Identification Of Natural Small Molecule Aquaporin-4Inhibitors By High Throughput Screening

Aquaporin (AQP) is a family of water channel proteins, which consist of13homologousmembers (AQP0-AQP12) in mammal, widely express in the nervous, respiratory, digestive,and urinary system. AQP4is highly expressed in the central nervous system (CNS), inducingwater fluxes in brain. Dysfunction or misregulation of AQP4is associated with somedisorders of brain, such as stroke, hydrocephalus, brain edema, infection, tumor, epilepsy, andtraumatic brain injury. This suggests that AQP4is a possible drug target of these disease, butfew AQP4inhibitor for functional and therapeutic studies are reported.We established the cell models of V79-V163S-AQP4-mCherry andFRT-V163S-AQP4. Cl-is one of the main anions in cell. EYFP-H148Q-V163S is a GFPmutant which is sensitive to Cl-. When permeable fluid flow happen across the cellmembrane, the rapid change of the osmotic pressure of cell cause the instant change of the Cl-concentration. Certain Cl-concentration in cell can quench the fluorescence ofEYFP-H148Q-V163S. So this GFP mutant could serve as an indicator of the cell membranewater permeability. When measuring the water permeability, we made the cell in thecircumstance of hyperosmotic.According to our comparation tests, we confirmed that V79-V163S-AQP4-mCherry cellline is more sensitive and accurate suitable for high throughput screening of AQP4inhibitorby measuring the water permeability. Using this cell model, we screened600Chinese herbs,and found61Chinese herbs can inhibit AQP4. A natural inhibitor named Gleditsia SaponinsEwas purified from the best activity ingredients. The inhibitory effects are dose-dependent withIC501.034μM. Gleditsia SaponinsE is a kind of pentacyclic triterpenoids. Pentacyclictriterpenoids distribute widely in plants, usually have various pharmacological activities.Further studies are required to find the therapeutic mechanism and evaluate cytotoxicit. Inconclusion, this research not only provide new drug for brain edema, but also supply proofs ofthe Chinese herbs development.