TMEM16A Promotes The Development Of Acute Pancreatitis Through The IP₃R-Ca²⁺-NFκB Signaling Pathway

Objective:TMEM16A is widely expressed in various mammalian cells and is involved in the regulation of human physiological functions and the development of diseases.Current research on TMEM16A has focused on its crystal structure and its pathophysiological role in diseases such as tumours,cystic fibrosis and hypertension,but whether it is involved in the development of acute pancreatitis has not been reported.Acute pancreatitis is an acute chemical inflammation of the pancreas.The current clinical treatment for acute pancreatitis is symptomatic support only and there is a lack of specific therapeutic agents.There is considerable evidence that elevated concentrations of free calcium ions in pancreatic alveolar cells are the initiating factor in the development of acute pancreatitis,but the pathogenesis of this is not yet fully understood.The organic combination of TMEM16A,IP₃R,calcium ions,NFκB and other signaling molecules was designed to elucidate the development of acute pancreatitis through activation of the IP₃R-Ca²⁺-NFκB signaling pathway by highly expressed TMEM16A in a cerulein-induced acute pancreatitis cell model,and the study provides theoretical support for the development of new drug targets for the treatment of acute pancreatitis.Methods:1.Application of the whole-cell recording mode of the membrane clamp technique to record the effect of different calcium ion concentrations on TMEM16A currents by cerulein under experimental conditions.2,Endogenous TMEM16A calcium-activated chloride currents in TMEM16A-scramble expression vector and AR42J cells transfected with TMEM16A-sh RNA expression vector in calcium-free internal and external solutions and changes in calcium-activated chloride currents after addition of TMEM16A inhibitor T16Ainh-A01.3,The intracellular binding of IP₃R to TMEM16A was observed using protein-protein immunoprecipitation technique.4,Real-time calcium release from the endoplasmic reticulum of AR42J cells transfected with overexpression of TMEM16A vector or incorporation of T16Ainh-A01 was observed under confocal microscopy using Fluo-4 calcium-binding fluorescent dye.5,Western-Blot technique was used to detect TMEM16A protein expression and the ratio of NFκB nuclear expression to cytoplasmic expression under different experimental conditions.Results:whole-cell membrane clamp recording patterns,the results showed that in AR42J cells,a higher concentration of Ca²⁺was required for TMEM16A activation;knockdown of TMEM16A in AR42J cells reduced cerulein-induced increase in calcium-activated chloride currents;addition of T16Ainh-A01 treatment to AR42J cells reduced cerulein-induced increase in calcium-activated chloride currents.Protein-protein immunoprecipitation results indicated the presence of protein interactions between IP₃R and TMEM16A.Application of FLUO-4 calcium-binding fluorescent dye under confocal microscopy revealed that Ca²⁺release from the endoplasmic reticulum was inhibited using T16Ainh-A01;AR42J cells overexpressing TMEM16A were able to promote increased IP₃R-mediated calcium release.western blot results showed that NFκB nuclear translocation was increased by high expression of TMEM16A.In the AP model induced by cerulein:NFκB nuclear translocation was increased;NFκB nuclear translocation was decreased by adding BAPTA;NFκB nuclear translocation was decreased by knocking down TMEM16A.Conclusions:In this experiment,we aimed to elucidate that high expression of TMEM16A in a cerulein-induced acute pancreatitis cell model promotes the development of acute pancreatitis through activation of the IP₃R-Ca²⁺-NFκB signalling pathway:1)there is protein binding between IP₃R and TMEM16A:TMEM16A High expression in acute pancreatitis cell models can regulate intracellular calcium ion concentration through IP₃R;2)elevated Ca²⁺in pancreatic alveolar cells can activate the NFκB signaling pathway.