Effects of acupuncture plus mild hypothermia on apoptosis-related factors in rats with cerebral ischemia-reperfusion

Objective

To investigate the effect of acupuncture plus mild hypothermia on neurological function impairment score, cerebral infarct size and apoptosis-related factors in cerebral ischemia reperfusion injury (CIRI) rats.

Methods

Sixty healthy male Sprague-Dawley (SD) rats were routinely reared for 1 week. Ten rats were randomly selected as the sham operation group and 10 rats as the blank control group, while the remaining 40 rats were subjected to preparing the middle cerebral artery occlusion (MCAO) model by modified filament occlusion method. The 40 MCAO rats were further randomly divided into a model group, an acupuncture group, a mild hypothermia group and an acupuncture plus mild hypothermia group, with 10 rats in each group. Rats in the sham operation group, the blank control group and the model group did not accept treatment except binding; rats in the acupuncture group received acupuncture treatment; rats in the mild hypothermia group received mild hypothermia treatment; rats in the acupuncture plus mild hypothermia group received acupuncture and mild hypothermia treatment. 72 h after the treatment, neurological function impairment score was performed; the infarct area ratio was determined by 2,3,5-tripheyl tetrazolium chloride (TTC) staining; apoptosis of brain cells was observed by TUNEL method; the expressions of Bcl-2, Bax and Caspase-3 were detected by immunohistochemistry.

Results

Compared with the blank control group and the sham operation group, the neurological function impairment score, cerebral infarct area ratio, apoptosis, and the expressions of Bax and Caspase-3 in the model group were significantly increased, while the expression of Bcl-2 was significantly decreased, and there were significant between-group differences (all P<0.05). After the treatment, there were statistically significant differences among the treatment groups in the neurological function impairment score, cerebral infarct area ratio and apoptosis in the ischemic side of rats, as well as the expressions of Bcl-2, Bax and Caspase-3 (all P<0.05), and from the figures, tables and statistical analysis, it was found that a better tendency in the acupuncture plus mild hypothermia group than the acupuncture group or mild hypothermia group.

Conclusion

Acupuncture plus mild hypothermia can protect the brain cells by improving neurological function impairment, decreasing cerebral infarct area ratio, reducing the number of apoptotic cells in the ischemic area and regulating the expressions of apoptosis related proteins to inhibit apoptosis.

     

针刺联合亚低温对脑缺血再灌注损伤大鼠脑组织MAPK/ERK通路及凋亡相关因子的影响

目的：观察针刺联合亚低温对脑缺血再灌注大鼠脑组织MAPK/ERK通路及凋亡相关因子的影响,探讨 针刺联合亚低温治疗缺血性中风的机制。方法：参照Longa 线拴法复制并改良大脑中动脉缺血模型,将90只SD大鼠 随机分为空白组、假手术组、模型组、针刺组、亚低温组、针刺联合亚低温组,针刺及亚低温治疗72 h后,观察神 经功能缺损评分,采用2, 3, 5-苯氯化四氮唑(2, 3, 5-triphenyl-2H-tetrazolium chloride,TTC)染色检测梗死面积百分比, 免疫组织化学法检测Bcl-2及Bax表达水平,TUNEL法检测凋亡细胞,Western印迹检测大鼠缺血侧海马组织MEK-2, ERK1/2蛋白的磷酸化水平。结果：造模后,模型组大鼠神经功能缺损评分增加,梗死面积百分比升高,凋亡细胞及 Bax表达增多,Bcl-2表达减少,MEK-2,ERK1/2蛋白的磷酸化水平明显增高,与空白组及假手术组比较差异有统计 学意义(P<0.05或P<0.01)。经针刺及亚低温治疗后,各治疗组神经功能缺损评分、梗死面积百分比、Bax表达减少, 凋亡细胞及MEK-2和ERK1/2蛋白的磷酸化水平均明显降低,Bcl-2表达升高,与模型组比较差异有统计学意义(P<0.05 或P<0.01)。与针刺组比较,针刺联合亚低温组神经功能缺损评分,MEK-2和ERK1/2蛋白的磷酸化水平下降(P<0.05或 P<0.01)。与亚低温组比较,针刺联合亚低温组MEK-2和ERK1/2蛋白的磷酸化水平下降(P<0.01)。结论：针刺及亚低 温均可改善神经功能缺损,减少脑梗死面积百分比、细胞凋亡、Bax表达及升高Bcl-2表达,对脑组织起保护作用。其机 制可能是通过MAPK/ERK通路,降低MEK-2和ERK1/2蛋白的磷酸化水平及调节凋亡相关因子Bcl-2表达和Bax表达来实现 的。针刺联合亚低温在降低神经功能评分及下调脑缺血再灌注损伤大鼠MEK-2,ERK1/2 蛋白的磷酸化水平上优于针刺 或亚低温单独应用。     

Effect of acupuncture plus mild hypothermia on MAPK/ERK pathway of brain tissues in rats with cerebral ischemia-reperfusion injury

Objective

To observe the protective effect of acupuncture plus mild hypothermia on brain tissues in rats with cerebral ischemia-reperfusion injury (CIRI), and the influence on protein expression levels of phosphorylated Raf-1, MEK-2 and ERK1/2 in the mitogen-activated protein kinase (MAPK)/extracellular regulated protein kinases (ERK) pathway, and to explore the mechanism of acupuncture plus mild hypothermia therapy for the ischemic stroke.

Methods

Ninety Sprague-Dawley (SD) rats were randomly divided into a blank control group, a sham operation group, a model group, an acupuncture group, a mild hypothermia group and an acupuncture plus mild hypothermia group, 15 rats in each group. Except the rats in the blank control group, the remaining rats were used to prepare the middle cerebral artery occlusion (MCAO) models according to the modified occlusion method using lines, while only the occlusion lines were inserted without blocking the brain arteries of rats in the sham operation group. When the vital signs of rats were stable, rats in the blank control group did not receive any intervention; rats in the sham operation group and the model group received fastening without treatment; rats in the acupuncture group, the mild hypothermia group, and the acupuncture plus mild hypothermia group were treated with the corresponding therapeutic methods. 72 h later, observed neurologic injury score, evaluated infarction area ratio by 2,3,5-tripheyl tetrazolium chloride (TTC) staining, determined apoptosis by TUNEL assay, and measured the phosphorylated Raf-1, MEK-2 and ERK1/2 protein expression levels in rat ischemic hippocampal tissues by Western blot assay.

Results

Compared with the blank control group and the sham operation group, after modeling, the neurologic injury score, infarction area ratio and apoptotic cells were increased, and phosphorylated Raf-1, MEK-2 and ERK1/2 protein expression levels were significantly increased in the model group; the differences were statistically significant (P<0.05 or P<0.01). Compared with the model group, after acupuncture or mild hypothermia therapy, neurologic injury score and infarction area ratio were decreased; apoptotic cells and phosphorylated Raf-1, MEK-2 and ERK1/2 protein expression levels were significantly decreased; the differences were statistically significant (P<0.05 or P<0.01). Compared with the acupuncture group, neurologic injury score and phosphorylated Raf-1, MEK-2 and ERK1/2 protein expression levels were decreased in the acupuncture plus mild hypothermia group; differences between the groups were statistically significant (P<0.05 or P<0.01). Compared with the mild hypothermia group, phosphorylated Raf-1, MEK-2 and ERK1/2 protein expression levels decreased in the acupuncture plus mild hypothermia group, and differences were statistically significant (P<0.01).

Conclusion

Acupuncture or mild hypothermia therapy can improve neurologic injury, reduce infarction area and apoptosis, which brought about protective effect on the brain tissues, in the MCAO model. The protective effect of acupuncture plus mild hypothermia group is the strongest. The mechanism may involve the MAPK/ERK pathway, by reducing the phosphorylated Raf-1, MEK-2 and ERK1/2 protein expression levels.

     

针刺联合亚低温对脑缺血再灌注损伤大鼠脑组织p-Raf1、p-ERK1/2的影响

目的 观察针刺联合亚低温疗法对脑缺血再灌注大鼠神经功能缺损评分、 细胞凋亡及p-Raf1、p-ERK1/2的影响.方法 参照ZeaLonga线拴法复制大脑中动脉缺血模型(MCAO)并加以改良,50只SD大鼠随机分为空白组、假手术组、模型组、针刺组、针刺联合亚低温组,每组10只,针刺及针刺联合亚低温治疗72 h后,观察神经功能缺损评分、TUNEL法检测凋亡细胞,Western Blot法检测大鼠缺血侧脑组织磷酸化Raf-1、ERK1/2蛋白的表达水平.结果 造模后,模型组大鼠神经功能缺损评分升高、凋亡细胞增多,磷酸化Raf1、ERK1/2蛋白的表达水平明显增高,与空白组及假手术组比较差异有统计学意义(P<0.05或P<0.01).经治疗后,各治疗组神经功能缺损评分减少、凋亡细胞及磷酸化Raf1、ERK1/2蛋白的表达水平均明显降低,与模型组比较差异有统计学意义(P<0.01).与针刺组比较,针刺联合亚低温组神经功能缺损评分,磷酸化Raf-1、ERK1/2蛋白的表达水平下降,差异有统计学意义(P<0.05或P<0.01).结论 针刺及针刺联合亚低温均可改善神经功能缺损,调节p-Raf1、p-ERK1/2,减少细胞凋亡,对脑组织起保护作用,且联合组的脑保护作用更强.     

针刺联合亚低温对脑缺血/再灌注损伤大鼠Bcl-2、Bax、Caspase-3蛋白表达的影响

目的 探讨针刺联合亚低温方法对脑缺血/再灌注(ischemia/reperfusion, I/R)损伤大鼠梗死面积比及Bcl-2、Bax、Caspase-3 蛋白表达的影响. 方法 将 50 只 SD 健康雄性大鼠常规饲养 1 周后, 随机选取假手术组 10 只, 余 40 只用Zea Longa 线栓法制作大鼠大脑中动脉闭塞局灶脑I/R模型,待造模成功后,再将40只SD大鼠随机分为模型组、针刺组、亚低温组、针刺联合亚低温组,每组各10 只. 治疗72 h 后,使用TTC 染色检测脑梗死面积比、免疫组化法检测 Bcl-2、Bax、Caspase-3蛋白的阳性细胞数. 结果 与假手术组比较,模型组大鼠梗死面积比、Bax、Caspase-3蛋白表达水平明显增高,Bcl-2表达水平显著下降,差异有统计学意义(P<0.05 或P<0.01);与模型组比较,各治疗组大鼠梗死面积比、Bax、Caspase-3 蛋白表达水平明显降低,Bcl-2表达水平明显升高,差异有统计学意义(P<0.05或P<0.01);各治疗组之间Bcl-2、Bax、Caspase-3的差异无统计学意义(P>0.05),但脑梗死面积比针刺联合亚低温组较针刺组和亚低温组低,差异有统计学意义(P<0.05 或 P<0.01).结论 针刺联合亚低温治疗可通过减少脑梗死面积比、提高Bcl-2表达水平、降低Bax与Caspase-3蛋白表达从而实现对脑细胞的保护作用.