杨桃根DMDD靶向干预TLR4信号通路治疗糖尿病肾病的作用及机制

Toll like receptor 4 (TLR4) plays an important role in the development of diabetic nephropathy (DN); the occurrence of insulin resistance (IR), interstitial fibrosis, and renal tubular epithelial cell transdifferentiation mediated by TLR4 is the crucial mechanism of DN. In our previous study, we firstly isolated an active ingredient named 2-dodecyl-6-methoxycyclohexa-2, 5-diene-1, 4-dione (DMDD) from Averrhoa carambola L. (Oxalidaceae) roots; and we found that it could significantly improve renal function in diabetic mice. Recently, our pilot experiment showed that it markedly inhibited the expression of TLR4. These results suggest that the protective effect of DMDD on DN may be related to the regulation of the TLR4 pathway. However, its exact mechanism remains unclear. Thus, based on our previous works, this study will establish the DN animal model in TLR4 knockout (TLR4-/-) mice by using high sugar, high fat diet and low dose of STZ. The role of TLR4 pathway in the occurrence of IR, interstitial fibrosis, and renal tubular epithelial cell transdifferentiation will be studied and subsequently the protective mechanism of DMDD on DN in vivo will be analyzed. Mouse podocytes induced by high glucose and renal tubular epithelial cell transdifferentiation will be used in vitro to explore the mechanism of DMDD on podocytes and renal tubular epithelial cell transdifferentiation by targeting the TLR4 signaling pathway, which is important to further clarify the molecular mechanism of DMDD for treatment of DN. This study will roundly illustrate the action mechanism of anti-DN effect of DMDD, and it will provide the experimental basis and theoretical support for the treatment of DN in clinical.

Toll样受体4（TLR4）信号通路与糖尿病肾病（diabetic nephropathy，DN）密切相关，其介导的胰岛素抵抗、间质纤维化及肾小管上皮细胞转分化是DN发病机制中的关键环节。本课题组前期研究发现杨桃根活性成份2-十二烷基-6-甲氧基-2,5-二烯-1,4-环己二酮（DMDD）可改善糖尿病小鼠肾功能，近期试验发现其可抑制TLR4表达，提示，DMDD治疗DN的作用可能与调控TLR4通路有关，但机制尚未完全明确。本项目拟在前期研究的基础上，用敲除TLR4基因（TLR4-/-）小鼠建立DN动物模型，探讨TLR4信号通路对DN小鼠IR、微炎症状态及间质纤维化的影响，分析DMDD保护作用机制；在体外采用高糖诱导小鼠足细胞和肾小管上皮细胞转分化，从TLR4信号通路活化角度进一步阐明DMDD减轻DN的分子机制。本研究将较全面地阐明DMDD抗DN的作用机理，为其临床治疗提供实验依据和理论支持。

本项目主要研究杨桃根活性单体2-十二烷基-6-甲氧基-2，5-二烯-1，4-环己二酮（DMDD）对链脲佐菌素诱导的糖尿病肾病小鼠和高糖所致的足细胞（MPC5）损伤及肾小管上皮细胞（HK-2）发生上皮间质转分化（EMT）的保护作用及机制。.⑴体内实验：选取TLR4野生型（WT）和敲除型（KO）小鼠为对象，高糖高脂饲料联合低剂量STZ100mg.kg-1，构建糖尿病小鼠模型。将造模成功的WT型和KO型小鼠随机分为5组：模型组（DN组）、阳性组（G，予格列喹酮10 mg.kg-1.d-1）、DMDD高、中、低剂量组（H、M、L，予DMDD50、25、12.5 mg.kg-1.d-1）。另设立正常对照组（NC组）。予DMDD 4周后，拔眼球取血，颈椎脱臼处死小鼠，取肾脏组织。ELISA测定血清肌酐（Scr）、尿素氮（BUN）、胱抑素-C（Cys-C），生化仪测定TC、TG、LDL等，ELISA测定炎症因子IL-1β、IL-6、IL-10、TNF-α、MCP-1等；HE、Masson染色观察肾脏组织病理、纤维化情况，电镜观察肾超微结构变化。RT-PCR、WB和免疫组化观察TLR4、MyD88、NF-κB、BAMBI、TGFβ1、Smad2/3 mRNA和蛋白表达情况。.（2）体外实验：采用高糖（30mmol/L）构建MPC5细胞损伤模型，高糖（60mmol/L葡萄糖）刺激HK-2细胞转分化。CCK-8法测定DMDD对MPC5、HK-2细胞活性，确定IC50及DMDD给药浓度。AO/EB方法及流式细胞分析术检测MPC5细胞凋亡情况，ELISA测定IL-6和TNF-α分泌水平，PCR和Western Blot测定TLR4、MyD88、NF-κB、TGFβ/Smad的mRNA和蛋白变化情况。慢病毒转染过表达及沉默BAMBI蛋白，RT-PCR和Western-blot检测TLR4和Smad2/3以及转分化相关指标E-cadherin和Vimentin的mRNA和蛋白表达的变化。.结论：DMDD对糖尿病肾病小鼠及高糖所致的MPC5细胞损伤和HK-2的EMT具有保护作用，可能通过抑制TLR4-MyD88-NF-κB和TLR4-TGFβ/SMad2/3 信号通路发挥作用。