【作者】 胥伶杰；

【导师】 王仲； 蒋澄宇；

【作者基本信息】 北京协和医学院 ， 急诊医学（专业学位）， 2014， 博士

【摘要】 目的百草枯是一种广泛运用于农业生产中的除草剂。百草枯经口误服或自服中毒后急性起病,无特效解毒剂,救治成功率低,病死率高。我国百草枯中毒致死患者呈逐年急剧递增趋势。国内外研究发现,百草枯能在肺组织中被肺泡I型上皮细胞、Ⅱ型上皮细胞和Clara细胞主动摄取,因此在肺内异常高浓度聚集,肺中浓度可达血浆浓度的6-10倍,该聚集机制与多胺转运系统相关。百草枯吸收后可导致急性呼吸窘迫综合征,并发展为急性呼吸衰竭,后期形成肺纤维化。目前,百草枯致急性呼吸窘迫综合征机制仍不清,且动物模型多采用腹腔注射百草枯建立,与人体经口摄入而中毒的途径不符。氯喹是一种广泛运用于临床的抗疟药,因其免疫调节作用,也被用于治疗免疫系统疾病、恶性肿瘤、病毒感染等疾病中。目前尚无相关研究,探索氯喹是否能用于百草枯中毒的解救。氯喹的治疗作用被认为可能与抑制细胞自噬反应相关。本实验研究采用人肺泡Ⅱ型上皮细胞A549细胞建立百草枯中毒细胞模型,并模拟临床中毒途径,通过百草枯灌胃建立稳定的急性呼吸窘迫综合征小鼠模型。同时,通过体外实验研究氯喹对百草枯攻毒后A549细胞存活率、蛋白表达情况的影响,分析百草枯致急性呼吸窘迫综合征可能的分子生物学机制,探讨自噬反应在百草枯中毒所致肺损伤过程中的作用及其机制。方法(一)百草枯灌胃致小鼠急性呼吸窘迫综合征模型的建立及其致病机制探讨采用不同浓度百草枯(3、10、30、100、150、300mg/kg)灌胃建立小鼠急性呼吸窘迫综合征模型。根据小鼠临床表现,确定建立稳定模型的百草枯浓度。采用该浓度百草枯灌胃建立小鼠模型后,在1d、2d、3d、4d时观察小鼠临床表现、测量肺湿/干比、行HE病理染色以进行研究分析。使用western-blot检测LC-3蛋白表达情况。(二)百草枯攻毒并氯喹解救百草枯攻毒A549细胞模型的建立和分子生物学机制探讨1、采用MTS法检测不同浓度百草枯(0.1、0.3、1、3、10、30、100、300、1000μmol/L)对A549细胞生存率的影响情况,确定能建立稳定细胞模型的百草枯浓度。2、使用不同浓度CQ(O.3、1、3、10、30μmol/L)、3-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)干预PQ100μmol/L浓度攻毒A549细胞,分别于攻毒后12h、24h、48h使用MTS法检测细胞存活率情况。3、采用不同浓度氯喹(0.3、1、3、10、30μmol/L)在PQ100μmol/L浓度攻毒0h、1h、3h、6h后对其进行干预,分别于攻毒后12h、24h、48h使用MTS法检测细胞存活率情况,并使用western-blot检测LC-3、LAMP-1、LAMP-2等蛋白表达情况,并与PQ攻毒组进行对比。结果(一)成功建立百草枯灌胃致小鼠急性呼吸窘迫综合征模型一次性使用百草枯灌胃可以建立小鼠急性呼吸窘迫综合征模型,建立稳定模型的百草枯浓度为300mg/kg。灌胃后,小鼠表现为体重下降,毛发蓬松无光泽,进食水差,呼吸困难,活动困难。肺湿／干比明显升高。肺组织充血出血水肿,广泛炎症细胞浸润、肺泡间隔增宽、肺泡结构破坏表现。PQ攻毒组小鼠LC-3表达含量较对照组有先升高后降低的趋势。(二)成功建立百草枯致A549细胞死亡模型,成功建立氯喹解救百草枯所致的A549细胞死亡模型百草枯能导致A549细胞发生时间、剂量依赖性细胞死亡。能建立稳定细胞模型、且使用药物解救可能有效的百草枯浓度为100μmol/L。经过不同浓度氯喹解救后,百草枯攻毒A549细胞存活率明显升高。各浓度3-MA、 LY294002、wortmannin、necrostatin-1、rolipram处理后细胞存活率无改善。PQ攻毒后A549细胞LC-3表达先下调后上调,LAMP-1、LAMP-2表达量有上调趋势,CQ解救组LC-3、LAMP-1、LAMP-2表达均上调。结论1.百草枯能导致肺泡Ⅱ型上皮细胞死亡和小鼠急性呼吸窘迫综合征。2.百草枯致急性呼吸窘迫综合征过程可能存在自噬反应的参与。3.氯喹能解救百草枯攻毒所致的肺泡Ⅱ型上皮细胞死亡。4.氯喹解救百草枯攻毒所致的肺泡Ⅱ型上皮细胞死亡可能并非经过阻断或抑制自噬反应途径、细胞坏死途径或通过抑制磷酸二酯酶-4(PDE-4)诱导的炎症反应途径而发挥作用,而是通过上调自噬反应或稳定溶酶体膜发生。更多还原

【Abstract】 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.更多还原