黄蜀葵花源性细胞外囊泡样颗粒靶向调控"线粒体-内质网耦联"抑制糖尿病肾脏疾病足细胞氧死亡的作用与机制OA

Abelmoschus manihot-derived extracellular vesicle-like particles(AM-EVLPs),as a novel natural nano-delivery system,have a precise protective effect on podocytes.This study aims to verify the effects in vivo and in vitro of AM-EVLPs in protecting against podocyte injury using a modified rat model with diabetic kidney disease(DKD)and a high-glucose(HG)-induced podocyte model,and then to explore its therapeutic targets based on the pathological mechanisms of"mitochondria-endoplasmic reticulum coupling"and tumor protein 53(P53)-mediated oxeiptosis.The first,AM-EVLPs were extracted from fresh flower petals of Abelmoschus manihot using ultracentrifugation combined with density gradient centrifugation.The second,in the in vivo study,the modified DKD rat model was established.After successful modeling,the DKD model rats were randomly divided into 4 groups,including the model group,the low-dose AM-EVLPs(L-AM-EVLPs)-treated group,the high-dose AM-EVLPs(H-AM-EVLPs)-treated group,and total flavones of Abelmoschus manihot(TFA)-treated group,receiving corresponding drugs for consecutive 4 weeks.In the in vitro study,mouse podocytes(MPC5 cells)were exposed to HG and subsequently treated with L-AM-EVLPs,H-AM-EVLPs,empagliflozin(EMPA),P53 small interfering RNA(P53-siRNA),or P53-overexpressing lentivirus(P53-OE),respectively.Using the DKD model rats,blood glucose,urinary albumin,liver and kidney function-related indicators were observed.Additionally,glomerular histopathological features,foot process and mitochondrial ultrastructure,the protein expression levels of podocyte-specific structural molecules(podocin and nephrin),renal reactive oxygen species(ROS)levels,as well as the protein expression levels of oxeiptosis-related proteins including P53,Kelch-like ECH-associated protein 1(KEAP1),phosphoglycerate mutase family member 5(PGAM5),and apoptosis-inducing factor/mitochondria-associated 1(AIFM1)were detected,respectively.Furthermore,the differential expression proteomic profile of diabetic kidneys was also analyzed.Using the HG-induced podocyte model,the protein expression levels of podocin and nephrin,the degree of mitochondria-endoplasmic reticulum coupling,the binding intensity of the IP3R1-GRP75-VDAC1 complex,mitochondrial calcium ion(Ca2+)concentration,mitochondrial reactive oxygen species(mtROS)levels,and the protein expression levels of key signaling molecules in the KEAP1/PGAM5/AIFM1 signaling axis were detected.The in vivo results showed that,for the DKD model rats,AM-EVLPs dose-dependently improved urinary albumin,blood urea nitrogen(BUN),and serum creatinine(Scr)levels;reduced glomerular cell proliferation,ECM deposition,and collagen deposition;ameliorated foot process fusion and mitochondrial ultrastructural damage;upregulated the protein expression levels of podocin and nephrin;reduced mtROS levels;and reversed the aberrant protein expression levels of key signaling molecules in the oxeiptosis pathway,including P53,KEAP1,PGAM5,and p-AIFM1.In which,the ameliorative effect of H-AM-EVLPs was superior to that of TFA.Additionally,proteomic analysis of diabetic kidneys revealed that differentially expressed proteins were enriched in pathways related to oxidative stress,programmed cell death,endoplasmic reticulum-mitochondria coupling,and P53-related signaling pathways.The in vitro results showed that AM-EVLPs improved the protein expression levels of podocin and nephrin in podocytes,dissociated the IP3R1-GRP75-VDAC1,restored mitochondria-endoplasmic reticulum coupling homeostasis,inhibited mitochondrial calcium ion overload and mtROS generation.In which,the ameliorative effects of H-AM-EVLPs were superior to those of EMPA.More importantly,the treatment of AM-EVLPs or P53-siRNA could downregulate the protein expression levels of KEAP1 and PGAM5,and upregulate the protein expression level of p-AIFM1 under HG conditions in the in vitro study,while P53-OE reversed the regulatory effects of AM-EVLPs on the protein expression levels of the above-mentioned signaling molecules,suggesting that AM-EVLPs could inhibit podocyte oxeiptosis by targeting P53 to regulate the KEAP1/PGAM5/AIFM1 signaling axis activation.In conclusion,this study demonstrated that AM-EVLPs precisely ameliorate HG-induced podocyte injury,and their effects are achieved by targeted regulation of"mitochondria-endoplasmic reticulum coupling"and P53-mediated oxeiptosis.These findings provide pharmacological evidence for AM-EVLPs as a novel natural nano-delivery system for precision therapy of podocyte injury in DKD.