Role Of Transient Receptor Potential Canonical Channel 6 And Thioredoxin Interacting Protein In The Pathogenesis Of Chronic Rhinosinusitis With Nasal Polyps

Background:Previous studies have demonstrated that inflammation and oxidative stress play critical roles in the pathogenesis of chronic rhinosinusitis with nasal polyps（CRSwNP）.Transient receptor potential canonical channel 6（TRPC6）,a member of the nonselective Ca²⁺-permeable transient receptor potential（TRP）family,contributes to the inflammatory response in bronchial epithelial cells.Thioredoxin interacting protein（TXNIP）plays a crucial role in the process of triggering oxidative stress.However,their role and mechanism in CRSwNP remain unclear.The present study sought to explore the role and mechanism of TRPC6 and TXNIP in the pathogenesis of CRSwNP,respectively.Methods:（1）Western blot,Real time-PCR and immunohistochemistry（IHC）were employed to assess expressions of TRPC6,stromal interaction molecule 1（STIM1）,calcium release activated calcium channel protein 1（Orai1）in nasal tissue samples from CRSwNP patients and control subjects.The concentrations of inflammatory mediators,including interleukin（IL）-1β,IL-5,and IL-25,were assayed by enzyme-linked immunosorbent assay（ELISA）.In experiments on human nasal polyp epithelial cell（HNEC）culture and stimulation,the mean fluorescence intensity（MFI）of intracellular Ca²⁺was assayed by flow cytometry.Western blot,Real time-PCR,and ELISA were also conducted to assess the effects and mechanisms of TRPC6 activator1-oleoyl-2-acetyl-sn-glycerol（OAG）and TRPC6 inhibitor 1-[2-（4-methoxyphenyl）-2-[3-（4-methoxyphenyl）propoxy]ethyl-1H-imidazole（SKF96365）on HNECs.（2）IHC,western blot,Real time-PCR were employed to assess expressions of TXNIP and thioredoxin（TRX）in nasal tissue samples from CRSwNP patients and control subjects.Malondialdehyde（MDA）levels and superoxide dismutase（SOD）activities in nasal tissue homogenates were measured using MDA and SOD Assay Kit.To evaluate the mechanism of TXNIP in CRSwNP,HNECs were cultured and stimulated using TXNIPsi RNA,with or without N-acetylcysteine（NAC,an ROS scavenger）.Western blot,Real time-PCR,ROS detecting dye 2’,7’-Dichlorofluorescin Diacetate（DCFH-DA）,and SOD Assay Kit were performed to assess the effects and mechanisms of stimulators on HNECs.Results:（1）Upregulation of TRPC6,STIM1,and Orai1 m RNA levels was found in CRSwNP patients.TRPC6 m RNA was positively correlated with STIM1 and Orai1m RNA levels,respectively.Moreover,TRPC6-positive cells correlated positively with the numbers of eosinophils and neutrophils,respectively.The concentrations of inflammatory mediators,including IL-1β,IL-5,and IL-25,were elevated in CRSwNP.In cultured HNECs,TRPC6,STIM1,Orai1,Ca²⁺MFI levels,and inflammatory mediators were upregulated by lipopolysaccharide（LPS）and OAG,but were inhibited by SKF96365.（2）Significantly increased levels of TXNIP and decreased levels of TRX protein expression,positive cells and m RNA,increased MDA level and decreased SOD activity were found in CRSwNP patients,compared with control subjects.In vitro study,significantly altered levels of TXNIP,TRX,SOD and ROS in HNECs were found following treatment of TXNIP si RNA,with or without NAC on HNECs.Conclusion:（1）TRPC6 plays a pro-inflammatory role via regulating Ca²⁺flow during the pathogenesis of CRSwNP.TRPC6 was elevated by LPS or OAG and inhibited by SKF96365.（2）Additionally,upregulation of TXNIP facilitates oxidative stress via decreasing TRX expression and SOD activities,and promoting MDA activities and ROS levels in CRSwNP.