Intervention Effects Of Emodin On Rats With Nonalcoholic Fatty Liver Disease Induced By Fat-rich Diet And Its Mechanisms

ObjectiveTo observe emodin’s intervention to rat model of nonalcoholic fatty liver disease induced by high fat diet.To approach emodin’s effect to the expression of TNF-a and IL-1βand possible effect to signal transduction of LPS-TLR4-NF-KB in liver of NASH rats.Initially approach the effect of emodin’s prevention and cure to NAFLD and possible mechanism of it’s pharmacologic action,supply experimental evidence for emodin’s clinic treatment to NAFLD. Methods42 healthy male Spraque-Dawley rats were numbered According to the weight after being adaptive feeding for a week. They were completely randomly divided into normal diet group(group A,n=8) which were feed on normal diet, and high fat diet modeling group(n=34),which were feed on high fat diet with free water intake.At the end of 4th week,2 rats were randomly drew off from model group to get their liver tissues for histopathological examination to identify if hepatic steatosis appeared. After the success of modeling, the 8 rats of nomal group (group A) continue to be fed with ordinary diet and with nomal sodium chloride saline by 10 mL-kg-1·d-1 by intragastric administration at the same time. The remaining rats in modeling group were indvided randomly into 4 groups with 8 in every group, they were named model group (group B), low-dose emodin group (group C), high-dose emodin (group D) and metformin group (group E). They continue to be fed with high-fat diet. At the same time, model group were fed with nomal sodium chloride saline by 10 mL·kg-1·d-1, group C with emodin by 20 mg·kg-1·d-1, group D with emodin by 40 mg·kg-1·d-1 and group E (the positive group) with metformin by 150 mg·kg-1·d-1. After being fed by intragastric administration for 8 weeks, a day before the rats were executed at the end of 12th week, the tail blood was taken by shearing tail method for mearuring blood glucose levels. At the end of 12th week, all rats were executed. Blood was draw from portal vein and inferior caval vein under pyrogen-free conditions. Right lobe of liver tissue was obtained, the rats’weight and their wet liver weight were got to count the liver index. Gross morphology of rats liver was observed. HE staining under light microscopy was used to observe liver histopathology, the degree of liver steatosis and liver inflammation grade classification were calculated. The serum ALT、AST、TG、TC and glucose were measured with automatic biochemieal analyzer. Oral glucose tolerance test (OGTT) was calculated. The expression of TNFa, NF-kB in liver tissue was detected by immunohistochemical method. RT-PCR method was adopted to detect the rat liver IL-1βmRNA, TLR4 mRNA expression. LAL method was used to monitor the level of portal plasma endotoxin detection. TLR4 expression in liver tissue was assayed by Western-blotting. Enzyme-linked immunosorbent assay (ELISA) double antibody sandwich method was adopted for the determination of liver tissue IL-1β. ResultsRat models of NAFLD were successfully replicated after 12 weeks of fat-rich diet consumption. There was no significant difference in the body weight of rats among group B、C、D (p>0.05), with significant body weight increase in group B compared with group A (P<0.05). The liver indexs of rats in group B were increased than that in group A (P<0.01), while the liver indexs in group C、group D and group E were lower than that in the model group (p<0.01). For the serum lipid detection, total serum cholesterol (TC) and triglycerol (TG) level in group B were higher than that in group A (p<0.01), and lower than that in either group C or D (p<0.01). Compared with group B, there was no significant improvement of liver steatosis in group C or D (P>0.05)。Compared with group B, the hepatic inflammatory activities in group C and D were dramatically reduced(p<0.05, p<0.01, respectively) with much lower in group D (p<0.01). Serum ALT and AST were lowered in group C and D compared with group B (p<0.01) with much lower in group D (p<0.01). For the immunohistologic analysis, expression of rat TNFa was significantly decreased in group D compared with group B (p<0.05). Correlation analysis showed that rat TNFa was positively correlated with serum ALT and AST(p<0.05). High dosage emodin reduced liver IL-βexpression in both mRNA and protein level (p<0.01), the effect was dose-dependent. The expression of NF-KB was increased in group B compared with that in group A (P<0.01), with much lower NF-KB expression in group D compared with group B based on the immunohistologic analysis (P<0.01) The portal endotoxin level in group B was significantly higher than that in group A (p<0.01), while significantly lowered in group D compared with group B (p<0.01). The portal endotoxin level in group C was decreased than that in group B with no significant difference (p>0.05). mRNA expression of TLR4 in group D was dramatically downregulated compared with that in group B, while the TLR4 expression in the high dosage group was significantly lowered than that in the model group based on the Western blotting detection (P<0.05) Conclusions1.Emodin can effectively intervene the formation of rat’s model of nonalcoholic fatty liver disease.2.Emodin can decrease the serum level of TG and TC of rats model of NAFLD.3.Emodin can apparently alleviate the inflammation of NASH.4.Inhibiting the expression of TNF-a and IL-1βin rat’s liver maybe one of the mechanism of emodin’s anti-inflammation.5.Emodin can effectively reduce the production of endotoxin in NASH rats.6.Emodin inhibits inflammatory factor’s production in NASH rats possibly by affecting the signaling molecule’s expression of TLR-4 and NF-KB.