Momordica antiviral protein 30 kD(MAP30)is a type I ribosome-inactivating protein(RIP)with antibacterial,anti-HIV and antitumor activities but lacks the ability to target tumor cells.To increase its tumor-targeting ab...Momordica antiviral protein 30 kD(MAP30)is a type I ribosome-inactivating protein(RIP)with antibacterial,anti-HIV and antitumor activities but lacks the ability to target tumor cells.To increase its tumor-targeting ability,the arginine-glycine-aspartic(RGD)peptide and the epidermal growth factor receptor interference(EGFRi)peptide were fused with MAP30,which was named ELRL-MAP30.The efficiency of targeted therapy for triple-negative breast cancer(TNBC)MDA-MB-231 cells,which lack the expression of estrogen receptor(ER),Progesterone receptor(PgR)and human epidermal growth factor receptor-2(HER2),is limited.In this study,we focus on exploring the effect and mechanism of ELRL-MAP30 on TNBC MDA-MB-231 cells.First,we discovered that ELRL-MAP30 significantly inhibited the migration and invasion of MDA-MB-231 cells and induced MDA-MB-231 cell apoptosis.Moreover,ELRL-MAP30 treatment resulted in a significant increase in Bax expression and a decrease in Bcl-2 expression.Furthermore,ELRL-MAP30 triggered apoptosis via the Fak/EGFR/Erk and Ilk/Akt signaling pathways.In addition,recombinant ELRL-MAP30 can inhibit chicken embryonic angiogenesis,and also inhibit the tube formation ability of human umbilical vein endothelial cells(HUVECs),indicating its potential therapeutic effects on tumor angiogenesis.Collectively,these results indicate that ELRL-MAP30 has significant tumor-targeting properties in MDA-MB-231 cancer cells and reveals potential therapeutic effects on angiogenesis.These findings indicate the potential role of ELRL-MAP30 in the targeted treatment of the TNBC cell line MDA-MB-231.展开更多
Objective To investigate the effect of mucin 1(MUC1)on the proliferation and apoptosis of nasopharyngeal carcinoma(NPC)and its regulatory mechanism.Methods The 60 NPC and paired para-cancer normal tissues were collect...Objective To investigate the effect of mucin 1(MUC1)on the proliferation and apoptosis of nasopharyngeal carcinoma(NPC)and its regulatory mechanism.Methods The 60 NPC and paired para-cancer normal tissues were collected from October 2020 to July 2021 in Quanzhou First Hospital.The expression of MUC1 was measured by real-time quantitative PCR(qPCR)in the patients with PNC.The 5-8F and HNE1 cells were transfected with siRNA control(si-control)or siRNA targeting MUC1(si-MUC1).Cell proliferation was analyzed by cell counting kit-8 and colony formation assay,and apoptosis was analyzed by flow cytometry analysis in the 5-8F and HNE1 cells.The qPCR and ELISA were executed to analyze the levels of TNF-αand IL-6.Western blot was performed to measure the expression of MUC1,NFкB and apoptosis-related proteins(Bax and Bcl-2).Results The expression of MUC1 was up-regulated in the NPC tissues,and NPC patients with the high MUC1 expression were inclined to EBV infection,growth and metastasis of NPC.Loss of MUC1 restrained malignant features,including the proliferation and apoptosis,downregulated the expression of p-IкB、p-P65 and Bcl-2 and upregulated the expression of Bax in the NPC cells.Conclusion Downregulation of MUC1 restrained biological characteristics of malignancy,including cell proliferation and apoptosis,by inactivating NF-κB signaling pathway in NPC.展开更多
AIM:To explore the influence of ethyl(2,4,6-trimethylbenzoyl)phenylphosphinate(TPOL)on cell apoptosis and its potential mechanism.METHODS:HEK293T cells sensitive to TPOL were treated with different concentrations of T...AIM:To explore the influence of ethyl(2,4,6-trimethylbenzoyl)phenylphosphinate(TPOL)on cell apoptosis and its potential mechanism.METHODS:HEK293T cells sensitive to TPOL were treated with different concentrations of TPOL with or without exposure to light radiation,before treatment with various inhibitors,N-acetyl-Lcysteine(NAC),pifithrin-αand Z-DVED-FMK.Cell viability was measured by CCK-8 assay.Annexin V/propidium iodide staining was used to count the number of apoptotic cells.DCFH-DA staining was used to detect reactive oxygen species(ROS)levels,and JC-1 staining was used to assess mitochondrial membrane potential by flow cytometry.The expression of apoptosis-related proteins and cell cycle-regulated molecules was measured by Western blot.RESULTS:TPOL enhanced the apoptosis of HEK293T cells in a dose-dependent manner(P<0.05),with a decrease in Bcl-2 and increases in Bax and cytochrome C(Cyto C),followed by up-regulation of activated caspase-9 and caspase-3,and the cleavage of PARP(P<0.05).The TPOL-enhanced cleavage of caspase-3 and PARP was rescued by Z-DVED-FMK(P<0.01).TPOL also led to a rapid increase in ROS,a reduction in mitochondrial membrane potential,and the release of Cyto C(P<0.01),all of which could be reversed by the ROS scavenger NAC.Moreover,the TPOL-caused alterations in p21,p27,Rb,and CDK2 were also recovered by the p53 inhibitor pifithrin-α(P<0.05).The TPOL-induced changes in Bax,Bcl-2,cleaved caspase-9,activated caspase-3,and cleaved PARP were subsequently rescued by pretreatment with pifithrin-α(P<0.05).CONCLUSION:TPOL can induce cellular apoptosis with ROS-mediated mitochondrial membrane damage through the activation of a ROS-dependent p53/p21/p27/Rb/Bax/Cyto C/caspase-mediated signal axis.展开更多
Objective To observe the apoptosis of osteosarcoma MG-63 cells induced by As2O3 and to explore its possible mechanisms. Methods The flowcytometric analysis and transmission electronmicroscope were performed to investi...Objective To observe the apoptosis of osteosarcoma MG-63 cells induced by As2O3 and to explore its possible mechanisms. Methods The flowcytometric analysis and transmission electronmicroscope were performed to investigate the inducing apoptosis and inhibitative of As2O3 on osteosarcoma MG-63 cells. In order to study mechanism of apoptosis in MG-63 cells treated with As2 O3, microarray was performed. The down-regulated gene was confirmed by RT-PCR, Northern-blotting. Results After treated with As2O3, hypodiploid peak before G0/G1 phase was observed in MG-63 cells through FCM analysis. Loss of microvilli, condensation and fragmentation of nuclear chromatin, condensation of cytoplasmic organelles, dilatation of the endoplasmic reticulum shrinkage of cells and alterations in cell membranes and apoptosis bodies which were observed in MG-63 cells treated with As2O3 by transmission electronmicroscope. The results of microarray show that As2 O3 induced MG-63 cell apoptosis involves down-regulation of IEX-1 and the down-regulated gene is confirmed by RT-PCR and Northern-blotting.Conclusion The results show that As2 O3 selectively inhibits growth of the solid tumor MG-63 cells by triggering apoptosis and indicates MG-63 induced by As2O3 cell apoptosis may through the IEX-1 pathway.展开更多
The hallmark of apoptosis, in suspension cultures of Taxus spp. cells induced by fungal extractive or by abiotic means, was studied by total DNA agarose gel electrophoresis and in situ end-labeling. The cleavage of nu...The hallmark of apoptosis, in suspension cultures of Taxus spp. cells induced by fungal extractive or by abiotic means, was studied by total DNA agarose gel electrophoresis and in situ end-labeling. The cleavage of nuclear DNA (nDNA) into oligonucleosomal fragments(DNA laddering) was a characteristic of apoptosis, which involved cell shrinkage, condensation of cytoplasm and tracheary elements differentiation. Terminal deoxynucleotidy transferase-mediated dUTP nick end in situ labeling (TUNEL) assay of Taxus spp. cells showed that fungal extractive or abiotic elicltors (Ce4+, Taxol, H2O2) induced TUNEL positive. Also, the increase of the apoptotic cell ratio was accompanied by the increase of secondary metabolites (especially Taxol). These results suggest that apoptosis may have some coincidence with biosynthesis of Taxol. The implication of apoptosis for the production of secondary metabolites in plant cell cultures is discussed.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-(1use 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
OBJECTIVE The present study was designed to investigate anticancer effect of zeylenone(Zey)on K562 cells derived from chronic myelogenous leukemia(CML)both in vitro and in vivo,followed by exploring the underlying mec...OBJECTIVE The present study was designed to investigate anticancer effect of zeylenone(Zey)on K562 cells derived from chronic myelogenous leukemia(CML)both in vitro and in vivo,followed by exploring the underlying mechanisms.METHODS Initially,the effects of Zey on cel viability,proliferation,and apoptosis were measured in K562 cells by MTT,soft agar assay,AO/EB staining,hoechst 33258 staining and flow cytometric analysis after they were treated with Zey for indicated time,the involving signaling pathways were then investigated by JC-1,real-time quantitative polymerase chain reaction(RT-q PCR),Western blotting and immunofluorescence analysis.Furthermore,the in vivo anti-tumoractivity of Zey was assessed with nude xenografts and the involving mechanism was confirmed by immunohistochemical(IHC)and histopathological analysis.RESULTS We identified that Zey dose-dependently decreased cell viability,colony formation and expression of Proliferating Cell Nuclear Antigen(PCNA),and significantly induced K562 cell apoptosis via regulating Bcl-2 family members,decreasing mitochondrial transmembrane potential,and activating caspase-3,caspase-9,and caspase-8(P<0.05 or P<0.01).Further study revealed that Zey significantly inhibited phosphorylation of Jak2 and Src and downregulated their downstream proteins,including stat3,PI3K/AKT/m TOR,and ERK1/2 signaling pathways(P<0.05 or P<0.01).Zey also suppressed tumor growth with low toxicity in mouse xenograft model of K562cells through decreasing expression of Jak2 and Src.CONCLUSION Our data demonstrated that Zey substantially suppressed K562 cells both in vitro and in vivo through Jak2 and Src pathways.These findings suggest the potential of Zey as an effective anticancer agent in CML treatment.展开更多
OBJECTIVE The pathological characteristics of nonalcoholic steatohepatitis(NASH)include liver steatosis,inflammation,and fibrosis.Fibrosis is the most severe and significant pathological feature in NASH.Effective drug...OBJECTIVE The pathological characteristics of nonalcoholic steatohepatitis(NASH)include liver steatosis,inflammation,and fibrosis.Fibrosis is the most severe and significant pathological feature in NASH.Effective drug treatment could reverse early liver fibrosis and is of significance to prevent NASH from progressing into cirrhosis and liver cancer.Identification of drug targets for NASH treatment has been an active research area and is essential for the development of anti-NASH medications.Naringenin(NGN)is a flavonoid compound rich in citrus fruits.Our preliminary data demonstrated that NGN reduced diet-induced lipid accumulation and inflammation in the mouse liver,but whether NGN can attenuate liver fibrosis of NASH is not known.METHODS To study the effect of NGN on NASH fibrosis.WT mice were fed with high fat diet(HFD)and injected intraperitoneally 20%carbon tetrachloride at the same time for 8 weeks to induce NASH,and NGN was administrated by gavage in the meantime.In vitro,LO2 cells and LX2 cells were stimulated by oleic acid(OA)combined with lipopolysaccharide(LPS),respectively.RESULTS Treating the WT mice with NGN 100 mg·kg^(-1)·d-1 significantly attenuated hepatic lipid accumulation,hepatic fibrosis,plasma ALT and AST levels,inhibited protein expression of p-ERK,p-FoxO3a in the mouse livers.In vitro,on OA and LPS stimulated LO2 or LX2 cells,NGN significantly promoted apoptosis of activated hepatic stellate cells while inhibited apoptosis of hepatocytes.Mechanism study indicated that NGN inhibited MAPK pathway and promoted activation of FoxO3a,consequently promoted apoptosis of the activated LX2 cells and inhibited liver fibrosis.CONCLUSION NGN preventes NASH fibrosis via regulating MAPK/FoxO3a pathway,thus promoting apoptosis of the activated hepatic stellate cells.展开更多
The purpose of this study was to explore the effects of recombinant human intestinal alkaline phosphatase(recIAP) on human neutrophils in vitro, and the migration, phagocytosis, apoptosis in presence and absence of LP...The purpose of this study was to explore the effects of recombinant human intestinal alkaline phosphatase(recIAP) on human neutrophils in vitro, and the migration, phagocytosis, apoptosis in presence and absence of LPS. In this study, freshly extracted human neutrophils were used to establish an inflammatory cell model, and the control group, recIAP group, LPS group and recIAP +LPS group were set up to stimulate the model. The migration of neutrophils was detected by agarose gel drop method. Fluorescent particles and fluorescent probes were added to different treatment groups, and the phagocytic rate of neutrophils and the release of reactive oxygen species(ROS) from neutrophils were detected by flow cytometry. The apoptosis rate of neutrophils was detected by flow cytometry according to Annexin V-FITC apoptosis detection kit. The results showed that regardless of the presence or absence of LPS, recIAP could inhibit the migration of neutrophils, phagocytosis and the release of ROS. In addition, recIAP could weaken the inhibitory effect of LPS on neutrophils apoptosis.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
Osteoarthritis(OA)is an inflammatory disease involving the joints that is prevalent in the global aging population.The purpose of this study is to determine whether irisin can attenuate osteoarthritis(OA)progression i...Osteoarthritis(OA)is an inflammatory disease involving the joints that is prevalent in the global aging population.The purpose of this study is to determine whether irisin can attenuate osteoarthritis(OA)progression in anterior cruciate ligament transection(ACLT)mice models and the mechanism of irisin therapy effect on OA by increase the resistance of apoptosis in MLO-Y4 cells induced by mechanical stretch in vitro.Methods For in vivo study,3-month-old male C57BL/6 J mice were randomized to three groups,sham-operated,anterior cruciate ligament transection(ACLT)-operated treated with vehicle,and ACLT-operated treated with irisin by intraperitoneal injection once a week.Cartilage erosion was observed by HE staining.Osteoarthritis Research Society International(OARSI)scores were evaluated according to the safranin O stai-ning.The microstructure of tibia cortical bone,trabecular bone,and subchondral bone was analyzed by micro-CT and the bone histomorphometry has been administrated including mineral apposition rate(MAR).Edu staining and cck-8 were used for the detection of the proliferation of MLO-Y4 cells.For mechanical stress,cells were seeded on the collagen-I coated chamber subjected with a peak biaxial stretch of 20%at 1 Hz for 16 hours to induce apoptosis.Flow cytometry was used for the detection of apoptosis and cell cycle.TUNNEL was used for staining the apoptotic cells and rt-PCR was applied for quantifying the expression of mRNA such as Bax,Bcl-2,SOST,c-myc,Opg.Western blot was utilized to confirm the mechanism of how irisin decrease the osteocyte apoptosis.Results In vivo,irisin can attenuate articular cartilage degeneration.Irisin maintains the proportion of hyaline cartilage and calcified cartilage and keep fewer cartilage erosions in ACLT-operated mice.For immunohistochemical(IHC)staining,irisin reduced the expression of caspase3,Bax and matrix metalloproteinase-13 in both cartilage and subchondral bone.Irisin-treated ACLT group shows higher Trabecular number(Tb.N)and bone volume fraction(BV/TV)compared to the vehicle-treated ACLT group.In vitro, irisin significantly increased the proliferation of MLO-Y4 cells detected by Edu and Ki67 staining,and irisin can protect the cells from both mechanical stretchinduced apoptosis detected by FITC-PI flow cytometry and maintain the cell activity by regulating the expression of Bax,Bcl-2,and c-myc.Transcriptome sequencing shows that irisin significantly activates the MAPK signaling pathway and we confirm the result by western blot:irisin effectively activates the Erk signaling pathway through phosphorylation and has a certain activation effect on p38 signaling pathway,no activation was observed for FAK signaling pathway.Conclusions Irisin can attenuate the progression of OA by decrease the apoptosis of osteocyte,which can improve the microarchitecture of subchondral bone.Erk pathway activation plays an important role in reducing the apoptosis of osteocyte.展开更多
Based on published sequences for chicken Bcl-2,three siRNAs（small interfering RNA）were designed,and expression vectors were constructed and transfected into goose granulosa cells cultured in vitro.Bcl-2 protein,apop...Based on published sequences for chicken Bcl-2,three siRNAs（small interfering RNA）were designed,and expression vectors were constructed and transfected into goose granulosa cells cultured in vitro.Bcl-2 protein,apoptosis and proliferation of granulosa cells,48 h after the transf ection,were analyzed by flow cytometry,and progesterone（P）secreted into the culture medium was measured by radioimmunoassay.In addition,apoptosis and Bcl-2 protein level were assessed in untreated granulosa cells from the four largest preovulatory follicles（F<sub>1</sub><sup>F</sup><sub>4</sub>）,the smallest preovulatory follicles（SPF）,small yellow follicles（SYF）and atretic follicles.The highest level of Bcl-2 protein was observed in granulosa cells from SPF,and levels in cells from healthy follicles were significantly higher than those of atretic follicles（P【0.05）.Bcl-2 protein levels in cells subjected to RNAi were significantly lower than those of controls（P【0.05）,while apoptosis indices（AI）,proliferation indices（PI）and P secretion in the RNAi treatments were higher than those of controls（P【0.05）.展开更多
OBJECTIVE Alzheimer disease(AD) is a progressive neurodegenerative disorder involving a gradual decline in many cognitive processes and in neurons.The endoplasmic reticulum(ER) is involved in several crucial cellular ...OBJECTIVE Alzheimer disease(AD) is a progressive neurodegenerative disorder involving a gradual decline in many cognitive processes and in neurons.The endoplasmic reticulum(ER) is involved in several crucial cellular functions,eg protein folding and quality control.Massive misfolded or unfolded proteins in ER can disturb the function of ER and induce ER stress,which results in neuronal death in AD.Icariin(ICA) has a wide range of neuro protection and has been researched in AD treatment.However,whether ICA has the effect on ER stress in AD condition,and how ICA affects ER stress remains stil unclear.Therefore,the current study aimed to investigate the mechanism of ICA against cognitive impairments in AD model through ER stress pathway and apoptosis.METHODS Twelve months male APP/PS1 or wild-type(WT)mice were randomly divided into four groups:APP/PS1,and APP/PS1+ICA,WT and WT+ICA groups.The treated mice were given ICA60 mg·kg-1 per day and control mice were received the same volume distilled water for consecutive 3 months.The Morris water maze and novel object recognition were used to detect animals′ behavior.Nissl staining was used to observe the neuronal morphology in hippocampus area.The protein and(or) phosphorylation level of GRP78,p-PERK,PERK,p-IRE1,IRE1,ATF6,p-e IF2α,eIF2α,ATF4,CHOP,the level of cleaved-casepase 3,Bax and Bcl-2 were examined by Western blotting.RESULTS The behavior performance testing by Morris water maze and novel object recognition deteriorated in APP/PS1 mice compared with WT mice,however,ICA significantly improved the behavior performance when compared with APP/PS1.The neuron impairments in APP/PS1 mice also were ameliorated after ICA treatment.The protein expression of GRP78,ATF4,CHOP,and the level of p-PERK and p-eI F2α were higher in APP/PS1 mice than that in WT mice.The Bax/Bcl-2 ratio elevation and caspase 3 activation have been found in APP/PS1 mice.After treated with ICA,those above-mentioned parameters were decreased compared with APP/PS1.However,the levels of IRE1,p-IRE1 and ATF6 were not change among other groups.CONCLUSION ICA may decrease the ER stress and apoptosis in AD model,and may be through inhibiting the PERK/eI F2α pathway,not IRE and ATF6 pathways.展开更多
Aim Aralia elata （Miq.） Seem is a medicinal plant with a variety of biological activities. Several trier- pene saponins extracted from the leaves of Aralia elata （Miq.） Seem possess potent cytotoxic activity, but ...Aim Aralia elata （Miq.） Seem is a medicinal plant with a variety of biological activities. Several trier- pene saponins extracted from the leaves of Aralia elata （Miq.） Seem possess potent cytotoxic activity, but the mechanisms remain unclear. Methods In the present study, we examined the anti-tumor activity of total saponins of Aralia elata （Miq.） Seem leaves （TSAESL） using both in vitro and in vivo experiments, and investigated the underlying molecular mechanisms. Results Briefly, TSAESL significantly inhibited the proliferation of five human cancer cell lines and induced apoptosis of MCF-7 cells in a concentration-dependent manner. TSAESL also in- creased the expression level of active caspase-3 and cleaved PARP. In addition, TSAESL dramatically activated the phosphorylation of ERK1/2, p38 and JNK in a concentration-dependent manner. Treatment with the ERK1/2 in- hibitor U0126, p38 inhibitor SB203580, or JNK inhibitor SP600125 prior to TSAESL exposure markedly attenuated TSAESL-induced phosphorylation of ERK1/2 and p38. In mice bearing MCF-7 xenografl, TSAESL markedly inhib- ited tumor growth without significantly affecting spleen coefficient and hematological parameters. Conclusion These results strongly suggest that TSAESL has anti-tumor effect via activating the MAPK-mediated signaling path- ways.展开更多
Objective The apoptosis of vascular smooth muscle cells(VSMCs)influenced by abnormal cyclic stretch is crucial for vascular remodeling during hypertension.We explored that the causes of mechano-responsive lamin A/C ch...Objective The apoptosis of vascular smooth muscle cells(VSMCs)influenced by abnormal cyclic stretch is crucial for vascular remodeling during hypertension.We explored that the causes of mechano-responsive lamin A/C changingin aonormai cyclic stretcn and its roles in VSMC apoptosis.Methods and results Our previous vascular proteomics study revealed that LaminA/C is mechano-sensitive molecule.When VSMCs are subjected to cyclic stretch,the expression of LaminA/C is significantly changed which participates dysfunctions of VSMCs during hypertension.However,the molecular mechanism involved in regulation of LaminA/C expression and the role of LaminA/C in the VSMC apoptosis during cyclic stretch application are still unclear.In the present study,VSMCs were subjected to different amplitudes of cyclic steetch in vitro:5%cyclic stretch(physiological strain)or 15%cyclic stretch(pathological strain).The expression of 2 different selective cleavage isomers of LaminA/C,i.e.LaminA and LaminC,and the apoptosis of VSMCs were detected.The results showed that compared with 5%group,15%cyclic stretch significantly decreased the expression of LaminA and LaminC,and promoted the apoptosis of VSMCs.Using specific small interfering RNA(siRNA)transfection which targets on LMNA the encoding gene of LaminA/C,the expression of LaminA and LaminC in VSMCs was significantly decreased,and the apoptosis was significantly increased.In order to study the molecular mechanism involved in cyclic stretch regulating the expression of LaminA/C,we focused on the microRNA(miR).Bioinformatics analysis showed that the 3’untranslated region(3’UTR)of LMNA has two potential binding sites to miR-124-3p.Double luciferase reported system revealed that both sites have binding abilities to miR-124-3p.Under static condition,miR-124-3p inhibitor significantly up-regulated the expression levels of LaminA and LaminC,while the miR-124-3p mimics significantly down-regulated them.RT-PCR results showed that 15%cyclic stretch significantly up-regulated the expression of miR-124-3p compared with 5%cyclic stretch.Furthermore,in order to study the role of changeed LaminA/C in VSMC apoptosis,LMNA-specific siRNA was transfected to repress the expression of LaminA/C in VSMCs,and Protein/DNA microarray was used to detecte the activity of transcription factors.The transcription factors whose activity were changed significantly(increase or decrease more than 2 times)were analyzed by cluster analysis and ingenurity pathway analysis(IPA).Six transcription factors associated with apoptosis were screened,in which TP53 was activated by the specific siRNA transfection and the other 5 were inavtived,including TP53,CREB1,MYC,STAT1/5/6 and JUN.Using abdominal aorta coarctation hypertensive model,the change of miR-124-3p in VSMCs was explored in vivo.A marked increase of miR-124-3p in thoracic aorta was revealed compared with the sham-operated controls,and in situ FISH revealed that this increase was mainly in the VSMCs.Conclusions The present study suggest that abnormally increased cyclic stretch(15%)up-regulates the expression of miR-124-3p in VSMCs,which subsequently targets on the 3’UTR of LMNA and decreases the expression of nuclear envelope protein LaminA/C;the repressed LaminA/C may play an important role in the apoptosis of VSMCs by regulating the activity of virious transcription factors,such as TP53,CREB1,MYC,STAT1/5/6 and JUN.The present study may provide a new insight into understanding the molecular mechanisms of vascular remodeling.展开更多
Progressive beta cell apoptosis is a major cause leading to a decline in beta cell mass in type 2 diabetes. While it has been discovered that mitochondrial protein Sirtuin 5 （ SIRT5 ） functions as the primary site o...Progressive beta cell apoptosis is a major cause leading to a decline in beta cell mass in type 2 diabetes. While it has been discovered that mitochondrial protein Sirtuin 5 （ SIRT5 ） functions as the primary site of oxidative metabolism and plays crucial roles in apoptosis and intracellular signaling, the contribution of SIRT5 in beta cell re- mains to be fully defined. Given the potential benefit of SIRT5 activation in cell metabolism, this study aimed to examine whether overexpression of SIRT5 in beta cells is sufficient to prevent saturated fatty acid induced impair- ment in insulin secretion and apoptosis, thus elucidating the role of SIRT5 in beta cell protection. In line with our previous study, two kinds of pancreatic beta cell lines were selected for the detection. Mouse-derived NIT-1 cells as well as human-derived PANC-1 cells were transfected with GFP-SIRT5-Ad plasmid and cell apoptosis was induced by palmitic acid （0.5 mM） , validated with TUNEL-DAPI double staining and RTCA iCelligence cell growth moni- tor system. Compared with control group, it was shown that SIRT5 overexpression could significantly reduce the quantity of apoptotic beta cells under chronic exposure to palmitic acid, accompanied with decreased Caspase 3 and Caspase 9 activities. Accordingly, cytochrome C oxidase activity in cells was also suppressed. Meanwhile, palmitic acid suppressed glucose-stimulated insulin secretion, but SIRT5 overexpression could recover the beta cell insulin secretion capacity against glucose fluctuation. Moreover, it is discovered that novel binding relationship exists be- tween SIRT5 and Bcl-XL, providing a reliable explanation for the anti-apoptosis role of SIRT5. Together, these re- sults reveal a potential role of SIRT5 in improvement of saturated fatty acid -induced beta cell dysfunction and apop- tosis. Considering the role of beta cell apoptosis in T2DM, overexpression or activation of SIRT5 may provide an e-rumpent approach as a potential target for beta cell protection. This approach might actually reverse the disease to a degree rather than just palliate glycemia.展开更多
Aim Solasodine, a steroidal alkaloid isolated from solanaceous species, has been reported to exhibit anticancer activity on several cell lines. The aim of this study is to explore the antitumor potential of solasodine...Aim Solasodine, a steroidal alkaloid isolated from solanaceous species, has been reported to exhibit anticancer activity on several cell lines. The aim of this study is to explore the antitumor potential of solasodine on ovarian cancer HEY cells. Methods MTT assay, Hoechst 33342 staining, JC-1 mitochondrial membrane potential assay, and LDH release assay were used to study the anti-proliferative and apoptosis-inducing effects of solasodine. MDC staining was to label the acidic puncta in the cells. Meanwhile, wound healing assay and transwell assay were performed to determine whether solasodine exhibits anti-metastasis effect on HEY cells. Gelatin zymography assay was used to determine the activities of MMP-2 and MMP-9 and Western blot was used to examine the expression of the relevant proteins. Results Solasodine inhibited the cell viability on HEY cells in time- and dose-dependent manners, causing shrinking of nuclei, damage of cell membrane, and appealing apoptotic body on high dosage and reducing cell mitochondrial membrane potential. It also inhibited autophagic flux that interfere autolysosome degra- dation. Furthermore, solasodine suppressed the migration and invasion abilities of HEY cells ascribable to down- regulating the expression and attenuating the activities of MMP-2 and MMP-9. Conclusion The inducing apoptosis and anti-metastasis activity of solasodine on HEY cells suggest it appears certain potential on ovarian cancer treat- ment.展开更多
Aim Oxaloacetate （OA） is one of the intermediates in the Krebs cycle. In addition to its role in the metabolism of energy production, OA may have other effects on the cell. We report in the present study that OA cou...Aim Oxaloacetate （OA） is one of the intermediates in the Krebs cycle. In addition to its role in the metabolism of energy production, OA may have other effects on the cell. We report in the present study that OA could have a cell type dependent cytoto^ic effect on the human hepatic carcinoma cell line HepG2 through induction of apoptosis and ROS accumulation. In our study, OA decreased the viability and colony formation of HepG2 cell and induced cell death. Caspase-3 activity was increased, pro-apoptotic protein Bax was up-regulated, and anti-ap- optotic protein Bcl-2 was clown-regulated in OA-treated HepG2 cells indicating that apoptosis through the intrinsic pathway was involved in the death of the cell. The level of reactive oxygen species （ROS） in OA-treated HepG2 cells was increased. Anti-oxidant N-acetylcysteine （NAC） and glutathione （GSH） prevented the viability of the cell induced by OA from decrease but could not alleviate the enhanced level of apoptotic Bax/Bcl-2 mRNA expres- sion ratio, which suggests that the OA-induced apoptosis of HepG2 cell is not driven by oxidative damage and at least two distinct mechanisms, one mediated by ROS and one involving apoptosis, lead to the cytotoxic effect of OA on HepG2 cells. These studies expand the biological functional repertoire of OA and provide a mechanism by which hepatocellular carcinoma may be targeted by OA to kill the cancer cells.展开更多
The YiQiFuMai powder injection （ YQFM）, a Traditional Chinese Medicine （TCM） prescription re-de- veloped based on the well-known TCM formula Sheng-maisan, showed a wide range of pharmacological activities in ca...The YiQiFuMai powder injection （ YQFM）, a Traditional Chinese Medicine （TCM） prescription re-de- veloped based on the well-known TCM formula Sheng-maisan, showed a wide range of pharmacological activities in cardiovascular diseases in clinic. However, its role in protection against myocardial ischemia/reperfusion （MI/R） injury has not been elucidated. The present study not only evaluated the eardioprotective effect of YQFM from MI/ R injury but also investigated the potential molecular mechanisms in vivo and in vitro. The mouse model of MI/R injury was induced by a transient vessel occlusion for 30 rain and reperfusion for 24 h. The myocardium infarct size, production of lactate dehydrogenase （LDH）, creatine kinase （CK）, TUNEL staining and easpase-3 activity were measured. AMPKeα and phospho-AMPKα was analyzed by Western blot. We further verified the protective effect and potential molecular mechanisms of YQFM in an in vitro model of simulated ischemia and reperfusion （ SI/ R） in H9c2 cardiomyocytes. Cell viability was determined, and cell apoptosis were measured by Hoechst 33342 staining and Flow cytometry. Mitochondrial membrane potential （△ψFm） was measured, and ATP content was quantified by biolumineseent assay. Expression of apoptosis-related proteins including Caspase-3, Bcl-2, Bax, AMPKα and phospho-AMPKα was analyzed by Western blot. AMPKoL siRNA transfection was also applied to the mechanism elucidation. YQFM significantly reduced myocardium infarct size and the production of LDH, CK in se- rum, and also produced a significant decrease of apoptotic index which was confirmed by TUNEL staining and the changes of caspase-3 activity. In addition, pretreatment with YQFM markedly improved cell viability and decreased LDH release. Moreover, YQFM inhibited H9c2 apoptosis, blocked the expression of easpase-3 and modulated Bcl- 2 and Bax proteins, leading to an increased mitochondrial membrane potential and cellular ATP content. Mechanis- tically, YQFM activated AMPK signaling pathways while pretreatment with AMPK inhibitor compound C and appli- cation of transfection with AMPKα siRNA attenuated the anti-apoptotie effect of YQFM. Our results indicated that YQFM could provide significant cardioproteetion against MI/R injury, and potential mechanisms might to suppres- sion of cardiomyocytes apoptosis at least in part through activating the AMPK signaling pathways.展开更多
文摘Momordica antiviral protein 30 kD(MAP30)is a type I ribosome-inactivating protein(RIP)with antibacterial,anti-HIV and antitumor activities but lacks the ability to target tumor cells.To increase its tumor-targeting ability,the arginine-glycine-aspartic(RGD)peptide and the epidermal growth factor receptor interference(EGFRi)peptide were fused with MAP30,which was named ELRL-MAP30.The efficiency of targeted therapy for triple-negative breast cancer(TNBC)MDA-MB-231 cells,which lack the expression of estrogen receptor(ER),Progesterone receptor(PgR)and human epidermal growth factor receptor-2(HER2),is limited.In this study,we focus on exploring the effect and mechanism of ELRL-MAP30 on TNBC MDA-MB-231 cells.First,we discovered that ELRL-MAP30 significantly inhibited the migration and invasion of MDA-MB-231 cells and induced MDA-MB-231 cell apoptosis.Moreover,ELRL-MAP30 treatment resulted in a significant increase in Bax expression and a decrease in Bcl-2 expression.Furthermore,ELRL-MAP30 triggered apoptosis via the Fak/EGFR/Erk and Ilk/Akt signaling pathways.In addition,recombinant ELRL-MAP30 can inhibit chicken embryonic angiogenesis,and also inhibit the tube formation ability of human umbilical vein endothelial cells(HUVECs),indicating its potential therapeutic effects on tumor angiogenesis.Collectively,these results indicate that ELRL-MAP30 has significant tumor-targeting properties in MDA-MB-231 cancer cells and reveals potential therapeutic effects on angiogenesis.These findings indicate the potential role of ELRL-MAP30 in the targeted treatment of the TNBC cell line MDA-MB-231.
文摘Objective To investigate the effect of mucin 1(MUC1)on the proliferation and apoptosis of nasopharyngeal carcinoma(NPC)and its regulatory mechanism.Methods The 60 NPC and paired para-cancer normal tissues were collected from October 2020 to July 2021 in Quanzhou First Hospital.The expression of MUC1 was measured by real-time quantitative PCR(qPCR)in the patients with PNC.The 5-8F and HNE1 cells were transfected with siRNA control(si-control)or siRNA targeting MUC1(si-MUC1).Cell proliferation was analyzed by cell counting kit-8 and colony formation assay,and apoptosis was analyzed by flow cytometry analysis in the 5-8F and HNE1 cells.The qPCR and ELISA were executed to analyze the levels of TNF-αand IL-6.Western blot was performed to measure the expression of MUC1,NFкB and apoptosis-related proteins(Bax and Bcl-2).Results The expression of MUC1 was up-regulated in the NPC tissues,and NPC patients with the high MUC1 expression were inclined to EBV infection,growth and metastasis of NPC.Loss of MUC1 restrained malignant features,including the proliferation and apoptosis,downregulated the expression of p-IкB、p-P65 and Bcl-2 and upregulated the expression of Bax in the NPC cells.Conclusion Downregulation of MUC1 restrained biological characteristics of malignancy,including cell proliferation and apoptosis,by inactivating NF-κB signaling pathway in NPC.
基金Supported by the National Natural Science Foundation of China(No.81172824)。
文摘AIM:To explore the influence of ethyl(2,4,6-trimethylbenzoyl)phenylphosphinate(TPOL)on cell apoptosis and its potential mechanism.METHODS:HEK293T cells sensitive to TPOL were treated with different concentrations of TPOL with or without exposure to light radiation,before treatment with various inhibitors,N-acetyl-Lcysteine(NAC),pifithrin-αand Z-DVED-FMK.Cell viability was measured by CCK-8 assay.Annexin V/propidium iodide staining was used to count the number of apoptotic cells.DCFH-DA staining was used to detect reactive oxygen species(ROS)levels,and JC-1 staining was used to assess mitochondrial membrane potential by flow cytometry.The expression of apoptosis-related proteins and cell cycle-regulated molecules was measured by Western blot.RESULTS:TPOL enhanced the apoptosis of HEK293T cells in a dose-dependent manner(P<0.05),with a decrease in Bcl-2 and increases in Bax and cytochrome C(Cyto C),followed by up-regulation of activated caspase-9 and caspase-3,and the cleavage of PARP(P<0.05).The TPOL-enhanced cleavage of caspase-3 and PARP was rescued by Z-DVED-FMK(P<0.01).TPOL also led to a rapid increase in ROS,a reduction in mitochondrial membrane potential,and the release of Cyto C(P<0.01),all of which could be reversed by the ROS scavenger NAC.Moreover,the TPOL-caused alterations in p21,p27,Rb,and CDK2 were also recovered by the p53 inhibitor pifithrin-α(P<0.05).The TPOL-induced changes in Bax,Bcl-2,cleaved caspase-9,activated caspase-3,and cleaved PARP were subsequently rescued by pretreatment with pifithrin-α(P<0.05).CONCLUSION:TPOL can induce cellular apoptosis with ROS-mediated mitochondrial membrane damage through the activation of a ROS-dependent p53/p21/p27/Rb/Bax/Cyto C/caspase-mediated signal axis.
文摘Objective To observe the apoptosis of osteosarcoma MG-63 cells induced by As2O3 and to explore its possible mechanisms. Methods The flowcytometric analysis and transmission electronmicroscope were performed to investigate the inducing apoptosis and inhibitative of As2O3 on osteosarcoma MG-63 cells. In order to study mechanism of apoptosis in MG-63 cells treated with As2 O3, microarray was performed. The down-regulated gene was confirmed by RT-PCR, Northern-blotting. Results After treated with As2O3, hypodiploid peak before G0/G1 phase was observed in MG-63 cells through FCM analysis. Loss of microvilli, condensation and fragmentation of nuclear chromatin, condensation of cytoplasmic organelles, dilatation of the endoplasmic reticulum shrinkage of cells and alterations in cell membranes and apoptosis bodies which were observed in MG-63 cells treated with As2O3 by transmission electronmicroscope. The results of microarray show that As2 O3 induced MG-63 cell apoptosis involves down-regulation of IEX-1 and the down-regulated gene is confirmed by RT-PCR and Northern-blotting.Conclusion The results show that As2 O3 selectively inhibits growth of the solid tumor MG-63 cells by triggering apoptosis and indicates MG-63 induced by As2O3 cell apoptosis may through the IEX-1 pathway.
文摘The hallmark of apoptosis, in suspension cultures of Taxus spp. cells induced by fungal extractive or by abiotic means, was studied by total DNA agarose gel electrophoresis and in situ end-labeling. The cleavage of nuclear DNA (nDNA) into oligonucleosomal fragments(DNA laddering) was a characteristic of apoptosis, which involved cell shrinkage, condensation of cytoplasm and tracheary elements differentiation. Terminal deoxynucleotidy transferase-mediated dUTP nick end in situ labeling (TUNEL) assay of Taxus spp. cells showed that fungal extractive or abiotic elicltors (Ce4+, Taxol, H2O2) induced TUNEL positive. Also, the increase of the apoptotic cell ratio was accompanied by the increase of secondary metabolites (especially Taxol). These results suggest that apoptosis may have some coincidence with biosynthesis of Taxol. The implication of apoptosis for the production of secondary metabolites in plant cell cultures is discussed.
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-(1use 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The project supported by PUMC Youth Fund(3332015047)Fundamental Research Funds for the Central Universities,Beijing Key Laboratory of Innovative Drug Discovery of Traditional Chinese Medicine(Natural Medicine)and Translational Medicine,Institute of Medical Plant Development,Peking Union Medical College and Chinese Academy of Medical Sciencesby the National Science and Technology Major Project and Scientific Researchers Aiding Enterprise Item(2012ZX09301-002-001-026 and 2012ZX09501001-004)from the Ministry of Science and Technology of China
文摘OBJECTIVE The present study was designed to investigate anticancer effect of zeylenone(Zey)on K562 cells derived from chronic myelogenous leukemia(CML)both in vitro and in vivo,followed by exploring the underlying mechanisms.METHODS Initially,the effects of Zey on cel viability,proliferation,and apoptosis were measured in K562 cells by MTT,soft agar assay,AO/EB staining,hoechst 33258 staining and flow cytometric analysis after they were treated with Zey for indicated time,the involving signaling pathways were then investigated by JC-1,real-time quantitative polymerase chain reaction(RT-q PCR),Western blotting and immunofluorescence analysis.Furthermore,the in vivo anti-tumoractivity of Zey was assessed with nude xenografts and the involving mechanism was confirmed by immunohistochemical(IHC)and histopathological analysis.RESULTS We identified that Zey dose-dependently decreased cell viability,colony formation and expression of Proliferating Cell Nuclear Antigen(PCNA),and significantly induced K562 cell apoptosis via regulating Bcl-2 family members,decreasing mitochondrial transmembrane potential,and activating caspase-3,caspase-9,and caspase-8(P<0.05 or P<0.01).Further study revealed that Zey significantly inhibited phosphorylation of Jak2 and Src and downregulated their downstream proteins,including stat3,PI3K/AKT/m TOR,and ERK1/2 signaling pathways(P<0.05 or P<0.01).Zey also suppressed tumor growth with low toxicity in mouse xenograft model of K562cells through decreasing expression of Jak2 and Src.CONCLUSION Our data demonstrated that Zey substantially suppressed K562 cells both in vitro and in vivo through Jak2 and Src pathways.These findings suggest the potential of Zey as an effective anticancer agent in CML treatment.
文摘OBJECTIVE The pathological characteristics of nonalcoholic steatohepatitis(NASH)include liver steatosis,inflammation,and fibrosis.Fibrosis is the most severe and significant pathological feature in NASH.Effective drug treatment could reverse early liver fibrosis and is of significance to prevent NASH from progressing into cirrhosis and liver cancer.Identification of drug targets for NASH treatment has been an active research area and is essential for the development of anti-NASH medications.Naringenin(NGN)is a flavonoid compound rich in citrus fruits.Our preliminary data demonstrated that NGN reduced diet-induced lipid accumulation and inflammation in the mouse liver,but whether NGN can attenuate liver fibrosis of NASH is not known.METHODS To study the effect of NGN on NASH fibrosis.WT mice were fed with high fat diet(HFD)and injected intraperitoneally 20%carbon tetrachloride at the same time for 8 weeks to induce NASH,and NGN was administrated by gavage in the meantime.In vitro,LO2 cells and LX2 cells were stimulated by oleic acid(OA)combined with lipopolysaccharide(LPS),respectively.RESULTS Treating the WT mice with NGN 100 mg·kg^(-1)·d-1 significantly attenuated hepatic lipid accumulation,hepatic fibrosis,plasma ALT and AST levels,inhibited protein expression of p-ERK,p-FoxO3a in the mouse livers.In vitro,on OA and LPS stimulated LO2 or LX2 cells,NGN significantly promoted apoptosis of activated hepatic stellate cells while inhibited apoptosis of hepatocytes.Mechanism study indicated that NGN inhibited MAPK pathway and promoted activation of FoxO3a,consequently promoted apoptosis of the activated LX2 cells and inhibited liver fibrosis.CONCLUSION NGN preventes NASH fibrosis via regulating MAPK/FoxO3a pathway,thus promoting apoptosis of the activated hepatic stellate cells.
基金the Heilongjiang Natural Science Fund Project (C2017035)。
文摘The purpose of this study was to explore the effects of recombinant human intestinal alkaline phosphatase(recIAP) on human neutrophils in vitro, and the migration, phagocytosis, apoptosis in presence and absence of LPS. In this study, freshly extracted human neutrophils were used to establish an inflammatory cell model, and the control group, recIAP group, LPS group and recIAP +LPS group were set up to stimulate the model. The migration of neutrophils was detected by agarose gel drop method. Fluorescent particles and fluorescent probes were added to different treatment groups, and the phagocytic rate of neutrophils and the release of reactive oxygen species(ROS) from neutrophils were detected by flow cytometry. The apoptosis rate of neutrophils was detected by flow cytometry according to Annexin V-FITC apoptosis detection kit. The results showed that regardless of the presence or absence of LPS, recIAP could inhibit the migration of neutrophils, phagocytosis and the release of ROS. In addition, recIAP could weaken the inhibitory effect of LPS on neutrophils apoptosis.
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.
基金supported by the National Natural Science Foundation of China ( 31670957)
文摘Osteoarthritis(OA)is an inflammatory disease involving the joints that is prevalent in the global aging population.The purpose of this study is to determine whether irisin can attenuate osteoarthritis(OA)progression in anterior cruciate ligament transection(ACLT)mice models and the mechanism of irisin therapy effect on OA by increase the resistance of apoptosis in MLO-Y4 cells induced by mechanical stretch in vitro.Methods For in vivo study,3-month-old male C57BL/6 J mice were randomized to three groups,sham-operated,anterior cruciate ligament transection(ACLT)-operated treated with vehicle,and ACLT-operated treated with irisin by intraperitoneal injection once a week.Cartilage erosion was observed by HE staining.Osteoarthritis Research Society International(OARSI)scores were evaluated according to the safranin O stai-ning.The microstructure of tibia cortical bone,trabecular bone,and subchondral bone was analyzed by micro-CT and the bone histomorphometry has been administrated including mineral apposition rate(MAR).Edu staining and cck-8 were used for the detection of the proliferation of MLO-Y4 cells.For mechanical stress,cells were seeded on the collagen-I coated chamber subjected with a peak biaxial stretch of 20%at 1 Hz for 16 hours to induce apoptosis.Flow cytometry was used for the detection of apoptosis and cell cycle.TUNNEL was used for staining the apoptotic cells and rt-PCR was applied for quantifying the expression of mRNA such as Bax,Bcl-2,SOST,c-myc,Opg.Western blot was utilized to confirm the mechanism of how irisin decrease the osteocyte apoptosis.Results In vivo,irisin can attenuate articular cartilage degeneration.Irisin maintains the proportion of hyaline cartilage and calcified cartilage and keep fewer cartilage erosions in ACLT-operated mice.For immunohistochemical(IHC)staining,irisin reduced the expression of caspase3,Bax and matrix metalloproteinase-13 in both cartilage and subchondral bone.Irisin-treated ACLT group shows higher Trabecular number(Tb.N)and bone volume fraction(BV/TV)compared to the vehicle-treated ACLT group.In vitro, irisin significantly increased the proliferation of MLO-Y4 cells detected by Edu and Ki67 staining,and irisin can protect the cells from both mechanical stretchinduced apoptosis detected by FITC-PI flow cytometry and maintain the cell activity by regulating the expression of Bax,Bcl-2,and c-myc.Transcriptome sequencing shows that irisin significantly activates the MAPK signaling pathway and we confirm the result by western blot:irisin effectively activates the Erk signaling pathway through phosphorylation and has a certain activation effect on p38 signaling pathway,no activation was observed for FAK signaling pathway.Conclusions Irisin can attenuate the progression of OA by decrease the apoptosis of osteocyte,which can improve the microarchitecture of subchondral bone.Erk pathway activation plays an important role in reducing the apoptosis of osteocyte.
基金supported by National Natural Science Foundation of China(30300253)Chen guang youth technology program of Wuhan(20065004116-25)
文摘Based on published sequences for chicken Bcl-2,three siRNAs（small interfering RNA）were designed,and expression vectors were constructed and transfected into goose granulosa cells cultured in vitro.Bcl-2 protein,apoptosis and proliferation of granulosa cells,48 h after the transf ection,were analyzed by flow cytometry,and progesterone（P）secreted into the culture medium was measured by radioimmunoassay.In addition,apoptosis and Bcl-2 protein level were assessed in untreated granulosa cells from the four largest preovulatory follicles（F<sub>1</sub><sup>F</sup><sub>4</sub>）,the smallest preovulatory follicles（SPF）,small yellow follicles（SYF）and atretic follicles.The highest level of Bcl-2 protein was observed in granulosa cells from SPF,and levels in cells from healthy follicles were significantly higher than those of atretic follicles（P【0.05）.Bcl-2 protein levels in cells subjected to RNAi were significantly lower than those of controls（P【0.05）,while apoptosis indices（AI）,proliferation indices（PI）and P secretion in the RNAi treatments were higher than those of controls（P【0.05）.
基金National Natural Science Foundation of China(81560594).
文摘OBJECTIVE Alzheimer disease(AD) is a progressive neurodegenerative disorder involving a gradual decline in many cognitive processes and in neurons.The endoplasmic reticulum(ER) is involved in several crucial cellular functions,eg protein folding and quality control.Massive misfolded or unfolded proteins in ER can disturb the function of ER and induce ER stress,which results in neuronal death in AD.Icariin(ICA) has a wide range of neuro protection and has been researched in AD treatment.However,whether ICA has the effect on ER stress in AD condition,and how ICA affects ER stress remains stil unclear.Therefore,the current study aimed to investigate the mechanism of ICA against cognitive impairments in AD model through ER stress pathway and apoptosis.METHODS Twelve months male APP/PS1 or wild-type(WT)mice were randomly divided into four groups:APP/PS1,and APP/PS1+ICA,WT and WT+ICA groups.The treated mice were given ICA60 mg·kg-1 per day and control mice were received the same volume distilled water for consecutive 3 months.The Morris water maze and novel object recognition were used to detect animals′ behavior.Nissl staining was used to observe the neuronal morphology in hippocampus area.The protein and(or) phosphorylation level of GRP78,p-PERK,PERK,p-IRE1,IRE1,ATF6,p-e IF2α,eIF2α,ATF4,CHOP,the level of cleaved-casepase 3,Bax and Bcl-2 were examined by Western blotting.RESULTS The behavior performance testing by Morris water maze and novel object recognition deteriorated in APP/PS1 mice compared with WT mice,however,ICA significantly improved the behavior performance when compared with APP/PS1.The neuron impairments in APP/PS1 mice also were ameliorated after ICA treatment.The protein expression of GRP78,ATF4,CHOP,and the level of p-PERK and p-eI F2α were higher in APP/PS1 mice than that in WT mice.The Bax/Bcl-2 ratio elevation and caspase 3 activation have been found in APP/PS1 mice.After treated with ICA,those above-mentioned parameters were decreased compared with APP/PS1.However,the levels of IRE1,p-IRE1 and ATF6 were not change among other groups.CONCLUSION ICA may decrease the ER stress and apoptosis in AD model,and may be through inhibiting the PERK/eI F2α pathway,not IRE and ATF6 pathways.
文摘Aim Aralia elata （Miq.） Seem is a medicinal plant with a variety of biological activities. Several trier- pene saponins extracted from the leaves of Aralia elata （Miq.） Seem possess potent cytotoxic activity, but the mechanisms remain unclear. Methods In the present study, we examined the anti-tumor activity of total saponins of Aralia elata （Miq.） Seem leaves （TSAESL） using both in vitro and in vivo experiments, and investigated the underlying molecular mechanisms. Results Briefly, TSAESL significantly inhibited the proliferation of five human cancer cell lines and induced apoptosis of MCF-7 cells in a concentration-dependent manner. TSAESL also in- creased the expression level of active caspase-3 and cleaved PARP. In addition, TSAESL dramatically activated the phosphorylation of ERK1/2, p38 and JNK in a concentration-dependent manner. Treatment with the ERK1/2 in- hibitor U0126, p38 inhibitor SB203580, or JNK inhibitor SP600125 prior to TSAESL exposure markedly attenuated TSAESL-induced phosphorylation of ERK1/2 and p38. In mice bearing MCF-7 xenografl, TSAESL markedly inhib- ited tumor growth without significantly affecting spleen coefficient and hematological parameters. Conclusion These results strongly suggest that TSAESL has anti-tumor effect via activating the MAPK-mediated signaling path- ways.
基金supported by grants from the National Natural Science Foundation of China( 11572199 and 11625209)
文摘Objective The apoptosis of vascular smooth muscle cells(VSMCs)influenced by abnormal cyclic stretch is crucial for vascular remodeling during hypertension.We explored that the causes of mechano-responsive lamin A/C changingin aonormai cyclic stretcn and its roles in VSMC apoptosis.Methods and results Our previous vascular proteomics study revealed that LaminA/C is mechano-sensitive molecule.When VSMCs are subjected to cyclic stretch,the expression of LaminA/C is significantly changed which participates dysfunctions of VSMCs during hypertension.However,the molecular mechanism involved in regulation of LaminA/C expression and the role of LaminA/C in the VSMC apoptosis during cyclic stretch application are still unclear.In the present study,VSMCs were subjected to different amplitudes of cyclic steetch in vitro:5%cyclic stretch(physiological strain)or 15%cyclic stretch(pathological strain).The expression of 2 different selective cleavage isomers of LaminA/C,i.e.LaminA and LaminC,and the apoptosis of VSMCs were detected.The results showed that compared with 5%group,15%cyclic stretch significantly decreased the expression of LaminA and LaminC,and promoted the apoptosis of VSMCs.Using specific small interfering RNA(siRNA)transfection which targets on LMNA the encoding gene of LaminA/C,the expression of LaminA and LaminC in VSMCs was significantly decreased,and the apoptosis was significantly increased.In order to study the molecular mechanism involved in cyclic stretch regulating the expression of LaminA/C,we focused on the microRNA(miR).Bioinformatics analysis showed that the 3’untranslated region(3’UTR)of LMNA has two potential binding sites to miR-124-3p.Double luciferase reported system revealed that both sites have binding abilities to miR-124-3p.Under static condition,miR-124-3p inhibitor significantly up-regulated the expression levels of LaminA and LaminC,while the miR-124-3p mimics significantly down-regulated them.RT-PCR results showed that 15%cyclic stretch significantly up-regulated the expression of miR-124-3p compared with 5%cyclic stretch.Furthermore,in order to study the role of changeed LaminA/C in VSMC apoptosis,LMNA-specific siRNA was transfected to repress the expression of LaminA/C in VSMCs,and Protein/DNA microarray was used to detecte the activity of transcription factors.The transcription factors whose activity were changed significantly(increase or decrease more than 2 times)were analyzed by cluster analysis and ingenurity pathway analysis(IPA).Six transcription factors associated with apoptosis were screened,in which TP53 was activated by the specific siRNA transfection and the other 5 were inavtived,including TP53,CREB1,MYC,STAT1/5/6 and JUN.Using abdominal aorta coarctation hypertensive model,the change of miR-124-3p in VSMCs was explored in vivo.A marked increase of miR-124-3p in thoracic aorta was revealed compared with the sham-operated controls,and in situ FISH revealed that this increase was mainly in the VSMCs.Conclusions The present study suggest that abnormally increased cyclic stretch(15%)up-regulates the expression of miR-124-3p in VSMCs,which subsequently targets on the 3’UTR of LMNA and decreases the expression of nuclear envelope protein LaminA/C;the repressed LaminA/C may play an important role in the apoptosis of VSMCs by regulating the activity of virious transcription factors,such as TP53,CREB1,MYC,STAT1/5/6 and JUN.The present study may provide a new insight into understanding the molecular mechanisms of vascular remodeling.
文摘Progressive beta cell apoptosis is a major cause leading to a decline in beta cell mass in type 2 diabetes. While it has been discovered that mitochondrial protein Sirtuin 5 （ SIRT5 ） functions as the primary site of oxidative metabolism and plays crucial roles in apoptosis and intracellular signaling, the contribution of SIRT5 in beta cell re- mains to be fully defined. Given the potential benefit of SIRT5 activation in cell metabolism, this study aimed to examine whether overexpression of SIRT5 in beta cells is sufficient to prevent saturated fatty acid induced impair- ment in insulin secretion and apoptosis, thus elucidating the role of SIRT5 in beta cell protection. In line with our previous study, two kinds of pancreatic beta cell lines were selected for the detection. Mouse-derived NIT-1 cells as well as human-derived PANC-1 cells were transfected with GFP-SIRT5-Ad plasmid and cell apoptosis was induced by palmitic acid （0.5 mM） , validated with TUNEL-DAPI double staining and RTCA iCelligence cell growth moni- tor system. Compared with control group, it was shown that SIRT5 overexpression could significantly reduce the quantity of apoptotic beta cells under chronic exposure to palmitic acid, accompanied with decreased Caspase 3 and Caspase 9 activities. Accordingly, cytochrome C oxidase activity in cells was also suppressed. Meanwhile, palmitic acid suppressed glucose-stimulated insulin secretion, but SIRT5 overexpression could recover the beta cell insulin secretion capacity against glucose fluctuation. Moreover, it is discovered that novel binding relationship exists be- tween SIRT5 and Bcl-XL, providing a reliable explanation for the anti-apoptosis role of SIRT5. Together, these re- sults reveal a potential role of SIRT5 in improvement of saturated fatty acid -induced beta cell dysfunction and apop- tosis. Considering the role of beta cell apoptosis in T2DM, overexpression or activation of SIRT5 may provide an e-rumpent approach as a potential target for beta cell protection. This approach might actually reverse the disease to a degree rather than just palliate glycemia.
文摘Aim Solasodine, a steroidal alkaloid isolated from solanaceous species, has been reported to exhibit anticancer activity on several cell lines. The aim of this study is to explore the antitumor potential of solasodine on ovarian cancer HEY cells. Methods MTT assay, Hoechst 33342 staining, JC-1 mitochondrial membrane potential assay, and LDH release assay were used to study the anti-proliferative and apoptosis-inducing effects of solasodine. MDC staining was to label the acidic puncta in the cells. Meanwhile, wound healing assay and transwell assay were performed to determine whether solasodine exhibits anti-metastasis effect on HEY cells. Gelatin zymography assay was used to determine the activities of MMP-2 and MMP-9 and Western blot was used to examine the expression of the relevant proteins. Results Solasodine inhibited the cell viability on HEY cells in time- and dose-dependent manners, causing shrinking of nuclei, damage of cell membrane, and appealing apoptotic body on high dosage and reducing cell mitochondrial membrane potential. It also inhibited autophagic flux that interfere autolysosome degra- dation. Furthermore, solasodine suppressed the migration and invasion abilities of HEY cells ascribable to down- regulating the expression and attenuating the activities of MMP-2 and MMP-9. Conclusion The inducing apoptosis and anti-metastasis activity of solasodine on HEY cells suggest it appears certain potential on ovarian cancer treat- ment.
文摘Aim Oxaloacetate （OA） is one of the intermediates in the Krebs cycle. In addition to its role in the metabolism of energy production, OA may have other effects on the cell. We report in the present study that OA could have a cell type dependent cytoto^ic effect on the human hepatic carcinoma cell line HepG2 through induction of apoptosis and ROS accumulation. In our study, OA decreased the viability and colony formation of HepG2 cell and induced cell death. Caspase-3 activity was increased, pro-apoptotic protein Bax was up-regulated, and anti-ap- optotic protein Bcl-2 was clown-regulated in OA-treated HepG2 cells indicating that apoptosis through the intrinsic pathway was involved in the death of the cell. The level of reactive oxygen species （ROS） in OA-treated HepG2 cells was increased. Anti-oxidant N-acetylcysteine （NAC） and glutathione （GSH） prevented the viability of the cell induced by OA from decrease but could not alleviate the enhanced level of apoptotic Bax/Bcl-2 mRNA expres- sion ratio, which suggests that the OA-induced apoptosis of HepG2 cell is not driven by oxidative damage and at least two distinct mechanisms, one mediated by ROS and one involving apoptosis, lead to the cytotoxic effect of OA on HepG2 cells. These studies expand the biological functional repertoire of OA and provide a mechanism by which hepatocellular carcinoma may be targeted by OA to kill the cancer cells.
文摘The YiQiFuMai powder injection （ YQFM）, a Traditional Chinese Medicine （TCM） prescription re-de- veloped based on the well-known TCM formula Sheng-maisan, showed a wide range of pharmacological activities in cardiovascular diseases in clinic. However, its role in protection against myocardial ischemia/reperfusion （MI/R） injury has not been elucidated. The present study not only evaluated the eardioprotective effect of YQFM from MI/ R injury but also investigated the potential molecular mechanisms in vivo and in vitro. The mouse model of MI/R injury was induced by a transient vessel occlusion for 30 rain and reperfusion for 24 h. The myocardium infarct size, production of lactate dehydrogenase （LDH）, creatine kinase （CK）, TUNEL staining and easpase-3 activity were measured. AMPKeα and phospho-AMPKα was analyzed by Western blot. We further verified the protective effect and potential molecular mechanisms of YQFM in an in vitro model of simulated ischemia and reperfusion （ SI/ R） in H9c2 cardiomyocytes. Cell viability was determined, and cell apoptosis were measured by Hoechst 33342 staining and Flow cytometry. Mitochondrial membrane potential （△ψFm） was measured, and ATP content was quantified by biolumineseent assay. Expression of apoptosis-related proteins including Caspase-3, Bcl-2, Bax, AMPKα and phospho-AMPKα was analyzed by Western blot. AMPKoL siRNA transfection was also applied to the mechanism elucidation. YQFM significantly reduced myocardium infarct size and the production of LDH, CK in se- rum, and also produced a significant decrease of apoptotic index which was confirmed by TUNEL staining and the changes of caspase-3 activity. In addition, pretreatment with YQFM markedly improved cell viability and decreased LDH release. Moreover, YQFM inhibited H9c2 apoptosis, blocked the expression of easpase-3 and modulated Bcl- 2 and Bax proteins, leading to an increased mitochondrial membrane potential and cellular ATP content. Mechanis- tically, YQFM activated AMPK signaling pathways while pretreatment with AMPK inhibitor compound C and appli- cation of transfection with AMPKα siRNA attenuated the anti-apoptotie effect of YQFM. Our results indicated that YQFM could provide significant cardioproteetion against MI/R injury, and potential mechanisms might to suppres- sion of cardiomyocytes apoptosis at least in part through activating the AMPK signaling pathways.
