上海交通大学学报（医学版）

上海交通大学学报（医学版） ›› 2020, Vol. 40 ›› Issue (3): 303-.doi: 10.3969/j.issn.1674-8115.2020.03.005

• 论著·基础研究 • 上一篇    下一篇

柴胡皂苷d对自身免疫性肝炎小鼠差异表达基因CTLA-4、IL-10和IL-17的影响

郝健亨，陈 浩，高 艳，徐慧超，高玉亭，苗宇船，郝慧琴，刘 杨   

  1. 山西中医药大学基础医学院， 晋中 030619
  • 出版日期:2020-03-28 发布日期:2020-04-09
  • 通讯作者: 刘 杨，电子信箱：syfy2004@163.com。
  • 作者简介:郝健亨（1993—），男，硕士生；电子信箱：651194582@qq.com。
  • 基金资助:
    国家自然科学基金（81470190）； 基于炎症反应的重大疾病创新药物山西省重点实验室开放课题基金（SXIDL-2018-07）；山西省卫生健康委员会科研课题计划（2018002）；山西中医药大学“肝脏炎性疾病中西医结合基础研究创新团队”资助项目（2018TD-016）。

Effects of Saikosaponin d on differentially expressed genes CTLA-4, IL-10 and IL-17 in mice with autoimmune hepatitis

HAO Jian-heng, CHEN Hao, GAO Yan, XU Hui-chao, GAO Yu-ting, MIAO Yu-chuan, HAO Hui-qin, LIU Yang   

  1. School of Basic Medical Sciences, Shanxi University of Traditional Chinese Medicine, Jinzhong 030619, China
  • Online:2020-03-28 Published:2020-04-09
  • Supported by:
    National Natural Science Foundation of China, 81470190; Open Project Fund of Shanxi Province Key Laboratory of Innovative Drugs for Major Diseases Based on Inflammatory Responses, SXIDL-2018-07; Scientific Research Project of Shanxi Provincial Health Committee, 2018002; “Innovative Team of Integrated Traditional Chinese and Western Medicine for Liver Inflammatory Diseases” Shanxi University of Traditional Chinese Medicine, 2018TD-016.

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摘要： 目的·选用Agilent-085631芯片筛选自身免疫性肝炎（autoimmune hepatitis，AIH）小鼠差异表达基因，并观察柴胡皂苷d（Saikosaponin d，SS-d）对部分差异表达基因的影响，探讨SS-d治疗AIH的作用机制。方法·健康雄性SPF级C57BL/6小鼠40只，分为芯片组（n8）和SS-d治疗机制组（n32）。芯片组分为正常组和模型组[按15 mg/kg剂量给予尾静脉注射刀豆蛋白 A（concanavalin A，Con A） ]（每组n4），12 h后处死，进行肝脏差异表达基因的筛选，并采用qRT-PCR技术对部分基因进行验证。SS-d治疗机制组分为正常组、模型组（处理同芯片组）、SS-d低剂量组和SS-d高剂量组（参照文献及预实验结果，分别按2.5和5.0 mg/kg剂量腹腔注射SS-d，8 h后按15 mg/kg剂量给予尾静脉注射Con A）（每组n8）。12 h后收集小鼠静脉血及肝脏总蛋白、总RNA，采用ELISA法检测血清谷丙转氨酶（glutamic pyruvic transaminase，GPT）、谷草转氨酶（glutamic oxaloacetic transaminase，GOT）及白介素（interleukin，IL） -10、IL-17的含量，Western blotting检测细胞毒T淋巴细胞相关抗原（cytotoxic T lymphocyte-associated antigen，CTLA） -4蛋白的表达量，qRT-PCR技术检测IL-10、IL-17和CTLA-4 mRNA表达量。结果·共筛查差异表达基因11 512个（上调5 189个，下调6 323个），与138条信号通路相关；差异表达基因IL-10、IL-17及CTLA-4的qRT-PCR检测结果与芯片筛查结果一致。与正常组比较：模型组小鼠血清GPT、GOT含量升高；IL-17 mRNA表达量升高，IL-10 mRNA和CTLA-4 mRNA表达量降低；血清IL-17含量升高，血清IL-10含量降低，肝组织CTLA-4蛋白表达量降低。与模型组比较：SS-d低剂量组和SS-d高剂量组小鼠血清GPT、GOT含量降低；IL-17 mRNA表达量降低，IL-10 mRNA和CTLA-4 mRNA表达量升高；血清IL-17含量降低，血清IL-10含量升高，肝组织CTLA-4蛋白表达量升高。结论·多条信号通路参与AIH的发病，而SS-d可通过调节IL-10、CTLA-4、IL-17的表达，改善AIH小鼠肝脏炎症反应。

关键词: 自身免疫性肝炎, 柴胡皂苷-d, 细胞毒T淋巴细胞相关抗原-4, 白介素-10, 白介素-17

Abstract:

Objective · To explore the effects of Saikosaponin d (SS-d) on autoimmune hepatitis (AIH)observing the changes of some differentially expressed genes screened with the Agilent-085631 gene chip in the liver of AIH mice. Methods · Forty healthy male SPF C57BL/6 mice were divided into chip group (n8) and SS-d treatment group (n32). The mice in the chip group were divided into the normal group and the model group [concanavalin A (Con A) was administered to the tail vein at a dose of 15 mg/kg] (both n4). The mice were sacrificed after 12 h. The differentially expressed genes of liver were screened, some of which were verifiedqRT-PCR. The SS-d treatment group was further divided into the normal group, the model group (treatment was the same with the chip group), SS-d low-dose group and SS-d high-dose group [according to the literature and pre-experiment results, 2.5 and 5.0 mg/kg dose of intraperitoneal injection of SS-d were given respectively, and 15 mg/kg of Con A was administered to the tail vein 8 h later] (all n8). After 12 h, total venous blood, liver total protein and total RNA of mice were collected. The levels of serum glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), interleukin (IL)-10 and IL-17 were detectedELISA, and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) was detectedWestern blotting. qRT-PCR technology was used to detect the mRNA levels of IL-10, IL-17 and CTLA-4. Results · A total of 11 512 differentially expressed genes were screened (up 5 189, down 6 323), which were related to 138 signal pathways. The qRT-PCR results of IL-10, IL-17 and CTLA-4 gene were consistent with the results of chip screening. Compared with the normal group, the serum levels of GPT and GOT in the model group increased, IL-17 mRNA level increased, IL-10 mRNA and CTLA-4 mRNA levels decreased, the content of serum IL-17 increased, the content of serum IL-10 decreased, and the level of CTLA-4 protein in the liver tissues decreased. Compared with the model group, the serum GPT and GOT levels of SS-d in the low-dose and high-dose groups decreased, IL-17 mRNA level decreased, the levels of IL-10 mRNA and CTLA-4 mRNA increased, the content of serum IL-17 decreased, the content of serum IL-10 increased, and the level of CTLA-4 protein in the liver tissue increased. Conclusion · Multiple signaling pathways are involved in the pathogenesis of AIH, and SS-d can alleviate the liver inflammation in AIH miceregulating the of IL-10, CTLA-4, and IL-17.

Key words: autoimmune hepatitis (AIH), saikosaponin d (SS-d), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), interleukin-10, interleukin-17