从多胺/HuR信号通路探讨四君子汤促进胃肠上皮细胞增殖作用机制研究

Gastrointestinal mucosa has effect on protecting the body from damage, but it’s easy to be damaged by various factors including non-steroid anti-inflammatory drug, smoking, ethyl alcohol, and so on. Previous studies found that polyamines played an important role in regulating gastrointestinal mucosal injuries repair which included epithelial cell migration, cell proliferation and cell differentiation, polyamines promoted epithelial cell proliferation by regulating RNA binding protein HuR and its interaction with genes which related to growing. Gastrointestinal mucosal injuries was the main pathological basis of spleen-deficiency syndrome and Yiqi Jianpi method is the main therapeutic method for spleen-deficiency syndrome, Sijunzi Decoction is the representative formula for herbs of strengthening spleen and replenishing Qi. Our team’s researches found that Sijunzi Decoction promoted gastrointestinal mucosal injuries repair by activating polyamines signaling pathway. Most studies focus on potential mechanisms of Sijunzi Decoction on promoting cell migration by regulating polyamines signaling pathways at present, but there are few researches of its role on the following repair of mucosa injuries. In the current research, lots of methods including western blotting, qPCR and immunoprecipitation will be carried out to detect the role of Sijunzi Decoction active components on the expressions and interactions of genes and proteins depending on polyamines/HuR signaling pathways, we investigate potential targets of Sijunzi Decoction on treatment of spleen deficiency syndrome related to gastroenteric disease, to further elucidate the underlying mechanisms of Sijunzi Decoction promoting the following repair of mucosa injuries and provide the scientific basis for treatment of gastroenteric disease.

胃肠粘膜对机体有保护作用，易受到抗炎药物、吸烟、酒精等的刺激而诱发胃肠粘膜损伤。研究表明，多胺是胃肠粘膜损伤修复（上皮细胞迁移、增殖及分化等）过程的重要调控因素之一，多胺通过调控HuR以及HuR蛋白与生长相关基因间的相互作用而促进上皮细胞增殖的粘膜修复过程。胃肠粘膜损伤是脾虚证的病理基础之一，益气健脾法是脾虚证的主治法则，四君子汤为其代表方剂。课题组研究发现四君子汤可通过激活多胺信号通路促进胃肠粘膜损伤修复，但前期仅探讨其对细胞迁移过程多胺信号通路的影响，对于其对粘膜损伤后续修复作用机制研究较少。本研究拟在整体动物和细胞水平，以四君子汤活性组分为受试药，运用蛋白质印迹、荧光定量PCR、免疫沉淀等手段，检测多胺/HuR信号通路中基因蛋白的表达及相互作用，研究四君子汤治疗脾虚证相关胃肠病变的潜在靶点，力求达到进一步阐明四君子汤促进后续粘膜损伤修复的作用机制的目的，为其治疗胃肠病变提供科学依据。

胃肠黏膜对机体有保护作用，易受到药物、吸烟、酒精等的刺激而诱发胃肠黏膜损伤。多胺是胃肠黏膜损伤修复（细胞迁移、增殖等）过程的重要调控因素之一，多胺通过调控HuR与生长相关基因间的相互作用而促进上皮细胞增殖的黏膜修复过程。胃肠黏膜损伤是脾虚证的病理基础之一，益气健脾法是脾虚证的主治法则，四君子汤为其代表方剂。研究发现四君子汤可通过激活多胺信号通路促进胃肠黏膜损伤修复，但前期仅探讨其对细胞迁移过程多胺信号通路的影响，对于其对黏膜损伤细胞增殖过程作用机制研究较少。本研究在细胞水平，观察四君子汤水提液含药血清及人参皂苷Re对小肠上皮细胞（IEC-6）增殖过程多胺/HuR信号通路中生长相关基因蛋白的表达及相互作用的影响；在整体动物水平，观察四君子汤水提液对吲哚美辛模型大鼠胃肠黏膜组织病理学变化、HuR细胞内移位以及生长相关基因蛋白表达的影响，进一步探讨四君子汤促进胃肠黏膜损伤修复机制。细胞水平研究结果显示，在正常或α-二氟甲基鸟氨酸(多胺合成抑制剂，DFMO)负荷下，四君子汤含药血清可提高细胞内多胺（腐胺及精脒）含量，降低胞浆内RNA结合蛋白HuR蛋白表达，提高细胞核HuR蛋白表达，降低HuR结合ATF-2、JunD、NPM、p53 mRNA含量，降低ATF-2、JunD mRNA稳定性以及ATF-2/JunD复合物含量，提高Chk2、c-Myc、CDK4、CDK2及RhoA的mRNA及蛋白表达和Chk2、CDK2活性(p-Chk2、p-CDK2蛋白的表达)，抑制ATF-2、JunD、NPM、p53及p21mRNA及蛋白表达，减少细胞周期中G0/G1期细胞占比，提高S期细胞占比，最终促进IEC-6细胞增殖。此外，四君子汤含药血清中有效成分之一人参皂苷Re作用及作用机制与四君子汤含药血清相似。动物水平研究结果发现四君子汤水提液可促使HuR更多在细胞核堆积，提高模型大鼠胃肠黏膜c-Myc蛋白表达及小肠黏膜CDK4蛋白表达，降低小肠黏膜ATF-2、NPM、p53及JunD蛋白表达，最终改善吲哚美辛所致的胃肠黏膜损伤。本研究重点探讨四君子汤促进胃肠黏膜损伤后续修复的潜在靶点，进一步阐明四君子汤促进胃肠黏膜损伤修复的作用机制，为其治疗胃肠病变提供科学依据。