冠通方含药血清通过miR-223/NLRP3信号通路调控血管内皮细胞焦亡的作用及机制研究

Effect and Mechanism of Guantong Decoction-Containing Serum in Regulating Vascular Endothelial Cell Pyroptosis through miR-223/NLRP3 Signaling Pathway

目的:探究冠通方含药血清通过microRNA-223(miR-223)/NOD样受体家族3(NLRP3)信号通路调控血管内皮细胞焦亡的作用及机制研究。方法:将血管内皮细胞系人脐静脉血管内皮细胞(HUVECs)分为对照组、模型组、空白血清组和含药血清组。对照组仅进行常规培养,其余3组采用氧化型低密度脂蛋白(ox-LDL)诱导HUVECs细胞损伤模型,模型组不进行任何干预,空白血清组采用空白血清干预,含药血清组则采用冠通方含药血清进行干预。实时荧光定量聚合酶链式反应(qRT-PCR)检测细胞中miR-223 mRNA表达,蛋白质印迹法(Western Blot)检测NLRP3、含半胱氨酸的天冬氨酸蛋白水解酶(Caspase1)的蛋白表达,酶联免疫吸附实验(ELISA)检测白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)的含量,细胞计数试剂盒(CCK8)检测细胞活性,并计算乳酸脱氢酶(LDH)释放水平。碘化丙啶(PI)染色荧光显微镜下观察细胞膜完整性和细胞焦亡情况。结果:与对照组相比,模型组HUVECs细胞活力明显降低(P<0.05);与模型组相比,空白血清组细胞活力差异无统计学意义(P>0.05),含药血清组的细胞活力则明显增强(P<0.05)。与对照组相比,模型组LDH释放水平明显升高(P<0.05);与模型组相比,空白血清组LDH释放水平差异无统计学意义(P>0.05),含药血清组LDH释放水平则明显下降(P<0.05)。与对照组相比,模型组PI染色程度明显增加;与模型组相比,空白血清组PI染色程度变化不明显,含药血清组PI染色程度明显减少。与对照组相比,模型组炎性因子IL-1β、IL-18含量明显升高(P<0.05);与模型组相比,空白血清组IL-1β、IL-18含量差异无统计学意义(P>0.05),含药血清组IL-1β、IL-18含量明显下降(P<0.05)。与对照组相比,模型组Caspase1蛋白表达明显上调;与模型组相比,空白血清组Caspase1蛋白表达差异无统计学意义(P>0.05),含药血清组Caspase1蛋白表达明显下调。与空白血清组相比,含药血清组HUVECs细胞内miR-223的表达明显上调(P<0.05),NLRP3的表达明显下调(P<0.05)。结论:冠通方含药血清通过上调miR-223下调NLRP3,抑制炎性因子IL-1β、IL-18水平,抑制Caspase1诱导的血管内皮细胞焦亡,提示冠通方对于冠心病病人的血管内皮细胞具有较好的保护作用。

Objective:To explore the effect and mechanism of Guantong Decoction-containing serum in regulating the pyroptosis of vascular endothelial cells through microRNA-223(miR-223)/NOD-like receptor family 3(NLRP3)signaling pathway.Methods:Human umbilical vein endothelial cells(HUVECs)of vascular endothelial cell line were divided into control group,model group,blank serum group,and drug-containing serum group.The control group received routine culture,and the remaining three groups were treated with oxidized low density lipoprotein(ox-LDL)to induce HUVECs cell injury model,and the model group did not undergo any intervention,the blank serum group was intervened with blank serum,and the drug-containing serum group was treated with Guantong Decoction-containing serum.Quantitative real-time PCR(qRT-PCR)was used to detect the mRNA expression of miR-223 in cells,and Western Blot was applied to measure the protein expressions of NLRP3 and cysteinyl aspartate specific proteinase 1(Caspase1).Enzyme linked immunosorbent assay(ELISA)was adopted to detect the levels of interleukin-1β(IL-1β)and interleukin-18(IL-18),and cell counting kit-8(CCK8)was used to detect cell viability,and the release level of lactate dehydrogenase(LDH)was calculated.The cell membrane integrity and cell pyroptosis were observed under fluorescence microscopy with propidium iodide(PI)staining.Results:The HUVECs cell viability was significantly reduced in model group compared with that in control group(P<0.05),and the cell viability in blank serum group hardly changed compared with that in model group(P>0.05),but it was significantly enhanced in drug-containing serum group(P<0.05).In addition,compared with control group,the LDH release level of model group was significantly increased(P<0.05),while compared with model group,the LDH release level of blank serum group showed no significant change(P>0.05),and the LDH release level of drug-containing serum group was significantly decreased(P<0.05).The PI staining degree was significantly increased in model group compared with that in control group,and hardly changed in blank serum group compared with that in model group,and the PI staining degree in drug-containing serum group was significantly reduced.The levels of inflammatory factors IL-1βand IL-18 in model group were significantly increased compared with that in control group(P<0.05),while the IL-1βand IL-18 levels in blank serum group showed almost no changes compared with that in model group(P>0.05),and the levels of IL-1βand IL-18 were reduced significantly in drug-containing serum group(P<0.05).Compared with control group,the protein expression of Caspase1 was significantly up-regulated in model group,but it was not significantly changed in blank serum group compared with that in model group(P>0.05),and the Caspase1 protein expression in drug-containing serum group was significantly down-regulated.Compared with blank serum group,the expression of miR-223 in HUVECs cells was significantly up-regulated(P<0.05),while the expression of NLRP3 was significantly down-regulated in drug-containing serum group(P<0.05).Conclusion:Guantong Decoction-containing serum can inhibit the levels of inflammatory factors IL-1βand IL-18 and inhibit Caspase1-induced vascular endothelial cell pyroptosis by up-regulating miR-223 and down-regulating NLRP3,suggesting that Guantong Decoction shows better protective effect on vascular endothelial cells in patients with coronary heart disease.