红花清肝十三味丸通过激活HNF4α改善肝功能的保肝机制研究

doi:10.12160/j.issn.1672-5190.2024.01.002

摘要/Abstract

摘要： [目的]探讨红花清肝十三味丸（HHQG）通过激活肝细胞核因子4α（hepatocyte nuclear factor 4α,HNF4α）改善肝功能的保肝作用机理。[方法]将45只C57BL/6J小鼠随机分为对照组、CCl₄损伤组、HHQG组,每组15只;HHQG组小鼠灌胃剂量为0.616 5 g/（kg·BW）的HHQG,每天1次,持续7 d,对照组和CCl₄损伤组灌胃等体积的无菌蒸馏水;末次灌胃4 h后,对照组小鼠腹腔注射剂量为5 mL/（kg·BW）的橄榄油,CCl₄损伤组和HHQG组小鼠腹腔注射相同剂量的CCl₄与橄榄油混合液（CCl₄∶橄榄油=1∶4,V/V）进行肝损伤造模;分别在造模后第2、5、7天采集各组小鼠血液及肝脏组织样本;记录各组小鼠在试验期间的体重;测定血清谷草转氨酶（AST）、谷丙转氨酶（ALT）及总超氧化物歧化酶（SOD）活力;采用HE染色法及Masson染色法观察肝脏病理组织学变化;应用转录组测序（RNA-seq）技术分析差异基因富集通路;利用免疫组织化学技术检测HNF4α蛋白的表达情况。[结果]与对照组相比,在造模后第1~4天以及第6~7天,CCl₄损伤组小鼠体重显著（P<0.05）或极显著（P<0.01或P<0.001）降低;与CCl₄损伤组相比,在造模后第4天以及第6~7天,HHQG组小鼠体重显著（P<0.05）或极显著（P<0.01）增加。与对照组相比,在造模后第2天,CCl₄损伤组小鼠血清ALT和AST活力极显著（P<0.001）升高;与CCl₄损伤组相比,在造模后第2天,HHQG组小鼠血清ALT和AST活力显著（P<0.05）降低。HE染色及Masson染色观察结果显示,在造模后第2、5、7天,与CCl₄损伤组相比,HHQG组小鼠肝脏组织损伤程度明显改善。KEGG信号通路富集分析表明,与CCl₄损伤组相比,HHQG组小鼠肝脏组织中过氧化物酶体增殖物激活受体、脂肪酸降解、脂肪酸代谢等信号通路上调,核糖体、甘油磷脂代谢、甲状腺癌症等信号通路下调。与CCl₄损伤组相比,HHQG组小鼠肝脏组织中与肝脏分化相关的基因群（HNF4α、ATF5、Cebpb等）显著上调。免疫组织化学分析显示,与CCl₄损伤组相比,在造模后第2、5天,HHQG组小鼠肝脏组织中HNF4α表达量明显增加。[结论]HHQG通过激活HNF4α,抑制肝脏氧化损伤、促进肝脏组织修复以及维持肝脏功能,改善小鼠肝损伤,从而发挥保肝效应。

关键词: 红花清肝十三味丸, 肝损伤, 抗氧化, 保肝作用, 肝细胞核因子4α

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

中图分类号:

R285.5
R-332

肖海军, 卞康坤, 包旭, 包玉龙, 王利. 红花清肝十三味丸通过激活HNF4α改善肝功能的保肝机制研究[J]. 畜牧与饲料科学, 2024, 45(1): 11-19.

XIAO Haijun, BIAN Kangkun, BAO Xu, BAO Yulong, WANG Li. Improving Liver Function by Activating HNF4α Is a Mechanism Underlying the Hepatoprotective Effects of Honghua Qinggan 13 Flavor Pill,a Traditional Mongolian Medicine[J]. Animal Husbandry and Feed Science, 2024, 45(1): 11-19.

参考文献

[1] ZAHIRA A, SULTANA S, RASUL A, et al.Hepatoprotective effects of almond shells against carbon tetrachloride induced liver injury in albino rats[J].Saudi Journal of Biological Sciences,2023,30(11):103811.
[2] MUSANA K A, YALE S H, ABDULKARIM A S.Tests of liver injury[J].Clinical Medicine and Research,2004,2(2):129-131.
[3] BERNAL W, AUZINGER G, DHAWAN A, et al.Acute liver failure[J].The Lancet,2010,376(9736):190-201.
[4] TACKE F, ZIMMERMANN H W.Macrophage heterogeneity in liver injury and fibrosis[J].Journal of Hepatology,2014,60(5):1090-1096.
[5] BAO Y L, WANG L, PAN H T, et al.Animal and organoid models of liver fibrosis[J].Frontiers in Physiology,2021,12:666138.
[6] WU T, XIE Y Q, WU Z K, et al.β-carotene protects mice against lipopolysaccharide and D-galactosamine induced acute liver injury via regulation of NF-κB, MAPK, and Nrf2 signaling[J].Journal of Oleo Science,2023,72(11):1027-1035.
[7] KAMIYA A, KAKINUMA S, YAMAZAKI Y, et al.Enrichment and clonal culture of progenitor cells during mouse postnatal liver development in mice[J].Gastroenterology,2009,137(3):1114-1126.
[8] SCHREM H, KLEMPNAUER J, BORLAK J.Liver-enriched transcription factors in liver function and development. Part Ⅰ:The hepatocyte nuclear factor network and liver-specific gene expression[J].Pharmacological Reviews,2002,54(1):129-158.
[9] LAZAREVICH N L, AL′PERN D V. Hepatocyte nuclear factor 4 (HNF4) in epithelial development and carcinogenesis[J].Molekuliarnaia Biologiia,2008,42(5):786-797.
[10] 阿拉坦图雅,巴日格其.蒙药古日古木-13考[J].中国民族医药杂志,2010,16(5):36-37.
[11] 夏瑶宾,朱明,赵秀清.《中华人民共和国卫生部药品标准·蒙药分册》质量标准研究概况[J].中国民族医药杂志,2006,12(1):44-45.
[12] 扎拉嘎. 蒙药治疗100例慢性乙肝的体会[J].中国民族医药杂志,2012,18(10):21.
[13] 鲍云,张更丰,姜昊.蒙药治疗乙型肝炎18例[J].中国民族医药杂志,2006,12(2):16.
[14] 包春梅. 蒙药治疗中毒性肝炎12例体会[J].中国民族民间医药,2014,23(7):9.
[15] 娜仁花. 蒙医蒙药十三味红花散治疗酒精性肝炎40例[J].中国中医药现代远程教育,2013,11(8):30-31.
[16] 乌力吉图,阿拉坦.古日古木-13味丸治疗酒精性肝病51例观察[J].中国民族医药杂志,2006,12(5):14-15.
[17] 乌吉木. 蒙药古日古木-13味丸治疗非酒精性脂肪肝疗效观察[J].中国民族医药杂志,2019,25(3):4-6.
[18] 额日登其木格.红花清肝-十三味丸治疗蒙医热盛型肝病42例临床疗效观察[J].中国民族民间医药,2013,22(20):5.
[19] DENG J S, CHANG Y C, WEN C L, et al.Hepatoprotective effect of the ethanol extract of Vitis thunbergii on carbon tetrachloride-induced acute hepatotoxicity in rats through anti-oxidative activities[J].Journal of Ethnopharmacology,2012,142(3):795-803.
[20] WEBER L W D, BOLL M, STAMPFL A. Hepatotoxicity and mechanism of action of haloalkanes: Carbon tetrachloride as a toxicological model[J].Critical Reviews in Toxicology,2003,33(2):105-136.
[21] 锡林其木格,吴玉小.古日古木-13味丸在蒙医临床中的应用[J].中国民族医药杂志,2020,26(7):72-73.
[22] 卢任玲,马丽杰.红花清肝十三味丸对急性肝损伤大鼠的影响[J].中成药,2022,44(2):564-568.
[23] 杨宏昕,白音夫,常亮,等.红花清肝十三味对CCl₄诱导大鼠肝损伤纤维化的作用[J].西北药学杂志,2018,33(3):349-352.
[24] 叶超凡,卢任玲,谢军,等.古日古木-13对CCl₄致慢性肝损伤小鼠保护作用的研究[J].时珍国医国药,2016,27(6):1364-1366.
[25] ZARET K S.Regulatory phases of early liver development:Paradigms of organogenesis[J].Nature Reviews Genetics,2002,3(7):499-512.
[26] HWANG-VERSLUES W W, SLADEK F M. HNF4α:Role in drug metabolism and potential drug target?[J].Current Opinion in Pharmacology,2010,10(6):698-705.
[27] LI J, NING G, DUNCAN S A.Mammalian hepatocyte differentiation requires the transcription factor HNF-4alpha[J].Genes and Development,2000,14(4):464-474.
[28] WU H, REIZEL T, WANG Y J, et al.A negative reciprocal regulatory axis between cyclin D1 and HNF4α modulates cell cycle progression and metabolism in the liver[J].Proceedings of the National Academy of Sciences of the United States of America,2020,117(29):17177-17186.
[29] INOUE Y, HAYHURST G P, INOUE J, et al.Defective ureagenesis in mice carrying a liver-specific disruption of hepatocyte nuclear factor 4alpha (HNF4alpha). HNF4alpha regulates ornithine transcarbamylase in vivo[J].The Journal of Biological Chemistry,2002,277(28):25257-25265.