Water-soluble fraction of Herb-B28 ameliorates nonalcoholic steatohepatitis (NASH) in high fat/high cholesterol diet-fed hamsters

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Nonalcoholic fatty liver disease (NAFLD) is one of the most common non-viral chronic liver disease worldwide. NAFLD has a histological spectrum ranging from simple steatosis, nonalcoholic steatohepatitis (NASH), to fibrosis and cirrhosis. However, there is little available therapy for NASH. Some Chinese herbal medicines (CHM) are believed to have liver-protective effects and our previous studies have identified a CHM B28 (B28) has protective effects against high fat/high cholesterol (HFC) diet-induced NAFLD and NASH in the golden Syrian hamster. The aim of this study is to test whether the water-soluble fraction of B28 can also exert protective effects against HFC diet-induced NASH in hamsters. In animal study I, B28 was sequentially partitioned using solvents with different polarity, including hexane, ethyl acetate and butanol; and the water-soluble extracts were collected. Hamsters were separated into 4 groups. The control group was fed with HFC diets with 0.5% cholesterol and gavaged with distilled water for 6 weeks. The other 3 groups, designated as HFC+B28, HFC+W-a and HFC+W-na were also fed with the HFC diets and gavaged with B28、B28 water-soluble extracts (autoclaved) and B28 water-soluble extracts (non-autoclaved), respectively, for 6 weeks. The results show that B28 and its water-soluble extracts decreased plasma and liver total cholesterol levels and lowered plasma AST and ALT activities compared to the HFC group. Gel filtration analysis revealed that cholesterol levels in LDL fractions is lower in the HFC+B28, HFC+W-a and HFC+W-na groups than in the HFC group. Histopathological analyses of liver sections show that B28 and its water-soluble extracts can alleviate liver inflammation and fibrosis, and also prevent neutrophil infiltration induced by the HFC diet. These results suggest that the liver-protective effects of B28 and its water-soluble extracts are independent of the autoclave sterilization. In animal study II the B28 was partitioned with ethyl acetate, and the water-soluble and ethyl acetate-soluble extracts were collected. Hamsters were assigned into 5 groups designated as HFC, HFC+B28, HFC+W, HFC+3x W and HFC+EA and all fed with HFC diets with 0.5% cholesterol and gavaged with distilled water, B28、B28 water-soluble extracts, B28 water-soluble extracts (triple-dose) and B28 ethyl acetate extracts, respectively, for 6 weeks. The results show that HFC+B28 and HFC+3x W significantly lowered levels of plasma and liver cholesterol and lowered AST activity compared to the HFC group. Gel filtration and ultracentrifugation analyses of lipoprotein profiles showed that HFC+B28 and HFC+3x W groups have significantly lower levels of cholesterol in LDL fractions. Histopathological analyses of liver sections revealed that HFC+B28 and HFC+3x W groups have minor inflammation and fibrosis state and less neutrophil infiltration compared to the HFC group. The HFC+W and HFC+EA groups have no such effects. These results demonstrate that it will require triple doses of water-soluble extracts to have protective effects against NASH when the partition procedures are changed. Still, our results of these two animal studies indicate that water-soluble extracts preserved the active compounds of B28. In animal study III, hamsters were separated into 3 groups designated as HFC, HFC+3x B28 and HFC+3x W. They were gavaged with distilled water, B28 (triple-dose) and B28 water-soluble extracts (triple-dose), respectively, for 6 weeks. All 3 groups were treated with chow diets during the first 2 weeks than the diets were substituted with HFC diets with 1% cholesterol for another 4 weeks. B28 significantly lowered plasma cholesterol and LDL-C levels and decreased plasma LDL(-) contents. B28 water-soluble extracts lowered plasma cholesterol, LDL-C levels and LDL(-) contents to a lesser degree of B28. In vitro studies show that B28 and its water-soluble extracts lowered the LDL(-)-induced secretion of proinflammatory cytokines by rat Kupffer cells. In the future, we aim to obtain the active compounds from the water-soluble fraction of B28 by refining the partition procedures, and it will be of great help for new drug development against NASH.