摘要
目的探讨氧化应激时成纤维细胞(NIH-3T3)中NALP3炎性复合体、p38丝裂原活化蛋白激酶(MAPK)的变化及阿魏酸钠的干预机制。方法将NIH-3T3细胞分为6组:对照组;H_2O_2(200μmol/L)刺激组;阿魏酸钠(400μg/mL)组;H_2O_2+N-乙酰半胱氨酸(H_2O_2200μmol/L+NAC 5mmol/L)组(抗氧化组);H_2O_2+p38 MAPK抑制剂(H_2O_2200μmol/L+SB203580 5μmol/L)组(p38 MAPK阻断剂组);阿魏酸钠干预(H_2O_2200μmol/L+阿魏酸钠400μg/mL)组。培养24h后,荧光定量PCR检测各组NIH-3T3细胞中NALP3、Caspase-1及p38αmRNA的表达;培养48h后,Western blot检测各组NIH-3T3细胞中NALP3、p-p38和p38蛋白的表达量(p-p38为p38活化表示形式,p38 MAPK信号通路的活化用p-p38/p38表示);培养24h后,ELISA检测各组细胞的上清液中白细胞介素(IL)-1β的含量。结果相比于对照组,H_2O_2的刺激可以上调NIH-3T3细胞中NALP3、Caspase-1及p38αmRNA的表达,NALP3及p-p38/p38蛋白的表达量,以及IL-1β分泌均增加(P均<0.05);抗氧化组、p38 MAPK阻断剂组及阿魏酸钠干预组相较于H_2O_2刺激组,NALP3、Caspase-1及p38αmRNA的表达量以及NALP3、p-p38/p38蛋白的表达量均降低,IL-1β的释放也减少(P均<0.05);而抗氧化组、p38MAPK阻断剂组及阿魏酸钠干预组间上述指标的差异无统计学意义(P>0.05)。结论阿魏酸钠可能通过抑制p38 MAPK通路的活化降低NALP3炎性复合体、Caspase-1和IL-1β的表达,从而下调炎症级联反应。
Objective To investigate the changes of NACHT-PYD-containing protein 3 （NALP3） inflammasome and p38 mitogen-activated protein kinase （MARK）, and the interventional mechanism of sodium ferulate （SF） in mouse embryonic fibroblasts （NIH-3T3 cells） under oxidative stress. Methods NIH-3T3 cells cultured in vitro were divided into 6 groups, including control group, H2O2 stress group （H2O2 200 μmol/L）, SF group （SF 400 μg/mL）, antioxidant group （H2O2 200 μmol/L＋NAC 5mmol/L）, p38 MAPK blockers group （H2O2 200 μmol/L＋SB203580 5 μ mol/L）, H2O2 ＋SF group （H2O2 200μmol/L＋SF 400 μg/mL）. The mRNA expression levels of NALP3, Caspase-1 and p38a were evaluated by fluorescent quantitative real-time PCR （qRT- PCR） at 24 h after cell culture; Western blot was performed to detect the expressions of NALP3, p-p38 to p38 protein （the activation of p38 MAPK signaling pathway expressed by the ratio of p-p38 to p38） at 48 h after cell culture; The levels of interleukin-lbeta （IL-1β） in different groups were detected by ELISA at 2 h after cell culture. Results Compared with control group, H2O2 could not only increase the expression levels of NALP3, Caspase-1, p38a mRNAs and NALP3, p-p38/p38 proteins but also the secretion of IL-1β in NIH-3T3 cells when compared with the control group （P〈0.05）; Antioxidant NAC, p38 MAPK blockers and H2O2 ＋SF group could partly resisted the effects of H2 O2 on NIH-3T3 cells, that decreased the level of mRNA expressions of NALP3, Caspase-1, p38a and protein expressions of NALP3 and p-p38/p38, and reduced the secretion of IL-1β when compared to the H2O2 stress group （P〈0. 05）; However, H2O2 ＋NAC group, SB203580 group and H2O2 ＋SF group showed no statistical difference of those indicators （P〈0. 05）. Conclusion The mechanism of sodium ferulate attenuated oxidative stress induced inflammation may be through blunting p38 MAPK signal pathway, the expression of NALP3 inflammasome, Caspase-1 and the secretion of IL-1β are reduced.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2018年第2期209-214,共6页
Journal of Sichuan University(Medical Sciences)
基金
四川省科技厅科技支撑计划项目(No.2013SZ0001)资助
关键词
H2O2
NALP3炎性复合体
P38
MAPK
阿魏酸钠
IL-1β
H2O2
NACHT-PYD-containing protein 3 inflammasome
p38 mitogen-activatedprotein kinase
Sodium ferulate
Interleukin-lbeta
