CD40siRNA调控c-Jun氨基末端激酶对自身免疫性心肌炎大鼠心肌细胞自噬的影响

doi:10.6040/j.issn.1671-7554.0.2018.1344

摘要/Abstract

摘要： 目的探讨CD40siRNA调控c-Jun氨基末端激酶(JNK)对自身免疫性心肌炎(EAM)大鼠心肌细胞自噬的影响。方法 6~8周健康雄性Lewis大鼠32只随机均分为EAM组、CD40siRNA治疗组、siRNA对照组和正常对照组。实验第0天和第7天于前3组大鼠后足垫区注射充分混合的猪心免疫球蛋白,0.2 mL/只,建立EAM大鼠模型;正常对照组大鼠后足垫区注射无菌磷酸盐缓冲液(PBS),0.2 mL/只。第7天,CD40siRNA治疗组尾静脉注射25 μL CD40siRNA慢病毒表达载体,siRNA对照组尾静脉注射25 μL siRNA慢病毒载体。每3天测量1次大鼠体质量,第21天处死所有大鼠,光学显微镜观察大鼠心肌组织的病理变化并计算病理积分,免疫组化染色JNK,采用Western blotting法检测自噬相关蛋白Beclin1和微管相关蛋白轻链3-Ⅱ/Ⅰ比值(LC3-Ⅱ/Ⅰ)。结果各组均无死亡,正常对照组体质量增长速度超过EAM组(P<0.05)。心肌组织病理积分CD40siRNA治疗组明显低于EAM组(P<0.001)。免疫组化染色显示,CD40siRNA治疗组和NC组的p-JNK明显低于EAM组。Western blotting法检测显示,CD40siRNA治疗组和NC组的LC3-Ⅱ/Ⅰ比值、Beclin1明显低于EAM组,且JNK与LC3-Ⅱ和内参比值具有正相关性斜率(r=0.985, P<0.001)。结论 CD40siRNA可减轻EAM大鼠的炎症,其机制可能与下调JNK抑制自身免疫性心肌炎大鼠心肌细胞自噬有关。

关键词: 自身免疫性心肌炎, 自噬, CD40-CD40配体, c-Jun氨基末端激酶, 微管相关蛋白轻链3-Ⅱ/Ⅰ

Abstract: Objective To investigate the effects of CD40siRNA regulated c-Jun N-terminal kinase(JNK)pathway on the autophagy of cardiomyocytes in experimental autoimmune myocarditis(EAM). Methods A total of 32 healthy male Lewis rats aged 6 to 8 weeks were randomly divided into the EAM group, EAM+CD40siRNA group, EAM+siRNA control group, and normal control group(NC group), with 8 rats in each group. The EAM models were established by injecting mixed porcine heart immunoglobulin(0.2 mL/rat)into the hind paw pad of rats in the first 3 groups on day 0 and day 7. Rats in the NC group were injected with 0.2 mL/rat of phosphate buffered saline(PBS). On day 7, rats in the EAM+CD40siRNA group were injected with 25 μL of CD40siRNA lentiviral vector in the tail vein, and rats in the EAM+siRNA control group were injected with 25 μL of LsiRNA lentiviral vector. The body weight of rats was measured every 3 days. All rats were sacrificed on day 21. Pathological changes of the myocardial tissues were observed 山东大学学报 (医学版)57卷4期 -孙红林,等.CD40siRNA调控c-Jun氨基末端激酶对自身免疫性心肌炎大鼠心肌细胞自噬的影响 \=-with a light microscopy and pathological scores were calculated. The JNK was immunohistochemically stained. The autophagy-related protein Beclin1 and microtubule-associated protein light chain 3-Ⅱ/Ⅰ ratio(LC3-Ⅱ/Ⅰ)were detected with Western blotting. Results During the experiment, no rats died. The body mass of rats in the NC group increased faster than that in the EAM group(P<0.05). The EAM+CD40siRNA group had significantly lower pathological scores than the EAM group(P<0.001). The EAM+CD40siRNA group and NC group had significantly lower LC3-Ⅱ/Ⅰ and Beclin1 than the EAM group, and there was a positive correlation between JNK and LC3-Ⅱ(r=0.985, P<0.001). Conclusion The CD40siRNA can relieve the inflammation of EAM rats, possibly by downregulating the JNK pathway and inhibiting the autophagy of cardiomyocytes.

Key words: Autoimmune myocarditis, Autophagy, CD40-CD40 ligand, C-Jun N-terminal kinase, Microtubule-associated protein light chain 3-II/I

中图分类号:

R725.4

孙红林,韩波,王静,高聆,朱梅,姜殿东,吕建利. CD40siRNA调控c-Jun氨基末端激酶对自身免疫性心肌炎大鼠心肌细胞自噬的影响[J]. 山东大学学报 (医学版), 2019, 57(4): 9-14.

SUN Honglin, HAN Bo, WANG Jing, GAO Ling, ZHU Mei, JIANG Diandong, LÜ Jianli. Effects of c-Jun N-terminal kinase regulated by CD40siRNA on the autophagy of cardiomyocytes in rats with autoimmune myocarditis[J]. Journal of Shandong University (Health Sciences), 2019, 57(4): 9-14.

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