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(n=8)和SS-d治疗机制组(n=32)。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n=4),12 h后处死,进行肝脏差异表达基因的筛选,并采用q RT-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n=8)。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,q RT-PCR技术检测IL-10、IL-17和CTLA-4 m RNA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q RT-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 m RNA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。
Objective · To explore the effects of Saikosaponin d(SS-d) on autoimmune hepatitis(AIH) by observing the expression changes of some differentially expressed genes screened with the Agilent-085631 gene chip in the liver of AIH mice. Methods · Forty healthy male SPF C57 BL/6 mice were divided into chip group(n=8) and SS-d treatment group(n=32). 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 n=4). The mice were sacrificed after 12 h. The differentially expressed genes of liver were screened, some of which were verified by qRT-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 preexperiment 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 n=8). 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 detected by ELISA, and cytotoxic T lymphocyteassociated antigen-4(CTLA-4) was detected by Western 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 m RNA level increased, IL-10 m RNA and CTLA-4 m RNA levels decreased, the content of serum IL-17 increased, the content of serum IL-10 decreased, and the level of CTLA-4 protein expression in the liver tissues decreased. Compared with the model g
作者
郝健亨
陈浩
高艳
徐慧超
高玉亭
苗宇船
郝慧琴
刘杨
HAO Jian-heng;CHEN Hao;GAO Yan;XU Hui-chao;GAO Yu-ting;MIAO Yu-chuan;HAO Hui-qin;LIU Yang(School of Basic Medical Sciences,Shanxi University of Traditional Chinese Medicine,Jinzhong 030619,China)
出处
《上海交通大学学报（医学版）》
CAS
CSCD
北大核心
2020年第3期303-309,共7页
Journal of Shanghai Jiao tong University:Medical Science
基金
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)。
