Xiaoyao San attenuates hepatic steatosis through estrogen receptor α pathway in ovariectomized ApoE−/− mice

Xiaoyao San (XYS) is a famous prescription in traditional Chinese medicine, which is used in the treatment of "liver depression and spleen deficiency" syndrome. It is often used clinically to treat chronic hepatitis, liver cirrhosis, various symptoms of postmenopausal women, especially mental disorders and digestive system diseases. However, the effect of XYS on hepatic steatosis in postmenopausal women remains unclear. In this research, we investigated the effects of XYS on hepatic steatosis in ovariectomized (OVX) apolipoprotein E knockout (ApoE-/-) mice, as well as the molecular mechanisms in vitro and in vivo.Fifty female ApoE-/- mice were divided into 5 groups: control group (Sham), model group (OVX), OVX + β-estradiol (E2, 0.4 mg/kg) group, OVX + XYS (13.0 g/kg) group, and OVX + XYS (6.5 g/kg) group. The control group received a standard diet, while the other groups received a high-fat diet (HFD). The hepatic pathologies of the mice were examined with Oil red O staining and HE staining after 12 week treatment. Blood and liver variables were determined enzymatically. Transmission electron microscopy was used to examine the ultrastructure of hepatocytes. The expression of estrogen receptor α (ERα) and lipid metabolism genes was analyzed by real-time PCR and/or Western blot. In in vitro studies, we investigated the effect of XYS-medicated serum on the expression and activity of ERα in L02 cells by immunofluorescence and luciferase reporter assays, and examined the protection of XYS-medicated serum against free fatty acid (FFA)-induced steatosis of L02 cells. Intracellular lipid accumulation were measured by Oil red O staining and Nile red staining assay. Finally, the influence of ICI 182,780, a specific antagonist of ERα, on the protective effect of XYS-medicated serum on FFA-induced steatosis of L02 cells was investigated.Treatment of Ovx/ApoE-/- mice with XYS significantly decreased HFD-induced increases in hepatic steatosis and triglyceride (TG) content, accompanied by inhibition of liver X receptor α (LXRα), sterol regulatory element binding protein (SREBP)-1c and its target lipogenic genes transcription. Similarly, XYS-medicated serum reduced the size and number of lipid droplets and the cellular TG content in FFA-induced L02 cells. In addition, XYS significantly increased the ERα expression in hepatocytes in vivo and in vitro and enhanced the transcriptional activity of ERα promoter in L02 cells. And these effects could be partly reversed by the antiestrogen ICI 182,780.These findings suggest that XYS has an estrogen-like effect and inhibits steatosis in postmenopausal animal models by reducing the expression of genes related to TG synthesis through ERα pathway.