Study On The Mechanisms Of Regulating Collagen Degradation In Rat Alcoholic Liver Fibrosis With Man-Gan-Xiao

1 AimsThe prescription of Man-Gan-Xiao (MGX) which based on the theory of traditional Chinese medicine and clinical experiences, is developed by professor Tian De-Lu. It has good effects on alcoholic liver fibrosis (ALF). In order to approach the mechanisms involved in the treatment to ALF with MGX, its effects on collagen degradation in ALF rats were observed.2 Methods2.1 Induction of alcoholic liver fibrosis in rats(1) Animals groups Male Wistar rats weighing between 140 and 160g were randomly divided into normal group , model 4wk, model 9wk, model 14wk. There were 8 rats in each group.(2) Model making Model rats were given ethanol-pyrazole-com oil gastrically once a day, and in the meantime rats were injected acetaldehyde-hemoglobin adducts intraperitoneally at 10-day intervals, for 4wk, 9wk, 14wk respectively.(3) Serumal measurement and histological examinations Liver tests were measured by biochemical method; levels of serum procollagen III (PCIII) and hyaluronic acid (HA) were detected by radioimmunoassay; liver sections were made from the paraffin-embedded tissues and stained with hematoxylin and eosin and Mallory staining; histological changes were observed under light microscope.2.2 Effects of MGX on pathologic histology in ALF rats(l)Animals groups Male Wistar rats weighing 140 ~ 160g were randomly divided into normal group, model group, treatment group, treatment control group. There were 8 rats in each group.(2) Model making Model group, treatment group and treatment control group rats were given ethanol-pyrazole-corn oil gastrically once a day, and in the meantime were injected acetaldehyde-hemoglobin adducts intraperitoneally at 10-day intervals for 14wk respectively.(3) Treatment of the rats Beginning with the 15th week, normal group and model group were given normal saline Iml/lOOg weight once a day for 4 weeks; treatment group and treatment control group were given Man-Gan-Xiao Iml/lOOg weight and Gan-Pi-Kang Iml/lOOg weight once a day for 4 weeks respectively.(4) Serumal measurement and histological grading Liver tests were measured by biochemical method; levels of serum PCIII and HA were detected by radioimmunoassay; liver sections were made from the paraffin-embedded tissues and stained with hematoxylin and eosin and Mallory's staining; the liver sections were graded numerically based on Fatty Metamorphosis Scores Standard and Collagen Fiber Deposition Standard.2.3 Effects of MGX on collagen!, Ill degradation in ALF rats(1) Liver sections Liver sections were made from Test 2.2(2) Histological observations Liver sections were stained with Sirius red. Effects of MGX on collagen I and III degradation were observaed under polarizing microscope.Semi-quantitative data for collagen I and III were obtained by colourful video imaging and image analysis software.2.4 Dynamic expressions of MMP-13mRNA and TIMP-lmRNA in ALF rats(1) Animals groups Male Wistar rats weighing between 140g and 160g were randomly divided into normal group and model group. There were Owk, 4wk, 9wk, 14wk,18wk time points in each group. Ther were 8 rats at each time point.(2)Model making Induction of ALF rats accorded to Test2.1(3) Expressions detection Expressions of MMP-13mRNA and TIMP-lmRNA at each time point were examined by RT-PCR. Semi-quantitative data for PCR products were obtained using G3PDH cDNA as internal control and comparing the intensity of the PCR band of MMP-13, TIMP-1 with that of the G3PDH respectively by video imaging and analyzed with image analysis software.3 Results3.1 Induction of alcoholic liver fibrosis in rats(1) Serum indices The serum levels of ALT, AST, GGTP, PCIII, HA in model group were significantly higher, Alb was low as compared with normal group (P < 0.001).(2) Pathlogic histology The livers of model 4wk showed fatty metamorphosis and no collagen fiber deposition was observed. The livers in model 9wk showed necrosis of hepatocytes around central veins and portal area, accomp...