金钗石斛生物碱激活自噬抑制谷氨酸诱导的大鼠海马神经元轴突变性

Alkaloids of Dendrobium nobile Lindl inhibits axonal degeneration induced by glutamate through activating autophay in hippocampus neurons of rats

目的观察金钗石斛生物碱(ADNL)对谷氨酸诱导的大鼠海马神经元轴突变性的保护作用,并探讨该保护作用与影响细胞自噬的关系。方法体外培养的大鼠海马神经元被随机分为对照组、模型组、ADNL(3.5、35、350μg·L^(-1)组、ADNL(350μg·L^(-1)+3^(-1)甲基腺嘌呤(3MA,1 mmol·L^(-1)或羟氯喹(HCQ,50μmol·L^(-1)组。谷氨酸(50μmol·L^(-1)诱导神经元轴突变性,βⅢTubulin免疫荧光观察轴突变性形态学改变并作定量分析,MTT比色法检测细胞活力,神经元过表达GFP^(-1)LC3B观察自噬体数量的变化,DQ^(-1)RED^(-1)BSA检测自噬体的降解,Western blot检测突触后密度蛋白^(-1)95(PSD95)、突触素(SYN)、LC3BⅠ、LC3BⅡ和p62蛋白水平。结果谷氨酸可诱导大鼠海马神经元轴突变性,变性指数显著增高,细胞活力、PSD95和SYN蛋白水平显著降低(P<0.05或P<0.01),除ADNL(3.5μg·L^(-1)对变性指数(72 h)、细胞活力和SYN蛋白水平未见显著影响(P>0.05)外,谷氨酸诱导的上述各指标的改变均被ADNL显著抑制,而且ADNL(350μg·L^(-1)对轴突变性的保护作用可被自噬抑制剂3MA拮抗。在谷氨酸诱导的轴突变性神经元中GFP^(-1)LC3B荧光斑点数量减少(P<0.01)、LC3BⅡ/LC3BⅠ比值降低(P<0.01),p62降解受阻(P<0.01),表明自噬功能被抑制。不同浓度的ADNL均可增加GFP^(-1)LC3B荧光斑点数量,增高LC3BⅡ/LC3BⅠ比值,促进p62和DQ^(-1)RED^(-1)BSA降解,其中对GFP^(-1)LC3B荧光斑点的影响呈浓度依赖性。ADNL(350μg·L^(-1)对自噬功能的影响可被自噬抑制剂3MA或HCQ拮抗。结论 ADNL通过激活自噬改善谷氨酸诱导的大鼠海马神经元轴突变性。

AIM To investigate the protective effects of alkaloids of Dendrobium nobile Lindl （ADNL） on axonal degeneration induced by glutamate （Glu） and it＇s potential mechanisms focusing on autophagy in hippocampus neurons of rats. METHODS Hippocampus neurons were divided randomly into control group, Glu （50μmol·L^-1） model group, ADNL （3.5, 35, 350μg·L^-1） group, ADNL （350 μg·L^-1） ＋ 3-methyladenine （3MA, 1 mmol·L-L） or hydroxychloroquine （50μmol·L^-1） group in vitro. The protective effects of ADNL （3.5, 35, 350 ng·mL-1） on axonal degeneration induced by Glu （50μmol·L^-1） in primary hippocampus neurons of rats in vitro were evaluated by measuring cell viability with MTT assay, degeneration index with immunofluorescence staining. The effects of ADNL on autophagy flux were checked by counting the number of autophagosome in hippocampus neurons overexpressing GFP-LC3B, measuring the degradation of DQ- RED- BSA. LC3B Ⅰ, LC3BⅡI , p62, postsynaptic density 95 （PSD95） and synaptophysin （SYN） protein levels were measured by Western blot. RESULTS Glu incubation led significantly to axonal degeneration, enhancement of degeneration index, decrease of cell viability and PSD95, SYN protein levels （P 〈 0.05 or P 〈 0.01） , which could be attenuated by ADNL pretreatment at the 3.5, 35, 350 μg ·L^-1 dose levels except for degeneration index （ADNL treatment for 72 h） , cell viability and SYN protein level at the 3.5 μg ·L^-1 level. Moreover, the protection of ADNL （350 μg ·L^-1） on axonal degeneration induced by Glu could be significantly inhibited by 3MA coincubation. In axonal degeneration model of neurons induced by Glu, there were a remarkable reduction of GFP- LC3B dots, the ratio of LC3B II to LC3B I protein levels, p62 protein degradation （P 〈 0.01）. ADNL pretreatment with concentration of 3.5, 35, 350 μg ·L^-1 prevented decrease of LC3B 11/LC3B I ratio in a concentration-dependent manner, the number of GFP-LC3B dots, promoted degradation of p62 and DQ- RED- BSA, which could also be inhibited significantly by 3MA and HCQ, respectively. CONCLUSION ADNL attenuates axonal degeneration induced by Glu through activating autophagy in hippocampus neurons of rats.