电针对颅脑损伤大鼠神经功能及凋亡相关蛋白Cyt-C、Caspase-9表达的影响

Impacts of electroacupuncture on neurological function and protein expressions of apoptosis-related Cyt-C and Caspase-9 in rats with traumatic brain injury

目的:观察电针对颅脑损伤(TBI)大鼠神经功能、脑组织病理形态、细胞凋亡水平及凋亡相关蛋白细胞色素C(Cyt-C)、半胱氨酸天冬氨酸蛋白酶-9(Caspase-9)表达的影响,探索电针治疗TBI的可能机制。方法:将70只清洁级SD大鼠随机分为空白组(8只)、假手术组(8只)、模型组(27只)和电针组(27只)。模型组及电针组再按干预3、7、14 d分为3个亚组,每亚组9只,并采用改良式Feeney自由落体法制备大鼠TBI模型;假手术组仅暴露颅骨、钻孔,不行自由落体打击。于造模后1 d对电针组大鼠行电针干预,穴取"百会""水沟"及患侧"内关""足三里",断续波,频率2 Hz,每天1次,每次10 min,分别干预3、7、14 d。分别于干预3、7、14 d采用改良神经功能缺损量表(m NSS)评定各组大鼠神经功能损伤程度;HE染色法和尼氏染色法观察脑组织病理形态改变;TUNEL法观察脑组织细胞凋亡水平;免疫组织化学法、Western blot法检测脑组织Cyt-C、Caspase-9蛋白表达。结果:与假手术组比较,模型组大鼠干预3、7、14d时m NSS评分均明显升高(P<0.01);与模型组比较,电针组大鼠干预3、7、14d时mNSS评分均降低(P<0.05)。干预3d时模型组及电针组大鼠脑损伤区域均可见组织坏死,核破碎、固缩及明显空泡,尼氏体减少、分布松散;干预7、14d时模型组及电针组均可见新生结缔组织填充和正常细胞,尼氏体增加,且电针组整体修复情况、尼氏体数量优于模型组。与假手术组比较,模型组大鼠干预3、7、14 d时脑组织凋亡细胞数均明显增多(P<0.01);与模型组比较,电针组大鼠干预3、7、14 d时脑组织凋亡细胞数均减少(P<0.05)。与假手术组比较,模型组大鼠干预3、7、14 d时损伤脑组织Cyt-C、Caspase-9蛋白表达均明显升高(P<0.01);与模型组比较,电针组大鼠干预3、7、14 d时损伤脑组织Cyt-C、Caspase-9蛋白表达均降低(P<0.05)。结论:电针可明显改善TBI模型大鼠神经功能损伤情况,降低细胞凋亡水平,其机制可能与下调损伤脑组织中Cyt-C、Caspase-9蛋白表达,进而影响线粒体介导的细胞凋亡进程有关。

Objective To observe the impacts of electroacupuncture(EA)on neurological function,the pathological morphology in brain tissue,apoptosis level and the protein expressions of apoptosis-related cytochrome C(Cyt-C)and cysteine aspartic acid protease-9(Caspase-9)in the rats with traumatic brain injury(TBI)and explore the potential mechanism of EA in treatment of TBI.Methods A total of 70 clean-grade SD mice were randomized into a blank group(8 rats),a sham-operation group(8 rats),a model group(27 rats)and an EA group(27 rats).In terms of interventions of 3,7 and 14 days,3 subgroups were divided in the model group and the EA group successively,9 rats in each subgroup.The modified Feeney free-fall percussion method was adopted to establish TBI models of rats.In the sham-operation group,only the skull was exposed and drilled and no free-fall percussion was exerted.One day after modeling,EA was given in the rats of EA group at"Shuigou"(GV 26),"Baihui"(GV 20)and"Neiguan"(PC 6)and"Zusanli"(ST 36)on the affected side,with intermittent wave,2 Hz in frequency,once daily,10 min each time,for 3,7 and 14 days successively.Separately,on the day 3,7 and 14 of intervention,the modified neurological severity scale(mNSS)was used to evaluate the degree of neurological function injury in the rats,HE staining and Nissl staining were to observe the pathological and morphological changes in brain tissue,TUNEL method was to observe the level of apoptosis in brain tissue and immunohistochemistry(IHC)method and Western blot were to determine the protein expressions of Cyt-C and Caspase-9 in brain tissue.Results Compared with the sham-operation group,on the day 3,7 and 14 of intervention,mNSS scores were increased obviously in the rats of the model group respectively(P<0.01).Compared with the model group,on the day 3,7 and 14 of intervention,mNSS scores were reduced in the rats of the EA group respectively(P<0.05).On day 3 of intervention,in brain injury region of the rats in the model group and the EA group,gross tissue necrosis,nuclear fragmentation,consolidation and obvious vacuolar changes,reduced Nissl bodies and scattered arrangement were found.On day 7 and 14 of intervention,in the model group and the EA group,the new connective tissue filling and normal cells were visible and Nissl bodies increased.The overall repair and Nissl body quantity in the EA group were better than the model group.Compared with the sham-operation group,on day 3,7 and 14 of intervention,the numbers of apoptotic cells were increased obviously in the model group(P<0.01)and they were reduced in the EA group as compared with the model group(P<0.05).Compared with the sham-operation group,on day 3,7 and 14 of intervention,the protein expressions of Cyt-C and Caspase-9 in damaged brain tissue were all increased obviously in the model group(P<0.01)and they were all reduced in the EA group as compared with the model group successively(P<0.05).Conclusion Electroacupuncture remarkably improves the condition in the neurological function injury and reduces apoptosis degree in TBI model rats,which is likely related to the down-regulation of the protein expressions of Cyt-C and Caspase-9 in damaged brain tissue and further to bring the impacts on mitochondria mediated apoptosis process.