Inhibition Of Chloride Channel In Intestinal Epithelial CaCC Is A Molecular Pharmacological Research Mechanism Of Resina Draconis Against Anti-rotavirus-induced Secretory Diarrhea

Secretory diarrhea is a clinical medical problem that affects the health of millions of people around the world every year.In recent years,it has been found that Ca²⁺-activated chloride channels?CaCCs?play an important role in the pathogenesis of secretory diarrhea caused by rotavirus and bacterial toxins,and are potential drug targets.Small molecule modulators are important pharmacological tools for studying ion channel gating mechanisms.CaCCs not only can distinguish the activity of known and unknown CaCCs on cell and ex vivo tissue models,but also provide effective molecular probes for the study of physiological functions and pathophysiological mechanisms of CaCCs.Finding CaCCs selective small molecule regulators is one of the key points in related research.The colon cancer cell line HT29 expresses a CaCC?intestinal epithelial CaCC?with unknown molecular identity.In this study,we used a HT29 cell stably expressing the green fluorescent protein mutant YFP-H148Q/I152L to establish a fluorescence quenching function screening model?HT29/YFP-H148Q/I152L?,the main components of Resina Draconis,Loureirin A,Loureirin B and Loureirin C have inhibitory effects on the intestinal epithelial CaCCchloridechannel.OntheHT29/YFP-H148Q/I152L,FRT/TMEM16A/YFP-H148Q/I152LandFRT/CFTR/YFP-H148Q/I152L,thebasic molecular pharmacological properties of the active compounds were analyzed using fluorescence quenching experiments and short-circuit current experiments.The in vivo activity of the compound was analyzed by short-circuit current experiment of mouse colon isolated tissue,rat rotavirus diarrhea model experiment,mouse gastrointestinal peristalsis test and mouse intestinal smooth muscle contraction experiment.The purpose of this study was to obtain an intestinal epithelial CaCC chloride channel inhibitor with high affinity and good activity in vivo,and to provide a small molecular probe for revealing the physiological and pathological function of the intestinal epithelial CaCC chloride channel,targeting the intestinal epithelial CaCC chloride channel.The treatment of secretory diarrhea provides lead compounds and new therapeutic ideas.Experimental results:1.The HT29/YFP-H148Q/I152L functional assay screening model was used to detect that Loureirin A,Loureirin B and Loureirin C inhibited intestinal epithelial CaCC chloride channel activity in a dose-dependent manner.The results of short-circuit current analysis on HT-29 cells showed that Resina Draconis,Loureirin A,Loureirin B and Loureirin C can inhibit the intestinal chloride CaCC chloride channel activity in the apical membrane side of HT-29 cells..2.The results of short-circuit current measurement of colonic mucosa in mice showed that Resina Draconis,Loureirin A,Loureirin B and Loureirin C inhibited the CaCC chloride channel in the colonic mucosa of mice.3.Rotavirus-induced model of diarrhea in young rats showed that both Resina Draconis and Loureirin C can inhibit watery diarrhea in newborn mice infected with rotavirus and gastrointestinal motility in young rats.The results of mouse intestinal smooth muscle contraction test showed that Loureirin A had no effect on smooth muscle contraction,while Resina Draconis,Loureirin B and Loureirin C can weaken the contractile strength of smooth muscle.4.Using the FRT/TMEM16A/YFP-H148Q/I152L functional assay model,it was found that Loureirin C can inhibit the TMEM16A chloride channel in a dose-dependent manner,and the inhibition is rapid and reversible;further short-circuit current measurement results show Loureirin C is able to inhibit TMEM16A-mediated chloride currents.5.Using FRT/CFTR/YFP-H148Q/I152L assay model,it was found that Loureirin C can activate CFTR chloride channel in a dose-dependent manner,and the activation activity is fast and reversible;further short-circuit current measurement results show that Loureirin C has a slight activation of CFTR-mediated chloride currents.The short-circuit current of colonic mucosa in mice showed that Loureirin C had an activation effect on the CFTR chloride channel of mouse colonic mucosa epithelium but was not significant.6.Loureirin C has no effect on Na⁺-K⁺-ATPase in the serosal side of mice,but has a slight inhibitory effect on K⁺channels in the serosal side of mice.7.The results of Ca²⁺concentration measurement showed that Loureirin C can inhibit the increase of intracellular calcium concentration caused by ATP,carbachol?CCh?and ionomycin,indicating that Loureirin C is responsible for intestinal epithelial CaCC chloride.The inhibition of ion channels is achieved by inhibiting the increase in intracellular calcium ion concentration.In summary,the results suggest that the inhibition of intestinal chloride CaCC chloride channel is one of the molecular pharmacological mechanisms of Resina Draconis against rotavirus-induced secretory diarrhea.The study reveals the material basis and molecular pharmacology mechanism of Chinese medicine activity.It has important theoretical significance.The Resina Draconis and Loureirin A,Loureirin B,and Loureirin C found in this study can exert anti-diarrhea effect by inhibiting the intestinal chloride CaCC chloride channel activity and TMEM16A on ICC.