Experimental Study On Inhibition Of NO In Process Of Glycochenodeoxycholate Inducing A549Cell Apoptosis

Background and ObjectiveNeonatal respiratory distress syndrome (NRDS) was also known as hyaline membranedisease (hyaline membrane disease, HMD), caused by lack of pulmonary surfactant (pulmonarysurfactant, PS).The condition of respiratory distress was progressively increased soon afterbirth. Prone to the premature infants, the smaller the gestational age, the higher the incidence ofcommon emergency. NRDS was a common severe disease in neonatal intensive care unit. Alarge number of clinical cases have shown that the pregnant women who had intrahepaticcholestasis of pregnancy (ICP) would give the birth to neonates with NRDS, with the incidenceof1times higher than normal newborns. Bile acid has been suggested to be the mostdisease-causing critical damage factor. There have been a few clinical reports on the relationshipbetween ICP and NRDS, but lack of pathogenesis would lead to difficulties in the preventionand treatment of ICP related NRDS. More studies are needed to investigate the pathogenesisand relationship between ICP and NRDS.Alveolar type2epithelium,(AECⅡ) has been found to be the main place of secretionsynthetic pulmonary surfactant. The apoptosis of AECII could directly cause inadequatesecretion of PS, leading to alveolar collapse, atrophy, and finally NRDS. The fetal AECIIproliferated and differentiated in the amniotic fluid infiltration for a long time. It has beensupposed that bile acid induced AECII apoptosis might be one of the reasons of neonatalNRDS. Previous researches have suggested that cholic acid was the richest bile acid, whichcould induce apoptosis of A549cells. Whether glycyl chenodeoxycholic acid wouldinfluence the AECII apoptotic necrosis and reduce alveolar AECII synthetic lung surfactantsynthesis need further studied.In recent years, nitric oxide (NO) has been widely used in NICU and has achieved insome fatal diseases such as neonatal pulmonary hypertension, bronchopulmonary dysplasiaamong premature infants, and congenital heart disease. NO is a soluble gas, which relies on the L-arginine nitric oxide synthase catalyzed. Under the pathological conditions, induciblenitric oxide synthase (iNOS) may be the key meaningful reason to produce NO.Multi-centered study has found that NO inhalation therapy could reduce the utilization ofexogenous PS and high-frequency mechanical ventilation among newborns with severerespiratory failure. More ever, it has been found that apoptosis of ACEⅡ in the course ofNRDS had a deep influence on respiratory failure.MethodsSection1：The pancreatic enzyme digestion type training alveolar Ⅱ epithelial cellswas used.0.25%trypsinization lung tissue contains10%fetal calf serum high glucosemedium could satisfy the need of in vitro studies of alveolar type II epithelial cell function.Section2：Rat model of IPC by the injection of estrogen was established. Pregnant ratshad been injected with the concentration of2.5mg/kg body weight estradiol at the16th dayof pregnancy, which could make an animal model of intrahepatic cholestasis of pregnancylevy.Section3：The experiment using lung cancer epithelial cell line A549and adoptingimmunofluorescence experimental method was to prove that glycodeoxycholic acid inducedA549cell apoptosis might rely on caspase-3way apoptosis. Western Blot method was usedto test iNOS expression level. In the model of inducing A549cell apoptosis by GCDCA,changes of expression of iNOS that might be caused by GCDC concentration and the effectof exogenous NO on cell apoptosis were investigated.Results1.0.25%trypsinization lung tissue contains10%fetal calf serum high glucose mediumcould satisfy the need of in vitro studies of alveolar type II epithelial cell function.2. Pregnant rats had been injected with the concentration of2.5mg/kg body weightestradiol at the16th day of pregnancy, which could make an animal model of intrahepaticcholestasis of pregnancy levy.3. After GCDC processing, A549could rely on caspase-3way apoptosis. GCDC couldrestrain the expression level of iNOS in cells and GCDC concentration was positivelyrelated with restraining iNOS expression level. When dealing with A549singly by NO donor SNAP0.2mmol, apoptosis would not occur to cells, but, after dealing with A549bySNAP and GCDC, the apoptosis degree of cells would decrease. Cholic acid could inducethe apoptosis of A549cells, furthermore, glycyl chenodeoxycholic acid has been confirmedto induce A549cells apoptosis relying on classic capease-3apoptosis pathway, and NOcould inhibit the glycocholic chenodeoxycholic acid-induced apoptosis of A549cells.Conclusion:Cholic acid could induce the apoptosis of A549cells, furthermore, glycylchenodeoxycholic acid has been confirmed to induce A549cells apoptosis relying on classiccapease-3apoptosis pathway, and NO could inhibit the glycocholic chenodeoxycholic acid-induced apoptosis of A549cells.