葫芦巴碱调节PI3K/Akt/NF-κB信号通路对变应性接触性皮炎大鼠免疫反应的影响

Impact of trigonelline on the immune response of rats with allergic contact dermatitis by regulating the PI3K/Akt/NF-κB signaling pathway

目的探讨葫芦巴碱对变应性接触性皮炎(ACD)大鼠免疫、炎性反应及PI3K/Akt/NF-κB信号通路的影响。方法60只SD大鼠随机分为正常组、ACD组、葫芦巴碱(低、中、高)剂量组(20、40、80 mg/kg)和PI3K抑制剂(LY294002)组(40 mg/kg),每组10只。除正常组外,其余组大鼠采用2,4二硝基氟苯(DNFB)诱导ACD模型。给药结束后,通过录像观察大鼠挠痒行为;HE染色检测大鼠耳皮肤组织病理学变化;ELISA检测大鼠血清IgE及Th1、Th2、Th17型细胞因子(IFN-γ、IL-4、IL-17)水平;Western blot检测大鼠耳皮肤组织中炎性因子(IL-1β、IL-6)蛋白及PI3K/Akt/NF-κB信号通路相关蛋白表达。结果与正常组比较,ACD组大鼠挠痒次数增加,血清IgE、IFN-γ、IL-4、IL-17水平及耳皮肤组织中IL-1β、IL-6蛋白表达和p-PI3K/PI3K、p-Akt/Akt、p-NF-κB/NF-κB比值升高(P<0.05),耳皮肤组织角化过度且可见大量炎性细胞浸润;与ACD组比较,葫芦巴碱(低、中、高)剂量组和LY294002组大鼠挠痒次数减少,血清IgE、IFN-γ、IL-4、IL-17水平及耳皮肤组织中IL-1β、IL-6蛋白表达和p-PI3K/PI3K、p-Akt/Akt、p-NF-κB/NF-κB比值降低(P<0.05),耳皮肤组织病理损伤均有所改善,且葫芦巴碱各给药组呈剂量依赖效应;葫芦巴碱高剂量组和LY294002组大鼠上述指标比较,差异无统计学意义(P>0.05)。结论葫芦巴碱可抑制ACD大鼠皮肤组织中PI3K/Akt/NF-κB信号通路激活,调控Th1、Th2、Th17型免疫应答并减轻皮肤局部和全身炎性反应。

Objective To investigate the impacts of trigonelline on immune,inflammatory response and the p-phosphatidylinositol 3-kinase(PI3K)/p-Protein kinase B(AKT)/p-nuclear factor kappa B(NF-kappaB)signaling pathway in rats with allergic contact dermatitis(ACD).Methods Sixty Sprague-Dawley(SD)rats were randomly grouped into normal group,ACD group,low-dose trigonelline group(20mg/kg),medium-dose trigonelline group(40mg/kg),high-dose trigonelline group(80mg/kg)and PI3K inhibitor group(LY29400240mg/kg),with 10 rats in each group.Except for those in normal group,rats in the remaining groups were subjected to ACD modeling by 2,4-dinitrofluorobenzene(DNFB)induction.After administration of trigonelline or LY294002,the scratching behavior of rats was observed by watching video.The hematoxylin and eosin(H&E)staining was applied to detect the histopathological changes of rat ear skin.Serum levels of immunoglobulin E(IgE)and T-helper type 1(Th1),Th2 and Th17 cytokines(interferon-gamma[IFN-γ],interleukin 4[IL-4],interleukin 17[IL-17])were detected by Enzyme-linked immunosorbent assay(ELISA).Protein expressions of inflammatory factors(interleukin-1beta[IL-1β],interleukin 6[IL-6])and key proteins involved in the PI3K/Akt/NF-κB signaling pathway in rat ear skin tissues were detected by Western blot.Results Compared with normal group,rats in ACD group presented significantly higher times of scratching,higher serum levels of IgE,IFN-γ,IL-4 and IL-17,higher protein levels of IL-1β,IL-6 in rat ear skin tissues,and higher p-PI3K/PI3K,p-Akt/Akt and p-NF-κB/NF-κB(P<0.05),as well as excessive keratinization of ear skin tissue and abundant infiltration of inflammatory cells.Compared with ACD group,rats in low-dose,medium-dose and high-dose trigonelline group and PI3K inhibitor group presented significantly lower times of scratching,lower serum levels of IgE,IFN-γ,IL-4 and IL-17,lower protein levels of IL-1β,IL-6 in rat ear skin tissues,and lower p-PI3K/PI3K,p-Akt/Akt and p-NF-κB/NF-κB(P<0.05),as well as alleviated ear skin damages.Moreover,the protective effect of trigonelline on rats was dose-dependent.The above indicators were comparable between rats of high-dose trigonelline group and PI3K inhibitor group(P>0.05).Conclusion Trigonelline alleviates skin local reaction and systemic inflammatory reactions in ACD rats by inhibiting the activation of the PI3K/Akt/NF-κB signaling pathway in the skin tissue,and regulating Th1,Th2,Th17-type immune responses.