基于NF-κB P65/IκBα信号通路探讨养阴益气活血方治疗干燥综合征作用机制

Study on the Mechanism of Yangyin Yiqi Huoxue Recipe in Treating Sj?gren’s Syndrome Based on NF-κB P65/IκBαSignal Pathway

目的研究养阴益气活血方对干燥综合征非肥胖糖尿病(NOD)小鼠颌下腺组织核因子-κB P65蛋白(NF-κB P65)/核因子κB抑制蛋白α(IκBα)信号通路的影响。方法取NOD小鼠24只随机分为4组,每组6只。模型组灌服去离子水0.4 mL(3 mg/mL),西药组灌服羟氯喹0.4 mL(2.5 mg/mL),中药组灌服中药养阴益气活血方0.4 mL,中西药组灌服中药养阴益气活血方及羟氯喹各0.4 mL,选取6只同龄Balb/c小鼠作为正常组灌服去离子水0.4 mL。给药8周后摘取双侧颌下腺组织进行PCR检测,镜下观察各组小鼠颌下腺组织免疫组化后蛋白变化。结果与正常组比较,模型组小鼠颌下腺组织P65mRNA表达著增高,IκBαmRNA表达降低(P<0.05)。与模型组比较,西药组、中药组、中西药组P65 mRNA表达降低,IκBαmRNA表达升高(P<0.05)。与西药组比较,中西药组P65 mRNA表达降低(P<0.05),中药组IκBαmRNA相对表达量降低(P<0.05)。镜下,模型组小鼠颌下腺组织P65蛋白呈高表达,IκBα蛋白呈低表达,中药组、西药组和中西药组P65蛋白散在表达,正常组表达最弱;IκBα蛋白在其余三组高表达,正常组表达最强。与正常组比较,模型组P65平均吸光度增高,IκBα平均吸光度减少(P<0.05);与模型组比较,中药组、西药组和中西药组P65平均吸光度降低,IκBα平均吸光度增加(P<0.05)。从药效比较,中西药组抑制NF-κB/IκBα通路中P65表达的疗效最好,西药组和中药组次之,且疗效相近;中西药组抑制IκBα磷酸化的效果最佳,其次是西药组,中药组最次。结论联合使用养阴益气活血方和羟氯喹能通过减少P65 mRNA及蛋白表达,维持IκBαmRNA及蛋白高表达,抑制NF-κB P65/IκBα信号通路,减少颌下腺炎症反应,从而发挥治疗干燥综合征的作用。

Objective To investigate the effect of Yangyin Yiqi Huoxue Recipe on NF-κB P65 nuclear factor kappa-B(NF-κB P65)inhibitor alpha of NF-κB(IκBα)signal pathway in submandibular gland tissue of no obesity diabetes(NOD)mice with Sj?gren’s syndrome.Methods Twenty-four NOD mice were selected as the control group,and the NOD mice were randomly divided into four groups,with six mice in each group.The model group was administered intragastrically with 0.4 mL deionized water,the Western medicine(WM)group was administered intragastrically with 0.4 mL hydroxychloroquine,the Chinese medicine(CM)group was administered intragastrically with 0.4 mL Yangyin Yiqi Huoxue Recipe,and the Chinese and Western medicine(CWM)group was administered intragastrically with 0.4 mL Yangyin Yiqi Huoxue Recipe and 0.4 mL hydroxychloroquine respectively.Selected six balb/c mice of the same age as the normal group,and 0.4 mL deionized water was perfused.After 8 weeks of administration,bilateral submandibular gland tissues were extracted for PCR detection,and the protein changes of submandibular gland tissues of mice in each group after immunohistochemistry were observed under microscope.Results Compared with the normal group,the mRNA expression of P65 increased,and the mRNA expression of IκBαdecreased in the submandibular gland tissue of the mice in the model group(P<0.05).Compared with the model group,the mRNA expression of P65WM decreased,and the mRNA expression of IκBαincreased in the WM group,the CM group and the CWM group(P<0.05).Compared with the WM group,the mRNA expression of P65 was significantly decreased in the CWM group(P<0.05),and the mRNA expression of IκBαwas decreased in the CM group(P<0.05).Microscopically,P65 protein was highly expressed and IκBαprotein was lowly expressed in the submandibular gland tissue of mice in the model group,and P65 protein was scattered in CM,WM and the CWM groups,with the weakest expression in the normal group.IκBαprotein was highly expressed in the other three groups,and the normal group had the strongest expression.Compared with the normal group,the mean absorbance of P65 was significantly increased and the mean absorbance of IκBαwas significantly decreased in the model group(P<0.05).Compared with the model group,the mean absorbance of P65 was significantly decreased and the mean absorbance of IκBαwas significantly increased in the CM,WM and the Chinese and Western medicine groups(P<0.05).In terms of efficacy comparison,the CWM group had the best efficacy in inhibiting P65 expression in the NF-κB/IκBαpathway,followed by the WM group and the CM group with similar efficacy.The CWM group had the best efficacy in inhibiting IκBαphosphorylation,followed by the WM group and the CM group the least.Conclusion The combination of Yangyin Yiqi Huoxue Recipe and hydroxychloroquine can play a role in the treatment of Sj?gren’s syndrome by reducing P65 mRNA and protein expression,maintaining high IκBαmRNA and protein expression,inhibiting NF-κB P65/IκBαsignaling pathway and reducing the inflammatory response of submandibular gland.