Effects of extracts from soothing-liver and invigorating-spleen formulas on the injury induced by oxidative stress in the hepatocytes of rats with non-alcoholic fatty liver disease induced by high-fat diet

Abstract

OBJECTIVE

To investigate effects of the extracts from soothing-liver and invigorating-spleen formulas on the injury due to oxidative stress, mediated by the Nuclear factor-like 2 (Nrf2)-Antioxidant response element (ARE) pathway, in the hepatocytes of rats with non-alcoholic fatty liver disease (NAFLD) induced by high-fat diet.

METHODS

Soothing-liver and invigorating-spleen formula mixtures were prepared for five groups: normal, model, soothing-liver formula group (SLG), invigorating-spleen formula group (ISG), integrated formula group (IG). The rat model of NAFLD was induced by feeding rats a high-fat diet (HFD). After 16 weeks, the hepatic tissue was examined following Haematoxylin-Eosin (H&E) staining and with Transmission electron microscopy (TEM). Levels of hepatic lipids, serum lipids and hepatic functions were measured using a biochemical analyser. Hepatocytes were isolated from the livers of rats and were identified by cellar immunohistochemistry, cellular immunofluorescence and flow cytometry. The expression levels of Nrf2, Kelch-like epichlorohydrin-associated protein 1 (Keap-1), haeme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) mRNAs were assessed by real-time fluorescence quantitative PCR. Nrf2, Keap-1, HO-1 and NQO1 proteins were measured by Western blotting. Finally, the levels of oxidative stress factors Superoxide Dismutase (SOD), malonaldehyde (MDA) and Glutathione peroxidase (GSH-Px) in hepatocytes were measured by WST-1, TBA and colorimetry.

RESULTS

The H & E and TEM results showed that the NAFLD model rats successfully reproduced typical pathogenetic and histopathological features. The liver function and levels of hepatic lipids and serum lipids from the model rats were dramatically increased. Compared with the model group, the levels of hepatic lipids, serum lipids and hepatic function in the treatment groups were ameliorated to different degrees. The yields of purified hepatocytes in each rat were 4-5 × 10⁸. The viability of the isolated hepatocytes was higher than 95%, with a purity over 93.2%. Cellular immunohistochemistry analysis showed that the hepatocytes were brown, while in the cellular immunofluorescence analysis, the hepatocytes showed green fluorescence. The expression levels of Nrf2, Keap-1, HO-1 and NQO1 mRNA and protein in the hepatocytes were significantly higher in the model group than in the normal group (P < 0.05, P < 0.01). Compared with the model group, the expression of Nrf2, Keap-1, HO-1 and NQO-1 mRNAs and proteins in all treatment groups increased, especially in the IG (P <0.01).

CONCLUSION

The extracts from soothing-liver and invigorating-spleen formulas may protect the liver against the injury induced by oxidative stress in hepatocytes by influencing the Nrf2-ARE pathway, which may be the mechanism having the potential for prevention and treatment of NAFLD.