Fetuin A介导游离脂肪酸与TLR4-JNK信号通路串话的脂毒性机制研究

Induction of proinflammatory responses in β cells is an important mediator of lipotoxicity, but this molecular mechanisms of lipotoxicity is not clear and thus can not target for therapeutic interventions. Previous studies have shown that free fatty acids (FFA) can activate pancreatic islet inflammation through Toll-like receptor 4 (TLR4). However, FFA does not bind directly to TLR4. We have found that Fetuin A（FetA）could increase the activity of TLR4 to promote FFA-induced lipoapoptosis in β cells, indicating that FetA may act as the medium that allows FFA-TLR4 crosstalk. So, this study is going to investigate the mediated effect of FetA by using the technology of RNAi and gene overexpression to regulate gene expression in vitro and FetA or TLR4 gene knockout mice models in vivo, as the following five experiments: at first, we will observe the association among FFA, FetA ,TLR4 and β-cell function;then, we want to analyze whether FetA participates in lipotoxicity damage of β-cell; third, we intend to investigate whether FFA-induced TLR4 signaling requires FetA; fourth, we will try to find the evidence of the interaction among FFA, FetA and TLR4; at last ,we will further investigate whether the effect of FFA-FetA-TLR4 complexes is conducted by PKC-JNK-AP-1-PANDER- caspase-3 pathway.In summary,this study is to search for the new target of lipotoxicity on β-cell.

促发炎症反应是脂毒性发挥β细胞损害的重要途径，但分子机制未完全阐明，故无法靶向干预。已知游离脂肪酸（FFA）通过Toll 样受体4（TLR4）介导胰岛炎性损伤，但FFA并不直接绑定TLR4。我们前期研究发现胎球蛋白A（FetA）可激活β细胞的TLR4而协同脂毒性损害，推测FetA可能具有介导FFA与TLR4串话的作用。为此，我们拟体外采用基因干扰和过表达技术调控细胞基因表达，体内运用FetA、TLR4基因敲除小鼠模型等方法，分5个层次验证假设：首先观察FFA、FetA和TLR4与β细胞功能的关联；接着分析FetA是否参与脂毒性细胞损害；进而研究FFA激活TLR4是否需要FetA介导；并验证FFA，FetA，TLR4蛋白间的相互作用；最后探讨FFA-FetA-TLR4复合物发挥作用是否经由PKC-JNK-AP-1-PANDER-caspase-3通路。以期为干预β细胞脂毒性损害提供新的靶点。

胎球蛋白A（FetA）是一种分泌性糖蛋白，可影响肥胖个体和动物的炎症和胰岛素抵抗（IR）。已知慢性脂毒性诱导的胰腺β细胞损害是糖尿病的重要致病机制。本项目的研究目的是确定FetA是否通过调节Toll样受体4（TLR4）炎症信号传导通路促进脂毒性作用，以及吡格列酮（PIO）/ Exendin-4 / GPR40激动剂对脂毒性的保护作用。我们的研究结果显示，FetA可呈剂量依赖性加剧棕榈酸（PA）诱导的βTC6细胞凋亡，胰岛素分泌障碍以及抑制胰岛素相关基因G蛋白偶联受体40（GPR40）和胰腺十二指肠同源框 - 1（PDX-1）的表达。联合FetA + PA方可诱导β细胞TLR4表达活化。抑制TLR4信号或表达，可遏制FetA对PA诱导的βTC6细胞脂毒性的增强作用；TLR4基因敲除亦可减弱饮食诱导的肥胖大鼠胰岛功能的损伤和胰腺细胞的凋亡。FetA + PA诱导β细胞p-JNK和核因子-κB（NF-κB）亚基P65表达，而分别抑制p-JNK、NF-κB可减弱PA + FetA诱导的βTC6细胞脂毒性损害。PIO / Exendin-4 / GPR40激动剂可以改善PA + FetA诱导的βTC6细胞损伤。同样，PIO / Exendin-4能改善饮食诱导的肥胖大鼠胰岛β细胞胰岛素分泌紊乱，减少胰腺细胞凋亡，降低FetA，TLR4，p-JNK，NF-κB亚基P65和cleaved caspase 3的表达，增加GPR40和PDX-1表达。相关的双变量分析表明，FetA增加与脂毒性β细胞损伤的发展成正比。总之，我们发现FetA可以通过TLR4-JNK-NF-κB信号通路促进β细胞的脂毒性损害。PIO / Exendin-4 / GPR40激动剂对β细胞脂毒性的保护作用可能涉及抑制FetA和TLR4信号通路的活化。我们的研究不仅有助于区分FetA在脂毒性中所起的作用，为抑制β细胞脂毒性损伤提供潜在靶点；同时有助于进一揭示PIO / Exendin-4 / GPR40激动剂减轻胰腺β细胞脂毒性损伤的分子机制。