黄连素通过CagA介导的NF-κB信号通路抑制幽门螺杆菌感染小鼠胃粘膜炎症因子分泌的研究

Berberine Inhibits the Secretion of Inflammatory Factors in Gastric Mucosa of Helicobacter Pylori Infected Mice Through CagA-mediated NF-κB Signaling Pathway

目的探讨黄连素对幽门螺杆菌(Helicobacter pylori,Hp)感染小鼠胃粘膜的治疗作用及其抑制胃粘膜炎性因子分泌的作用机制。方法将60只SPF级环境饲养昆明(Kunming,KM)小鼠随机分为空白组、模型组、三联组、低剂量黄连素治疗组、中剂量黄连素治疗组和高剂量黄连素治疗组。不对空白组干预,其它5组小鼠Hp感染后依次予无菌生理盐水、兰索拉唑/甲硝唑/克拉霉素、低剂量黄连素、中剂量黄连素、高剂量黄连素灌胃,观察胃粘膜组织的病理改变。苏木精-伊红(hematoxylin-eosin staining,HE)染色法检测血清炎性因子肿瘤坏死因子α(tumor necrosis factor alpha,TNF-α)、白细胞介素6(interleukin 6,IL-6)、IL-8、环氧化酶2(cyclooxygenase 2,COX-2)等的表达。采用实时荧光逆转录定量聚合酶链反应(real-time reverse transcription quantitative polymerase chain reaction,RT-qPCR)、免疫组化方法检测细胞毒素相关蛋白A(cytotoxin associated gene A,CagA)和核因子κB(nuclear factor kappa B,NF-κB)信号通路p65/p50的表达。Hp感染小鼠原代细胞进行培养,将细胞分为非治疗组,抗Hp治疗组,高剂量黄连素组,中剂量黄连素组,低剂量黄连素组,依次加入相应的无药或含药血清的培养基中继续培养后,检测各组细胞炎症因子、CagA和NF-κB信号通路的表达水平。结果动物实验结果表明,高剂量黄连素治疗组治疗效果最佳,胃粘膜组织炎症得到明显控制,Hp根治率明显提高,同时,血清TNF-a、IL-6、IL-8、COX-2的表达明显降低,与其余组比较差异均有统计学意义(P<0.05)。与模型组以及三联组比较,黄连素各剂量组的CagA、NF-κB p65、NF-κB p50的表达均明显降低(P<0.05)。细胞实验表明,高剂量黄连素组各种炎症因子表达水平最低,且CagA、NF-κB p65、p50表达水平也最低,差异具有统计学意义(P<0.05)。结论黄连素能抑制Hp阳性胃粘膜组织炎症,减轻炎性损伤,其机制可能是通过抑制CagA-NF-κB信号通路的过度活化而调节炎症有关。

Objective To investigate the therapeutic effect of berberine on the gastric mucosa of Helicobacter pylori(HP)-infected mice and its mechanism of inhibiting the secretion of gastric mucosal inflammatory factors.Methods Sixty Kunming(KM)mice raised in an SPF environment were randomly divided into a blank group,a model group,a triplet group,a low-dose berberine treatment group,a middle-dose berberine and a high-dose berberine treatment group.Without any intervention in the blank group.After Hp infection,mice in the other 5 groups were given sterile saline,lansoprazole/metronidazole/clarithromycin,low-dose berberine,middle-dose berberine and high-dose of berberine.The pathological changes of gastric mucosa were observed after intragastric administration.Hematoxylin-eosin staining(HE)was used to detect expression of tumor necrosis factor alpha(TNF-α),interleukin 6(IL-6),IL-8 and cyclooxygenase 2(COX-2).Real-time reverse transcription quantitative polymerase chain reaction(RT-qPCR)and immunohistochemical method were used to detect the expression of cytotoxin associated gene A(CagA)and nuclear factor kappa B(NF-κB)signaling pathway p65/p50.The primary cells of Hp-infected mice were cultured and divided into non-treatment group,anti-Hp treatment group,high dose berberine group,medium dose berberine group and low dose berberine group.The expression levels of inflammatory factors,CagA and NF-κB signaling pathway in each group were detected after cultured in corresponding medium without drug or containing drug serum.Results The animal experiment results showed that the treatment effect of the high-dose berberine treatment group was the best,the inflammation of the gastric mucosal tissue was significantly controlled,and the Hp radical cure rate was significantly improved.At the same time,the expressions of serum TNF-a,IL-6,IL-8,COX-2 were significantly reduced,and the difference was statistically significant compared with the other groups(P<0.05).Compared with the model group and the triple group,the expressions of CagA,NF-κB p65,and NF-κB p50 in each dose group of berberine were significantly reduced(P<0.05).Cell experiments showed that the expression levels of various inflammatory factors in the high-dose berberine group were the lowest,and the expression levels of Cag A,NF-κB p65,and p50 were also the lowest.The difference was statistically significant(P<0.05).Conclusion Berberine can inhibit the inflammation of Hp-positive gastric mucosal tissues and reduce inflammatory damage.The mechanism may be related to the regulation of inflammation by inhibiting the excessive activation of the CagA-NF-κB signaling pathway.