Inhibitory Effect Of Matrine On Mesenchymal Transition Of Human Peritoneal Mesothelial Cells Induced By TGF-β1 Via Regulating Hsa-miR-4485-3p

Objective Peritoneal dialysis(PD)can cause mesenchymal to mesenchymal transition(EMT)of peritoneal mesothelial cells,then cause peritoneal fibrosis,and finally lead to ultrafiltration failure and treatment interruption.Transforming growth factor-β1(TGF-β1)is the core element of EMT.Recent years,studies have confirmed that micro RNA is an important regulator of EMT in peritoneal mesothelial cells induced by TGF-β1.Matrine has unique anti fibrosis and EMT effects,and can regulate the expression of micro RNA.Previous studies found that matrine can inhibit EMT in human peritoneal mesothelial cells induced by LPS or TGF-β1.However,it is not known whether matrine plays an anti EMT role by regulating the expression of micro RNA.This study is committed to find out the micro RNAs involved in the regulation effect of matrine on the mesenchymal transition of human peritoneal mesothelial cells induced by TGF-β 1,and its specific mechanism were deeply studied.Methods In the chapter 1,the effect of matrine on micro RNA expression profile in human peritoneal mesothelial cells induced by TGF-β1 was studied.Taking human peritoneal mesothelial cells(HPMCs)as the research object.(1)Different concentrations of matrine(0,0.5,0.8,1.1mg/ml)stimulated human HPMCs for 24 h and 48 h respectively,and the cell viability was detected by CCK8.(2)Using TGF-β1(5 ng/ml)to construct HPMCs EMT model.Different concentrations of matrine(0,0.5,0.8,1.1)mg/ml)were used to intervene.The changes of cell morphology were observed by inverted microscope.Expression of m RNA and protein of the EMT markers,E-cadherin,α-SMA,N-cadherin and fibronectin were detected by quantitative real-time PCR and western blot respectively.(3)Three study groups were established: control group(Group C),TGF-β1(5 ng/ml)induction group(group T),TGF-β 1(5 ng/ml)+ matrine(0.8mg/ml)intervention group(group TM),intervention time was 48 h.The total RNA of each group was extracted and purified,and the differential micro RNA of each group was obtained by high-throughput microarray technology and difference analysis technology.The online database is used to predict the target genes of the significantly different micro RNAs obtained from the analysis,and the GO / KEGG pathway enrichment analysis is carried out for the predicted target genes to understand the molecular pathways that the target genes may participate in.In the chapter 2,the expression and function of hsa-miR-4485-3p were verified,and the downstream mechanism of hsa-miR-4485-3p on EMT in human peritoneal mesothelial cells induced by TGF-β1 was investigated.Taking HPMCs as the research object and using TGF-β1(5 ng/ml)to construct HPMCs EMT model.The experiment object was divided into five groups,control group,TGF-β1 group,TGF-βl + matrine group,TGF-β1+ miR-4485-3p mimics group(TGF-β1 + mimics group),TGF-β1 + negative control group(TGF-β1+NC group).In the TGF-β1+miR-4485-3p mimics group,HPMCs was intervened with miR-4485-3p mimics for 24 hours in advance and then intervened with TGF-β1 5 ng/ml for 48 hours.In the TGF-β1+ NC group,HPMCs was intervened with negative control for 24 hours in advance and then intervened with TGF-β1 5 ng/ml for 48 hours.The expression of hsa-miR-4485-3p in HPMCs was detected by quantitative real-time PCR after TGF-β1 stimulation and matrine intervention.The transfection effect of miR-4485-3p mimics was detected by quantitative real-time PCR.Quantitative real-time PCR and western blot were used to detect the m RNA and protein expression of E-cadherin,α-SMA,N-cadherin,FN and Snail2.The expressions of ERK1/2 and p-ERK1/2were detected by Western blot.In the Chapter 3,the expression of Snail2 in the peritoneum of uremic peritoneal dialysis rats induced by LPS and the effect of matrine intervention on its expression were studied.The uremia rat model was established by 5/6nephrectomy,and then peritoneal dialysis catheter was indwelled.After successful catheterization,they were randomly divided into three groups which were injected with NS+PDF,LPS+PDF and LPS+PDF+matrine respectively intraperitoneally through peritoneal dialysis catheter once every other day for 7times.The peritoneal structure and mesothelial cells were observed by HE staining.The peritoneal thickness and collagen deposition were observed by Masson staining.Immunohistochemical staining and PCR were used to observe the expression of E-cadherin,α-SMA and Snail2.Results In the chapter 1 we found(1)Different concentrations of matrine(0.5,0.8,1.1mg / ml)had no significant effect on cell viability after stimulating human HPMCs for 24 h or 48 h.(2)TGF-β1(5 ng/ml)stimulated human HPMCs for 48 hours can promoted EMT.Matrine(0.8,1.1 mg / ml)could significantly reverse EMT in HPMCs induced by TGF-β 1(5 ng/ml).(3)Taking the expression difference of 2 times or more and P value < 0.05 as the screening standard,TGF-β1(5 ng/ml)stimulated for 48 hours could make 49 differences in the expressions of micro RNAs in human peritoneal mesothelial cells(41 upregulated and 8 down-regulated).There were 61 differences in micro RNA expressions(30 up-regulated and 31 down regulated)comparing TM group and T group.11 micro RNAs were up-regulated after induced by TGF-β1,while were down regulated after matrine intervention,and 7 micro RNAs including hsamiR-4485-3p were down-regulated after induced by TGF-β1,while were upregulated after matrine intervention.The target genes of hsa-miR-4485-3p,which can be significantly up-regulated by matrine and expressed abundantly,were predicted,and 1379 potential target genes of hsa-miR-4485-3p were collected.KEGG analysis showed that the target genes of hsa-miR-4485-3p were most enriched in MAPK signal pathway.In the chapter 2 we found(1)The expression of hsa-miR-4485-3p was down-regulated in human peritoneal mesothelial cells after induced by TGF-β1(P<0.01)by quantitative real-time PCR,while matrine(0.8mg/ml)significantly up-regulated the expression of hsa-miR-4485-3p(P<0.05).(2)After transfection of human peritoneal mesothelial cells with miR-4485-3p mimics,the expression of hsa-miR-4485-3p was significantly up-regulated(P<0.01),while there was no significant change in negative control group(P>0.05),suggesting successful transfection.(3)the expression of E-cadherin in human peritoneal mesothelial cells decreased significantly after TGF-β1 stimulation,while the expression of α-SMA,N-cadherin and FN increased significantly.Both Matrine and over-expression of hsa-miR-4485-3p could significantly reverse the changes of the above markers in human peritoneal mesothelial cells induced by TGF-β1.Correlation analysis showed that the relative expression level of miR-4485-3p was positively correlated with the relative expression of E-cadherin and negatively correlated with the relative expression of α-SMA,Ncadherin and FN.(4)The relative expression of p-ERK1/2 and Snail2 in human peritoneal mesothelial cells increased significantly after stimulated by TGF-β1,while both matrine and over-expression of hsa-miR-4485-3p could significantly down-regulated the relative expression of p-ERK1/2 and Snail2.(5)the relative expression of hsa-miR-4485-3p was negatively correlated with the relative expression of p-ERK1/2 and Snail2 m RNA.In the chapter 3 we found,3 months after 5/6 nephrectomy,the plasma urea and creatinine of rats increased to 2-3 times,suggesting that the model was successful.HE staining and Masson staining showed LPS stimulation can lead to peritoneal fibrosis in peritoneal dialysis rats,while matrine could significantly reduce the peritoneal fibrosis.Immunohistochemistry and PCR showed that the expression of E-cadherin in peritoneal tissue decreased significantly after LPS stimulation,while increased significantly after matrine intervention.The expression of α-SMA and Snail2 increased significantly after LPS stimulation,while decreased significantly after matrine intervention.Conclusion The present study indicates that matrine can reverse some abnormal expression of micro RNAs in human peritoneal mesothelial cells induced by TGF-β1,and Matrine can inhibit EMT of human peritoneal mesothelial cells induced by TGF-β1 via up-regulating the expression of hsamiR-4485-3p.ERK/Slug pathway is the downstream mechanism of hsa-miR-4485-3p when regulating EMT.