摘要目的 通过MTB细胞壁成分相对分子质量为19 000的脂蛋白(P19)和姜黄素作用于人巨噬细胞WBC264-9C细胞系,并对p38丝裂原活化蛋白激酶(p38 MAPK)信号通路进行药物干预,观察姜黄素对P19诱导巨噬细胞免疫反应的影响并探讨其可能的分子机制.方法 将人源性巨噬细胞WBC264-9C分为P19组、姜黄素组和P19+姜黄素组,每组5×108个细胞.用P19和姜黄素刺激WBC264-9C 48 h,观察其对巨噬细胞增殖和凋亡的影响及对IL-1β、IL-6、肿瘤坏死因子-α(TNF-α)和信号转导子与转录活化子3(STAT3)等分子表达的调控作用.用p38 MAPK阻断剂干预p38 MAPK活性后,观察姜黄素对P19诱导的巨噬细胞炎症细胞因子及凋亡相关分子表达的影响.组间比较采用方差分析,用Dunnett's法或独立样本t检验进行两两比较.结果 20和40 μmol/L姜黄素对WBC264-9C的增殖抑制率分别为(10.1±2.3)％和(19.0±2.7)％.对照组、P19组和P19+姜黄素组对巨噬细胞的抑制率分别为(6.7±4.2)％、(45.4±3.6)％和(32.1±3.0)％,提示姜黄素可显著降低P19所致巨噬细胞增殖抑制作用(q =9.75,P＜0.01).姜黄素+P19组IL-1β、IL-6、TNF-α和STAT3的mRNA吸光度值分别为13.2 ±2.7、33.5±1.1、3.3±2.2和0.9±1.7,均显著低于P19组(21.8±3.5、14.3 ±1.4、6.1±3.6和4.5±3.4),差异均有统计学意义(q值为7.18 ～3.22,均P＜0.05).姜黄素+P19组P53和Bax蛋白表达的吸光度值(94.3 ±0.2和70.8 ±0.7)均显著低于P19组(320.2±0.2和182.6±1.2),差异均有统计学意义(q值分别为7.05和7.66,均P＜0.01).应用p38 MAPK阻断剂后姜黄素+P19组IL-6的mRNA和Bax/Bcl2蛋白的吸光度值(34.9±1.5和0.36 ±0.09)均显著低于未应用p38 MAPK阻断剂组(69.9±1.8和0.71 ±0.11),差异均有统计学意义(q值分别为2.36和3.50,均P＜0.05).结论 P19可能通过激活p38 MAPK信号通路上调巨噬细胞中炎性因子的表达并促进细胞凋亡.低浓度姜黄素可能通过抑制p38 MAPK激活发挥对巨噬细胞的保护作用.

abstractsObjective By using the cell wall component of Mycobacterium tuberculosis 19 000 lipoprotein (P19) and curcumin (CUR) acting on the human macrophage cell line WBC264-9C,and by the blocking of the p38 mitogen-activated protein kinases (p38 MAPK) signaling pathway,we wanted to investigate the effect of curcumin on P19-induced inflammatory responses and apoptosis in human macrophages and the potential underlying molecular mechanisms.Methods P19 and CUR were used to stimulate human macrophages for 48 h.Their effects on growth inhibition and on apoptosis in macrophages were observed.The combination effect of CUR with P19 on the expression of IL-1β,IL-6,TNF-α,STAT3,P53,Bax,Bcl2 and phspho-p38 MAPK levels were also observed.By using the antagonist of p38 MAPK,the effect of CUR on P19-induced expression of inflammatory cytokines and apoptotic proteins were investigated.Results Twenty and 40 μmol/L of CUR inhibited the growth of macrophages by (10.1 ±2.3)％ and (19.0 ±2.7)％.The growth inhibition rate of maerophages in the controls,P19 and P19 + CUR treated groups were (6.7 ± 4.2) ％,(45.4 ± 3.6) ％ and (32.1 ± 3.0) ％,respectively.The P19-induced growth inhibition was significantly attenuated by CUR treatment (q =9.75,P ＜ 0.01).The expression of IL-1β,IL-6,TNF-α and STAT3 mRNA (13.2 ± 2.7,33.5 ± 1.1,3.3 ± 2.2,0.9 ± 1.7)were significantly lower than P19-treated groups (21.8 ± 3.5,14.3 ± 1.4,6.1 ± 3.6,4.5 ± 3.4)(q values were ranged from 7.18 to 3.22,all P ＜ 0.05).Furthermore,P53 protein (94.3 ± 0.2 ; q =7.05,P ＜0.01) and Bax (70.8 ±8.7; q =7.66,P ＜0.01) levels were decreased in CUR ±P19 group as compared with P19 group (320.2 ±0.2 and 182.6 ± 1.2).Blockade of p38 MAPK accompanied with CUR and P19 induced significantly lower levels of IL-6 mRNA (34.9 ± 1.5,q =2.36,P ＜0.05) and Bax/Bcl2 protein (0.36 ± 0.09; q =3.50,P ＜ 0.05) expression,as compared with the controls (69.9 ± 1.8 and 0.71 ± 0.11).Conclusions P19 increased the expression of inflammatory cytokines and promoted the apoptosis of macrophages possibly through the activation of p38 MAPK signaling pathway.Low concentration of curcumin may play a protective effect against P19-induced immune responses by inhibiting the p38 MAPK pathway in macrophages.