Experimental Study On The Effect And Mechanisms Of Tetramethylpyrazine On Collogen And Hepatocellular Gene Spectra Of Rat In Hepatic Fibrosis

Chronic liver disease is a severe disease to harm mankind health. Hepatic fibrosis is the feature owned by both chronic liver disease and the essential stage that develop into hepatic cirrhosis. Pathologically, it is characteristic of proliferation and deposit of fiber tissue which is mainly collagenⅠandⅢin portal canal area and liver lobule. It is well known that damaging factors bring about liver injury, then cause cellular necrosis of liver and inflammation. Cells, which are correlated with hepatic fibrosis secrete cytokines to activate HSC to develop mass of ECM. Subsequently the synthesis of ECM in liver is more than degradation of it, which leads to the occurrence of liver fibrosis.And it is popularly thought that by virtue of the participation of several kinds of cell and cytokines extracellular matrix deposits excessively. And a lot of study data indicate that the activation of hepatic stellate cell is central tache in the occurrence and development of liver fibrosis. Ever since a long time ago, HSC is the main study object about hepatic fibrosis . With the deep study , we realize the important position of cooperation of liver cells . The cells in liver contain hepatic parenchymal cells and hepatic nonparenchymal cells, hepatic parenchymal cells are hepatocytes, and hepatic nonparenchymal cells include SECs, KCs, HSCs and hepatic lymphocytes etc. Hepatocytes hold 80 percent in livervolume or so, and hepatic nonparenchymal cells only hold 6.5 percent. Hepatocytes complete multitude function of liver. The pathologicalchange of hepatocytes refer to cell injury, apoptosis and canceration. So the pathogenesy , prevention and cure of hepatic fibrosis should not ignore hepatocytes.ECM include collagen, non-collagen glycoprotein, proteoglycan, elastin, matrix metalloproteinases, tissue inhibitor of metalloproteinase, adhesion molecule, growth factor and cytokine . ECM have important effects on frame texture and attachment site, it can control attachment, migration, proliferation, differentiation and gene expression.Collagen is the essential component of ECM . There are twenty-seven species of collagens, and there mainly are collagen typeⅠand collagen typeⅢin liver, the ratio is 1:1. Collagen protein holds 5%-10% in total protein of normal liver, and the ratio is up to 50% when hepatic fibrosis happens. The ratio of collagen typeⅠand collagen typeⅢincrease, and in later period it can increase there times than before. So, collagen typeⅠand collagen typeⅢare the essential components of ECM when hepatic fibrosis happens. Collagen typeⅠis mainly secreted by HSC and produce a marked effect in middle and later period. It can promote the activation and proliferation of HSC, and inhibit the hyperplasia of HC and SEC in order to promote the development of hepatic fibrosis. Collagen typeⅢis a kind of fibroid collagen protein, and it increases mainly in the morning of hepatic fibrosis.When gene chip containing thousands of DNA or RNA sequence, which used as probes , on silica wafer or nylon membrane are hybridized to labeld sample, gene expression, DNA sequencing as well as DNA mutation and polymorphism can be analyzed in large scale. The major advance of this technology, as compared to conventional techniques, results from the small size of the array, which allows for a higher accurate, a quicker process and a higher sensitivity enables the parallel screening of large number of genes simultaneously on one chip and provides the opportunity to use smaller amp liation of startingmaterial. Now, its application in medical science has been expanded to research differential gene expression, discover new gene, diagnose disease and so on. Gene chip has a characteristic of high-flux, high sensitivity and high accuracy rating. It can be quick, all automatic, multiparametered and at equal pace to analyze the poly-gene even the whole-gene. Gene chip technique provides a new method and consideration to illuminate the development mechanism of hepatic fibrosis.Tetramethylpyrazine is the effective constituent of rhizome. Animal experiments and clinical researches all confirm that tetramethylpyrazine can activate blood circulation to dissipate blood stasis, inhibit platelet coacervating, broaden arteriola, improve microcirculation, inhibit xidation, rival Ca2+, and resist fibrosis. Our study deployed the n-atural extractive of rhizome, and investigated the change of collagen and gene spectra of hepatocyte.Ⅰ. Experimental study on the effect of tetramethylpyrazine on the expression of collagen typeⅠand collagen typeⅢin hepatic fibrosisOur study adopted special immunohistochemistry method---Van Gieson staining method to detect the proliferation degree of collagen, and semiquantitatively analyzed the proliferation degree. The result was that the collagen fibrils of hepatic fibrosis largely deposited and the proliferation degree was very high. But tetramethylpyrazine obviously reduced the collagen deposition, so, tetramethylpyrazine obviously reduced the proliferation degree of collagen fibrils in hepatic fibrosis.Collagen typeⅠand collagen typeⅢare the essential components of ECM when hepatic fibrosis happens. So, the kinesis change of collagen typeⅠand collagen typeⅢis the important parameters to judge the degree of hepatic fibrosis. In this experiment, we used immunocytochemical staining and image analysis techniques to investigate the effect of tetramethylpyrazine on the expression of collagen typeⅠand collagen typeⅢ. The result indicated that tetramethylpyrazine can remarkably reduce the collagen deposition and degree of injury.Ⅱ. Experimental study on the effect of tetramethylpyrazine on the expression of hepatocellular gene in hepatic fibrosisIn this experiment, we used gene chip technique to detect the differential expression genes with tetramethylpyrazine intervention. These genes included proto- carcinoma and anti-oncogene, ion channel and transport protein gene, cell cycle gene, cytoskeleton and movement protein gene, apoptosis-associated protein gene, DNA transcription and repair gene, cell receptor gene, cell signaling gene, metabolism and growth gene, protein translation and synthesis gene and or so. Gene related to the collagen are MMP, collagen typeⅢ, leptin r, TIMP and TGF-βmRNA. In this experiment, tetramethylpyrazine can remarkably downregulated the expression of leptin r, TIMP and TGF-βmRNA, so reduced the expression of collagen.In summary, tetramethylpyrazine is an effective medicine against liver fibrosis. According to experiments we have conducted, the major mechanisms of tetramethylpyrazine might be involved in the following several aspects :1. Tetramethylpyrazine significantly reduce the expression of collagen of hepatic tissue and the proliferation degree of collagen fibrils.With immunocytochemical staining method, we find tetramethylpyrazine remarkably reduce the expression of collagen typeⅠand collagen typeⅢ.2. With gene chip technique, we find tetramethylpyrazine has no effect on the collagen gene of hepatocyte, but has effect on the gene which related to the collagen, so tetramethylpyrazine might through regulating the expression of the gene which related to the collagen of hepatocyte, affect the expression of collagen in liver.In a word, through the experimental study, we can conclude that tetramethylpyrazine has a certain effect on preventing and treating hepatic fibrosis.