基于TLRs信号通路与"暴露-反应-机制"三联模式探讨桑叶抗糖尿病IR作用机理

Mulberry leaf has the exact effect in treating Insulin Resistance (IR), and its mechanisms include inhibiting the activity of alpha-glucosidase, improving Insulin (Ins) excresion and glucose metabolism, but no research concerns about the relationship between anti-IR and anti-inflammation. Our former study indicated that inflammation induced by TLRs participated in the pathological formation of IR, and mulberry leaf improved the symptoms of IR, its mechanism may be related to regulating the expression of TLR2 and TLR4, but its specificity and active targets remain unknown. In this study, we will establish the animal and cellular model of IR, use the technologies of real-time PCR, immunohistochemistry and Western blot to analyze the influence of mulberry leaf to TLRs and Ins signaling transduction pathways, and the activity of alpha-glucosaccharase, utilize the "exposure-response-mechanism" triple modes to clarify the active ingredient of mulberry leaf which affecting TLRs and Ins signaling transduction pathways. This research will reveal the new mechanisms of mulberry leaf in treating IR by anti-inflammation effect, further improve its molecular targets on the signaling transduction pathways of TLRs and Ins, and we believe that we will to have a key breakthrough in clarifing new mechanism, active principles and effective target organs of mulberry leaf in treating IR.

桑叶治疗糖尿病IR疗效确切，其机制与抑制a-糖苷酶活性、促进胰岛素（Ins）分泌及糖代谢等有关，但与抗炎作用关系研究尚属空白。本课题组先期研究表明，TLRs介导的免疫性炎症参与了IR形成，桑叶可能通过调节TLR2、TLR4表达发挥抗IR的药效学作用，但其特异性及作用靶点有待于进一步研究。本课题拟建立IR动物与细胞模型，采用定量PCR、免疫化学、Western blot等技术，从整体-细胞两个层次，在基因-蛋白两个水平探讨桑叶对TLRs、Ins通路信号元件及α-葡萄糖苷酶活性的影响，并利用"暴露-反应-机制"三联模式研究方法揭示桑叶作用于TLRs、Ins信号通路及抑制α-葡萄糖苷酶活性的有效部位及其效应靶器官。本课题将首次揭示桑叶抗炎治疗IR的新机制，系统阐明其基于TLRs、Ins信号通路与抑制α-糖苷酶活性的分子靶标，有望在桑叶抗IR新机制、有效组分及效应靶器官研究方面取得重大突破。

本项目根据既定的实验方案和研究计划，经过四年的研究，利用"暴露-反应-机制"三联模式揭示了桑叶作用于TLRs、Ins信号通路及抑制α-葡萄糖苷酶活性的有效部位及其效应靶器官。（1）本课题制备了桑叶的不同提取物，建立了指纹图谱，明确桑叶水提物的11个化学成分，并鉴定了桑叶含药血清中的30个成分（血中暴露成分）；（2）证明桑叶水提物具有确切的降糖、改善胰岛素抵抗和调节血脂的作用（反应）（3）明确了桑叶水提取物对α-葡萄糖苷酶的抑制作用（机制一）；（4）分别以糖尿病大鼠、小鼠为模型，发现桑叶水提物通过调节小鼠肝脏、肌肉中TLR通路和脂肪中Ins通路改善胰岛素抵抗（机制二、机制三）；（5）以C2C12、3T3-L1、HepG2细胞建立胰岛素抵抗模型为研究对象，明确桑叶含药血清通过调节TLR通路改善C2C12细胞和HepG2细胞胰岛素抵抗，通过调节Ins通路改善3T3-L1细胞胰岛素抵抗（机制二、机制三）。