Study On The Effect And The Mechanism Of Anti-hepatic Fibrosis Of β-glucan To Experimental SD Hepatic Fibrotic Rats

BackgroundHepatic fibrosis is the critical stage in the developing of chronic liver diseases. Factors including viruses, alcohol, parasites, copper and iron deposition, toxicants, cholestatis and malnutrition chronically affect liver, which cause chronic hepatic injury. The process develops and causes an increasing synthesis of the extracellular matrix(ECM) mainly including collagen, however, the degradation relatively insufficient. Excess deposition of ECM causes hepatic fibrosis.It is traditionally thought that hepatic fibrosis could not be reversed. Recent studies on reversion of hepatic fibrosis developed dramatically. Professor Roking pointed out that human typical hepatic fibrosis could be reversed totally. Basing on this viewpoint, studies have focused on the mechanism of hepatic fibrosis and treatment of hepatic fibrosis in this decade.There are few effective drugs to treat hepatic fibrosis. We still could not make sure of the therapeutic effect of colchicin, glucocorticoid, penicillamine on hepatic fibrosis. The studies on Chinese traditional herb to treat hepatic fibrosis have been highly evaluated. It is found some herbs could inhibit hepatic fibrosis.Studies have proved that the antioxidant nutriments including Vitamin A, Vitamin E, Vitamin C, selenium and taurine could prevent and inhibit the formation of hepatic fibrosis. Because of the advantage of safety, little adverse reaction, easily for long term using, nutriments are used to prevent and cure hepatic fibrosis. To research for nutriments and nutritional components which could prevent and inhibit hepatic fibrosis have potential benefits.β-glucan is a sort of glycans extracted from barley and oat. It belongs to soluble dietary fiber. Experiments have proved that 6-glucan have antioxidate effectivness.It can decrease blood fat, decrease blood sugar, protect liver cell , increase the facilitate the growing of intestinal beneficial bacteria, but whether 6-glucan could prevent and inhibit hepatic fibrosis is still not clear.Our research based on the newest researching of hepatic fibrosis. We adopted carbon tetrachloride(CC14) tea oil solution intraperitoneal injection to prepare the SD rat hepatic fibrosis model, then we performed the experiment of 6-glucan intervention for 8 weeks based on this model. We observed the effects of 6-glucan to hepatic fibrosis rats which includes ameliorating hepatic function, improving renal function, promoting nutrition state, changing pathology. We particularly focused on the antioxidation and inhibition of 6-glucan to hepatic febrosis rats. We used the real-time fluorogenetic quantitative PCR(FQ-PCR) technology to detect the regulation of 6-glucan to the expression of hepatic fibrosis rat liver type I collagen and type III collagen, we also investigated the regulation of smad3 mRNA, smad7 mRNA expression in the process of hepatic fibrosis signal conduction. To study the benefit effects of 6-glucan to experimental animal hepatic fibrosis and the mechanism will provide a theory foundation for us to develop functional and health protection food to prevent and inhibit hepatic fibrosis.Objectives1. To observe the hepatic function amelioration of 6-glucan to hepatic fibrosis rats.2. To investigate the fibrosis reverse effect of 6-glucan to hepatic fibrosis rats.3. To explore the mechanism of B-glucan inhibiting hepatic fibrosis.4. To find the theory foundation to develop functional and health protection food to prevent and inhibit hepatic fibrosis.5. Methods1. In Animal experiments(1) Animal model setting80 male SD rat was divided into 2 groups randomly: control group and model group. 70 rats in model group were given 2ml/kg CCI4 tea oil solution intraperitoneal injection twice a week to make model. 10 rats in control group were given equal volume sodium chloride intraperitoneal injection. 3 hepatic fibrosis model rats and 2 control group rats were selected randomly in the time of 4 week, 6 week and 8 week to perform ultrasound liver check to ascertain the formation of hepatic fibrosis.(2) Further group dividing in model group and experimental methods The hepatic fibrosis was ascertained by pathological check after made model for 8 weeks. 26hepatic fibrosis rats were randomly divided into positive control group (10 rats, hepatic fibrosis rats without treating), colchicin treating group (7 rats) and 6-glucan (lOrats) treating group. 9 rats were selected into control group. During experimental period, 6-glucan group rats were given lOml/kg, 5% 6-glucan intragastric administration every day, colchicin group rats were given 0.2g/kg colchicin intragastric administration, positive control group rats and control group rats were given lOml/kg, 0.9% sodium chloride intragastric administration.2. Biochemistry detectionTo observe the improve effects of 6-glucan to hepatic function, renal function and protein synthesis of hepatic fibrosis ratsWe checked serum ALT, AST, AKP, y-GT, total bile acid, conjugated bilirubin, total bilirubin, total protein, Albumin, prealbumin, retinol-binding protein, creatinine(Cr), urea nitrogen(BUN), K+, Na+, Cl", Ca2+, Mg2+, Fe2+ and Ph with automated biochemistry analysator(7600-020, HITACHI, Japen).3. Pathology observationWe observed the pathological changes of hepatic fibrosis rat liver caused by 6-glucan with HE stain and collagen stain.4. Antioxidation enzymology index Serum SOD and MDA were determined by detection kits.5. Determination of cytokinesRat serum TNF-a and IL-2 were checked with immunohistochemistry method.6. mRNA expression detectionThe regulation effect of 6-glucan to type I collagen and type III collagen mRNA expression of hepatic fibrosis rats' livers was determined with real-time fluorogenetic quantitative PCR, and the effects of B-glucan to regulate smad3 mRNA expression, smad7 mRNA expression PPAR-a nRNA expression and TNF-a mRNA expression which were in the pathway of hepatic fibrosis were also determined.Results1. Animal experiments(1) 8-glucan could improve hepatic function of hepatic fibrosis rats. Serum ALT, AST, AKP, Y-GT, total bile acid, conjugated bilirubin, total bilirubin, total protein, Albumin, prealbumin, retinol-binding protein, creatinine(Cr), urea nitrogen(BUN) of 6-glucan group were better than those of positive control group.(2)6-glucan can prevent and inhibit rat hepatic fibrosis caused by CCL4.2. Experiments on mechanism of 6-glucan against hepatic fibrosis(1) SOD of 6-glucan group and colchicin group was higher than that of hepatic fibrosis positive control group, whereas MDA of 6-glucan group and colchicin group was lower than that of hepatic fibrosis positive control group.(2) 6-glucan can inhibit serum IL-2 expression:Type I collagen and type III collagen mRNA expression levels of 6-glucan group were significantly lower than those of positive control group.Smad3 mRNA expression level of 6-glucan group was significantly lower than that of positive control group. Smad7 mRNA expression level of 6-glucan group was significantly higher than that of positive control group.TNF-a mRNA expression level of 6-glucan group was significantly lower than that of positive control group.Conclusions1. We successfully replicated the rat hepatic fibrosis model with 40% CCI4 tea oil solution intraperitoneal injection.2. 6-glucan can prevent and inhibit experimental rat hepatic fibrosis. The possible mechanism of 6-glucan to reverse hepatic fibrosis maybe via ways of improving and protecting hepatocyte function, inhibiting inflammation, down regulation expression hepatic fibrosis type I collagen and type III collagen, down regulation expression of smad3 mRNA, up regulation expression of smad7 mRNA and down regulation expression.