针康法调控细胞自噬改善脑缺血再灌注大鼠神经损伤的机制研究

Mechanism of Acupuncture-Rehabilitation Therapy in Regulating Autophagy to Improve Nerve Injury in Rats with Cerebral Ischemia Reperfusion

目的:观察针康法对脑缺血再灌注模型大鼠神经损伤的干预效果及对细胞自噬的影响。方法:选用清洁级雄性健康SD大鼠90只,随机分为假手术组、模型组、针刺组、康复组和针康组5组,每组各18只。Longa改良线栓法建立大鼠局灶性脑缺血再灌注模型(MCAO),评定造模成功后分组给予针刺、康复及针康法干预治疗。于治疗第6 h、3天、7天与14天分别进行改良的Bederson s神经功能评分,于治疗后TTC染色观察脑梗死灶面积,免疫组化检测脑组织Beclin1、LC3-Ⅱ阳性细胞分布情况,荧光实时定量PCR和Western blot检测脑组织Beclin1、LC3-Ⅱ蛋白及mRNA表达。结果:不同时间点模型组、针刺组、康复组和针康组大鼠的Bederson s神经功能评分均较假手术组明显升高,差异具有统计学意义(P<0.05);治疗后相同时间点针刺组、康复组和针康组大鼠的Bederson s神经功能评分均较模型组降低,并在第3天、7天和14天时呈现显著性差异,差异具有统计学意义(P<0.05),且随时间呈现下降趋势。TTC染色和免疫组化结果可见,模型组、针刺组、康复组和针康组大鼠的脑梗死百分比和Beclin1、LC3-Ⅱ阳性细胞计数均较假手术组明显增加,差异具有统计学意义(P<0.05);针刺组、康复组和针康组大鼠的脑梗死面积和Beclin1、LC3-Ⅱ阳性细胞计数均较模型组明显减少,差异具有统计学意义(P<0.05),其中以针康组的脑梗死百分比和阳性细胞计数为最低。Western blot和荧光实时定量PCR结果显示,模型组、针刺组、康复组和针康组大鼠的Beclin1、LC3-Ⅱ蛋白及mRNA表达均较假手术组明显升高,差异具有统计学意义(P<0.05);针刺组、康复组和针康组大鼠的Beclin1、LC3-Ⅱ蛋白及mRNA表达均较模型组明显降低,差异具有统计学意义(P<0.05),其中以针康组大鼠降低最为明显。结论:针康法可通过抑制细胞自噬改善MCAO大鼠神经损伤,发挥脑缺血再灌注后神经保护作用。

Objective:To observe the effect of acupuncture-rehabilitation therapy on nerve injury and autophagy in cerebral ischemia reperfusion model rats.Methods:90 clean male healthy SD rats were randomly divided into the sham operation group,the model group,the acupuncture group,the rehabilitation group and the acupuncture-rehabilitation group,with 18 rats in each group.The model of middle cerebral artery occlusion was established by modified Longa threading method.After successful modeling,the rats were correspondingly given the interventions of acupuncture,rehabilitation and acupuncture-rehabilitation.The modified Bederson s neurological score was assessed at 6h,3d,7d and 14d after the treatment,respectively.TTC staining was used to observe the area of cerebral infarction after the treatment.Immunohistochemistry was used to detect the distribution of Beclin1 and LC3-Ⅱpositive cells in brain tissues.Fluorescence real-time PCR and Western blot were used to detect the protein and mRNA expressions of Beclin1 and LC3-Ⅱin brain tissues.Results:The modified Bederson s neurological function scores were significantly higher in the model group,the acupuncture group,the rehabilitation group and the acupuncture-rehabilitation group than those in the sham operation group at different time points(P<0.05);the modified Bederson s neurological function scores were all lower in the acupuncture group,the rehabilitation group and the acupuncture-rehabilitation group than those in the model group at the same time point after the treatment,which showed significant differences on 3rd,7th and 14th days(P<0.05),and manifested as a decreasing trend over time.TTC staining and immunohistochemical results showed that the percentage of cerebral infarction and Beclin1 and LC3-Ⅱpositive cells were significantly increased in the model group,the acupuncture group,the rehabilitation group and the acupuncture-rehabilitation group compared with those in the sham operation group(P<0.05).The percentage of cerebral infarct area and Beclin1 and LC3-Ⅱpositive cells were significantly decreased in the acupuncture group,the rehabilitation group and the acupuncture-rehabilitation group compared with those in the model group(P<0.05),and the acupuncture-rehabilitation group was the lowest in terms of the percentage of cerebral infarction and positive cells.Western blot and fluorescence real-time PCR results showed that the protein and mRNA expressions of Beclin1 and LC3-Ⅱwere significantly increased in the model group,the acupuncture group,the rehabilitation group and the acupuncture-rehabilitation group compared with those in the sham operation group(P<0.05);whereas the protein and mRNA expressions of Beclin1 and LC3-Ⅱwere significantly decreased in the acupuncture group,the rehabilitation group and the acupuncture-rehabilitation group compared with those in the model group(P<0.05),and the decreases were most significant in the acupuncture-rehabilitation group.Conclusion:Acupuncture-rehabilitation therapy can improve the nerve injury of MCAO rats by inhibiting autophagy and play a neuroprotective role after cerebral ischemia reperfusion.