基于“肠道菌群-SCFAs-GPCR/HDAC-mTOR”研究虫草多糖治疗慢性肾病的作用机制

Cordyceps is a traditional Chinese medicine which has been proved as an effective medicine for chronic kidney disease (CKD) treatment. Based on phase II clinical trial showing effective treatment using Cordyceps polysaccharide (CPS), combining with the intestine ("Pi" in traditional Chinese medicine) - renal axis Theory, applicants investigate the effective substance for CKD treatment. Recent studies have shown that short chain fatty acids (SCFAs) metabolized by intestinal flora have anti-inflammatory and immunomodulatory effects. Preliminary results showed that the number of intestinal probiotics was significantly increased in CKD rats after administration of CPS, meanwhile，increased the content of acetic acid and butyric acid in the colon, and were directly related to the decrease of renal tissue inflammation. According to these findings, we therefore propose a hypothesis that the efficacy of CPS for CKD is most likely to be mediated by its regulatory effects on intestinal microbial flora and the consequent enhanced content of SCFAs. To test this hypothesis, we propose the following in vitro and in vivo studies: (1)mechanistic study on the regulatory effects of CPS on intestinal microbial flora and SCFA content by high throughput sequencing and metabolomic technologies; (2)study on the expression levels of SCFA-associated receptors and other factors in the kidney and other immune organs of the CPS-treated animals; and (3) study on the SCFA-related signaling pathways in renal and immune cells treated by SCFAs from the CPS-fed animals. These studies, we believe, not only provide a theoretical basis for the clinical application of CPS, but offer an important insight into the pharmacology of polysaccharides from traditional Chinese medicines.

申请人依据虫草多糖（CPS）治疗慢性肾病（CKD）临床有效，拟从肠—肾轴理论探索其作用机制。最新研究显示肠道菌群代谢的短链脂肪酸（SCFAs）具有抗炎和调节免疫作用。我们前期研究也揭示：CPS灌胃CKD大鼠后显著增加了肠道益生菌数量，提高了结肠中丁酸和乙酸含量，这种改变与肾组织炎症的降低直接相关。据此提出假说：CPS通过调控CKD大鼠结肠菌群，产生特定的SCFAs是其治疗CKD的重要药效机制。本项目拟从动物、细胞和分子水平印证假说：①通过高通量测序及代谢组学研究，探寻CPS藉由调控结肠菌群以及特定SCFAs治疗CKD的关键机制；②研究肾脏及免疫器官中与SCFAs作用的受体及相关因子的表达水平，探索与CPS药效的相关性；③用结肠SCFAs处理肾细胞及免疫细胞，阐明与SCFAs作用的信号通路及各通路间的相关性。本项目研究将为CPS临床合理应用提供理论依据，也为中药多糖药理学研究提供新的思路。

本课题前期经过大量实验证明，水提虫草多糖可以治疗多种急慢性肾病。在前期研究基础上，本课题确认了CPS对腺嘌呤诱导的慢性肾病大鼠的治疗作用，在此基础上，通过肠-肾轴理论，研究虫草多糖治疗腺嘌呤诱导的慢性肾病的作用机制。.结果显示，腺嘌呤诱导的慢性肾病大鼠，灌胃CPS两周后，血清中BUN及SCr含量显著降低；HE染色法观察组织切片，CPS给药组大鼠肾损伤显著降低，随着CPS给药剂量的增加，大鼠肾损伤得到了显著缓解；qPCR结果显示灌胃CPS可以显著提高慢性肾病大鼠结肠及盲肠内双歧杆菌属、乳酸杆菌属及产丁酸梭菌属含量，降低肠杆菌属含量；实验结果表明大鼠灌胃CPS后，可以增加其肠道有益菌群的数量，降低有害菌群的数量，从而减少有毒代谢产物的积累，缓解肾损伤；GC检测慢性肾病大鼠体内SCFAs含量。灌胃CPS后，显著提高慢性肾病大鼠体内乙酸及丁酸的含量；Western Blot和免疫组化结果显示灌胃CPS两周后，慢性肾病大鼠肾脏组织中 GPR41及 GPR43表达量显著回调；炎症相关因子 NF-κB 表达量显著下调，磷酸化S6核糖体蛋白表达量显著回调；ELISA 及流式细胞术检测结果显示，CPS给药后，慢性肾病大鼠体内 HDAC 活性显著下降；CPS给药高剂量组脾脏及肠系膜淋巴结中调节性 T 细胞的比例与模型组相比具有极显著性增加（P<0.01）。本研究表明，CPS对腺嘌呤诱导的慢性肾病大鼠具有良好的治疗作用；CPS可以影响肠道菌群的数量，改善肠道菌群的紊乱，从而增加了肠道内乙酸、丁酸的含量；乙酸、丁酸一方面通过作用于局部靶器官相应受体发挥作用，另一方面，通过表观遗传影响了全身免疫系统。综上所述，实验结果初步表明，CPS 对慢性肾病大鼠有治疗作用，其作用机制可能与以下两方面相关：①SCFAs 直接作用于肾脏上GPCR发挥抗炎作用；②SCFAs 通过抑制 HDAC 活性，调控 mTOR相关蛋白表达量影响调节性T细胞的比例，发挥抗炎作用。