芥子酸拮抗同型半胱氨酸诱导的血管内皮细胞凋亡及焦亡

Antagonistic effect of sinapic acid on homocysteine-induced apoptosis and pyroptosis in endothelial cells

目的研究同型半胱氨酸(homocysteine,Hcy)对casepase途径介导的内皮细胞凋亡和焦亡的促进作用,以及该过程中芥子酸(sinapic acid,SA)对Hcy的拮抗作用。方法分别利用0,0.1,0.3,0.5,1,3,5 mmol/L浓度的Hcy处理HUVEC-12人脐静脉内皮细胞24 h,检测细胞凋亡和细胞焦亡水平,筛选最适处理浓度;以最适浓度Hcy处理细胞24 h,同时分别添加0.1,0.5,1,5,10μmol/L的SA。采用Annexin-Ⅴ/PI法检测早期细胞凋亡,caspase-1 FLICA/PI法检测细胞的焦亡,Western blot检测细胞中焦亡关键调控蛋白NRLP3、ASC、caspase-1,凋亡相关蛋白caspase-8、caspase-3和抗凋亡蛋白Bcl-2的表达,通过ELISA方法检测细胞上清中内皮素(endothelin-1,ET-1)及炎性因子白细胞介素1β(interleukin-1β,IL-1β)的分泌量。结果细胞凋亡细胞焦亡水平随Hcy浓度升高而逐渐上升;与对照相比,当Hcy浓度分别≥0.5 mmol/L和≥1 mmol/L时,细胞凋亡和焦亡水平显著升高(P<0.05);3 mmol/L和5 mmol/L Hcy进一步促进细胞的凋亡、焦亡及相关调控蛋白的水平(P<0.05);选择3 mmol/L为最适浓度,用于后续研究。1μmol/L SA可以抑制上述改变并能拮抗Hcy诱导的ET-1和IL-1β的分泌(P<0.05),并且其拮抗作用跟泛caspase抑制剂Z-VAD-FMK的效果类似。结论 Hcy可通过casepases途径诱导血管内皮细胞的凋亡和焦亡;低浓度的SA可拮抗Hcy诱导的血管内皮细胞凋亡和焦亡。

Objective To investigate the effect of homocysteine（ Hcy） on the promotion of caspases-mediated apoptosis and pyroptosis in endothelial cells and the antagonistic effect of sinapic acid（ SA） during this process. Methods Hcy at the concentrations of 0,0. 1,0. 3,0. 5,1,3,5 mmol/L were respectively used to incubate human umbilical vein endothelial HUVEC-12 cells for 24 h. Cell apoptosis and pyroptosis were detected to determine the optimal concentration of Hcy. Then the cells were incubated with the optimal concentration of Hcy together with SA at the concentrations 0. 1,0. 5,1,5 and 10 μmol/L for 24 h,respectively. Early cell apoptosis was detected with the Annexin-Ⅴ/PI staining method. Cell pyroptosis was detected by the caspase-1-FLICA/PI method. Western blot was used to detect the expression of pyroptotic proteins NRLP3,ASC and caspase-1,the apoptotic proteins caspase-8 and caspase-3,and the anti-apoptotic protein Bcl-2. In the culture media,the production of ET-1 and IL-1β was determined by ELISA. Results Cell apoptosis and pyroptosis were both gradually increased with the elevation of the Hcy concentration. Compared with 0 mmol/L Hcy,the cell apoptosis and pyroptosis were significantly increased when Hcy concentration≥0. 5 mmol/L and ≥1 mmol/L,respectively（ P〈 0. 05）,and 3 mmol/L and 5 mmol/L Hcy further promoted cell apoptosis,pyroptosis and expression of related proteins（ P〈 0. 05）.So 3 mmol/L Hcy was selected as the optimal concentration for subsequent experiments. The 1 μmol/L SA antagonized the promoting effect of Hcy on cell apoptosis and pyroptosis and decreased the secretion of endothelin-1（ ET-1） and IL-1β. Its antagonistic effect was consistent with Z-VAD-FMK,a pan-caspase inhibitor. Conclusion Hcy can induce the apoptosis and pyroptosis of HUVEC-12 cells through the caspase pathways,which can be partially antagonized by low-dose of SA.