Effects And Mechanisms Of Autophagy In Paraquat Induced Acute Respiratory Distress Syndrome C57Mice Model And Paraquat Induced A549Cells Death

PurposeParaquat (PQ) is an organic heterocyclic herbicides which is widely used in agriculture. Nearly all paraquat poisonings resulted from intentional or accidental oral administration. With no specific anti-toxification drug, PQ has a reputation of high mortality. The prevalence of paraquat poisonings has increased dramatically in the recent two decades in China. Uptake by the polyamine uptake system in the alveolar type Ⅱ cells, the Clara cells, and very probably the alveolar type Ⅰ cells, PQ ends to accumulate in the lung tissue and the pulmonary concentration can be6to10times higher than that in the plasma. During the early phase of intoxication, clinical manifestations of the respiratory system mainly include acute respiratory distress syndrome(ARDS), followed by progressive pulmonary fibrosis after acute phase. Nowadays, the exact toxic mechanism remains unclear. What’s more, the establishment of PQ-induced ARDS animal models mostly by intraperitoneal injection, which is not accompanied with oral administration in clinical situations.Chloroquine (CQ), is widely used not only in the prophylactic treatment of malaria, but also in the treatment of rheumatic diseases, carcinomas, inflammations and viral infective diseases. Its therapeutic effect may be related to its inhibition effect to autophagy.In this study, we used A549type Ⅱ-like alveolar epithelial cells to establish cell model, and established PQ-induced ARDS mice model by gavage. Then we evaluated the potential role of CQ in reducing the cytotoxic effect of PQ in the A549cell line, and pursue the molecular mechanisms of PQ-induced ARDS.Methods1. ARDS mice models establishment by PQ gavage Mice poisoning models were established by PQ gavage with varies doses of3、10、30、100.150.300mg/kg respectively. The clinical manifestations, lung wet/dry (W/D), pathological manifestations by HE staining were observed and analyzed on1d.2d、3d and4d. Western-blot was used to detect proteins of LC-3.2. PQ-induced A549cells death model establishmentA549cell viability after exposed to different concentration of PQ (0.1.0.3.1.3.10.30、100.300、1000μmol/L) was measured by MTS assay.3. The rescuing effect of CQ to PQ-treated A549cells models and investigation of its molecular mechanisms.After0h,1h,3h,6h exposed to100μmol/L PQ, A549cells were treated by different concentrations of CQ (0.3,1,3,10,30μmol/L). After12h,24h,48h exposed to100μmol/L PQ, A549cell viability was measured by MTS assay. Western-blot was used to detect proteins of LC-3, LAMP-1, LAMP-2. Then we tried different concentrations of3-MA (0.3,1,3,5mmol/L), LY294002(0.3,1,3,10μmol/L), wortmannin (3,10,30,50,100μmol/L), necrostatin-1(0.1,0.3,1,3,10μmol/L), rolipram (1,3,10,30,100μmol/L) when0h exposed to100μmol/L PQ, after12h,24h,48h exposed to100μmol/L PQ, A549cell viability was measured by MTS assay.Results1. Successfully established ARDS mice models by PQ gavageThe stable ALI mice models can be successfully established by single dose of PQ gavage, with the concentration of300mg/kg. Clinical manifestations included weight loss, poor water and food ingestion, difficulty in breathing, limitation of motion. Lung W/D was increased dramatically. Congestion, edema, hemorrhage, alveolar structure damage, inflammation cells infiltration of lung tissue were observed. There is a slightly increasing trend followed by downward tendency of LC-3expression in lung tissue.2. Successfully established PQ-induced A549cells death modelWhen the concentration is≥100μmol/L, PQ causes A549cell death, with both concentration-dependence and time-dependence. 3. Successfully established CQ rescue PQ-induced A549cells death modelCQ can ameliorate PQ induced cell death in a dose-dependent manner.3-MA, wortmannin, LY294002, necrostatin-1and rolipram were not able to ameliorate A549cells death caused by PQ at all concentration levels. The LC-3expression increased after an initial decline, while both of LAMP-1and LAMP-2showed upward trend in PQ-treated cell line. Three mentioned proteins expression elevated after rescued by CQ.Conclusions1. PQ can induce A549cell death and ARDS to mice.2. Autophagy may play a role in PQ-induced ARDS.3. CQ can ameliorate PQ induced cell death.4. CQ ameliorated PQ-induced A549cells death not though inhibition of autophagy, necrosis or phosphodiesterase-4(PDE-4) induced inflammation, but through activation of autophagy, protection of lysosome membrane or some unknown molecular mechanisms.