The Role Of SOCE In Severe Acute Pancreatitis-associated Acute Lung Injury Of Rats And Rat Pulmonary Microvascular Endothelial Cells

Background: Calcium is an important messenger molecule in a variety of cell life activities, including proliferation, differentiation, gene transcription. Store-operated Ca2+ channels(SOCs) are plasma membrane Ca2+ channels that are activated by a decrease in Ca2+ content in the ER. STIM1, which is an ER membrane protein, is the calcium sensor that transmits ER Ca2+ store depletion signals to the plasma membrane, thus causing the opening of SOCs.Orai1 proteins appear to constitute pore-forming subunits of the SOCs. Store-operated calcium entry(SOCE) is a tightly regulated mechanism for controlling the influx of extracellular Ca2+ into cells to replenish depleted ER Ca2+ stores. Ca2+ is a ubiquitous second messenger; thus, SOCEplays an important role in a variety of cellular processes, in nearly all cell types.Severe acute pancreatitis(SAP) is an acute inflammatory process that is capable of inducing multiple organ system dysfunction syndromes. Acute lung injury(ALI) and its more severe form, acute respiratory distress syndrome(ARDS), are the most common critical complications of SAP-associated multiple organ dysfunction. It has recently been shown that PMVEC injury and apoptosis play a key role in ALI.The aim of this study was to determine whether Ca2+ entry through SOCs is involved in SAP-associated ALI and to investigate the role of SOCE in PMVEC apoptosis induced by SAP-associated ALI.Methods: Thirty male Sprague-Dawley rats were randomly divided into the SAP group(n=10), the 2-APB+SAP group(n=10) and the control group(n=10). The SAP model was established by the retrograde infusion of 1.5% sodium deoxycholate(1 mL/kg). In the 2-APB+SAP group, 2-aminoethoxydiphenyl borate(2-APB), an efficient antagonist of SOCs, was administered intraperitoneally(2.5mg/kg) 24 h before the induction of SAP. Tissue samples were obtained 24 h after sodium deoxycholate injection. Serum levels of amylase, TNF-α, and IL-6, histological scores for the pancreas and the lungs, the water content of the lungs, and the oxygenation index were determined. The protein expression levels of calcium release-activated calcium channel protein1(Orai1) and stromal interaction molecule1(STIM1), the two main molecular constituents of SOCs, were evaluated both in lung tissues and in PMVECs. The mRNA expression levels of Orai1, STIM1, Bax and caspase3 were also evaluated in lung tissues. PMVEC apoptosis and ultrastructural changes in PMVECs were analyzed in lung tissues.PMVECs were transfected with siRNA using lipofectamine 2000. The efficacy of RNA interference was determined with quantitative real-time PCR, PMVECs were divided into control group, LPS group(100 ng/mL), LPS+STIM1 interference group, LPS+Orai1 interference group and LPS+INCA-6 group(1.0uM).Cell viability and cell damage were detected by MTT, the mRNA expression levels of STIM1, Orai1, NFATc3, Bax and caspase3 were also evaluated.Results: Administration of 2-APB reduced the serum levels of amylase, TNF-α, and IL-6, the water content of the lungs, and the pathology injury scores for the lungs and the pancreas, all of which were elevated in the SAP group. 2-APB significantly suppressed Orai1, STIM1, Bax and caspase3 mRNA expression in lung tissues as well as Orai1 and STIM1 protein expression of both in lung tissues and in PMVECs, all of which were elevated in the SAP group. In addition, the decreased oxygenation index, increased level of apoptosis of PMVECs, and ultrastructural damage in PMVECs associated with SAP were ameliorated in the 2-APB+SAP group compared with the SAP group.SiRNA knockdown both of STIM1 and Orai1 resulted in a significant decrease in LPS increased cell viability and NFATc3, Bax and caspase3 mRNA expression, all of which were elevated in the LPS group. In addition, INCA-6, an efficient antagonist of NFATc3, had the same effect.Conclusions: In summary, the present study showed that SOCE may play a critical role in PMVEC apoptosis induced by SAP-associated ALI and that inhibition of SOCs can exert protective effects against SAP-associated ALI. The protective effects of inhibition of SOCs could be mediated by restraining mitochondrial associated apoptosis in NFAT signaling pathway.