The Influence Of Endoplasmic Reticulum Stress-related PERK Signaling Pathway On Ferroptosis In Diabetic Kidney Disease And Intervention Mechanism Of Placenta-derived Mesenchymal Stem Cells

Background and aims Diabetic kidney disease(DKD)is one of microvascular complications in diabetes,and also cause end-stage renal disease.The pathogenesis is complex and unknown,and the existing treatment methods and efficacy are limited.Ferroptosis and endoplasmic reticulum stress(ERS)levels were increased in patients with DKD.Mesenchymal stem cells(MSCs)are a multipotent stem cells with self-renewal capacity and multidirectional differentiation potential,showing great potential in the treatment of diabetic complications.MSCs have been reported to play a important role in regulating ERS and diabetic renal cell apoptosis.However,it is unclear whether MSCs can regulate the ferroptosis level in DKD kidney cells.This study intends to explore: 1.Whether high glucose-induced glomerular podocytes undergo ferroptosis;2.Whether placental mesenchymal stem cells(PMSCs)can alleviate ferroptosis and ERS level in DKD model;3.Whether ERS-related PERK signaling pathway can mediate high glucose-induced ferroptosis in glomerular podocytes;4.Whether PMSCs can regulate high glucose-induced glomerular podocyte ferroptosis through ERS-related PERK signaling pathway.Methods DKD model was established,1)In order to explore whether high glucose could induce ferroptosis in renal podocytes(MPC 5),the experiment was divided into three groups: normal control group,high-glucose treated group(50 m M),and high-glucose+ ferroptosis inhibitor group(Ferrostain-1,Fer-1 10 u M).2)In order to explore whether PMSCs can improve ferroptosis and ERS level in DKD model,the cell experiment was divided into three groups: normal control group,high glucose treated group,and high glucose + PMSCs coculture group;the animal experiment was divided into three groups: normal control group,DKD model group,and DKD model+ PMSCs injection group.3)To explore whether the ERS-related PERK signaling pathway mediated high glucose-induced ferroptosis in podocytes,the experiment was divided into three groups: normal control group,high glucose treated group,and high glucose + PERK inhibitor group(PERK inhibitor,PERK-i 10 u M).4)To explore whether PMSCs improve high glucose-induced ferroptosis by inhibiting ERS-related PERK signaling,the experiment was divided into four groups: normal control group,high glucose treated group,high glucose + PMSCs coculture group,and high glucose+ PMSCs coculture + PERK agonist group(PERK agonist,PERK-a 10 u M).The protein expression levels of p-PERK,GRP78,GPX4,FSP1,and COX2 in podocytes were determined by western blotting,The protein expression levels of p-PERK,GRP78,GPX4,FSP1 and COX2 in kidney tissues were evaluated by immunohistochemistry,GPX4,COX2 and FTH1 m RNA expression levels in podocytes were measured by real-time quantitative PCR,Cell microprototype glutathione(GSH)detection kit and malondialdehyde(MDA)detection kit detected MDA and GSH levels in podocytes,The level of podocyte ROS was measured by flow cytometry,mitochondrial ultrastructural changes were visualized by transmission electron microscopy.Results Cell experimental results showed that compared with control group,high glucose-induced podocytes increased expression of p-PERK,GRP78,COX2 protein,decreased expression of GPX4,FSP1 protein significantly,elevated levels of ROS and MDA,reduced levels of GSH,and altered the mitochondrial structure represented by a mitochondrial swelling,outer membrane disrupted,mitochondrial cristae reduced or disappeared.While cocultured with PMSCs,using PERK or ferroptosis inhibitor,these changes were reversed.In contrast,pretreatment of PERK agonists with podocytes abolished this effect of PMSCs.The results of animal experiments showed that compared with the DKD group,the expression of ERS-related proteins such as p-PERK and GRP78 were significantly increased,the expression of ferroptosis-related proteins GPX4 and FSP1 was significantly decreased,and COX2 expression was significantly increased.While the above indexes were reversed after injected PMSCs.Conclusion PMSCs effectively ameliorate the ferroptosis and ERS levels in the DKD model,and possibly through regulating the ERS-related PERK signaling pathway.This study provides new evidence for PMSCs treatment from animal,cellular and molecular levels,enriching the application of MSCs in the field of ferroptosis,while revealing a potential link between ferroptosis and ERS in the DKD model.