电针曲池穴及足三里穴对脑缺血再灌注损伤大鼠神经细胞线粒体凋亡途径的影响

Effect of electroacupuncture at Quchi and Zusanli acupoints on the regulation of neuronal mitochondrial apoptosis pathway in rats with cerebral ischemia-reperfusion injury

目的 观察电针干预对脑缺血再灌注损伤大鼠神经细胞线粒体凋亡途径的影响.方法 选取SD大鼠60只,按照随机数字表法将其分为假手术组、模型组和电针组,每组20只.模型组、电针组大鼠采用Zea Longa线栓法制备大脑中动脉闭塞（MCAO）模型,缺血2h后,再灌注3d.模型组与电针组各有18只大鼠造模成功.术后2h,在电针组大鼠“曲池”、“足三里”穴进行电针治疗,假手术组及模型组不作特殊干预.采用Zea Longa 5分评价法观察大鼠神经功能缺损的恢复程度;用2,3,5-氯化三苯基四氮唑染色,计算脑梗死体积;应用TUNEL试剂盒检测皮质区缺血周围神经细胞的凋亡情况;采用Western blot法、免疫组织化学法和逆转录-聚合酶链式反应（RT-PCR）检测脑组织中B淋巴细胞瘤-2基因（Bcl-2）、Bax蛋白、胱天蛋白酶（caspase）的表达水平.结果 与组内术后2h、1d及2d比较,模型组[（1.67±0.58）分]、电针组[（1.14±0.37）分]术后3d时的神经功能缺损评分较低（P＜0.05）.与假手术组同时间点比较,模型组、电针组大鼠术后2h、ld、2d及3d的神经功能缺损评分均较高,电针组术后2d及3d神经功能缺损评分低于模型组（P＜0.05）.术后3d,电针组大鼠脑梗死体积百分比明显减小（P＜0.05）.假手术组、模型组、电针组大鼠大脑皮质缺血半暗带区神经细胞的凋亡百分比分别为（1.07±0.02）％、（39.4±10.1）％、（15.1±4.2）％.与模型组比较,电针组凋亡细胞数量百分比明显较低（P＜0.05）.术后3d,模型组大鼠大脑皮质Bcl-2蛋白（0.11±0.02）、mRNA的相对含量（0.13±0.04）较假手术组明显下降（P＜0.05）,与模型组比较,电针组Bcl-2蛋白（0.36±0.11）、mRNA的相对含量（0.41±0.15）较高（P＜0.05）.术后3d,模型组、电针组大鼠大脑皮质Bax蛋白、mRNA的相对含量均高于假手术组（P＜0.05）,与模型组比较,电针组大鼠大脑皮质Bax蛋白（0.51±0.14）、mRNA的相对含量（0.37±0.13）较高（P＜0.05）.术后3d,电针组活化型胱天蛋白酶-3（cleaved caspase-3）免疫阳性细胞明显少于模型组,差异有统计学意义（P＜0.05）.结论 电针刺激“曲池”及“足三里”穴对脑缺血再灌注损伤大鼠的神经细胞具有保护作用,其机制可能与调控线粒体凋亡途径蛋白的表达水平有关.

Objective To investigate the effects of electroacupuncture （EA） at Quchi （LI11） and Zusanli （ST36） on the regulations of neuronal mitochondrial apoptosis pathway in rats with cerebral ischemia-reperfusion （IR） injury.Methods Sixty male adult Sprague-Dawley rats were randomly and evenly divided into three groups：a sham control group （SC）,an ischemia control group （IC）,and an electroacupuncture group （EA）,with 20 rats in each group.After 2 h of ischemia caused by middle cerebral artery occlusion （MCAO） followed by 3 d of reperfusion,IR model was established in IC group and EA group.Finally,there were 18 successful model rats in IC and EA groups,respectively.Rats in EA group received EA at Quchi and Zusanli acupoints beginning at 24 h after MCAO.Neurological deficit induced by IR injury were assessed by using Zea Longa method.The infarct volume was determined at 3 days after IR injury using TTC staining.Apoptotic cells in the cerebral cortex were quantified under confocal fluorescence microscope using TUNEL staining.The expressions of Bcl-2 and Bax were assessed by Western blotting and RT-PCR.Cleaved caspase-3-positive cells at ischemic penumbra of cerebral cortex were detected through immunohistoehemistry method.Results Comparison in the group showed that at the time of 3 d postoperation neurological deficit scores ofIC group （1.67±0.58） and EA group （1.14±0.37） were lower than at the time of 2 h,1 d and 2 d postoperation（P ＜ 0.05）.EA group scored significantly lower than IC group in terms of neurological deficit at the time of 2 d and 3 d postoperation（P ＜ 0.05）.At 3 d postoperation,infarct volume of EA group reduced significantly（P ＜ 0.05）.In SC,IC and EA group apoptotic nerve cells percentage were （1.07 ± 0.02） ％,（39.4 ±10.1）％,（15.1 ±4.2）％,respectively.Compared with IC group,the number of apoptotic nerve cells reduced greatly in EA group（P ＜ 0.05）.The expressions of Bcl-2 at the protein and mRNA levels in IC and EA groups were lower than SC group（P ＜ 0.05）,however those in EA group were higher than those in IC group（P ＜ 0.05）.On the contrary,the expressions of Bax at protein and mRNA levels in IC and EA groups were higher than that in SC group （P ＜ 0.05）.Compared with IC group the Bax expression was inhibited in EA group（P ＜ 0.05）.Moreover,the number of cleaved caspase-3-positive cells in EA group decreased significantly compared with IC group （P ＜ 0.05）.Conclusions EA treatment at acupoints of Zusanli （ST36） and Quchi （LI1 1） exerted the neuroprotective effect on the cerebral IR injury through the modulation of related factor of mitochondrial apoptosis pathway.