Grape Seed Procyanidin Extract Reduce Diabetic Nephropathy By Activating The Xc~-/GPX4 Pathway To Inhibit Cell Ferroptosis

Objective:ferroptosis is a newly discovered form of cell death in recent years.It has been pointed out that the occurrence and development of diabetic nephropathy（DN）is related to cell ferroptosis,but its mechanism has not been fully elucidated.Xc^-/GPX4 signaling pathway has been shown to play an important role in ferroptosis.Grape seed procyanidin extract（GSPE）has been shown to have a protective effect against DN,however,whether GSPE can improve cell ferroptosis by mediating the Xc^-/GPX4 pathway and thus inhibit or alleviate the occurrence and development of DN is unclear.The aim of this study was to establish the model of diabetic nephropathy（DN）rats and HK-2 cells cultured in high glucose,and to explore the protective effect of GSPE on DN and its mechanism from the perspective of cell ferroptosis.Methods:1.In vivo experiments:45 SD rats were randomly selected 10 as Control,the other 35 rats were used to establish the DN model with the combination of high glucose high fat diet and streptozotocin.and 31 rats were successfully.The DN rats were divided into DN Group（N=11）,GSPE group（N=10）and Ferrostatin-1（Fer-1）group（N=10）.The rats in GSPE or Fer-1 groups were given 250 mg/Kg GSPE for 12 weeks or 2.5μmol/Kg Fer-1 intraperitoneally for 12 weeks,respectively.The basic indexes such as blood glucose,urine,water intake,body weight,renal function indexes such as blood creatinine,urea nitrogen,urine albumin,oxidative and antioxidant indexes such as MDA,SOD,GSH were measured.The renal tissue sections were stained with HE,PAS,Lillie and Masson,and Western Blot was used to detect the expressions of fibrosis-related proteins COL I,TGF-β1 andα-SMA,ferroptosis critical proteins ACSL4,GPX4,TfR1 and Xc^-/GPX4pathway-related proteins SLC7A11,GSS and GCLM.2.In vitro experiment:HK-2 cells were used to simulate the diabetic environment in vivo.The model of hyperglycemic injury was established with 30 mmol/L glucose.Erastin（20μmol/L）,Fer-1（1.2μmol/L）and GSPE（10,20,30 mg/L）were given respectively,or GSPE intervention after inhibition of the XC^-/GPX4pathway with Erastin（20μmol/L）.Cell viability was measured by CCK-8,ROS level and mitochondrial membrane potential were detected by fluorescence,GSH and MDA content were measured by corresponding kits,and Fe²⁺level was detected by Lillie staining,at the same time,Western Blot was used to detect the expression of fibrosis-related proteins such as COL I,TGF-β1 andα-SMA,ferroptosis key proteins such as ACSL4,GPX4,TfR1,and Xc^-/GPX4 pathway-related proteins such as SLC7A11,GSS,and GCLM.Results:1.Blood glucose,water intake,urine output,serum creatinine,urea nitrogen and urine albumin were significantly increased in DN rats,while body weight was decreased（P<0.05）.The contents of SOD and GSH in renal tissue decreased,while the levels of MDA and Fe²⁺in renal tissue increased（P<0.05）.The results of HE and PAS showed that the basement membrane of the kidney thickened,the glomerulus expanded and the cystic cavity became smaller.Masson’s staining showed that the deposition of fibrinogen was significantly increased,and the expression of COL I,TGF-β1 andα-SMA were significantly increased（P<0.05）.The expression of ferroptosis related proteins ACSL4 and TfR1 were significantly increased,while the expression of GPX4 was significantly decreased（P<0.05）.Meanwhile,the expression of SLC7A11 and its downstream proteins GSS and GCLM were decreased（P<0.05）.After the intervention of Fer-1 or GSPE,the blood glucose,water intake,urine output,serum creatinine,urea nitrogen and urine albumin were decreased,and the body weight was increased（P<0.05）.The levels of SOD and GSH in kidney were increased,while the levels of MDA and Fe²⁺were decreased（P<0.05）.The expression of COL I,TGF-β1andα-SMA were all decreased（P<0.05）.The expression of ACSL4 and TfR1 was inhibited,the expression of GPX4 was enhanced,the expression of SLC7A11 and its downstream proteins GSS and GCLM were enhanced（P<0.05）.2.The cell viability of HK-2 cells cultured in high glucose environment was significantly decreased（P<0.05）.The levels of GSH and mitochondrial membrane potential decreased,while the levels of MDA,ROS and Fe²⁺increased significantly（P<0.05）.The expressions of COL I,TGF-β1 andα-SMA increased significantly（P<0.05）.The expressions of ACSL4 and TfR1 increased significantly,while the expressions of GPX4decreased significantly（P<0.05）.At the same time,the expression of SLC7A11 and its downstream proteins GSS and GCLM decreased（P<0.05）.However,Cell viability was improved after the administration of ferroptosis inhibitor fer-1 or different doses of GSPE（P<0.05）.The levels of GSH and mitochondrial membrane potential increased,while the levels of MDA,ROS and Fe²⁺decreased（P<0.05）.The expression of COL I,TGF-β1 andα-SMA were significantly decreased（P<0.05）,while the expression of ACSL4 and TfR1 were significantly decreased,at the same time the expression of GPX4 was significantly increased（P<0.05）,the expression of SLC7A11 and its downstream proteins GSS and GCLM were significantly increased（P<0.05）.After erastin inhibited Xc^-/GPX4,the cell survival rate was significantly decreased（P<0.05）,and the expression of COL I,TGF-β1 andα-SMA were significantly increased（P<0.05）,while the expression of ACSL4 and TfR1 were significantly increased,the expression of GPX4 was significantly decreased（P<0.05）,and the expression of SLC7A11 and its downstream proteins GSS,GCLM were all decreased（P<0.05）.At this time,different doses of GSPE could still effectively improve the cell survival rate（P<0.05）,the expression of COL I,TGFβ1 andα-SMA were significantly decreased（P<0.05）,while MDA,ROS and Fe²⁺were decreased（P<0.05）,at the same time,the expression of ACSL4 and TfR1decreased significantly,while the expression of GPX4 increased significantly（P<0.05）,and the expression of SLC7A11 and its downstream proteins GSS and GCLM increased significantly（P<0.05）.Conclusion:DN may present with kidney damage characterized by renal fibrosis,and ferroptosis is involved in the occurrence and development of DN.Inhibition of ferroptosis can significantly improve DN.GSPE can alleviate diabetic nephropathy characterized by fibrosis by activating the Xc^-/GPX4 signaling pathway to inhibit the occurrence of ferroptosis in renal tubular epithelial cells.