Explore The Protective Effect And Mechanism Of Autophagy Inhibitor 3-methyladenine On Retinal Ischemia Injury

ObjectiveThis study aims to investigate the possible mechanisms and protective effect of 3-MA against retina ischemia-reperfusion in rats.As result,we hope to find new directions and theoretical basis for treatment in glaucoma.Methods 1.Establish the model of retinal ischemia-reperfusion injury in ratsRetinal ischemia/reperfusion injury(IRI)was performed on the right eyes of male Sprague-Dawley rats,30 Male rats were randomly divided into three groups: normal control group(NC),Sham operated group(Sham),Retinal ischemia-reperfusion injury(IRI)group,at 24 hours after surgery,The morphological transformations of Retina and the number of RGCs were measured by hematoxylin-eosin staining.;the level of LC3(Microtubule-associated protein-1 light chain 3)、Beclin1 in retina were assessed by immunofluorescence staining and Western-blot 2.Explore The protective effect of 3-MA on retina ischemia-reperfusion in rats 40 Male rats were randomly divided into four groups: normal control group(NC),Sham-operated group(Sham),,Retinal ischemia/reperfusion injury(IRI)group,Retinal ischemia/reperfusion injury+3-MA(IRI+3-MA)group,As part 1 described,retinal IRI was performed,then 3-MA(10mg/ml)was injected in Vitreous,The morphological transformations of Retina and the number of RGCs were measured by hematoxylin-eosin staining.;the distribution of LC3 、Beclin1 in retina were assessed by immunofluorescence staining;the level of LC3、Beclin1、B cell lymphoma/leukemia-2(Bcl-2)、Caspase-3 were measured by Western-blot.Results Part one: Establish the model of retinal ischemia-reperfusion injury in rats(1)HE staining results showed that there was no significant difference in the number of RGC cells between Sham group and NC group(P >0.05).The number of RGC cells in IRI group was significantly lower than that in NC and Sham group(P <0.01).(2)Immunohistochemistry and Western-blot results showed that there was no difference in the expression of LC3 and Beclin1 between Sham group and NC group(P >0.05).Compared with NC group and Sham group,The expression of LC3 and Beclin 1 was significantly increased in IRI group(P <0.05)The protective effect of 3-MA against retina ischemia-reperfusion in rats Part two: The protective effect of 3-MA against retina ischemia-reperfusion injury(1)HE staining: There was no significant difference in the number of RGC cells between the Sham group and the NC group(P >0.05).The number of RGC cells in the 3-MA+IRI and IRI groups was significantly lower than that in the NC group.P <0.05);However,the number of RGC cells in the 3-MA+IRI group was increased compared with the IRI group(P <0.05).(2)Immunohistochemistry results showed that there was no significant difference in the AOD of LC3,Beclin1、Bcl-2 and Caspase-3 between the sham group and the NC group(P >0.05),IRI group and IRI+3-MA group.Compared with the N group,the AOD of LC3、Beclin 1、and Caspase-3 were significantly increased(P <0.01),while the expression of LC3,Beclin1,and Caspase-3 was decreased in the IRI+3-MA group than in the IRI group(P <0.01).(3)Western-blot results showed that there was no significant difference in the expression of LC3,Beclin1、Bcl-2 and Caspase-3 between the sham group and the NC group(P >0.05),IRI group and IRI+3-MA group.Compared with the NC group,the protein expression levels of LC3、Beclin1、and Caspase-3 were signi ficantly increased(P <0.05),while the expression of LC3,Beclin1,and Caspase-3 was decreased in the IRI+3-MA group than in the IRI group(P <0.05),but Bcl-2 protein expression increased(P <0.05).Conclusions 1.Rat retinal ischemia-reperfusion can cause apoptosis of retinal ganglion cells 2.Rat retinal ischemia-reperfusion can activate autophagy-related pathways in theretina and increase autophagy-related protein expression in the retina.3.3-MA can reduce the expression of autophagy-related proteins induced by reti-nal ischemia-reperfusion,and it has protective and anti-apoptotic effects on retina l ganglion cells.