丹参酮ⅡA抑制马兜铃酸诱导HUVECs凋亡及其可能机制

Tanshinone ⅡA inhibits the apoptosis of HUVECs induced by aristolochic acid and its mechanism

目的观察丹参酮ⅡA(TSN)对马兜铃酸(AA)诱导的人脐静脉血管内皮细胞(HUVECs)凋亡的保护作用及其可能机制。方法体外培养HUVECs,设对照组、AA刺激组(AA终浓度为10 mg/L)、TSN干预组(先加入0.2、0.4和0.8 mg/L TSN,1 h后再加入AA)和LY294002预处理组(20μmol/L的PI3K抑制剂LY294002预处理30 min后,再加入TSN)。24 h后,MTT法检测细胞增殖;Hoechst33258荧光染色观察细胞形态;Annexin V-FITC/PI双荧光染色流式细胞仪检测细胞凋亡率;Western blot法检测细胞Bcl-2、Bax和磷酸化Akt(p-Akt)蛋白表达及比色法测定细胞caspase-3活性。结果与对照组相比,AA引起细胞凋亡率显著增加(P<0.05),细胞Bcl-2和p-Akt表达降低(P<0.05),细胞Bax表达升高(P<0.05);TSN能减轻AA对细胞凋亡率及对细胞Bcl-2、Bax和p-Akt表达的作用(P<0.05);PI3K抑制剂LY294002可抑制TSN的抗凋亡作用(P<0.05)。结论 TSN可抑制AA诱导的HUVECs凋亡。

Objective To observe the protective effect of tanshinone Ⅱ A （TSN） on the apoptosis of human umbili- cal vein endothelial cells（HUVECs） stimulated by aristolochic acid（AA） and its possible mechanism. Methods Cultivated HUVECs were divided into four groups： control group, AA group （the final concentration of AA was 10 mg/L）, TSN treatment group（HUVECs was added with AA after incubation with 0. 2, 0.4 and 0. 8 mg/L of TSN for one hour） and LY294002 pretreatment group（ HUVECs was pretreated with 20 μmol/L of PI3K antagonist LY294002 for 30 min before TSN was added）. After 24 hours, cell viabilities were evaluated by the MTT assays. The morphological changes of apoptotic cells were observed by Hoechst33258 fluorescence staining. The proportions of apoptotic cells were assessed by flow cytometry. Western blot was used to determine the expression of Bcl-2, Bax and phophorylated Akt. Meanwhile, the caspase-3 activity was detected by colorimetric method. Results AA increased the proportions of apoptotic cells（ P 〈0.05 ） , inhibited the expressions of Bcl-2 and phosphorylated Akt（ P 〈 0. 05 ） , and promoted the expression of Bax（ P 〈 0.05 ）. The protective effects of TSN were partially inhibited by PI3K antagonist LY294002 （P 〈 0.05）. Conclusions TSN can inhibit the apoptosis of I-IUVECs stimulated by aristolochic acid.