冬虫夏草鞘氨醇衍生物靶向慢阻肺Cer-Sph-S1P代谢通路的免疫调节机制研究

Cordyceps Sinensis (CS) is a TCM for treating pulmonary diseases from ancient times and exhibits dual-directional immuno regulation effects, yet with not enough research data on effective components and respiratory pharmacology. We are now competent to produce large amount of CS for research use. CS sphingosine(sph) fraction was previously shown to significantly inhibit the proliferation of mouse lymphocytes which is similar to immunosuppressive drug FTY720, and reduce inflammatory mediators induced by cigarette smoke extraction. Because FTY720 exhibits dual-directional immuno regulation effects on Cer-Sph-S1P signaling, and shares similar structure with CS sph, CS sph fraction may have similar mechanism or has more potential targets. It is also possible that CS sph fraction may contain compounds with stronger pharmacological activity. In this study, we will isolate and enrich CS sph fraction, perform structural identification and chemical synthesis. Secondly, we will investigate the anti-inflammatory and phagocytic promoting effects of sph fraction on COPD and influenza virus induced exacerbation mice model, by using MojoSort Mouse WBCs Isolation and flow cytometry methods. We further explore how CS sph compounds act on Cer-Sph-S1P signaling by measuring enzyme activity, determining sphingolipid expression by LC-MS and generating siRNA. Overall, under the holistic concept of TCM, our study will first elucidate the active components from CS in preventing and combating COPD as well as their mechanism of action , which supports the pharmacological property of CS: dual-directional immuno regulation.

冬虫夏草（CS）是自古以来治疗肺病（归肺经）的有效中药，有免疫双向调节特色，但缺乏物质基础和呼吸药理。前期已完成生态孵育CS规模化生产，突破来源限制；首次发现CS鞘氨醇（Sph）组分显著抑制小鼠淋巴细胞增殖（与经典免疫抑制剂FTY720近似）和香烟提取物（慢阻肺模型）引起的炎症介质表达。因FTY720双向调节Cer-Sph-S1P代谢通路，而CS Sph与FTY720结构与作用相似，推测有类似机制和有更多潜在靶点，该天然组分可能含活性更高的单体。拟从CS提取富集Sph组分，鉴定结构和化学合成，用磁珠分选和流式细胞术等方法探讨Sph在慢阻肺及流感感染致急性加重小鼠模型的抗炎和提升固有免疫细胞吞噬功能的作用。用酶活性测定、LC-MS分析鞘脂类和RNA沉默等方法开展Cer-Sph-S1P通路的体外机制研究。项目在中医整体观指导下，首次阐明CS防治肺病慢阻肺的成分和作用机制，揭示其双向调节内涵。

冬虫夏草（CS）是治疗慢性阻塞性肺病（COPD）的有效中药，但仍缺乏药理机制和物质基础研究。本项目以Cer-Sph-S1P代谢通路、抗炎和免疫提升为切入点，研究CS中鞘氨醇（Sph）对COPD以及流感感染致COPD急性加重（AECOPD）的防治作用。本项目研究发现为：（1）从CS中提取分离获得鲜草冷水提物（WEC）；获得一系列长链烯烃骨架类组分，包括Sph和神经酰胺（Cer）组分以及不饱和脂肪酸（DDEA）；获得6种神经酰胺衍生物（环二肽化合物）；（2）证实WEC对香烟提取物（CSE）刺激气道细胞和巨噬细胞具有显著抗炎作用，对巨噬细胞吞噬功能缺陷无改善作用；明确WEC可改善香烟烟雾暴露致小鼠肺功能下降、肺部炎症和肺气肿形成；WEC可抑制猪胰蛋白酶刺激的肺部炎症介质、减少巨噬细胞募集以及防止胞外基质降解，从而改善肺气肿；（3）Sph和Cer组分对CSE刺激细胞有显著抗炎作用， Sph对Cer-Sph-S1P通路中S1PR1受体和SPHK1激酶有显著抑制作用，对通路中神经酰胺和鞘氨醇的表达有明显抑制活性，而Cer对上述通路无活性；（4）DDEA通过抑制MAPK通路的c-Jun和FAR1及其磷酸化从而抑制CSE刺激的炎症反应，并通过作用于TLR-3、 RIG-I 及 IFN 介导的信号通路从而抑制H1N1流感感染的炎症反应；（5）环二肽Cyclo-（Pro-Val）和Cyclo-（Pro-Leu）对H1N1感染引起TNF-α表达有抑制作用；（6）构建了H1N1与NTHi单一感染以及共感染的AECOPD小鼠模型，为下一步开展冬虫夏草及其鞘氨醇衍生物防治AECOPD的研究提供实验模型。总体而言，本项目明确了冬虫夏草总提物及其长链烯烃类化合物（鞘氨醇、神经酰胺和不饱和脂肪酸）等对COPD发生发展的防治作用以及靶向Cer-Sph-S1P和炎症信号通路的作用机制，对冬虫夏草临床有效性的确认具有重要参考价值。项目已发表SCI论文4篇（另在投2篇），已授权专利2项（另已受理1项）