黄芩抑制脂多糖/三磷酸腺苷诱导的内皮细胞NIMA相关蛋白激酶7(NEK7)表达和Nod样受体蛋白3(NLRP3)炎症小体激活

【目的】观察黄芩和黄芩苷对脂多糖(LPS)/三磷酸腺苷(ATP)诱导的人脐静脉内皮细胞(HUVECs)NIMA相关蛋白激酶7(NEK7)和Nod样受体蛋白3(NLRP3)小体表达的影响。【方法】将HUVECs随机分为空白对照组、模型组、阿托伐他汀组、黄芩苷组和黄芩血清组,按分组相应药物预处理细胞24 h,然后用1μg/mL LPS刺激细胞24 h,再用5.5 mmol/L ATP刺激细胞4 h后检测各指标。四甲基偶氮唑盐(MTT)法检测细胞活力,均相竞争法测定细胞上清液中内皮素1(ET-1)的含量,硝酸还原酶法测定细胞上清液中总硝酸盐(NOx)水平,蛋白免疫印迹法检测细胞内NEK7、NLRP3、凋亡相关斑点样蛋白(ASC)、裂解型胱天蛋白酶1(cleaved-Caspase-1)、白细胞介素1β(IL-1β)蛋白的表达。【结果】与模型组比较,黄芩血清组、黄芩苷组、阿托伐他汀组LPS/ATP诱导的细胞活力升高,ET-1水平降低、NOx水平升高,NEK7、NLRP3、ASC、cleaved-Caspase-1、IL-1β的蛋白表达水平降低(P<0.05或P<0.01),且黄芩血清组、黄芩苷组的作用效果优于阿托伐他汀组。【结论】黄芩和黄芩苷可以抑制LPS/ATP诱导HUVECs的NLRP3炎症小体激活引起的细胞焦亡而保护内皮,其机制可能与调控NEK7表达有关。

Radix Scutellariae Inhibits LPS/ATP-Induced NEK7 Expression and NLRP3 Inflammasome Activation in Endothelial Cells

Objective To observe the effects of Radix Scutellariae and baicalin on lipopolysaccharide(LPS)/adenosine triphosphate(ATP)-induced expression of NIMA-related kinase 7(NEK7)and Nod-like receptor protein 3(NLRP3)in human umbilical vein endothelial cells(HUVECs).Methods HUVECs were randomly divided into blank control group,model group,atorvastatin group,baicalin group and Radix Scutellariae-containing serum group.After pretreatment with corresponding drugs for 24 h,the cells were stimulated with LPS of 1μg/mL for 24 h,followed by 5.5 mmol/mL of ATP for 4 h.Cell viability was detected by methyl-thiazolyl-tetrazolium(MTT)assay,content of endothelin 1(ET-1)in cell supernatant was determined by homogeneous competition assay,levels of nitrite/nitrate(NOx)in cell supernatant was determined by nitrate reductase assay,and expression of NEK7,NLRP3,apoptosis-associated speck-like protein containing a CARD(ASC),cleaved-Caspase-1,interleukin 1β(IL-1β)proteins was detected by Western blotting assay.Results Compared with the model group,LPS/ATP-induced cell viability was increased in Radix Scutellariae-containing serum group,baicalin group and atorvastatin group,and ET-1 content was decreased,NOx level was increased,and expression levels of NEK7,NLRP3,ASC,cleaved-Caspase-1,IL-1βproteins were lowered(P<0.05 or P<0.01).Conclusion Radix Scutellariae and baicalin have effects on inhibiting pyroptosis resulted by LPS/ATP-induced activation of NLRP3 inflammasome in HUVECs and thus to protect endothelium,and the mechanism may be related with regulating and controlling NEK7 expression.