雌马酚通过CCDC92调控AMPK/mTOR通路对糖尿病肾病足细胞损伤的作用及机制研究OA

Objective To investigate the effects and mechanism of equol on podocyte injury in diabetic nephropathy(DN)by regulating the AMP-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)pathway by coiled-coil domain containing 92(CCDC92).Methods Mouse MPC5 cells were divided into five groups:the control group(without any treatment),the model group(treated with 30 mmol·L-1glucose for 48 h),the experimental group(treated with 1 μmol·L-1equol for 24 h on the basis of the model group),the AAV-NC group(transfected with AAV-NC on the basis of the experimental group)and the AAV-CCDC92 group(transfected with AAV-CCDC92 on the basis of the experimental group).Cell viability was detected by the methyl thiazolyl tetrazolium assay.The mRNA level of CCDC92 was determined by real-time fluorescence quantitative polymerase chain reaction.Cell invasive ability was assessed by the transwell assay.The protein relative expression levels of Podocin and Nephrin were detected by immunofluorescence assay.The protein relative expression levels of microtubule-associated protein 1 light chain 3Ⅱ/Ⅰ(LC3 Ⅱ/Ⅰ),Beclin-1,AMPK and mTOR related proteins were measured by Western blot.Results The cell viability rates of the control group,model group and experimental group were(100.00±2.84)％,(64.91±9.57)％and(81.36±13.19)％,respectively;the relative expression levels of CCDC92 mRNA in the control group,model group,experimental group,AAV-NC group and AAV-CCDC92 group were 1.00±0.12,2.26±0.35,1.42±0.19,1.48±0.22 and 2.05±0.34,respectively;the number of invasive cells were(79.35±11.62),(25.81±4.06),(53.69±7.95),(60.46±10.58)and(41.13±7.27)cells,respectively;the relative expression levels of Nephrin protein were 1.00±0.14,0.31±0.05,0.65±0.09,0.58±0.08 and 0.39±0.06,respectively;the relative expression levels of Podocin protein were 1.00±0.17,0.28±0.04,0.61±0.11,0.56±0.09 and 0.32±0.06,respectively;the relative expression levels of LC3 Ⅱ/Ⅰ protein were 1.00±0.17,0.26±0.04,0.74±0.12,0.69±0.11 and 0.41±0.08,respectively;the relative expression levels of Beclin1 protein were 1.00±0.15,0.32±0.06,0.68±0.10,0.75±0.12 and 0.46±0.07,respectively;the relative expression levels of phosphorylated(p)-AMPK/AMPK protein were 1.00±0.19,0.23±0.04,0.86±0.17,0.89±0.16 and 0.38±0.07,respectively;the relative expression levels of p-mTOR/mTOR protein were 1.00±0.16,3.16±0.53,1.72±0.31,1.65±0.29 and 2.91±0.52,respectively.There were statistically significant differences in the above indicators between the model group and the control group,between the experimental group and the model group and between the AAV-CCDC92 group and the AAV-NC group(P＜0.05,P＜0.01,P＜0.001).Conclusion Equol can alleviate podocyte injury in DN through CCDC92,and the effects may be related to regulating the AMPK/mTOR pathway and improving autophagy level.