葛根芩连方/Genistein通过Ca2+介导的IP3-DAG-PKC和CaMKII通路调控RV感染性腹泻中NHEs的机制

钠氢转运子（NHEs）在调节水钠吸收等多方面具有重要作用。前期研究发现葛根芩连方具有抗RV腹泻的作用，所含染料木素体外可干扰RV生物合成，该方可下调RV感染Caco-2细胞NHE1,6mRNA表达。目前NHEs在RV感染性腹泻中的作用与机制尚不清楚。基于此，拟建立RV及RV非结构白蛋白4感染乳鼠和Caco-2细胞模型，系统研究9种NHEs在肠道的分布及表达变化；制备稳定转染shNHEs的Transwell模型，并结合高效液相色谱、正电子核素22Na测定等技术，建立同时定量检测水钠吸收的方法，评价相关NHEs的功能；揭示通过Ca2+介导的IP3/DAG-PKC和CaMKII途径调控NHEs活性的机制；同时进行葛根芩连方及染料木素干预对比研究，阐明该方通过此途径抗RV感染性腹泻的机制及物质基础。该研究将为NHEs作为RV致泻新靶点提供可靠证据，为该方应用于抗RV感染性腹泻提供充分科学依据。

The mammalian Sodium hydrogen transfer proteins (NHEs) exert the importance on regulating water and sodium metabolism,but the function and mechanism of intestinal NHEs in diarrhea during rotavirus (RV) infection still remain unclear. Our previous work indicated that Gegen Qinlian Formula (GGQL) had potential activity to against rotavirus, which could also down-regulation the mRNA expression of NHE-1, NHE-6 in Caco-2 cells. Herein, we will prepare the recombinant fusion protein GST-NSP4T, then suckling mice or Caco-2 cells will be inoculated with the rotavirus or GST-NSP4T. In this project, we will investigate the expressions of some intestinal NHEs induced by RV and GST-NSP4T in suckling mice or Caco-2 cells. The stable transfection with short hairpin RNA (shRNA) mediated NHEs of Caco-2 cell monolayer will be use for determining the the functions of relevant NHEs, including water and sodium metabolis using HPLC and positron nuclide 22Na technique, and demonstrate GGQL/Genistein will through Ca+ - phospholipid - dependent protein kinase and CaMKII signaling pathways to regulate the NHEs activity. Our research will support NHEs as a new target in RV-induced diarrhea, and will provide adequate scientific basis for GGQL/Genistein used in anti-RV infectious diarrhea.