Efficacy Of Tetramethylpyrazine In Prophylaxis And Treatment On Liver Fibrosis In Rats And Related Mechanism

Object:In this study, the direct effect of TMP, an extraction of Chinese medicine chuanxiong, against chronicity liver fibrosis inducing by porcine serum in rats and the possible mechanism in vitro were investigated.Method:(1) One hundred and twenty clean famale Wistar rats were dividede into two equal group randomly: group I and group II. Sixty rats in group I were secondely divided into six groups: blank control group(10 rats), porcine serum model control group(18 rats), oxymatrine control group(8 rats), 10mg/kg TMP group(8 rats), 20mg/kg TMP group(8 rats)and 40mg/kg TMP group(8 rats). Sixty ras in group II were also devided into six groups just like the group I.(2) We established liver fibrosis model by intraperitoneal injection porcine serum into rats. Expect the rats in two blank control groups, the others were intraperitoneally injected 0.5ml procine serum twice a week for eight weeks. While we set up animal models, we selected one rat in blank control group and three rats in model control group in group I randomly and removed their livers for pathological studies at 4, 6, 7th weekends to assess if the liver fibrosis was formed.(3) Rats in OM control group, 10mg/kg TMP group, 20mg/kg TMP group and 40mg/kg TMP in group I were intraperitoneally injected differet agentes q.d. for eight weeks, while injected procine serum. Rats in blank control group and model control group in group I were intraperitoneally injected Sodium Chloride Injection 10ml/kg q.d. for eight weeks as control. Rats in group II were not injected procine serum from the 9^th week and were started intraperitoneal injection differet agentes respectively. The way of administration was like that of rats in group I. We randomly selected one rat in blank control group and three rats in model control group in group II and removed their livers for pathological studies at 11,13,15^th weekends to assess if the liver fibrosis was natural cure.(4) Before we collected targets, all rats were absolute diet for 12 h. We collected the blood then killed the rats in group I and removed their livers at the 8 weekend. The serum was separated and the livers was stored in 10% formaldehyde solution. The targets of rats in group II were collected at the 16^th weekend like the methods of rats in group I.(5) The degree of Liver fibrosis was assessed directly by hepatic morphometric analysis(HE); The level of alanine aminotransferase(ALT), aspartate aminotransferase(AST), alkaline phosphatase(ALP), albumin (A) and globulin(G) of each rat serum was measured; We detected the level of hyaluronic acid(HA), laminin(LN ), N-terminal peptide of procollagen type III(PIIINP) and the collagen type IV(C-IV) in each rats serum isolated by radioimmunity method; The expressions of transforming growth factor-beta 1 (TGF-β₁), tissue inhibitor of metalloproteinase-1 (TIMP-1) in liver tissue were detected by immunohistochemical techniques.(6) HSC-T₆ cells were cultured in vitro and were treated with different concentrations of TMP. Cell proliferation was examined by 3-(4, 5-simethylthiazoly)2, 5-diphenyl-tetrazolium bromide (MTT) and apoptosis was analyzed by acridine orange / ethidium Bromide (AO/EB) staining method and flow cytometry methods.Results:1. The degree of liver fibrosis indexes were declined significantly, the inflammatory infiltration and necrosis of hepar were relieved obviously, compaired with those of rats in modle control groups respectively (P<0.01).2. The serum ALT, AST, ALP level were depressed by intraperitoneal injection TMP 20 mg/kg, 40mg/kg compared with model control group, but the level of A/G was enhanced (P<0.01).3.The serum HA, LN, PIIINP, C-IV level in rats serum were depressed by intraperitoneal injection TMP 20 mg/kg, 40mg/kg, compared with model control group (P<0.01).4.The expressions of TGF-β1, TIMP-1 were degraded both in hepatic fibrosis forming couse and after hepatic fibrosis formed by TMP 10 mg/kg, 20 mg/kg, 40mg/kg injection (P< 0.01).5. TMP 0.1mg/ml could inhibit the proliferation and enhance apoptosis of HSC-T6 (P<0.05 or P<0.01). These effections had good dose and time dependablity.Conclusions:TMP has prevention and treament actions to rats with immunodamaged hepatic fibrosis. The mechanism of these effections is related with its actions of both anticytokine, inhibition of collagen synthesis, enhancement of collagen degradation, and supression of the HSC-T₆ proliferation and promotion of actived HSC-T₆ apoptosis.