磷酸氯喹对双链RNA诱导的血管内皮细胞焦亡的影响

Effect of chloroquine phosphate on vascular endothelial cell injury induced by double-stranded RNA

目的·探究磷酸氯喹(chloroquine phosphate,CQ)对病毒感染中双链RNA(double-stranded RNA,dsRNA)所致血管内皮细胞损伤的抑制作用及机制。方法·采用聚肌胞苷(polyinosinic-polycytidylic acid,poly I:C)刺激人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC),建立病毒性脓毒症中dsRNA诱导血管内皮细胞损伤的体外模型。将HUVEC随机分为正常对照组(NC组)、不同浓度poly I:C组和CQ+poly I:C组。采用细胞计数试剂盒8(cell counting kit-8,CCK-8)检测不同处理组HUVEC活力的变化,采用光学显微镜检测细胞形态变化,采用流式细胞术检测细胞碘化丙啶(propidium iodide,PI)阳性率(PI+)以观察细胞膜完整性,采用蛋白质印迹(Western blotting)检测焦亡相关蛋白中NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)、胱天蛋白酶-1活性剪切体(cleaved caspase-1)、gasdermin D蛋白N端(gasdermin D N-terminal,GSDMD-N)、白介素-1β(interleukin-1β,IL-1β)以及Toll样受体3(Toll-like receptor 3,TLR3)的蛋白表达水平。结果·与NC组相比,poly I:C组HUVEC活力明显降低,成熟IL-1β水平升高,细胞形态呈现膨胀“吐泡”现象,PI+率增加,焦亡相关蛋白NLRP3、cleaved caspase-1和GSDMD-N的表达升高(均P<0.05)。与poly I:C组相比,CQ+poly I:C组的细胞活力有所增加,成熟IL-1β表达减少,膨胀及破裂的细胞比例下降,PI+降低,焦亡相关蛋白cleaved caspase-1、GSDMD-N表达减少(均P<0.05)。CQ预处理并未显著改变dsRNA诱导的NLRP3表达水平,而抑制了TLR3的表达(P<0.05)。结论·dsRNA能够诱导HUVEC细胞焦亡;CQ能够抑制由dsRNA诱导的血管内皮细胞焦亡,该效应可能与调节TLR3表达有关。

Objective·To investigate the inhibitory effect of chloroquine phosphate(CQ)on vascular endothelial cell injury induced by double-stranded RNA(dsRNA)and its mechanism.Methods·An in vitro model of vascular endothelial cell injury induced by dsRNA in viral sepsis was established via stimulating human umbilical vein endothelial cells(HUVECs)with polyinosinic-polycytidylic acid(poly I:C).HUVECs were randomly divided into normal control group(NC group),poly I:C group with different concentrations and CQ+poly I:C group.The cell viability of HUVECs before and after poly I:C treatment was detected by using Cell Counting Kit-8(CCK-8).The morphological changes of HUVECs were observed by using optical microscope.The integrity of cell membrane was assessed by calculating the rate of propidium iodide(PI)positive cells by using flow cytometry.The expression level of mature proinflammatory cytokine interleukin 1β(IL-1β),and key proteins of pyroptosis were detected by Western blotting.Results·Compared with the NC group,HUVECs treated with poly I:C showed a decreased cell viability,more expanded"Vesicles"and more ruptured cells.An increased level of IL-1βexpression and positive rate of PI+cells were also observed.The expression levels of NOD-like receptor thermal protein domain associated protein 3(NLRP3),and cleaved caspase-1 and gasdermin D N-terminal(GSDMD-N)were increased significantly(all P<0.05)under the treatment of poly I:C and the changes were concentration-dependent.Interestingly,compared with poly I:C group,the cell viability of HUVEC pretreated by CQ was significantly increased,and a decreased IL-1βlevel were observed.The phenomena of cell swelling and rupture was improved,and a lower positive rate of PI+cells was detected.The expression levels of cleaved caspase-1 and GSDMD-N of cell pyrosis were also decreased(all P<0.05).CQ pretreatment did not reduce NLRP3 expression induced by dsRNA,but it did inhibit the expression of Toll-like receptor 3(TLR3).Conclusion·Pyroptosis is involved in vascular endothelial cells injury induced by dsRNA,and CQ inhibits dsRNA-induced pyroptosis,which could be related to TLR3 regulation.