通窍活血汤灌胃对小鼠颅脑损伤的改善作用及其机制

Effect and mechanism of Tongqiao Huoxue decoction on neurological function recovery for mice with traumatic brain injury

目的观察通窍活血汤灌胃对小鼠颅脑损伤的改善作用,并探讨其机制。方法60只小鼠随机分为假手术组、模型组和治疗组各20只,模型组和治疗组小鼠采用改良自由落体法建立颅脑损伤模型。建模后6 h时,三组均给予凝血酶、甘露醇尾静脉注射,治疗组同时给予通窍活血汤灌胃。建模后24 h时,使用改良的神经功能缺损评分(mNSS评分)评估各组小鼠神经功能损伤程度。评估后每组取3只小鼠处死,取损伤侧脑组织,分别称取湿重和干重后,计算小鼠脑组织水含量。评估后每组取3只小鼠处死,取损伤侧脑组织,TUNEL法检测小鼠脑组织细胞凋亡率,PCR法检测小鼠脑组织线粒体DNA(mtDNA),Western Blotting法检测小鼠脑组织中自噬相关蛋白LC3-Ⅱ、LC3-Ⅰ、p62及锰超氧化物歧化酶(MnSOD)、细胞色素C氧化酶Ⅳ亚型(COXⅣ)、切割活化的半胱天冬酶-3(cleaved Caspase-3)、凋亡抑制基因Bcl-2蛋白。结果假手术组、模型组、治疗组mNSS评分分别为(2.00±0.47)、(15.00±0.47)、(6.00±0.94)分,组间相比,P均<0.05。假手术组、模型组、治疗组脑组织水含量分别为74.4%±0.36%、84.2%±0.64%和79.5%±0.70%,组间相比,P均<0.05。假手术组、模型组、治疗组细胞凋亡率分别为9.60%±1.87%、35.70%±6.61%和21.50%±5.19%,组间相比,P均<0.05。假手术组、模型组、治疗组脑组织mtDNA相对表达量分别为1.028±0.233、0.040±0.003和0.510±0.201,组间相比,P均<0.05。与假手术组相比,模型组和治疗组LC3Ⅱ/Ⅰ、MnSOD、COXⅣ、Bcl-2蛋白相对表达量均显著降低,p62、cleaved Caspase-3蛋白相对表达量均显著升高;与模型组相比,治疗组LC3Ⅱ/Ⅰ、MnSOD、COXⅣ、Bcl-2蛋白相对表达量均显著升高,p62、cleaved Caspase-3蛋白相对表达量均显著降低。结论通窍活血汤灌胃可改善小鼠颅脑损伤情况,其作用机制可能与激活线粒体自噬、抑制细胞凋亡有关。

Objective To observe the effect of Tongqiao Huoxue decoction on traumatic brain injury mice and to explore its mechanism.Methods Sixty mice were randomly divided into the sham operation group,model group,and treatment group.The mice in the model group and the treatment group were modified with the free-fall method to establish the brain injury models.At 6 h after modeling,the mice in the three groups were given thrombin and mannitol via tail vein injection,while the mice in the treatment group were given Tongqiao Huoxue decoction at the same time.At 24 h after modeling,the neurological impairment of mice in each group was assessed by using the modified neurological deficit score(mNSS score).After the evaluation,3 mice in each group were sacrificed,and the injured lateral brain tissues were taken.The wet and dry weights of the mice were weighed and the water content in the brain tissues of the mice was calculated.After evaluation,3 mice in each group were sacrificed and the injured lateral brain tissue was taken.The TUNEL method was used to detect the apoptosis rate of mouse brain tissue cells.PCR was used to detect the mitochondrial DNA(mtDNA)in the mouse brain tissues.Western blotting was used to detect autophagy-related proteins LC3-Ⅱ,LC3-Ⅰ,p62 and manganese superoxide dismutase(MnSOD),cytochrome C oxidase subtype IV(COXⅣ),cleaved activated Caspase-3(cleaved Caspase-3),and Bcl-2 protein.Results The mNSS scores of the sham operation group,the model group,and the treatment group were(2.00±0.47),(15.00±0.47),and(6.00±0.94)points,respectively,with statistically significant differences between groups(all P<0.05).The water content in the brain tissues of the sham operation group,model group,and treatment group were 74.4%±0.36%,84.2%±0.64%and 79.5%±0.70%,respectively,with statistically significant differences between groups(all P<0.05).The apoptotic rates of the sham operation group,model group,and treatment group were 9.60%±1.87%,35.70%±6.61%and 21.50%±5.19%,respectively,with statistically significant differences between groups(all P<0.05).The relative expression levels of mtDNA in the brain tissues of sham operation group,model group,and treatment group were 1.028±0.233,0.040±0.003 and 0.510±0.201,respectively,with statistically significant differences between groups(all P<0.05).Compared with the sham operation group,the relative expression levels of LC3Ⅱ/Ⅰ,MnSOD,COXⅣ,and Bcl-2 proteins decreased significantly in the model group and the treatment group,and the relative expression levels of p62 and cleaved Caspase-3 proteins increased significantly.Compared with the treatment group,the relative expressions of LC3Ⅱ/Ⅰ,MnSOD,COXⅣ,and Bcl-2 proteins increased significantly,and the relative expression levels of p62 and cleaved Caspase-3 proteins decreased significantly.Conclusion Tongqiao Huoxue decoction may alleviate the brain injury in mice by activating mitochondrial autophagy and inhibiting apoptosis.