小檗碱调控痛风TLR4-NF-κB信号通路及NLRP3炎性体活化的分子机制研究

Monosodium urate crystals activating TLR4-NF-κB signaling pathway and NLRP3 inflammasome is a key factor in triggering inflammatory response of gout. Traditional Chinese Medicine holds that Phellodendron amurense has unique effect on the treatment of gout. Modern researches have proved that berberine (BBR) is the main active ingredients of phellodendron amurense, and plays an important role in regulating inflammation and immune. However, the study about whether berberine can regulate the inflammatory response of gout or not has not been reported. Our prophase researches have found that the expression of TLR4, NF-κB(p50/p65) and NLRP3 inflammasome were significantly abnormal in peripheral blood mononuclear cells (PBMCs) of patients with gout and BBR could regulate the abnormal expressions of NF-κB(p65), NLRP3 and IL-1β. Therefore, we hypothesize that the mechanism by which the BBR can inhibit the inflammatory response of gout may be closely related to the fact that it can regulate the activation of TLR4-NF-κB signaling pathway and NLRP3 inflammasome. To verify this hypothesis, the technologies of quantitative real-time polymerase chain reaction (qRT-PCR), Small interfering RNA (siRNA) interference and so on will be used in vitro cell culture and gout mouse model from the whole, cell and transcription level to study the regulation and the molecular mechanism of BBR activating TLR4-NF-κB signaling pathway and NLRP3 inflammasome . This project is expected to provide a new strategy for the diagnosis and treatment of gout and new drug development.

尿酸盐晶体活化TLR4-NF-κB信号通路及NLRP3炎性体是触发痛风炎症反应的关键因素。中医认为黄柏对痛风具有独特的治疗作用，现代研究证实黄柏主要有效成分小檗碱对炎症和免疫具有重要的调控作用，然而小檗碱是否调控痛风炎症尚未见相关报道。我们前期研究发现，痛风患者外周血单个核细胞TLR4、NF-κB(p50/p65)及NLRP3炎性体表达显著异常，而小檗碱可调控表达异常的NF-κB(p65)、NLRP3及IL-1β。为此，我们提出假说：小檗碱可抑制痛风炎症反应，其机制可能与其调控TLR4-NF-κB信号通路及NLRP3炎性体的活化密切相关。为验证这一假说，本项目拟通过体外细胞培养及痛风小鼠模型，采用qRT-PCR与siRNA抑制等技术分别从整体、细胞和转录水平探讨小檗碱对痛风TLR4-NF-κB信号通路及NLRP3炎性体活化的调控及其分子机制。本项目有望为痛风的诊治及新药开发提供新的思路。

目的：探讨小檗碱调控痛风患者TLR4-NF-κB-NLRP3炎症信号轴活化的分子机制。方法：用半定量聚合酶链反应（RT-PCR）法和/或实时荧光定量聚合酶链反应(qRT-PCR)法检测65例原发性痛风（PG）患者和30名健康对照（HC）PBMCs的NLRP3炎性体、TLR4、核因子κB（p65）[NF-κB（p65）]、IL-1β基因mRNA表达水平；用Western Blot法检测NLRP3炎性体、TLR4-NF-κB等蛋白表达水平；用ELISA检测血清IL-1蛋白表达水平；采用Spearman相关分析NF-κB(p50)与PYCARD、NLRP3、PYCARD及IL-1β相关性；培养THP-1源巨噬细胞，以尿酸盐晶体、不同浓度小檗碱（BBR）以及MSU+BBR刺激THP-1源巨噬细胞以及与健康人PBMCs，在0.5、3、6h后提取培养上清、THP-1细胞与PBMCs，采用相同的方法检测各组NLRP3炎性体、TLR4-NF-κB、IL-1表达水平。结果：PG患者PBMCs的NLRP3炎性体、TLR4、NF-κB（p65）、IL-1β mRNA与蛋白表达与HC组相比具有统计学意义（P＜0.05, P＜0.01）；Spearman相关性分析显示NF-κB(p50)与PYCARD mRNA表达、NLRP3与IL-1β mRNA表达、PYCARD与IL-1β mRNA表达具有相关性。经MSU、BBR、MSU+LPS刺激THP-1源巨噬细胞后NLRP3炎性体、TLR4-NF-κB信号通路相关蛋白表达与对照组相比均具有统计学意义（P＜0.05,P＜0.01），同时经MSU、BBR、MSU+LPS刺激健康人PBMCs后培养上清IL-1与对照组相比具有统计学意义（P＜0.01）。结论：在PG患者的炎症反应中TLR4-NF-κB-NLRP3炎症信号轴的活化发挥重要的作用；BBR对TLR4-NF-κB-NLRP3炎症信号轴的活化具有重要的调控作用；BBR可能为调控痛风炎症反应中TLR4-NF-κB-NLRP3炎症信号轴活化的重要靶药物。