玄参环烯醚萜苷对氧糖剥夺再灌注诱导的原代皮层神经元细胞内质网应激的作用研究

Effect of iridoid glycosides of Scrophulariae Radix on endoplasmic reticulum stress induced by oxygen glucose deprivation and reperfusion in primary cortical neurons

目的探讨玄参环烯醚萜苷通过内质网应激介导的细胞凋亡通路对氧糖剥夺再灌注(OGD/R)诱导的原代皮层神经元细胞的作用及机制。方法分离SD大鼠原代皮层神经元,用玄参环烯醚萜苷(50、100、200 μg/mL)对原代皮层神经元进行预处理24 h,采用OGD/R诱导制备脑缺血再灌注细胞模型,倒置显微镜下鉴定细胞纯度,观察细胞形态变化;MTT法检测细胞存活率;试剂盒检测细胞内乳酸脱氢酶(LDH)水平和超氧化物歧化酶(SOD)活性;流式细胞术检测细胞凋亡率;Western blotting法检测C/EBP同源蛋白(CHOP)、葡萄糖调节蛋白-78(GRP78)和Caspase-12蛋白表达。结果培养的原代皮层神经元胞体饱满,状态良好,纯度较高。与对照组比较,经OGD/R处理后原代皮层神经元整体回缩变圆,胞体表面变粗糙;细胞存活率、SOD活性显著降低;LDH水平、细胞凋亡率显著升高;CHOP、Caspase-12和GRP78蛋白表达水平显著升高。与模型组比较,玄参环烯醚萜苷预处理能改善细胞损伤情况;提高细胞存活率、SOD活性;降低LDH水平和细胞凋亡率;降低CHOP、Caspase-12和GRP78蛋白表达水平。结论玄参环烯醚萜苷能拮抗OGD/R诱导的原代皮层神经元神经损伤,其作用机制与抑制内质网应激介导的细胞凋亡有关。

Objective To investigate the effects and mechanisms of iridoid glycosides of Scrophulariae Radix (IGRS) via endoplasmic reticulum stress-mediated apoptosis pathway on the primary cortical neurons induced by oxygen glucose deprivation/reperfusion (OGD/R). Methods Newborn SD rats were performed primary cortical neurons culture. And the primary cortical neurons were pretreated with IGRS (50, 100, and 200 μg/mL) for 24 h, and the in vitro model of oxygen-glucose deprivation/reoxygenation (OGD/R) was applied. The neurons purity and morphology were observed under inverted microscope, the cell viability was detected by MTT assay;the intracellular lactate dehydrogenase (LDH) level and superoxide dismutase (SOD) activity were detected by commercial kit. The apoptotic rate was detected by flow cytometry. The expression of C/EBP homologous protein (CHOP), glucose-regulated protein-78 (GRP78) and Caspase-12 protein were detected by western blotting. Results The cultured primary cortical neurons were plump with high purity in good condition. Compared with the control group, the primary cortical neurons were retracted and rounded after OGD/R treatment, and the surface of the neurons became rough;The cell viability and SOD activity were significantly decreased;The LDH level and apoptotic rate were evidently increased;The expression of CHOP, Caspase-12, and GRP78 were significantly increased. Compared with the model group, IGRS could relieve neurons damage, increase cell viability and SOD activity, decrease LDH level and apoptotic rate, and down-regulate the expression of CHOP, Caspase-12, and GRP78. Conclusion IGRS can antagonize the neuronal damage induced by OGD/R in primary cortical neurons, and its mechanism is related to the inhibition of endoplasmic reticulum stress-mediated apoptosis.