Mechanism Of TSPO/VDAC1-mediated Mitophagy In Diabetic Retinopathy

Purpose:Diabetic Retinopathy(DR)is one of the most common microvascular complications of diabetes.It is a degenerative disease of the retina,which causes severe visual impairment and blindness.Studies have confirmed that its pathological process is closely related to mitochondrial autophagy.DR patients are often accompanied by changes in mitochondrial autophagy-related proteins,and these autophagy-related proteins can affect a series of photoreceptor activities of the retina.Based on this,this topic starts from the important intracellular adaptive event of mitochondrial autophagy,and further explores its mechanism of action and regulatory mechanism in diabetic retinopathy.Translocation protein(TSPO)and voltage anion channel protein 1(VDAC1)are both components of the mitochondrial permeability transport pore.The interaction of the two proteins helps to improve the efficiency of mitochondrial quality control,regulate the structure and function of mitochondria,and its expression ratio is Cell homeostasis is critical.TSPO and VDAC1 form a protein complex.When the ratio of TSPO/VDAC1 increases,it reduces mitochondrial coupling and promotes the excessive production of reactive oxygen species(ROS),thereby inhibiting the mitochondrial autophagy downstream of the PINK1/Parkin pathway and limiting the mitochondria of SQSTM1/p62 Recruitment and abolition of the mitochondrial rearrangement of the autophagy marker LC3II resulted in the accumulation of dysfunctional mitochondria.This subject aims to explore the role of TSPO/VDAC1 in the pathogenesis of DR using STZ-induced diabetic retinopathy rats,and further explores whether TSPO/VDAC1 may play a role in regulating DR through mitochondrial autophagy through in vitro experiments.Clarify the mechanism of TSPO/VDAC1 in DR.Methods1.The animal model of DR was constructed with STZ.The rats were divided into two groups:normal control group and DR model group.HE staining was used to observe the pathological damage of the retina of the rat.The expression of VDAC1 protein and key molecules of mitochondrial autophagy PINK1,Parkin,LC3II,SQSTM1/P62 were detected.The expression of TSPO protein was detected by immunohistochemistry and Western Blot.2.In vitro simulation of DR process,primary HRCECs were cultured in high glucose(30mM)for Oh,3h,12h,24h,48h,real-time fluorescence quantitative PCR detection TSPO,VDAC1,TXNIP,DNM1L,PINK1,Parkin,P62/SQSTM1,LC3II MRNA level,Western Blot detection of TSPO,VDAC1,PINK1,Parkin,SQSTM1/P62,LC3II protein expression,CCK8 detection of cell activity and proliferation capacity of each group,Annexin V/PI flow cytometry detection of cell apoptosis Mitochondrial ROS detection kit detects mitochondrial ROS level,and ELISA kit detects cell supernatant IL-1 and IL-18 levels.3.After silencing TSPO with lentivirus in primary HRCECs,TSPO protein expression decreased,VDAC1 protein expression increased,TSPO/VDAC1 ratio decreased,the expression level of mitochondrial autophagy key protein PINK1,Parkin increased significantly,and the autophagy substrate P62/SQSTM1 was significantly consumed.The cell activity increased,the apoptosis rate,and the level of inflammatory factors decreased compared with the empty carrier group.After adding autophagy inhibitor 3-MA to primary HRCECs that stably and lowly express TSPO under high glucose environment,it was found that 3-MA inhibitor can inhibit the increase of autophagy activity caused by silencing TSPO;the same ELISA results showed that autophagy was inhibited The expression of inflammatory factors IL-1 and IL-18 increased after the level;the results of flow cytometry experiment showed that the percentage of apoptosis increased after the inhibition of autophagy level.Results1.The expression of mitochondrial autophagy-related protein in the retina of DR rats was reduced,and the activity of mitochondrial autophagy was inhibited;the expression of TSPO protein was increased,the expression of VDAC1 protein was decreased,and the ratio of TSPO/VDAC1 was increased;the retinal inflammation of DR rats was increased.2.After high glucose treatment of primary HRCECs,cell viability and proliferation capacity decreased,apoptosis rate increased,intracellular ROS and TXNIP mRNA levels increased,inflammatory factors IL-1 and IL-18 expression increased,and mitochondrial cleavage protein DNM1L mRNA levels increased High,the expression of key proteins of mitochondrial autophagy PINK1,Parkin decreased,the accumulation of autophagy substrate P62/SQSTM1,TSPO protein expression increased,VDAC1 protein expression decreased,TSPO/VDAC1 ratio increased.3.Under high glucose conditions,after silencing TSPO with lentivirus in primary HRCECs,the expression of VDAC1 protein increases,the ratio of TSPO/VDAC1 shrinks,the expression level of mitochondrial autophagy key protein PINK1,Parkin increases significantly,and the autophagy substrate P62/SQSTM1 is consumed Obviously,the cell activity increased,the apoptosis rate,and the level of inflammatory factors were higher than those in the sugar group.Conclusions1.High glucose conditions in vivo and in vitro can lead to increased TSPO expression,decreased VDAC1 expression,increased TSPO/VDAC 1 ratio,and suppressed mitochondrial autophagy;excessive ROS production,increased secretion of inflammatory factors and increased apoptosis.2.Downregulation of TSPO expression in primary HRCECs under high glucose conditions will increase VDAC1 expression and decrease TSPO/VD AC 1 ratio3.TSPO/VD AC1 promotes the generation of ROS and secretion of inflammatory factors by inhibiting the mitochondrial autophagy PINK 1/Parkin pathway under high glucose,thereby participating in the occurrence and development of DR.