Role Of Microecologics In The Progression Of Nonalcoholic Steatohepatitis

Objectives: Non-alcoholic steatohepatitis (NASH) is an inheritance-environment-metablism clinico-pathological syndrome that characterized by hepatocyte steatosis, lobular and periportal inflammation and even focal necrosis. NASH is commonly observed in individuals with metabolic syndrome comprising obesity, type 2 diabetes and hyperlipidemia. It is estimated that the hepatic fibrosis of different degrees is detectable histologically in approximately 30% patients with NASH, so NASH becomes an important cause of cryptogenic liver cirrhosis. The mechanism of NASH has not been well elucidated up to now. According to the"two Hits"theory put forward by Dye and James, insulin resistance (IR), endotoxemia, and inflammatory cytokines are thought to place an important role in the pathogenesis of NASH. The clinical treatment is currently unideal. We established an animal model of NASH by high-fat diet. At the same time, rats in the treated group were treated by microecologics(Golden Bifid). The levels of TNF-α, LPS, adiponectin in serum and the expression AdipoR-2 protein in the liver was detected. The purpose of this study is to investigate the effect of microecologics in NASH, in order to reveal the pathogenesis of NASH and provide a new idea for clinical treatment.Methods : Thirty male Wistar rats weighing 140±10g were purchased from Hebei Medical University, and housed in cages under standard conditions with free access to water. After a week of adaptive feeding, the rats were randomly divided into five groups: normal control group (n = 6, fed with normal diet), model group [n = 6, fed with high-fat diet(normal diet plus 2% cholesterol, 10% lard and 5% corn oil)], Golden Bifid group(n=6, fed with high-fat diet 12 weeks later, Golden Bifid 400mg/kg·d was administered by gastric perfusion for 4 weeks), normal+LPS group(n = 6, fed with normal diet 16 weeks later, LPS 1mg/kg·d was administered by intraperitoneal injection 6 hours before killed), HFD+LPS group(n = 6, fed with high-fat diet 16 weeks later, LPS 1mg/kg·d was administered by intraperitoneal injection 6 hours before killed). At the end of the 16th week, when the NASH model of rats were set up, all rats were killed to collect serum to measure the levels of total cholesterol(TC), triglyceride(TG), alanine aminotransferase(ALT), aspartate aminotransferase(AST), tumor necrosis factor-α(TNF-α), lipopolysaccharide(LPS) and adiponectin. The specimens of liver were stained with HE, Masson, SudanⅣand then studied microscopically. The expression of AdipoR-2 protein was determined by western blot.Results:1. The general manifestation of rats: Normal control rats all have good appetite, their fur was bright, psychosis was good and body weight increased. Model rats have bad appetite, less movement, their fur has no shine and psychosis was bad. Rats in model group were abdominal obesity, the body weight and liver index increased significantly than those in the normal control group(P<0.01). In Golden Bifid group, the psychosis, appetite and movement of rats were between the normal rats and model rats. the body weight and liver index were lower than those in the model group(P<0.01). Rats in LPS group, have extremely apathetic psychosis, slow response, cyanosis lips and dry fur. No significant difference were found in the body weight and liver index between normal+LPS group and normal group(P>0.05). In HFD+ LPS group, the body weight and liver index significantly increased compared to those in normal +LPS group, but have no difference compared to model group.2. The examination of biochemical indexes in serum: The levels of serum TC, TG, ALT, and AST in the model group increased significantly than those in the normal control group(P<0.01). In Golden Bifid group,the levels of serum TC, TG, ALT, and AST decreased significantly than those of the model group(P values were<0.01, <0.05, <0.05, <0.01). No significant difference were found in comparison with the normal+LPS group and the normal control group(P>0.05). In HFD+LPS group, the levels of serum TC, TG have no difference compared to model group, but the levels of serum ALT and AST increased significantly(P<0.01). However all of the biochemical indexes increased significantly compared to those in the normal +LPS group(P<0.01).3. Serum adiponectin, plasma endotoxin and serum TNF-αlevels: In NASH model group, serum adiponectin levels were significantly decreased (P <0.01), plasma endotoxin (P<0.05) and serum TNF-α(P<0.01) levels were significantly increased compared with the normal control group; Golden Bifid group compared with model group, serum adiponectin increased, however endotoxin and TNF-αlevels decreased than before (P values were<0.01, < 0.05, <0.01); Normal+LPS group rats after injection of LPS, plasma endotoxin levels increased compared with normal control group (P<0.01), whereas TNF-αand adiponectin levels to maintain the same level; In contrast, HFD+ LPS group rats plasma endotoxin and serum TNF-αlevels were significantly higher than model group (P<0.01), adiponectin levels were no significant difference (P> 0.05); compared to normal + LPS group, the level of serum adiponectin decreased (P <0.01), plasma endotoxin and serum TNF-αwere significantly increased (P values were<0.05, <0.01).4. Liver histopathological observation: Examined by the unaided eye, the normal livers were reddish-brown and bright. The model rat livers were buff and significantly increased volume, which adhered to the surrounding tissues. The sections were greasy and dim. The livers in Golden Bifid group looked between the normal and model groups. No significant differences were found in comparison with the normal+LPS group and normal control group, as well as the HFD+LPS group and model group. HE staining showed the normal hepatocyte arranged in radiation by the center of the central veins under light microscope. There are very little collagen in central veins and portal areas with Masson staining. Liver cytoplasmic have no lipid droplets with SudanⅣstaining. In hepatic tissue of the model rats, all hepatocytes presented moderate to severe steatosis, cloudy swelling with the inflammation of the interlobular and portal area, most obviously in hepatic acinar zoneⅢ. Masson triple staining showed periportal deposition of collagen fibers and fibrosis around the hepatic sinusoids and venofibrosis could also presented in some rats. Orange-red lipid droplets could be found in hepatocyte cytoplasm with SudanⅣstaining, and the liver tissue showed diffuse steatosis. The fatty degeneration, inflammatory activity and fibrosis were significantly lower in Golden Bifid rat livers. No significant differences were found in comparison with the normal +LPS group and normal control group. Compared to the model group, the HFD+ LPS group showed increased hepatic lobule inflammation. Piecemeal necrosis and bridging necrosis, blood cells within the sinusoidal can be seen. Masson and SudanⅣstaining showed that the degree of liver fibrosis and steatosis have no significant difference.5. The expression of AdipoR-2 protein in liver tissues: comparison with the normal control group, model group rats AdipoR-2 protein expression in the liver tissue was significantly lower (P <0.01); Golden Bifid can up-regulate its expression (P<0.05); No significant difference was found in normal+LPS group and the normal control group (P> 0.05); HFD+LPS group and model group showed no significant difference (P>0.05), but significantly lower compared with normal + LPS group (P <0.01).6. Correlation analysis: NASH model group, serum TNF-αlevel and ALT, AST levels was positively correlated (r values were 0.940, 0.970, P values were <0.01, <0.01); the expression of serum adiponectin level and serum TG levels were negatively correlated (r = -0.890, P<0.05), and liver AdipoR-2 protein expression was positively correlated (r = 0.978, P <0.01).Conclusion:1. Rat model of NASH could be established by feeding with long-term high-fat and high-cholesterol food gradually. Lipid metabolism disorder is an important mechanism for the development of NASH.2. TNF-αand endotoxemia play an important role in the process of NASH, and the NASH model rats significantly increased sensitivity to LPS.3. The decreased serum adiponectin level and adipoR-2 expression in the liver play an important role in the liver injury of NASH rats.4. Microecologics may have preventive and therapeutic effect on NASH by complementing normal intestinal flora, inhibiting intestinal endotoxemia and TNF-αsynthesis and release. Increased expression of adiponectin and its receptors are the middle part of this mechanism.