CAJ | 학술논문

目的:探讨在脂多糖(LPS)诱导的条件下中国蜂胶对血管内皮细胞(VECs)磷脂酰胆碱特异性磷脂酶C (PC-PLC)活性和TLR4表达的影响.方法:将100 μg/L LPS 加入到含0.5%血清的培养液中培养VECs,经12.5 mg/L的中国蜂胶分别处理12 h和24 h后,SRB法测定细胞存活率;化学法测定NO含量;以L-α-卵磷脂为底物测定PC-PLC的活性;Western blotting检测TLR4、核因子κB p65(NF-κB p65)和p53的表达;细胞内活性氧(ROS)和线粒体膜电位分别通过荧光探针DCHF和JC-1检测.结果:中国蜂胶处理LPS诱导的血管内皮细胞24 h并不影响细胞存活率,但降低NO的含量和ROS的水平;处理12 h后降低PC-PLC活性和NF-κB p65表达;处理12 h和24 h后降低TLR4和p53的表达.此外,中国蜂胶不影响细胞内线粒体膜电位的水平.结论:中国蜂胶通过降低PC-PLC活性和TLR4表达,抑制其下游信号分子NF-κB p65、p53的表达和ROS的水平,以及抑制NO的释放,从而发挥抗炎功效.
목적:탐토재지다당(LPS)유도적조건하중국봉효대혈관내피세포(VECs)린지선담감특이성린지매C (PC-PLC)활성화TLR4표체적영향.방법:장100 μg/L LPS 가입도함0.5%혈청적배양액중배양VECs,경12.5 mg/L적중국봉효분별처리12 h화24 h후,SRB법측정세포존활솔;화학법측정NO함량;이L-α-란린지위저물측정PC-PLC적활성;Western blotting검측TLR4、핵인자κB p65(NF-κB p65)화p53적표체;세포내활성양(ROS)화선립체막전위분별통과형광탐침DCHF화JC-1검측.결과:중국봉효처리LPS유도적혈관내피세포24 h병불영향세포존활솔,단강저NO적함량화ROS적수평;처리12 h후강저PC-PLC활성화NF-κB p65표체;처리12 h화24 h후강저TLR4화p53적표체.차외,중국봉효불영향세포내선립체막전위적수평.결론:중국봉효통과강저PC-PLC활성화TLR4표체,억제기하유신호분자NF-κB p65、p53적표체화ROS적수평,이급억제NO적석방,종이발휘항염공효.
AIM: To investigate the effect of Chinese propolis on the activity of phosphatidylcholine-specific phospholipase C (PC-PLC) and the expression of Toll-like receptor 4 (TLR4) in LPS-treated vascular endothelial cells (VECs). METHODS: Confluent VECs were stimulated with LPS at the concentration of 100 μg/L in the presence of 0.5% fetal bovine serum. The cells were treated with Chinese propolis at the concentration of 12.5 mg/L for 12 h and 24 h. The viability of VECs and the level of nitric oxide (NO) were detected by sulforhodamine B (SRB) assay and chemical method, respectively. The activity of PC-PLC was measured using L-α-phosphatidylcholine as substrate. The protein levels of TLR4, nuclear factor-Κb p65 (NF-Κb p65) and p53 were determined by Western blotting. The level of intracellular reactive oxygen species (ROS) was examined using a fluorescent probe, 2',7'-dichlorodihydrofluorescin (DCHF). For the measurement of mitochondrial membrane potential, the fluorescent dye JC-1 was used. RESULTS: Treatment with Chinese propolis for 24 h had no effect on the viability of VECs. However, the levels of NO and ROS were significantly decreased by Chinese propolis. PC-PLC activity and NF-Κb p65 expression were significantly depressed by Chinese propolis treated for 12 h, and the expression of TLR4 and p53 were dramatically decreased by Chinese propolis treated for 12 and 24 h. No effect of Chinese propolis on mitochondrial membrane potential was observed. CONCLUSION: Chinese propolis depresses the activity of PC-PLC and the expression of TLR4, and then inhibits the downstream signal molecules such as NF-Κb p65, p53, ROS and NO in VECs.