Identification And Anti-diarrheal Study Of Natural Small Molecule Calcium-activated Chloride Channel Inhibitors

Rotaviral diarrhea is one of the leading causes of mortality in newborn. Although death rate has been reduced by antiviral vaccine used worldwide, Diarrhea in early infection still leads to severe dehydration, stunted growth, even death. Effective antidiarrheal drugs should be valuable in inhibiting dehydration and prolonging treatment time. Vibrio cholerae and Escherichia coli endotoxin are considered activating CFTR through elevating c AMP and c GMP, leading to Clsecretion and subsequent water and Na+ transport. However, rotaviral diarrhea might be induced by activating intestinal epithelial Ca²⁺-activated chloride channels（Ca CC）, leading to massive fluid secretion in the intestine; or enhancing contraction of intestinal smooth muscle by activating TMEM16A-Ca CC. The exact mechanisms remain controvercial. Inhibition of chloride channel by Ca CC selective inhibitors should contribute to elucidate what role Ca CC plays in rotaviral diarrhea, also Ca CC inhibitors could be therapeutic drugs for rotaviral diarrhea and diarrhea caused by other factors that activate Ca CC. Selective Ca CC inhibitors are important pharmacological tools in Ca CC functional researches, current inhibitors have limitations due to low affinity and poor selectivity, looking for Ca CC inhibitors with high affinity and high selectivity is still a problem remaining to be solved in this fieldIn the present study, we identified resveratrol oligomers trans-ε-viniferin（TV）, r-2-viniferin（RV） and fraction D5 of Bistortae Rhizoma with Ca CC inhibitory effect from natural small molecule library and traditional fraction library of Chinese medicinal herbs using HT-29 cells stably transfected with halide sensitive YFP fluorescent proteins and Fischer rat thyroid（FRT） epithelial cells transfected with human TMEM16 A and YFP proteins. TV and RV inhibited ATP and CCh-induced Ca CC-mediated I- influx in a dose-dependent manner with IC50 values of 1.1 μM and 12.3 μM in HT-29 cells. Fraction D5 inhibited TMEM16A-mediated I- influx dose-dependently in FRT cells with IC50 values of 32.2 μg/ml, the maximal effect was at 200 μg/ml.Next we used short-circuit current measurement, ex-vivo and in-vivo experiments to analyze pharmacological characteristics and antidiarrheal effects of TV, RV and fraction D5. First, the dose-response manner, binding site and characteristics of TV and RV were analyzed by short-circuit current measurements. Results showed that:（1） TV and RV inhibited ATP-induced Cl^- current in HT-29 cells in a dose-dependent way with maximal inhibitory effect at 5 and 50 μM, respectively; IC50 values were 1 μM and 20 μM.（2） TV and RV inhibited Ca CC chloride channel activity in the apical side of enterocytes rather than basolateral side.（3） TV（5 μM） blocked ATP-induced sustained short-circuit current, but had little inhibitory effect on transient short-circuit current in HT-29 cells; RV（50 μM） inhibited both sustained and transient short-circuit currents induced by ATP in HT-29 cells.（4） TV（5 μM） didn’t inhibit basal or ATP-induced cytoplasmic Ca²⁺ increase, RV manifested slight inhibitory effect at 50 μM（inhibition rate ¹7%）.CFTR and TMEM16 A are the known chloride channels in the enterocytes. Selectivity analysis showed that:（1） Apical applications of TV（200 μM） or RV（50 μM） completely inhibited Eact-induced TMEM16A-mediated Cl^- currents, IC50 values were 50 and 100 μM. Fraction D5 completely inhibited Cl^- currents at 200 μg/ml apically.（2） TV and RV inhibited CFTR-mediated Cl^- current dose-dependently in HT-29 cells, IC50 values were 3 and 80 μM, respectively.Inhibitory effect of TV and RV on Ca CC chloride channel activity was analyzed by ex-vivo short-circuit current measurements in the isolated mouse colon. The results showed that（1） TV and RV inhibited short-circuit currents induced by carbachol in mouse colonic mucosa with maximal inhibitory rates of 57% and 47% respectively, remaining currents were abolished by CFTR inhibitor BPO-27 and K+ channel inhibitor clotrimazole.（2） Fraction D5 inhibted carbachol-induced short-circuit currents in the mucosal side with maximal inhibitory rate of 50%, however no inhibitory effect was seen with serosal application of D5.（3） Antidiarrheal drug crofelemer（1 m M） didn’t inhibit forskolin-induced CFTR-mediated Cl^- current in mouse colonic epithelia, but fully inhibited carbachol-induced Ca CC-mediated Cl^- current mucosally.Intestinal motility affects intestinal net fluid secretion apart from Cl^- secretion thus mouse intestinal smooth muscle contraction and gastrointestinal peristalsis were measured for the regulatory effects of TV and RV on intestinal motility. Results revealed taht（1） TV at 5-200 μM and RV at 10-100 μM inhibited mouse ileal smooth muscle contraction which were in accordance with the concentrations that inhibited TMEM16 A chloride channel activity.（2） Orally administration of TV and RV（1 m M） reduced mouse gastrointestinal peristaltic rates; both orally and intraperitoneally administratioon of fraction D5 inhibited mouse gastrointestinal peristaltic rate.Current perspective considered non-structural protein NSP4 and the active fragments as rotavirus-secreted endotoxin that cause secretory diarrhea via activation of Ca CC chloride channel activity, rotaviral diarrhea mainly occurred in infants and young children. Therefore we set up a rotaviral diarrhea neonatal mice model to investigate the anti-diarrheal effects of Ca CC inhibitors TV and RV using watery stool content measurements. Results revealed that TV and RV prevented watery stools of rotaviral infectious neonatal mice in two methods of administration, and the anti-diarrheal effects were better than Ca CCinh-A01. Histology showed that large vacuoles were seen in enterocytes at day 3 after rotavirus infection, with swelling of the villus tips. Similar vacuolisation appeared in rotavirus-infected mice receiving TV. Vacuoles were not seen in control mice. Compared to untreated group, TV didn’t affect jejunal fluid absorption at 30 min.Chromatographic analysis showed that TMEM16 A inhibitory effect of rhizoma bistortae components D5 had no relationship with compound ECG, EGCG, TV and RV.In conclusion, TV, RV and fraction D5 of Bistortae Rhizoma showed Ca CC inhbitory effects, TV is the inhibitor of epithelial Ca CC chloride channel with highest affinity so far. Short-circuit current measurement demonstrated that TV and RV acted in the apical membrane of enterocytes, confirming the existence of non-TMEM16 A gastrointestinal epithelial Ca²⁺-activated chloride channel（Ca CCGI）, Ca CCGI should be the predominant Ca CC chloride channel of intestinal epithelia. Ex-vivo short-circuit current measurements demonstrated that TV, RV and fraction D5 could inhibit colonic mucosal Ca CCGI activity induced by carbachol, CFTR and K+ channels are also involved in the generation of carbachol-induced short-circuit current. Crofelemer inhibited Ca CCGI chloride channel activity rather than CFTR at tissue level. TV, RV and fraction D5 reduced gastrointestinal motility at high concentration through inhibiting TMEM16 A. TV and RV could inhibited watery stools of rotaviral infectious neonatal mice effectively without affecting virus infection and fluid absorption, proving that rotavirus induced secretory diarrhea through activating Ca CC chlordice channel of enterocyte thus increasing net fluid secretion. Our present study provided theretical significance to set forth the roles of Ca CCGI and TMEM16 A chloride channels in intestinal fluid secretion and intestinal motiliy in diarrheal pathology, and clarifed Ca CCGI and TMEM16 A as new potential drug targets for antidiarrheal therapy. Moreover, our study provides new therapeutic strategy and leading drugs for the treatment of chloride channel-targeted secretory diarrhea.