白术黄芪汤乙酸乙酯提取物对巨噬细胞NLRP3炎症小体的作用研究

摘要

目的:观察白术黄芪汤乙酸乙酯提取物对巨噬细胞NOD样受体-3(NLRP3)炎症小体的影响.方法:用佛波酯(PMA) (10 ng·mL-1)刺激U937细胞48 h,诱导其成为巨噬细胞,观察不同剂量白术黄芪汤乙酸乙酯提取物(0,3.125,6.25,12.5,25,50,100 μg·mL-1)对细胞活力的作用,并选择合适的浓度.采用实时荧光定量(RT-PCR)和免疫印迹法(Western blot)测定细胞中NLRP3,半胱天冬酶-1(caspase-1)的含量,采用酶联免疫吸附测定(ELISA)检测细胞上清液中白细胞介素(IL)-1β的含量.结果:细胞计数试剂盒(CCK-8)实验表明,当药物浓度低于25 μg· mL-1时,对细胞活力没有影响,当药物浓度高于50 μg· mL-1时,对细胞活力有抑制作用(P＜0.05),选择25 μg·mL-1浓度进行后续实验.与空白组相比,LPS组细胞中NLRP3,caspase-1表达明显增加(P＜0.01),细胞培养上清中的IL-1β浓度也明显增高(P＜0.01).白术黄芪汤乙酸乙酯提取物作用后,NLRP3,caspase-1,IL-1β表达均明显下降(P＜0.05).结论:白术黄芪汤乙酸乙酯提取物对LPS刺激的巨噬细胞NLRP3炎症小体有抑制作用.%This study was aimed to observe the effect of Bai-Zhu Huang-Qi (BZHQ) decoction ethyl acetate extract on NOD like receptor family,pyrin domain-containing 3 (NLRP3) inflammasome in macrophages.The U937 cells were pretreated with phorbol 12-myristate 13-acetate (PMA,10 ng· mL-1) for 48 hours to induce macrophages.Effects on cell viability by different doses of BZHQ decoction ethyl acetate extract (0,3.125,6.25,12.5,25,50,100 μg· mL-1) were observed to select the appropriate concentration.Contents of NLRP3 and caspase-1 in cells were detected by real-time PCR and western blot.The concentration of interleukin-1β (IL-1β) in cell supernatant was detected by enzyme linked immunosorbent assay (ELISA).The cell counting kit-8 (CCK-8) assay showed that when the drug concentration was lower than 25 μg· mL-1,there was no impact on cell viability;when the drug concentration was higher than 50 μg· mL-1,there was inhibition on cell viability (P ＜ 0.05).The concentration of 25 μg· mL-1 was used to conduct the following experiment.Compared to the blank group,the expression of NLRP3 and caspase-l in cells of the LPS group were significantly increased (P ＜ 0.01).The concentration of IL-1β in cell supernatant was also significantly increased (P ＜ 0.01).After treated with BZHQ decoction ethyl acetate extract,levels of NLRP3,caspase-1 and IL-1β were significantly decreased (P ＜ 0.05).It was concluded that BZHQ decoction ethyl acetate extract can inhibit the production of NLRP3 inflammasome in LPS-stimulated macrophages.