The Effects Of Acanthus Ilicifolius Alkaloid A On Extracellular Matrix And Cytokines In Hepatic Fibrosis Rats

Objective:Previous research had synthesized and identified Acanthus Ilicifolius Alkaloid A(4-hydroxy-2-benzoxazolone, HBOA). Based on the previous research, this study discussed from the aspects of extracellular matrix and related cytokines of hepatic fibrosis in rats induced by carbon tetrachloride by the treatment of HBOA, and to explore its action mechanism.Method:Sprague Dawley rats were randomly divided into normal control group and CCl4 induced chemical hepatic fibrosis model group. Hepatic fibrosis model group received 50% CCl4 oil solution, with the volume of 2 ml/kg by intra-gastric administration, twice per week for the duration of 8 weeks to induce hepatic fibrosis. In the same time, the normal control group(10 rats) received the same volume of normal saline. After the formation of hepatic fibrosis was confirmed by pathology at the end of 8 weeks administration, the rats of hepatic fibrosis were randomly divided into model control group, positive control group (received colchicine), high-dose of HBOA-treated group and low-dose of HBOA-treated group. The dosages of high-dose of HBOA-treated group were 100 mg/kg and the low-dose were 50mg/kg, in addition, the dosage of colchicine were 0.4 mg/kg. In the same time, normal control group and model control group were received 0.6%CMC-Na solution 10mL/kg. Each group of rats were administrated by intra-gastric gavage once a day for 4 weeks. During the treatment, in order to prevent the hepatic fibrosis automatically reversed, in addition to the normal control group, each treated group of rats were given 50% CCl4 twice a week, and rats of normal control group only given corresponding volume of normal saline.24 hours after the last administration, abdominal aortic blood was taken from the rats after 12h fasting in a state of anesthesia. Separated the upper serum, and then took part of the upper serum for automatic biochemical analyzer. Alanine aminotransferase(ALT) and aspartate aminotransferase(AST) of serum were measured. Enzyme linked immunosorbent assay(ELISA) was used to measure the levels of hyaluronic acid(HA), laminin(LN), tumor necrosis factor-α(TNF-α) and transforming growth factor-β1(TGF-β1). Weight of liver of each rat was weighed to calculate the liver index. Hepatic fibrosis pathological damages were observed by HE staining and Masson trichromatic dyeing. The collagen type Ⅰ(COL-Ⅰ), collagen type Ⅲ(COL-Ⅲ), matrix metallopeptidase-9(MMP-9) and tissue inhibitor of metalloproteinases-1(TIMP-1) protein expression of hepatic fibrosis were detected by immune histochemical.The COL-Ⅰ, COL-Ⅲ, TNF-α, TGF-β1, PPARγ, ERK1, ERK2 mRNA expression of the livers were detected by reverse trans-PCR.Results:Serum biochemical results demonstrated that in comparison to the normal control group, the activities of ALT and AST of model control group were distinctly increased(P<0.05), the levels of HA, LN, TNF-a and TGF-β1 were obviously increased in model control group (P＜0.01). Compared to the model control group, the activities of ALT and AST in HBOA-H and HBOA-L groups were significantly decreased (P<0.05 or P<0.01); the levels of HA, LN, TNF-a and TGF-01 in HBOA-H and HBOA-L groups were clearly decreased(P<0.05 or P＜0.01).Pathological results showed that HBOA could obviously reduce the degree of hepatic fibrosis in rats induced by CCl4. Compared with normal control group, liver index of model control group was increased significantly(P<0.01). In addition, HBOA-H and HBOA-L dosages could obviously decrease the liver index(P<0.05 or P<0.01).Immune histochemical results displayed that in comparison to the normal control group, the protein expression of COL-I, COL-III and TIMP-1 in model control group significantly increases and color deepened, but the protein expression of MMP-9 obviously decreased (P＜0.05 or P<0.01). Compared with model control group, HBOA-H and HBOA-L dosages can obviously decrease the protein expression of COL-I, COL-III and TIMP-1, but the protein expression of MMP-9 was significantly increases(P<0.05 or P＜0.01).Reverse trans-PCR results showed that compared with normal control group, the mRNA expression of COL-I, COL-III, TNF-a, TGF-β1, ERK1 and ERK2 in model control group were obviously increased, but the mRNA expression of PPARy obviously decrease(P<0.05 or P<0.01); Compared with model control group, HBOA-H dosage inhibited the mRNA expression of COL-I, COL-III, TNF-a, TGF-β1, ERK1 and ERK2(P<0.05 or P＜0.01); HBOA-L dosage inhibited the mRNA expression of COL-I, COL-III, TGF-β1, ERK1 and ERK2(P<0.05 or P<0.01), inhibited the mRNA expression of TNF-a but no statistical significance. Both HBOA-H and HBOA-L dosages could obviously increased the mRNA expression of PPARy(P<0.05).Conclusion:HBOA regulate and control the production of the related cytokines such as MMP-9, TIMP-1, PPARγ and ERK1, ERK2 which can decrease the proliferation of extracellular matrix(ECM). These effects may account for its mechanism of anti-hepatic fibrosis.