白花丹参带药血浆预处理对氧糖剥夺损伤的神经元的保护效应:抑制细胞凋亡

Protective effect of pretreatment of Salvia miltiorrhiza Bunge. f. alba plasma against oxygen-glucose deprivation-induced injury of cultured rat hippocampal neurons by inhibiting apoptosis

本文旨在观察白花丹参带药血浆预处理对乳鼠海马神经元氧糖剥夺(oxygen-glucose deficiency,OGD)损伤的影响及机制。培养乳鼠(出生12h)海马神经元,分为正常血浆组、低、中、高浓度白花丹参血浆组及空白对照组,前4组细胞经生理盐水或白花丹参灌胃的大鼠血浆(2.5%,5%and10%)预处理后,用含1mmol/L Na2S2O4的无糖Earle's液培养大鼠新生鼠海马神经元1.5h建立OGD模型,空白对照组正常血浆培养,不复制OGD模型。应用免疫荧光、激光共聚焦显微镜等检测神经元生存率、凋亡、Bcl-2/Bax表达水平。MTT及Annexin V/PI检测显示:空白对照组细胞凋亡少见,与正常血浆组、低浓度白花丹参血浆组相比,中、高浓度白花丹参血浆组细胞生存率提高(P<0.05),凋亡减少(P<0.05),其中高浓度白花丹参血浆组细胞生存率提高、凋亡减少更明显。中、高浓度白花丹参血浆组Bcl-2表达显著高于正常血浆组、低浓度白花丹参血浆组和空白对照组,而Bax表达则相反,且中、高浓度白花丹参血浆两组间Bcl-2、Bax表达无显著差别;各组Bcl-2、Bax阳性细胞数量呈一致性变化趋势;高浓度白花丹参血浆组Bcl-2/Bax阳性细胞数比值高于其余各组(P<0.05或0.01)。以上结果提示,白花丹参带药血浆预处理能提高OGD损伤后神经元生存率,该效应与上调Bcl-2表达、下调Bax表达,提高Bcl-2/Bax比值,抑制细胞凋亡有关。

The present study was to investigate the effect of Salvia miltiorrhiza Bunge. f. alba （SMA） pharmacological pretreatment on apoptosis of cultured hippocampal neurons from neonate rats under oxygen-glucose deprivation （OGD）. Cultured hippocampal neurons were randomly divided into five groups （n = 6）： normal plasma group, low dose SMA plasma （2.5%） group, middle dose SMA plasma （5%） group, high dose SMA plasma （10%） group and control group. The hippocampal neurons were cultured and treated with plasma from adult Wistar rats intragastrically administered with saline or aqueous extract of SMA. The apoptosis of neurons was induced by glucose-free Earle’s solution containing 1 mmol/L Na2S2O4 and labeled by MTT and Annexin V/PI double staining. Moreover, protein expressions of Bcl-2 and Bax were detected by immunofluorescence. The results showed that few apoptotic cells were observed in control group, whereas the number of apoptotic cells was greatly increased in normal plasma group and low dose SMA plasma group. Both middle and high dose SMA plasma could protect cultured hippocampal neurons from apoptosis induced by OGD （P 0.05）. The protective effect of high dose SMA plasma was stronger than that of middle one （P 0.05）. Compared to control, normal plasma and low dose SMA plasma groups, middle and high dose SMA plasma groups both showed significantly higher levels of Bcl-2 （P 0.05 or 0.01）, whereas expressions of Bax was opposite. There were no significant differences of Bcl-2 and Bax expressions between middle and high dose SMA plasma groups. Number of Bcl-2and Bax-positive cells had similar tendency. Bcl-2/Bax （number of positive cells） ratio was higher in high dose SMA plasma group than those of all the other groups （P 0.05 or 0.01）. These results suggest that pharmacological pretreatment of blood plasma containing middle and high dose SMA could raise viability and inhibit apoptosis of OGD-injured hippocampal neurons by up-regulating the expression of Bcl-2 and down-regulating the expression of Bax.