Mechanisms Of Emodin Against Renal Fibrosis By Regulating The Expression Of MiRNA-490

ObjectivesIn this experiment,the mechanism of emodin anti-kidney fibrosis was explored through in vivo and in vitro experiments;the role of miRNA-490 in renal fibrosis was explored through miRNA-490 overexpression and cell inhibition experiments;the intervention experiment through emodin and miRNA-490 overexpression Emodin regulates the expression of miRNA-490 and exerts anti-renal fibrosis effect.Methods1.To investigate the mechanism of emodin against renal fibrosis1.1 In vivo experiment: SPF healthy male C57BL/6 mice were randomly divided into 6 groups according to the number table method:(1)sham operation group;(2)model group;(3)low-dose emodin group;(4)emodin medium-dose group;(5)emodin high-dose group Dosage group;(6)Losartan group,10 rats in each group.An animal model of renal fibrosis with unilateral ureteral ligation was constructed,and the drug interventions were gavage with emodin(10mg/kg,20mg/kg,40mg/kg).The dose of losartan in the losartan group was 10 mg/kg,once a day,for 14 consecutive days.The serum creatinine,blood urea nitrogen and blood uric acid of the mice were detected before and after treatment.The degree of renal tubular damage and interstitial fibrosis was observed by HE and Masson staining;the expression levels of fibrosis-related proteins α-SMA,Collagen IV,Fibronectin,TGF-β1and E-cadherin were detected by Western blot;Elisa The expressions of inflammatory factors IL-1β,MCP-1nd TNF-α were detected;the gene expression levels of miRNA-490,HMGA2 and TGF-β1 were detected by RTPCR.1.2 In vitro experiments: NRK-52 E cells induced by TGF-β1 were treated with emodin to construct a kidney injury model.Experimental group(1)NRK-52 E cells;(2)NRK-52 E cells + TGF-β1;(3)NRK-52 E cells + TGF-β1+ emodin low-dose group(40 μM)+ TGF-β1;(4)NRK-52 E cells + TGF-β1+ high-dose hyperflavin group(80 μM).CCK8 assay to detect cell viability;the expression levels of fibrosis-related proteins α-SMA,Collagen IV,Ecadherin and HMGA2 were detected by Western blot;The gene expression levels of miRNA-490 were detected by RT-PCR.2.Investigate miRNA-490 targeting HMGA2 agaists EMT in renal tubular epithelial cellsThe experiment was divided into two parts: overexpression and inhibition:the overexpression experiment was divided into 4 groups:(1)NRK-52E+NC;(2)NRK-52E+NC+TGF-β1;(3)NRK-52E+miRNA-490 mimic;(4)NRK-52E+miRNA-490 mimic+TGF-β1.The inhibition experiments were divided into 4 groups:(1)NRK-52 E + NC;(2)NRK-52 E +NC+TGF-β1;(3)NRK-52E+miRNA-490 inhibitor;(4)NRK-52E+miRNA-490 inhibitor + TGF-β1.Western blot was used to detect the expression levels of fibrosis-related proteins α-SMA,Collagen IV,E-cadherin and HMGA2.The gene expression levels of miRNA-490 was detected by RT-PCR.Use Targetscans target gene prediction software to predict the possible target genes of miRNA-490.Recombinant plasmid preparation: prepare a recombinant plasmid containing the gene to be detected HMGA2 for cotransfection,and co-transfect the reporter gene and target gene with HMGA2 marker into HEK-293 cells,usually 24h;cell processing: double luciferase detection The kit was operated according to the protocol;it was divided into the following three groups;(1)HEK293+HMGA2-UTRWT vector;(2)HEK293+HMGA2-UTRWT vector;(3)HEK293+HMGA2-UTRWT vector+miRNA-490 NC,the target gene was verified by dual-luciferase reporter gene assay.3.To Investigate emodin regulates miRNA-490 against renal fibrosisTGF-β1-induced NRK-52 E cell model and TGF-β1-induced NRK-52 E cell model transfected with miRNA-490 mimics were treated with emodin.Experimental grouping: The experiment was divided into 4 groups:(1)NRK-52E+NC;(2)NRK-52E+TGF-β1;(3)NRK-52E+TGF-β1+emodin;(4)NRK-52E+TGF-β1+emodin+miRNA-490 mimic.Western blot was used to detect the expression levels of fibrosis-related proteins α-SMA,E-cadherin and target protein HMGA2.Results1.Emodin can reduce serum creatinine,blood urea nitrogen and serum uric acid in UUO mice,improve the pathological damage of kidney tissue,reduce the expression of fibrosis indicators α-SMA,Collagen IV,Fibronectin protein and HMGA2 protein,and increase the expression of E-cadherin protein;2.The miRNA-490 mRNA in the kidney tissue of the mice in the UUO group was significantly decreased,and the expression of HMGA2 mRNA was significantly increased,both were negatively correlated;3.Emodin can improve the expression of fibrosis index in NRK-52 E cells induced by TGF-β1;4.Emodin can increase the expression of miRNA-490 in TGF-β1-induced NRK-52 E cells and inhibit HMGA2;5.MiRNA-490 targeting HMGA against renal fibrosis.ConclusionsMiRNA-490 targets HMGA2 against renal fibrosis,and it was found that the protective effect of emodin may be achieved by up-regulating the expression of miRNA-490.