The Mechanism Of Metformin Regulating Mitochondrial Dynamics Via AMPK Signaling In Retinal Ischemic/Reperfusion Injury

Objective Retinal ischemia is one of the main causes of visual impairment and loss,and is closely related to the underlying pathological mechanisms of many ophthalmic diseases.Further research on the underlying pathological and molecular mechanisms of retinal ischemia injuries has been become an important issue in the world.Metformin is a biguanide drug widely used for glucose metabolism-related and diabetes-related complications.In recent years,it has been found that in addition to lowering blood sugar and controlling diabetic complications,metformin also has antiaging,anti-tumor,anti-inflammation and other beneficial effects.However,the effetct of metformin on retinal ischemia-reperfusion injury is unclear,and its related molecular mechanism needs to be further investigated.The purpose of this study was to determine the effect of metformin on ischemia-reperfusion retinal injury,to clarify the specific molecular mechanism of metformin regulating mitochondrial dynamics and its beneficial effect on retinal ischemia-reperfusion injuries,so as to provide a new direction and theoretical basis for clinical treatment of ischemic retinal diseases.Methods A mouse model of retinal ischemia-reperfusion injury was established,and the effects of intravitreal injection of metformin at different doses on retinal ischemiareperfusion injury were detected by retinal paraffin section and H&E staining.The CCK8 cell viability assay was used to detect the effect of metformin on the cell viability after simulated ischemia-reperfusion injury in retinal R28 cells;Western blot assay was used to detect the mitochondrial morphology regulatory proteins including Drp1,Drp1 phosphorylation,OPA1 and Mfn2 at different time points after retinal ischemia-reperfusion injury in mice retinas;Western blot assay was used to detect the expression alterations of mitochondrial morphology regulatory proteins Drp1,Drp1 phosphorylation,Mfn2 and OPA1 in mice retina with retinal ischemia-reperfusion injury after metformin application;Western blot assay was used to detect the level changes of mitochondrial morphology regulatory proteins Drp1,Drp1 phosphorylation,Mfn2 and OPA1 in R28 cells with simulated ischemia reperfusion after metformin treatment;TMRE staining and flow cytometry was used to detect the effect of metformin on mitochondrial membrane potential after simulate ischemiareperfusion injury in R28 cells;Western blot was used to detect the activity of AMPK signaling in R28 cells after simulated ischemia-reperfusion injury with or with out metformin;Western Blot was used to examine the effect of metformin on the level of mitochondrial morphology regulation protein after SIR injury after inhibiting AMPK;The effect of AMPK-mediated expression alterations of mitochondrial morphology regulatory protein on the increase of intracellular ROS production and mitochondrial membrane potential decrease caused by SIR injury was detected by DHE staining and TMRE staining followed by flow cytometry.The effects of AMPK-mediated expression changes of mitochondrial morphology regulatory protein on retinal ischemia-reperfusion injury-induced RGC cell loss were detected by whole retinal mounting and immunofluorescence staining.Results Metformin significantly alleviated the reduction of retinal thickness and the number of ganglion cells caused by retinal ischemia-reperfusion injury in vivo.Metformin significantly attenuated the decrease of cell viability of R28 cells caused by simulated ischemia-reperfusion injury in vitro.The expression of mitochondrial fusion proteins OPA1 and Mfn2 were inhibited in the early stage of injury(on days 1and 3 after IR),and returned to normal levels on day 7.The expression alterations of mitochondrial fission protein Drp1 was not observed after retinal ischemiareperfusion injury.However,the phosphorylation of Drp1 at S616 was significantly upregulated on day 1 after ischemia-reperfusion injury.Intravitreal injection of metformin in mice or administration or metformin in R28 cells reversed the ischemiareperfusion injury-induced inhibition of OPA1 and Mfn2 expression,but had no effect on the phosphorylation levels of Drp1 and Drp1 phosphorylation at S616.Metformin significantly inhibited the mitochondrial fission and mitochondrial membrane potential decrease caused by simulated ischemia-reperfusion injury in R28 cells.AMPK was mildly inhibition after ischemia-reperfusion injury,while administration of metformin significantly activated AMPK signaling.Application of AMPK inhibitor Compound C,knockdown of OPA1 or Mfn2 abolished the protective effect of metformin on the reduction of mitochondrial membrane potential and ROS generation in R28 cells induced by simulated ischemia-reperfusion injury in R28 cells.Application of AMPK inhibitor,Compound C or knockdown of OPA1 or Mfn2 abolished the protective effect of metformin on RGC cell loss induced by retinal ischemia-reperfusion injury in mice.Conclusions Metformin significantly alleviated retinal ischemia-reperfusion injury,manifested in a significant improvement in retinal thickness reduction and RGC cell loss.Metformin ameliorated retinal ischemia-reperfusion injury-induced retinal damage and RGC loss dependent on AMPK-mediated upregulation of OPA1 and Mfn2 proteins.OPA1 and Mfn2 upregulation mediated by AMPK inhibited mitochondrial fission,cellular oxidative stress and mitochondrial dysfunction.Our study shows that metformin may become a potential drug for the clinical treatment of ischemic retinal diseases,and further exploration of molecules or drugs targeting to AMPK,OPA1 and Mfn2 may have important scientific and clinical value significance.