连翘脂素通过调控P2X7R/NF-κB/NLRP3炎性小体信号通路缓解LPS/ATP诱导的L02细胞炎症  

Phillygenin Mitigates LPS/ATP-induced L02 Cell Inflammation by Regulating P2X7R/NF-κB/NLRP3 Inflammasome Signaling Pathway

作　　者：邓英 赵兴桃 周梦婷 薛鑫妍 廖利[1] 王晶 李芸霞[1] DENG Ying;ZHAO Xingtao;ZHOU Mengting;XUE Xinyan;LIAO Li;WANG Jing;LI Yunxia(State Key Laboratory of Southwestern Chinese Medicine Resources,School of Pharmacy,Chengdu University of Traditional Chinese Medicine,Chengdu 611137,China)

机构地区：[1]成都中医药大学药学院西南中药资源国家重点实验室,成都611137

出　　处：《中国实验方剂学杂志》2022年第14期61-69,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81630101);四川省科技计划项目(2021JDRC0041);成都中医药大学杏林学者研究推进项目(CXTD2018019)。

摘　　要：目的:为研究连翘脂素(Phillygenin,PHI)对脂多糖(LPS)和腺嘌呤核苷三磷酸(ATP)联合诱导L02细胞中的抗炎药效和对P2X7受体(P2X7R),NOD样蛋白结构域受体3(NLRP3)和核转录因子-κB(NF-κB)表达的影响。方法:本研究采用先给予100μg·L^(-1)LPS处理24 h后,再使用5 mmoL·L^(-1)ATP 5 h建立L02细胞炎症模型。给药组在给予LPS处理的同时给予不同浓度PHI(100、50、25 mg·L^(-1))培养6 h,随后换液,继续用LPS处理18 h,ATP处理5 h后,采用实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)检测L02细胞中白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)、P2X7R、NLRP3、胱天蛋白酶-1前体(pro-Caspase-1)、裂解胱天蛋白酶-1(cleaved Caspase-1)、NF-κB、NF-κB抑制蛋白α(IκBα)mRNA和蛋白表达。通过分子对接实验预测P2X7R与PHI能否结合,以及DCFH-DA活性氧(ROS)荧光探针检测细胞中ROS的累积。采用小干扰核糖核酸(siRNA)沉默P2X7R,并随后通过Real-time PCR检测IL-1β、IL-18、P2X7R、NLRP3、Caspase-1、NF-κB、IκBα mRNA表达情况。结果:Real-time PCR和Western blot分析显示,与空白组比较,模型组IL-1β、IL-18的表达均有所升高(P<0.05);与模型组比较,给药组明显下调了IL-1β、IL-18 mRNA和蛋白的表达(P<0.05)。分子对接结果显示PHI与P2X7R有较好的结合作用。Real-time PCR和Western blot分析显示,与空白组比较,模型组P2X7R的表达明显上调(P<0.05);与模型组比较,给药组下调了P2X7R mRNA和蛋白表达(P<0.05)。ROS荧光探针分析显示,与空白组比较,ROS的含量明显升高(P<0.05);与模型组比较,给药组明显降低了ROS的积累(P<0.05)。Real-time PCR和Western blot分析显示,与空白组比较,模型组NLRP3炎性小体,NF-κB的表达明显升高(P<0.05);与模型组比较,给药组明显降低NLRP3、cleaved-Caspase-1和上调NF-κB、IκBα mRNA及蛋白表达(P<0.05)。Real-time PCR分析显示,与模型组比较,siRNA沉默P2X7R后,IL-1β、IL-18�Objective: To explore the effects of phillygenin(PHI)on the inflammation in L02 cells induced by lipopolysaccharide(LPS)and adenosine triphosphate(ATP)and the expression of purinergic 2X7receptor(P2X7R),NOD-like receptor family pyrin domain containing 3(NLRP3),and nuclear factor kappa B(NF-κB)expression. Method: In this study,the inflammation model was induced in L02 cells by 100 μg·L^(-1)LPS treatment for 24 h and 5 mmoL·L^(-1)ATP treatment for 5 h. The cells in the PHI groups were cultured with PHI(100,50,25 mg·L^(-1))for 6 h in the LPS treatment period,followed by LPS treatment for another 18 h.After ATP treatment for 5 h,the mRNA and protein expression of interleukin-1β(IL^(-1)β),interleukin-18(IL^(-1)8),P2X7R,NLRP3,Caspase-1 precursor(pro-Caspase-1),cleaved Caspase-1,NF-κB,and NF-κB inhibitor protein α(IκBα)in L02 cells was detected by real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)and Western blot,respectively. Molecular docking was used to predict whether P2X7R could bind to PHI,and DCFH-DA was employed to detect the accumulation of reactive oxygen species(ROS)in cells. P2X7R was silenced by small interfering ribonucleic acid(siRNA),and then the mRNA expression of IL^(-1)β,IL^(-1)8,P2X7R,NLRP3,Caspase-1,NF-κB,and IκBα was detected by Real-time PCR.Result: Real-time PCR and Western blot showed that compared with the normal group,the model group showed increased expression of IL^(-1)β and IL^(-1)8(P<0.05),and compared with the model group,the PHI groups showed down-regulated IL^(-1)β,IL^(-1)8 mRNA and protein expression(P<0.05). Molecular docking suggested a good binding effect of PHI to P2X7R. Real-time PCR and Western blot analysis showed that the expression of P2X7R in the model group was significantly up-regulated compared with that in the normal group(P<0.05),and compared with the model group,the PHI groups showed down-regulated mRNA and protein expression of P2X7R(P<0.05). DCFH-DA results showed that compared with the normal group,the model group showed in

关 键 词：连翘脂素 P2X7R L02细胞 NOD样蛋白结构域受体3(NLRP3)炎性小体 核转录因子-κB(NF-κB) 

分 类 号：R2-0[医药卫生—中医学] R22R285.5R289R33