针刺“水沟”“内关”对脑出血大鼠血肿周围脑组织细胞凋亡相关因子表达的影响

Effect of manual acupuncture of“Shuigou”(GV26)“Neiguan”(PC6)on neurological function and expression of apoptosis-related factors in brain tissues surrounding hematoma in intracerebral hemorrhage rats

目的:观察大鼠脑出血急性期血肿周围脑组织中含半胱氨酸的天冬氨酸水解酶Caspase-3、Caspase-9表达随时间的动态变化及针刺干预对其影响,探讨针刺“水沟”“内关”对大鼠脑出血急性期神经细胞凋亡的影响及其可能的神经保护机制。方法:健康雄性大鼠随机分为对照组、模型组、穴位组、非穴组,每组24只。采用自体非肝素抗凝动脉血二次注入大鼠尾状核制作急性脑出血模型,符合纳入标准的大鼠再按出血后6、24、48、72h分为4个时间点亚组。穴位组用提插捻转泻法刺激双侧“内关”,雀啄法强刺激“水沟”,两穴留针30min;非穴组用提插捻转泻法刺激双侧腋中线下5mm的2个非穴点,雀啄法强刺激尾骨尖左侧旁开3mm的非穴点。6、24h组于造模后大鼠清醒即刻干预1次,对应时间点取材;48、72h组每日干预1次,对应时间点取材。采用神经损伤评分(NSS)法对大鼠行为学进行评分;用免疫组织化学法检测血肿周围脑组织Caspase-3、Caspase-9蛋白表达情况。结果:与同时点对照组比较,模型组大鼠出血后各时点NSS、血肿周围脑组织中Caspase-3及Caspase-9蛋白表达均明显升高(P<0.01,P<0.05);与同时点模型组比较,穴位组72h亚组NSS、各时点血肿周围脑组织中Caspase-3及Caspase-9蛋白表达均明显降低(P<0.05);与同时点穴位组比较,非穴组72h亚组NSS、各时点血肿周围脑组织Caspase-3及Caspase-9蛋白表达均明显升高(P<0.05)。结论:针刺“水沟”“内关”能改善出血后大鼠神经功能缺损体征,还可能通过降低出血后脑组织中Caspase-3、Caspase-9蛋白表达水平,抑制出血后由Caspase家族介导的神经元凋亡的发生,拮抗出血后脑组织损伤,保护神经元功能。

Objective To observe the effect of manual acupuncture stimulation of“Shuigou”(GV26)and“Neiguan”(PC6)on neurological function and expression of Caspase-3and Caspase-9in brain tissues around the intracerebral hematoma in rats with acute intracerebral hemorrhage(ICH),so as to explore its possible mechanisms underlying improvement of ICH.Methods Ninety-six male SD rats were randomly divided into 4groups:control,model,acupoint and non-acupoint(24rats in each group).The ICH model was established by injection of the rat’s autologous blood into the caudate nucleus.According to the time-points of 6,24,48and 72hafter ICH,each of the 4groups was further divided into 4subgroups.For rats of the acupoint group,the PC6on both sides was manually stimulated by manipulating the needle with lifting-thrusting-twisting reducing techniques,while the GV26was stimulated with strong“sparrow-pecking”method for 10times,then,left the needles in the acupoints for 30min.For rats of the non-acupoint group,two non-acupoints:mid-spot below the bilateral axilla and the spot 3mm above the left side of the coccyx tip were stimulated with the same methods to PC6and GV26,respectively.For rats of the 6h and 24hsubgroups,the intervention was given once after waking up from modeling,and for those of the 48and 72hsubgroups,the intervention was conducted once a day for 2or 3times,respectively.The neurological severity score(NSS)was used to evaluate the degree of neurological function.The immunoactivity(expression)of Caspase-3and Caspase-9proteins of the hematoma focus of the brain was detected by immunohistochemistry.Results Following modeling,the NSS and the expression levels of Caspase-3and Caspase-9proteins in the brain tissues surrounding the hematoma at each time-points(6,24,48and 72h)after modeling were significantly increased in the model group relevant to the control group(P<0.01,P<0.05).Compared with the model group,the NSS at 72hand the expression levels of Caspase-3and Caspase-9proteins at 6,24,48and 72hwere significantly down-regulated in the acupoint group(P<0.05)rather than in the non-acupoint group(P>0.05).Conclusion Acupuncture of GV26and PC6can improve the neurological function in rats with ICH,which may be related to its function in reducing the expression of Caspase-3and Caspase-9proteins(apoptosis-related proteins)in the brain.