Protection Of Autophagy In Ischemic Brain Injury And The Neuroprotective Mechanism Of Salvianolic Acid B Base On AMPK/mTOR/ULK1 Pathway

Background:Autophagy is an important process in resisting stress and improving survival ability.Some unnecessary and damaged components in the cell can be degraded and recycled by autophagy after ischemic stroke occurs,thus producing sufficient metabolic substrates for the cell to maintain energy homeostasis and improve survival.As the main cell type in the central nervous system,astrocytes can change their survival and function by activating autophagy,which plays an important role in brain injury and neurological function recovery after stroke.Unc-51 like kinase 1(ULK1)is a key protein for initiating autophagy.Mammalian target of rapamycin(m TOR)can inhibit the activation of ULK1 by phosphorylating the serine 757 site of ULK.Previous studies have shown that activated AMP-activated protein kinase(AMPK)can release the inhibitory effect of m TOR on ULK1 under stress conditions,thereby promoting autophagy.However,there are few studies on the regulation of autophagy by the AMPK/m TOR/ULK1 pathway in astrocytes after ischemia.In addition,selective autophagy is the specific degradation of specific components within the cell.There are many studies on the regulation of autophagy by p S757-ULK1,but few studies explore the effect of autophagy on the degradation of p S757-ULK1.Therefore,we attempted to explore the significance of the AMPK/m TOR/ULK1 pathway in astrocytes after ischemia and its regulation of autophagy,in order to provide a new target for the treatment of ischemic stroke.Salvianolic acid B(Sal B),the main active ingredient in Salvia miltiorrhiza,has been reported to promote autophagy in acute myocardial infarction models,LPS-induced depression models,liver cancer cells,and human umbilical vein endothelial cells.However,the regulation of autophagy and the mechanism of Sal B in astrocytes after cerebral ischemia has not yet been examined.Purpose:To clarify the neuroprotective effect of autophagy on ischemic brain injury,the regulation and mechanism of the AMPK/m TOR/ULK1 pathway on autophagy after ischemic brain injury,and the neuroprotective effect of Sal B.Methods:In vivo,adult male C57BL/6 mice weighing 20-23 g were divided into Sham group,middle cerebral artery occlusion(MCAO)group,Sal B group,bafilomycin A1(Baf A1)group and Sal B+Baf A1 group.After MCAO injury,the cerebral infarction volume was evaluated by Triphenyl tetrazolium chloride(TTC)staining and the ethology was scored by Longa’s standard.In vitro,based on the model of oxygen glucose deprivation(OGD),cultured astrocytes were divided into normal,OGD,Sal B,Baf A1,Sal B+Baf A1,and Dorsomorphin groups.First,the survival rate of astrocytes in each group was determined by methyl thiazolyl tetrazolium(MTT)assay,and cell apoptosis was detected by flow cytometry.Next,the release of inflammatory cytokines was detected using a cytometric bead array(CBA).Western blotting was used to detect the expression of autophagy markers Beclin 1 and LC3 B as well as proteins related to the AMPK/m TOR/ULK1 pathway.The si RNA technique was used to knock down Atg5,NDP52,OPTN,NBR1,and p62 in mouse astrocytes.We also compared the expression of p S757-ULK1 in gene knockdown astrocytes in each group.Co-immunoprecipitation(Co-IP)was used to evaluate the binding of p S757-ULK1 to the autophagy receptors NDP52 and OPTN in astrocytes.Finally,immunofluorescence(IF)was used to observe the co-localization of p S757-ULK1 with exogenous NDP52 and OPTN in He La cells.Results:1.Histomorphological and neurological deficit scores: Compared with the MCAO group,the volume of the cerebral infarction in the group given Sal B was significantly lower(p<0.0001),and the Longa score showed a decreasing trend,but the difference was not statistically significant.2.Cell survival and functional status: Compared with the normal group,the cell survival rate was lower(p<0.001)and the apoptosis rate was higher(p<0.001)in the OGD group.The levels of inflammatory factors IFN-γ,IL-2,and IL-6 in the supernatant of OGD group were higher than those in the normal group(p<0.01),while the levels of anti-inflammatory factors IL-4 and IL-10 were lower than those in the normal group(p<0.05).In addition,the Sal B intervention significantly improved cell survival rate(p<0.01),reduced apoptosis rate(p<0.001),reduced IFN-γ,IL-2,and IL-6(p<0.01),and increased IL-4 and IL-10(p<0.05)release,while the autophagy inhibitor Baf A1 can reverse the protective effect of Sal B on cell survival and function.3.Autophagy and its pathway changes: Compared with the control group,the expression of Beclin 1,p T172-AMPK and LC3 B II/LC3 B I ratio were significantly higher(p<0.01),and the expression of p S2448-m TOR and p S757-ULK1 was significantly lower(p<0.05)in the OGD group.Compared with the OGD group,THE Sal B treatment further increased the expression of Beclin 1,p T172-AMPK,and LC3 B II/LCB I ratio(p<0.05),and decreased the expression of p S2448-m TOR and p S757-ULK1(p<0.01),indicating that it activated the AMPK/m TOR/ULK1 pathway and promoted autophagy.Furthermore,treatment of the Sal B intervention group with dorsomorphin,an AMPK inhibitor,reversed these changes.4.Degradation of p S757-ULK1: for the OGD group and the Sal B intervention group,p S757-ULK1 accumulated in astrocytes treated with the autophagy inhibitors Baf A1 and Atg5 knockdown(p<0.05),and the accumulation was more obvious in the Sal B intervention group.Furthermore,we found that p S757-ULK1 accumulated significantly in astrocytes with respective knockdown of NDP52 and OPTN(p<0.01)after hypoxia and Sal B treatment.The results of Co-IP demonstrated that Sal B treatment increased the binding of p S757-ULK1 with endogenous OPTN or NDP52 in astrocytes after hypoxia.The IF results showed that Sal B treatment increased the co-localization of p S757-ULK1 with exogenous OPTN or NDP52 in astrocytes after hypoxia.Conclusion:1.Enhanced autophagy activity in astrocytes after ischemia can improve the survival of astrocytes,reduce the release of proinflammatory factors and increase the release of anti-inflammatory factors.2.Activation of the AMPK/m TOR/ULK1 signaling pathway can enhance the autophagy activity in astrocytes after ischemia,and autophagy promotes the selective degradation of p S757-ULK1 mediated by NDP52 and OPTN,froming a positive feedback protection loop.3.Sal B promotes the autophagy of astrocytes after ischemia by activating the AMPK/m TOR/ULK1 pathway and promoting the degradation of p S757-ULK1,thereby improving astrocytes survival and regulating their release of inflammatory cytokines.