肠道菌群孔道形成毒素和三叶因子复合物在血管内皮衰老中的作用机制

Human ageing processes are largely influenced by gut flora. Gastrointestinal bacteria can produce pore-forming toxins. We previously found that a host derived pore-forming toxin-like protein and trefoil factor complex βγ-CAT can induce destabilization of lysosomes, thus activate Nod-like receptor protein 3 (NLRP3) inflammasome and promote endothelial cell senescence. Recently, we discovered that the serum trefoil factor 2 (TFF2) can covalently bind to bacteria derived ε-toxin (ETX) forming ε-toxin-TFF2 (ETX-TFF2) complex. Its structure is similar to βγ-CAT. Preliminary results suggested that both βγ-CAT and ε-toxin-TFF2 can facilitate the senescence of Human Umbilical Vein Endothelial Cells. These results strongly suggest that gut flora can influence ageing processes especially endothelial senescence by ε-toxin-TFF2 complex. But its mechanism is largely unknown. This proposal aims to unveil the underlying mechanism. The following studies are planned: 1)searching for the microenviroment for the formation of ε-toxin-TFF2 complex, 2)identifying the membrane receptor ofε-toxin-TFF2, 3) exploring the mechanisms how βγ-CAT and ε-toxin-TFF2 promote endothelial cell senescence, including the involvement of lysosome destabilization and autophagy, 4) studying the in vivo role and mechanism of ε-toxin-TFF2 on vascular ageing of rats. The proposed studies not only help to gain insight into mechanism on ε-toxin-TFF2 complex signaling pathways in endothelial cell senescence but also have clinical implication in prevention and confrontation of ageing and age-related vascular diseases.

肠道菌群影响个体衰老。我们发现孔道形成毒素样蛋白与三叶因子复合物βγ-CAT可增加溶酶体不稳定性、激活NLRP3炎性小体（PNAS，2014）、促进衰老。我们最近发现人血液中存在的三叶因子TFF2与βγ-CAT一样，可以和肠道菌群生成的孔道形成毒素ε-toxin结合形成ε-toxin-TFF2复合物。我们还发现βγ-CAT和ε-toxin-TFF2均可促进人脐静脉内皮细胞衰老。说明肠道菌群可能通过ε-toxin-TFF2影响衰老，但机制不清。本项目拟研究：(1)ε-toxin-TFF2复合物形成的微环境基础；（2）ε-toxin-TFF2的膜受体鉴定；（3）溶酶体和自噬参与βγ-CAT和ε-toxin-TFF2引起衰老的机制；（4）ε-toxin-TFF2在大鼠血管衰老中的作用。拟通过这些研究，明确孔道形成毒素和三叶因子复合物在血管衰老中的作用机制，为延缓和对抗衰老提供新的靶点。

项目基于前期研究发现肠道菌群来源的孔道形成毒素ε-toxin（ETX）可以和TFF2相互作用，形成ETX-TFF2复合物，影响细胞衰老。项目拟： (1)ETX-TFF2复合物形成的微环境基础；（2）ETX-TFF2的膜受体鉴定；（3）溶酶体和自噬参与βγ-CAT和ETX-TFF2引起衰老的机制；（4）ETX-TFF2在大鼠血管衰老中的作用。拟通过这些研究，明确孔道形成毒素和三叶因子复合物在血管衰老中的作用机制，为延缓和对抗衰老提供新的靶点。.在项目实际执行中，完成了：（1）明确了胃肠道分泌的三叶因子TFF2和TFF3可以和肠道产气荚膜梭菌分泌的ETX协同作用，促进ETX的细胞毒性。细胞在受到刺激后产生的活性氧则可以促进ETX和TFF2的结合。二者的结合可以降低其细胞毒性，但促进其向细胞内运输。（2）发现蛋白酶激活受体PAR1和PAR4介导了TFF2促进ETX毒性的作用。PAR1和PAR4并不直接影响ETX的毒性作用，但是PAR1和PAR4的敲低可以降低TFF2对ETX毒性的促进作用。（3）ETX和TFF2相互作用被内皮细胞内吞并定位到溶酶体，引起溶酶体去酸化。溶酶体去酸化进一步导致内质网应激（ER stress）及线粒体未折叠蛋白反应（Mitochondrial unfolded protein response，UPRmt），促进内皮细胞衰老。线粒体未折叠蛋白反应对细胞存活起到保护作用。（4）在整体动物水平，发现TFF2和TFF3均可以提高ETX的毒性，降低ETX对小鼠的LD50值，TFF2和TFF3抗体则可以降低ETX的毒性。另外，线虫内源性孔道形成毒素类似蛋白lin-24参与溶酶体功能调节，参与线虫抗感染免疫。.此外，在探究细胞炎症过程中，发现细胞膜通透性的增加导致的钾离子外流是NLRP3炎症小体激活的核心途径（Cytotechnology, 2018）。另外，也发现细胞在受到高渗刺激时，Hsp70的活化和自噬的激活对细胞起到保护作用（Cell stress and cheperones, 2020）。本项目的研究揭示了孔道形成毒素及孔道形成毒素类似蛋白通过增加细胞膜、溶酶体膜对离子通透性的调节内质网非折叠蛋白反应和线粒体非折叠蛋白反应参与细胞应激保护和细胞衰老的机制。