Lianhe Xiaozhi ointment ameliorates metabolic dysfunction-associated steatotic liver disease via peroxisome proliferator-activated receptor alpha pathway activation

Figure 1 Schematic representation of the therapeutic efficacy and pharmacological mechanisms of Lianhe Xiaozhi ointment in high-fat diet-induced metabolic dysfunction-associated steatotic liver disease. UPLC-Q-TOF-MS: Ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry; LXO: Lianhe Xiaozhi ointment; HIF: Hypoxia inducible factor; PPAR: Peroxisome proliferator-activated receptor; MASLD: Metabolic dysfunction-associated steatotic liver disease; ND: Normal diet; L-LXO: Low-dose Lianhe Xiaozhi ointment; H-LXO: High-dose Lianhe Xiaozhi ointment; IL: Interleukin; KEGG: Kyoto Encyclopedia of Genes and Genomes; ECM: Extracellular matrix; HFD: High-fat diet; RT-PCR: Quantitative real-time polymerase chain reaction.

Figure 2 Ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry analysis of the Lianhe Xiaozhi ointment decoction. A: Base peak ion chromatograms of the Lianhe Xiaozhi ointment (LXO) decoction in positive ion mode; B: Base peak ion chromatograms of the LXO decoction in negative ion mode.

Figure 3 Prediction of key targets, mechanistic pathways impacted, and bioactive components of Lianhe Xiaozhi ointment relevant to metabolic dysfunction-associated steatotic liver disease treatment. A: Venn diagram of Lianhe Xiaozhi ointment (LXO) targets key to metabolic dysfunction-associated steatotic liver disease (MASLD) treatment; B: Protein-protein interaction network diagram of intersecting targets (top 50); C: Gene Ontology functional enrichment analysis; D: Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis; E: “Herb-ingredient-disease-pathway-target” network. The pink symbol denotes the five herb constituents of LXO; turquoise indicates bioactive components; green labels indicate molecular targets of LXO in MASLD; yellow highlights the top 20 enriched pathways; orange represents disease. BP: Biological process; CC: Cellular component; MF: Molecular function; HIF: Hypoxia inducible factor; PPAR: Peroxisome proliferator-activated receptor; CR: Coptidis Rhizoma; LL: Lotus leaf; PT: Pericarpium trichosanthis; IBO: Immature bitter orange; TP: Tangerine peel.

Figure 4 Lianhe Xiaozhi ointment normalized hepatic lipid metabolism and ameliorated inflammation in metabolic dysfunction-associated steatotic liver disease. A: Representative images of mice and liver morphology; B: Lianhe Xiaozhi ointment (LXO) reduced high-fat diet-induced weight gain (n = 12); C: Food intake post-LXO treatment (n = 12); D: Oral glucose tolerance test (OGTT) curves (n = 8); E: Area under the curve of the OGTT (n = 8); F: Hepatic non-esterified fatty acid levels (n = 7); G: Representative Hematoxylin and eosin and Oil red O staining of liver tissue, with 100 μm scale bars (n = 5); H: Detection of interleukin (IL)-1β and IL-18 levels in liver tissue by enzyme-linked immunosorbent assay (n = 5); I and J: Representative western blot images (I) and quantitative analyses (J) of IL-1β and IL-18 in liver tissue (n = 6). One-way analysis of variance was employed, and the data are presented as mean ± SEM. ^aP < 0.05 vs normal diet group. ^bP < 0.01 vs normal diet group. ^cP < 0.001 vs normal diet group. ^dP < 0.05 vs high-fat diet group. ^eP < 0.01 vs high-fat diet group. ^fP < 0.001 vs high-fat diet group. ND: Normal diet; HFD: High-fat diet; L-LXO: Low-dose Lianhe Xiaozhi ointment; H-LXO: High-dose Lianhe Xiaozhi ointment; IL: Interleukin; AUC: Area under curve; OGTT: Oral glucose tolerance test; NEFA: Non-esterified fatty acid; HE: Hematoxylin and eosin.

Figure 5 Lianhe Xiaozhi ointment influence on hepatic gene expression in mice with metabolic dysfunction-associated steatotic liver disease. A: Heatmap presenting the clustered differentially expressed genes; B: Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis; C-E: Validation of Pparα, Pparγ, Pparδ, Hmgcs2, Cpt1a, Acox1, Fxr, Fgf21, and Srebp1c through quantitative real-time polymerase chain reaction (n = 5); F and G: Representative western blot images (F) and quantitative analysis (G) of peroxisome proliferator-activated receptor (PPAR) α, carnitine palmitoyl transferase 1a, acyl-CoA oxidase 1, and 3-hydroxy-3-methylglutaryl-CoA synthase 2 in liver tissue (n = 6); H: Serum β-hydroxybutyrate concentrations (n = 6). One-way analysis of variance was employed, and the data are presented as mean ± SEM. ^aP < 0.05 vs normal diet group. ^bP < 0.01 vs normal diet group. ^cP < 0.05 vs high-fat diet group. ^dP < 0.01 vs high-fat diet group. ^eP < 0.001 vs high-fat diet group. ND: Normal diet; HFD: High-fat diet; H-LXO: High-dose Lianhe Xiaozhi ointment; KEGG: Kyoto Encyclopedia of Genes and Genomes; ECM: Extracellular matrix; PPAR: Peroxisome proliferator-activated receptor; L-LXO: Low-dose Lianhe Xiaozhi ointment; BHB: Β-hydroxybutyrate; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; Acox1: Acyl-CoA oxidase 1; CPT1A: Carnitine palmitoyl transferase 1a; HMGCS2: 3-hydroxy-3-methylglutaryl-CoA synthase 2.

Figure 6 Peroxisome proliferator-activated receptor alpha-dependent mechanism of Lianhe Xiaozhi ointment in HepG2 cells. A: Cell viability under different treatments: Lianhe Xiaozhi ointment-derived serum, oleic acid/palmitic acid, and GW6471 [peroxisome proliferator-activated receptor alpha (PPARα) inhibitor, 10 μM]; B: Intracellular triglyceride levels (n = 5); C: Oil red O staining of lipid droplets, with a scale bar of 50 μm (n = 3); D: Representative western blot images of PPARα, carnitine palmitoyl transferase 1a, acyl-CoA oxidase 1, and 3-hydroxy-3-methylglutaryl-CoA synthase 2 (n = 3); E: Extracellular β-hydroxybutyrate levels (n = 5). One-way analysis of variance was employed, and the data are presented as mean ± SEM. ^aP < 0.001 vs normal control group. ^bP < 0.05 vs palmitic acid + oleic acid group. ^cP < 0.001 vs palmitic acid + oleic acid group. ^dP < 0.05 vs palmitic acid + oleic acid + 10.0% Lianhe Xiaozhi ointment-derived serum group. ^eP < 0.05 vs palmitic acid + oleic acid + 5.0% Lianhe Xiaozhi ointment-derived serum group. LXO: Lianhe Xiaozhi ointment; OA: Oleic acid; PA: Palmitic acid; PPAR: Peroxisome proliferator-activated receptor; BHB: Β-hydroxybutyrate; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; Acox1: Acyl-CoA oxidase 1; CPT1A: Carnitine palmitoyl transferase 1a; HMGCS2: 3-hydroxy-3-methylglutaryl-CoA synthase 2.

Figure 7 Molecular docking of peroxisome proliferator-activated receptor alpha with key bioactive components from Lianhe Xiaozhi ointment. A-F: Three-dimensional (left) and two-dimensional (right) interaction diagrams of quercetin-3-O-β-D-glucuronide (A), hesperetin (B), coclaurine (C), palmatine (D), armepavine (E), and arvenin I (F) with peroxisome proliferator-activated receptor alpha.

Figure 8 Modulation of gut microbiota composition by Lianhe Xiaozhi ointment in mice with metabolic dysfunction-associated steatotic liver disease. A and B: Taxonomic profiles at phylum (A) and genus (B) levels in the cecal contents of mice from different experimental groups (n = 6); C: Spearman correlations between key microbial genera and biochemical parameters; D-F: Hepatic gamma-linolenic acid (D), docosahexaenoic acid (E), and linolenic acid (F) levels (n = 4). One-way analysis of variance was employed. Data are presented as mean ± SEM. ^aP < 0.05 vs normal diet group. ^bP < 0.01 vs normal diet group. ^cP < 0.05 vs high-fat diet group. ND: Normal diet; HFD: High-fat diet; L-LXO: Low-dose Lianhe Xiaozhi ointment; H-LXO: High-dose Lianhe Xiaozhi ointment; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; FBG: Fasting blood glucose; TC: Total cholesterol; TG: Triglyceride; LDL-C: Low-density lipoprotein cholesterol; HDL-C: High-density lipoprotein cholesterol; DHA: Docosahexaenoic acid; GLA: Gamma-linolenic acid; LA: Linolenic acid.

Figure 9 Proposed Lianhe Xiaozhi ointment mechanism in metabolic dysfunction-associated steatotic liver disease treatment. Solid black arrows signify promotional effects. Inverted blue arrows denote a reduction in content or activity, and the T-shaped solid line without an arrow represents inhibitory effects. LXO: Lianhe Xiaozhi ointment; PPAR: Peroxisome proliferator-activated receptor; CPT1A: Carnitine palmitoyl transferase 1a; NEFA: Non-esterified fatty acid; TG: Triglyceride; IL: Interleukin; NF-κB: Nuclear factor kappa-B; Acox1: Acyl-CoA oxidase 1; HMGCS2: 3-hydroxy-3-methylglutaryl-CoA synthase 2.