摘要
目的探讨肝X受体α(LXRα)激动剂对巨噬细胞中Nod样受体蛋白3(NLRP3)炎症小体活化导致成熟白细胞介素1β(IL-1β)水平升高的影响及其机制,为探索同时具有抗炎和调脂能力的防治动脉粥样硬化药物新靶点提供实验支持。方法 THP-1细胞经佛波酯活化成为人源性巨噬细胞,在培养基中加入胆固醇晶体(1μg/L)为模型组,其它各组在模型组基础上分别加入高剂量(30μmol/L)、中剂量(10μmol/L)、低剂量(3.3μmol/L)的LXRα激动剂GW3965以及GW3965 10μmol/L+GGPP 10μmol/L、GGPP 10μmol/L、GW3965 10μmol/L+ABCA1抗体(1∶200)、GW3965 10μmol/L+载脂蛋白E抗体(1∶200),48 h后提取总RNA、总蛋白、核蛋白和培养基蛋白。利用real-time PCR和Western blot对相应指标的mRNA和蛋白水平进行检测。结果模型组成熟IL-1β蛋白水平、NLRP3和Caspase-1的mRNA和蛋白水平与对照组有显著差异(P<0.05);与模型组相比,GW3965中、高剂量组上述指标均有显著减低(P<0.05),LXRα抑制剂GGPP能显著抑制GW3965的作用(P<0.05),载脂蛋白E抗体及ABCA1抗体均对GW3965中剂量的活性无显著影响,单独使用GGPP处理的细胞各项指标与模型组无显著差异。结论 LXRα激动剂能够显著抑制巨噬细胞模型中由胆固醇晶体激活NLRP3炎症小体导致的IL-1β水平升高,且此作用与抑制核因子κB(NF-κB)信号通路有关。
Aim To explore the role and mechanism of liver X receptor α( LXRα) agonist on increased cleaved interleukin-1β( IL-1β) expression caused by Nod-like receptor protein-3( NLRP3) inflammasome in macrophages.Methods THP-1 cells were activated into macrophages by phorbol 12-myristate 13-acetate( PMA),and then were divided into different groups: model group( 1 μg / L cholesterol crystal),GW3965 high,middle and low concentration groups( added30 μmol / L,10 μmol / L,3. 3 μmol / L GW3965 based on model group),GW3965 + GGPP group( 10 μmol / L GW3965 plus10 μmol / L GGPP),GGPP group( 10 μmol / L GGPP),ABCA1 antibody and apolipoprotein E( Apo E) antibody groups( 10μmol /L GW3965 plus 1∶ 200 ABCA1 or Apo E respectively). Total RNA,total protein,nuclear protein and supernatant protein were extracted after 48 h treatment. mRNA levels of ABCA1,NLRP3,Caspase-1 and IL-1β were detected by realtime PCR,and the protein levels of ABCA1,NLRP3,Caspase-1,nuclear NF-κB p65,IL-1β and cleaved IL-1β were measured by Western blot. Results Compared with control group,cleaved IL-1β,NLRP3 and Caspase-1 levels were significantly increased after the stimulation of cholesterol crystal( P < 0. 05) in model group. The treatments of middle and high concentration GW3965 attenuated the alterations( P < 0. 05). Compared with GW3965 middle concentration group,there were significant statics differences of the above biomarker levels in GW3965 + GGPP group,but not in ABCA1 antibody or Apo E antibody group. Conclusion LXRα agonist GW3965 can attenuate the increased level of cleaved IL-1β caused by NLRP3 Inflammasome,and NF-κB p65 pathway may be involved in the mechanism.
出处
《中国动脉硬化杂志》
CAS
北大核心
2015年第1期17-23,共7页
Chinese Journal of Arteriosclerosis
基金
国家自然科学基金(81102448)
