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.