Ca^(2+)/NLRP3/Caspase-1信号通路对动脉粥样硬化内皮功能紊乱的调节作用

Regulation of Ca^(2+)/NLRP3/Caspase⁃1 signaling pathway in atherosclerotic endothelial dysfunction

目的 探讨Ca^(2+)/NLRP3/Caspase-1信号通路对动脉粥样硬化(atherosclerosis,AS)内皮功能紊乱的调节作用。方法 将16只雄性ApoE^(-/-)小鼠随机分为普通饮食组和高脂饮食组,每周空腹测量小鼠体质量;持续喂养20周,处死小鼠,收集动脉组织;生化分析仪检测血清胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL-C)及高密度脂蛋白(HDL-C)水平;油红O染色评估主动脉管腔斑块及内膜厚度。将人脐静脉内皮融合细胞EA.hy926分为对照组、高糖高脂组和Ca^(2+)抑制剂组,流式细胞术检测不同时间段Ca^(2+)含量;免疫荧光法检测NLRP3表达;Western blot法检测主动脉组织和细胞中NLRP3、Caspase-1/p10/p20、VCAM-1、ICAM-1蛋白表达水平。结果 与普通饮食组相比,高脂饮食组小鼠体质量明显增高(F=3.333,P=0.026),血清TC、TG、LDL-C水平显著增高,HDL-C水平显著降低(F分别为1.852、2.410、1.920和2.917,P分别为0.000、0.004、0.002和0.003),小鼠主动脉斑块表面积/主动脉表面积显著增加(F=3.256,P=0.000),斑块横断面积/主动脉管腔面积明显增高(F=6.433,P=0.008),NLRP3、Caspase-1/p10/p20、VCAM-1和ICAM-1蛋白表达水平明显增高(F分别为8.997、4.664、4.486和9.949,P分别为0.036、0.022、0.005和0.018)。与对照组相比,高糖高脂处理6、12、24 h的EA.hy926细胞Ca^(2+)含量均显著增高(F分别为0.310、5.649和5.580,P分别为0.006、0.009和0.004),6 h组含量最高;高糖高脂组NLRP3表达显著增高,Caspase-1/p10/p20、VCAM-1及ICAM-1蛋白表达水平显著增高(F分别为10.476、5.310和9.306,P分别为0.029、0.030和0.018)。与高糖高脂组相比,Ca^(2+)抑制剂组EA.hy926细胞的NLRP3、Caspase-1/p10/p20蛋白表达水平显著降低(F分别为2.196、2.882和0.035,P分别<0.01、<0.05和<0.05)。结论 NLRP3炎性小体诱导血管内皮细胞功能紊乱,加重AS进展,其发生过程可能与Ca^(2+)及下游信号通路密切相关。

Objective To explore the regulation of Ca^(2+)/NLRP3/Caspase⁃1 signaling pathway on endothelial dysfunction in atherosclerosis(AS).Methods 16 male Apo^(E⁃/⁃) mice were randomly divided into a normal diet group and a high⁃fat diet group and measured for the weight on an empty stomach every week;The mice were fed continuously for 20 weeks and then sacrificed,of which the arterial tissues were collected;Serum cholesterol(TC),triglyceride(TG),low density lipoprotein(LDL⁃C)and high density lipoprotein(HDL⁃C)were detected by biochemical analyzer;The thickness of aortic luminal plaque and intima was assessed by oil red O staining.Human umbilical vein endothelial fusion cells EA.hy926 were divided into control group,high⁃sugar and high⁃fat group,and Ca^(2+)inhibitor group,which were detected for Ca^(2+) content by flow cytometry at different time periods,for the expression of NLRP3 by immunofluorescence,and for the protein expression levels of NLRP3,Caspase⁃1/p10/p20,VCAM⁃1 and ICAM⁃1 in aortic tissue and cells by Western blot.Results Compared with the normal diet group,the weight of the mice in high⁃fat diet group increased significantly(F=3.333,P=0.026),the serum TC,TG and LDL⁃C significantly increased,while HDL⁃C significantly decreased(F=1.852,2.410,1.920 and 2.917,P=0.000,0.004,0.002 and 0.003,respectively);The aortic plaque surface area/aortic surface area increased significantly(F=3.256,P=0.000),plaque cross⁃sectional area/aortic lumen area increased significantly(F=6.433,P=0.008),and NLRP3,Caspase⁃1/p10/p20,VCAM⁃1 and ICAM⁃1 protein expressions increased significantly(F=8.997,4.664,4.486 and 9.949,P=0.036,0.022,0.005 and 0.018,respectively).Compared with the control group,the Ca 2+content of endothelial cells in high⁃sugar and high⁃fat group increased in the 6 h,12 h,and 24 h(F=0.310,5.649 and 5.580,P=0.006,0.009 and 0.004,respectively),especially in the 6 h group;The expression of NLRP3 in high⁃sugar and high⁃fat group increased significantly;VCAM⁃1,ICAM⁃1 and Caspase⁃1/p10/p20 protein expression increased significantly(F=10.476,5.310 and 9.306,P=0.029,0.030 and 0.018,respectively).Compared with the high⁃sugar and high⁃fat group,the expression of NLRP3 and Caspase1/p10/p20 protein in EA.hy926 cells of Ca^(2+)inhibitor group decreased significantly(F=2.196,2.882 and 0.035,P<0.01,<0.05 and<0.05,respectively).Conclusion NLRP3 inflammasome induced dysfunction of vascular endothelial cells and aggravated the progression of AS,which might be closely related to Ca^(2+)and downstream signaling pathways.