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目的:观察镇肝熄风汤含药血清对6-羟基多巴胺(6-hydroxydopamine,6-OHDA)诱导PC12细胞凋亡的影响,并探讨其是否通过抗氧化活性发挥神经保护作用。方法:将40只Wistar大鼠随机分为空白组、镇肝熄风汤低、中、高剂量组,每组10只。镇肝熄风汤按照8,16,32 g·kg~(-1)灌胃,空白组灌胃等体积生理盐水,采血制备含药和空白血清。常规培养PC12细胞,分为空白组、模型组、镇肝熄风汤低、中、高剂量组,空白组和模型组给予空白血清,其他3组给予10%含药血清,孵育1 h,再加100μmol·L~(-1)6-OHDA共孵育24 h后收集细胞。采用MTS法检测细胞活性,流式细胞术检测细胞凋亡,分光光度法检测脂质过氧化物(lipid peroxidation,LPO)水平,实时荧光定量PCR(Real-time PCR)检测醌氧化还原酶-1(NADPH quinine oxidoreductase 1,NQO-1)mRNA表达。结果:与空白组比较,模型组细胞活力明显降低,而细胞凋亡和LPO水平明显增加(P<0.05)。与模型组比较,10%的镇肝熄风汤低、中、高剂量含药血清组细胞活力显著增加,而细胞凋亡和LPO水平明显减少(P<0.05)。与模型组比较,10%的镇肝熄风汤低、中、高剂量含药血清组NQO-1 mRNA表达明显上调(P<0.05),而空白组与模型组NQO-1mRNA表达无显著性差异。结论:镇肝熄风汤含药血清具有保护6-OHDA诱导PC12细胞损伤的作用,其机制可能与上调PC12细胞NQO-1 mRNA表达进了提高了细胞抗氧化能力有关。
OBJECTIVE: To observe the effect of Zhengan Xifeng Decoction-containing serum on the apoptosis of PC12 cells induced by 6-hydroxydopamine (6-OHDA) and to explore whether it can play a neuroprotective role through antioxidant activity. Methods: Forty Wistar rats were randomly divided into blank group, Zhengan Xifeng Decoction low, middle and high dose group, 10 rats in each group. Zhengan Xifeng Decoction was given to rats at the doses of 8, 16, and 32 g · kg -1. The rats in the blank group were given gavage with equal volume of normal saline and blood was collected to prepare medicated and blank serum. The PC12 cells were routinely cultured and divided into blank group, model group, Zhengan Xifeng decoction low, medium and high dose groups, blank group and model group were given blank serum, the other three groups were given 10% serum, incubated for 1 h, and then Cells were harvested after incubation with 100 μmol·L -1 6-OHDA for 24 h. The cell viability was detected by MTS assay, apoptosis was detected by flow cytometry, the levels of lipid peroxidation (LPO) were detected by spectrophotometry and the levels of quinone oxidoreductase-1 by real-time PCR (NADPH quinine oxidoreductase 1, NQO-1) mRNA expression. Results: Compared with the blank group, the viability of the model group was significantly decreased, while the apoptosis and LPO levels were significantly increased (P <0.05). Compared with the model group, the cell viability of 10% Zhengan Xifeng decoction group at low, medium and high doses of serum was significantly increased, while the apoptosis and LPO levels were significantly decreased (P <0.05). Compared with the model group, NQO-1 mRNA expression was significantly up-regulated in 10% Zhengan Xifeng decoction group (P <0.05), but no significant difference was found between the blank group and the model group . CONCLUSION: Zhengan Xifeng Decoction-containing serum can protect PC12 cells from damage induced by 6-OHDA. The mechanism may be related to the up-regulation of NQO-1 mRNA expression in PC12 cells and the enhancement of antioxidant capacity.