| Background: Peritoneal dialysis is one of the main methods of renal replacement therapy(RRT)in patients with end-stage renal disease(ESRD).Some studys found that in patients with the treatment of peritoneal dialysis chronic inflammation in the body induced peritoneal mesothelium cells to occur epithelial-mesenchymal transition(EMT),which is an important cause of peritoneal fibrosis,structural and functional change of the peritoneum and will eventually lead to technical failure and patients ’ droping out of treatment.Peritoneal dialysis assosiated peritonitis caused by Gram-negative bacteria such as Escherichia coli is common in the southern region of China.Matrine has many kinds of pharmacological action such as antibacterial,anti-inflammatory and anti-fibrosis and so on.Our preliminary experimental study has found that matrine could inhibit the biofilm formation of Escherichia coli.However,the role of matrine on the EMT of peritoneal mesothelium cells on chronic inflammatory conditions is not yet clear.So we chose lipopolysaccharide(LPS),the main ingredients of the wall of Escherichia coli,to induce the EMT model of peritoneal mesothelium cells,combining with relevant EMT markers,miRNAs,to study the effect of matrine on the EMT of HPMC in this experiment.Aims:We chosed the EMT model of human peritoneal mesothelium cells(HPMCs)induced by LPS in vitro to study the effect of matrine on the EMT of HPMCs through cellular level,and investigated the changes of expression level of miR-29,miR-129-5p at the same time.Methods: MTT assay and the cell morphology observation were employed to determine the growth character of HPMCs stimulated with LPS or matrine and explore the appropriate concentration of the EMT model of HPMCs induced by LPS and the effective concentration of matrine in the EMT model of HPMCs induced by LPS.The Wound Scratch experiment was chosen to assess the migration ability of the control group,the LPS group,the matrine group(medium dosage)after drug intervention 0 h,8 h,16 h and 24 h.Quantitative Real-Time PCR was used to measuring the expression levels of the epithelial marker “E-cadherin”,the mesenchymal markers “alpha smooth muscle actin(α-SMA)”,miR-29 b,and mi R-129-5p in control group,the LPS group,the LPS + small dose matrine group,the LPS + medium dose of matrine,the LPS +large dose of matrine these 5 groups at 24 h.Western blot analysis was used to measuring the proteinic levels of E-cadherin,α-SMA in control group,the LPS group,the LPS + small dose matrine dose of matrine group,the LPS +medium dose of matrine,the LPS + large dose of matrine at 72 h.Results: Determined by MTT experiment we found that 0.1 g/l ~ 2.0g/l matrine or 0.5ug/l ~20.0ug/l LPS did not affect the growth of HPMCs.According to results of MTT assay and the reference literatures,the concentration of LPS was chosen as10 ug/ml,the concentration of matrine was chosen as 0.2 g/l(lowdose),0.4 g/l(moderate dose),0.6 g/l(high dose).Through the microscope we found that the growth of HPMCs was not affected after stimulated by LPS or LPS with matrine(0.6 g/l)for 72 h.The wound scratch experiments showed that the migration ability of HPMCs was enhanced after stimulated by LPS,while matrine could inhibit the enhancement of migration ability induced by LPS.10ug/ml LPS could induce HPMCs to occur EMT,characterized by the expression of E-cadherin was decreased,alpha SMA was increased as compared with the control group.Matrine could inhibit the EMT of HPMCs,as the expression of E-cadherin was increased,α-SMA was decreased in a dosage-dependent manner.We also found that LPS could induce the expression levels of miR-29 b and miR-129-5P of HPMCs to reduce,while matrine could promote the expression level of miR-29 b and miR-129-5P.Conclusions: LPS could induce HPMCs to occur EMT,reducing the expression of miR-29 b,mi R-129-5 p of HPMCs.Matrine could inhibit EMT of HPMCs induced by LPS,increasing the expression of miR-29 b,miR-129-5p. |
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