丹参酮IIA磺酸钠不同给药时段对其内皮保护作用的影响

Influence of Different Administration Timepoints on the Endothelial Protective Effects of Sodium Tanshinone IIA Sulfonate

关键词:  丹参酮ⅡA磺酸钠  给药时段  人脐静脉内皮细胞  脂多糖  内皮功能异常  cleaved caspase-3  cleaved caspase-9

Key words:  sodium tanshinone IIA sulfonate  administration timepoints  human umbilical vein endothelial cells  LPS  endothelial dysfunction  cleaved caspase-3  cleaved caspase-9

摘要:

目的 研究不同给药时段对丹参酮IIA磺酸钠（sodium tanshinone IIA sulfonate，STS）内皮保护作用的影响。方法 将人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVEC）分组刺激，分为5组： DMEM组、脂多糖（lipopolysaccharide，LPS）处理组、STS预处理+LPS组、STS+LPS共处理组、STS后处理+LPS组。其中，LPS终浓度为1 μg·mL^-1，STS终浓度为25 μg·mL^-1，LPS刺激时间为12 h。处理完成后，CCK-8法测定HUVEC活力； ELISA检测细胞培养上清中炎症因子白细胞介素-1β（interleukin-1β，IL-1β）的蛋白浓度； Western blotting检测细胞裂解液中核因子κB （nuclear factor κB，NF-κB）的蛋白水平；划痕试验检测HUVEC的迁移能力；光镜观察细胞凋亡状态，并用Western blotting检测凋亡相关分子cleaved caspase-3和cleaved caspase-9的蛋白水平。结果 与DMEM组相比，LPS处理后HUVEC增殖活力减低；IL-1β和NF-κB蛋白水平增高；细胞迁移能力降低、细胞凋亡增加并伴有cleaved caspase-3和cleaved caspase-9蛋白水平的升高（P<0.05）； STS处理能够部分逆转LPS引起的上述现象，其中以STS预处理的效果最为明显，而STS后处理的效果相对较弱。结论 STS预处理、共处理和后处理均能部分抑制LPS诱导的HUVEC功能异常和凋亡，且以预处理时的效果最为明显。

Abstract:

OBJECTIVE To investigate the influence of different administration timepoints on the endothelial protective effects of sodium tanshinone II A sulfonate(STS). METHODS Human umbilical vein endothelial cells(HUVEC) were divided into five groups, individually stimulated, including DMEM group, lipopolysaccharide(LPS) group, STS pre-treatment+LPS group, STS co-treatment+LPS group, STS post-treatment+LPS group. The final concentration of LPS in the experiment was 1 μg·mL^-1, and that of STS was 25 μg·mL^-1, LPS stimulation time was 12 h. After that, HUVEC viability was determined by CCK-8 assay. The protein concentration of inflammatory cytokine interleukin-1β(IL-1β) in HUVEC culture supernatant was detected by ELISA. The protein levels of nuclear factor κB(NF-κB) in HUVEC lysate were detected by Western blotting. The migration capacity of HUVEC was detected by wound-healing test. HUVEC apoptosis was observed under an optical microscopy, and the expression of apoptosis-related proteins including cleaved caspase-3 and cleaved caspase-9 were detected by Western blotting. RESULTS Compared with DMEM group, after LPS treatment, HUVEC cell viability was decreased; IL-1β and NF-κB protein expression increased; migration capacity impaired; increased apoptosis with the higher protein expression of cleaved caspase-3 and cleaved caspase-9(P<0.05). STS treatments could partially reverse these LPS-induced phenomena, among which the STS pre-treatment was most effective, while the STS post-treatment was with relative lower efficacy. CONCLUSION STS pre-treatment, co-treatment and post-treatment can partially reverse LPS-induced HUVEC dysfunction and apoptosis, among which the STS pre-treatment is most effective.