Integrating proteomics, metabolomics, and molecular docking to elucidate the mechanism of Jiangtang Qingre formula against metabolic dysfunction-associated steatohepatitis

Figure 1 Jiangtang Qingre formula ameliorates hepatic function and lipid profiles in methionine-choline-deficient diet-induced metabolic dysfunction-associated steatohepatitis mice (n = 8). A: Study design and experimental timeline; B: Body weight; C: Representative gross appearance of livers; D: Liver weight and liver-to-body weight ratio; E: Serum alanine aminotransferase and aspartate aminotransferase; F: Serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein, and low-density lipoprotein; G: Hepatic TC and TG. ^aP < 0.05 vs methionine-choline-sufficient (MCS) group; ^bP < 0.01 vs MCS group; ^cP < 0.001 vs MCS group; ^dP < 0.05 vs methionine-choline-deficient (MCD) group; ^eP < 0.01 vs MCD group; ^fP < 0.001 vs MCD group. MCD: Methionine-choline-deficient; MCS: Methionine-choline-sufficient; PPC: Phosphatidylcholine capsules; JQF: Jiangtang Qingre formula; JQFH: Jiangtang Qingre formula-high; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; TC: Total cholesterol; TG: Triglycerides; HDL: High-density lipoprotein; LDL: Low-density lipoprotein.

Figure 2 Jiangtang Qingre formula attenuates hepatic pathology in methionine-choline-deficient-induced metabolic dysfunction-associated steatohepatitis mice (n = 3). A: Representative images of hematoxylin and eosin, oil red O, Sirius red, and α-smooth muscle actin immunohistochemistry; B: Semi-quantification of staining results. MCD: Methionine-choline-deficient; MCS: Methionine-choline-sufficient; PPC: Phosphatidylcholine capsules; JQF: Jiangtang Qingre formula; JQFH: Jiangtang Qingre formula-high; H&E: Hematoxylin and eosin; α-SMA: Α-smooth muscle actin.

Figure 3 Jiangtang Qingre formula mitigates hepatic oxidative stress in methionine-choline-deficient diet-induced metabolic dysfunction-associated steatohepatitis mice (n = 8). A: Serum and hepatic free fatty acids; B: Hepatic malondialdehyde; C: Hepatic total superoxide dismutase; D: Hepatic glutathione peroxidase. ^aP < 0.001 vs methionine-choline-sufficient group; ^bP < 0.05 vs methionine-choline-deficient (MCD) group; ^cP < 0.01 vs MCD group; ^dP < 0.001 vs MCD group. MCD: Methionine-choline-deficient; MCS: Methionine-choline-sufficient; PPC: Phosphatidylcholine capsules; JQF: Jiangtang Qingre formula; JQFH: Jiangtang Qingre formula-high; FFA: Free fatty acids; MDA: Malondialdehyde; T-SOD: Total superoxide dismutase; GSH-Px: Glutathione peroxidase.

Figure 4 Jiangtang Qingre formula modulates the hepatic proteome in methionine-choline-deficient diet-induced metabolic dysfunction-associated steatohepatitis mice (n = 5). A: Volcano plots depicting differentially expressed proteins (DEPs) in the methionine-choline-deficient (MCD) vs methionine-choline-sufficient and MCD + Jiangtang Qingre formula-high vs MCD comparisons; B: Venn diagram of DEP overlap; C: Gene Ontology biological process enrichment analysis (top 20 terms) of the 247 DEPs; D: Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis (top 20 pathways) of the 247 DEPs; E: Clustered heatmap of the expression profiles of the 26 core DEPs. MCD: Methionine-choline-deficient; MCS: Methionine-choline-sufficient; JQF: Jiangtang Qingre formula; JQFH: Jiangtang Qingre formula-high; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes.

Figure 5 Jiangtang Qingre formula modulates the hepatic metabolome in methionine-choline-deficient diet-induced metabolic dysfunction-associated steatohepatitis mice (n = 8). A: Partial least squares discrimination analysis score plots depicting group separation; B: Volcano plots of differential metabolites [methionine-choline-deficient (MCD) vs methionine-choline-sufficient; MCD + Jiangtang Qingre formula-high vs MCD]; C: Human metabolome database compound classification of the differential metabolites; D: Top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways from the topological analysis of differential metabolites; E: Top 20 shared KEGG pathways enriched by differentially expressed proteins (DEPs) and differential metabolites; F: Correlation heatmap of the top 20 differential metabolites and metabolic dysfunction-associated steatohepatitis-related phenotypes; G: Correlation network of the 26 core DEPs and the 20 key differential metabolites. MCD: Methionine-choline-deficient; MCS: Methionine-choline-sufficient; JQFH: Jiangtang Qingre formula-high; KEGG: Kyoto Encyclopedia of Genes and Genomes.

Figure 6 Expression levels of the 31 significantly differential metabolites in the three experimental groups (n = 8). ^aP < 0.05 vs methionine-choline-sufficient (MCS) group; ^bP < 0.01 vs MCS group; ^cP < 0.001 vs MCS group; ^dP < 0.01 vs methionine-choline-deficient (MCD) group; ^eP < 0.001 vs MCD group. MCD: Methionine-choline-deficient; MCS: Methionine-choline-sufficient; JQFH: Jiangtang Qingre formula-high.

Figure 7 Molecular docking analysis. A: Heatmap of the binding affinities between 15 major Jiangtang Qingre formula compounds and the key targets; B: Representative structures of the target-compound docking complexes.

Figure 8 Targeted metabolomic analysis of timosaponin B II, timosaponin A III, and sarsasapogenin (n = 6). A: Serum levels; B: Hepatic levels; C: Fecal levels. ^aP < 0.01 vs control group. Con: Control; JQF: Jiangtang Qingre formula.

Figure 9 Experimental validation of key target expression. A: Real-time quantitative PCR validation of key targets (n = 5); B: Western blot validation of key targets (n = 4). ^aP < 0.05 vs methionine-choline-sufficient (MCS) group; ^bP < 0.001 vs MCS group; ^cP < 0.05 vs methionine-choline-deficient (MCD) group; ^dP < 0.01 vs MCD group; ^eP < 0.001 vs MCD group. MCD: Methionine-choline-deficient; MCS: Methionine-choline-sufficient; JQF: Jiangtang Qingre formula; JQFH: Jiangtang Qingre formula-high.

Figure 10  Validation of Lmod3 knockdown by small interfering RNA in AML12 cells (n = 3). A: Lmod3 protein expression level; B: Intracellular triglycerides content; C: Malondialdehyde level; D: Α-smooth muscle actin protein expression level; E: Cell viability. ^aP < 0.01 vs AML12 group; ^bP < 0.01 vs AML12 + free fatty acids + negative control small interfering RNA (siRNA) group; ^cP < 0.01 vs AML12 + free fatty acids + siRNA targeting mouse Lmod3 + control group. MDA: Malondialdehyde; TG: Triglycerides; si-NC: Negative control small interfering RNA; si-Lmod3: Small interfering RNA targeting mouse Lmod3; Con: Control; JQF: Jiangtang Qingre formula; FFA: Free fatty acids; α-SMA: Α-smooth muscle actin.

Figure 11  Schematic illustration of the mechanisms underlying Jiangtang Qingre formula-mediated amelioration of metabolic dysfunction-associated steatohepatitis. JQF: Jiangtang Qingre formula; FFA: Free fatty acids; ROS: Reactive oxygen species; OS: Oxygen species; MDA: Malondialdehyde; T-SOD: Total superoxide dismutase; GSH-Px: Glutathione peroxidase.