| Objective: To study the modulation of mitochondrial autophagy in cerebral ischemiareperfusion injury(CIRI)by Taohong Siwu Tang(THSWD)and its relationship with NLRP3 inflammasome activation,and to elucidate the neuroprotective mechanism of THSWD.Methods: 1.Middle cerebral artery occlusion reperfusion(MCAO/R)model in rats was built to simulate I/R.Adult male SD rats(220-270 g)were randomly divided into the following four groups: the sham group,the MCAO/R group,the MCAO/R + THSWD group,and the MCAO/R + THSWD + Mitochondrial division inhibitor 1(Mdivi-1)group.Neurological defect scores were used to evaluate neurological function.2,3,5-Triphenyltetrazolium chloride(TTC)staining was conducted to measure cerebral infarct volume.Nissl staining,H&E staining and TUNEL staining were executed to detect ischemic cortical neuronal cell viability and apoptosis.Electron microscopy was used to observe the ultrastructural changes of mitochondria.Total Reactive Oxygen Species(ROS)in tissue were measured by fluorescence spectrophotometry,and the activation status of microglia was evaluated by Iba-1/CD16 immunofluorescence staining.The levels of mitophagy-related proteins(LC3,Parkin,PINK1),NLRP3 inflammasomerelated proteins(NLRP3,ASC,Pro-caspase-1,Cleaved-caspase-1),and inflammatory cytokines(Pro-IL-18,Pro-IL-1β,IL-18,IL-1β)were evaluated by western blotting.2.Oxygen glucose deprivation/ reperfusion(OGD/R)model of PC12 cells was used to simulate I/R injury of nerve cells in vitro.The experiment was grouped as follows:control,OGD/R,OGD/R+THSWD(5%,10% and 15%)group,OGD/R+THSWD+DMSO group and OGD/R+THSWD+Mdivi-1 group.Oxygen and glucose was restored for 24 hours after 4-6 h of deprivation.The severity of damage to PC12 cells was evaluated by CCK8,flow cytometry and lactate dehydrogenase(LDH).Mitochondrial morphology and function were examined by transmission electron microscopy(TEM),ATP and mitochondrial membrane potential(MMP)assay kits..Cellular autophagy and NLRP3 inflammasome-associated proteins were detected by Western blot and immunofluorescence staining.Results: 1.THSWD treatment reduced brain infarct volume(P < 0.001),decreased neurological function scores(P < 0.05),and reduced neuronal cell injury(P < 0.05)in MCAO/R rats;enhanced the expression of autophagic markers(LC3-II/LC3-I and Beclin1)and mitochondrial autophagic markers(Parkin and PINK1)(P < 0.05);reduced ROS production(P < 0.01),inhibited the expression of NLRP3 inflammasome-related proteins(NLRP3,ASC,Cleaved-caspase-1)and inflammatory factors(IL-18,IL-1β)(P < 0.05).In addition,THSWD treatment inhibited the activation of M1 microglia(P <0.001).2.THSWD enhanced the viability(P < 0.05),reduced apoptosis(P < 0.01)and damage(P < 0.01)of OGD/R-injured PC12 cells;improved mitochondrial structural disruption,increased mitochondrial membrane potential and ATP production(P < 0.05);and inhibited ROS release(P < 0.01)and NLRP3 inflammasome activation(P < 0.05).In contrast,the mitochondrial autophagy inhibitor Mdivi-1 inhibited the above beneficial effects of THSWD(P < 0.05).Conclusions: THSWD was able to exert neuroprotective effects on MCAO/R-injured rats and OGD/R-induced PC12 cells,and its mechanism of action was associated with enhanced mitochondrial autophagy to inhibit NLRP3 inflammasome activation. |
PDF Full Text Request