脑心通胶囊对缺糖缺氧损伤脑微血管内皮细胞的保护作用及其机制

Protective effects and mechanism of Naoxintong Capsules on rat brain microvascular endothelial cells(rBMECs) induced by oxygen-glucose deprivation

目的探讨脑心通胶囊(NXT)对乳鼠缺糖缺氧损伤脑微血管内皮细胞(rBMECs)的体外保护作用及其机制。方法原代培养rBMECs,进行兔抗鼠VⅢ因子鉴定,MTT筛选出NXT肠吸收液体外保护rBMECs的质量浓度范围,优选出3个质量浓度进行实验。实验设对照组、模型组、尼莫地平(200μg/mL)组、不同质量浓度(62.50、125.00、250.00 mg/L)NXT肠吸收液组、NXT肠吸收液(250.00 mg/L)和LY294002(20μmol/L,PI3K/Akt通路抑制剂)共作用组。倒置显微镜下观察rBMECs形态,按照ELISA试剂盒方法检测细胞上清液中乳酸脱氢酶(LDH)、基质金属蛋白酶9(MMP-9)的表达水平,Hoechst33342染色荧光显微镜观察细胞凋亡情况,流式细胞仪检测各组rBMECs的早期凋亡率,Western blotting法检测PI3K/Akt信号通路关键蛋白的表达情况。结果与模型组相比,NXT不同质量浓度给药组均能显著改善rBMECs的形态,降低细胞上清液中LDH、MMP-9的量,显著降低rBMECs的凋亡数和早期凋亡率,可显著抑制PI3K/Akt通路中p-Akt、Bcl-2的表达上调及Bax的表达下降,抑制Caspase-3活性。PI3K/Akt信号通路的特异性抑制剂LY294002的加入,阻断了该通路信号的传导,显著降低了NXT的保护作用。结论 NXT对缺糖缺氧致rBMECs损伤具有抗凋亡保护作用,其作用机制与PI3K/Akt信号通路有关。

Objective To investigate the protective effects and mechanism of Naoxintong Capsules（NXT） on primary cultured neonate rat brain microvascular endothelial cells（rBMECs） induced by oxygen-glucose deprivation. Methods The primary cultured rBMECs model was established and the identification of rabbit anti-rat VⅢ factor was carried out. MTT was used to screen out the concentration range of NXT intestinal absorption solution to pretect rBMEC in vitro, three doses were selected for experiment. The experimental groups were divided into control group, model group, nimodipine group（200 μg/mL, NXT intestinal absorption solution group（62.50 mg/L, 125.00 mg/L, and 250.00 mg/L）, and NXT intestinal absorption solution（250.00 mg/L） and LY294002（20 μmol/L, PI3 K/Akt pathway inhibitor） co-administration group. The morphology of rBMECs was observed under inverted microscope. The expression of lactate dehydrogenase（LDH） and matrix metalloproteinase 9（MMP-9） in the cell supernatant was detected by ELISA kit. The apoptosis was observed by Hoechst33342 staining fluorescence microscope. The early apoptotic rate of rBMECs in each group was detected by FCM, and the expression of PI3 K/Akt signaling pathway key proteins was detected by Western blotting. Results Compared with model group, the administration of NXT could significantly improve the morphology of rBMECs, decrease the intracellular levels of LDH and MMP-9, significantly reduce the number of apoptotic cells and early apoptotic rate of rBMECs, and inhibit the expression of p-Akt, Bcl-2 upregulation, decrease the expression of Bax, and inhibit caspase-3 activity. The addition of LY294002, a specific inhibitor of PI3 K/Akt signaling pathway, blocked the signal transduction of this pathway and significantly reduced the protective effect of NXT. Conclusion NXT have protective effects on rBMECs induced by oxygen-glucose deprivation, and its mechanism is related to the PI3 K/Akt signaling pathway.