The Mechanism Of Matrine Inhibiting TGF-β1-induced Epithelial Mesenchymal Transition Of Human Peritoneal Mesothelial Cells

Objective: Peritoneal dialysis is one of the main renal replacement therapy for end-stage renal disease(ESRD).Compared with hemodialysis,peritoneal dialysis has better protection of residual renal function and more advantages in blood pressure and fluid control in the first few years.Besides,the quality of life for ESRD patients who choose peritoneal dialysis will be higher compare to those who choose hemodialysis.The number of patients who accept the peritoneal dialysis is increasing in recent years.Therefore,to improve the technology of peritoneal dialysis and to find how to prevent its complications has become an urgent scientific problem.Peritoneal fibrosis is one of the main complications of peritoneal dialysis.continuous physical and inflammatory stimulation will cause peritoneal fibrosis in the process of long-term peritoneal dialysis,which will cause the ultimately ultrafiltration failure.It will let the ESRD patients fail to use peritoneal dialysis for renal replacement treatment.The mechanism of peritoneal fibrosis and the development of drugs for the prevention and treatment of peritoneal fibrosis have always been the research focus.At present,the molecular mechanism of peritoneal fibrosis is not clear yet.It has been confirmed that EMT is an important early process of the occurrence and development of peritoneal fibrosis.Therefore,inhibition of EMT transformation of peritoneal mesothelial cells is the key to prevention and treatment of peritoneal fibrosis.In this study,we studied the effect of matrine on transforming growth factor β1(TGF-β1)-induced EMT in human peritoneal mesothelial cells,and hoped to provide scientific basis for the exploration of drugs to prevent and treat peritoneal fibrosis.Methods: The human peritoneal mesothelial cells were induced to produce EMT by 5ng/ml TGF-β1,and treated with matrine at different concentrations(0.4,0.6,0.8 and 1.0mg/ml).Then,the EMT related genes(E-cadherin,α-SMA,Col Ⅲ and Fibronectin)were detected by real-time fluorescent quantitative PCR and Western blotting(WB)respectively.According to the results of real-time fluorescence quantitative PCR and WB,the inhibition of Matrine on EMT of human peritoneal mesothelial cells was analyzed.If matrine has a significant inhibitory effect on the EMT of human peritoneal mesothelial cells induced by TGF-β1,the optimal concentration of matrine is selected to inhibit the EMT of human peritoneal mesothelial cells.Then used the 5ng/ml TGF-β1 to induce the EMT of human peritoneal mesothelial cells.The optimal concentration of Matrine is used to treat the human peritoneal mesothelial cells at the same time.The total RNA of cells is extracted for RNA-seq sequencing analysis of the whole genome of mRNA.The main signal pathway was analyzed by KEGG pathway to explore the specific molecular mechanism of matrine inhibiting EMT in human peritoneal mesothelial cells.Finally,the real-time fluorescent quantitative PCR and WB were used to verify the specific molecular mechanism of matrine inhibiting EMT in human peritoneal mesothelial cells.Results:(1)The mRNA and protein expression levels of α-SMA,col Ⅲ and Fibronectin was up-regulated in human peritoneal mesothelial cells after being stimulated by 5ng/ml TGF-β1.The mRNA and protein expression levels of E-cadherin was down-regulated in human peritoneal mesothelial cells after being stimulated by 5ng/ml TGF-β1.Matrine(0.4,0.6,0.8and 1.0mg/ml)could down-regulate the mRNA and protein expression levels ofα-SMA,Fibronectin and Col Ⅲ after being stimulated by 5ng/ml TGF-β1 in human peritoneal mesothelial cells.Matrine(0.4,0.6,0.8 and 1.0mg/ml)could up-regulate the mRNA and protein expression levels of E-cadherin after being stimulated by TGF-β1 in human peritoneal mesothelial cells.Among them,0.8and 1.0mg/ml matrine had the most significant effect to inhibit the TGF-β1-induced EMT of the human peritoneal mesothelial cells.(2)The results of RNAseq sequencing showed that there were 154 genes which were significant difference in expression related to the inhibition of matrine on TGF-β1-induced EMT in human peritoneal mesothelial cells.Further analysis of KEGG pathway showed that the 154 genes were mainly enriched in MAPK signaling pathway and anther nine signaling pathways.The expression of Snail2 was significant difference found among the 154 genes which was the common downstream factor in Smad dependent and Smad independent signaling pathways.(3)Western blotting was used to detect the phosphorylation expression levels of ERK1/2which was the key proteins in the ERK1/2 MAPK signaling pathway.It was found that TGF-β1 could increase the phosphorylation expression levels of ERK1/2,while matrine could decrease the phosphorylation expression levels of ERK1/2after being stimulated by 5ng/ml TGF-β1 in human peritoneal mesothelial cells.Conclusion: Matrine inhibits TGF-β1-induced EMT by inhibiting the expression of snail2 in human peritoneal mesothelial cell,which may through the ERK1/2MAPK signaling pathway to exert an inhibitory effect.