红花清肝十三味丸通过抑制炎性细胞浸润改善小鼠肝损伤的研究

doi:10.12160/j.issn.1672-5190.2024.01.001

摘要/Abstract

摘要： [目的]研究蒙药红花清肝十三味丸（HHQG）通过抑制炎性细胞浸润改善小鼠肝损伤的作用机制。[方法]选取6~8周龄的雄性C57BL/6小鼠54只,随机分为对照组、模型组和HHQG保肝组,每组18只。HHQG保肝组连续7 d每天灌胃1次HHQG,剂量为0.616 5 g/（kg·BW）,对照组和模型组每天灌胃1次等体积的生理盐水;第7天灌胃4 h后,对照组腹腔注射剂量为0.1 mL/（10 g·BW）的橄榄油,模型组和HHQG保肝组均腹腔注射等剂量的CCl₄与橄榄油混合物（CCl₄∶橄榄油=1∶4,V/V）。肝损伤造模后第2、5、7天,通过检测血清谷草转氨酶（AST）和谷丙转氨酶（ALT）活力、观察肝脏组织病理学变化（HE染色及Masson染色）,评价肝损伤模型制备是否成功以及HHQG对肝损伤的缓解作用;通过免疫组织化学技术检测肝脏组织中性粒细胞和单核/巨噬细胞浸润情况;应用免疫组织化学技术和反转录实时荧光定量PCR（RT-qPCR）技术分析肝脏组织中炎性因子TNF-α的表达情况;运用转录组测序（RNA-seq）及RT-qPCR技术分析HHQG对肝脏组织中黏附分子基因表达的影响。[结果]综合多项评价指标,模型组小鼠出现明显的肝损伤症状,提示CCl₄诱导的肝损伤模型制备成功。与模型组相比,肝损伤后第2天,HHQG保肝组小鼠血清中AST和ALT活力极显著（P<0.01）降低。HHQG可减少肝损伤区域中性粒细胞和单核/巨噬细胞等炎性细胞浸润,缓解胶原纤维物质沉积,通过促进肝细胞增殖进行组织修复。HHQG保肝组小鼠肝损伤区域TNF-α表达量减少;与模型组相比,肝损伤后第2、7天以及第5天,HHQG保肝组的肝脏组织中TNF-α基因的mRNA相对表达量分别极显著（P<0.01）和显著（P<0.05）降低。转录组数据分析表明,HHQG可显著下调肝损伤后与中性粒细胞和单核/巨噬细胞浸润相关的黏附分子基因群;RT-qPCR验证试验结果显示,与模型组相比,肝损伤后第5、7 天,HHQG保肝组的肝脏组织中VCAM-1基因的mRNA相对表达量极显著（P<0.01）降低;肝损伤后第2天以及第5、7天,HHQG保肝组的肝脏组织中ICAM-1基因的mRNA相对表达量分别显著（P<0.05）和极显著（P<0.01）降低。[结论]HHQG通过减少以中性粒细胞和单核/巨噬细胞为主的炎性细胞浸润,下调肝损伤区炎性因子TNF-α的表达,以及抑制黏附分子的表达,减轻肝损伤后发生的炎症,从而发挥抗炎保肝作用。

关键词: 红花清肝十三味丸, 肝损伤, 黏附分子, 中性粒细胞, 单核/巨噬细胞

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

中图分类号:

R285.5
R-332

包旭, 卞康坤, 肖海军, 王利, 包玉龙. 红花清肝十三味丸通过抑制炎性细胞浸润改善小鼠肝损伤的研究[J]. 畜牧与饲料科学, 2024, 45(1): 1-10.

BAO Xu, BIAN Kangkun, XIAO Haijun, WANG Li, BAO Yulong. Honghua Qinggan 13 Flavor Pill Alleviates Liver Injury in Mice through Inhibiting Inflammatory Cell Infiltration[J]. Animal Husbandry and Feed Science, 2024, 45(1): 1-10.

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