Protection And Mechanism Of Astrocytic Connexin43-mediated Mitophagy On Cerebral Ischemia-reperfusion Injury

Background:Ischemic stroke is mainly caused by sudden reduction or complete interruption of local blood flow in brain,resulted in damage to the neurovascular unit(NVU)composed of neurons,glial cells and vascular endothelial cells.As an important component of NVU,astrocytes protect the structure and function of neurons through various mechanisms.Hemichannels and gap junctions composed of astrocytic connexin43(C_x43)have been shown to play an important role in the transmission of various beneficial or harmful information in cerebral ischemia/reperfusion injury.Recent studies have shown that C_x43 can regulate autophagy,which is not related to its function of intercellular communication.Our previous study has also revealed that C_x43 can protect mitochondrial function after cerebral ischemia.As an important way for cells to clear damaged or dysfunctional mitochondria,the role of mitophagy in cerebral ischemia/reperfusion injury has caused increasing attention.However,whether mitophagy participates in the regulation of C_x43 on mitochondrial function has not been reported.This study was designed to investigate the effect and mechanism of astrocytic C_x43 on mitophagy and to clarify its effect on neuron survival after cerebral ischemia/reperfusion,in order to provide a new target for ischemic stroke.Object:To observe the changes of astrocytic mitophagy in cerebral ischemia/reperfusion injury;to explore the effect and mechanism of astrocytic C_x43 on mitophagy and to explicit the effect of C_x43-mediated mitophagy on ischemic neurons.Methods:Astrocytes and neurons were obtained from the cerebral cortex of newborn C57BL/6mice and cultured in vitro.A co-culture system of astrocytes and neurons was constructed with the Transwell chamber.The oxygen-glucose deprivation/reoxygenation(OGD/R)model was made to simulate cerebral ischemia/reperfusion injury.1.Neurons were randomly divided into the neuron group and the co-culture group,and the viability at each OGD/R time point was detected by CCK-8.Astrocytes were randomly divided into various OGD/R time groups,and the LC3-II/I ratio,p62 and TOMM20 expression was detected by Western blot.OGD4h/R24 h was selected as the experimental time,and an autophagy activator rapamycin(RAPA)was used.The expression of TOMM20 and C_x43 in astrocytes was detected by Western blot.Co-localization of LC3 B and C_x43 or TOMM20 was observed by immunofluorescence staining.Mitochondrial autophagosomes were observed by transmission electron microscopy(TEM),and the viability of astrocytes was measured by CCK-8.2.Astrocytes were randomly divided into the control(Con)group,C_x43 knockdown(C_x43 KD)group,glucose and oxygen-glucose deprivation/reoxygenation(OGD/R)group,oxygen-glucose deprivation/reoxygenation + C_x43 knockdown(OGD/R + C_x43 KD)group.The astrocytic viability was tested by CCK-8,and mitochondrial membrane potential(MMP)was detected by the laser confocal microscope and microplate reader.Intracellular reactive oxygen species(ROS)and extracellular nitric oxide(NO)was detected by microplate reader.The viability and apoptosis of neurons in the co-culture system were detected by CCK-8 and TUNEL.Astrocytic mitophagy was detected by examining TOMM20 and TIMM23 expression,mitochondrial autophagosomes and the colocalization of mitochondria and lysosomes.Astrocytic mitophagy pathway PINK1 and FUNDC1 was examined by Western blot.3.Astrocytes were randomly divided into the oxygen-glucose deprivation/reoxygenation(OGD/R)group,oxygen-glucose deprivation/reoxygenation + C_x43overexpression(OGD/R + C_x43 OE)group,oxygen-glucose deprivation/reoxygenation +PINK1 knockdown(OGD/R + PINK1 KD)group,oxygen-glucose deprivation/reoxygenation + CX43 overexpression + PINK1 knockdown(OGD/R + C_x43 OE + PINK1KD)group.Western blot,TEM,and live cell probes were used to detect mitophagy in astrocytes.The astrocytic viability,MMP,intracellular ROS and extracellular NO were measured,and the viability and apoptosis of neurons in the co-culture system were examined.Results:1.Co-culture of astrocytes and neurons significantly improved the viability of neurons after OGD/R(P<0.01);at OGD4h/R24 h,co-culture improved the viability of neurons from32% to about 50%(P<0.001).2.Within OGD8 h,the LC3-II/I ratio in astrocytes significantly increased(P<0.05),and the expression of p62 and TOMM20 reduced(P<0.01).During OGD4h/R(0-24)h,the LC3-II/I ratio increased to varying degrees,and the expression of p62 and TOMM20 decreased significantly(P<0.01).At OGD4h/R24 h,the expression of TOMM20 and C_x43 in astrocytes significantly reduced(P<0.001).Immunofluorescence staining showed that LC3 B expression and the colocalization of TOMM20 and LC3 B enhanced,the distribution of C_x43 changed from cell membrane to cytoplasm,and the colocalization of C_x43 and LC3 B enhanced.TEM showed that mitochondrial autophagosomes significantly increased in astrocytes.Compared with the OGD/R group,RAPA further reduced the expression of TOMM20 and C_x43(P<0.05),significantly increased the number of mitochondrial autophagosomes,enhanced the colocalization of TOMM20 and LC3 B,and improved astrocytic viability(P<0.01).3.Compared with the Con group,the astrocytic viability and MMP in the OGD/R group decreased significantly(P<0.001),and intracellular ROS and extracellular NO increased significantly(P<0.001).Compared with the OGD/R group,the viability and MMP of the astrocytes with C_x43 KD further reduced after OGD/R(P<0.05),while ROS and NO increased(P<0.01).In the co-culture system,compared with the Con group,the viability of neurons in the OGD/R group significantly reduced(P<0.001)and the apoptosis increased(P<0.01),which further enhanced(P<0.01)in neurons co-cultured with C_x43 KD astrocytes after OGD/R.4.Compared with the Con group,the expression of TOMM20 and TIMM23 in astrocytes after OGD/R significantly reduced(P<0.001).Mitochondrial autophagosomes,colocalization of mitochondria and lysosomes,and the expression of PINK1 and FUNDC1 were observed significantly increased(P<0.01).Compared with the OGD/R group,the above changes further enhanced in astrocytes with C_x43 KD after OGD/R(P<0.05),while there was no significant change in expression of FUNDC1(P> 0.05).5.The expression of TOMM20 and TIMM23 significantly reduced in astrocytes with C_x43 OE after OGD/R(P<0.01).A significant increase in mitochondrial autophagosomes and colocalization of mitochondria and lysosomes was observed,and the expression of PINK1 significantly increased(P<0.001).The above changes were reversed by PINK1 KD in astrocytes with C_x43 OE after OGD/R(P<0.01).6.In the OGD/R + C_x43 OE group,the astrocytic viability and MMP increased significantly(P<0.05),while ROS and NO significantly reduced(P<0.01)compared with the OGD/R group.In the co-culture system,astrocytes with C_x43 OE increased the viability of neurons(P<0.001)and reduced the apoptosis(P<0.01)after OGD/R.Whereas PINK1 KD reversed the above effects of C_x43 OE after OGD/R(P<0.05).Conclusions:1.Astrocytic mitophagy was activated by OGD/R,which provided protection for astrocytes against OGD/R injury.2.Astrocytic C_x43 protected the survival of themselves and neurons after OGD/R.This protection of C_x43 was achieved by up-regulating PINK1 pathway to activate mitophagy and ultimately stabilize mitochondrial function.