P2X7 receptor （P2X7r） is important in inflammation and fibrosis. The aim of the present study was to investigate the effect of P2X7r inhibition, using a specific inhibitor （A438079） to prevent the development of l...P2X7 receptor （P2X7r） is important in inflammation and fibrosis. The aim of the present study was to investigate the effect of P2X7r inhibition, using a specific inhibitor （A438079） to prevent the development of liver fibrosis on human hepatic stellate cells, LX-2. The supernatant from lipopolysaccharide （LPS）-stimulated RAW 264.7 mouse macrophages was supplemented to LX-2 cells for 24 h. LX-2 cells were primed with LPS for 4 h and subsequently stimulated for 30 rain with 3 mmol · L^-1 of adenosine 5＇-triphosphate （ATP）. A438079 （ 10 μmol · L^-1） was supplemented to LX-2 cells 10 rain prior to ATP. Directly treated with LPS on LX-2 cells, mRNA ex- pressions of IL-1β, IL-18 and IL-6 were increased, as well as P2X7r. And caspase-1, ASC and NLRP3 mRNA ex- pressions were increased with LPS stimulation. LPS stimulation also increased oL-SMA and collagen I mRNA expres- sions. Interestingly treatment of LX-2 cells with mediums from LPS-primed RAW 264.7 mouse macrophages exhibi- ted greater increase of mRNA expressions of above genes than those in LX-2 directly treated with LPS. Pretreatment of directly or indirectly LPS-stimulated LX-2 cells with A438079 both suppressed IL-1β mRNA expression. In addi- tion treatment of LPS-primed LX-2 cells with 3 mM ATP induced the significant increase of IL-1β, IL-6, caspase- 1, pannexin-1, α-SMA and collagen I mRNA expression, the increasing of oL-SMA protein expression and cleavage of IL-1β. These events were significantly suppressed by pretreatment with P2X7r antagonist A438079. P2XTr blockade also significantly reduced the protein expression of oL-SMA. Our results suggest that the involvement of the P2X7r-NLRP3 inflammasome pathway in the secretion of IL-1β from extracellular ATP/LPS-stimulated human he- patic stellate cells. This study demonstrated that repression of the P2XTr represents a novel potential therapeutic ap- proach to control liver fibrosis.展开更多
OBJECTIVE To research the effect of naringenin(NAR) on LPS-induced dopaminergic neurons damage and its potential mechanism.METHODS Rats were randomly divided into the following six groups(n=10):control(0.9% NaCl),NAR ...OBJECTIVE To research the effect of naringenin(NAR) on LPS-induced dopaminergic neurons damage and its potential mechanism.METHODS Rats were randomly divided into the following six groups(n=10):control(0.9% NaCl),NAR alone(100 mg·kg-1),LPS(5 μg),LPS+NAR(50 mg·kg-1) and LPS+NAR(100 mg·kg-1).Rats were received a single LPS unilateral injection into the SN pars compacts,after seven daily intragastric administration of NAR,rats′ behavior was analyzed by rotarod test.Then,the expression of TH,IBA-1 and NLRP3 inflammasome were analyzed by Western blotting and immunofluorescence.In vitro experiments,BV-2 cel s were treated with different doses of NAR,and 1 h later,LPS(1 g·L^(-1)) was added to the medium for 24 h,then collect the culture medium and protein for later experiments.The production of IL-1β and IL-18 in culture medium were tested by ELISA,and the production of NO was detected by Griess reagent.The expression of IBA-1,NLRP3 and p-caspase 1 were detected by Western blotting.MN9 D cells were co-cultured with BV2 cells to mimic the animal experiments.MTT assay was used to analyzed the viability of MN9 D cells,and the expression of TH was detected by Western blotting.RESULTS NAR(100 mg · kg-1) could significantly improve the time of rats on the rotating(116.73 s vs 185.45 s,P<0.05).The result of the pathological analysis also suggested that NAR could decrease the activation of microglia as well as the expression of NLRP3 Inflammasome.In addition,NAR also could suppress the expression of pro-inflammatory factor levels,such as IL-1β(P<0.05),IL-18(P<0.05),and the protection of NAR could be inhibited by siR NA NLRP3.Moreover,an in vitro co-culture system with BV2 and MN9 D cells wasused to find the protection of NAR must via microglia,while there is no effect of NAR were directly added to MN9 D cells.CONCLUSION NAR protection of LPS-induced dopaminergic neurons damage might be through mediating NLRP3 inflammasome.展开更多
OBJECTIVE Recent studies have demonstrated that the Nlrp3 inflammasome serve as a central role in the pathogenesis of cardiovascular diseases and endothelial dysfunction occurs in association with several cardiovascul...OBJECTIVE Recent studies have demonstrated that the Nlrp3 inflammasome serve as a central role in the pathogenesis of cardiovascular diseases and endothelial dysfunction occurs in association with several cardiovascular risk factors.Given the demonstrated anti-inflammatory effects of aspirin,the present study was designed to test whether aspirin diminish NLRP3 inflammasome activation and prevent endothelium injury and associated coronary artery damage during LPS.METHODS Mouse carotid arterial endothelial cells(CAECs)were cultured and treated with 0.1-3 mmol·L^(-1) of aspirin in response to LPS(2μg·mL^(-1))stimuli.After 24 h,the Nlrp3 inflammasome complexes consist of varied proteins were analyzed by WB.NO and T-AOC in the supernatant was detected by ELISA.Intracellular reactive oxygen species(ROS)generation for 24 h was observed by DCF fluorescence.The mice were treated with aspirin(12.5 mg·kg^(-1) per day,62.5 mg·kg^(-1) per day,125 mg·kg^(-1) per day)and dexamethasone(0.0182 mg·kg^(-1)per day)for 7 d.The level of IL^(-1)β,IL^(-1)8 protein was detected by ELISA.RESULTS Immunofluorescence results showed the colocalization of Nlrp3 with ASC or caspase 1decrease in a concentration-dependent manner.Meanwhile,the expression of Nlrp3 and caspase 1protein was decreased with the concentration of aspirin,but no changes the expression of ASC protein.Nlrp3 protein levels in CAECs were 0.33-0.8-fold and cle-caspase 1 protein levels in CAECs were 0.48-1-fold compared to those in LPS stimulation when treated with 0.1-3 mmol·L^(-1) aspirin for 24 h(P<0.01).Aspirin significantly antagonized the effect of LPS on NO(1.22-1.91-fold that of LPS stimulation,P<0.01)and T-AOC expression(1.02-1.90-fold that of LPS stimulation,P<0.01).As the different concentration of aspirin treated,the generation of ROS was 0.51-1.10-fold that of LPS stimulation(P<0.01).In vivo data shown the level of IL^(-1)β,IL^(-1)8 protein from serum are in concordance with the level of Nlrp3 inflammasome activation.CONCLUSION We conclude that aspirin has anti-inflammatory properties,protecting CAECs fromLPS-induced injury by inhibition of NLRP3 inflammasome activation through ROS pathway.展开更多
OBJECTIVE To identify the role of GSK3 isoform inhibition on inflammasome activation.METHODS The NLRP3 inflammasome was activated by typical LPS/ATP and host-derived metabolites in primary mouse macrophages.The pharma...OBJECTIVE To identify the role of GSK3 isoform inhibition on inflammasome activation.METHODS The NLRP3 inflammasome was activated by typical LPS/ATP and host-derived metabolites in primary mouse macrophages.The pharmacological inhibition of GSK3 isoforms on inflammasome activation was assayed by quantifying IL-1βin the supernatant,and activated caspase-1in cell lysates using highly selective inhibitors.Further molecular mechanisms were investigated by protein pulldown assay,confocal imaging using forced gene expression system and endogenous protein tagged mouse macrophages.RESULTS Pharmacological inhibition of GSK3-β,but not GSK3-αisoform suppressed NLRP3 inflammasome activation in response to ATP,urate crystal and the microbial alkaloid toxin staurosporine.GSK3-βinhibition did not inhibit melanoma 2(AIM2)inflammasome activation in response to double-stranded DNA(dsDNA)and did not affect non-canonical caspase-11 inflammasome activation.GSK3-βinhibition suppressed high glucose mediated NLRP3 inflammasome activation.Mechanistically,GSK3-βinhibition blocked NLRP3 inflammasome by preventing pro IL-1βtranscription,reducing caspase-1 activation and ASC speck formation.GSK3-βinhibition blocked NLRP3 inflammasome activation without affecting the level of reactive oxygen species(ROS)which is a crucial component in initialing inflammasome activation.Further studies revealed that GSK3-βdirectly binds to ASC by both co-forced expression and endogenous protein level.Interestingly,we found ASC can be glycosylated in response to inflammasome activation,and GSK3-βinhibition reduced ASC glycosylation.Consistently,the O-Glc NAc transferase(OGT)deficient mouse macrophages showed the significant reduction of mature IL-1βsecretion in response to NLRP3 inflammasome activation.CONCLUSION Our results demonstrate a critical role of metabolism-sensing GSK3-βpathway in mediating NLRP3 inflammasome activation,thus defining a new therapeutic target for sterile inflammation.展开更多
OBJECTIVE Interleukin(IL)-1β,one of the principal inflammatory cytokines mainly secreted by monocytes and macrophages,is produced by cleavage of the inactive pro-IL^(-1)βprecursor by caspase-1 via the NLRP3 inflamma...OBJECTIVE Interleukin(IL)-1β,one of the principal inflammatory cytokines mainly secreted by monocytes and macrophages,is produced by cleavage of the inactive pro-IL^(-1)βprecursor by caspase-1 via the NLRP3 inflammasome complex.The fruits of Garcinia cambogia(Clusiaceae)are widely developed as health products for anti-obese purpose.14-deoxygarcinol(DOG)is a polyisoprenylated benzophenone from the fruits of G.cambogia,which showed potent anti-inflammatory effect in our previous study.The objective of this study was to explore the anti-inflammatory mechanism of DOG and its roles in alleviating adipose tissue inflammation and insulin resistance.METHODS The anti-inflammatory effect of DOG was evaluated on LPS plus nigericin-induced THP-1 macrophages.The expression of NLRP3 inflammasome complex proteins was analyzed by Western blotting,immunofluorescence staining and co-immunoprecipitation.The pro-inflammatory cytokines levels were determined by ELISA kits.RESULTS DOG increased the expression of Sirtuin 2(SIRT2)deacetylase and enhanced its deacetylating activity to suppress the NLRP3 inflammasome activation and IL^(-1)βsecretion in THP-1 macrophages.Moreover,DOG attenuated macrophage conditioned medium-induced inflammatory responses in adipocytes and blocked THP-1 macrophages migration towards 3T3-L1 adipocytes.CONCLUSION DOG attenuated the inflammatory crosstalk between macrophages and adipocytes through SIRT2-mediated NLRP3 inflammasome inhibition,which might be used for the treatment of adipose tissue inflammation-related metabolic disorders.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 investigate the neuroprotective effects and exact mechanisms of myrrh extract following cerebral ischemic stroke.METHODS Male rats were randomly divided into three groups:sham group,middle cerebral artery...OBJECTIVE To investigate the neuroprotective effects and exact mechanisms of myrrh extract following cerebral ischemic stroke.METHODS Male rats were randomly divided into three groups:sham group,middle cerebral artery occlusion(MCAO)group and myrrh group.Morphological changes were assessed after 7 d of myrrh treatment.Microarray analysis with circulating mRNA was performed to identify differential gene expression profile,gene ontology and pathway enrichment analyses were carried out to predict the gene function.Gene co-expression and pathway networks were constructed to identify the potential targets.The markers of oxidative stress,inflammatory reaction and ferroptosis in the cerebral cortex were detected by ELISA assays.The identified hub pathways and genes were validated by western blotting,immunofluorescence and immunohistochemistry analyses.Neurons were exposed to transient oxygen-glucose deprivation(OGD)to model ischemia-like conditions.siRNA-TXNIP were transfected in OGD-induced neurons to explore the mechanism.RESULTS Myrrh extract significantly alleviated neurological deficits,infarct volume and histo⁃pathological damage in MCAO rats.A total of 2200 differentially expressed genes were identified among the three groups.Oxidation-reduction process,inflammatory response,ferroptosis were enriched as the significant gene ontology items.NOD-like receptor signaling were identified as the hub pathway based on the pathway relation network.TXNIP and NLRP3 were screened as the potential targets by a time sequence profile analysis.The levels of IL-1β,IL-18,TNF-α,MDA and TFR in brain tissues were increased while the CAT,SOD,GSH-px and GPX4 levels were significantly decreased in MCAO group.As expected,myrrh extract greatly reversed these changes.The similarly results were also observed in OGD treated neuron cells.The elevated expressions of TXNIP and NLRP3 induced by OGD were success⁃fully inhibited by myrrh treatment.Knockdown of TXNIP significantly alleviated OGD-induced ROS accumulation and oxidative stress,but the antioxidative effect of myrrh was impaired when TXNIP was absent in neuron cells.In addition,knockdown of TXNIP significantly decreased the expression of NLRP3 and increased the expression of GPX4 in OGDinduced neuron cells.However,myrrh treatment scarcely changed the expressions of NLRP3 and these ferroptosis markers in siRNA-TXNIP pretreated cells,compared with the siRNA-TXNIP alone treatment group.Therefore,these data demonstrated that the neuroprotective effect of myrrh extract was dependent on TXNIP-NLRP3 axis.CONCLU⁃SION Thatmyrrh extract exerts neuroprotective property through alleviated ROS-mediated ferroptosis by regulating the TXNIP/NLRP3 axis in ischemic stroke.Myrrh extract could be considered as a promising candidate for the treatment of ischemic stroke.展开更多
Aim To study the effects of baicalein （BC）, a phenolic flavonoid extracted mainly from Scutellaria ba- icalensis Georgi, on lipopolysaccharide （LPS）-induced acute lung injury （ALI） in mice and the molecular mech...Aim To study the effects of baicalein （BC）, a phenolic flavonoid extracted mainly from Scutellaria ba- icalensis Georgi, on lipopolysaccharide （LPS）-induced acute lung injury （ALI） in mice and the molecular mecha- nisms underlying. Methods Mice were administrated intranasally with LPS （20 mg · kg^-1/body weight） to estab- lish the ALI model. Then the mice were treated twice with BC （50,100 and 200 mg · kg^-1, p. o. ） 0. 5 hour and 12 hours after LPS stimulation, following another 12 hours, the lungs were collected for histological study. Results LPS caused marked inflammatory cell infiltration and myeloperoxidase activation in lungs, accompanied by significantly in- creased lung W/D ratio, from 7.97±0. 60 in normal group to 12. 49 ± 1.49 in the model. 77.88% reduction in the lung W/D ratio was observed in 200 mg· kg^-1 dose of baicalein. The myeloperoxidase activity was reduced to 40. 14% in mice treated with 200 mg · kg^-1. The number of total cells, neutrophils, and macrophages in BALF de- creased with increasing concentration of baicalein. Inflammatory cytokines level in serum declined significantly while insignificant changes of the same in BALF was observed in mice treated with 50,100 and 200 mg · kg^-1 doses of ba- icalein. Furthermore, LPS induced markedly the expression of inflammasomes and other inflammation-related genes in lung tissue. Treatment of LPS-exposed mice with BC significantly reduced the expression levels of these genes and al- leviated the pathological changes in lungs. Moreover, 1 μmol · L^-1 and 10 μmol · L^-1 BC inhibited remarkably the nuclear translocation of NF-kappaB p65 in Raw264.7 cells. Conclusion Baicalein alleviates LPS-induced acute lung injury in mice by suppressing NF-KB-mediated inflammatory responses and downregulation of inflammasomes.展开更多
文摘P2X7 receptor （P2X7r） is important in inflammation and fibrosis. The aim of the present study was to investigate the effect of P2X7r inhibition, using a specific inhibitor （A438079） to prevent the development of liver fibrosis on human hepatic stellate cells, LX-2. The supernatant from lipopolysaccharide （LPS）-stimulated RAW 264.7 mouse macrophages was supplemented to LX-2 cells for 24 h. LX-2 cells were primed with LPS for 4 h and subsequently stimulated for 30 rain with 3 mmol · L^-1 of adenosine 5＇-triphosphate （ATP）. A438079 （ 10 μmol · L^-1） was supplemented to LX-2 cells 10 rain prior to ATP. Directly treated with LPS on LX-2 cells, mRNA ex- pressions of IL-1β, IL-18 and IL-6 were increased, as well as P2X7r. And caspase-1, ASC and NLRP3 mRNA ex- pressions were increased with LPS stimulation. LPS stimulation also increased oL-SMA and collagen I mRNA expres- sions. Interestingly treatment of LX-2 cells with mediums from LPS-primed RAW 264.7 mouse macrophages exhibi- ted greater increase of mRNA expressions of above genes than those in LX-2 directly treated with LPS. Pretreatment of directly or indirectly LPS-stimulated LX-2 cells with A438079 both suppressed IL-1β mRNA expression. In addi- tion treatment of LPS-primed LX-2 cells with 3 mM ATP induced the significant increase of IL-1β, IL-6, caspase- 1, pannexin-1, α-SMA and collagen I mRNA expression, the increasing of oL-SMA protein expression and cleavage of IL-1β. These events were significantly suppressed by pretreatment with P2X7r antagonist A438079. P2XTr blockade also significantly reduced the protein expression of oL-SMA. Our results suggest that the involvement of the P2X7r-NLRP3 inflammasome pathway in the secretion of IL-1β from extracellular ATP/LPS-stimulated human he- patic stellate cells. This study demonstrated that repression of the P2XTr represents a novel potential therapeutic ap- proach to control liver fibrosis.
基金National Natural Science Foundation of China(8146055681760658).
文摘OBJECTIVE To research the effect of naringenin(NAR) on LPS-induced dopaminergic neurons damage and its potential mechanism.METHODS Rats were randomly divided into the following six groups(n=10):control(0.9% NaCl),NAR alone(100 mg·kg-1),LPS(5 μg),LPS+NAR(50 mg·kg-1) and LPS+NAR(100 mg·kg-1).Rats were received a single LPS unilateral injection into the SN pars compacts,after seven daily intragastric administration of NAR,rats′ behavior was analyzed by rotarod test.Then,the expression of TH,IBA-1 and NLRP3 inflammasome were analyzed by Western blotting and immunofluorescence.In vitro experiments,BV-2 cel s were treated with different doses of NAR,and 1 h later,LPS(1 g·L^(-1)) was added to the medium for 24 h,then collect the culture medium and protein for later experiments.The production of IL-1β and IL-18 in culture medium were tested by ELISA,and the production of NO was detected by Griess reagent.The expression of IBA-1,NLRP3 and p-caspase 1 were detected by Western blotting.MN9 D cells were co-cultured with BV2 cells to mimic the animal experiments.MTT assay was used to analyzed the viability of MN9 D cells,and the expression of TH was detected by Western blotting.RESULTS NAR(100 mg · kg-1) could significantly improve the time of rats on the rotating(116.73 s vs 185.45 s,P<0.05).The result of the pathological analysis also suggested that NAR could decrease the activation of microglia as well as the expression of NLRP3 Inflammasome.In addition,NAR also could suppress the expression of pro-inflammatory factor levels,such as IL-1β(P<0.05),IL-18(P<0.05),and the protection of NAR could be inhibited by siR NA NLRP3.Moreover,an in vitro co-culture system with BV2 and MN9 D cells wasused to find the protection of NAR must via microglia,while there is no effect of NAR were directly added to MN9 D cells.CONCLUSION NAR protection of LPS-induced dopaminergic neurons damage might be through mediating NLRP3 inflammasome.
基金supported by National Natural Science Foundation of China(81603587,81603668)Science and Technology Development Plan of Guangdong Province(2017A020211016)Youth Medical Talent Fund of Guangzhou University of Chinese Medicine(QNYC20170105)
文摘OBJECTIVE Recent studies have demonstrated that the Nlrp3 inflammasome serve as a central role in the pathogenesis of cardiovascular diseases and endothelial dysfunction occurs in association with several cardiovascular risk factors.Given the demonstrated anti-inflammatory effects of aspirin,the present study was designed to test whether aspirin diminish NLRP3 inflammasome activation and prevent endothelium injury and associated coronary artery damage during LPS.METHODS Mouse carotid arterial endothelial cells(CAECs)were cultured and treated with 0.1-3 mmol·L^(-1) of aspirin in response to LPS(2μg·mL^(-1))stimuli.After 24 h,the Nlrp3 inflammasome complexes consist of varied proteins were analyzed by WB.NO and T-AOC in the supernatant was detected by ELISA.Intracellular reactive oxygen species(ROS)generation for 24 h was observed by DCF fluorescence.The mice were treated with aspirin(12.5 mg·kg^(-1) per day,62.5 mg·kg^(-1) per day,125 mg·kg^(-1) per day)and dexamethasone(0.0182 mg·kg^(-1)per day)for 7 d.The level of IL^(-1)β,IL^(-1)8 protein was detected by ELISA.RESULTS Immunofluorescence results showed the colocalization of Nlrp3 with ASC or caspase 1decrease in a concentration-dependent manner.Meanwhile,the expression of Nlrp3 and caspase 1protein was decreased with the concentration of aspirin,but no changes the expression of ASC protein.Nlrp3 protein levels in CAECs were 0.33-0.8-fold and cle-caspase 1 protein levels in CAECs were 0.48-1-fold compared to those in LPS stimulation when treated with 0.1-3 mmol·L^(-1) aspirin for 24 h(P<0.01).Aspirin significantly antagonized the effect of LPS on NO(1.22-1.91-fold that of LPS stimulation,P<0.01)and T-AOC expression(1.02-1.90-fold that of LPS stimulation,P<0.01).As the different concentration of aspirin treated,the generation of ROS was 0.51-1.10-fold that of LPS stimulation(P<0.01).In vivo data shown the level of IL^(-1)β,IL^(-1)8 protein from serum are in concordance with the level of Nlrp3 inflammasome activation.CONCLUSION We conclude that aspirin has anti-inflammatory properties,protecting CAECs fromLPS-induced injury by inhibition of NLRP3 inflammasome activation through ROS pathway.
文摘OBJECTIVE To identify the role of GSK3 isoform inhibition on inflammasome activation.METHODS The NLRP3 inflammasome was activated by typical LPS/ATP and host-derived metabolites in primary mouse macrophages.The pharmacological inhibition of GSK3 isoforms on inflammasome activation was assayed by quantifying IL-1βin the supernatant,and activated caspase-1in cell lysates using highly selective inhibitors.Further molecular mechanisms were investigated by protein pulldown assay,confocal imaging using forced gene expression system and endogenous protein tagged mouse macrophages.RESULTS Pharmacological inhibition of GSK3-β,but not GSK3-αisoform suppressed NLRP3 inflammasome activation in response to ATP,urate crystal and the microbial alkaloid toxin staurosporine.GSK3-βinhibition did not inhibit melanoma 2(AIM2)inflammasome activation in response to double-stranded DNA(dsDNA)and did not affect non-canonical caspase-11 inflammasome activation.GSK3-βinhibition suppressed high glucose mediated NLRP3 inflammasome activation.Mechanistically,GSK3-βinhibition blocked NLRP3 inflammasome by preventing pro IL-1βtranscription,reducing caspase-1 activation and ASC speck formation.GSK3-βinhibition blocked NLRP3 inflammasome activation without affecting the level of reactive oxygen species(ROS)which is a crucial component in initialing inflammasome activation.Further studies revealed that GSK3-βdirectly binds to ASC by both co-forced expression and endogenous protein level.Interestingly,we found ASC can be glycosylated in response to inflammasome activation,and GSK3-βinhibition reduced ASC glycosylation.Consistently,the O-Glc NAc transferase(OGT)deficient mouse macrophages showed the significant reduction of mature IL-1βsecretion in response to NLRP3 inflammasome activation.CONCLUSION Our results demonstrate a critical role of metabolism-sensing GSK3-βpathway in mediating NLRP3 inflammasome activation,thus defining a new therapeutic target for sterile inflammation.
基金National Natural Science Foundation of China(81872754)Research Fund of University of Macao(MYRG2018-00037-ICMS)and Science and Technology Development Fund,Macao SAR(FDCT 0031/2019/A1)。
文摘OBJECTIVE Interleukin(IL)-1β,one of the principal inflammatory cytokines mainly secreted by monocytes and macrophages,is produced by cleavage of the inactive pro-IL^(-1)βprecursor by caspase-1 via the NLRP3 inflammasome complex.The fruits of Garcinia cambogia(Clusiaceae)are widely developed as health products for anti-obese purpose.14-deoxygarcinol(DOG)is a polyisoprenylated benzophenone from the fruits of G.cambogia,which showed potent anti-inflammatory effect in our previous study.The objective of this study was to explore the anti-inflammatory mechanism of DOG and its roles in alleviating adipose tissue inflammation and insulin resistance.METHODS The anti-inflammatory effect of DOG was evaluated on LPS plus nigericin-induced THP-1 macrophages.The expression of NLRP3 inflammasome complex proteins was analyzed by Western blotting,immunofluorescence staining and co-immunoprecipitation.The pro-inflammatory cytokines levels were determined by ELISA kits.RESULTS DOG increased the expression of Sirtuin 2(SIRT2)deacetylase and enhanced its deacetylating activity to suppress the NLRP3 inflammasome activation and IL^(-1)βsecretion in THP-1 macrophages.Moreover,DOG attenuated macrophage conditioned medium-induced inflammatory responses in adipocytes and blocked THP-1 macrophages migration towards 3T3-L1 adipocytes.CONCLUSION DOG attenuated the inflammatory crosstalk between macrophages and adipocytes through SIRT2-mediated NLRP3 inflammasome inhibition,which might be used for the treatment of adipose tissue inflammation-related metabolic disorders.
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National Natural Science Foundation of China(8167363181603385)+2 种基金China Postdoctoral Science Foundation(2018M643843)Natural Science Foundation of Shaanxi Province(2017JM8056)Key Research and Development Foundation of Shaanxi province(2018SF-241)
文摘OBJECTIVE To investigate the neuroprotective effects and exact mechanisms of myrrh extract following cerebral ischemic stroke.METHODS Male rats were randomly divided into three groups:sham group,middle cerebral artery occlusion(MCAO)group and myrrh group.Morphological changes were assessed after 7 d of myrrh treatment.Microarray analysis with circulating mRNA was performed to identify differential gene expression profile,gene ontology and pathway enrichment analyses were carried out to predict the gene function.Gene co-expression and pathway networks were constructed to identify the potential targets.The markers of oxidative stress,inflammatory reaction and ferroptosis in the cerebral cortex were detected by ELISA assays.The identified hub pathways and genes were validated by western blotting,immunofluorescence and immunohistochemistry analyses.Neurons were exposed to transient oxygen-glucose deprivation(OGD)to model ischemia-like conditions.siRNA-TXNIP were transfected in OGD-induced neurons to explore the mechanism.RESULTS Myrrh extract significantly alleviated neurological deficits,infarct volume and histo⁃pathological damage in MCAO rats.A total of 2200 differentially expressed genes were identified among the three groups.Oxidation-reduction process,inflammatory response,ferroptosis were enriched as the significant gene ontology items.NOD-like receptor signaling were identified as the hub pathway based on the pathway relation network.TXNIP and NLRP3 were screened as the potential targets by a time sequence profile analysis.The levels of IL-1β,IL-18,TNF-α,MDA and TFR in brain tissues were increased while the CAT,SOD,GSH-px and GPX4 levels were significantly decreased in MCAO group.As expected,myrrh extract greatly reversed these changes.The similarly results were also observed in OGD treated neuron cells.The elevated expressions of TXNIP and NLRP3 induced by OGD were success⁃fully inhibited by myrrh treatment.Knockdown of TXNIP significantly alleviated OGD-induced ROS accumulation and oxidative stress,but the antioxidative effect of myrrh was impaired when TXNIP was absent in neuron cells.In addition,knockdown of TXNIP significantly decreased the expression of NLRP3 and increased the expression of GPX4 in OGDinduced neuron cells.However,myrrh treatment scarcely changed the expressions of NLRP3 and these ferroptosis markers in siRNA-TXNIP pretreated cells,compared with the siRNA-TXNIP alone treatment group.Therefore,these data demonstrated that the neuroprotective effect of myrrh extract was dependent on TXNIP-NLRP3 axis.CONCLU⁃SION Thatmyrrh extract exerts neuroprotective property through alleviated ROS-mediated ferroptosis by regulating the TXNIP/NLRP3 axis in ischemic stroke.Myrrh extract could be considered as a promising candidate for the treatment of ischemic stroke.
文摘Aim To study the effects of baicalein （BC）, a phenolic flavonoid extracted mainly from Scutellaria ba- icalensis Georgi, on lipopolysaccharide （LPS）-induced acute lung injury （ALI） in mice and the molecular mecha- nisms underlying. Methods Mice were administrated intranasally with LPS （20 mg · kg^-1/body weight） to estab- lish the ALI model. Then the mice were treated twice with BC （50,100 and 200 mg · kg^-1, p. o. ） 0. 5 hour and 12 hours after LPS stimulation, following another 12 hours, the lungs were collected for histological study. Results LPS caused marked inflammatory cell infiltration and myeloperoxidase activation in lungs, accompanied by significantly in- creased lung W/D ratio, from 7.97±0. 60 in normal group to 12. 49 ± 1.49 in the model. 77.88% reduction in the lung W/D ratio was observed in 200 mg· kg^-1 dose of baicalein. The myeloperoxidase activity was reduced to 40. 14% in mice treated with 200 mg · kg^-1. The number of total cells, neutrophils, and macrophages in BALF de- creased with increasing concentration of baicalein. Inflammatory cytokines level in serum declined significantly while insignificant changes of the same in BALF was observed in mice treated with 50,100 and 200 mg · kg^-1 doses of ba- icalein. Furthermore, LPS induced markedly the expression of inflammasomes and other inflammation-related genes in lung tissue. Treatment of LPS-exposed mice with BC significantly reduced the expression levels of these genes and al- leviated the pathological changes in lungs. Moreover, 1 μmol · L^-1 and 10 μmol · L^-1 BC inhibited remarkably the nuclear translocation of NF-kappaB p65 in Raw264.7 cells. Conclusion Baicalein alleviates LPS-induced acute lung injury in mice by suppressing NF-KB-mediated inflammatory responses and downregulation of inflammasomes.
