A breakthrough in paraquat-induced pulmonary fibrosis

首先，我们研究了一氧化氮（NO）和谷胱甘肽（GSH）水平对暴露于PQ的A549细胞和大鼠器官PQ毒性的影响。我们的体外模型表明，根据产生的浓度和使用的模型系统，NO显示出有益和有害的作用。在体内模型中，我们检测到肝脏组织iNOS mRNA水平和硝基酪氨酸沉积显著增加，而肺组织中细胞因子基因表达增加，提示PQ暴露在不同器官中的中毒机制不同。我们发现谷胱甘肽水平、谷胱甘肽合成限速酶的活性和表达水平明显增加；γ-谷氨酰半胱氨酸合成酶（γ-GCS）和γ-谷氨酰转肽酶（γ-GT）。PQ孵育后，A549细胞GSH水平略有升高。接下来，通过分析胆固醇衍生的氢过氧化物，我们发现低剂量PQ暴露的大鼠肝脏和肾脏中有7 α-OOH和7 β-OOH的积累，特别是它们在肾脏病理中反映了更大的氧化应激。最后，为了鉴定PQ引起的氧化应激反应中差异表达的基因，我们使用20 mg/kg PQ处理3小时的大鼠肺部的cdna进行了基于RT-PCR的差异显示实验。大量cDNA条带被鉴定为上调和下调；少数克隆与已知基因同源，其余克隆为未知基因。对未知的6个不同的cDNA克隆进行了分离和测序。利用RT-PCR方法验证了这些克隆的mRNA表达差异。这些结果可能有助于阐明PQ摄入引起的肺纤维化过程中的分子变化。

First, we studied the effect of nitric oxide (NO) and glutathione (GSH) levels on PQ toxicity in A549 cells and rat organs exposed to PQ.Our in vitro models demonstrated that NO showed both beneficial and deleterious actions, depending on the concentrations produced and model system used. We detected, in vivo models, a remarkable increase in iNOS mRNA level and deposit of nitrotyrosine in liver tissues, whereas gene expressions for cytokines increased in lung tissues, suggesting that the mechanisms of PQ poisoning are different among organs exposed to PQ.We found a specific increase in GSH levels, the activities and the expression levels of the rate-limiting enzymes in GSH synthesis ; γ-glutamylcysteine synthetase (γ-GCS) and γ-glutamyl transpeptidase (γ-GT) in lungs exposed to PQ.Also, the GSH levels in A549 cells slightly increased by incubation with PQ.Next, by analyzing cholesterol-derived hydroperoxide, we found 7 α-OOH and 7 β-OOH accumulations in rat liver and kidney exposed to low dose of PQ, especially they reflected greater oxidative stress in the pathology of kidney. Finally, to identify genes that are differentially expressed in response to oxidative stress due to PQ, we carried out RT-PCR based differential display experiments using cDNAs from rat lungs that were treated for 3 hrs with 20 mg/kg PQ.Numerous cDNA bands were identified that were up-regulated and down-regulated ; a few clone were homologous to known genes and the remainders were unknown. Unknown six different cDNA clones were isolated and sequenced. The differential mRNA expression of these clones could be verified using RT-PCR method. These results may prove useful in elucidating the molecular changes during pulmonary fibrosis evoked by PQ intake.