PINK1-Parkin线粒体自噬在创伤性脑损伤小鼠中的作用

Role and mechanism of the PINK1-Parkin mediated mitophagy in the traumatic brain injury in mice

目的探讨PINK1-Parkin线粒体自噬在创伤性脑损伤(TBI)中减轻神经细胞线粒体功能障碍及凋亡的保护作用。方法36只小鼠随机分为野生对照组(野生小鼠接受假手术)(n=6);基因对照组(Parkin-/-小鼠接受假手术)(n=6);野生模型组(野生小鼠接受TBI)(n=18),并将其分为TBI 1、3、5 d组,各6只小鼠;基因模型组(Parkin-/-小鼠接受TBI)(n=6)。采用控制性皮层损伤法建立TBI模型。Western blot检测线粒体自噬相关蛋白(PINK1表达、Parkin移位)及凋亡相关蛋白(Bax、Bcl-2、caspase-3);JC-1法检测线粒体膜电位;TUNEL法检测神经细胞凋亡;干湿重比法检测脑水肿;mNSS评分评价小鼠神经功能缺损。结果与野生对照组比较,野生模型组PINK1及线粒体Parkin表达显著增加,细胞质Parkin表达显著下降,其中在第3天最为明显,分别为(326.0±21.9)%、(300.0±23.1)%及(57.0±6.6)%;与野生对照组比较,野生模型组第3天脑组织含水量、mNSS评分、神经细胞线粒体JC-1单体含量、凋亡率、cleaved caspase-3及Bax表达显著增加为(77.3±3.5)%、(9.7±1.5)分、(143.5±22.6)%、(16.3±3.9)%、(184.0±19.5)%及(201.0±21.7)%,Bcl-2表达显著下降为(77.0±5.1)%。与野生模型组比较,基因模型组第3天脑组织含水量、m NSS评分、神经细胞线粒体JC-1单体含量、凋亡率、cleaved caspase-3及Bax表达进一步显著增加为(83.0±4.3)%、(13.8±1.9)分、(194.5±26.1)%、(34.2±6.9)%、(297.0±15.0)%及(303.0±28.5)%,Bcl-2进一步显著下降为(57.0±7.0)%。结论在TBI小鼠中,PINK1-Parkin线粒体自噬通过减轻神经细胞线粒体功能障碍及凋亡发挥神经保护作用,针对PINK1-Parkin线粒体自噬的干预可能作为治疗TBI的潜在治疗靶点。

Objective To explore the protective effect of the PINK1-Parkin mediated mitophagy against mitochondrial dysfunction and neuronal apoptosis in the traumatic brain injury(TBI)in mice.Methods Mice were randomly divided into wild-control group(wild type mice received sham operation),gene-control group(Parkin-/- mice received sham operation),wild-model group(wild type mice received TBI)and gene-model group(Parkin-/- mice received TBI).The TBI model was established by the controlled cortical damage method.The mitopahgy related proteins(PINK1 and Parkin translocation)and apoptosis(Bax,Bcl-2 and caspase-3)related proteins were detected by western blot.The mitochondrial membrane potential was detected by JC-1.The water content of brain tissue was detected by dry/wet weight ratio method.The neurological deficit of mice was evaluated by mNSS score.The neuronal apoptosis was evaluated by TUNEL staining.Results Compared with the mice of the wild-control group,the expression of PINK1 and mitochondrial Parkin were significantly increased and the expression of cytoplasmic Parkin was significantly decreased in wild type mice;those changes were most obvious on the third day after TBI,respectively were(326.0±21.9)%,(300.0±23.1)% and(57.0±6.6)%.Compared with the mice of the wild-control group,the water content of brain tissue,mNSS score,JC-1 monomer content,neuronal apoptosis ratio,cleaved caspase-3 and Bax expression in mice of the wild-model group 3 days after TBI were respectively increased to(77.3±3.5)%,(9.7±1.5),(143.5±22.6)%,(16.3±3.9)%,(184.0±19.5)% and(201.0±21.7)%,and the Bcl-2 expression was decreased to(77.0±5.1)%.Compared with the mice of the wild-model group,the water content of brain tissue,mNSS score,JC-1 monomer content,neuronal apoptosis ratio,cleaved caspase-3 and Bax expression in the Parkin-/- mice 3 days after TBI respectively further increased to(83.0±4.3)%,(13.8±1.9),(194.5±26.1)%,(34.2±6.9)%,(297.0±15.0)% and(303.0±28.5)%,and the Bcl-2 expression was further decreased to(57.0±7.0)%.Conclusions PINK1-Parkin mediated mitophagy could protect against mitochondrial dysfunction and neuronal apoptosis in the mice of TBI.Targeting mitophagy might be a potential therapy for TBI.