基于肠道菌群对肠道短链脂肪酸及β-葡萄糖醛酸酶的影响探讨生姜泻心汤预防伊立替康迟发性腹泻的机制研究

Delayed-onset diarrhea is a common adverse reaction in cancer patients accepting chemotherapy involving irinotecan, which leads to a reduction in the dose of chemotherapy, delays or interruptions in chemotherapy schedules, and life-threatening dehydration and electrolyte imbalances in severe cases. At present, there is no standard and effective prevention and treatment for Western medicine. In our previous studies, it was proved that Shengjiang Xiexin Decoction(SXD) can improve irinotecan-induced diarrhea by protecting and repairing intestinal mucosal cells. We plan to make a further research to analyze the effect of SXD on the structure of intestinal microflora in irinotecan-induced diarrhea rat models using 16s RNA sequencing technology. Simultaneously, UHPLC-MS/MS, immunohistochemistry, ELISA, Western blotting, PCR are used to detect the concentrations of intestinal short-chain fatty acid, β-glucuronidase activity, the concentrations of irinotecan metabolites SN-38/SN-38G, intestinal permeability, expression of tight junction/mucoprotein and the content of inflammatory/anti-inflammatory factor. And the regulating effect of intestinal flora on TLR4—MyD88—JNK/NF-κB signal pathway through affecting short-chain fatty acid is explored further. Finally, the mechanism of action above will be verified again using pseudo-germfree mice model by the technique of fecal microbiota transplantation, to illuminate the action of SXD in alleviating enterotoxicity, is due to the effect of SXD on intestinal short-chain fatty acid and β-glucuronidase activity through regulating the structure of intestinal flora. It will provide the new scientific evidence for pharmacodynamic mechanism of TCM in preventing the adverse reactions of chemotherapy.

迟发性腹泻是化疗药伊立替康常见不良反应，会导致化疗剂量减少，化疗推迟或中断，严重者出现危及生命的脱水和电解质紊乱，目前西医尚无有效防治手段。在前期研究中，本课题组已证实生姜泻心汤通过保护和修复肠上皮细胞改善伊立替康迟发性腹泻。本研究拟采用16sRNA基因测序技术，分析生姜泻心汤对迟发性腹泻大鼠模型肠道菌群的影响；并采用UHPLC-MS/MS、免疫组化、ELISA、Western blotting、PCR等技术检测肠道短链脂肪酸浓度、β-葡萄糖醛酸酶活性、SN-38/SN-38G浓度、肠道通透性、肠道紧密连接/黏蛋白表达及炎性/抗炎因子含量，并进一步探索肠道菌群通过影响短链脂肪酸对TLR4—MyD88信号通路的调控作用。最后通过菌群移植技术以伪无菌小鼠为模型对上述作用机制加以验证，阐明生姜泻心汤通过调节肠道菌群影响肠道短链脂肪酸代谢及β-葡萄糖醛酸酶活性，从而减轻伊立替康肠毒性的作用机制。

迟发性腹泻是化疗药伊立替康常见不良反应，会导致化疗剂量减少，化疗推迟或中断，严重者出现危及生命的脱水和电解质紊乱，目前西医尚无有效防治手段。本研究采用16sRNA基因测序技术，分析生姜泻心汤对迟发性腹泻大鼠模型肠道菌群的影响；并采用UHPLC-MS/MS、免疫组化、ELISA、Western blotting、PCR等技术检测肠道短链脂肪酸浓度、β-葡萄糖醛酸酶活性、SN-38/SN-38G浓度、肠道通透性、肠道紧密连接/黏蛋白表达及炎性/抗炎因子含量，并进一步探索肠道菌群通过影响短链脂肪酸对TLR4—MyD88信号通路的调控作用。结果显示，伊立替康可造成大鼠肠道菌群紊乱，菌群结构发生改变。模型组厚壁菌门增加，拟杆菌门减少；生姜泻心汤可增加肠道中疣微菌门、拟杆菌门，且显著升高了拟杆菌属、Muri菌属、普雷沃氏菌NK3B31_group菌属、阿克曼氏菌属等。中药生姜泻心汤可通过调节肠道菌群结构，降低肠道代谢酶葡萄糖醛酸酶活性，升高大鼠肠道短链脂肪酸浓度，促进肠道短链脂肪酸代谢，下调TLR4、MyD88、JNK、NF-κB信号通路蛋白表达，提高肠道黏蛋白表达，进而保护肠道机械屏障，下调大鼠空肠炎性因子IL-1β、IL-6、TNF-α表达，增强抗炎因子IL-10、TGF-β1的表达，促进肠道免疫屏障功能，从而预防伊立替康所引起的肠毒性，对大鼠腹泻级别及体重、进食量具有改善作用。研究证实生姜泻心汤对伊立替康迟发性腹泻的预防作用，可能与该方通过调节肠道菌群结构，升高肠道短链脂肪酸含量有关。