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.

参考文献

[1] Kawai C. From myocarditis to cardiomyopathy: mechanisms of inflammation and cell death: learning from the past for the future[J]. Circulation, 1999, 99(8): 1091-1100.
[2] Kodama M, Matsumoto Y, Fujiwara M, et al. A novel experimental model of giant cell myocarditis induced in rats by immunization with cardiac myosin fraction[J]. Clin Immunol Immunopathol, 1990, 57(2): 250-262.
[3] Fuse K, Kodama M, Ito M, et al. Polarity of helper T cell subsets represents disease nature and clinical course of experimental autoimmune myocarditis in rats[J]. Clin Exp Immunol, 2003, 134(3): 403-408.
[4] Kodama M, Zhang S, Hanawa H, et al. Immunohistochemical characterization of infiltrating mononuclear cells in the rat heart with experimental autoimmune giant cell myocarditis[J]. Clin Exp Immunol, 1992, 90(2): 330-335.
[5] Vallerie SN, Hotamisligil GS. The role of JNK proteins in metabolism[J]. Sci Transl Med, 2010, 2(60): 60.
[6] Solinas G, Becattini B. JNK at the crossroad of obesity, insulin resistance, and cell stress response[J]. Mol Metab, 2017, 6(2): 174-184.
[7] Zhou YY, Li Y, Jiang WQ, et al. MAPK/JNK signalling: a potential autophagy regulation pathway[J]. Biosci Rep, 2015, 35(3): e00199. doi: 10.1042/BSR20140141.
[8] Bachmaier K, Toya S, Malik AB. Therapeutic administration of the chemokine CXCL1/KC abrogates autoimmune inflammatory heart disease[J]. PLoS One, 2014, 9(2): e89647. doi: 10.1371/journal.pone.0089647.
[9] Matsui Y, Inobe M, Okamoto H, et al. Blockade of T cell costimulatory signals using adenovirus vectors prevents both the induction and the progression of experimental autoimmune myocarditis[J]. J Mol Cell Cardiol, 2002, 34(3): 279-295.
[10] 杨文巍. CD40siRNA对自身免疫性心肌炎大鼠Th17细胞及IL-17与IL-23的作用[D]. 济南: 山东大学, 2013.
[11] 张荣军, 韩波, 高聆, 等. siRNA沉默CD40基因对实验性自身免疫性心肌炎大鼠的作用及 IL-22表达的影响[J]. 山东大学学报(医学版), 2015, 53(5): 36-40. ZHANG Rongjun, HAN Bo, GAO Ling, et al. Effects of CD40 knockdown by siRNA on rats with experimental autoimmune myocarditis and IL-22 expression[J]. Journal of Shandong University(Health Sciences), 2015, 53(5): 36-40.
[12] Cui Y, Jiang L, Shao Y, et al. Anti-alcohol liver disease effect of Gentianae macrophyllae extract through MAPK/JNK/p38 pathway[J]. J Pharm Pharmacol, 2019, 71(2): 240-250.
[13] Hommes DW, Peppelenbosch MP, van Deventer SJ.Mitogen activated protein(MAP)kinase signal transduction pathways and novel anti-inflammatory targets[J]. Gut, 2003, 52(1): 144-151.
[14] Zanotti S, Kumar A, Kumar A. Cytokine modulation in sepsis and septic shock[J]. Expert Opin Investig Drugs, 2002, 11(8): 1061-1075.
[15] Dai Q, Zhang D, Yu H, et al. Berberine Restricts Coxsackievirus B Type 3 Replication via Inhibition of c-Jun N-Terminal Kinase(JNK)and p38 MAPK Activation In Vitro[J]. Med Sci Monit, 2017, 23(25): 1448-1455.
[16] Johnson JC, Martinez O, Honko AN, et al. Pyridinyl imidazole inhibitors of p38 MAP kinase impair viral entry and reduce cytokine induction by Zaire ebolavirus in human dendritic cells[J]. Antiviral Res, 2014, 107: 102-109.doi: 10.1016/j.antiviral.2014.04.014.
[17] Kim SM, Park JH, Chung SK, et al. Coxsackievirus B3 infection induces cyr61 activation via JNK to mediate cell death[J]. J Virol, 2004, 78(24): 13479-13488.
[18] Kim EK, Choi EJ. Pathological roles of MAPK signaling pathways in human diseases[J]. Biochim Biophys Acta, 2010, 1802(4): 396-405.
[19] Cargnello M, Roux PP. Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases[J]. Microbiol Mol Biol Rev, 2011, 75(1): 50-83.
[20] Tan YQ, Zhang J, Zhou G. Autophagy and its implication in human oral diseases[J]. Autophagy, 2017, 13(2): 225-236.
[21] Wong J, Zhang J, Si X, et al. Autophagosome supports coxsackievirus B3 replication in host cells[J]. J Virol, 2008, 82(18): 9143-9153.
[22] Eskelinen EL. Maturation of autophagic vacuoles in Mammalian cells[J]. Autophagy, 2005, 1(1): 1-10.
[23] Klionsky DJ, Baehrecke EH, Brumell JH, et al. A comprehensive glossary of autophagy-related molecules and processes(2nd edition)[J]. Autophagy, 2011, 7(11): 1273-1294.
[24] Aoki H, Kang PM, Hampe J, et al. Direct activation of mitochondrial apoptosis machinery by c-Jun N-terminal kinase in adult cardiac myocytes[J]. J Biol Chem, 2002, 277(12): 10244-10250.
[25] Gao F, Yue TL, Shi DW. p38 MAPK inhibition reduces myocardial reperfusion injury via inhibition of endothelial adhesion molecule expression and blockade of PMN accumulation[J]. Cardiovasc Res, 2002, 53(2): 414-422.
[26] Jeong CW, Yoo KY, Lee SH, et al. Curcumin protects against regional myocardial ischemia/reperfusion injury through activation of RISK/GSK-3beta and inhibition of p38 MAPK and JNK[J]. J Cardiovasc Pharmacol Ther, 2012, 17(4): 387-394.
[27] Qi L, Xin Q, Wenjun J. Inhibition of iNOS protects cardiomyocytes against coxsackievirus B3-induced cell injury by suppressing autophagy[J]. Biomed Pharmacother, 2017, 91: 673-679. doi: 10.1016/j.biopha.2017.04.123.
[28] Reddy KB, Nabha SM, Atanaskova N. Role of MAP kinase in tumor progression and invasion[J]. Cancer Metastasis Rev, 2003, 22(4): 395-403.
[29] Xu YF, Liu M, Peng B, et al.Protective effects of SP600125 on renal ischemia-reperfusion injury in rats[J]. J Surg Res, 2011, 169(1): 77-84.
[30] Sarkar S, Rubinsztein DC. Huntingtons disease: degradation of mutant huntingtin by autophagy[J]. FEBS J, 2008, 275(17): 4263-4270.
[31] Wu ZZ, Zhang JJ, Gao CC, et al. Expression of autophagy related genes mTOR, Becline-1, LC3 and p62 in the peripheral blood mononuclear cells of systemic lupus erythematosus[J]. Am J Clin Exp Immunol, 2017, 6(1): 1-8.
[32] Zhai X, Bai B, Yu B, et al. Coxsackievirus B3 Induces Autophagic Response in Cardiac Myocytes in vivo[J]. Biochemistry(Mosc), 2015, 80(8): 1001-1009.

相关文章 15

[1]	孟婷婷,王淑亚,吴会会,陈嘉敏,郑燕,李莹,苏国海. 脂联素通过分泌型卷曲相关蛋白2及相关通路缓解AngⅡ诱导的心肌肥厚[J]. 山东大学学报 (医学版), 2021, 59(8): 44-52.
[2]	徐兵,李勇,刘明,刘永辉. 沉默PRRX1基因表达可增强前列腺癌耐药细胞株PC-3/DTX对多西他赛的敏感性[J]. 山东大学学报 (医学版), 2021, 59(6): 103-110.
[3]	张小红,周云,杜秋莹,任慧欣,王超群. Atg7-siRNA通过调节精氨酸循环干扰食管癌ECA109细胞放疗敏感性[J]. 山东大学学报 (医学版), 2021, 59(4): 28-34.
[4]	张华宇,殷思源,刘健,马嘉旭,宋茹,曹国起,王一兵. 氧糖剥夺条件下培养表皮干细胞的定量蛋白质组学分析[J]. 山东大学学报 (医学版), 2021, 59(4): 17-27.
[5]	索东阳,申飞,郭皓,刘力畅,杨惠敏,杨向东. Tim-3在药物性急性肾损伤动物模型中的表达及作用机制[J]. 山东大学学报 (医学版), 2020, 1(7): 1-6.
[6]	熊超,刘力,冯建国,魏继承. 七氟醚预处理对H9C2心肌细胞缺氧/复氧后转录沉默信息调节器3的表达及乙酰化水平的影响[J]. 山东大学学报 (医学版), 2019, 57(3): 25-30.
[7]	蒿魁元,赵圣,张宇,崔迪,荆翌峰,夏术阶,韩邦旻. 雄激素阻断对膀胱癌UM-UC-3细胞自噬与凋亡的影响[J]. 山东大学学报 (医学版), 2018, 56(3): 41-47.
[8]	赵姗姗,李晓梅,梁婷,张超,宋静,侯桂华. 自噬对¹²⁵I-anti-TLR5同种移植排斥靶向显像的影响[J]. 山东大学学报（医学版）, 2017, 55(9): 46-52.
[9]	殷雷,殷睿,李文佳,刘帅,吕家驹. CYLD抑制自噬提高膀胱癌细胞吉西他滨化疗敏感性[J]. 山东大学学报（医学版）, 2017, 55(8): 1-6.
[10]	任宝鑫,马云峰,刘殿伟,李卓,姜勇. Wnt3a在大鼠蛛网膜下腔出血后早期脑损伤中对神经细胞自噬和凋亡的影响[J]. 山东大学学报（医学版）, 2016, 54(10): 11-15.
[11]	黄智龙, 刘帅, 王建伟, 韩立平, 王晓庆, 李新, 毕东滨, 解放, 牛志宏. 乌苯美司诱导肾癌细胞死亡的作用及机制[J]. 山东大学学报（医学版）, 2015, 53(9): 58-64.
[12]	张荣军, 韩波, 高聆, 朱梅, 丁国玉, 梁燕. siRNA沉默CD40基因对实验性自身免疫性心肌炎大鼠的作用及IL-22表达的影响[J]. 山东大学学报（医学版）, 2015, 53(5): 36-40.
[13]	赵雪莲, 于君, 谢兆宏, 曹彦军, 刘震, 王晓, 徐琳琳, 杨慧, 郑晓磊, 沈阳, 毕建忠. 线粒体自噬在阿尔茨海默病细胞模型中的作用机制[J]. 山东大学学报（医学版）, 2015, 53(10): 1-5.
[14]	赵永强, 谢晓烨, 陈雪梅, 冯慧伟, 贾涛, 张辉, 范献良. COX-2选择性抑制剂诱导人喉癌Hep-2细胞凋亡及自噬的体外研究[J]. 山东大学学报（医学版）, 2014, 52(9): 39-43.
[15]	郝凤成1,2，苏中华1,3. 自噬在ox-LDL诱导炎症反应中的保护作用[J]. 山东大学学报（医学版）, 2014, 52(4): 30-34.

多维度评价

Viewed

Full text

Abstract

Cited

Shared

Discussed