轮状病毒NSP4蛋白诱导细胞锚定连接相关蛋白下调引起腹泻的分子机制研究

Previous studies have shown that rotavirus (RV) enterotoxin non-structural protein (NSP4) caused by intracellular and extracellular Ca2 + imbalance, activated chloride channel lead to diarrhea. However, knockout mice that chloride ion channels blocked can still induce diarrhea, suggesting another pathogenic mechanism. Anchor connection are important structures in the intestinal epithelial cells (IEC). Destruct anchor connection could accelerate the immature IEC update, and ion exchange imbalance, seriously affect the intestinal absorption, lead to diarrhea. The latest research shows that calcium imbalance Cdc42 signaling pathways to promote activation of the Rho GTPase family of molecules anchored to connect protein internalization and degradation. We found that IEC anchor connector protein decreased significantly after RV infection, and the Ca2 + chelator can effectively inhibit the decrease. Thus, we propose that the main cause of diarrhea is Ca2 + imbalance caused by RV infection could undermine the IEC anchor connections. And lots of mature IEC were brushed off under the mechanical action of the intestinal contents, then caused by absorption dysfunction. The project is planned based on previous work, aim to further explore the mechanism of calcium ion imbalance after RV infection, the Cdc42 and, EGFR, of Src signaling pathway and anchorage connector impact caused by diarrhea. This study will deepen the understanding of the mechanisms of RV diarrhea occur, provide the experimental basis for new anti-RV drug development.

以往研究证明，轮状病毒（RV）肠毒素NSP4能引起细胞内外Ca2+失衡，激活氯离子通道引发腹泻。但基因敲除小鼠证明氯离子通路阻断仍能诱发腹泻，提示另有致病机制。锚定连接是肠上皮细胞(IEC)间重要结构，其破坏加速不成熟IEC更新和离子交换失衡，严重影响肠吸收功能，导致腹泻。最新研究表明钙离子失衡激活Rho GTP酶家族分子Cdc42信号通路促进锚定连接蛋白内化与降解。我们发现RV感染后IEC锚定连接蛋白显著下降，且Ca2+螯合剂可有效抑制其下降。由此，我们提出RV感染引起Ca2+失衡可能破坏IEC间锚定连接，成熟IEC在肠道内容物机械作用下大量脱落引起吸收功能障碍是导致腹泻的主要原因。本项目拟在前期工作基础上，研究RV感染后钙离子失衡对Cdc42、EGFR、Src信号通路及锚定连接影响的致腹泻机制。本研究将深化对RV腹泻发生机制的认识，为新型抗RV药物开发提供实验依据。

轮状病毒感染是5岁以下婴幼儿腹泻主要病原感染原因，机制研究一直未能明确。本课题立足于病毒感染主要宿主细胞IEC进行体内外感染分子机制研究。通过定量蛋白组学及定量PCR\WB、激光共聚焦等技术，明确了RV感染引起连接蛋白磷酸化激酶信号通路PI3K通路活化及FN1蛋白降解是其关键腹泻机制。首先建立了腹泻动物模型，观察到轮状病毒感染后小鼠肠上皮细胞脱落程度与腹泻密切相关。进而从细胞模型进行相关机制研究。定量蛋白组学研究提示感染后宿主细胞连接蛋白下调，肠上皮细胞感染RV也同样观察到肠上皮细胞失巢脱落，而不是凋亡。进一步采用分泌蛋白组学分析FN1表达下调可能是导致肠上皮细胞失巢脱落的关键分子。进一步的IF、WB、ELISA、qPCR分析显示RV感染细胞内外FN1下降。进一步WB分析显示RV感染主要通过PI3K蛋白磷酸化信号通路引起FN1降解。FN1抑制剂进行动物实验证实抑制羧肽酶活性可以有效缓解RV感染引起肠上皮细胞的失巢及脱落。