p38 MAPK介导MSK1/2-CREB通路关键分子在溃疡性结肠炎肠黏膜微环境中的响应及温补脾肾法干预效应

Based on chronic recurrent UC viscera pathogenesis regularity and clinical practice, combined with the theoretical background of inflammatory lesions related to the imbalance of intestinal mucosal homeostasis caused by p38 MAPK-mediated signaling pathways and previous basis, the study hypothesis of the response of p38 MAPK-mediated Key Molecules of MSK1/2-CREB signaling pathway in the colonic mucosal microenvironment of ulcerative colitis and the possible mechanism of intervention by warming and tonifying the spleen and kidney were established. UC rats with spleen and kidney yang deficiency were used as research objects. On the basis of evaluating the pathological damage of colonicmucosa, immunohistochemistry, qPCR and western blotting were adopted to determine protein and mRNA expression of p38 MAPK, MSK1/2, CREB in p38 MAPK-mediated MSK1 / 2-CREB signaling pathway and c-jun, c-fos, cox-2, IL-10, IL-17 and IL-33 in the downstream of the signaling pathway in UC colonic mucosal tissue. At the same time, through the intervention of Jiuxieling Decoction by warming and tonifying the spleen and kidney and through the correlation analysis, the pathological mechanism of UC and intervention effect of warming and tonifying the spleen and kidney were revealed from the perspective of p38 MAPK-mediated Key Molecules of MSK1/2-CREB signaling pathway, providing theoretical support for the clinical application of the preparation and the development of the key technologies for the prevention and treatment of UC proprietary Chinese medicinals.

本研究基于慢性复发性UC脏腑病机规律及临床实践基础，结合p38 MAPK介导相关信号通路诱发肠黏膜稳态失衡而导致炎性病变发生的理论背景以及前期工作基础，建立“p38 MAPK介导MSK1/2-CREB通路关键分子在UC病程中结肠黏膜微环境中的响应及温补脾肾法干预效应的可能机制”工作假说，以脾肾阳虚型UC大鼠为研究对象，在评估结肠黏膜病理损伤基础上，采用qPCR、免疫组化和蛋白免疫印迹技术检测p38 MAPK介导MSK1/2-CREB信号通路上p38 MAPK、MSK1/2、CREB以及下游c-jun，c-fos、cox-2、IL-10、IL-17、IL-33基因蛋白在结肠组织中的表达规律；并应用以温补脾肾法为指导的久泻灵进行干预，通过相关性分析，拟从p38 MAPK介导MSK1/2-CREB信号通路关键分子角度揭示UC的病理机制以及温补脾肾法的干预效应，为该制剂的临床应用提供理论支撑。

本研究通过网络药理学预测以温补脾肾法为指导的理中汤合四神丸对UC的潜在作用机制及核心靶点，并用分子对接技术以及动物实验验证其核心基因与相关通路中关键基因和蛋白的表达，探讨温补脾肾法的干预效应，为该制剂的临床应用提供理论支撑。网络药理学与分子对接研究显示，理中汤合四神丸的活性成分对UC的核心靶点有很强的关联性，以MAPK家族基因占比最多。动物实验验证分为四部分。采用大黄水煎液灌胃+皮下注射氢化可的松+DNBS/乙醇溶液灌肠建立脾肾阳虚型UC大鼠模型。模型建立成功后，理中汤合四神丸（低、中、高）剂量组、美沙拉嗪组按照相应剂量给予治疗，空白组与模型组灌服等剂量蒸馏水，持续治疗14天。实验一探讨理中汤合四神丸对脾肾阳虚UC大鼠一般状态、结肠组织病理形态及病理学评分的影响。结果显示，模型组大鼠结肠长度缩短变粗，CMDI及HE病理学评分显著升高，理中汤合四神丸在改善大鼠一般状态和结肠组织病理形态等方面体现出与美沙拉嗪类似的功效，且呈计量依赖趋势，以理中汤合四神丸高剂量组效果最为显著，其可明显改善脾肾阳虚UC大鼠的结肠黏膜组织损伤情况，恢复肠黏膜细胞形态以及减少肠组织炎性浸润合腺体的破坏。实验二检测大鼠结肠组织中P38 MAPK-MSK1-CREB 信号通路关键因子的表达。WB法检测相关蛋白含量表达，RT-qPCR 法检测对应mRNA表达量。结果显示，理中汤合四神丸可能通过抑制 P38MAPK/MSK1/CREB 信号通路及其下游C-jun、C-fos、COX-2 蛋白及基因表达水平，对UC大鼠起到治疗作用。实验三检测UC大鼠免疫相关指标。ELISA 检测大鼠血清 IL-10、IL-17 含量，流式细胞法检测大鼠脾脏细胞 CD3+CD4+CD8 Th细胞与CD4+CD25+FOXP3 Treg 细胞表达情况。实验四检测细胞凋亡情况。Tunnel 法检测大鼠结肠组织细胞凋亡情况。实验三和实验四结果显示，理中汤合四神丸可抑制促炎因子表达，降低免疫反应和凋亡阳性细胞表达率，其可能通过平衡免疫反应，抑制过度的凋亡进而缓解炎症反应，改善结肠黏膜膜病理状态，对UC起到治疗作用。