一贯煎对M1型骨髓巨噬细胞治疗肝硬化大鼠模型效果的影响及其机制分析

宗梦瑶; 简迅; 王丹阳; 许燕楠; 郑欣瑞; 邢飞飞; 陈高峰; 陈佳美; 刘平; 慕永平

doi:10.12449/JCH240817

一贯煎对M1型骨髓巨噬细胞治疗肝硬化大鼠模型效果的影响及其机制分析

DOI: 10.12449/JCH240817

上海中医药大学附属曙光医院，上海市中医药研究院肝病研究所，肝肾疾病病证教育部重点实验室，上海市中医临床重点实验室，上海 201203

基金项目:

国家自然科学基金面上项目 (81874390);

上海市科委自然科学基金面上项目 (21ZR1464100);

上海市科委2022年度“科技创新行动计划”生物医药科技支撑专项 (22S11901700);

上海市临床重点专科建设项目 (shslczdzk01201)

伦理学声明：本研究于2019年11月22日经上海中医药大学动物伦理委员会审批同意，批号：PZSHUTCM191122004，符合实验室动物管理与使用准则。

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：慕永平、刘平等负责课题设计；宗梦瑶、简迅负责实验实施，资料分析以及撰写论文；王丹阳、许燕楠、郑欣瑞、邢飞飞参与收集数据，修改论文；陈佳美、陈高峰负责医学统计；慕永平负责拟定写作思路，指导撰写文章并最后定稿。宗梦瑶和简迅对本文贡献等同，为共同第一作者。

详细信息

通信作者:
慕永平， ypmu8888@126.com （ORCID： 0000-0002-6808-8243）

计量
- 文章访问数: 112
- HTML全文浏览量: 197
- PDF下载量: 13
- 被引次数: 0
出版历程
- 收稿日期: 2023-11-24
- 录用日期: 2024-01-29
- 出版日期: 2024-08-25

Effect of Yiguan Decoction on the efficacy of M1 bone marrow-derived macrophages in treatment of liver cirrhosis rats and its mechanism

Key Laboratory of Liver and Kidney Diseases，Ministry of Education，Institute of Liver Diseases，Shuguang Hospital，Shanghai University of Traditional Chinese Medicine，Shanghai Key Laboratory of Traditional Chinese Clinical Medicine，Shanghai 201203，China

Research funding:

General Project of National Natural Science Foundation of China (81874390);

Shanghai Natural Science Foundation (21ZR1464100);

Special Project for Biomedical Science and Technology Support of the “Science and Technology Innovation Action Plan” of Shanghai Science and Technology Commission in 2022 (22S11901700);

Shanghai Key Specialty of Traditional Chinese Clinical Medicine (shslczdzk01201)

More Information

Corresponding author: MU Yongping， ypmu8888@126.com （ORCID： 0000-0002-6808-8243）

摘要

摘要: 目的观察一贯煎对M1型骨髓巨噬细胞（M1-BMDM）治疗2-AAF/CCl₄诱导的大鼠肝硬化作用的影响与机制。方法分离BMDM，脂多糖诱导分化为M1-BMDM。雄性Wistar大鼠随机分为正常组（n=5）和造模组（n=45）。造模组大鼠予以50% CCl₄每周2次皮下注射。第7周开始，将造模大鼠随机分为模型组（M组）、一贯煎组（YGJD组）、M1-BMDM组、M1-BMDM+YGJD组、索拉非尼组（SORA组），改用30% CCl₄皮下注射以维持肝硬化进展，同时以2-AAF灌胃，且饮水中加入CCR2抑制剂，分组干预，9周末取材。观察血清肝功能、肝组织病理、肝组织羟脯氨酸（Hyp）含量、肝星状细胞活化、肝纤维化及炎症相关因子、Wnt信号通路相关分子表达水平等。计量资料多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。结果与M组比较，M1-BMDM+YGJD组大鼠血清ALT、AST、TBil水平均明显降低（P值均<0.05），Alb含量显著增加（P<0.05）；与M1-BMDM组比较，M1-BMDM+YGJD组血清TBil含量显著降低（P<0.05）、Alb含量显著升高（P<0.05）。与M1-BMDM组比较，M1-BMDM+YGJD组CD68、TNF-α表达显著降低（P<0.05）。与M1-BMDM组比较，M1-BMDM+YGJD组Hyp含量及天狼星红胶原阳性染色面积均显著降低（P值均<0.05）。M1-BMDM+YGJD组非经典Wnt信号通路分子Wnt5a mRNA和蛋白表达均显著低于M1-BMDM组（P值均<0.05），Fzd2 mRNA表达水平显著低于M1-BMDM组（P<0.05），且Wnt4、Wnt5b和Fzd3 mRNA表达水平亦显著降低（P值均<0.05），而经典Wnt信号通路分子β-catenin、LRP5、LRP6、Fzd5和TCF mRNA表达水平均无明显改变。结论一贯煎可提高M1-BMDM对2-AAF/CCl₄诱导的大鼠肝硬化的治疗效应，其机制可能与抑制非经典Wnt5a/Fzd2信号通路有关，为中医药协同M1-BMDM治疗肝硬化提供了新思路。
- 肝硬化 /
- 巨噬细胞 /
- 一贯煎 /
- Wnt信号通路 /
- 大鼠， Wistar
Abstract: Objective To investigate the effect and mechanism of Yiguan Decoction （YGJD） on the efficacy of M1 bone marrow-derived macrophages （M1-BMDMs） in the treatment of rats with liver cirrhosis induced by 2-AAF/CCl₄. Methods BMDMs were isolated and induced into M1-BMDMs by lipopolysaccharide. A total of 50 male Wistar rats were randomly divided into normal group with 5 rats and model group with 45 rats. The rats for modeling were given subcutaneous injection of 50% CCl₄ twice a week. Since week 7， the rats for modeling were randomly divided into model group （M group）， YGJD group， M1-BMDM group， M1-BMDM+YGJD group， and sorafenib （SORA） group， and they were given subcutaneous injection of 30% CCl₄ to maintain the progression of liver cirrhosis and intragastric administration of 2-AAF. CCR2 inhibitors were added to the drinking water， and each group was given the corresponding intervention. Related samples were collected at week 9. The rats were observed in terms of serum liver function parameters， liver pathology， hydroxyproline （Hyp） content in liver tissue， hepatic stellate cell activation， hepatic fibrosis and inflammation factors， and the expression levels of molecules associated with the Wnt signaling pathway. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. Results Compared with the M group， the M1-BMDM+YGJD group had significant reductions in the serum levels of alanine aminotransferase， aspartate aminotransferase， and total bilirubin （TBil）（all P<0.05） and a significant increase in the content of albumin （Alb）（P<0.05）， and compared with the M1-BMDM group， the M1-BMDM+YGJD group had a significant reduction in the serum level of TBil （P<0.05） and a significant increase in the serum level of Alb （P<0.05）. Compared with the M1-BMDM group， the M1-BMDM+YGJD group had significant reductions in the expression levels of CD68 and TNF-α （P<0.05）. Compared with the M1-BMDM group， the M1-BMDM+YGJD group had significant reductions in Hyp content and Sirius red positive area （P<0.05）. As for the non-canonical Wnt signaling pathway molecules， compared with the M1-BMDM group， the M1-BMDM+YGJD group had significantly lower mRNA and protein expression levels of Wnt5a （P<0.05） and mRNA expression level of Fzd2 （P<0.05）， as well as significant reductions in the mRNA expression levels of Wnt4， Wnt5b， and Fzd3 （P<0.05）， while there were no significant changes in the mRNA expression levels of the canonical Wnt signaling pathway molecules β-catenin， LRP5， LRP6， Fzd5， and TCF. Conclusion YGJD can enhance the therapeutic effect of M1-BMDMs on rats with liver cirrhosis induced by 2-AAF/CCl₄， possibly by inhibiting the non-canonical Wnt5a/Fzd2 signaling pathway， which provides new ideas for the synergistic effect of traditional Chinese medicine on M1-BMDMs in the treatment of liver cirrhosis.
- Liver Cirrhosis /
- Macrophages /
- Yi Guan Jian /
- Wnt Signaling Pathway /
- Rats， Wistar

HTML全文

图 1 一贯煎对M1-BMDM改善肝脏炎症反应的作用

注： a，HE染色；b、c，CD68和CD206免疫组化染色；d，血清生化学指标；e，CD68、CD206、TNF-α免疫印迹及灰度积分比值；f，CD68、CD206、TNF-α、IL-6、IL-10 mRNA表达水平。*P<0.05；**P<0.01。

Figure 1. YGJD can ameliorate the effect of M1-BMDM on improving liver inflammatory

下载: 全尺寸图片幻灯片

图 2 一贯煎对M1-BMDM治疗肝硬化的作用

注： a，天狼星红胶原染色；b，α-SMA免疫组织化学染色；c，肝组织Hyp含量；d，天狼星红胶原染色半定量分析；e，α-SMA免疫印迹和灰度积分比值及mRNA水平；f，TGF-β1 mRNA水平。*P<0.05；**P<0.01。

Figure 2. YGJD can enhance the therapeutic effect of M1-BMDM on liver cirrhosis

下载: 全尺寸图片幻灯片

图 3 一贯煎与M1-BMDM联用对肝脏非经典Wnt信号通路的影响

注： a，Wnt5a免疫组化染色；b，Fzd2免疫组化染色；c，Wnt5a、Fzd2免疫印迹及灰度积分比值；d，Wnt5a、Fzd2、Wnt4、Wnt5b、Fzd3、Fzd6、ATF、c-Jun mRNA表达水平。*P<0.05；**P<0.01。

Figure 3. The effect of combination of YGJD and M1-BMDM on non classical Wnt signaling pathway in the liver

下载: 全尺寸图片幻灯片

图 4 一贯煎与M1-BMDM联用对肝脏经典Wnt信号通路的影响

注： a，β-catenin免疫组化染色；b，β-catenin、LRP5、LRP6、Fzd5、LEF、TCF mRNA表达水平。*P<0.05；**P<0.01。

Figure 4. The effect of combination of YGJD and M1-BMDM on the classic Wnt signaling pathway in the liver

下载: 全尺寸图片幻灯片

参考文献(27)

[1]	Chinese Society of Hepatology, Chinese Medical Association. Chinese guidelines on the management of liver cirrhosis[J]. J Clin Hepatol, 2019, 35( 11): 2408- 2425. DOI: 10.3969/j.issn.1001-5256.2019.11.006. 中华医学会肝病学分会. 肝硬化诊治指南[J]. 临床肝胆病杂志, 2019, 35( 11): 2408- 2425. DOI: 10.3969/j. issn.1001-5256.2019.11.006.
[2]	FREEMAN RB Jr, STEFFICK DE, GUIDINGER MK, et al. Liver and intestine transplantation in the United States, 1997-2006[J]. Am J Transplant, 2008, 8( 4 Pt 2): 958- 976. DOI: 10.1111/j.1600-6143.2008.02174.x.
[3]	ZHANG YT, LI YW, ZHANG LL, et al. Mesenchymal stem cells: Potential application for the treatment of hepatic cirrhosis[J]. Stem Cell Res Ther, 2018, 9( 1): 59. DOI: 10.1186/s13287-018-0814-4.
[4]	SICA A, INVERNIZZI P, MANTOVANI A. Macrophage plasticity and polarization in liver homeostasis and pathology[J]. Hepatology, 2014, 59( 5): 2034- 2042. DOI: 10.1002/hep.26754.
[5]	LIU ZR, WAN XY, WANG ZL, et al. Electroactive biomaterials and systems for cell fate determination and tissue regeneration: Design and applications[J]. Adv Mater, 2021, 33( 32): e2007429. DOI: 10.1002/adma.202007429.
[6]	HEISSIG B, DHAHRI D, EIAMBOONSERT S, et al. Role of mesenchymal stem cell-derived fibrinolytic factor in tissue regeneration and cancer progression[J]. Cell Mol Life Sci, 2015, 72( 24): 4759- 4770. DOI: 10.1007/s00018-015-2035-7.
[7]	LANZONI G, OIKAWA T, WANG Y, et al. Concise review: clinical programs of stem cell therapies for liver and pancreas[J]. Stem Cells, 2013, 31( 10): 2047- 2060. DOI: 10.1002/stem.1457.
[8]	TACKE F. Targeting hepatic macrophages to treat liver diseases[J]. J Hepatol, 2017, 66( 6): 1300- 1312. DOI: 10.1016/j.jhep.2017.02.026.
[9]	JIAN X, WANG DY, XU YN, et al. Effect of polarized bone marrow-derived macrophage transplantation on the progression of CCl₄-induced liver fibrosis in rats[J]. J Clin Hepatol, 2021, 37( 12): 2830- 2837. DOI: 10.3969/j.issn.1001-5256.2021.12.020. 简迅, 王丹阳, 许燕楠, 等. 极化骨髓巨噬细胞移植对CCl₄诱导的肝纤维化大鼠模型的影响[J]. 临床肝胆病杂志, 2021, 37( 12): 2830- 2837. DOI: 10.3969/j.issn.1001-5256.2021.12.020.
[10]	XU Y, XU W, LIU W, et al. Yiguanjian decoction inhibits macrophage M1 polarization and attenuates hepatic fibrosis induced by CCl₄/2-AAF[J]. Pharm Biol, 2021, 59( 1): 1150- 1160. DOI: 10.1080/13880209.2021.1961820.
[11]	DAVIS BK. Derivation of macrophages from mouse bone marrow[J]. Methods Mol Biol, 2019, 1960: 41- 55. DOI: 10.1007/978-1-4939-9167-9_3.
[12]	WATANABE Y, TSUCHIYA A, SEINO S, et al. Mesenchymal stem cells and induced bone marrow-derived macrophages synergistically improve liver fibrosis in mice[J]. Stem Cells Transl Med, 2019, 8( 3): 271- 284. DOI: 10.1002/sctm.18-0105.
[13]	MILY A, KALSUM S, LORETI MG, et al. Polarization of M1 and M2 human monocyte-derived cells and analysis with flow cytometry upon mycobacterium tuberculosis infection[J]. J Vis Exp, 2020. DOI: 10.3791/61807.
[14]	GAO Z, JIANG JN, HOU LJ, et al. Dysregulation of miR-144-5p/RNF187 axis contributes to the progression of colorectal cancer[J]. J Transl Int Med, 2022, 10( 1): 65- 75. DOI: 10.2478/jtim-2021-0043.
[15]	CHEN L, MU YP, ZHANG H, et al. Research progress on mechanism of Yiguanjian intervention in liver cirrhosis[J]. Chin J Exp Med Formul, 2020, 26( 24): 186- 192. DOI: 10.13422/j.cnki.syfjx.20202437. 陈龙, 慕永平, 张华, 等. 一贯煎干预肝硬化作用机制研究进展[J]. 中国实验方剂学杂志, 2020, 26( 24): 186- 192. DOI: 10.13422/j.cnki.syfjx.20202437.
[16]	XU LW, DENG XJ, ZHU LD, et al. Research progress of differentiating and treating lung cancer from the liver with Yiguanjian[J]. Chin J Exp Med Formul, 2022, 38( 1): 114- 117. DOI: 10.13463/j.cnki.cczyy.2022.01.027. 徐丽伟, 邓小静, 朱立德, 等. 一贯煎从肝论治肺癌的研究进展[J]. 长春中医药大学学报, 2022, 38( 1): 114- 117. DOI: 10.13463/j.cnki.cczyy.2022.01.027.
[17]	ESHGHJOO S, KIM DM, JAYARAMAN A, et al. Macrophage polarization in atherosclerosis[J]. Genes(Basel), 2022, 13( 5): 756. DOI: 10.3390/genes13050756.
[18]	MA PF, GAO CC, YI J, et al. Cytotherapy with M1-polarized macrophages ameliorates liver fibrosis by modulating immune microenvironment in mice[J]. J Hepatol, 2017, 67( 4): 770- 779. DOI: 10.1016/j.jhep.2017.05.022.
[19]	TSUCHIDA T, FRIEDMAN SL. Mechanisms of hepatic stellate cell activation[J]. Nat Rev Gastroenterol Hepatol, 2017, 14( 7): 397- 411. DOI: 10.1038/nrgastro.2017.38.
[20]	SCHWABE RF, TABAS I, PAJVANI UB. Mechanisms of fibrosis development in nonalcoholic steatohepatitis[J]. Gastroenterology, 2020, 158( 7): 1913- 1928. DOI: 10.1053/j.gastro.2019.11.311.
[21]	LIASKOU E, ZIMMERMANN HW, LI KK, et al. Monocyte subsets in human liver disease show distinct phenotypic and functional characteristics[J]. Hepatology, 2013, 57( 1): 385- 398. DOI: 10.1002/hep.26016.
[22]	KISSELEVA T, BRENNER D. Molecular and cellular mechanisms of liver fibrosis and its regression[J]. Nat Rev Gastroenterol Hepatol, 2021, 18( 3): 151- 166. DOI: 10.1038/s41575-020-00372-7.
[23]	BAARSMA HA, KÖNIGSHOFF M.‘WNT-er is coming’: WNT signalling in chronic lung diseases[J]. Thorax, 2017, 72( 8): 746- 759. DOI: 10.1136/thoraxjnl-2016-209753.
[24]	ZENG G, AWAN F, OTRUBA W, et al. Wnt’er in liver: Expression of Wnt and frizzled genes in mouse[J]. Hepatology, 2007, 45( 1): 195- 204. DOI: 10.1002/hep.21473.
[25]	SAHA S, ARANDA E, HAYAKAWA Y, et al. Macrophage-derived extracellular vesicle-packaged WNTs rescue intestinal stem cells and enhance survival after radiation injury[J]. Nat Commun, 2016, 7: 13096. DOI: 10.1038/ncomms13096.
[26]	LIU WT, JING YY, GAO L, et al. Lipopolysaccharide induces the differentiation of hepatic progenitor cells into myofibroblasts constitutes the hepatocarcinogenesis-associated microenvironment[J]. Cell Death Differ, 2020, 27( 1): 85- 101. DOI: 10.1038/s41418-019-0340-7.
[27]	ZHANG Y, ZHU Y. Efficacy studies of Yiguanjian treatment in mice fibrosis induced by dimethylnitrosamine[J]. J Dalian Med Univ, 2014, 36( 6): 527- 535, 596. DOI: 10.11724/jdmu.2014.06.04. 张媛, 朱英. 一贯煎治疗二甲基亚硝胺致小鼠肝纤维化的机制研究[J]. 大连医科大学学报, 2014, 36( 6): 527- 535, 596. DOI: 10.11724/jdmu.2014.06.04.