NO抑制体外IFN-γ刺激成肌细胞/肌管NLRP3炎性小体活化

Inhibition of nitric oxide on activation of NLRP3 inflammasome of myoblast/myotubes stimulated by IFN-γ in vitro

目的观察一氧化氮供体硝普钠(SNP)、一氧化氮合酶抑制剂(L-NAME)对体外炎性培养(IFN-γ刺激)成肌细胞/肌管NLRP3炎症小体活化的影响。方法利用IFN-γ刺激小鼠C2C12成肌细胞/肌管,q PCR和Western blot分析炎性小体(ASC、NLRP3、caspase-1)的形成及活化、ELISA检测细胞培养上清IL-1β的分泌。进一步利用L-NAME和SNP处理IFN-γ诱导的C2C12细胞/肌管,分析炎性小体的形成、活化及IL-1β的分泌。结果 IFN-γ刺激培养后,成肌细胞/肌管中NLRP3、ASC和mature-caspase-1表达水平上调(P<0.01)。较之未刺激的细胞,IFN-γ诱导会显著上调成肌细胞/肌管培养基中的IL-1β浓度(P<0.01)。SNP处理后6 h,分化肌管(IFN-γ刺激48 h)内炎性小体(ASC、NLRP3、caspase-1)mRNA和蛋白水平较单纯刺激细胞显著下调(P<0.01),L-NAME则上调ASC、NLRP3、caspase-1表达水平(P<0.05)。与上述结果一致,SNP和L-NAME处理同时分别下调或上调培养基中IL-1β浓度(P<0.05)。结论在炎性条件下,成肌细胞/肌管具备合成并活化NLRP3炎性小体的能力。NO对炎性小体的形成及活化有一定的抑制作用。

Objective To observe the effect of sodium nitroprusside （SNP） or nitro L arginine acid methyl ester （L-NAME） on the NLRP3 inflammasome activation of C2C12 myoblast or differentiated myotubes in inflammatory culture in vitro. Methods C2C12 myoblasts were stimulated by IFN-γ, then the formation and activation of NLRP3 inflammasome were analyzed by qPCR and Western blot, and the secretion of IL- 1β in the cell culture supernatant was detected by ELISA. Further, C2C12 myoblasts induced by IFN-γwere treated by L-NAME or SNP respectively, then the formation and activation of NLRP3 inflammasome and the secretion of IL-1 [3 were analyzed. Results qPCR andWestern blot detection confirmed that expression of NLRP3, ASC and mature-caspase-I in C2C12 myoblasts or differ- entiation myotubes induced by IFN-γwas up-regulated（P 〈0. 01 ）. The IL-1β concentration in the medium of C2C12 induced by IFN-γwas up-regulated as compared with that of the non stimulating cells（P 〈0. 01 ）. At 6 h after SNP treatment, a significant down-regulation of mRNA and protein levels of differentiation myotube inflammasome （ASC and NLRP3, caspase-1 ） was found （P 〈 0. 01 ） and a contrary result was obtained after L-NAME treatment （P 〈 0. 05 ）. Consistent with the above results, the expression of IL- 115 in the culture supernantent was down-regulated or up-regulated by SNP or L-NAME respectively （P 〈 0. 05）. Conclusions In inflammatory conditions, C2C12 myo- blasts or differentiation myotubes possess the ability to synthesize inflammasome NLRP3. NO may inhibit the NLRP3 inflammasome formation and activation.