Discovery And Molecular Pharmacological Mechanism Of Sesquiterpenoid Lactones On Calcium-Activated Chloride Channel In Intestinal Epithelium

Calcium-activated chloride channels?CaCCs?is the basis of many cellular functions,It plays an important regulatory role in various physiological processes,such as intestinal dynamic regulation,smooth muscle contraction,epithelial fluid transport,neuronal excitation and injury sensation.The study of selective regulators can provide theoretical basis for revealing the mechanism of action of CaCCs and the molecular identity of CaCCs in various physiological and pathological processes.Therefore,screening of high affinity and strong selectivity CaCCs inhibitors has become a key research object.Colon cancer cell line HT-29 endogenous express TMEM16A andintestinal epithelial CaCC with unknown molecular identity,this study used HT29/YFP-H148Q/I152L as a screening model,selected from natural small molecule compounds which have inhibitory effect of the drug on intestinal epithelial CaCC,found sesquterpenoid lactone of costunolide,dehydroxylinolactone,alantolactone and isoalantolactone has significantinhibitory activityon intestinal epithelial CaCC.The selectivity,mechanism of action and molecular pharmacology of the compounds were investigated by cell biology experiments and electrophysiological methods.The in vivo activity of the compound was analyzed in mice.The purpose of this study was to screen intestinal epithelial CaCC inhibitors with good affinity and strong targeting from natural products and conduct molecular pharmacological studies on them,so as to provide theoretical basis for revealing the molecular identity of intestinal epithelial CaCC.Experimental results:1.HT29/YFP-H148Q/I152L was used as the screening model,sesquterpenoid lactones of costunolide,dehydroxylinolactone,alantolactone and isoalantolactone were found to inhibit intestinal epithelial CaCC in a dose-dependent manner on HT-29 cells,respectively with apparent IC₅₀0 values of 10.6?M,29.3?M,13.5?M and19.8?M,and their inhibitory effects were reversible.2.Ht-29 short-circuit current test results showed that four sesquterpenoid lactones all in a dose-dependent manner inhibited intestinal epithelial CaCC,with IC₅₀0 values of 24.7?M,12.5?M,5.3?M and 9.2?M.3.Sesquterpenoid lactone could significantly increase ATP,CCh and ionomycin induced Ca²⁺concentration in HT-29 cells.4.FRT-TMEM16A short-circuit current experimental results show that apical applicationoffoursesquterpenoidlactonescoulddose-dependentlyinhibit TMEM16A-mediated currents,IC₅₀0 value respectively of 18.9?M?55.4?M?34.2?M and38.5?M.The results showed that the concentration range of the inhibitory effect of four sesquterpenoids on TMEM16A was different from that on CaCCs on ht-29 cells which endogenous express TMEM16A and a CaCC(CaCC_gie)with unknown molecular identity,so the inhibitory effect of four sesquterpenoid lactones on intestinal epithelial CaCC could be determined.5.T84 cell short-circuit current experimental results show that apical application of four sesquterpenoid lactone could dose-dependently inhibit CFTR choride channel,IC₅₀0 value concentration respectively of 20.3?M,9.5?M,11.1?M and 14.6?M.6.Short-circuit current experimental of colonic mucosa in mice results show that:four sesquterpenoid lactones had no inhibitory effect on CFTR of colonic mucosa and Na⁺-K⁺-ATPase of colonic serous membrane in mice.7.Short-circuit current experimental of serous membrane in mice results that four sesquiterpenoid lactones had inhibitory effects on Ca²⁺-activated-K⁺channels.8.In vitro and in vivo studies,mice colonic mucosa short-circuit current experimental results show that four sesquterpenoid lactones also inhibit CaCCs in in mice colonic mucosal with a dose-dependent manner.The rotavirus diarrhea model test of newborn showed that both alantolactone and isoalantolactone could inhibit the secretory diarrhea caused by rotavirus.At the same time,four sesquiterpenoid lactones can significantly inhibit intestinal motility in mice.Conclusion:four sesquterpenoid lactones were found to inhibit intestinal epithelial CaCC_gieie in this study,our sesquterpenoid lactones of CaCC,TMEM16A and K⁺channels have inhibitory activity,and the inhibition dose concentration range of TMEM16A and CaCCs on HT-29cells are different,HT-29 cells endogenous expres TMEM16A and a unknown molecular identity intestinal epithelial CaCC,therefore four sesquterpenoid lactones can inhibit intestinal epithelial CaCC.Four sesquterpenoid lactones also inhibit intracellular calcium ion concentration,so the four sesquterpenoid lactones may inhibit CaCCs and K⁺channels by targeting Ca²⁺concentration.Four sesquterpenoid lactones also inhibit CaCC-mediated fluid secretion and intestinal motility.This discovery provides a theoretical basis for the pathogenesis and treatment of secretory diarrhea and the determination of the molecular identity of intestinal epithelial CaCC.