection.Knockdown of autophagy related proteins(ATG)7,ATG 5,or beclin 1by siRNA,the expression of LC3-Ⅱand GFP-LC3 punt atriggered by GA were decreased.Furthermore,the c-Jun N-terminal kinase(JNK)pathways were activated after treatment with GA,and pretreatment with JNK inhibitor SP600125 or silence of JNK pathway by siRNA of JNK or c-jun obviously reduced GA-induced LC3-Ⅱexpression and GFP-LC3 punta formation.GA also stimulate dendoplasmic reticulum stress response by triggering inositol-requiring enzyme 1α(IRE1α)pathway,and knockdown of IRE1αinhibited the activation of JNK pathway and autophagy induced by GA.In addition,GA-induced cell proliferative inhibition and apoptosis were both enhanced when silence of autophagy as well as JNK pathway.CONCLUSION Our study demonstrated,for the first time,that GA induced a cytoprotective autophagy in non-small cell lung cancer cells by activating the IRE1α-JNK pathway,which might decreased the anti-cancer effects of GA.
基金supported by National Natural Science Foundation of China(81220108025,81473141) NSFC-TRF collaboration projects(81561148011,DBG5980001)+3 种基金 Drug Innovation Project of SIMM(CASIMM0120154035) Personalized Medicine-Molecular Signature-based Drug Discovery and Development, Strategic Priority Research Program of Chinese Academy of Sciences (XDA12040303) the Project Funded by the Priority Aca-demic Program Development of Jiangsu Higher Education Institutions (Integration of Chinese and Western Medi-cine17KJA350002) National Natural Science Foundation for Young Scientists of China (81703806)
文摘OBJECTIVE To explore the potential effect and mechanisms of protopanaxadiol deriva.tive 1-(3,4-dimethoxyphenethyl)-3-(3-dehydroxyl-20(s)-protopa-naxadiol-3 b-yl)-urea(DDPU) in the treatment of Alzheimer disease.METHODS ELISA assay was performed in both HEK293-APPswe and CHO-APP cells to demonstrate the efficacy of DDPU in reducing Ab level.SH-SY5 Y,primary neurons and astrocyte cellswereused to study the regulation of DDPU against the signaling pathways involved in Aβ/ER-stress pathology.APP/PS1 transgenic mice wereusedto study the regulation of DDPU against ADL and cognitive deficits.APP/PS1 transgenic mice were randomly placed into three groups(n=10):The two 6-month transgenic groups were administrated with 30 mg·kg^(-1) DDPU or vehicle and the 6-month non-transgenic group was administrated with vehicle for 100 days by intraperitonealinjec.tion.After 100-day administration,nest construction assay and Morris water maze(MWM) assay were applied to evaluate the daily living activities and cognitive abilities of the mice with continuous DDPU treatment.Upon completion of behavior assays,mice were euthanized,and the brains were removed and bisected in mid-sagittal plane.The right hemispheres were frozen and stored at-80°C,and the left hemispheres were fixed in 4% paraformaldehyde.RESULTS DDPU effectively improved learning and memory impairments in APP/PS1 transgenic mice,and the underlying mechanisms have been inten.sively investigated.DDPU reduced Ab production by inhibiting the PERK/eIF2 a signaling-mediated BACE1 translation,while promoted Ab clearance as a PI3K inhibitor thus negatively regulating PI3K/AKT/mTOR signaling in promotion of autophagy.Moreover,DDPU also exhibited neuroprotective effect by attenuating ER stress.Therefore,all findings have clearly demonstrated the crosstalk between Ab and ER stress,and confirmed that targeting ER stress should be a potential target for innovative anti-AD drug development,while highlighted the potential of DDPU in the treatment of AD.
文摘Tau oligomers are the etiologic molecules of Alzheimer disease(AD), and correlate strongly with neuronal loss and exhibit neurotoxicity. Recent evidence indicates that small tau oligomers are the most relevant toxic aggregate species. The aim of the present study was to investigate the mechanisms of cornel iridoid glycoside(CIG) on tau oligomers and cognitive functions. We injected wortmannin and GF-109203 X(WM/GFX, 200 μmol·L-1 each) into the lateral ventricles to induce tau oligomer and memory impairment in rats. When oral y administered with CIG at 60 and 120 mg·kg-1 per day for 14 d, CIG decreased the escape latency in Morris water maze test. We also found that CIG restored the expression of presynaptic p-synapsin, synaptophysin, and postsynaptic density-95(PSD-95) decreased by WM/GFX in rat cortex. CIG reduced the accumulation of tau oligomers in the brain of WM/GFX rats and in cells transfected with wild type glycogen synthase kinase-3β(wt GSK-3β). In addition, CIG up-regulated the levels of ATG7, ATG12, Beclin-1, and LC3 II in vivo and in vitro, suggesting the restoration of autophagy function. These results suggest that CIG could ameliorate memory deficits and regulate memory-associated synaptic proteins through the clearance of tau oligomers accumulation. Moreover, CIG clears tau oligomers by restoring autophagy function.
基金The project supported by National Natural and Science Foundation of China(81341141)Yichang Qingqianliu Biotechnology Co.,Ltd.(SDHZ201602)Science and Technology Bureau of Yichang city(A15301-25)
文摘OBJECTIVE To explore the protective effects of total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja on STZ-stimulated INS-1 cells through regulating of autophagy and apoptosis.METHODS INS-1cells were cultured in media containing 3n M STZ and different doses of total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja.The proliferation of cells was examined by MTT assay,ROS content were detected by fluorescence enzyme label.The levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),hydrogen peroxidase(CAT),malondialdehyde(MDA)were also measured by colorimetry.The activities of caspase-3,9 were also observed by Caspase colorimetric assay kit in each group.The expressions of Bcl-2 m RNA and Bax m RNA were detected by Real time PCR,protein expression of LC3-Ⅱand PARP were detected by Western blotting.RESULTS Compared with the control group,total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja could promote the proliferation of INS-1 cells no matter with STZ or not when its concentration lower than 25μg·m L^(-1);but when its concentration higher than 100μg·m L^(-1)(use individually)or 50μg·m L^(-1)(combined use),total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja might significantly inhibite the growth of the cells whether STZ existed or not.Total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja(6.25,12.5,25μg·m L^(-1))might inhibit INS-1cell apoptosis,decrease intra-cellular ROS contents,improve the s upernatant liquid SOD,GSH-PX,CAT activities,decrease MDA level,promote INS-1 cell secreting insulin,decrease the protein expressions of autophagy protein LC3Ⅱand apoptosis regulating protein cleaved PARP protein,up-regulate the anti-apoptotic protein Bcl-2 m RNA expression,down-regulate the pro-apoptotic protein Bax m RNA expression and Bcl-2and Bax,reduce the activity of caspase-9 and caspase-3(P<0.05 or P<0.01).CONCLUSION Total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja has good protective effect on STZ-stimulated INS-1cells.It can inhibit STZ injured INS-1 cells to overproduce ROS production,enhance endogenous antioxidant enzymes(GSH-Px,SOD,CAT activities),reduce the expression of autophagy protein LC3Ⅱand apoptosisregulating protein cleaved PARP,up-reglute the antiapoptosis protein Bcl-2 expression,down-regulate the pro-apoptotic protein Bax expression,decrease the caspase-9 and caspase-3 activities,and improve the INS-1cell survival rate,and then play a protective effect on damaged INS-1 cells.
基金Supported by the National Natural Science Foundation of China(31372438,31201911)
文摘Porcine parvovirus(PPV) is one of the major causes of reproductive failure in pigs, which poses a great threat to the pig breeding industry and results in tremendous economic losses worldwide. Autophagy is the biological process of cell self-defense and self-protection. Despite many viruses can cause cell autophagy, when they enter cell or copied, the relationship between autophagy and PPV infection has not been reported. In this study, impact of autophagy after swine testicular(ST) cells infected by PPV was studied. Autophagy was demonstrated by the effective replication of PPV through transmission electron microscopy, immunofluorescence and western blot analysis. Moreover, autophagy was confirmed to benefit PPV replication by real-time fluorescence quantitative PCR and determination of median tissue culture infective dose(TCID). For the first time, the complex interaction between PPV infection and autophagy was explored in this study. It indicated that PPV could induce autophagy in ST cells, which in turn facilitated its own replication, which might be one of the mechanisms of the virus infection. These findings could facilitate the study of the pathogenesis of PPV infection and provide new insight into the development of effective therapeutic strategies.
基金The project supported by National Natural Science Foundation of China(81373384)
文摘OBJECTIVE Inhibition of phosphodiesterase 4(PDE4) improves the learning and memory abilities in Alzheimer disease animal models. The cognition-enhancing effects of PDE4 inhibition involve reduced inflammatory responses in the brain. However,the underlying mechanisms are illunderstood. cA MP induces autophagy,and deficiency of autophagy leads to elevated inflammatory factors.In the present study,we aimed to investigate the contribution of autophagy to the anti-inflammatory effect of PDE4 inhibitor ROF. METHODS Acidic vesicles were traced by Lysotracker(LYT) red and acridine orange(AO) staining. Autophagosomes in BV-2 cells was observed by immunofluorescence staining of microtubule-associated protein 1 light chain 3(LC3). Aβ_(25-35) or lipopolysaccharide(LPS) with ATP were used to activate microglial cells and inflammasome. Cytokine levels were measured by ELISA method. The levels of pro-inflammatory factors and essential proteins involved in the formation of autophagosome were detected by Western blotting. RESULTS ROF increased the level of LC3-Ⅱ,while the level of p62 was decreased. Enhanced fluorescent signals were observed in BV-2 cells treated with ROF by AO and LYT red staining. In addition,immunofluorescence indicated a significant increase in punctate LC3. Both LPS plus ATP and Aβ_(25-35) enhanced the conversion of pro-caspase-1 to cleaved-caspase-1 and increased the production of mature IL-1β. Interestingly,these effects were blocked by the treatment of ROF. Moreover,ROF decreased the apoptosis of neuronal N2 a cells in conditioned media from BV-2 microglia. These effects were reversed by inhibition of microglial autophagy.Treatment with ROF also showedenhanced autophagy in mcie treated with LPS. CONCLUSION PDE4 inhibitor ROF inhibits inflammasome activities and reduces the release of IL-1β by inducing autophagy.
基金supported by National Natural Science Foundation of China(81602953)China Postdoctoral Special Science Foundation(2017T100706)China Postdoctoral Science Foundation(2016M590893)
文摘OBJECTIVE To discover a small-molecule activator of ULK1 for Parkinson disease treatment and exploreits potential mechanisms.METHODS Candidate ULK1 activator was found by using structure-based design and high-through put screening,then modified by chemical synthesis and screened by kinase and autophgic activities.The amino acid residues that key to the activation site of the best candidate ULK1 activator(BL-918) were determined by site-directed mutagenesis,as well as in vitro kinase assay,ADP-Glo kinase assay and surface plasmon resonance(SPR) analysis.The mechanisms of BL-918 induced cytoprotective autophagy were investigated by electron microscopy,fluorescence microscopy,Western blotting,co-immunoprecipitation assay,si RNA and GFP-LC3 plasmid transfections.The therapeutic effect of BL-918 was determined by MPTP-mouse model,including behavioral tests,the levels of dopamine and its derivatives,as well as immunofluorescence and Western blotting.The toxicity of BL-918 was assessed by blood sample analysis and hematoxylin-eosin staining.RESULTS We discovered a small molecule(BL-918) as a potent activator of ULK1 by structure-based drug design.Subsequently,some key amino acid residues(Arg18,Lys50,Asn86 and Tyr89) were found to be crucial to the binding pocket between ULK1 and BL-918,by site-directed mutagenesis.Moreover,we found that BL-918 could induce autophagy via the ULK complex in neuroblastoma SH-SY5Y cells.Intriguingly,this activator displayed a cytoprotective effect on MPP+-treated SH-SY5Y cells,as well as protected against MPTP-induced motor dysfunction and loss of dopaminergic neurons by targeting ULK1-modulated autophagy in mouse models of PD.CONCLUSION We discovered a novel ULK1 activator(BL-918) that potently activated ULK1.This activator could induce cytoprotective autophagy via the ULK1 complex in SH-SY5Y cells,and also exerted its neuroprotective effects by targeting ULK1-modulated autophagy in a MPTP-induced PD mouse model,which may serve as a candidate drug for future PD therapy.
基金supported by the Key Funding Project of Hunan Provincial Health Commission(A202303057091)the Nephrology Medical Development Research Fund Project(20220316ZN),China.
文摘Objective:IgA nephropathy(IgAN)is the most common primary glomerular disease in China,but its pathogenesis remains unclear.This study aims to explore the regulatory role of the mammalian target of rapamycin(mTOR)signaling pathway in autophagy and mesangial proliferation during renal injury in IgA.Methods:The activity of mTOR and autophagy was evaluated in kidney samples from IgAN patients and in an IgAN mouse model induced by oral bovine serum albumin and carbon tetrachloride(CCl4)injection.mTOR inhibitors(rapamycin)and activators[bpV(phen)]were administered to the IgAN mouse model to observe the effects of mTOR on autophagy and renal lesions.In human mesangial cells treated with polymeric IgA1(p IgA1)and mTOR modulators,the expression and distribution of cell cycle proteins were assessed,along with the effects of mTOR on mesangial cell proliferation and autophagy.Results:Increased mTOR activity and decreased autophagy were observed in kidney tissues from IgAN patients and the mouse model,as evidenced by elevated phosphorylated mTOR(p-mTOR)levels and reduced LC3 expression.In the IgAN mouse model,rapamycin inhibited mTOR,restored autophagy,reduced mesangial IgA deposition,alleviated mesangial cell proliferation,and decreased proteinuria(all P<0.05).In contrast,bpV(phen)activated mTOR,further suppressed autophagy,exacerbated kidney damage,and increased proteinuria(all P<0.05).In vitro,p-IgA1 induced mesangial cell proliferation and inhibited autophagy,effects that were reversed by rapamycin and aggravated by bpV(phen)(all P<0.05).mTOR regulated mesangial cell proliferation by altering cell cycle distribution,with rapamycin inducing G1 phase arrest and bpV(phen)promoting cell cycle progression.Additionally,cyclinD1 expression in renal cortex was up-regulated in the IgAN mouse model,further increased by bpV(phen),and reduced by rapamycin(all P<0.05).Conclusion:Inhibition of the mTOR signaling pathway enhances renal autophagy,reduces mesangial cell proliferation,and improves renal injury in IgAN.
基金The project supported by Science and Technology Development Fund,Macao S.A.R(FDCT)(039/2014/A1)the Research Fund of University of Macao(MYRG2016-00043-ICMS-QRCM)
文摘OBJECTIVE Reactive oxygen species(ROS)mediated both apoptosis and protective autophagy in response to natural products while the detailed mechanisms remain unclear.Cucurbitacin B(Cuc B),a natural tetracyclic triterpene,induced both protective autophagy and apoptosis mediated by ROS.This study was designed to explore the mechanism of Cuc B-induced apoptosis and autophagy in hepatocellular carcinoma BEL-7402cells.METHODS AND RESULTS Cuc B decreased cell viability in concentration-and time-dependent manners.Cuc B caused long comet tails and increased expression ofγHA2X,phosphorylation of ATM/ATR,and Chk1/Chk2.This DNA damage response was inhibited by KU55933 and caffeine.Cuc B induced autophagy as evidenced by monodansylcadaverine(MDC)staining,increased expression of LC3Ⅱ,phosphorylated ULK1and decreased expression of phosphorylated AKT,mT OR.Cuc B induced apoptosis mediated by Bcl-2 family proteins and caspase activation.Furthermore,Cuc B induced ROS formation,which was inhibited by N-acetylL-cysteine(NAC).NAC pretreatment dramatical y reversed Cuc B-induced DNA damage,autophagy,and apoptosis.Cuc B-induced apoptosis was reversed by NAC but enhanced by 3-methyladenine(3-MA),chloroquine(CQ),and silencing phosphatase and tensin homolog(PTEN).3-MA and CQ showed no effect on Cuc B-induced DNA damage.In addition,Cuc B increased PTEN phosphorylation.Silence PTEN restored Cuc B-induced autophagic protein expressions without affecting DNA damage.CONCLUSION Cuc B induced DNA damage,apoptosis and protective autophagy mediated by ROS.PTEN activation in response to DNA damage bridged apoptosis and pro-survival autophagy.These observations provide insights for better understanding the crosstalk between apoptosis and autophagy.
文摘Aim This study is aimed to determine whether SPK2 （Sphingosine kinase-2） is involved in isoflurane preconditioning induced autophagy activation in primary cultured neurons. We also examined whether SPK2 pro- tects neurons from ischemic injury by activating autophagy, and explored the molecular mechanism of SPK2 contrib- uting to the autophagy activation in neurons. Methods Isoflurane preconditioning （ISO） and oxygen glucose dep- rivation （OGD） model was established in primary cultured murine cortical neurons. Neurons were transfected by siRNA to interfere SPK2 and Beclin 1, or lentivirus to overexpress SPK2. Protein expression of SPK2, LC3, and Beclinl were detected with immunofluorescence and Western blot analysis. The neurons were treated with lysosomal inhibitor ammonium chloride （NH4 C1） to test the autophagy flux. The protection of SPK2 on OGD/R induced neu- ronal death was detected with CCK-8 （cell counting kit-8） and LDH cytotoxicity assay kit. Autophagy inhibitor 3- MA （3-Methyladenine） was used to detect the protection of autophagy on SPK2 induced isehemie tolerance. Co-im- munoprecipitation was used to detect the interaction between Beclin 1 and Bel-2. Results In primary cultured neu- ISO enhanced SPK2 and LC3 immunofluorescenee. SPK2 siRNA inhibits LC3II upregulation induced by rons, ISO. Beclin 1 siRNA also inhibits LC3II upregulation induced by ISO. Lentivirus-indueed SPK2 overexpression in- creased LC3II/LC3I ratio and enhanced the autophagy flux in neurons. SPK2 overexpression also exerted neuropro- teetion against OGD model in cortical neurons, as evidenced by improvement of neuronal morphology, increased cellular viability and reduced LDH leakage, while 3-MA partly abolished the SPK2-induced neuroprotection. After SPK2 overexpression, Beclin 1 siRNA inhibited SPK2 induced LC3II upregulation, and the coimmunoprecipitation of Beclin 1 and Bcl-2 was reduced.. Conclusion ISO increases SPK2 and activates autophagy in neurons. SPK2 or Beclin 1 interference cancels ISO induced autophagy activation. SPK2 overexpression activates autophagy, and protects the neurons against ischemic injury. SPK2 may induce autophagy by disrupting Beclin 1/Bcl-2 interaction.
文摘Aim To investigate the nephroprotective activity of berberine in diabetic nephropathy （DN） mice. Methods Diabetic nephropathy was induced by intraperitoneal injection with 55 mg · kg^-1 streptozotocin（STZ） , Berberine was administered at daily doses of 50, 100 and 200 mg· kg^-1 by gavage for 8 weeks. To detect serum creatinine and blood urea nitrogen （BUN） levels, blood were collected after the last dose of berberine, renal cortex was separated on ice after heart peffusion by precooled normal saline. The specimen was stored in -80℃ for the next experiments, and some of the kidney tissue were immobilized by 4% paraformaldehyde solution and 3% glut- araldehyde solution for the preparation of paraffin tissue slides and electron microscope biopsy respectively. After that, PAS staining and electron microscope were used to observe the glomerular morphology changes; ELISA was used to measure proinflammatory chemokines and cytokines levels in renal cortex. Real time RT-PCR was taken to detect the level of nucleotide binding oligomerzation domain 2 （NOD2） mRNA, Western blot was used to test the ex- pression of NOD2 and autophagy marker light chain 3 （LC3） in renal cortex. Results Histopathological changes and the increase in serum creatinine and BUN in DN mice were significantly ameliorated by berberine in a dose-de- pendent manner. Additionally, The expression of tumor necrosis factor-or （TNF-α）, interleukin-6 （IL-6） and in- tercellular adhesion molecule-1 （ICAM-1） was markedly suppressed by berberine, indicating the inhibition of in- flammatory response. Treatment of DN mice with berberine also significantly reduced the expression of NOD2 and LC3 in the kidneys. Conclusion The current study showed the nephroprotective activity of berberine in DN mice could be attributed to the inhibition of inflammation and
文摘Aim Endoplasmic reticulum （ER） stress is increasingly recognized as an important contributor to the pathophysiology of many diseases, and therapeutic interventions that target ER stress response emerge as new thera- peutic modalities to treat cardiovascular diseases driven by prolonged ER stress. Ginkgolides K （GK） is a diterpene lactone constituent isolated from the leaves of Ginkgo biloba and has been found to possess potent neuroprotective properties. This study is aimed to investigate the cytoprotective effect of GK in cultured cardiomyocytes subjected to ER stress injury. Neonatal rat cardiomyocytes （NRCMs） were treated with ER stress inducer tunicamycin to mimic the ER stress injury. We demonstrated that GK pre-treatment mitigated ER stress-induced apoptosis in tunicamycin treated NRCMs. We observed that the activation of ER-associated degradation （ERAD） and autophagy were in- volved in the ER stress inhibition exerted by GK. These beneficial effects of GK were nearly abolished by the addi- tion of specific short interfering RNA （siRNA） for IRElα and XBP-1. Therefore, we conclude that GK might be a promising therapeutic agent for ER stress-mediated cardiovascular diseases, and ER-associated degradation （ERAD） and autophagy play a vital role in GK mediated cytoprotection.
文摘Autophagy is a highly evolutionarily conserved pathway that depends on lysosome to degrade misfolded proteins and damaged organelles. Besides canonical autophagy, studies have shown some chemicals could bypass several core genes to induce autophagy, but the targets and regulatory mechanism is still unclear. In this work one novel chemical, G1, was screened out which could trigger both canonical autophagy and non-canonical autophagy by recruiting Atgl6L1 to pre-autophagosomal site and causing LC3 lipidation. The Gl-induced non-canonical auto- phagy was ULK1, and Beclinl-independent but ubiquitin-like conjugation system-dependent, indicating G1 might target the upstream of Atgl6L1. Moreover, inhibition of V-ATPase by specific V-ATPase inhibitiors could suppress the formation of Gl-indueed autophagosomes in FIP200-defieient MEF cells. While other classic lysosomal inhibi- tors could not block the puneta of Atgl2, Atgl6L1 and LC3, in different stages, suggesting V-ATPase activity in- stead of lysosome function is required for Gl-indueed non-canonical autophagy. These studies broaden the under- standing of different working pattern of autophagy and the crucial roles of V-ATPase in the regulation of different au- tophagy.
文摘Aim p53 up-regulated modulator of apoptosis （PUMA） is a known apoptosis inducer; however its role in microglial survival remains poorly understood. In addition to the classical transcription factor p53, microRNA- 143 （miR-143） is involved in PUMA expression at the post-transcriptional level. Furthermore, they identify unique roles of miR-143/PUMA in mediating microglial survival via the regulation of apoptosis and autophagy interplay. Results Blockage of autophagy accelerated methamphetamine-induced apoptosis, whereas the induction of autoph- agy attenuated the decrease in microglial survival. Moreover, anti-miR-143-dependent PUMA up-regulation re- versed the methamphetamine-induced decrease in microglial survival via the regulation of apoptosis and autophagy. The in vivo relevance of these findings was confirmed in mouse models, which demonstrated that the microinjection of anti-miR-143 into the hippocampus ameliorated the methamphetamine-induced decrease in microglia as well as that observed in heterozygous miR-143 ^＋/- mice. Conclusion These findings provided new insight for the specific contributions of miR-143/PUMA to microglial survival in the context of drug abuse.
文摘Aim Leptin, a 16 ku hormone from the ob gene, has been identified as an important protein involved obesity. As a chronic metabolic disorder, Obesity is associated with a high risk of developing cardiovascular and metabolic diseases, such as heart failure. The main aim of this paper is to probe the effects of leptin on contractile function of cardiomyocyte in adult rat. And the mechanism of the action involved of oxidative stress and autophagy were investigated. Methods Isolated adult rat cardiomyocytes were exposed to leptin （1, 10, and 100 nmol · L^-1） for 1 hour. The calcium transients and contraction in adult rat cardiomyocytes were recorded with SoftEdge MyoCam system. Using the corresponding agents such as apocynin, tempol and rapamycin, the underlying mecha- nism of leptin involved of oxidative stress and autophagy was determined. Werstern blot was employed to evaluate the expression of LC3B and Beclin-1. Results While perfusing the cardiomyocytes with leptin, peak shortening （PS） and maximal velocity of shortening/relengthening （ ± dL/dtmax） of cell shortening were markedly decreased, and prolonged time to 50% relengthening （TR50%）. As well as, baseline （bl） , peak and time to 50% baseline （TB50%） of calcium transient were markedly depressed. Leptin attenuated autophagy as evidenced by decreased LC3-Ⅱ and Beclin-1. All of the abnormalities were significantly attenuated by apocynin, tempol or rapamyein. Conclusion Leptin has depressing effects on intracellular free calcium and myocardial systolic function. Apocy-nin, tempol or rapamycin could block the effects of leptin. The mechanism involved oxidative stress and autophagy.
文摘Aim It has been widely accepted that autophagy plays a key role in some human diseases such as Par- kinson＇ s disease （PD）. UNC-51-1ike kinasesl （ ULK1 ） has been widely reported to initiate autophagy via its com- plex ULKl-mAtg13-FIP200 at the first stage; however, targeting ULK1 as a therapeutic strategy in PD still remains in its infancy. This study aimed at developing a novel ULK1 activator as candidate drugs for PD therapy and valida- ting the possible mechanism and efficacy in vitro and in vivo. Methods Sequence alignment, phylogenetic analy- sis, homology modeling, molecular dockingand structure modificationwere applied forscreening of candidate com- pounds. Surface plasmon resonance （SPR） analysis and molecular dynamics （MD） simulations were carried outto prove the binding betweenULKland BL-UA07. Observations of cell morphology were executed through several methods including MDC staining and GFP-LC3 transfection. Flow cytometric analysis of MDC was used for quantifi- cation of autophagy ratio. Western blot and RNAi transfection were used to explore the detailed mechanisms of BL- UA07-induced autophagy. Furthermore, an in vivo PD mouse model was established for validating the PD treatment efficacy of BL-UA07. Results After a series of screening and structure modification, a novel compound BL-UA07 targeting ULK1 was obtained, which couldeffectivelybind with its target. Then, our results showed that BL-UA07 could induce autophagy via ULK1 complex and decrease damage induced by MPP ~ in SH-SY5Y cells. In addition, in vivomouse model was established to evaluate the protective effect of BL-UA07. The results demonstrated that BL- UA07 has a therapeutic effect on the in vivomouse model without apparent toxicity, which is dependent on the cyto- protective autophagy mediated by ULK1. Conclusion In this study, a novel specific ULK1 activator （BL-UA07） was computationally designed, chemically synthesized and biologically validatedthat could induce cytoprotective au- tophagy in neuroblastoma SH-SYSY cells and in vivo mouse models. Together, these results may uncover this small-molecule compound BL-UA07 as a novel ULK1 activator in autophagy and thus would provide a new clue for exploring more candidate drug targeting ULK1 for future PD therapy.
