Study On The Protective Effects And Mechanism Of Effective Compounds From Corni Fructus And Radix Rehmanniae In Diabetic Nephropathy Based On Mesangial Cells Injury

Objectives:With the number of diabetic patients is increasing worldwide,diabetic nephropathy(DN),as one of the most common microvascular complications of diabetes mellitus,has become a leading factor in end-stage renal disease.Glomerular mesangial cells are renal innate cells,which play a key role in maintaining glomerular filtration barrier composition and function.In this study,glumerular mesangial cells were used to study the effects and the mechanism of loganin and catalpol on the GMC injury model induced by advanced glication end products(AGEs)in vitro to provide a new method of DN treatment.Methods:In vitro cell experiments:(1)the protective effect of loganin and catalpol on AGEs-induced mesangial cell injury:Based on AGEs-induced mesangial cell injury model,add loganin and catalpol.MTS assay was used to detect the proliferation rate of GMC.Cell cycle changes of GMC were detected by flow cytometry.Western blotting was used to detect the activationof NF-?B,and the secretion of ECM(LN,Col I,CTGF)was detected by Elisa.The morphological changes of GMC were observed by transmission electron microscopy.(2)Effect of loganin and catalpol on endoplasmic reticulum stress induced by AGEs in mesangial cells:AGEs-induced mesangial cell injury in vitro was used to observe the effects of loganin and catalpol on the molecular mechanism of endoplasmic reticulum stress.Western blotting was used to detect the expression of GRP78,IRE1,XBP1 and CHOP in endoplasmic reticulum.Add 4-PBA to verify the target of loganin and catalpol on endoplasmic reticulum stress in mesangial Cells.(3)Effects of loganin and catalpol on mitophagy induced by AGEs in GMC:AGEs-induced mesangial cell injury in vitro was used to observe the molecular mechanism of the protection of mitophagy of GMC.The expressions of Keapl,Nrf2,PINK1,Parkin,p62 and LC-3 were detected by Western blotting.Immunofluorescence was used to detect the mitochondrial membrane potential of GMC.Add 3-MA,to verify the target of loganin and catalpol on mitophagy of GMC.Results:(1)The protective effect of loganin and catalpol on AGEs-induced mesangial cell injury:?MTS results showed that administration of loganin and catalpol could significantly inhibit the proliferation of AGEs-induced GMC in a concentration-dependent(P<0.05,P<0.01).?The proliferation of GMC was inhibited in G0/G1 phase,and the cells were prevented from G0/G1 phase to S phase,and the cell proliferation was inhibited in G0/G1 phase.? Loganin and catalpol can effectively inhibit the activation of NF-?B.?Elisa results showed that loganin and catalpol could down-regulate the levels of LN,Col I and CTGF in supernatant of GMC,reduce ECM expression compared with the model group(P<0.05,P<0.01),and the concentration was related.?Loganin and catalpol can effectively improve the subcellular organelles injury induced by AGEs,reduce the swelling of endoplasmic reticulum lumen and mitochondrial crest,reduce the endoplasmic reticulum and mitochondrial lesions of mesangial cells,and improve DN.(2)The protective effect of loganin and catalpol on endoplasmic reticulum stress in AGEs-induced mesangial cells:?The expression of GRP78,IRE1 and CHOP proteins in GMC were significantly different from that of model group(P<0.05),as well as the differentiation of XBP1 were significantly different from that of model group(P<0.05)P<0.05,P<0.01).?Addthe inhibitor of endoplasmic reticulum stress 4-PBA,loganin and catalpol did not decrease the effect compared with the inhibitor,indicating that the effects of loganin and catalpol by regulating the endoplasmic reticulum stress of GMC to release the injury of GMC induced by AGEs.(3)The protective effect of loganin and catalpol on mitophagy induced by AGEs in mesangial cells:(1)Loganin and catalpol can up-regulate mitochondrial ATPase activity in GMC and increase the mitochondrial membrane potential,which is significantly different from that of model group P<0.05,P<0.01),and showed a concentration correlation.The expression of Keap1,Nrf2,PINK1,Parkin and LC-3 protein of GMC were decreased,and also the nuclear translocation of Nrf2 protein and the phosphorylation of p62.(P<0.05,P<0.01).?Add the inhibitor of autophagy 3-MA,loganin and catalpol did not decrease the effect compared with the inhibitor,indicating that loganin and catalpol reduced AGEs-induced GMC injury by reducing mitophagy of GMC.Conclusion:The results indicated that loganin and catalpol could significantly improve the damage of mesangial cells induced by AGEs.The mechanism may be related to the improvement of endoplasmic reticulum stress,the reduction of mitophagy and the inhibition of cell proliferation.