人参皂苷Rg1调控Epac1/Rap1信号通路对缺血性脑卒中大鼠神经保护作用机制研究

Mechanism of neuroprotective effect of ginsenoside Rg1 regulating Epac1/Rap1 signaling pathway in rats with ischemic stroke

目的:研究人参皂苷Rg1对缺血性脑卒中大鼠的神经保护作用并探究其作用机制。方法:将84只13周龄左右的SPF级SD雄性大鼠随机分为7组(n=12):假手术组、模型组、Rg1低剂量组、Rg1中剂量组、Rg1高剂量组、Epac1激动剂组、Epac1抑制剂组。模型组、Rg1低、中、高剂量组、Epac1激动剂组、Epac1抑制剂组均采用线栓法建立永久性局灶脑缺血大鼠模型。Rg1低中高剂量组大鼠每天上午固定时间进行灌胃给药,Rg1低剂量组、Rg1中剂量组和Rg1高剂量组分别使用60、120、240μmol/L Rg1处理;Epac1激动剂组和Epac1抑制剂组大鼠每天上午固定时间进行腹腔注射给药,Epac1激动剂8-CPT使用浓度为1.0×10^(4)μmol/L,抑制剂ESI-09使用浓度为1.0×10^(5)μmol/L。连续给药两周后,将各组大鼠施以断头处死。分别采用TTC染色、尼氏染色、TUNEL染色、微量酶标法、Western blot法检测各组大鼠脑梗死体积、完整神经元数目、氧化损伤指标、细胞凋亡情况,以及NOX2、Epac1、Rap1、caspase3的蛋白表达水平。结果:与假手术组相比,模型组大鼠脑梗死体积明显增大,脑组织中完整的神经元数量明显减少,脑组织中神经元氧化损伤明显加重,神经细胞凋亡率明显升高,NOX2、Epac1、Rap1、Caspase3蛋白表达明显升高,差异有统计学意义(P<0.05);与模型组相比,Rg1低、中、高剂量组和Epac1抑制剂组大鼠脑梗死体积明显减少,脑组织中完整的神经元数量明显增多,神经细胞凋亡率明显降低,NOX2、Epac1、Rap1、Caspase3蛋白表达明显降低,差异有统计学意义(P<0.05)。结论:人参皂苷Rg1可以调控缺血性脑卒中后的Epac1/Rap1信号通路,减轻脑神经元的氧化应激,从而减轻神经功能损害,发挥对神经细胞的保护作用。

AIM:To investigate the neuroprotective effect of ginsenoside Rg1 on rats with ischemic stroke and to investigate its mechanism of action.METHODS:Eighty-four SPF-grade SD male rats at about 13 weeks of age were randomly divided into 7 groups(n=12):sham-operated group,model group,Rg1 low-dose group,Rg1 medium-dose group,Rg1 high-dose group,Epac1 agonist group,and Epac1 inhibitor group.The model group,Rg1 low,medium and high dose groups,Epac1 agonist group and Epac1 inhibitor group were all used to establish a permanent focal cerebral ischemia rat model.Rats in the Rg1 low,medium and high dose groups were treated with 60,120 and 240μmol/L Rg1 administered by gavage at a fixed time every morning.The rats in the Epac1 agonist and Epac1 inhibitor groups were administered intraperitoneally at a fixed time each morning with a concentration of 1.0×10^(4)μmol/L for the Epac1 agonist 8-CPT and 1.0×10^(5)μmol/L for the inhibitor ESI-09.After two weeks of continuous administration,the rats in each group were decapitated.The brain infarct volume,number of intact neurons,oxidative damage index,apoptosis,and protein expression levels of NOX2,Epac1,Rap1,and caspase3 in each group of rats were detected by TTC staining,Nissler staining,TUNEL staining,microenzyme labeling,and Western blotting method,respectively.RESULTS:Compared with the sham-operated group,the brain infarct volume of rats in the model group and Epac1 agonist group was significantly larger,the number of intact neurons in brain tissue was significantly reduced,the oxidative damage of neurons in brain tissue was significantly aggravated,the apoptosis rate of neuronal cells was significantly higher,and the expression of NOX2,Epac1,Rap1,and caspase3 was significantly higher,with statistically significant differences(P<0.05);compared with the model group,the brain infarct volume was significantly reduced in the Rg1 low,medium and high dose groups and Epac1 inhibitor group,the number of intact neurons in brain tissue was significantly increased,the apoptosis rate of neuronal cells was significantly reduced,and the expression of NOX2,Epac1,Rap1 and Caspase3 was significantly reduced,and the differences were statistically significant(P<0.05).CONCLUSION:Ginsenoside Rg1 can regulate the Epac1/Rap1 signaling pathway after ischemic stroke and attenuate the oxidative stress of brain neurons,thus reducing neurological impairment and exerting a protective effect on neuronal cells.