Abstract: [Objective] The aim of this study was to assess the mechanism by which Honghua Qinggan 13 Flavor Pill （HHQG）, a traditional Mongolian medicine, alleviated liver injury in mice through inhibiting inflammatory cell infiltration. [Method] A total of 54 male C57BL/6 mice aged 6 to 8 weeks were selected and randomly divided into control group （n=18）, model group （n=18） and HHQG liver protection group （n=18）. The HHQG liver protection group was intragastrically administered 0.616 5 g/（kg·BW） of HHQG once a day for 7 consecutive days, while the control group and model group were intragastrically administered the same volume of physiological saline once a day. On the 7^th day, 4 hours after the administration, the control group received 0.1 mL/（10 g·BW） of olive oil by intraperitoneal injection, while the model group and HHQG liver protection group received an equal dose of a CCl₄ and olive oil mixture （CCl₄∶olive oil=1∶4, V/V） in the same manner. On the 2^nd, 5^th and 7^th days after modeling, the prepared liver injury model and the alleviating effect of HHQG on liver injury were evaluated by detecting serum aspartate aminotransferase （AST） and alanine aminotransferase （ALT） activities as well as observing liver tissue pathological changes （HE staining and Masson staining）. The neutrophil and monocyte/macrophage infiltration in liver tissue was observed using immunohistochemical assay. The expression of the inflammatory factor TNF-α in liver tissue at both protein and gene levels were assessed using immunohistochemical assay and reverse transcription real-time fluorescence quantitative PCR （RT-qPCR）. The effects of HHQG on the expression levels of adhesion molecule genes in liver tissue were analyzed by transcriptome sequencing （RNA-seq） and RT-qPCR. [Result] According to the multiple evaluation indicators, the model group mice exhibited obvious symptoms of liver injury, indicating the successful preparation of CCl₄ induced liver injury model. Compared with the model group, on the 2^nd day after modeling, the serum AST and ALT activities in the HHQG liver protection group were extremely significantly （P<0.01） reduced. HHQG intervention inhibited the inflammatory cell infiltration such as neutrophil and monocyte/macrophage in liver injury areas, alleviated collagen fiber deposition and promoted liver tissue repair by cell proliferation. HHQG liver protection group had reduced expression level of TNF-α in the liver injury areas. Compared with the model group, on the 2^nd and 7^th, as well as the 5^th days after modeling, the relative mRNA expression levels of TNF-α gene in the liver tissue of HHQG liver protection group were extremely significantly （P<0.01） and significantly （P<0.05） reduced, respectively. RNA-seq analysis showed that HHQG intervention significantly downregulated the gene groups of adhesion molecules associated with neutrophil and monocyte/macrophage infiltration in liver injury mice. RT-qPCR validation results demonstrated that HHQG liver protection group had extremely significantly （P<0.01） reduced relative mRNA expression levels of VCAM-1 gene in the liver tissue than the model group on the 5^th and 7^th days after modeling. In addition, in comparison to the model group, significantly （P<0.05） and extremely significantly （P<0.01） decreased relative mRNA expression levels of ICAM-1 gene in the liver tissue of the HHQG liver protection group were observed on the 2^nd as well as the 5^th and 7^th days after modeling, respectively. [Conclusion] Through inhibiting the inflammatory cell infiltration dominated by neutrophil and monocyte/macrophage, HHQG downregulated the expressions of the inflammatory factor TNF-α and adhesion molecules in the liver injury areas, and alleviated inflammation after liver injury, thereby exerting anti-inflammatory and hepatoprotective effects in mice.

Key words: Honghua Qinggan 13 Flavor Pill, liver injury, adhesion molecule, neutrophil, monocyte/macrophage