| Background and Objective:Hepatic fibrosis is regarded as a pathologically repair reac-tion followed various kinds of chronic liver diseases. Fibrosis mainly presents the imbalance of the ECM compound production and degradation, which makes the ECM deposit pervadingly in liver. Hepatic fibrosis is a common stage of most chronic liver diseases regardless of the etiology, and its progression may lead to hepatic cirrhosis or hepatocelluar carcinoma (HCC). Although hepatic fibro-sis is thought to be a reversible pathological state, there is no established effective therapy for hepatic fibrosis yet.Because of its special feature of poly-target, poly-lint, poly-channel, and its compound effects, the Chinese medicine can aim to the complicated mechanisms of hepatic fibrosis accordingly, which makes it have traditional and elearly prevalence against the western medicine. The topic is through the establishment of immune animal model of hepatic fibrosis, to get a preliminary understanding the inhibition of Artemisia argyi extracting solution on rat hepatic fibrosis, and through its rat liver tissue TGFβ-Smad signal transduction pathway in the key signal transduction molecules TGFβ1, Smad 3, Smad 4, Smad 7 expression level changes, as well as against the apoptosis gene bcl-2 and cell cycle proteins, to explore Artemisia argyi extracting solution the molecular mechanism of anti-fibrosis.Methods:1. Use of pig serum-induced liver fibrosis model,72 Wistar rats were fed 1 week after adaptation,were randomly divided into normal control group, liver fibrosis model group, high dose, middle dose and low-dose treatment of Artemisia argyi extracting solution grou-ps,12 rats in each. In addition to the normal control group, the rest of the rats by intraperitoneal injection of sterile body weight 100g pig serum 0.3ml,2 times per week, a total of 10 weeks,20 times. Artemisia argyi extracting solution large, medium and low-dose treatment group were given 6ml/only,3ml/only and 1.5ml/only with Artemisia argyi extracting solution fed to pray daily 1; model group were given 6ml/only normal saline gavage, every day 1 times; the normal control group was given intraperitoneal injection of saline,2 times per week,10 weeks a total of 20 times, and to give 6ml/only normal saline gavage, day 1 times. At 10 over the weekend after the last injection of pig serum 48h, animals in each group were decapitated, eyes, enough to take blood specimens from blood, separa-tion of serum, and take part of the liver,4% formalin-fixed, conven-tional paraffin-embedded sections.Part of the gradient of fresh liver tissue placed in -70℃to save, for real-time fluorescent quantitative reverse transcription-polymerase chain reaction(RT-PCR) method and protein blot (Western blot).2. Serological test:The rats in each group were detected in serum indicators of liver fibrosis serum hyaluronic acid (HA),laminin (LN),Ⅲprocollagen (PCⅢ),Ⅳprocollagen (Ⅳ-C)content, as well as indicators of lipid peroxidation superoxide dismutase(SOD), malondialdehyde (MDA). ELISA, serum TGFβ1 levels。3. Liver histology tests:using HE staining and Masson staining of collagen triple-pathological changes in rat liver.4.Immunohistochemistry:using of immunohistochemistry SABC method, combined with microscope to observe the rat liver tissue TGFβ1, Smad3, Smad4, Smad7 and Bcl-2, cyclinDl of expression.5. Using real-time fluorescent quantitative reverse transcription-poly mer-ase chain reaction (RT-PCR) method and Western blot test (Western blot) were measured TGFβ1, Smad3, Smad4, Smad7 and Bcl-2, cyclinDl mRNA and protein expression.Results:1. Artemisia argyi extract solution inhibited rat liver fibrosis in rats.Compared with the normal control group, the serum markers of liver fibrosis HA,PCⅢ,LN,Ⅳ-C levels of model group,were signi-ficantly higher(P＜0.01), serum TGF-β1 levels are significantly higher (P＜0.05), increase in serum MDA concentration, SOD activity was significantly decreased(P＜0.01);with the model group,Artemi-sia argyi extracting solution treatment for all groups HA, PCIII, LN,Ⅳ-C levels were significantly lower (P＜0.05), serum TGF-β1 content also decreased significantly(P＜0.05), serum indicators of lipid peroxidation MDA contents were significantly lower(P＜0.05), SOD activity was significantly increased (P＜0.05), and Artemisia argyi extracting solution treatment group and normal control group had no significant difference (P＞0.05).Optical microscope observation:model group can be seen part of the organizational structure of liver damage, liver cells a wide range of variability of edema, water balloon-like degeneration or degeneration, but also shows the extent, scope, ranging from liver cell necrosis; liver disorder board arrangement, some of loss of normal structure of hepatic lobule; periportal fibrous septum in a large number of eosinophils, monon-uclear cell infiltration of lymphocytes and fibroblasts, a large number of collagen deposition, there are more obvious fibrous tissue hyperplasia, collagen fibers along the periportal areas and around the central vein to the hepatic lobule within the extension,we can see the connection between fiber split hepatic lobule, and even some regions the performance of early liver cirrhosis. Artemisia argyi extracting solution therapy in liver of rats in each group in the color, textu-re, size, surface smoothness in a significant improvement compared with model group, cell degeneration and necrosis significantly reduced hepatic lobule was not obvious structural damage, inflame-matory cell infiltration and fibrous tissue reduced proliferation, the central veins and periportal extension of a small amount of fibrous tissue, different doses of Artemisia argyi extracting solution between the treatment group no significant difference.2. Artemisia argyi extracting solution expect rat liver tissue TGFβ-Smad signal transduction pathway protein expression.Immunohistochemistry results showed that, TGFβ1, smad3, smad4 liver cells in the normal group no positive cells, liver expression was significantly increased in model group, mainly seen in interstitial cells, inflammatory cells, injury of liver cells, the positive substances mainly concentrated on the membrane, there is a little expression in the cytoplasm; Artemisia argyi extracting solution each dose group significantly reduced the number of positive cells, positive staining is reduced significantly.RT-PCR and Western blot test results showed, TGFβ1, smad3, smad4 mRNA and protein expression in the normal control group, significantly lower, while the smad7 mRNA and protein expression was significantly increased; Artemisia argyi extracting solution high, medium and low dose group TGFβ1, smad3, smad4 mRNA and protein expression compared with model group gradually reduced, smad7 mRNA and protein expression compared with model group were increased gradually, in which middle and high dose group TGFβ1, smad3, smad7 mRNA and protein and model group and there was significant difference (P＜0.05, P＜0.01), compared with the SM group was no significant difference (P＞0.05). 3. Artemisia argyi extracting solution against apoptosis gene Bcl-2 and cell cycle protein expression of cyclinDl.Immunohistochemistry results showed that, Bcl-2 and cyclinDl protein in normal liver cells there is no positive cells, liver expression was significantly increased in model group, mainly seen in portal area,hepatic cells and stromal cells'membrance and cytoplasm, positive substance is mainly concentrated in the membr-ance; Artemisia argyi extracting solution each dose group signifi-cantly reduced the number of positive cells, positive staining is reduced significantly.RT-PCR and Western blot test results showed that the normal control group, Bcl-2, CyclinDl mRNA and protein expression compared with model group, low, Artemisia argyi extracting solution every dose group Bcl-2, CyclinD1 mRNA and protein expression compared with model group gradually reduced, which high-dose group compared with the model group were significantly different (P＜0.05), with the normal group and the SM group showed no significant difference (P＞0.05).Conelusions:1. Artemisia argyi extracting solution can significantly improve the rat liver fibrosis histopathological damage induced by immune pig serum, reduce fibrous tissue proliferation and decreased collagen fibers interval,liver cell necrosis,degeneration,and inflammatory cell infiltration,and can effectively reduce serum markers of liver fibrosis and its mechanism may be anti-fibrosis and anti-lipid peroxidation, reducing the concentration of serum TGF-β1.2. Artemisia argyi extracting solution can significantly inhibit rat liver tissue TGFβ1, Smad3, Smad4 protein and mRNA expression levels, increase the level of Smad7 protein and mRNA expression, indicating that it can effectively regulate TGFβ-Smad signal transduction pathway, thereby suppressing rat liver fibrosis.3. Artemisia argyi extracting solution can reduce the rat liver tissue of anti-apoptotic gene Bcl-2 protein and mRNA expression levels, suggesting that it can effectively induce apoptosis in rat HSC to achieve the anti-fibrosis.4. Artemisia argyi extracting solution could be reduced in rat liver tissue of cyclin CyclinDl protein and mRNA expression levels to play on cell cycle regulation, so that stagnation in the G0/G1 phase cell replication, inhibit cell proliferation. |
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