| Objective: Diabetic nephropathy(DN),also known as diabetic glomerulosclerosis,is the most common microvascular complication of diabetes mellitus,bring about death in end-stage renal disease and diabetic patients.The pathogenesis of DN is complicated,and there are many pathways involved in regulation.The diagnosis of DN mainly depends on urinary microalbumin and renal biopsy.There is no effective,stable and non-invasive detection method.Therapeutically,the development of DN can only be delayed by lowering blood sugar and blood pressure.Artificial dialysis and kidney transplantation are not only expensive,but also limited in application.With the in-depth study of DN,it has been found that autophagy is particularly closely related to DN renal fibrosis.On the other hand,as a stable and highly tissue-specific small molecule RNA,microRNAs play an important role in the development of DN.The aim of this study was to investigate the expression of microRNA-130 b,fibrosis-related proteins and autophagic activity by establishing db/db diabetic nephropathy model in mice and culturing human glomerular mesangial cells,to explore the effects of microRNA-130 b on autophagy and damage in DN.The target genes were predicted to reveal the potential mechanism of diabetic nephropathy fibrosis and provide new ideas for the prevention and treatment of DN renal fibrosis.Methods:Animal experiment used db/db mice and C57 BLKS mice to establish diabetic nephropathy model and control.The general situation and indexes of each group were monitored.After feeding,the corresponding samples were collected to detect the fasting blood glucose and serum creatinine of mice.The pathological changes of kidney tissues were observed by HE and Masson staining,and the ultrastructure of kidney cells was observed by transmission electron microscopy.The autophagy and fibrosis-related factors were detected by qRT-PCR and Western blotting.In cell experiment,HMC cells were treated with instantaneous transfection of microRNA-130 b and 0 or10 ng/ml TGF-beta 1.Quantitative RT-PCR,Western blotting and JC-1 were used to detect the autophagy and fibrosis related factors in each group,in order to clarify the effects of over-expression or down-regulation of microRNA-130 b on autophagy and fibrosis of HMC cells,to clarify the effects of TGF-beta 1 on autophagy and fibrosis of HMC cells,and to clarify the relationship betweenmicroRNA-130 b,TGF-beta 1,autophagy and diabetic renal fibrosis.Predict the target gene of microRNA-130 b and identify its association with microRNA-130 b.Results: The fasting blood sugar and serum creatinine in db/db mice were evidently higher than those in the control group.The pathological changes of renal interstitial fibrosis and basement membrane thickening were observed in the model group.The expressions of microRNA-130 b,fibrosis factor type I collagen and FN mRNA in the kidneys of the db/db model group were superior than those of the normal control group(P < 0.01,P < 0.05,P < 0.01).The expression quantity of FN and col I protein in kidney tissue of db/db model mice was significantly higher than that of normal control mice by WB,and the difference between the two groups was significant(P < 0.001,P< 0.01);the expression of ACVR1 in kidney tissue of db/db model mice was lower than that of normal control mice(P < 0.01);and the expression of autophagy-related proteins Beclin1 and LC3 was lower than that of normal control mice(P < 0.01,P <0.01),at the same time,expression of p62 was better than that of the control group(P <0.01).Under transmission electron microscopy,the glomerular basement membrane of db/db model mice was obviously thickened.In cell experiments,qRT-PCR results showed that the expression of microRNA-130 b,FN and ColⅠmRNA in HMC cells cultured with high glucose increased(P<0.01,P<0.01,P<0.01)compared with normal control cells while the ACVR1 decreased(P<0.01).Compared with the microRNA-130 b mimics NC group,the expression of microRNA-130 b,FN and ColⅠincreased significantly in the microRNA-130 b mimimics group,but the ACVR1 mRNA decreased.On the contrary,compared with the group of microRNA-130 b inhibitor NC,the expression of microRNA-130 b FN and ColⅠdecreased in the group of microRNA-130 b inhibitor,and the ACVR1 mRNA increased.Compared with the group of microRNA-130 b mimics+TGF and the group of microRNA-130 b mNC+TGF,the expression of microRNA-130 b and its target gene ACVR1 showed an upward and downward trend,while the expression of FN and Col I decreased.The results of comparison between the microRNA-130 b inhibitor+TGF group and the microRNA-130 b iNC+TGF group showed that the expression of microRNA-130 b FN and Col I in the former group was lower than that in the latter group,accompanied by the increase of ACVR1 expression.Western blotting results showed that the expression of autophagy-inducible protein Beclin1 and LC3II/I in model group was significantlylower than that in normal control group(P < 0.001)(P < 0.001),while the expression of another autophagy-related protein p62 in model group showed an upward trend,and the discrepancy was statistically significant(P < 0.001).Meanwhile,the expression of ACVR1 in model set was lower than that in normal group(P < 0.001)and the expression of FN and ColⅠare higher.Compared with NC group,the expression of autophagy-related protein Beclin1 and LC3II/I reduced markedly(P < 0.001)and the expression of p62 protein increased significantly(P < 0.001)in the microRNA-130 b mimics group.The expression of ACVR1 in the microRNA-130 b mimics group was also lower than that in the NC group(P < 0.001).The expression levels of Beclin1 and LC3II/I in the microRNA-130 b inhibitor group were significantly higher than those in the microRNA-130 b iNC group,while the expressions of p62 protein were significantly lower than those in the microRNA-130 b iNC group.However,the fibrosis was enhanced in microRNA-130 b inhibitor group.Compared with the microRNA-130 b mNC+TGF group,the expression levels of Beclin1 and LC3II/I in the microRNA-130 b mimics+TGF group decreased significantly,while the expression levels of FN,ColⅠ and p62 increased.The expression of Beclin 1 in the group of microRNA-inhibitor+TGF-beta 1 was increased compared with that in the group of microRNA-iNC+TGF-beta 1,as was the case in the group of LC3II/I and ACVR1.The expression levels of autophagy inhibitory marker protein p62 and fibrosis factors decreased.JC-1 mitochondrial membrane potential showed that the mitochondrial membrane potential of the normal control group was lower than that of the model group,indicating that the mitochondrial autophagy of HMC cells in the model group might be inhibited.The membrane potential of the microRNA-130 b mimics group was higher than that of the microRNA-130 b mimics NC group.In contrast,the mitochondrial membrane potential of the cells transfected with microRNA-130 b inhibitor was lower than that of the cells transfected with microRNA-130 b inhibitor NC.Conclusion: In Db/db diabetic model group,the expression of microRNA-130 b was up-regulated,while the expression of fibrosis-related factors-collagen type I and adhesion protein was up-regulated,but the autophagy level was down-regulated.Mitochondrial autophagy in human mesangial cells was inhibited and fibrosis was induced by microRNA-130 b.MiR-130 b may have a co-effect on fibrosis andautophagy of HMC cells by targeting ACVR1 and TGF-beta 1. |
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