Abstract
Secretory diarrhea remains a global health burden and causes major mortality in children. There have been some focuses on antidiarrheal therapies that may reduce fluid losses and intestinal motility in diarrheal diseases. In the present study, we identified shikonin as an inhibitor of TMEM16A chloride channel activity using cell-based fluorescent-quenching assay. The IC50 value of shikonin was 6.5 μM. Short-circuit current measurements demonstrated that shikonin inhibited Eact-induced Cl- current in a dose-dependent manner, with IC50 value of 1.5 μM. Short-circuit current measurement showed that shikonin exhibited inhibitory effect against CCh-induced Cl- currents in mouse colonic epithelia but did not affect cytoplasmic Ca2+ concentration as well as the other major enterocyte chloride channel conductance regulator. Characterization study found that shikonin inhibited basolateral K+ channel activity without affecting Na+/K+-ATPase activities. In vivo studies revealed that shikonin significantly delayed intestinal motility in mice and reduced stool water content in a neonatal mice model of rotaviral diarrhea without affecting the viral infection process in vivo. Taken together, the results suggested that shikonin inhibited enterocyte calcium-activated chloride channels, the inhibitory effect was partially through inhbition of basolateral K+ channel activity, and shikonin could be a lead compound in the treatment of rotaviral secretory diarrhea.
Highlights
Secretory diarrhea remains a global health burden and causes major mortality in children under 5 years old (Walker et al, 2013)
TMEM16A was the first calcium-activated chloride channels (CaCCs) identified, and the effect of shikonin (Figure 1A) on TMEM16A chloride channel activity was studied using a previously set up cell-based fluorescent quenching assay
Short-circuit current measurement showed that inhibition of TMEM16A chloride currents by shikonin was concentration-dependent, and the highest concentration (10 μM) of shikonin used could only abolish about 65% of the Cl− current stimulated by
Summary
Secretory diarrhea remains a global health burden and causes major mortality in children under 5 years old (Walker et al, 2013). One of the major chloride channels, cystic fibrosis transmembrane conductance regulator (CFTR), has been shown to be responsible for the enterotoxin-induced secretory diarrhea in cholera and Travelers’ diarrhea (Chao et al, 1994; Grubb, 1995; Thiagarajah et al, 2004; Sonawane et al, 2007). Another enterocyte chloride channel, calcium-activated chloride channels (CaCCs), is thought to be associated with rotavirus-induced and probably other virusrelated secretory diarrhea (Morris et al, 1999). It is considered that a non-structural protein NSP4 activates TMEM16A/CaCC in the enterocytes (Ball et al, 1996; Morris et al, 1999), but the exact mechanism of this remains to be elucidated
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