槲皮素调控p38 MAPK/NF-κB/NLRP3信号通路对大鼠分泌性中耳炎的作用机制

Mechanism of quercetin regulating p38 MAPK/NF-κB/NLRP3 signaling pathway in rats with secretory otitis media

目的 探究槲皮素(quercetin, Que)对大鼠分泌性中耳炎(secretory otitis media, SOM)的作用及机制。方法 选取雄性SD大鼠48只,随机性分为对照组(Control)、模型组(Model)、Que组(100 mg·kg^(-1))、Que+p38 MAPK激动剂组(100 mg·kg^(-1)Que+2 mg·kg^(-1)Anisomycin),每组各12只。除Control组外,其余各组大鼠均采取腹腔注射卵清蛋白复制分泌性中耳炎大鼠模型,造模成功后,各组大鼠进行相应干预21 d。给药干预结束后,苏木精-伊红(HE)染色观察各组大鼠中耳组织形态学变化;酶联免疫吸附法(ELISA)测定血清TNF-α、IL-6、IL-1β、IL-18及中耳灌洗液中IFN-γ、IL-4水平;实时定量PCR (RT-qPCR)和Western blot检测中耳黏膜组织p38 MAPK/NF-κB/NLRP3信号通路相关mRNA及蛋白表达变化。结果 与Control组相比,Model组大鼠表现中耳内黏膜层增厚,伴有大量炎性细胞浸润,血清炎性因子TNF-α、IL-6、IL-1β、IL-18及中耳灌洗液中IL-4增加,IFN-γ降低(P <0.01),中耳黏膜组织p-p38MAPK/MAPK、p-NF-κB/NF-κB、NLRP3、ASC、pro-caspase-1、cleaved-caspase-1、IL-1β、IL-18蛋白及mRNA表达水平均明显升高(P <0.01)。与model组相比,Que能够明显改善中耳内黏膜增厚,抑制炎性细胞浸润,降低TNF-α、IL-6、IL-1β、IL-18、IL-4及中耳黏膜组织p38 MAPK/NF-κB/NLRP3通路mRNA和蛋白表达,升高IFN-γ(P <0.05或P <0.01)。与Que组相比,Anisomycin能够逆转Que对SOM大鼠的保护作用。结论 Que能够有效改善SOM发生、发展,其机制与抑制p38 MAPK/NF-κB/NLRP3信号通路介导的炎性反应相关。

Aim To investigate the effect of quercetin(Que)on secretory otitis media(SOM)rats and its mechanism.Methods Forty-eight male SD rats were selected and randomly divided into control group(control),model group(model),Que group(100 mg·kg^(-1)),Que+p38 MAPK agonist group(100 mg·kg^(-1)),Que+2 mg·kg^(-1) Anisomycin),with 12 rats in each group.Except for the control group,the rats in the other groups were given intraperitoneal injection of ovalbumin to replicate the rat model of secretory otitis media.After successful modeling,rats in each group were given corresponding interventions for 21 days,and hematoxylin-eosin(HE)staining was used to observe the morphological changes of the middle ear tissue of the rats in each group.TNF-α,IL-6,IL-1β,IL-1β,IL-18 were measured in serum and the levels of IFN-γand IL-4 in the middle ear lavage fluid by enzyme-linked immunosorbent assay(ELISA).The changes of mRNA and protein expression related to the p38 MAPK/NF-κB/NLRP3 signaling pathway in the middle ear mucosa were detected by real-time quantitative PCR(RT-qPCR)and Western blot.Results Compared with the control group,the rats in the model group showed thickening of the inner ear mucosa,accompanied by a large number of inflammatory cell infiltration,serum inflammatory factors TNF-α,IL-6,IL-1β,IL-18 and IL-4 levels in middle ear lavage fluid significantly increased,IFN-γdecreased(P<0.01),and the protein and mRNA expression levels of p-p38MAPK/MAPK,p-NF-κB/NF-κB,NLRP3,ASC,pro-caspase-1,cleaved-caspase-1,IL-1β,IL-18 all significantly increased(P<0.01).Compared with the model group,Que could significantly improve the thickening of the inner ear mucosa,inhibit the infiltration of inflammatory cells,reduce TNF-α,IL-6,IL-1β,IL-18,IL-4 and p38 MAPK/NF-κB/NLRP3 pathway mRNA and protein expression in the middle ear mucosa and elevate IFN-γ(P<0.05 or P<0.01).Compared with Que group,Anisomycin could reverse the protective effect of Que on SOM rats.Conclusions Que could effectively improve the occurrence and development of SOM,and its mechanism is related to the inhibition of the inflammatory response mediated by the p38 MAPK/NF-κB/NLRP3 signaling pathway.