柯诺辛碱对创伤性脑损伤大鼠线粒体自噬的影响

Effects of Corynoxine on Mitophagy in Rats with Traumatic Brain Injury

目的:探究柯诺辛碱(Cor)对创伤性脑损伤(TBI)大鼠的治疗作用及其对线粒体自噬的影响。方法:将80只大鼠随机分为假手术组(n=10)、造模组(n=70)。假手术组大鼠仅切开皮肤,不进行造模;造模组大鼠建立TBI大鼠模型。将58只造模成功大鼠随机分为模型组(n=12)、低剂量组(n=12)、中剂量组(n=12)、高剂量组(n=11)、抑制剂组(n=11)。低、中、高剂量组大鼠分别予低[5 mg/(kg·d)]、中[10 mg/(kg·d)]、高[20 mg/(kg·d)]剂量柯诺辛碱治疗,抑制剂组大鼠予柯诺辛碱[20 mg/(kg·d)]和自噬抑制剂3-甲基腺嘌呤(3-MA)[15 mg/(kg·d)]治疗。给药14 d后,采用改良神经功能缺损评分法进行神经功能评分,采用Morris水迷宫实验评价认知功能,采用蔗糖偏好实验和旷场实验评价行为学。通过苏木素-伊红(HE)染色和TUNEL染色评价脑组织损伤和神经元凋亡。检测脑组织含水量、脑组织氧化应激指标[超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)和丙二醛(MDA)]水平。采用Western blotting检测脑组织Bax、Bcl-2、cleaved Caspase-3、LC3B、p62和Beclin-1蛋白表达。结果:低、中、高剂量组大鼠的神经功能评分、逃避潜伏期、脑组织含水量、TUNEL阳性率、MDA水平,以及Bax、cleaved Caspase-3、p62蛋白表达水平均低于模型组(P<0.05);低、中、高剂量组大鼠穿越平台次数、蔗糖偏好率、水平活动分数、垂直活动分数,SOD、CAT和GSH-Px水平,以及Bcl-2、LC3B-Ⅱ/LC3B-Ⅰ、Beclin-1蛋白表达水平均高于模型组(P<0.05)。抑制剂组大鼠的神经功能评分、逃避潜伏期、脑组织含水量、TUNEL阳性率、MDA水平,以及Bax、cleaved Caspase-3、p62蛋白表达水平均高于高剂量组(P<0.05);抑制剂组大鼠穿越平台次数、蔗糖偏好率、水平活动分数、垂直活动分数,SOD、CAT和GSH-Px水平,以及Bcl-2、LC3B-Ⅱ/LC3B-Ⅰ、Beclin-1蛋白表达水平均低于高剂量组(P<0.05)。结论:柯诺辛碱可有效减轻TBI后的继发性损伤,抑制神经元凋亡和氧化应激,其机制可能与激活线粒体自噬有关。

Objective:To reveal the therapeutic effect of Corynoxine(Cor)on traumatic brain injury(TBI)rats and its effect on mitophagy.Methods:Totally 80 rats were randomly divided into sham group(n=10)and modeling group(n=70).Rats in sham group only had skin incisions without modeling,while rats in modeling group established a TBI rat model.The 58 rats with successful modeling were randomly divided into model group(n=12),low dose group(n=12),middle dose group(n=12),high dose group(n=11)and inhibitor group(n=11).Rats in low,middle and high dose groups were treated with low[5 mg/(kg·d)],middle[10 mg/(kg·d)]and high[20 mg/(kg·d)]doses of corynoxine respectively.Rats in inhibitor group were treated with corynoxine[20 mg/(kg·d)]and autophagy inhibitor 3-MA[15 mg/(kg·d)].After 14 days of administration,the improved neurological deficit scoring method was used to score the neurological function,and the Morris water maze test was used to evaluate the cognitive function.The sucrose preference test and the open field test were used to evaluate the behavior.Brain tissue injury and neuronal apoptosis were evaluated by hematoxylin and eosin(HE)staining and TUNEL staining.The water content of brain tissue and the levels of oxidative stress indicators[superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GSH-Px)and malondialdehyde(MDA)]in brain tissue were detected.The protein expressions of Bax,Bcl-2,cleaved Ccaspase-3,LC3B,p62 and Beclin-1 in brain tissue were detected by Western blotting.Results:The neurological function score,escape latency,brain water content,TUNEL positive rate,MDA level,Bax,cleaved Caspase-3,and p62 protein levels in low,middle and high dose groups were lower than those in model group,while the times of crossing the platform,sucrose preference,horizontal activity score,vertical activity score,SOD,CAT,GSH-Px,Bcl-2,LC3B-Ⅱ/LC3B-Ⅰand Beclin-1 protein levels were higher than those in model group(P<0.05).The neurological function score,escape latency,brain water content,TUNEL positive rate,MDA,cleaved Caspase-3 and p62 protein levels in inhibitor group were higher than those in high dose group,while the platform crossing times,sucrose preference,horizontal activity fraction,vertical activity fraction,SOD,CAT,GSH-Px,Bcl-2 Bcl-2,LC3B-Ⅱ/LC3B-Ⅰand Beclin-1 protein levels in the inhibitor group were lower than those in high dose group(P<0.05).Conclusion:Corynoxine can effectively reduce the secondary injury after TBI,inhibit neuronal apoptosis and oxidative stress,and its mechanism may be related to the activation of mitophagy.