To Investigate Autophagic Function On Mitochondrial And Apoptosis And Relative Mechanisms In Müller Glia Cell

Retinal degeneration is the irreversible damage of retinal cells caused by disturbance of retinal microenvironment.Müller glial cells(MGC)are the most predominant glial cells in the retina,supporting other cell components in the retina.Several evidences have shown that the damaged Müller glial cells can be found in some degenerative retinas.Autophagy is a process to renewal of cellular components.Recent studies have shown that autophagy is involved in the pathophysiological process of retinal degeneration,but the role of autophagy in the Müller glial cells is still obscure.Objective: This study mainly investigated the effects of autophagy on the mitochondrial morphology,reactive oxygen species generation,mitochondrial membrane potential and apoptosis of Müller glia cells under normal conditions and oxidative stress conditions respectively,and explored the related molecular mechanisms of SIRT4-mediated autophagic pathway in Müller glia cells.This research will open a therapeutic window for retinal degenerative disease.Methods: Müller glial cells originated from the rat(r-MC)were divided into six group: Normal control group,rapamycin(RAPA)treatment group,3-methyladenine(3-MA)treatment group,oxidative stress group,oxidative stress plus rapamycin treatment group,oxidative stress plus 3-MA treatment group.The expressional changes of Optic atrophy gene1(OPA1)and dynamin-related protein1(DRP1)were detected by western blotting,and the morphological changes of mitochondria were detected by transfected with mitochondrial plasmids(Mito-Ds Red).Reactive oxygen species(ROS)kit was used to detect changes in ROS production,mitochondrial membrane potential was detected by flow cytometry,and apoptosis was detected by western blotting and the living/dead staining.Then we transfected of SIRT4 RNAi virus to knockdown the expression of SIRT4 gene.Western blotting was used to quantitative detect the expressional pattern of Microtubule-associated protein 1 light Chain-3B(LC3),P62,mammalian target of rapamycin(m TOR),Adenosine 5’-monophosphate-activated protein kinase,AMPK)to explore autophagy function and expressional pattern of p-AMPK and its downstream signaling pathways.Adenosine triphosphate(ATP)kit was used to detect ATP production of r-MC cells in normal control group and SIRT4 knockdown group.Finally,Compound C was used to treat r-MC normal control group and SIRT4 knockdown group respectively to inhibit AMPK phosphorylation and explore the expressional pattern of autophagy related proteins.Results: Under normal circumstances,the up-regulation and down-regulation of autophagy induced by rapamycin and 3-methyladenine respectively did not cause morphological changes and apoptosis of r-MC.Under oxidative stress,autophagic dysfunction can increase the apoptosis of r-MC.In addition,down-regulation of autophagy by 3-methyladenine can cause mitochondrial fission and fusion disorder and reduce the expression level of OPA1 under oxidative stress,and rapamycin treatment can reduce mitochondrial fission by reducing the expression level of DRP1.Under oxidative stress,3-methyladenine mediated down-regulation of autophagy can lead to increased ROS production and depolarization of mitochondrial membrane potential.Quantitative detection by western blotting showed that SIRT4 knockdown group of r-MC cells could activate AMPK phosphorylation,inhibit m TOR phosphorylation,up-regulate LC3 I/II ratio,down-regulate P62 expression level and reduce ATP production compared with r-MC normal control group.In vitro experiments showed that Compound C inhibition of p-AMPK could reverse the expression patterns of m TOR,LC3 I/II and P62 induced by SIRT4 knockout.Conclusions: In this study,we concluded that under normal circumstances,r-MC can show great resistance to the dysfunctional autophagy,so the down-regulation of autophagy cannot induce the apoptosis of r-MC under normoxia.However,under oxidative stress,down-regulation of autophagy can cause mitochondrial dynamics disorder of r-MC and induce apoptosis of r-MC.Up-regulation of autophagy can protect r-MC by partially alleviating mitochondrial disorder caused by oxidative stress.Finally,our results indicate that SIRT4 knockdown can activate the autophagic function of r-MC by reducing ATP production and inhibiting AMPK-MTOR signaling pathway.