Mechanisms Underlying Astrocytic Connexin-43 Autophagy Degradation During Cerebral Ischemia Injury And The Effect On Neuroinflammation And Cell Apoptosis

Background:In the central nervous system,gap junction protein 43(connexin,Cx43)is the main component of gap junction protein,Cx43 can achieve cell-to-cell communication(especially neurons and astrocytes,astrocytes Plastid cells and oligodendrocytes),and play an important role in ion transmission,substrate exchange and information transmission.After the occurrence of ischemic stroke,the gap junction function is abnormal,which can transmit not only death signals and calcium overload,but also some neuroprotective factors.This role is very important,but the mechanism is not clear.Studies in recent years have shown that the content of Cx43 on the astrocyte cell membrane decreases and internalization increases after ischemia.In addition,studies have confirmed that Cx43,which is internalized under physiological conditions,is selectively degraded by autophagy.The results of the previous studies provided a new idea for this study,namely,to investigate whether Cx43 internalized after ischemia is selectively autophagy-degraded and its detailed degradation mechanism,and to intervene in the autoimmune degradation of Cx43 for neuroimmunity and apoptosis.Effects,which allows us to find more targets for therapeutic intervention by understanding the detailed molecular mechanisms.Object:To explore the fate of Cx43 after cerebral ischemia and to observe whether Cx43 is degraded by cell autophagy pathway;To clarify the molecular mechanism of Cx43 autophagy degradation after cerebral ischemia;To observe the neuroimmunity and apoptosis induced by Cx43 autophagy degradation to cerebral ischemia and to determine whether intervention of Cx43 autophagy degradation could become a new target for the treatment of ischemic brain injury.Methods:In vitro experiments were performed using primary cultured newborn C57 BL / 6 mice astrocytes and HeLa cell lines,and a model of Oxygen-glucose deprivation(OGD)was used to simulate cerebral ischemic injury.Adult in vivo C57 BL / 6 male mice weighing 20-25 g were used to simulate cerebral ischemic injury using the Middle cerebral artery occlusion model(MCAO).1.Cx43 is degraded by autophagy under ischemic conditions: control groups of mouse primary astrocytes and OGD were set up,and Western blotting(WB)was used to study the deficiency of mouse astrocytes.Expression of Cx43 and phosphorylated Cx43(S368)before and after blood.Immunofluorescence(IF)was used to study the colocalization of Cx43 and LC3 b in astrocytes of mice before and after ischemia;siRNA was used to knock down the autophagy-related protein Atg5 For expression in primary mouse astrocytes,the control group,Atg5 knockdown group,OGD group and Atg5 knockdown + OGD group were set,and the expressions of Cx43 and phosphorylated Cx43(S368)before and after ischemia were detected by Western blotting;The sham operation group and MCAO group were set up in vivo.The co-localization of Cx43 and LC3 b in frozen brain sections of mice before and after MCAO was studied by IF.2.The autophagy degradation of Cx43 after ischemia is achieved through two autophagy receptors,OPTN and NDP52: In vitro experiments use siRNA to knock down the expression of OPTN or NDP52 in primary mouse astrocytes.The expressions of Cx43 and phosphorylated Cx43(S368)before and after ischemia were detected by Western blotting in the low group,OGD group and knockdown + OGD group.The control group and OGD group were set up,and the astrocytes in mice before and after OGD were studied by IF.Co-localization of Cx43 with autophagy receptors OPTN and NDP52,and the use of co-immunoprecipitation(Co-IP)to study Cx43 and autophagy receptors OPTN and NDP52 before and after ischemia in mouse astrocytes Ligation situation;using plasmid overexpression technology to overexpress RFP-OPTN or RFP-NDP52 in HeLa cell lines,set normal overexpression group and OGD + overexpression group,and apply Co-IP to study Cx43 and autophagy in HeLa cells before and after ischemia The connection of the receptors RFP-OPTN and RFP-NDP52,and the co-localization of Cx43 and autophagy receptors RFP-OPTN and RFP-NDP52 before and after ischemia in HeLa cells were observed by IF;in vivo experiments were set up in a sham operation group and a MCAO group Before researching MCAO through IF Frozen sections of Cx43 and autophagy receptor co-localization OPTN NDP52 case mouse brain.3.The autophagy degradation of Cx43 under ischemic conditions is related to PINK1,and PINK1 is an upstream protein: siRNA technology was used to knock down the expression of PINK1 in primary mouse astrocytes.The control group,knockdown group,and OGD group were set up.And knockdown + OGD group,the expression of Cx43 and phosphorylated Cx43(S368)before and after ischemia were detected by Western blotting;the control group,knockdown group,OGD group and knockdown + OGD group were set,and co-immunoprecipitation was applied.,Co-IP)to study the connection of Cx43 to OPTN,NDP52,and PINK1 before and after ischemia in astrocytes in mice;using plasmid overexpression technology to simultaneously overexpress RFP-OPTN and PINK1-Myc or RFP-NDP52 and PINK1-Myc.Colocalization of Cx43,RFP-OPTN or RFP-NDP52 and PINK1-Myc in HeLa cells before and after ischemia was observed by IF.4.The autophagy degradation of Cx43 after ischemia is related to ubiquitination: use plasmid overexpression,overexpress HA-Ub in HeLa cells,set overexpression group and overexpression + OGD group,and study the pre-and post-ischemia by Co-IP Cx43 in HeLa cells and the connection between phosphorylated Cx43(S368)and HA-Ub.5.Phosphorylation of Cx43 is the starting point of autophagy degradation after ischemia: Amino acid point mutation technology was used to construct the GFP-Cx43,GFP-Cx43(368A)and GFP-Cx43(247A + 265A)plasmids,and passed through HeLa cells The expression of GFP-Cx43,GFP-Cx43(368A)and GFP-Cx43(247A + 265A)and OPTN,NDP52 and PINK1 before and after ischemia were studied by Co-IP.6.Intervention of the effects of Cx43 autophagy degradation on neuroimmunity and apoptosis: siRNA technology was used to knock down the expression of Atg5,OPTN,NDP52 and PINK1 in mouse astrocytes,and grouped into knockdown groups and corresponding knockdowns.In the low + OGD group,the supernatants of each group before and after ischemia were collected,and the expressions of TNF and IL-10 in the supernatants were measured by flow cytometry.The expression of Cx43 in mouse astrocytes was knocked down by siRNA,and the group was set as The control group,autophagy agonist group,OGD group,and OGD + autophagy agonist group were used to determine the apoptotic ratio of mouse astrocytes by flow cytometry.Results:1.Western blotting(WB)showed that the level of Cx43 in astrocytes after hepoxia decreased with ischemia time,while phosphorylated Cx43(S368)remained basically unchanged.In Hela cells with overexpression of Parkin,the level of Cx43 under hypoxic conditions also showed a downward trend.The results of immunofluorescence(IF)demonstrated that co-localization of Cx43 and LC3 b in mouse astrocytes increased after ischemia.Cx43 and phosphorylated Cx43(S368)accumulated after ischemia in mouse astrocytes with the knockdown of Atg5.In mouse astrocytes with the addition of Baf A1,Cx43 and phosphorylated Cx43(S368)accumulated after ischemia.2.In mouse astrocytes with the respective knockdown of OPTN and NDP52,the level of Cx43 and phosphorylated Cx43(S368)accumulated after ischemia.The results of immunofluorescence(IF)demonstrated that the co-localization of Cx43 with OPTN or NDP52 improved in mouse astrocytes after ischemia.The results of co-immunoprecipitation(Co-IP)demonstrated the binding of Cx43 with OPTN or NDP52 in mouse astrocytes after hypoxic had increased.3.In mouse astrocytes knocked down by PINK1,the level of Cx43 and phosphorylated Cx43(S368)accumulated after ischemia.In mouse astrocytes with the knockdown of PINK1,there was uncoupling of Cx43 with OPTN or NDP52 after ischemia.4.The results of co-immunoprecipitation(Co-IP)showed that the level of ubiquitin modification of Cx43 and phosphorylated Cx43(S368)in Hela cells transfected with ubiquitin protein increased after ischemia.The S65 modification also increased.5.Co-IP analysis showed that NDP52,OPTN,PINK1,p Ub(S65)bound to GFP-Cx43 had increased following OGD,but these connections decreased in the overexpressed GFP-Cx43(368A)HeLa cells or overexpressed GFP-Cx43(247A+265A)HeLa cells.6.Results of Flow cytometry suggested that blocking the Cx43 autophagy degradation pathway can reduce pro-inflammatory cytokine concentrations(TNF)and increase anti-inflammatory cytokine concentrations(IL-10)during OGD.Additionally,knockdown of Cx43 or accelerating its degradation protects astrocytes from apoptosis under ischemia stress.Conclusion:Cx43 is degraded by autophagy under cerebral ischemia.When ischemia occurs,the S368,Y265,and Y247 sites of Cx43 are phosphorylated,while the S65 site of ubiquitin molecule is phosphorylated by PINK1.Plain.Cx43,carrying phosphorylation and ubiquitination,binds to autophagy receptors OPTN and NDP52,and is targeted for degradation in autophagosomes.Intervention of the Cx43 autophagy degradation pathway has confirmed that Cx43 may have the potential to promote apoptosis and inhibit inflammation in ischemia,thereby providing new treatment ideas for cerebral ischemia treatment.