基于nNOS途径的天麻素注射液改善甲基苯丙胺诱导大鼠神经毒性损伤的作用机制研究

Study on the Mechanism of Improvement Effects of Gastrodin Injection on Methamphetamine Induced Neurotoxic Damage in Rats via nNOS Pathway

目的:研究天麻素注射液通过神经型一氧化氮合酶(nNOS)途径改善甲基苯丙胺诱导大鼠神经毒性损伤的作用机制。方法:将SD大鼠随机分为对照组、甲基苯丙胺组、常规剂量天麻素组、加倍剂量天麻素组、阴性对照(NC)腺病毒组、NC腺病毒+甲基苯丙胺组、NC腺病毒+天麻素组、n NOS腺病毒+天麻素组,每组10只。对照组大鼠腹腔注射生理盐水,每日2次;甲基苯丙胺组大鼠腹腔注射甲基苯丙胺(7.5 mg/kg),每日2次;常规剂量、加倍剂量天麻素组大鼠提前30 min分别腹腔注射不同剂量天麻素注射液(10、20 mg/kg),每日1次,再按甲基苯丙胺组方法注射甲基苯丙胺。NC腺病毒组大鼠在纹状体一次性注射NC腺病毒(4.8×107PFU)3μL+腹腔注射生理盐水,每日2次;NC腺病毒+甲基苯丙胺组大鼠同法注射NC腺病毒,然后腹腔注射甲基苯丙胺(7.5 mg/kg),每日2次;NC腺病毒+天麻素组大鼠同法注射NC腺病毒+甲基苯丙胺,同时在注射甲基苯丙胺之前30 min腹腔注射天麻素注射液(20 mg/kg),每日1次;nNOS腺病毒+天麻素组大鼠同法注射nNOS腺病毒和甲基苯丙胺,同时在注射甲基苯丙胺之前30 min腹腔注射天麻素注射液(20 mg/kg),每日1次。各组大鼠每次腹腔注射体积均为1 mL/100 g,均连续注射给药3 d。观察大鼠刻板行为并评分,检测大鼠纹状体的细胞凋亡率、凋亡相关因子[Bcl-2、Bax、活化胱天蛋白酶-3(Cleaved caspase-3)]蛋白表达量、氧化应激相关因子[丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GPx)]水平,以及一氧化氮(NO)水平和nNOS蛋白表达量。结果:与对照组比较,甲基苯丙胺组大鼠的刻板行为评分和纹状体的细胞凋亡率、Bax、Cleaved caspase-3、nNOS蛋白表达量及MDA、NO水平均显著升高,Bcl-2蛋白表达量及SOD、GPx水平均显著降低(P<0.05或P<0.01);与甲基苯丙胺组比较,常规剂量、加倍剂量天麻素组大鼠的刻板行为评分和纹状体的细胞凋亡率、Bax、Cleaved caspase-3、n NOS蛋白表达量及MDA、NO水平均显著降低,Bcl-2蛋白表达量及SOD、GPx水平均显著升高,且加倍剂量天麻素组大部分上述指标均显著优于常规剂量组(P<0.05或P<0.01)。与NC腺病毒组比较,NC腺病毒+甲基苯丙胺组大鼠纹状体的细胞凋亡率、Bax、Cleaved caspase-3、n NOS蛋白表达量及MDA、NO水平均显著升高,Bcl-2蛋白表达量及SOD、GPx水平均显著降低(P<0.01);与NC腺病毒+甲基苯丙胺组比较,NC腺病毒+天麻素组大鼠纹状体中细胞凋亡率,Bax、Cleaved caspase-3、n NOS蛋白表达量以及MDA、NO水平均显著降低,Bcl-2蛋白表达量以及SOD、GPx水平均显著升高(P<0.01);与NC腺病毒+天麻素组比较,n NOS腺病毒+天麻素组大鼠纹状体中的细胞凋亡率、Bax、Cleaved caspase-3、n NOS蛋白表达量及MDA、NO水平均显著升高,Bcl-2蛋白表达量及SOD、GPx水平均显著降低(P<0.01)。结论:天麻素注射液对甲基苯丙胺诱导的大鼠神经毒性损伤具有明显的保护作用,且这一作用与抑制n NOS介导的细胞凋亡及氧化应激反应有关。

OBJECTIVE:To study the mechanism of improvement effects of Gastrodin injection on methamphetamine induced neurotoxic damage in rats via n NOS pathway.METHODS:SD rats were randomly divided into control group,methamphetamine group,regular-dose of gastrodin group,double-dose of gastrodin group,negative control(NC)adenovirus group,NC adenovirus+methamphetamine group,NC adenovirus+gastrodin group and nNOS adenovirus+gastrodin group,with 10 rats in each group.Control group was given normal saline intraperitoneally,twice a day.Methamphetamine group was given methamphetamine intraperitoneally(7.5 mg/kg),twice a day.Regular-dose and double-dose of gastrodin groups were respectively given different doses of Gastrodin injection(10,20 mg/kg)intraperitoneally 30 min earlier,once a day,and then given methamphetamine intraperitoneally by the same way as methamphetamine group.NC adenovirus group was given NC adenovirus(4.8×107 PFU)3μL once in the striatum and normal saline intraperitoneally,twice a day.NC adenovirus+methamphetamine group was given NC adenovirus by the same way and methamphetamine(7.5 mg/kg)intraperitoneally,twice a day.NC adenovirus+gastrodin group was given NC adenovirus+methamphetamine by the same way,meanwhile given Gastrodin injection intraperitoneally(20 mg/kg)30 min before methamphetamine,once a day.nNOS adenovirus+gastrodin group was given nNOS adenovirus and methamphetamine by the same way,meanwhile given Gastrodin injection intraperitoneally(20 mg/kg)30 min before methamphetamine,once a day.Each group was given relevant medicine intraperitoneally 1 m L/100 g,for consecutive 3 days.The stereotyped behavior of rats were observed and scored;the apoptotic rate,the protein expression of apoptotic factors(Bcl-2,Bax,Cleaved caspase-3),the levels of oxidative stress factors(MDA,SOD,GPx)and NO,the protein expression of nNOS were detected.RESULTS:Compared with control group,stereotyped behavior score,cell apoptosis rate of striatum,protein expression of Bax,Cleaved caspase-3 and nNOS,the levels of MDA and NO were increased significantly in methamphetamine group;while the protein expression of Bcl-2 and the levels of SOD and GPx were decreased significantly(P<0.05 or P<0.01).Compared with methamphetamine group,stereotyped behavior score,cell apoptosis rate of striatum,protein expression of Bax,Cleaved caspase-3 and nNOS,the levels of MDA and NO were decreased significantly in regular-dose and double-dose of gastrodin groups;while the protein expression of Bcl-2,the levels of SOD and GPx were increased significantly,and most above indexes in double-dose of gastradin group were better than regular-dose of gastrodin group(P<0.05 or P<0.01).Compared with NC adenovirus group,cell apoptosis rate of striatum,protein expression of Bax,Cleaved caspase-3 and nNOS,the levels of MDA and NO were increased significantly in NC adenovirus+methamphetamine group;while the protein expression of Bcl-2,the levels of SOD and GPx were decreased significantly(P<0.01).Compared with NC adenovirus+methamphetamine group,cell apoptosis rate of striatum,protein expression of Bax,Cleaved caspase-3 and nNOS,the levels of MDA and NO were decreased significantly in NC adenovirus+gastrodin group;while the protein expression of Bcl-2,the levels of SOD and GPx were increased significantly(P<0.01).Compared with NC adenovirus+gastrodin group,cell apoptosis rate of striatum,protein expression of Bax,Cleaved caspase-3 and nNOS,the levels of MDA and NO were increased significantly in nNOS adenovirus+gastrodin group;while the protein expression of Bcl-2,the levels of SOD and GPx were decreased significantly(P<0.01).CONCLUSIONS:Gastrodin injection can protect rats against neurotoxic damage induced by methamphetamine,and the effect is related to the inhibition of nNOS-mediated apoptosis and oxidative stress.