硼替佐米调节miR-223/NLRP3轴对脂多糖诱导的人单核细胞炎症反应的影响

Bortezomib attenuates LPS-induced inflammatory response of human monocytes through regulating miR-223/NLRP3 signaling pathway

目的:探讨硼替佐米(BTZ)调节微小RNA-233(miR-223)/核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)轴对脂多糖(LPS)诱导的人单核细胞炎症反应的影响。方法:从类风湿性关节炎(RA)患者外周血中分离、纯化单核细胞。采用酶联免疫吸附测定(ELISA)法检测单核细胞上清液中白细胞介素6(IL-6)、IL-1β和肿瘤坏死因子α(TNF-α)水平,并根据IL-6、IL-1β和TNF-α水平筛选LPS的最佳诱导时间和BTZ的最佳处理浓度。实验分为对照组、LPS诱导组和BTZ组。RT-qPCR法测定各组单核细胞中miR-223水平;Western blot法测定单核细胞中NLRP3、caspase-1、细胞因子信号抑制物1(SOCS1)和含SH2结构域的肌醇磷酸酶1(SHIP-1)水平。结果:成功从RA患者外周血中分离、纯化出单核细胞,其呈类球形,分布均匀;经LPS诱导24 h后,细胞多呈梭形、聚集状。根据IL-6、IL-1β和TNF-α水平筛选出LPS的最佳诱导时间为24 h,BTZ的最佳处理浓度为50 nmol/L。与对照组相比,LPS诱导组miR-223、SOCS1和SHIP-1水平显著降低(P<0.05),NLRP3和caspase-1水平显著升高(P<0.05);与LPS诱导组相比,BTZ组miR-223、SOCS1和SHIP-1水平显著升高(P<0.05),NLRP3和caspase-1水平显著降低(P<0.05)。结论:BTZ可能通过阻断miR-223/NLRP3轴活化,减少LPS诱导的人单核细胞炎症因子的分泌。

AIM:To investigate the effects of bortezomib(BTZ)on microRNA-223(miR-223)/nucleotidebinding oligomerization domain-like receptor protein 3(NLRP3)signaling pathway and lipopolysaccharide(LPS)-induced inflammatory response of human monocytes.METHODS:Monocytes were isolated and purified from peripheral blood of rheumatodid arthritis(RA)patients. The levels of interleukin-6(IL-6),IL-1β and tumor necrosis factor-α(TNF-α)in supernatants of the monocytes were determined by ELISA,and the optimal induction time of LPS and the optimal treatment concentration of BTZ were selected according to the levels of IL-6,IL-1β and TNF-α. The monocytes were divided into control group,LPS induced group and BTZ group. The level of miR-223 in the monocytes was measured by RT-qPCR,and the protein levels of NLRP3,caspase-1,suppressor of cytokine signaling 1(SOCS1)and SH2 domain-containing inositol phosphatase-1(SHIP-1)in the monocytes were determined by Western blot.RESULTS:The monocytes successfully isolated and purified from the peripheral blood of RA patients were spherical,evenly distributed and regular in shape.And after LPS induction for 24 h,the cells were mostly spindle-shaped and aggregated. According to the levels of IL-6,IL-1β and TNF-α,the optimal induction time of LPS was 24 h,and the optimal concentration of BTZ was 50 nmol/L. Compared with control group,the levels of miR-223,SOCS1 and SHIP-1 in LPS induction group were significantly decreased(P<0. 05),and the levels of NLRP3 and caspase-1 were significantly increased(P<0. 05). Compared with LPS induction group,the levels of miR-223,SOCS1 and SHIP-1 in BTZ group were significantly increased(P< 0. 05),and the levels of NLRP3 and caspase-1 were significantly lowered(P<0. 05).CONCLUSION:Bortezomib may block the activation of miR-223/NLRP3 signaling pathway,thus reducing the secretion of inflammatory factors in LPS-induced human monocytes.