雷公藤二萜类化合物通过抑制多条信号通路缓解巨噬细胞炎症反应

Diterpenoids of Tripterygium wilfordii decrease inflammatory response of macrophage by inhibiting multiple signaling pathways

目的研究雷公藤二萜类化合物对脂多糖诱导的小鼠单核巨噬细胞炎症模型的抗炎活性及其作用机制。方法MTT法检测化合物对细胞活力的影响;Griess法检测化合物对脂多糖诱导的RAW264.7细胞中NO产生的影响;ELISA法检测细胞培养上清中白介素6(IL-6)、肿瘤坏死因子α(TNF-α)、白介素1β(IL-1β)、白介素18(IL-18)的含量;Western blot法检测细胞内相关蛋白的表达和信号通路蛋白的磷酸化;流式细胞技术检测化合物对RAW264.7细胞吞噬功能的影响。结果雷公藤二萜类化合物hypoglicin H(1)和ent-pimara-8(14),15-diene-19-ol(6)能够有效抑制LPS诱导的RAW264.7细胞中的炎性介质(COX-2和iNOS)和细胞炎性因子(IL-6、IL-1β、IL-18)的生成,并能够明显抑制IκBα、JNK、ERK、p38、STAT3的磷酸化和NLRP3的生成,但是对IκBα、JNK、ERK、p38、STAT3的表达没有明显的影响。同时,雷公藤二萜类化合物1和6还抑制巨噬细胞的吞噬功能。结论雷公藤二萜类化合物1和6可能是通过抑制MAPK和NF-κB信号通路以及STAT3的激活,从而抑制NLRP3炎症小体、炎性介质(COX-2和iNOS)和细胞炎性因子(IL-6、IL-1β、IL-18)的生成来发挥其抗炎作用。

Aim To study the anti-inflammatory activity of diterpenes from Tripterygium wilfordii on lipopolysaccharide(LPS)-induced macrophage and its mechanism.Methods MTT assay was used to detect the cytotoxicity of compounds.The Griess method was used to detect the NO on LPS-induced RAW264.7 cells.ELISA was applied to determine the contents of interleukin 6(IL-6),tumor necrosis factorα(TNF-α),interleukin 1β(IL-1β)and interleukin 18(IL-18)in cell culture supernatant.Western blot was used to detect IκBα,JNK,ERK,p38,STAT3 and their phosphorylation in LPS-induced RAW264.7,as well as the effect on COX-2,iNOS,NLRP3,caspase-1,cleaved-caspase-1.Flow cytometry was employed to detect the effects of compounds on the phagocytosis of RAW264.7 cells.Results Hypoglicin H(1)and ent-pimara-8(14),15-diene-19-ol(6),two diterpenoid compounds from Tripterygium wilfordii could effectively inhibit the expression of inflammatory mediators(COX-2 and iNOS)and inflammatory cytokines(IL-6,IL-1β,IL-18)in LPS-induced RAW264.7 cells.Further research found that the phosphorylation of IκBα,JNK,ERK,P38,STAT3 and NLRP3 was all inhibited;however,there was no significant effect on the expression of IκBα,JNK,ERK,P38 and STAT3.At the same time,they also inhibited the phagocytosis of macrophages.Conclusions The anti-inflammatory mechanism of Tripterygium wilfordii diterpenoids 1 and 6 might be through inhibiting the production of NLRP3 inflammatory bodies,inflammatory mediators(COX-2 and iNOS)and inflammatory cytokines(IL-6,IL-1βand IL-18),which is closely related to inhibiting the activation of MAPK,NF-κB and STAT3 pathway.