Abstract: [Objective] This study was conducted to reveal the molecular mechanism underlying the hepatoprotective effects of Honghua Qinggan 13 Flavor Pill （HHQG）, a traditional Mongolian medicine, by improving liver function via activating hepatocyte nuclear factor 4α （HNF4α）. [Method] A total of 45 C57BL/6J mice were randomly assigned into control group, CCl₄ injury group and HHQG group, with 15 mice in each group. The mice in HHQG group were given HHQG at a dose level of 0.616 5 g/（kg·BW） by gavage once daily for 7 consecutive days, and those in control group and CCl₄ injury group were given an equal volume of sterile distilled water by gavage. Four hours after the last gavage, the mice in control group were intraperitoneally injected with 5 mL/（kg · BW） of olive oil, and those in CCl₄ injury group and HHQG group were intraperitoneally injected with the same dose of a CCl₄ and olive oil mixture （CCl₄∶olive oil=1∶4, V/V） for liver injury modeling. Blood and liver tissue samples from mice in each group were collected on the 2^nd, 5^th and 7^th days after modeling. The body weights of the mice were monitored during the experiment period. The serum activities of aspartate aminotransferase （AST）, alanine aminotransferase （ALT） and total superoxide dismutase （SOD） were measured. HE staining and Masson staining were used for observing liver histopathological changes. Differential gene enrichment pathways were analyzed by transcriptome sequencing （RNA-seq）. The expression of HNF4α protein in liver tissue was detected using immunohistochemical assay. [Result] Compared with control group, the body weight of CCl₄ injury group significant （P<0.05） or extremely significant （P<0.01 or P<0.001） dropped on the 1^st to 4^th and 6^th to 7^th days after modeling, respectively. In comparison to CCl₄ injury group, the body weight of HHQG group significant （P<0.05） or extremely significant （P<0.01） rose on the 4^th and 6^th to 7^th days after modeling, respectively. On the 2^nd day after modeling, CCl₄ injury group had extremely significantly （P<0.001） increased serum activities of ALT and AST than control group, while significantly （P<0.05） decreased serum activities of ALT and AST were observed in HHQG group compared with CCl₄ injury group. HE staining and Masson staining demonstrated that the liver damages in HHQG group were obviously ameliorated compared with CCl₄ injury group on the 2^nd , 5^th and 7^th days after modeling. KEGG signaling pathway enrichment analysis showed that the signaling pathways such as peroxisome proliferator-activated receptor, fatty acid degradation and fatty acid metabolism in the liver tissue of HHQG group mice were upregulated, while ribosome, glycerophospholipid metabolism, thyroid cancer and some others were downregulated compared with CCl₄ injury group. In comparison to CCl₄ injury group, the gene groups associated with liver differentiation （such as HNF4α, ATF5 and Cebpb） in the liver tissue of HHQG group were significantly upregulated. Immunohistochemical analysis showed that HHQG group had obviously elevated HNF4α expression level in liver tissue than CCl₄ injury group on the 2^nd and 5^th days after modeling. [Conclusion] As a mechanism underlying the hepatoprotective effects, HHQG ameliorated liver injury in mice by inhibiting liver oxidative damage, promoting liver tissue repair and maintaining liver function via activating HNF4α .

Key words: Honghua Qinggan 13 Flavor Pill, liver injury, anti-oxidation, hepatoprotective effect, hepatocyte nuclear factor 4α