基于PI3K/AKT通路探讨水蛭、地龙提取物对MCAO/R小鼠脑缺血半暗带神经元的保护作用

Exploration of the protective effects of Leech and Earthworm extracts on cerebral ischemic penumbra neurons in MCAO/R mice based on PI3K/AKT pathway

[目的]探究水蛭和地龙提取物对MCAO/R小鼠脑缺血半暗带神经元的保护作用及机制。[方法]选用40只8周龄健康C57BL/cnc雄性小鼠,采用大脑中动脉线栓法制备脑缺血再灌注小鼠模型,造模成功后采用随机数字表法分为假手术组(Sham组)、脑缺血再灌注组(MCAO/R组)、水蛭提取物组、地龙提取物组。各给药组每日尾静脉注射给药1次,连续给药7 d。mNSS评分评价小鼠神经功能损伤,苏木精-伊红(HE)染色检测各组小鼠脑缺血半暗带组织病理变化,TUNEL染色检测小鼠脑缺血半暗带细胞凋亡率,Nissl染色观察神经元受损伤程度,蛋白免疫印迹(Western-Blot)法检测B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、Cleaved Caspase-3、Caspase-3、磷脂酰肌醇3激醇(PI3K)、蛋白激酶B(AKT)、p-AKT、神经元核蛋白(NeuN)以及微管相关蛋白2(MAP2)的蛋白表达情况,免疫荧光检测Bax,Bcl-2表达情况。[结果]与MCAO/R组比较,水蛭提取物组、地龙提取物组,神经功能缺损、组织形态、神经元损伤情况均有不同程度改善,上调脑缺血半暗带PI3K、Bcl-2、NeuN、MAP2蛋白表达及升高p-AKT/AKT和Bcl-2/Bax比率。[结论]水蛭、地龙提取物可以改善MCAO/R小鼠脑缺血半暗带神经元损伤,其机制可能是通过激活PI3K/AKT信号通路。

[Objective]To explore the protective effect and mechanism of leeches and earthworm extracts on cerebral ischemic penumbra neurons in MCAO/R mice.[Methods]Forty 8-week-old healthy C57BL/cnc male mice were selected to prepare cerebral ischemiareperfusion mouse models using the middle cerebral artery suture method.After successful modeling,they were divided into sham operation group(Sham)and brain by random number table method.Ischemia-reperfusion group(MCAO/R),leech extract group,earthworm extract group.Each administration group was administered by tail vein injection once a day for 7 consecutive days.mNSS score to evaluate the neurological damage of mice,HE staining to detect the pathological changes in the cerebral ischemic penumbra of each group of mice,TUNEL staining to detect the cell apoptosis rate in the cerebral ischemic penumbra of mice,Nissl staining to observe the degree of neuronal damage,Western Blot method to detect B lymphoma-2(Bcl-2),Bcl-2 related X protein(Bax),activated Caspase-3(Cleaved Caspase-3),Caspase-3,PI3K,AKT,p-AKT,neuron nuclear protein(NeuN)and microtubule-associated protein 2(MAP2)protein expression,immunofluorescence detection of Bax,Bcl-2 expression.[Results]Compared with the MCAO/R group,the neurological deficit,tissue morphology,and neuron damage in the leech extract group and ground dragon extract group were improved to varying degrees,and the cerebral ischemic penumbra PI3K and Bcl-2 were up-regulated,NeuN,MAP2 protein expression and increase the ratio of p-AKT/AKT and Bcl-2/Bax.[Conclusion]The extracts of leech and earthworm can ameliorate neuronal damage in the cerebral ischemic penumbra of MCAO/R mice,and the mechanism may be through activation of the PI3K/AKT signaling pathway.