NALP3炎性复合体及p38 MAPK信号通路在氧化应激中的作用及阿魏酸钠的干预机制

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；H2O2 （200 μmol/L）刺激组；阿魏酸钠（400 μg/mL）组；H2O2 +N-乙酰半胱氨酸（H2O2 200 μmol/L+ NAC 5 mmol/L）组（抗氧化组）；H2O2 + p38 MAPK抑制剂（H2O2 200 μmol/L+ SB203580 5 μmol/L）组（p38 MAPK阻断剂组）；阿魏酸钠干预(H2O2 200 μmol/L+阿魏酸钠 400 μg/mL)组。培养24 h后，荧光定量PCR检测各组NIH-3T3细胞中NALP3、Caspase-1及P38α mRNA的表达；培养48 h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24 h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，H2O2 的刺激可以上调NIH-3T3细胞中NALP3、Caspase-1及P38α mRNA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H2O2 刺激组，NALP3、Caspase-1及P38α mRNA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、p38 MAPK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。

Sodium Ferulate Attenuates Oxidative Stress Induced Inflammation via Suppressing NALP3 Inflammasome and p38 MAPK Signal Pathway

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 5 mmol/L), p38 MAPK blockers group (H2O2 200 μmol/L+SB203580 5 μmol/L), H2O2+SF group (H2O2 200 μmol/L+SF 400 μg/mL). The mRNA expression levels of NALP3, Caspase-1 and p38α 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-1beta (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, p38α 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 H2O2 on NIH-3T3 cells, that decreased the level of mRNA expressions of NALP3, Caspase-1, p38α 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.