The Effects Of Cynomorium Songaricum On Hepatic Fibrosis Mice And Its Hedgehog Signaling Pathway

ObjectiveTo investigate the effect of Cynomorium on the model of liver fibrosis in mice treated with carbon tetrachloride(CCl4),and to observe the effect of the drug on Hedgehog signaling pathway.The aim of this study was to investigate the possible mechanism of Cynomorium serotonus on liver fibrosis in mice.Method1.The male C57 mice(n=24)of 6 weeks old were randomly divided into 4groups:normal control(n=6),two weeks model group(n=6),three weeks model group(n=6),four weeks model group(n=6).Using the mixture of CCl4 and olive oil by 1: 3ratio,according to 4ml/kg dose intraperitoneal injection twice a week to replicate liver fibrosis models.Respectively,in the first 2,3,4 weeks drawn,taking the liver and serum.To observe the general morphology of the mice,the liver changes in general,and testing ALT and AST in the serum and the content of hydroxyproline(Hyp)in the liver tissue of the mice.The expression of transforming growth factor-β(TGF-β1)and platelet-derived factor(PDGF)in serum of mice were measured by enzyme-linked immunosorbent assay.HE,Masson and Sirius red staining were used to evaluate the degree of liver fibrosis in different periods.Fluorescence real-time quantitative PCR was used in the liver to analyze the expression of α-SMA and Gli1 in Hedgehog signaling pathways.2.The male C57 mice(n=40)of 6 weeks old were randomly divided into 6groups:normal control group(N=8),model group(n=8),colchicine group(0.1mg/kg)(n= 8),low dose group of Cynomorium(2.77mg/kg)(n=8)and high dose group(27.7mg/kg)(n=8).The experiment started from the first week,except for the normal control group,the other three groups of modeling,was used the mixture of CCl4 and olive oil by 1: 3 ratio,according to 4ml/kg dose intraperitoneal injection twice a week.The normal control group was given equal olive oil 4ul/g for five weeks.From the third week of administration,colchicine group and Cynomorium low dose,Cynomorium high dose group once a day for gavaging,continued to the fifth week,taking liver and serum.The changes of ALT and AST in the mice were observed.The levels of TGF-β1 and PDGF in serum were measured by enzyme-linked immunosorbent assay(ELISA).HE,Masson and Sirius red staining were used to observe the pathological changes of hepatic fibrosis in different groups and quantitatively analyzed.Changes of Hyp Content in Liver Tissue by Alkaline Water Cracking.Fluorescence Quantitative PCR was used to analyze the expression of α-SMA and Gli1 in the Hedgehog signaling pathway in the liver.Immunofluorescence staining was used to observe the expression of α-SMA and Gli1 in each group.Results1.CCl4 intraperitoneal injection to the second week,the liver has a slight sense of fiber.At the third week,the hepatic fibrosis model group showed obvious deposition of collagen fibers,vacuoles appeared in the cytoplasm,and the fibrosis was obvious.At 4th,the liver grain was significant and the fibrosis was significantly increased.The levels of ALT and AST and Hyp in the liver were significantly higher than those in the normal control group(p <0.05).Fluorescence real-time quantitative PCR results showed that the expression of α-SMA and Gli1 mRNA in the liver of the mice were significantly higher than the normal group control group,and distributed around the portal area.The fibrosis was more obvious with the extension of liver fibrosis model cycle.2.After the experiment to the fifth week,it was found that the liver of the mice in the treated group was significantly improved,and the degree of fibrosis was decreased in the model group,especially in the colchicine and Cynomorium high dose group.The levels of AST and ALT and Hyp and the expression of TGF-β1 and PDGF in low dose and high dose group of Cynomorium were lower than those in model group.Among them,the difference was more obvious between the high dose group of Cynomorium and the model.The results of immunofluorescence showed that the expression ofα-SMA and Gli1 cells in Cynomorium group was lower than that in model group,and the effect of high dose group was more obvious Conclusion1.Male C56 mice were intraperitoneally injected with 0.8ml/kg CC14 twice a week.The liver fibrosis model was successfully replicated in the third week.The Hedgehog signaling pathway is inhibited in the normal state of the human body,andwhen subjected to external damage and stimulation,the Hedgehog pathway is activated,thereby increasing the expression of α-SMA and Gli1 in the liver.Therefore,Hedgehog signaling pathway may be closely related to the occurrence of liver fibrosisgroup(p <0.01).The results of fluorescence quantitative PCR showed that the expression of α-SMA and Gli1 in the liver was lower than that in the model group,and the high dose of Cynomorium reductions was more obvious.2.Cynomorium has an improved effect on liver fibrosis in mice,and the high dose of Cynomorium is effective.Its mechanism may be related to the inhibition of Hedgehog signaling pathway.Cynomorium may be a new drug target for the treatment of liver fibrosis.Cynomorium low-dose treatment of its role is not very significant,it should be accurate and reasonable to grasp the dose.