眼针对脑缺血再灌注大鼠半暗带脑组织中Raf/MEK/Erk通路调控机制的实验研究

Experimental Study on Mechanism of Eye Acupuncture Regulating Raf/MEK/Erk Pathway in Cerebral Penumbra Tissue of Cerebral Ischemia-Reperfusion Rats

目的 观察眼针疗法对脑缺血再灌注(CIRI)大鼠半暗带脑组织中细胞凋亡和Bcl-2蛋白表达水平的影响,同时检测眼针对CIRI大鼠半暗带脑组织中Raf、p-MEK1/2、p-Erk1/2、p-p90RSK、p-CREB表达的影响,探讨眼针抑制CIRI大鼠半暗带脑神经元凋亡的作用机制。方法 SPF级SD大鼠60只,首先分为两组:假手术组(15只),造模组(45只),采用线栓法对造模组45只大鼠制备CIRI模型,模型评价后,将模型复制成功的大鼠随机分为3组:模型对照组、眼针组、穴区外对照组。模型复制后即刻开始进行干预:假手术组不做处理;模型对照组大鼠进行捆绑固定,但不进行针刺干预;眼针组大鼠分别在眼周肝胆区、肾膀胱区、上焦区、下焦区进行针刺干预;穴区外对照组大鼠分别在眼周相应穴区外的2 mm处进行针刺干预,连续干预1周。末次针刺后1 h,采用Zea Longa法评价各组大鼠神经功能缺损评分。然后过量麻醉法处死大鼠,每组大鼠随机选取6只,取全脑,4%多聚甲醛溶液固定;剩余大鼠,取全脑,并分离半暗带脑组织,冻存于-80℃冰箱,备用。采用免疫印迹(Western blot)法检测各组大鼠半暗带脑组织中Bcl-2、Raf、p-MEK1/2、p-Erk1/2、p-p90RSK、p-CREB的表达水平;采用TUNEL法及免疫组织化学法检测各组大鼠半暗带脑脑组织中细胞凋亡及Bcl-2表达的水平。结果 与假手术组比较,模型对照组、眼针组、穴区外对照组大鼠神经功能缺损评分及半暗带脑组织中细胞凋亡数、Raf、p-MEK1/2、p-Erk1/2、p-p90RSK、p-CREB蛋白表达水平显著升高(P<0.01),半暗带脑组织中bcl-2蛋白表达水平显著降低(P<0.01);与模型对照组比较,眼针组大鼠神经功能缺损评分及半暗带脑组织中细胞凋亡数、Raf、p-MEK1/2、p-Erk1/2、p-p90RSK、p-CREB蛋白表达水平显著降低(P<0.01),半暗带脑组织中Bcl-2蛋白表达水平显著升高(P<0.01);模型对照组与穴区外对照组大鼠神经功能缺损评分、细胞凋亡数、Bcl-2、Raf、p-MEK1/2、p-Erk1/2、p-p90RSK、p-CREB表达水平差异均无统计学意义。结论 眼针疗法能够改善CIRI大鼠神经功能缺损评分,减少CIRI大鼠半暗带脑组织细胞凋亡的发生,该作用可能是通过抑制Raf/MEK/Erk信号通路,上调Bcl-2蛋白的表达而实现的。

Objective To observe the effects of eye acupuncture therapy on apoptosis and Bcl-2 protein expression in cerebral penumbra tissue of cerebral ischemia-reperfusion(CIRI) rats and detect the effects of eye therapy on expressions of Raf,P-Mek1/2,P-Erk1/2,P-P90 RSK and P-CREb in cerebral penumbra tissue of CIRI rats.To investigate the mechanism of eye acupuncture inhibiting apoptosis of brain neurons in CIRI rats penumbra.Methods Sixty SPF SD rats were first divided into two groups:sham operation group(n=15) and model group(n=45).Forty-five rats in the model group were used to make CIRI model by wire occlusion method.After model evaluation,rats with successful model were randomly divided into three groups:model control group,eye acupuncture group and outside the acupoint area control group.The intervention was initiated immediately after model replication.The sham operation group was not treated.The rats in the model control group were bound and fixed without acupuncture intervention.In the eye acupuncture group,acupuncture intervention was performed in the liver and gallbladder area around the eye,the kidney and bladder area,the upper coke area and the lower coke area.The rats in the outside the acupoint area control group were treated with acupuncture intervention at 2 mm outside the corresponding acupoint area around the eye,and the intervention lasted for 1 week.1 hour after the last acupuncture,Zea Longa method was used to evaluate the neurological deficit score of rats in each group.Then the rats were sacrificed by excessive anesthesia.Six rats were randomly selected from each group.The whole brain was taken out and fixed with 4% paraformaldehyde solution.In the remaining rats,the whole brain was removed,and the penumbra brain tissue was separated,and frozen in the-80 ℃ refrigerator for future use.The expression levels of Bcl-2,Raf,P-MeK1/2,P-ErK1/2,P-P90 RSK and P-CREb in penumbra were detected by Western blot.Apoptosis and expression of Bcl-2 in penumbra were detected by TUNEL and immunohistochemistry.Results Compared with those of the sham operation group,the nerve function deficit score,the number of apoptotic cells and the protein expression levels of Raf,P-Mek1/2,P-Erk1/2,P-P90 RSK and P-CREB in the brain tissue of the model control group,eye acupuncture group and outside the acupoint area control group were significantly increased(P<0.01).The expression level of Bcl-2 protein in penumbra brain tissue was significantly decreased(P<0.01),and the difference was statistically significant.Compared with those of the model control group,the nerve function deficit score,the apoptotic cell number,and expression levels of Raf,P-Mek1/2,P-ERk1/2,P-P90 RSK and P-CREB protein in penumbra brain tissue of rats in the eye acupuncture group were significantly decreased(P<0.01).The expression level of Bcl-2 protein in penumbra brain tissue was significantly increased(P<0.01),and the difference was statistically significant.There were no significant differences in nerve function deficit score,apoptosis number,the expressions of Bcl-2,Raf,P-Mek1/2,P-ERk1/2,P-P90 RSK or P-CREB between the model control group and the outside the acupoint area control group.Conclusions Eye acupuncture therapy can improve the neurological deficit score of CIRI rats and reduce the occurrence of apoptosis in the penumbra brain tissue of CIRI rats,which may be achieved by inhibiting the Raf/MEK/Erk signaling pathway and up-regulating the expression of Bcl-2 protein.