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扶正化瘀方通过改变急性肝损伤小鼠模型肝脏CD8+T淋巴细胞表型功能预防肝纤维化的价值分析
DOI: 10.3969/j.issn.1001-5256.2022.02.017
利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。
作者贡献声明：徐列明、周扬、平键负责课题设计，拟定写作思路；黄辉参与收集数据，资料分析，撰写论文；徐列明、周扬负责修改论文，指导撰写文章并最后定稿。
Value of Fuzheng Huayu prescription in preventing liver fibrosis by altering the phenotypic function of CD8+ T lymphocytes in the liver of mice with acute liver injury
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摘要:
目的 观察应用扶正化瘀方后急性肝损伤小鼠模型肝脏CD8+ T淋巴细胞对共培养的肝星状细胞的影响，探讨扶正化瘀方预防肝纤维化的作用机制。 方法 18只SPF级雄性C57BL/6NCrl Vr小鼠随机分为正常组、模型组、扶正化瘀方组，每组6只。扶正化瘀方组提前给予扶正化瘀方灌胃5 d。实验前12 h以2 mL/kg体质量的剂量腹腔注射10%的CCl4，腹主静脉取血，留取血清并检测ALT、AST，留取部分肝脏做病理观察；应用流式细胞术分选小鼠肝脏CD8+ T淋巴细胞，预先小鼠体内用药，再与小鼠肝星状细胞株(JS 1)以2∶ 1比例共培养于96孔板中24 h、48 h，使用qPCR方法检测各组Col.Ⅰ mRNA和α-SMA mRNA表达变化。符合正态分布的计量资料多组间比较采用方差分析，进一步两两比较采用SNK-q或LSD-t检验。 结果 模型组的ALT和AST水平均显著高于正常组(P值均＜0.000 1)。扶正化瘀方组小鼠的ALT升高的幅度明显小于模型组(P＜0.001)。HE染色显示，扶正化瘀方组小鼠肝细胞变性、坏死程度较模型组有所减轻。与正常组比较, 模型组总淋巴细胞、CD45、CD4-CD8+T、CD8+CD28-T显著上升, 而扶正化瘀方组与模型组相比较, 上述淋巴细胞比例显著降低(P值均＜0.001)。自各组小鼠肝脏分离的CD8+ T淋巴细胞与JS 1共培养48 h，模型组与对照组(JS 1单培养)和正常组相比α-SMA mRNA的表达明显增加(P值均＜0.01)；Col.Ⅰ mRNA表达也明显高于对照组和正常组(正常小鼠肝脏CD8+ T淋巴细胞与JS 1共培养)(P值均＜0.001)。扶正化瘀方组α-SMA mRNA和Col.Ⅰ mRNA表达显著低于模型组(P值均＜0.01)。 结论 扶正化瘀方能通过改变小鼠肝脏CD8+ T淋巴细胞功能表型间接抑制活化的肝星状细胞。 -
关键词:
- 肝硬化 /
- 扶正化瘀方 /
- 肝星状细胞 /
- CD8阳性T淋巴细胞
Abstract:Objective To investigate the effect of liver CD8+ T lymphocytes on co-cultured hepatic stellate cells (HSCs) after the application of Fuzheng Huayu prescription in a moues model of acute liver injury, as well as the mechanism of action of Fuzheng Huayu prescription in preventing liver fibrosis. Methods A total of 18 specific pathogen-free male C57BL/6NCrl Vr mice were randomly divided into normal group, model group, and Fuzheng Huayu prescription group, with 6 mice in each group. The mice in the Fuzheng Huayu prescription group were given Fuzheng Huayu prescription for 5 days in advance. At 12 hours before the experiment, 10% CCl4 was injected intraperitoneally at a dose of 2 mL/kg body weight. Blood was collected from the main abdominal vein, and the serum was separated to measure the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Part of the liver was used for pathological observation. After the mice were pretreated with medication in vivo, flow cytometry was used for the sorting of mouse liver CD8+ T lymphocytes, which were then co-cultured with the mouse HSC cell line (JS 1) in a 96-well plate at a ratio of 2∶ 1, and after co-culture for 24 and 48 hours, qPCR was used to measure the changes in the mRNA expression of Col.I and α-SMA. An analysis of variance was used for comparison of normally distributed continuous data between multiple groups, and the SNK-q test or the least significant difference t-test was used for further comparison between two groups. Results The model group had significantly higher activities of ALT and AST than the normal group (both P < 0.000 1), and compared with the model group, the Fuzheng Huayu prescription group had a significantly lower degree of increase in ALT activity (P < 0.001). HE staining showed that the Fuzheng Huayu prescription group had a significantly lower degree of hepatocyte degeneration and necrosis compared with the model group. Compared with the normal group, the total lymphocytes, CD45, CD4-CD8+ T and CD8 + CD28-T in the model group increased significantly, while the proportion of the above lymphocytes in the Fuzheng Huayu formula group decreased significantly compared with the model group (P < 0.001). CD8+ T lymphocytes isolated from the liver of mice in each group were co-cultured with JS 1 for 48 hours, and compared with the control group (JS 1 cultured alone) and the normal group, the model group had a significant increase in the mRNA expression of α-SMA (both P < 0.01) and significantly higher mRNA expression of Col.I than the control group and the normal group (normal mouse liver CD8+ T lymphocytes co-cultured with JS 1) (both P < 0.001). The Fuzheng Huayu prescription group had significantly lower mRNA expression levels of α-SMA and Col.I than the model group (both P < 0.01). Conclusion Fuzheng Huayu prescription can indirectly inhibit activated HSCs by altering the functional phenotype of CD8+ T lymphocytes in mouse liver. -
表 1 qPCR引物列表
Cat.No. TM值 名称 上游引物 下游引物 Tnt0338 85.1 GAPDH 5′AAA TGG TGA AGG TCG GTG TG 3′ 5′AGG TCA ATG AAG GGG TCG TT 3′ Tnt1328 81.1 α-SMA 5′CAC ACA CCT TTT AGA ACC CA 3′ 5′GTT TTC CCC CTT CCT CTT ACT 3′ Tnt1680a 85.9 Col.Ⅰ 5′TGA CTG GAA GAG CGG AGA GTA 3′ 5′GAC GGC TGA GTA GGG AAC AC 3′ 
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表 2 各组肝脏相关细胞占总淋巴细胞比例
分组 动物数(只) 总淋巴细胞(个/mL) CD45(%) CD4-CD8+T(%) CD8+CD28-T(%) 正常组 6 8.62×106±0.425×106 40.20±1.31 3.96±0.152 3.73±0.251 模型组 6 2.53×107±0.157×107 58.80±1.90 1.37±0.152 1.27±0.153 扶正化瘀方组 6 1.69×107±0.447×107 29.60±1.43 1.63±0.115 1.67±0.152 F值 316.846 265.604 260.143 143.394 P值 ＜0.001 ＜0.001 ＜0.001 ＜0.001
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[1] DING BS, CAO Z, LIS R, et al. Divergent angiocrine signals from vascular niche balance liver regeneration and fibrosis[J]. Nature, 2014, 505(7481): 97-102. DOI: 10.1038/nature12681. [2] CAMPANA L, IREDALE JP. Regression of liver fibrosis[J]. Semin Liver Dis, 2017, 37(1): 1-10. DOI: 10.1055/s-0036-1597816. [3] IRSHAD M, GUPTA P, IRSHAD K. Immunopathogenesis of liver injury during hepatitis C virus infection[J]. Viral Immunol, 2019, 32(3): 112-120. DOI: 10.1089/vim.2018.0124. [4] XING YM. Current status and prevention of chronic hepatitis B infection[J]. J World Latest Med Inf, 2017, 69: 174-175. DOI: 10.19613/j.cnki.1671-3141.2017.69.130.邢亚明. 慢性乙型肝炎感染现状及预防[J]. 世界最新医学信息文摘, 2017, 69: 174-175. DOI: 10.19613/j.cnki.1671-3141.2017.69.130. [5] LIU CH, LIU P, LIU C, et al. Discussion on serum pharmacological methods of effective Chinese herbal compound prescriptions for anti-liver fibrosis[J]. Chin J Exp Med Formul, 1998, 2: 16-19. https://www.cnki.com.cn/Article/CJFDTOTAL-ZSFX802.006.htm刘成海, 刘平, 刘成, 等. 抗肝纤维化有效中药复方血清药理学方法探讨[J]. 中国实验方剂学杂志, 1998, 2: 16-19. https://www.cnki.com.cn/Article/CJFDTOTAL-ZSFX802.006.htm [6] KOYAMA Y, BRENNER DA. Liver inflammation and fibrosis[J]. J Clin Invest, 2017, 127(1): 55-64. DOI: 10.1172/JCI88881. [7] NOTAS G, KISSELEVA T, BRENNER D. NK and NKT cells in liver injury and fibrosis[J]. Clin Immunol, 2009, 130(1): 16-26. DOI: 10.1016/j.clim.2008.08.008. [8] SCHUSTER S, CABRERA D, ARRESE M, et al. Triggering and resolution of inflammation in NASH[J]. Nat Rev Gastroenterol Hepatol, 2018, 15(6): 349-364. DOI: 10.1038/s41575-018-0009-6. [9] ARRESE M, CABRERA D, KALERGIS AM, et al. Innate immunity and inflammation in NAFLD/NASH[J]. Dig Dis Sci, 2016, 61(5): 1294-1303. DOI: 10.1007/s10620-016-4049-x. [10] JENNE CN, KUBES P. Immune surveillance by the liver[J]. Nat Immunol, 2013, 14(10): 996-1006. DOI: 10.1038/ni.2691. [11] CHEN Y, OU Y, DONG J, et al. Osteopontin promotes collagen I synthesis in hepatic stellate cells by miRNA-129-5p inhibition[J]. Exp Cell Res, 2018, 362(2): 343-348. DOI: 10.1016/j.yexcr.2017.11.035. [12] WU P, TAN XY. The effect of Fuzheng Huayu capsule on immine regulation and anti-fibrosis in hepatic C virus infected patients with decompensated cirrhosis[J]. Chin J Integr Trad West Med Dig, 2016, 24(9): 658-660. DOI: 10.3969/j.issn.1671-038X.2016.09.02.吴平, 谭小燕. 扶正化瘀胶囊对丙型肝炎失代偿期肝硬化患者免疫调节以及抗纤维化作用研究[J]. 中国中西医结合消化杂志, 2016, 24(9): 658-660. DOI: 10.3969/j.issn.1671-038X.2016.09.02. [13] CHEN H, YANG BW, YUAN M, et al. Prevention and therapeutic effects of Fuzheng Huayu capsule on liver fibrosis and expression of connective tissue growth factor in rats[J]. J Sichuan Univ (Med Sci Edi), 2016, 47(2): 197-202. https://www.cnki.com.cn/Article/CJFDTOTAL-HXYK201602012.htm陈晗, 杨碧伟, 袁满, 等. 扶正化瘀胶囊对肝纤维化大鼠的防治作用及对结缔组织生长因子表达的影响[J]. 四川大学学报(医学版), 2016, 47(2): 197-202. https://www.cnki.com.cn/Article/CJFDTOTAL-HXYK201602012.htm [14] WANG J. The effect of Fuzheng Huayu capsule assisting western medicine on intestinal mucosal barrier function, immune function and liver function in patients with hepatitis B liver cirrhosis[J]. Mod J Integr Tradit Chin West Med, 2017, 26(27): 3051-3053. DOI: 10.3969/j.issn.1008-8849.2017.27.031.王军. 扶正化瘀胶囊辅助西医对乙肝肝硬化患者肠黏膜屏障功能、免疫功能及肝功能状态的影响[J]. 现代中西医结合杂志, 2017, 26(27): 3051-3053. DOI: 10.3969/j.issn.1008-8849.2017.27.031. [15] XU HY, YUN CX, WANG YX. The effects of PSP on the subset of T cell and apoptosis of the tumo-bearing mice[J]. J Qiqihar Med Coll, 2004, 25(5): 485-487. DOI: 10.3969/j.issn.1002-1256.2004.05.003.许惠玉, 运晨霞, 王雅贤. 桃仁总蛋白对荷瘤鼠T淋巴细胞亚群及细胞凋亡的影响[J]. 齐齐哈尔医学院学报, 2004, 25(5): 485-487. DOI: 10.3969/j.issn.1002-1256.2004.05.003. [16] MA YL. Study on the immune regulation mechanism of Fuzheng Huayu recipe against liver fibrosis based on the changes of CD8+ T lymphocyte subsets[D]. ShangHai: ShangHai University of Traditional Chinese Medicine, 2017.马亚丽. 基于CD8+T淋巴细胞亚群的变化探讨扶正化瘀方抗肝纤维化的免疫调节机制[D]. 上海: 上海中医药大学, 2017. [17] SPAHN J, PIERCE RH, CRISPE IN. Ineffective CD8+ T-cell immunity to adeno-associated virus can result in prolonged liver injury and fibrogenesis[J]. Am J Pathol, 2011, 179(5): 2370-2381. DOI: 10.1016/j.ajpath.2011.08.004. [18] XIE M, JIANG Q, ZHAO S, et al. Prognostic value of tissue-infiltrating immune cells in tumor microenvironment of follicular lymphoma: A Meta-analysis[J]. Int Immunopharmacol, 2020, 85: 106684. DOI: 10.1016/j.intimp.2020.106684. [19] KLAMETHA, NEUBAUER A, KELLER C, et al. Aberrant CD3-positive, CD8-low, CD7-negative lymphocytes may appear during viral infections and mimic peripheral T-cell lymphoma[J]. Diagnostics, 2020, 10(4): 204. DOI: 10.3390/diagnostics10040204. [20] ZHANG PX, BIAN PY, YE CT, et al. Changes in senescence-and function-related parameters of CD8+T cells after direct-acting antiviral treatment in patients with hepatitis C virus infection[J]. J Clin Hepatol, 2020, 36(7): 1496-1501. DOI: 10.3969/j.issn.1001-5256.2020.07.011.张沛欣, 边培育, 叶传涛, 等. HCV感染者直接抗病毒药物治疗前后CD8+T淋巴细胞衰老和功能相关指标的变化[J]. 临床肝胆病杂志, 2020, 36(7): 1496-1501. DOI: 10.3969/j.issn.1001-5256.2020.07.011. -
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