固肠胶囊调控p38MAPK/NF-κB信号通路治疗炎症性肠病机制

Mechanism of Guchang Capsule in treating inflammatory bowel disease by regulating p38MAPK/NF-κB signaling pathway

关键词:  固肠胶囊  炎症性肠病  lncRNA THRIL  p38MAPK/NF-κB信号通路  炎症因子

Key words:  Guchang Capsule  inflammatory bowel disease  lncRNA THRIL  p38MAPK/NF-κB signaling pathway  inflammatory factor

摘要:

[目的] 探究固肠胶囊对炎症性肠病（IBD）模型动物的治疗效果，明确其是否通过下调长链非编码RNA（lncRNA）THRIL、抑制p38丝裂原活化蛋白激酶（p38MAPK）/核转录因子-κB（NF-κB）信号通路，从而发挥改善IBD的作用机制。[方法] 将36只C57BL/6雄性小鼠随机分为对照组、模型组、固肠胶囊低剂量组[500 mg/（kg·d）]、固肠胶囊中剂量组[1 000 mg/（kg·d）]、固肠胶囊高剂量组[1 500 mg/（kg·d）]及美沙拉嗪组[200 mg/（kg·d）]，每组6只。除对照组外，其余各组通过饮用4%葡聚糖硫酸钠（DSS）水溶液连续7 d诱导IBD模型。造模成功后，各给药组连续灌胃给药10 d，对照组和模型组给予等量蒸馏水。观察各组小鼠疾病活动指数（DAI）评分；苏木精-伊红（HE）染色观察结肠组织病理变化；酶联免疫吸附（ELISA）法检测结肠组织中肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）、白细胞介素-1β（IL-1β）、白细胞介素-4（IL-4）、白细胞介素-10（IL-10）水平；实时荧光定量聚合酶链反应（qRT-PCR）法检测lncRNA THRIL表达；蛋白免疫印迹（Western blot）及免疫组织化学法检测p38MAPK/NF-κB通路相关蛋白[磷酸化p38MAPK（p-p38MAPK）、p38MAPK、磷酸化NF-κB p65（p-p65）、p65]表达。[结果] 与对照组比较，IBD模型组小鼠体质量降低（P<0.05），DAI评分升高（P<0.05）；结肠组织病理评分升高（P<0.05）；促炎因子TNF-α、IL-1β、IL-6水平升高（P<0.05），抗炎因子IL-4、IL-10水平降低（P<0.05）；lncRNA THRIL表达升高（P<0.05）；p-p38MAPK/p38MAPK、p-p65/p65蛋白比值升高（P<0.05）。与模型组比较，固肠胶囊中、高剂量组及美沙拉嗪组小鼠体质量升高（P<0.05），DAI评分、结肠组织病理评分及促炎因子（TNF-α、IL-1β、IL-6）水平降低（P<0.05），抗炎因子（IL-4、IL-10）水平升高（P<0.05）；结肠组织中lncRNA THRIL表达及p-p38MAPK/p38MAPK、p-p65/p65蛋白比值下调（P<0.05）。[结论] 固肠胶囊可以通过下调lncRNA THRIL、抑制p38MAPK/NF-κB信号通路激活，减轻肠道炎症反应，从而改善IBD症状。

Abstract:

[Objective] To investigate the therapeutic effect of Guchang Capsule on animal models of inflammatory bowel disease（IBD），and to clarify whether it improves IBD by downregulating long non-coding RNA（lncRNA） THRIL and inhibiting the p38 mitogen-activated protein kinase（p38MAPK）/nuclear factor-κB（NF-κB） signaling pathway. [Methods] A total of 36 male C57BL/6 mice were randomly divided into six groups（6 mice per group）：control group，model group，low-dose Guchang Capsule group [500 mg/（kg·d）]，medium-dose Guchang Capsule group [1 000 mg/（kg·d）]，high-dose Guchang Capsule group [1 500 mg/（kg·d）]，and mesalazine group [200 mg/（kg·d）]. Except for the control group，all other groups were induced with IBD by drinking 4% dextran sulfate sodium（DSS） aqueous solution for 7 consecutive days. After successful modeling，each administration group received intragastric administration for 10 consecutive days，while the control group and model group received an equal amount of distilled water. The disease activity index（DAI） score of mice in each group was observed. Hematoxylin-eosin（HE） staining was used to observe pathological changes in colon tissue. Enzyme-linked immunosorbent assay（ELISA） was used to detect the levels of tumor necrosis factor-α（TNF-α），interleukin-6（IL-6），interleukin-1β（IL-1β），interleukin-4（IL-4），and interleukin-10（IL-10） in colon tissue. Quantitative real-time polymerase chain reaction（qRT-PCR） was used to detect the expression of lncRNA THRIL. Western blot and immunohistochemistry were used to detect the expression of p38MAPK/NF-κB pathway-related proteins（p-p38MAPK， p38MAPK，p-p65，p65）. [Results] Compared with the control group，the model group showed significantly decreased body weight（P<0.05），increased DAI score（P<0.05），increased colon tissue pathological score（P<0.05），increased levels of pro-inflammatory factors TNF-α，IL-1β，and IL-6（P<0.05），decreased levels of anti-inflammatory factors IL-4 and IL-10（P<0.05），increased expression of lncRNA THRIL（P<0.05），and increased protein ratios of p-p38MAPK/p38MAPK and p-p65/p65（P<0.05）. Compared with the model group，the medium-dose and high-dose Guchang Capsule groups and the mesalazine group showed significantly increased body weight（P<0.05），decreased DAI score，colon tissue pathological score，and levels of pro-inflammatory factors（TNF-α，IL-1β，IL-6）（P<0.05），increased levels of anti-inflammatory factors（IL-4，IL-10）（P<0.05），and downregulated expression of lncRNA THRIL and protein ratios of p-p38MAPK/p38MAPK and p-p65/p65 in colon tissue（P<0.05）. [Conclusion] Guchang Capsule can improve IBD symptoms by downregulating lncRNA THRIL and inhibiting the activation of the p38MAPK/NF-κB signaling pathway，thereby reducing intestinal inflammatory responses.