Hepatoprotective effects of Xiaoyao San formula on hepatic steatosis and inflammation via regulating the sex hormones metabolism

Abstract

Background: The modified Xiaoyao San (MXS) formula is an adjuvant drug recommended by the National Health Commission of China for the treatment of liver cancer, which has the effect of preventing postoperative recurrence and metastasis of hepatocellular carcinoma and prolonging patient survival. However, the molecular mechanisms underlying that remain unclear.

Aim: To investigate the role and mechanisms of MXS in ameliorating hepatic injury, steatosis and inflammation.

Methods: A choline-deficient/high-fat diet-induced rat nonalcoholic steatohepatitis (NASH) model was used to examine the effects of MXS on lipid accumulation in primary hepatocytes. Liver tissues were collected for western blotting and immunohistochemistry (IHC) assays. Lipid accumulation and hepatic fibrosis were detected using oil red staining and Sirius red staining. The serum samples were collected for biochemical assays and NMR-based metabonomics analysis. The inflammation/lipid metabolism-related signaling and regulators in liver tissues were also detected to reveal the molecular mechanisms of MXS against NASH.

Results: MXS showed a significant decrease in lipid accumulation and inflammatory response in hepatocytes under metabolic stress. The western blotting and IHC results indicated that MXS activated AMPK pathway but inhibited the expression of key regulators related to lipid accumulation, inflammation and hepatic fibrosis in the pathogenesis of NASH. The metabonomics analysis systemically indicated that the arachidonic acid metabolism and steroid hormone synthesis are the two main target metabolic pathways for MXS to ameliorate liver inflammation and hepatic steatosis. Mechanistically, we found that MXS protected against NASH by attenuating the sex hormone-related metabolism, especially the metabolism of male hormones.

Conclusion: MXS ameliorates inflammation and hepatic steatosis of NASH by inhibiting the metabolism of male hormones. Targeting male hormone related metabolic pathways may be the potential therapeutic approach for NASH.

Keywords: Hepatic steatosis; Inflammation; Male hormone; Phosphatase and tensin homolog deleted on chromosome ten; Sex hormone metabolism.

Figure 1

Modified Xiaoyao San demonstrated its capacity to alleviate inflammation and hepatic steatosis in rats subjected to a choline-deficient/high-fat diet. A: The diagram illustrating the experimental procedure outlined the systematic investigation of the protective role of modified Xiaoyao San (MXS) in rats following a 12-week choline-deficient/high-fat (CDHF) diet regimen. Starting at week 13, rats that had been fed the CDHF diet received daily intragastric administration of either saline or MXS decoction for a duration of 6 weeks; B and C: The liver weight and liver/body weight ratio of the rat groups were presented; D and E: The images from hepatic sections were stained with hematoxylin and eosin and Oil Red O (ORO). Black arrows indicate the liver portal areas. The lipid droplets (indicated by blue arrows) in the tissue are orange-red and the nuclei are blue; F and G: Quantitative data pertaining to ORO-positive areas and the nonalcoholic fatty liver disease activity score for each group were displayed. Data were presented as mean ± standard error of mean (n = 10/group), and were analyzed by ANOVA. ^aP < 0.05 Control vs nonalcoholic steatohepatitis (NASH) group. ^bP < 0.05 NASH vs MXS group. MXS: Modified Xiaoyao San; NASH: Nonalcoholic steatohepatitis.

Figure 2

Significant changes in metabolites after modified Xiaoyao San treatment were mainly related to lipid metabolism. A: Principal component analysis score map of each group; B: Gene ontology enrichment analyses; C: Human Metabolome Database classification notes; D: Partial least squares discrimination analysis between nonalcoholic steatohepatitis and modified Xiaoyao San groups. The scores of the first-ranked principal component (PC1, horizontal) and the second-ranked principal component (PC2, vertical) were used to represent the data. Samples from various experimental cohorts were represented by differently colored scatter plots, while 95% confidence intervals were visually depicted using ellipses. PCA: Principal component analysis; PLS-DA: Partial least squares discrimination analysis; HMDB: Human Metabolome Database; MXS: Modified Xiaoyao San; NASH: Nonalcoholic steatohepatitis.

Figure 3

Steroid hormone synthesis was the primary target pathway affected by modified Xiaoyao San treatment. A: Statistical analysis of differential metabolites. Total Sig denoted the total count of metabolites that exhibited significant changes. Down/up Sig represented the overall number of metabolites that underwent significant downregulation or upregulation; B: A Venn diagram illustrated the comparative analysis of metabolites among the three groups; C and D: Volcano plots displayed significantly changed metabolites, indicated by red and blue-colored dots. The criteria for significance were fold change (FC) below than 0.667, FC greater than 1.5, or variable importance in the projection greater than 1.0, and P value below than 0.05; E and F: Pathway enrichment analysis of differential metabolites in different comparisons. The pathway impact value (X-axis) was denoted by the size of the circles, and the -log10 P value weight (ranging from white to red, Y-axis) was indicated by the color intensity. MXS: Modified Xiaoyao San; NASH: Nonalcoholic steatohepatitis.

Figure 4

Heatmap of the top 30 metabolites that decreased in the nonalcoholic steatohepatitis model group but recovered or increased after modified Xiaoyao San treatment. MXS: Modified Xiaoyao San; NASH: Nonalcoholic steatohepatitis.

Figure 5

Heatmap depicted the 30 most prominent metabolites that unusually increased in the nonalcoholic steatohepatitis model group but recovered or downregulated after modified Xiaoyao San treatment. MXS: Modified Xiaoyao San; NASH: Nonalcoholic steatohepatitis.

Figure 6

Modified Xiaoyao San inhibited steroid synthesis and inflammation-related metabolic pathways. A and B: Metabolite set enrichment and pathway analysis of the 30 identified metabolites that decreased in the nonalcoholic steatohepatitis (NASH) model group but recovered or increased after modified Xiaoyao San (MXS) treatment; C and D: Metabolite set enrichment and pathway analysis of the 30 identified metabolites that unusually increased in the NASH model group but recovered or were downregulated after MXS treatment; E: Histogram of the top 3 metabolites related to steroid hormones and the inflammation process. MXS: Modified Xiaoyao San; NASH: Nonalcoholic steatohepatitis.

Figure 7

Metabolites related to male hormones were the primary targets influenced by modified Xiaoyao San. A: Heatmap of metabolites related to steroid hormone metabolism; B and C: Heatmaps of metabolites related to male hormones and estrogen metabolism; D: Histogram of the top 4 metabolites related to male hormones metabolism. MXS: Modified Xiaoyao San; NASH: Nonalcoholic steatohepatitis.

Figure 8

Modified Xiaoyao San treatment regulated the signaling pathways and factors related to inflammation and lipid metabolism. A: In hepatic tissues, PPARγ and COX2 expressions were decreased by modified Xiaoyao San (MXS) treatment and MXS also activated the AMPK signaling; B: MXS treatment attenuated hepatic fibrosis and α-SMA expression. Collagen fibers appear red (black arrows) under normal light microscopy and other tissue components were dyed yellow; C: The MXS treatment inhibited the levels of FASN in liver tissues; D: The PTEN expression in liver tissues was restored after MXS treatment (immunohistochemistry detection reagents yielding a brown reaction product, indicated by black arrows). The brown portion is the portion of the antigen that expresses coloration. MXS: Modified Xiaoyao San; NASH: Nonalcoholic steatohepatitis.