The Effects Of Simvastatin And Ligustrazine On High Glucose-induced Type Ⅰ Collagen,MMP-1 And TIMP-1 Expressions In Cultured Human Peritoneal Mesothelial Cells

ObjectivePeritoneal fibrosis (PF) is a serious problem in long-term constinuous ambulatory peritioneal dialysis (CAPD) patient, which is characterized by extracellular matrix (ECM) accumulation in human peritoneal mesothelial cells (HPMCs).Type I collagen,the major extracellular matrix (ECM), play important roles in the process of PF. Our study is to investigate the effects of ligustrazine and simvastatin on the expression of type I collagen together with its collagenase system of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression in human peritoneal mesothelial cells (HPMCs) cultured in high glucose conditions.MethodsHPMCs were separated from human greater omenta (HGO) obtained at surgery by digestive method. Cultured HPMCs were divided into eight groups, RPMI1640 culture medium as control (N group),2.5% glucose as G group,2.5% glucose and 2.5,5 or 10μmol/L simvastatin respectively as S1, S2 and S3 group,2.5% glucose and 10,20, or 40 mg/L ligustrazine respectively as L1, L2 and L3 group, Each group was supplemented with equal volum of RPMI1640 culture medium containing 10% FCS. The cell viability of HPMCs was detected by MTT. Semi-quantitative reverse transcriptive-polymerase chain reaction (RT-PCR) was used to detect the mRNA expressions of type I collage,MMP-1 and TIMP-1 in HPMCs. Protein levels of type I collage,MMP-1 and TIMP-1 in culture supernatants were measured by enzyme linked immunosorbent assay (ELISA). Cells protein concentration measured by trace BCA method was to correct the ELISA assay results.Results(1) Identification of HPMC:The confluent cells showed a uniform cobblestone-like appearance, with a purity≥95%. Immunohistochemical studies showed positive staining for cytokeratin and vimentin, but negative staining for factorⅧassocialted antigen and CD45, indicating a pure population of HPMC.(2) The comparison of cell viability:Significant decreased cell viability was observed in G group compared with those in normal control group and drugs groups (P<0.01). To compared with high glucose group, Simvastatin and ligustrazine groups could significantly ameliorate the viability of HPMCs inhibited by high glucose conditions (P＜0.01).(3) Effect of simvastatin and ligustrazine on Type I collagen,MMP-1,TIMP-1 protein and mRNA expression:The expression of type I collagen and TIMP-1 was increased by the stimulation of glucose.Simvastatin and ligustrazine significantly attenuated high glucose-induced up-regulation of type I collagen and TIMP-1 in a dose-dependent manner both in protein and gene levels (P< 0.01). What's more,both high dose treatment of simvastatin and ligustrazine significantly increased MMP-1 expression inhibited by high glucose concentrations (P<0.05).Conclusions1. Under high glucose conditions, cell viability was decreased and the expression of type I collagen and TIMP-1 was increased and the expression of MMP-1 was decreased.2. Simvastatin and ligustrazine not only decreased up-regulation of type I collagen synthesis, but also promoted its degradation by modulating unbalanced MMP-1/TIMP-1 expression in HPMCs cultured under high glucose conditions, and thus resulted in decreased accumulation of ECM.and thus preventing and/or delaying the process of PF in patients with CAPD.