佛司可林对活化的人单核巨噬细胞分泌白细胞介素-1β的抑制作用  

Forskolin inhibited the secretion of interleukin -1βin activated human THP-1 macrophages

作　　者：陈燕[1,2] 冯锦锦[1,2] 闫少华[1] 李一冬[1] 李云龙[1,2] 文建国[1,2] Chen Yan;Feng Jinjin;Yah Shaohua;Li Yidong;Li Yunlong;Wen Jianguo(Institute of Clinic Medicine,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China;Peadiatric Urodynamic Center and Department of Urology,the First Affiliated Hospital of Zhengzhou University ,Zhengzhou450052,China)

机构地区：[1]郑州大学第一附属医院临床医学重点学科开放实验室,450052 [2]郑州大学第一附属医院小儿尿动力学中心&泌尿外科,450052

出　　处：《中华实用儿科临床杂志》2018年第23期1823-1827,共5页Chinese Journal of Applied Clinical Pediatrics

基　　金：国家自然科学基金青年基金(81500588).

摘　　要：目的 探讨佛司可林对人单核巨噬细胞(THP-1巨噬细胞)内核苷酸结合寡聚化结构域样受体3 (NLRP3)炎症小体活化及活化后白细胞介素-1β(IL-1β)炎性因子分泌的影响,从而为临床治疗小儿炎症性疾病提供参考.方法 利用佛波酯诱导人单核细胞系THP-1细胞分化成巨噬细胞,尼日利亚菌素活化巨噬细胞,佛司可林(10、50、100 μmol/L)刺激活化的巨噬细胞;实时荧光定量聚合酶链反应(qPCR)法检测细胞炎症小体核苷酸结合寡聚化结构域样受体3(NLRP-3)、炎性因子IL-1β和Caspase-1 mRNA表达;Western blot法检测细胞炎症小体NLRP-3、白细胞介素1β前体(pro-IL-1β)、半胱氨酸天冬氨酸蛋白酶1前体(pro-Caspase-1)和Caspase-1蛋白量的表达;酶联免疫吸附法(ELISA)检测炎性因子IL-1β蛋白的表达.结果 尼日利亚菌素使THP-1巨噬细胞的NLRP-3、IL-1β和Caspase-1蛋白的mRNA表达分别提高为未激活组的7.59倍(P<0.001)、579.10倍(P<0.001)和3.59倍(P<0.001),但佛司可林刺激对激活的THP-1巨噬细胞的炎症小体NLRP-3、炎性因子IL-1β和Caspase-1的mRNA表达无影响(P>0.05).Western blot结果显示,佛司可林对激活的THP-1巨噬细胞的pro-IL-1β和pro-Caspase-1的蛋白量表达无明显影响(P>0.05),但明显降低NLRP-3和Caspase-1的蛋白量表达(P<0. 001).ELISA结果提示,尼日利亚菌素使THP-1巨噬细胞的IL-1β的分泌由未激活量584.0 nmol/L升高到激活量2 695.6 nmol/L(t=16.031 1,P<0.001),而佛司可林10 μmol/L、50 μmol/L和100 μmol/L分别可使激活量降为1 858.4 nmol/L(t=5.365 5,P<0.001)、1 467.9 nmol/L(t=8.047 5,P<0.001)和1 246.7 nmol/L(t=10.199 1,P<0.05).结论 佛司可林不影响激活的THP-1巨噬细胞炎症相关蛋白前体的基因转录和蛋白翻译表达,但降低其炎性因子IL-1β翻译后的蛋白分泌量,从而抑制炎性反应,可用于治疗由IL-1β引起的炎性疾病.Objective To investigate the effects of Forskolin on the activation of nucleotide-binding oligome-rization domain like receptor family,pyrin domain-containing 3(NLRP-3)inflammasome and the secretion of inter-leukin-1β(IL-1β)in activated human THP-1 macrophages,which can provide evidence for clinical treatment of inflammatory diseases in children.Methods Human monocyte cell line THP-1 cells were induced to differentiate into macrophages by Phorbol-12-myristate-13-acetate(PMA),and Forskolin(10,50,100 μmol/L)stimulated activa-ted macrophages by nigericin.The mRNA of the inflammasome markers NLRP-3,IL-1β and Caspase-1 were detec-ted by real-time quantitative polymerase chain reaction(qPCR)method.The protein of NLRP -3,pro -IL -1β, pro-Caspase-1 and Caspase-1 were detected by Western blot.The secretion of inflammatory cytokines IL-1β was detected by enzyme-linked immuno-sorbent assay(ELISA).Results Nigericin activated the cells and the mRNA expression of NLRP-3,IL-1β and Caspase-1 increased by 7.59 times(P<0.001),579.10 times(P<0.001)and 3. 59 times (P <0. 001 ),compared to non - activated cells;Forskolin had no effect on the mRNA expression of NLRP-3,Caspase-1 and IL-1β on activated THP-1 macrophages(P>0.05).Western blot showed that Forskolin also had no effect on the protein expression of pro-Caspase-1 and pro-IL-1β in activated THP-1 macrophages (P>0. 05),but the expression of NLRP -3 and Caspase -1 decreased significantly (P <0.001). The results of ELISA showed that the IL -1β secretion increased from basal 584. 0 nmol/L to activated 2 695. 6 nmol/L (t =16.031 1,P<0. 001)on THP -1 macrophages by nigericin,but Forskolin(10,50,100 μmol/L)reduced it to 1 858. 4 nmol/L(t=5.365 5,P <0. 001),1 467.9 nmol/L(t =8.047 5,P <0.001)and 1 246.7 nmol/L(t =10.199 0,P<0.001)on the activated THP-1 macrophages.Conclusions Forskolin can not affect the expression of pro-cytokines at the gene transcription and protein translation levels in activated THP-1 cells,but inhibit the secre-tion of inflammatory cytokines IL-1β,so

关 键 词：炎症 佛司可林 巨噬细胞 炎症小体 白细胞介素-1Β 

分 类 号：R72[医药卫生—儿科]