The Inhibitory Effect And Mechanism Of Berberine On Ferroptosis Induced By High Glucose In Retinal Vascular Endothelial Cells

Objective:To explore whether ferroptosis occurs in human retinal microvascular endothelial cells（HRCECs）in high glucose environment and the inhibitory effect of Berberine（BBR）on ferroptosis in HRCECs induced by high glucose and its mechanism,in order to provide a new therapeutic target for Diabetic retinopathy（DR）.Methods:1.Normal human retinal microvascular endothelial cells（HRCECs）were cultured in vitro.When the cells grew to 70%,the serum was starved for 12h,and then the cultured HRCECs were stimulated with 30μM high glucose for 48h.The levels of iron death related genes GPX4,ACSL4 and FSP1 were detected by RT-q PCR and Western blot.CCK8 kit was used to detect cell activity.Reactive oxygen species（ROS）were detected with ROS kit.The levels of GSH,MDA and Fe²⁺were detected by GSH,MDA and Fe²⁺kit.The changes of mitochondria stimulated by hyperglycemia were observed under electron microscope.2.HRCECs cultured in vitro were stimulated with 30μM high glucose+gradient concentration BBR（0,7.5,15,30,60μM）for 48h,and the optimal intervention concentration of BBR was selected by using CCK8 kit to detect the cell activity at each concentration.3.Berberine intervention and experimental grouping:Group A:normal control group;Group B:high-glucose modeling group;Group C:modeling+DMSO group;Group D:modeling+Berberine group;Group E:Modeling+iron death inhibitor（FER1）group.The dynamic changes of iron-death related genes GPX4,ACSL4,FSP1 and core signaling pathway factors Nrf2 and HO-1 in each group were detected by RT-q PCR and Western blot.Reactive oxygen species（ROS）levels were detected by reactive oxygen species（ROS）kit.The levels of GSH,MDA and Fe²⁺were detected by GSH,MDA and Fe²⁺kit.The changes of mitochondria in each group were observed under electron microscope.Results:1.Under high glucose stimulation,the activity of HRCECs cells was decreased,the ROS level was increased（P<0.05）,the GSH level was decreased（P<0.05）,the MDA and Fe²⁺levels were increased（P<0.05）,the ACSL4 protein and m RNA levels were elevated,meanwhile the protein and m RNA levels of GPX4 and FSP1 were reduced（P<0.05）.2.After BBR intervention on HRCECs stimulated by high glucose,compared with high glucose treatment group alone,the ROS level of cells after BBR intervention was decreased（P<0.05）,the intracellular GSH level was increased（P<0.05）,the MDA and Fe²⁺levels were decreased（P<0.05）,and the protein and m RNA levels of ACSL4 were decreased.The protein and m RNA levels of GPX4 and FSP1were increased（P<0.05）,the protein and m RNA levels of Nrf2 were increased,and the protein and m RNA levels of HO-1 were increased（P<0.05）.The results of BBR intervention were consistent with those of iron death inhibitor intervention.Conclusion:1.Ferroptosis of retinal endothelial cells is involved in the occurrence and development of diabetic retinopathy2.Berberine inhibits ferroptosis in retinal endothelial cells by activating Nrf2/HO-1/GPX4 signaling pathway,thus delaying the process of DR,which provides a theoretical basis for the study of the pathogenesis of DR And drug therapy.