Objective To investigate the effect of vitamin B12(VB12)on the expression of zonula occludens-1(ZO-1)in house dust mite(HDM)-treated human airway epithelial cell line(Beas-2b)and its underlying mechanism.Methods Beas-2b cells were cultured in DMEM high-glucose medium containing 10%fetal bovine serum.The cells were divided into four groups:control,VB12,HDM,and VB12+HDM.Beas-2b cells were trans-fected with lentiviruses carrying NC-siRNA,ATG5-siRNA,BECN1-siRNA,and mCherry-EGFP-LC3.After 12 hours of transfection(MOI=20),the medium was replaced with fresh medium,and stable transfected cell lines were selected using puromycin(1 μg/mL).Cells were stimulated with VB12(20 μg/mL)and HDM(50 μg/mL)for 24 hours.The protein levels of ZO-1,autophagy-related protein 5(ATG5),BECN1 and microtubule-associated protein light chain 3(LC3)were detected by immunofluorescence and Western blot.Autophagy in human airway epithelial cells was observed using confocal microscopy.Results Compared with the control group,the expression of ZO-1 in the HDM group was lower(P<0.05),while the expressions of ATG5,BECN1,and LC3 were higher(P<0.05).Compared with the HDM group,the VB12+HDM group showed increased ZO-1 expression(P<0.05),decreased expressions of ATG5,BECN1,and LC3(P<0.01),and reduced autophagosome formation(P<0.05).In ATG5-and BECN1-knockdown cell lines,ZO-1 expression increased after HDM treatment(P<0.05).Conclusion Vb12 can enhance ZO-1 expression in HDM-treated human airway epithelial cells by down-regulating autophagy,and its mechanism is associated with the ATG5 and BECN1 signaling pathways.

Objective To investigate the effect of vitamin B12(VB12)on the expression of zonula occludens-1(ZO-1)in house dust mite(HDM)-treated human airway epithelial cell line(Beas-2b)and its underlying mechanism.Methods Beas-2b cells were cultured in DMEM high-glucose medium containing 10%fetal bovine serum.The cells were divided into four groups:control,VB12,HDM,and VB12+HDM.Beas-2b cells were trans-fected with lentiviruses carrying NC-siRNA,ATG5-siRNA,BECN1-siRNA,and mCherry-EGFP-LC3.After 12 hours of transfection(MOI=20),the medium was replaced with fresh medium,and stable transfected cell lines were selected using puromycin(1 μg/mL).Cells were stimulated with VB12(20 μg/mL)and HDM(50 μg/mL)for 24 hours.The protein levels of ZO-1,autophagy-related protein 5(ATG5),BECN1 and microtubule-associated protein light chain 3(LC3)were detected by immunofluorescence and Western blot.Autophagy in human airway epithelial cells was observed using confocal microscopy.Results Compared with the control group,the expression of ZO-1 in the HDM group was lower(P<0.05),while the expressions of ATG5,BECN1,and LC3 were higher(P<0.05).Compared with the HDM group,the VB12+HDM group showed increased ZO-1 expression(P<0.05),decreased expressions of ATG5,BECN1,and LC3(P<0.01),and reduced autophagosome formation(P<0.05).In ATG5-and BECN1-knockdown cell lines,ZO-1 expression increased after HDM treatment(P<0.05).Conclusion Vb12 can enhance ZO-1 expression in HDM-treated human airway epithelial cells by down-regulating autophagy,and its mechanism is associated with the ATG5 and BECN1 signaling pathways.

Calcium-activated chloride channels(CaCCs)are a class of channel proteins that trans-port chloride ions activated by intracellular calcium,which play a crucial role in regulating membrane potential,intracellular calcium balance,and cell excitability,particularly in neurons and muscle cells.In the Anoctamin(Ano)family,Ano1 is the most classic CaCC.Targeted modulators of Ano1 have poten-tial therapeutic effects against such diseases as cancer,cystic fibrosis,hypertension,diarrhea,and asthma.Since the discovery of Ano1 in 2008,several methods for screening CaCC-specific modulators have emerged including high-throughput primary screening of fluorescent proteins,electrophysiological patch clamp technique and virtual screening,and identification of small molecule modulators with diverse pharmacological effects.This paper summarizes the principles,advantages and disadvantages of the mainstream screening methods,and reviews the chemical structures and potential applications of Ano1-specific modulators discovered to date.