电针通过抑制下丘脑细胞外调节蛋白激酶5信号通路改善手术创伤大鼠脾脏自然杀伤细胞的功能  

Electroacupuncture Intervention Enhances Splenic Natural Killer Cell Activity via Inhibiting Phosphorylation of ERK 5in the Hypothalamus of Surgically Traumatized Rats

作　　者：陈焱[1] 李静 朱可蓥[1] 肖晟[1] 王彦青[1] 吴根诚[1] 汪军[1] 

机构地区：[1]复旦大学基础医学院中西医结合学系,上海200032

出　　处：《针刺研究》2015年第3期173-179,共7页Acupuncture Research

基　　金：国家自然科学基金项目(No.81202746);国家重点基础研究发展计划(No.2013CB 531906)

摘　　要：目的:观察下丘脑内细胞外调节蛋白激酶(ERK)5信号通路在电针改善手术创伤大鼠脾脏自然杀伤(NK)细胞功能中的作用及机制。方法:SD雄性大鼠随机分为对照组、创伤组、创伤+电针组、创伤+未电针组、创伤+低剂量BIX 02188组、创伤+高剂量BIX 02188组,每组6只。采用手术创伤模型,创伤+电针组选取双侧"足三里"穴,术后立刻电针,创伤+未电针组针刺双侧"足三里"穴,但不接电针仪,均持续30min,创伤+低剂量BIX 02188组、创伤+高剂量BIX 02188组于造模前1周进行侧脑室埋管,手术创伤前30min注射BIX 02188。免疫组化法和Western blot法观察下丘脑ERK 5磷酸化水平和蛋白表达,及促肾上腺皮质激素释放激素(CRF)蛋白表达,乳酸脱氢酶释放法和Real-time PCR法检测脾脏NK细胞杀伤功能及穿孔素(Perforin)和颗粒酶B(Granzyme B)的mRNA表达。结果:创伤组大鼠下丘脑内ERK 5信号通路显著激活,ERK 5磷酸化水平及蛋白表达均升高(P<0.01,P<0.05),电针及ERK 5抑制剂均可抑制ERK 5的磷酸化(P<0.05,P<0.01);手术创伤后,脾细胞Perforin及Granzyme B的mRNA明显下调,脾脏NK细胞的杀伤活力下降(P<0.01,P<0.05),电针治疗可增加Perforin及Granzyme B的基因表达,进而提高脾脏NK细胞的杀伤活力(P<0.05);下丘脑内CRF的表达在创伤后明显上升(P<0.01),给予ERK 5抑制剂或电针均可下调其表达(P<0.01)。结论:电针改善手术创伤引起的脾脏NK细胞杀伤活力降低的机制之一可能是下调下丘脑内ERK 5信号通路的激活,减少CRF的产生,从而抑制下丘脑-垂体-肾上腺轴激活。Objective To observe the effect of electroacupuncture（EA）on cytotoxic activity of splenic natural killer（NK）cells after surgical trauma via extracellular signal-regulated kinase（ERK）5pathway in the rats＇ hypothalamus,so as to explore its mechanism underlying improving immune disorders after surgery.Methods Sprague-Dawley rats were randomly divided into the following 6groups：control,trauma model,EA,sham EA,4nmol-BIX 02188（an inhibitor for ERK 5catalytic activity）and 20nmol-BIX 02188（n=6rats per group）.The surgical trauma model was established by making a longitudinal incision（6cm in length）along the median line of the back to expose the spinal column and another longitudinal incision along the abdominal median line.EA（2Hz/15 Hz,1-2mA）was applied to bilateral＂Zusanli＂（ST 36）for 30 min immediately after surgery.For rats of the BIX groups,intra-lateral ventricular microinjection of BIX 02188（10μL,4nmol or 20 nmol,or saline for control rats）was conducted 30 min before the surgery.The expression level and protein of phosphorylated ERK 5（p-ERK 5）and corticotropin-releasing factor（CRF）protein were measured by immunohistochemistry and Western blot,respectively.The cytotoxicity of splenic NK cells and the expression of splenic Perforin and Granzyme-B genes were measured by lactate dehydrogenase（LDH）release assay and real-time PCR,respectively.Results In comparison with the control group,hypothalamic p-ERK 5immunoactivity,p-ERK5 protein and CRF protein expression levels were significantly up-regulated in the model group（P〈0.01,P〈0.05）,while splenic NK cell cytotoxicity and Perforin mRNA and Granzyme-B mRNA expression levels were notably down-regulated in the model group（P〈0.05,P〈0.01）.Following EA and administration of ERK 5antagonist,the increased expression levels of p-ERK 5immunoactivity in the EA group,and p-ERK 5and CRF proteins in both EA and 20nmol-BIX 02188 groups were obviously down-regulated（P〈0.05,P〈0.01）,without changes in the sham

关 键 词：电针 手术创伤应激 下丘脑 细胞外调节蛋白激酶5 促肾上腺皮质激素释放激素 脾脏自然杀伤细胞 

分 类 号：R245.97[医药卫生—针灸推拿学]