Berberine Protects Against High Glucose-Induced Retinal Cone And Pigment Epithelial Cell Injury Via Activation Of AMPK/mTOR

Diabetic retinopathy(diabeticretinopathy,DR)is a unique and common eye complication of diabetes,which is an important cause of visual impairment,retinal exfoliation and blindness in patients with diabetes.In the past,it was believed that DR was only a diabetic microvascular complication.however,recent studies have found that in addition to microvascular lesions,there are neurodegenerative lesions in the process of DR lesions,even earlier than microvascular lesions.Among them,cones,as important photoreceptor cells in the retina,are responsible for vision,and retinal pigment epithelial cells,as an important part of the retinal blood-visual barrier,play an important role in supporting and nourishing photoreceptors.It plays an important role in maintaining visual and retinal function,and may become one of the cells invaded in the early stage of diabetes.Therefore,the study on the mechanism of cone and retinal pigment epithelial cell injury induced by high glucose and the research and development of therapeutic drugs as a target are of great significance for the prevention and treatment of DR.Previous studies have shown that the pathogenesis of DR is closely related to the imbalance between autophagy and apoptosis in retina and retinal supporting cells.AMPK,as an energy metabolic switch,upregulates autophagy by inhibiting mTOR,.It is proposed that AMPK/mTOR signal pathway,as an important signal pathway to regulate autophagy-apoptosis balance,plays a key role in the occurrence and development of DR.The drugs that regulate this pathway may play a role in protecting retinal neurodegenerative diseases.Berberine hydrochloride(Berberine,BBR),also known as berberine hydrochloride,is the main active component of Coptis chinensis,which is mainly used in the treatment of gastrointestinal diseases.In recent years,it has been found that berberine can be used as an AMPK agonist for the prevention and treatment of type 2 diabetes and its complications.The previous study of our group found that Huangkui solid dispersion(Huang-guisoliddispersion,HGSD)with berberine as the main active component could activate AMPK/mTOR pathway,increase autophagy of M ü ller cells,reduce apoptosis of M ü ller cells,and improve DR.However,it is not clear whether berberine can activate autophagy of cones and retinal pigment epithelial cells,reduce apoptosis and improve the protective mechanism of high glucose-induced injury of cones and retinal pigment epithelial cells by regulating AMPK/mTOR signal pathway.The aim of this study is to establish a model of cone and retinal pigment epithelial cell injury induced by high glucose.AMPK inhibitor siRNA and agonist AICAR,were given to verify the mechanism of down-regulation of AMPK activity and autophagy-apoptosis imbalance in cones and retinal pigment epithelial cells injured by high glucose.To clarify the mechanism of berberine promoting autophagy,reducing apoptosis and improving the damage of cones and retinal pigment epithelial cells induced by high glucose by regulating AMPK/mTOR signal pathway.The research contents of this thesis are as follows:Ⅰ.To explore the role of abnormal AMPK/mTOR pathway in the injury of cones and retinal pigment epithelial cells induced by high glucose.The injury model of cones and pigment epithelial cells was induced by high glucose(HG group),and AMPK inhibitor siRNA or agonist AICAR was given.Apoptosis was detected by Hoechst33258 staining,autophagy was detected by MDC staining,and the expression of AMPK/mTOR signal pathway related proteins p-AMPK/AMPK,pmurmTORand mTOR,apoptosis related proteins Bcl-2,Bax,Caspase-3,and autophagy related proteins Beclin-1,LC3I/II and ULK1 were detected by WesternBlot.Compared with Con,in HG group,nuclear condensation pyknosis,irregular margin,increased apoptotic cells,decreased yellow-green fluorescent granules and reduced autophagy,decreased anti-apoptotic protein Bcl-2,increased expression of Caspase-3 and pro-apoptotic protein Bax,decreased expression of autophagy-related proteins Beclin-1,LC3I/II and ULK1,decreased expression of p-AMPK/AMPK and increased expression of p-mTOR/mTOR in HG group.The level of autophagy and apoptosis and their protein expression in cones and pigment epithelial cells treated with siRNA were similar to those treated with HG,while the number of apoptotic cells increased and the number of apoptotic cells decreased when treated with AMPK agonist AICAR,which reversed the effects of high glucose on apoptosis-related proteins and autophagy-related proteins.The results showed that high glucose could inhibit the expression and activity of AMPK in cones and pigment epithelial cells,increase the expression of mTOR,reduce autophagy and induce apoptosis.Ⅱ.It is clear that berberine ameliorates the damage of cones and retinal pigment epithelial cells induced by high glucose by regulating AMPK/mTOR pathway.On the basis of the injury model of cone and pigment epithelial cells induced by high glucose,berberine(BBR group)or combined with AMPK inhibitor siRNA was used to detect the above indexes.The results showed that compared with HG group,the nuclear morphology of BBR group was significantly improved,nuclear pyknosis decreased,nucleus showed diffuse and uniform light blue,and MDC staining showed that yellow-green fluorescent particles were significantly increased.The expression of anti-apoptotic protein Bcl-2 increased significantly,while the expression of Caspase-3 and pro-apoptotic protein Bax decreased significantly,the expression of autophagy-related proteins Beclin-1,LC3I/II and ULK1 increased,the expression of p-AMPK protein increased,and the expression of p-mTOR protein decreased.The effect of berberine on autophagy and apoptosis was blocked after intervention with AMPKsiRNA.The results showed that berberine could promote autophagy and reduce apoptosis by activating AMPK signal pathway.It can protect cones and retinal pigment epithelial cells from damage induced by high glucose.In summary,abnormal AMPK/mTOR pathway plays an important role in the damage of cones and pigment epithelial cells induced by high glucose.Berberine promotes autophagy,inhibits apoptosis and improves the damage of cones and pigment epithelial cells induced by high glucose.Berberine can promote autophagy,inhibit apoptosis and improve the damage of cones and pigment epithelial cells induced by high glucose.