5-氨基水杨酸对急性百草枯中毒治疗作用的实验研究
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摘要
百草枯（paraquat, PQ），又名克芜综或对草快，其化学名称为1-1-二甲基-4-4-联吡啶阳离子盐，是一种广谱、高效、环境污染较小的除草剂，在全球140余个国家得到了广泛应用。但是，伴随PQ的应用所产生的中毒事件常见报道。近年来，我国因PQ中毒或死亡事件呈现上升趋势。PQ中毒已经成为急诊医学研究领域所面临的重大问题之一。
PQ可经胃肠道、皮肤、呼吸道和腹腔等吸收，引起肺、肝、肾、心以及中枢神经系统等多脏器损伤，其中肺脏是PQ中毒的最重要的靶器官，因肺内存在聚胺类物质摄取系统使得PQ在肺组织中异常聚集而引起严重的肺损伤，主要表现为间质内水肿、白细胞浸润、肺泡充血，成纤维细胞增殖以及胶原沉积增加。上述损伤最终可能导致死亡，存活者可遗留长期的肺功能障碍。因PQ作用机制复杂且不明了，故目前尚无特效解毒药，临床上对百草枯的治疗困难重重，疗效多不佳。因此，深入探讨其中毒机制及治疗策略，尤其是针对其引起的肺损伤的研究是中毒领域研究中的热点和难点。
在由多种细胞因子以及信号转导通路形成的参与肺损伤及纤维化的网络系统中，TGF-β1/Smads是重要的调节因子，有研究提出针对TGF-β信号系统进行治疗有望缓解PQ引起的脏器的纤维化。近年来研究表明，PPARγ激动剂可以阻断TGF-β信号传导通路，进而抑制主要脏器的慢性纤维化进程。5-氨基水杨酸（5-ASA）是最经典的抗炎剂，可以作为PPARγ的配体直接结合并激活PPARγ，进而影响TGF-β信号通路活性而发挥作用。目前有关PPARγ在百草枯引起的肺损伤中所起的作用，以及5-ASA在PQ中毒的治疗作用少见报道。
鉴于此，本研究通过建立PQ中毒大鼠模型，从整体水平上观察PQ对大鼠急性肺脏损伤及氧化应激的作用；PQ对大鼠肺组织纤维化以及PPARγ、TGF-β1、P-Smad3的表达的影响；同时观察5-ASA在上述过程中的治疗作用。在此基础上，进一步通过体外实验观察5-ASA在PQ对WI38VA13细胞PPARγ、TGF-β1、P-Smad3的表达的影响。旨在揭示PPARγ在百草枯引起的急性肺损伤及肺纤维化过程中的作用以及5-ASA对百草枯中毒的治疗作用。
第一部分5-ASA对PQ染毒大鼠肺组织的保护性抗损伤作用
目的：探讨5-ASA对PQ染毒大鼠肺组织的炎性损伤是否有保护作用。
方法：成年雄性Wister大鼠共125只，随机分为5组。对照组（空白对照组，Control组）给予蒸馏水灌胃，5-ASA组按75mg/kg灌胃，PQ组按80mg/kg灌胃，PQ+30mg5-ASA组按PQ80mg/kg+5-ASA30mg/kg灌胃，PQ+75mg5-ASA组按PQ80mg/kg+5-ASA75mg/kg灌胃。分别于试验开始后24h、72h、7d、14d、28d分批处死动物，每组5只，留取静脉血及肺组织待检。计算肺系数，测定血清中SOD、GSH、MDA含量。同时留取部分肺组织做病理切片，行HE染色后在光学显微镜下观察肺组织病理改变。
结果：
1染毒及干预后各组大鼠的一般状况观察
PQ组大鼠在染毒后2h即可出现明显的中毒表现，且在1~3天最明显，具体表现为：①一般情况：精神较差、倦怠、嗜睡、烦躁、易激惹、口鼻及眼周血性分泌物、竖毛、体重减轻、下降程度最大的一只达46g，进食水明显减少；②呼吸系统表现：呼吸急促、呼吸困难、腹式呼吸、口周紫绀，严重者可以听到喘鸣音；③消化系统：厌食水，稀便等；④泌尿系统：少尿或无尿，血尿等。PQ+30mg5-ASA组和PQ+75mg5-ASA组（染毒+治疗组）：大鼠经5-ASA治疗后，中毒表现较PQ组为轻，但不同剂量5-ASA两个治疗组之间没有明显差别。
2染毒及干预后各组大鼠的大体病理学变化观察
PQ组染毒早期（24h、72h、7d）可见双肺体积明显增大、充血、水肿、有点片状出血，部分大鼠胸腔有血性渗出物，胃肠道可见明显胀气、充血，粘膜可见散在出血点及溃疡；肝脏充血、肿胀，双肾肿大，颜色紫红，被膜紧张，偶可见出血点。染毒晚期（14d,28d）可见双肺表面不平，散在陈旧出血点，有点片状白色，偏僵硬感。PQ+30mg5-ASA组和PQ+75mg5-ASA组染毒早期各脏器病变表现均较PQ组为轻，双肺出血不明显，晚期双肺表面较光滑，有点片状白色区域，但明显少于PQ组。两个治疗组区别不明显。
综上可见5-ASA治疗可以改善PQ中毒引起的大体病理变化，且两种剂量无明显差别。
3相对体重的比较
给予75mg5-ASA组各实验动物相对体重随实验时间逐渐上升，与对照组相比无明显差异（P>0.05）。 PQ染毒后实验动物体重下降，明显低于对照组，72h时达到高峰（P<0.05），染毒14d时恢复至与对照组相同水平（P>0.05）。PQ+30mg和75mg5-ASA组动物72小时内相对体重也有下降，明显低于对照组和5-ASA组（P<0.05）；但7d时即可恢复至对照组和5-ASA组水平（P>0.05）。提示5-ASA治疗可以减轻PQ中毒对大鼠体重的影响，侧面反映其可减轻PQ的毒性反应。
4肺系数的比较
5-ASA组动物处理各时间段肺系数无明显变化，与对照组无明显差异（P>0.05）。PQ染毒后肺系数明显上升，至第7d升至高峰，14天时较第7d下降，但28d时再次升高，呈双相升高表现。各时间段肺系数均明显高于对照组和5-ASA组（P<0.05）。PQ+30mg5-ASA组和PQ+75mg5-ASA组动物24h和72h肺系数与对照组和5-ASA组相比均有明显增加，尤以72h时升高明显（P<0.05），但两个治疗组24h和72h肺系数及其后各时间点肺系数均明显低于PQ组（P<0.05），且恢复至与对照组和5-ASA组同一水平的时间均明显短于PQ组（7d vs28d）。但两治疗组之间相比无明显差异（P>0.05）。
综上表明，5-ASA治疗组肺系数明显较中毒组轻，说明5-ASA在一定程度上减轻了PQ中毒引起的充血水肿。
5血清学指标测定结果
5-ASA组各时间点血清SOD、GSH、MDA含量与对照组相比无明显差异（P>0.05），染毒后PQ组血清SOD、GSH活力明显下降，SOD活力在7d前均明显低于对照组和5-ASA组（P<0.05），以72h降低最明显，GSH含量在14d前均明显低于对照组和5-ASA组（P<0.05），以14d时含量最低，MDA含量则在染毒后呈上升趋势，7d前均较对照组和5-ASA组明显升高（P<0.05），其余时间点三个血清学指标均与对照组和5-ASA组相比无明显差异（P>0.05）。而两个治疗组血清学指标变化趋势与PQ组相一致，但程度均低于PQ组，SOD活力仅在72h时下降，与PQ组，对照组和5-ASA组相比均有显著差异（P<0.05），GSH含量仅24h时与对照组和5-ASA组相比有显著差异（P<0.05），24h～14d内均明显高于PQ组（P<0.05）；MDA含量在72h后逐渐下降，PQ+30mg5-ASA组在各时间段与对照组和5-ASA组相比均无显著差异（P>0.05），72h和7d时明显低于PQ组（P<0.05）； PQ+75mg5-ASA组在24h时明显高于对照组和5-ASA组（P<0.05），其在各时间段与PQ组相比均无显著差异（P>0.05）。结果表明PQ中毒早期会出现SOD、GSH含量下降和MDA含量上升，5-ASA治疗使这些变化在持续时间和程度上都有所减轻。
6肺组织病理改变（HE染色）
5-ASA组动物肺组织结构与对照组无明显差异，镜下可见肺泡结构清晰，肺泡壁薄，肺泡间隔无增宽及充血，肺泡腔无炎性细胞浸润及出血现象。PQ染毒后实验动物肺组织出现明显肺泡炎改变，24h可见肺内炎性细胞浸润，肺泡壁毛细血管扩张、瘀血，气管及血管周围有不同程度的水肿，72h、7d时肺间质和肺泡水肿继续进展，7d时达高峰，病变范围超过75%，肺泡间隔充血、增厚，肺泡腔变窄，部分肺泡腔充满粉染均质水肿液，可伴广泛血管内皮细胞损伤，弥漫性肺出血，部分有透明膜形成，肺泡腔内有游离的中性粒细胞和巨噬细胞，14d和28d炎症改变逐渐减轻，炎性细胞浸润以巨噬细胞、中性粒细胞为主，并有少量淋巴细胞，仍有出血及水肿，肺间隔明显增宽，组织增生明显，可见胶原纤维增生。
PQ+30mg5-ASA组和PQ+75mg5-ASA组实验动物肺组织病变较轻，肺泡内充血水肿和炎性细胞浸润程度低于PQ组，病变范围低于60%，后期胶原纤维增生亦较PQ组为轻。
提示：5-ASA的治疗减轻了PQ中毒引起的肺的早期以肺泡炎和晚期以增生和纤维化为主要表现的病理变化。
第二部分5-ASA对PQ引起的肺纤维化的保护作用
目的：TGF-β1/Smads信号途径是肺纤维化发生的关键环节，本部分研究目的旨在探讨作为PPARγ激动剂，5-ASA是否能通过抑制TGF-β1/Smads信号途径，进而减轻PQ中毒引起的肺纤维化。
方法：实验分组与处理同第一部分，留取肺组织测HYP含量，肺组织进行病理形态学观察及VG染色。免疫组化法检测PPARγ、TGF-β1在肺中的表达，蛋白免疫印迹测定P-smad3水平。
结果：
1肺组织HYP含量测定结果
5-ASA组实验动物各时间点肺组织HYP水平与对照组无明显差异（P>0.05）。PQ组于染毒后第7d实验动物肺组织HYP含量开始升高，7d、14d、28d均显著高于对照组和5-ASA组（P<0.05）。给予5-ASA治疗后实验动物肺组织HYP水平升高时间延后至第14d，与对照组和5-ASA组相比14d和28d时肺组织HYP含量增高，但两组动物肺组织HYP水平均明显均低于PQ组。结果表明HYP含量升高发生在PQ中毒后期，5-ASA的治疗使其升高的时间延后，程度减轻。
2Van Gieson胶原纤维染色
Van Gieson胶原纤维染色可见对照组和5-ASA组肺组织仅在大支气管壁、血管周围及肺泡间隔区见少量红色的胶原纤维。PQ染毒早期（24h、72h、7d）实验动物肺组织未见明显胶原纤维增生，但染毒14d以后胶原纤维着色明显增强，血管壁与支气管壁的内皮下与肌层下均有红染的胶原纤维出现，肺泡间隔呈纤维性增厚，部分区域胶原纤维不规则排列。
PQ+30mg5-ASA组和PQ+75mg5-ASA组动物染毒24h至14d时未见明显胶原纤维增生；仅在染毒28d时可见胶原纤维数量多于对照组，但较染毒组明显减轻，有部分肺泡间隔增厚。结果提示5-ASA的治疗减少了胶原纤维的合成。
3PPARγ和TGF-β1的免疫组织化学结果
3.1PPARγ在大鼠肺组织中的表达与分布
免疫组化染色结果表明，PPARγ免疫反应产物主要定位于细胞核，呈棕黄色颗粒状，胞质很少着色，正常组实验动物肺组织内可见PPARγ表达阳性的血管内皮细胞、肺泡上皮细胞和巨噬细胞。给予5-ASA后，实验动物肺组织PPARγ阳性表达明显增强，7天时达高峰（1.61±0.06），与用药时间相一致，较其他组明显升高（P<0.05）。14天后PPARγ表达水平下降，恢复至对照组实验动物水平（P>0.05）。PQ染毒后肺组织内PPARγ表达阳性的血管内皮细胞、肺泡上皮细胞和巨噬细胞均明显减少，尤以PQ染毒7d时最明显（0.44±0.07），14d之前与对照组相比均有显著差异（P<0.05）。至染毒28d时恢复至正常水平。5-ASA治疗组实验动物肺组织PPARγ表达阳性的细胞较对照组减少，但较PQ组多，如7d时染色指数分别为0.59±0.07、0.61±0.06，在14d之前与两组相比均有明显差异（P<0.05）。28d时恢复至正常水平。提示PQ使PPARγ表达下调，而5-ASA使这种作用减弱。
3.2TGF-β1在大鼠肺组织中的表达与分布
免疫组化染色结果表明，TGF-β1表达定位于细胞胞浆，主要表达于支气管上皮细胞、平滑肌细胞、成纤维细胞及散在的炎性细胞。PQ组实验动物染毒后TGF-β1表达明显增强，至14d时达高峰（0.976±0.063），各时间点染色指数均明显高于对照组（P<0.05）。28d时恢复至正常水平。
PQ+30mg5-ASA组和PQ+75mg5-ASA组：TGF-β1表达于染毒后增多，但染色指数介于对照组和PQ组之间，于7d时达高峰（0.557±0.078，0.612±0.056），与前两组相比均有显著差异（P<0.05）。28d时回落至正常。
由上可见5-ASA的治疗抑制了由于PQ中毒引起的肺内TGF-β1的表达上调。
4肺组织中P-smad3的免疫蛋白印迹结果
5-ASA组动物肺组织p-smad3蛋白表达在各时间段无明显变化，与对照组相比无明显差异（P>0.05）。PQ染毒后P-smad3蛋白表达逐渐上升，14d时达高峰（0.688±0.065），28d时下降，但仍高于正常水平，各时间点P-smad3蛋白表达均明显高于对照组（P<0.05）。PQ+30mg5-ASA组和PQ+75mg5-ASA组：P-smad3蛋白表达水平于染毒后升高，但介于对照组和PQ组之间，7d时达高峰（0.349±0.021,0.347±0.027），之后下降，14d前与两组差异均有意义（P<0.05）。28d时回落至正常水平（P>0.05）。结果表明PQ染毒后肺内smad3蛋白磷酸化水平上升，5-ASA的应用抑制了PQ的这种作用。
第三部分5-ASA对WI38VA13细胞PPARγ、TGF-β1、P-Smad3的表达的影响
目的：了解PQ是否对于成纤维细胞的TGF-β1、P-Smad3的分泌产生影响，及应用5-ASA后是否对TGF-β1/Smads信号通路的传导有调控作用。
方法：用MTT法确定试验用PQ浓度，Western Blot方法检测PQ、5-ASA、PQ+5-ASA处理WI38VA13细胞后，在12h、24h和48h时细胞中PPARγ、TGF-β1、P-Smad3含量。
结果：
1PQ对WI38VA13细胞存活率的影响
PQ对WI38VA13细胞的生长有明显的抑制作用。经PQ处理24h后，200、250、300、400、600、800、1000μmol/L PQ处理组WI38VA13细胞存活率均明显低于对照组（P<0.05）。在200-1000μmol/L浓度范围内，随着PQ浓度的升高，WI38VA13细胞的存活率明显降低。
25-ASA对PQ作用下WI38VA13细胞中PPARγ、TGF-β1、P-Smad3的影响
2.15-ASA对PQ作用下WI38VA13细胞PPARγ表达的影响
Western Blot检测结果显示，给予5-ASA处理后，WI38VA13细胞中PPARγ蛋白表达与对照组相比增强，各时间组与对照组相比均有明显差异（P<0.05）。单纯给予PQ处理，WI38VA13细胞中PPARγ蛋白表达与对照组相比明显下降，并随时间延长递减，各时间组与对照组相比均有明显差异（P<0.05）。5-ASA与PQ共同处理组，WI38VA13细胞中PPARγ蛋白与对照组相比表达下降，但介于5-ASA组与PQ组之间，与两组相比均有统计学意义（P<0.05）。提示PQ下调了WI38VA13细胞中PPARγ蛋白的表达，而5-ASA则有上调作用，共同作用下，5-ASA可以部分的逆转PQ对WI38VA13细胞中PPARγ蛋白表达的下调作用。
2.25-ASA对PQ作用下WI38VA13细胞TGF-β1表达的影响
Western Blot检测结果显示，给予5-ASA处理后，WI38VA13细胞中TGF-β1蛋白表达与对照组相比无明显差异（P>0.05），PQ处理后，TGF-β1蛋白表达与对照组相比明显增强，并随时间延长递增，与对照组相比有明显差异（P<0.05）。5-ASA与PQ共同处理组，TGF-β1蛋白表达逐渐增强，数值介于PQ组和对照组之间，与两组相比均有明显差异（P<0.05）。提示PQ激活WI38VA13细胞中TGF-β1蛋白表达，5-ASA可以部分逆转PQ对WI38VA13细胞中TGF-β1蛋白表达的上调作用。
2.35-ASA对PQ作用下WI38VA13细胞P-Smad3表达的影响
Western Blot检测结果显示，给予5-ASA处理后，WI38VA13细胞中Smad3蛋白磷酸化水平与对照组相比无明显差异（P>0.05），PQ处理后，Smad3蛋白磷酸化水平与对照组相比明显增强，并随时间延长递增，与对照组相比有明显差异（P<0.05）。5-ASA与PQ共同处理组，Smad3蛋白磷酸化逐渐增强，数值介于PQ组和对照组之间，与两组相比均有明显差异（P<0.05）。提示PQ作用后，WI38VA13细胞中Smad3蛋白磷酸化水平上调，5-ASA可以部分逆转PQ对WI38VA13细胞中Smad3蛋白磷酸化水平的上调作用。
结论：
1PQ灌胃染毒是建立急性肺损伤和肺纤维化动物模型的可靠方法，为研究PQ中毒机制及药物疗效奠定了基础。
2氧自由基的产生在PQ中毒所致肺损伤中起重要作用，表现为染毒后脂质过氧化产物MDA含量的升高，抗氧化物质SOD、GSH含量的下降。
3TGF-β1/Smads信号转导通路在PQ引起的大鼠肺纤维化中发挥重要作用。
4PQ处理可以引起WI38VA13细胞TGF-β1以及P-Smad3表达增强。
55-ASA有抗自由基及减轻脂质过氧化作用，对PQ引起的肺损伤有一定的保护性抗炎作用。
65-ASA作为PPARγ配体，通过干预TGF-β1/Smads信号通路一定程度上缓解PQ导致的肺纤维化。
75-ASA可以逆转PQ引起的细胞中PPARγ的表达下调，同时使TGF-β1/Smads信号通路受到抑制。
Paraquat dichloride (1,1’-dimethyl-4,4’-bipyridilium dichloride; methylviologen, PQ) is an effective and widely used herbicide in most countries. Theintentional and accidental ingestion of commercial liquid formulations ofparaquat has caused a large number of human fatalities. It has become one ofthe most important medical and social problems which can not be ignored inour country.
The toxicity of PQ was first described by Clark. It can cause injury tomany organs, including liver, kidney, thymus and lung. Most importantly, lungtissue is the major targeted organ for paraquat toxicity in rat and man, becauseit has active polyamine uptake transport systems that concentrate paraquatrapidly into the type II epithelial cells of the alveoli. The toxic effects ofparaquat on the lungs result in pulmonary edema, hypoxia, respiratory failure,and pulmonary fibrosis. Survivors of paraquat poisoning may be left with arestrictive type of long-term pulmonary dysfunction. Because of themechanism is complex and not fully understood, so the treatment of paraquatpoisoning is difficulty. So the mechanism of paraquat toxicity and treatment,especially for the study of lung injury caused by PQ, is hotspot and difficultyin the field of poisoning.
Transforming growth factor-β1(TGF-β1) is a key growth factor thatinitiates tissue repair and its sustained production underlies the developmentof tissue fibrosis. TGF-β signals are mediated by a family of Smad proteins.Inrecent years many studies have demonstrated that PPAR agonists could inhibitthe TGF-β signaling pathway, thereby inhibiting chronic fibrosis in majororgans. The effect of PPARγ in PQ-induced lung injure is seldom reported.Over the past decade,5-ASA has been first-line therapy for mild to moderateactive IBD. Recent researchs have demonstrated that, as a ligand for PPARγ, 5-ASA was found to reduce TGF-β1signalling as judged by the reduction inTGF-β specific reporter gene activity.
In this study, we aimed to investigate whether PPARγ played a regulatoryrole in paraquat induced acute lung injury and pulmonary fibrosis both in vitroand in vivo.
PartⅠThe protective anti-inflammatory effects of5-ASA on lungtissue of rats with PQ poisoning.
Objective: To explor whether5-ASA has therapeutic effect to PQ-induced lung injure of rat.
Methods:125adult healthy Wister male rats were randomly distribu-tioned to five groups.(1) Control group, n=25: animals were intragastricadministered with distilled water;(2)5-ASA group, n=25: animals were intra-gastric administered with5-ASA(75mg/kg);(3) PQ group, n=25: animalswere intragastric administered with PQ(80mg/kg);(4) PQ+30mg5-ASA group,n=25, animals were intragastric administered with PQ(80mg/kg)+5-ASA(30mg/kg);(5) PQ+75mg5-ASA group, n=25, animals were intragastric adminis-tered with PQ(80mg/kg)+5-ASA(75mg/kg). At24h,72h,7d,14d,28day ratswere sacrificed, venous blood were collected to detect SOD, GSH andMDA.Weight for lung coefficient. Optical microscope was performed toexamine pathological changes in lung tissues with HE.
Results:
1The general condition of each group
Intoxication manifestations occurred in rats only2hours after PQ ad-ministered, and were especially severe during the first three days. Intoxicationinvolves a combination of signs and symptoms including lethargy, hypoxia,dyspnea, tachyeardia, hyperpnea, hyperexcitability, convulsions, unhairing,nose and/or eyes bleeding, anorexia, diarrhea, oliguria, hematuria and markedlosses in body weight. In5-ASA treatment group, intoxication manifestationswere lighter than PQ group.
2Each group’s gross pathology changes
In PQ group, double lung’s volume increased significantly at the early stage(24h,72h,7d). Hyperemia, edema, hemorrhage could be observed.Thereis hemorrhagic exudate in rat pleural. Gastrointestinal obvious flatulence,hyperemia, mucous membrane can be seen ulcers; liver congestion, swelling,bilateral renal enlargement, the color is purple, film tension, even visiblebleeding. In the late stage(14d,28d), double lung’s surface uneven, oldhaemorrhage dot could be seen.In PQ+30mg5-ASA group and PQ+75mg5-ASA group, each organ manifestations were lighter than PQ group. Doublelung volume increases, congestion and edema, but no obvious bleeding inearly period. Lung surface is smooth，there is a bit flaky white on it, less thanthat of PQ group.
The PQ-induced pathological changes could be improved by5-ASA.There was no significant difference between the two treatment doses.
3Comparison of the relative weight
Relative body weight gradually increases with time in5-ASA group. Ithas no change compared with the Control group(P>0.05). After the PQexposure, the relative weight decreased to minimum at72h(P<0.05), thengradually increased to the control level at14d(P>0.05). In PQ+30mg5-ASAgroup and PQ+75mg5-ASA group, relative body weight decreased to aminimum at72h. It had statistical significance when compared with controlgroup and5-ASA group at72h(P <0.05).So the PQ-induced relative bodyweight decrease could be improved by5-ASA.
4Comparison of the lung coefficient
The lung coefficient did not change at all time points in5-ASA group. Ithad no statistical significance compared with control group(P>0.05).In PQgroup, the lung coefficient increased significantly after exposure, it rosed tothe highest position at7d, then decreased at14d, increased again at28d. Eachdata had statistical significance compared with control group(P<0.05). InPQ+30mg5-ASA group and PQ+75mg5-ASA group, The lung coefficientincreased after exposure. It had statistical significance when compared withcontrol group and5-ASA group at24h,72h(P<0.05). But it was significantlylower than PQ group at all time points(P<0.05). There was no difference between two treatment group(P>0.05).
The lung coefficients of5-ASA treatment group were lower than that ofthe PQ group. Which indicated that5-ASA reduced congestion and edemacaused by PQ poisoning.
5Serum measurement
In5-ASA group, the activities of serum SOD, GSH, MDA have nosignificantly difference compared with the control group(P>0.05). In PQgroup, the activities of serum SOD and GSH significantly decreased afterexposure. SOD down to the lowest point at72h, then began to increase. It hadstatistical significance when compared with control group and5-ASA group at24h,72h and7d(P<0.05). GSH were significantly lower than control groupand5-ASA group. The lowest point was at14d(P<0.05). The content of MDAin serum increased after exposure. It was much higher than control group and5-ASA group before7d(P<0.05). These three serum index had no differencecompared with control group and5-ASA group at other time points(P>0.05).The three serum index had a similar changing trend to PQ group in the twotreatment groups. But the level was lower than that of PQ group.SOD onlydecreased at72h(P<0.05).GSH had significantly difference with control and5-ASA groups only at24h(P<0.05). it was much higher than PQ group from24h to14d. The content of MDA in serum of PQ+30mg5-ASA group has nochange compared with the control group and5-ASA group at eachtime(P>0.05), it was lower than PQ group at72h and7d(P<0.05).There wasdifference between PQ+75mg5-ASA group and control group at24h(P<0.05).There was no difference between PQ+75mg5-ASA group and PQ group ateach time. The reduction of SOD and GSH activity and the increased of MDAoccurred in the early. Therapy with5-ASA reduced these changes in bothduration and extent.
6Histological changes: HE staining
The lung structure of5-ASA group had no significantly difference withthat of control group. The normal alveolar wall was thin and clear, with noinfiltration of inflammatory cells in the lumen of alveoluses or oceasionally only a few erythrocytes. In PQ group, there were alveolitis occurred in lungafter exposure.We can see inflammatory cell infiltration, telangiectasis inalveolar, pulmonary congestion, edema around the trachea and vessels at24h.The edema continued to increase, reached a peak at7d. Alveolar septalhyperemia, thickening, alveolar lumen narrowed, diffuse pulmonaryhemorrhage, hyaline membrane formation could be seen in72h and7d; At thelater stage, we also observed proliferation of collagen fibers, fibrousthickening of the alveolar wall and fibroblastial hyperplasia in differentdegrees.
PQ+30mg5-ASA group and PQ+75mg5-ASA group:5-ASA reducedthe pulmonary pathological changes caused by PQ.
PartⅡ Protective effect of5-ASA on PQ induced pulmonary fibrosis
Objective: TGF-β1/Smads Signaling pathway is a key link of pulmonaryfibrosis
To explore as a PPARγ agonist, whether5-ASA can inhibitTGF-β1/Smad3signal pathway, then reduce pulmonary fibrosis induced by PQpoisoning.
Methods: The experimental group and treatment same with the part I.Lung homogenate to detect HYP. Optical microscope was performed toexamine pathological changes in lung tissues with VG staining. To detect theexpress of PPARγ, TGF-β1in lung tissues by immunohistochemical staining.To detect the express of P-smad3by Western Blot.
Results:
1the content of HYP in lung tissue
The content of HYP in lung tissue of5-ASA group had no changecompared with that of the control group(P>0.05). In PQ group, HYP had asignificantly increased after the7thday. It was much higher than control groupand5-ASA group at7d,14d and28d(P<0.05). The lever of HYP were higherthan control group in the two5-ASA treatment groups at14d and28d(P<0.05),but they were also lower than PQ group(P<0.05).
2Van Gieson stainingSurrounding blood vessels and alveolar septal area,we could see little redcollagen fibers in bronchial wall in control group and5-ASA group. In PQgroup, collagen fiber hyperplasia was not obvious in24h,72h and7d. Afterthe14thday, fibrous thickening occurred in bronchia walls and the alveolusinterspacea, in some areas with an irregular collocation of collagen fibres.PQ+30mg5-ASA group and PQ+75mg5-ASA group: Treated with5-ASA, collagen fiber hyperplasia was not obvious in24h to14d. Collagenfiber was higher than that of control group, but less than PQ group at28d.Suggested that treated with5-ASA could reduce collagen synthesis.
3immunohistochemical staining
3.1The distribution and expression of PPARγ in lung tissues of ratIn control group, there was strong expression of PPARγ in vascularendothelial cells, Alveolar epithelial cells and macrophages, The nuclearstaining is deep brown. Single treated with5-ASA the positive expression ofPPARγ is stronger than other group before the7thday(P<0.05). After treatedwith PQ, the cell which positive express PPARγ is significantly less thancontrol group before the14thday(P<0.05). In PQ+30mg5-ASA group andPQ+75mg5-ASA group, the cell which positive express PPARγ is less thancontrol group but more than PQ group before the14thday(P<0.05).
3.2The distribution and expression of TGF-β1in lung tissues of ratTGF-β1is stain at cytoplasm. It was main expressed in bronchialepithelial cells, smooth muscle cells, fibroblasts and scattered inflammatorycells. The positive expression of TGF-β1in PQ group was much stronger thanthat of control group before the14thday(P<0.05).PQ+30mg5-ASA group and PQ+75mg5-ASA group: The cells whichpositive express TGF-β1were more than Control group but less than PQ groupbefore the14thday(P<0.05).
4Western blot detect the expression of P-smad3in lung tissues of ratThe5-ASA group has no change compared with the control group(P>0.05). The expression of P-smad3was significantly increased in comparedPQ group with the control group at all the time(P<0.05). In PQ+30mg5-ASA group and PQ+75mg5-ASA group: The expression of P-smad3wassignificantly increased compared with the control group before the14thday(P<0.05). But it was decreased compared with the PQ group at all thetime(P<0.05).
PartⅢ Effect of5-ASA on PPARγ、TGF-β1、P-Smad3in WI38VA13cellin vitro
Objective: To explore whether PQ could regulate TGF-β, P-Smad3inWI38VA13cell and whether5-ASA could regulate TGF-β/Smads signalpathway.
Method: Cell survival rates was tested by MTT assay. PPARγ, TGF-β1and P-Smad3in WI38VA13cell were detected at protein lever by WesternBlot, the cell respectively treated with PQ,5-ASA and PQ+5-ASA.
Results:
1Effects of PQ on the survival rates in WI38VA13cells in vitro
MTT assay showed that PQ had a potent inhibitory effect on the growthof WI38VA13cells in vitro (P<0.05). The cell survival rates in PQ treatedgroups displayed a significant decrease in a dose-dependent manner rangingfrom200to1000μmol/L.
2Effects of5-ASA on PPARγ, TGF-β1, P-Smad3in PQ-treated WI38-VA13cells in vitro
2.1Effects of5-ASA on PPARγ in PQ-treated WI38VA13cells in vitro
Western Blot results showed that the expresion of PPARγ protein wassignificantly increased in5-ASA treatment group as compared to the controlgroup in WI38VA13cells (P<0.05). But the PPARγ were significantlydecreased in PQ group and PQ+5-ASA group compared with the controlgroup(P<0.05).However, that in PQ+5-ASA group was higher than PQ group(P<0.05).
2.2Effects of5-ASA on TGF-β1in PQ-treated WI38VA13cells in vitro
Western Blot results showed that the expresion of TGF-β1protein has nosignificant difference in5-ASA treatment group compared with control groupin WI38VA13cells (P>0.05). But the TGF-β1were significantly increased in PQ group and PQ+5-ASA group as compared to the control group(P<0.05).However, that in PQ+5-ASA group was less than PQ group (P<0.05).
2.3Effects of5-ASA on P-Smad3in PQ-treated WI38VA13cells in vitroWestern Blot results showed that the expresion of P-Smad3protein hasno significant difference in5-ASA treatment group compared with controlgroup in WI38VA13cells (P>0.05). But the P-Smad3were significantlyincreased in PQ group and PQ+5-ASA group as compared to the controlgroup(P<0.05). However, that in PQ+5-ASA group was less than PQ group(P<0.05).
Conclusions:
1Intragastric administered with PQ is a reliable method to establish acutelung injury and pulmonary fibrosis animal model. It lay the foundation forthe research of PQ poisoning mechanism and drug therapy.
2The production of oxygen free radicals plays an important role inPQ-induced lung injury. It can induce the increase of MDA while decreasethe content of SOD and GSH.
3TGF-β1/Smads signal transduction pathway plays an important role in PQinduced pulmonary fibrosis in rat.
4Treatment with PQ can induce the expression of TGF-β1and P-Smad3inWI38VA13cell.
55-ASA has the effects of anti-free radical and resisting lipid peroxidation,which can inhibit PQ induced lung injury.
6As PPARγ ligand,5-ASA inhibits PQ induced pulmonary fibrosis incertain degree through regulate TGF-β1/Smads signaling pathway.
75-ASA can up-regulate the expression of PPARγ which was inhibited byPQ, while inhibites the TGF-β1/Smads signaling pathway at the same time.
PQ可经胃肠道、皮肤、呼吸道和腹腔等吸收，引起肺、肝、肾、心以及中枢神经系统等多脏器损伤，其中肺脏是PQ中毒的最重要的靶器官，因肺内存在聚胺类物质摄取系统使得PQ在肺组织中异常聚集而引起严重的肺损伤，主要表现为间质内水肿、白细胞浸润、肺泡充血，成纤维细胞增殖以及胶原沉积增加。上述损伤最终可能导致死亡，存活者可遗留长期的肺功能障碍。因PQ作用机制复杂且不明了，故目前尚无特效解毒药，临床上对百草枯的治疗困难重重，疗效多不佳。因此，深入探讨其中毒机制及治疗策略，尤其是针对其引起的肺损伤的研究是中毒领域研究中的热点和难点。
在由多种细胞因子以及信号转导通路形成的参与肺损伤及纤维化的网络系统中，TGF-β1/Smads是重要的调节因子，有研究提出针对TGF-β信号系统进行治疗有望缓解PQ引起的脏器的纤维化。近年来研究表明，PPARγ激动剂可以阻断TGF-β信号传导通路，进而抑制主要脏器的慢性纤维化进程。5-氨基水杨酸（5-ASA）是最经典的抗炎剂，可以作为PPARγ的配体直接结合并激活PPARγ，进而影响TGF-β信号通路活性而发挥作用。目前有关PPARγ在百草枯引起的肺损伤中所起的作用，以及5-ASA在PQ中毒的治疗作用少见报道。
鉴于此，本研究通过建立PQ中毒大鼠模型，从整体水平上观察PQ对大鼠急性肺脏损伤及氧化应激的作用；PQ对大鼠肺组织纤维化以及PPARγ、TGF-β1、P-Smad3的表达的影响；同时观察5-ASA在上述过程中的治疗作用。在此基础上，进一步通过体外实验观察5-ASA在PQ对WI38VA13细胞PPARγ、TGF-β1、P-Smad3的表达的影响。旨在揭示PPARγ在百草枯引起的急性肺损伤及肺纤维化过程中的作用以及5-ASA对百草枯中毒的治疗作用。
第一部分5-ASA对PQ染毒大鼠肺组织的保护性抗损伤作用
目的：探讨5-ASA对PQ染毒大鼠肺组织的炎性损伤是否有保护作用。
方法：成年雄性Wister大鼠共125只，随机分为5组。对照组（空白对照组，Control组）给予蒸馏水灌胃，5-ASA组按75mg/kg灌胃，PQ组按80mg/kg灌胃，PQ+30mg5-ASA组按PQ80mg/kg+5-ASA30mg/kg灌胃，PQ+75mg5-ASA组按PQ80mg/kg+5-ASA75mg/kg灌胃。分别于试验开始后24h、72h、7d、14d、28d分批处死动物，每组5只，留取静脉血及肺组织待检。计算肺系数，测定血清中SOD、GSH、MDA含量。同时留取部分肺组织做病理切片，行HE染色后在光学显微镜下观察肺组织病理改变。
结果：
1染毒及干预后各组大鼠的一般状况观察
PQ组大鼠在染毒后2h即可出现明显的中毒表现，且在1~3天最明显，具体表现为：①一般情况：精神较差、倦怠、嗜睡、烦躁、易激惹、口鼻及眼周血性分泌物、竖毛、体重减轻、下降程度最大的一只达46g，进食水明显减少；②呼吸系统表现：呼吸急促、呼吸困难、腹式呼吸、口周紫绀，严重者可以听到喘鸣音；③消化系统：厌食水，稀便等；④泌尿系统：少尿或无尿，血尿等。PQ+30mg5-ASA组和PQ+75mg5-ASA组（染毒+治疗组）：大鼠经5-ASA治疗后，中毒表现较PQ组为轻，但不同剂量5-ASA两个治疗组之间没有明显差别。
2染毒及干预后各组大鼠的大体病理学变化观察
PQ组染毒早期（24h、72h、7d）可见双肺体积明显增大、充血、水肿、有点片状出血，部分大鼠胸腔有血性渗出物，胃肠道可见明显胀气、充血，粘膜可见散在出血点及溃疡；肝脏充血、肿胀，双肾肿大，颜色紫红，被膜紧张，偶可见出血点。染毒晚期（14d,28d）可见双肺表面不平，散在陈旧出血点，有点片状白色，偏僵硬感。PQ+30mg5-ASA组和PQ+75mg5-ASA组染毒早期各脏器病变表现均较PQ组为轻，双肺出血不明显，晚期双肺表面较光滑，有点片状白色区域，但明显少于PQ组。两个治疗组区别不明显。
综上可见5-ASA治疗可以改善PQ中毒引起的大体病理变化，且两种剂量无明显差别。
3相对体重的比较
给予75mg5-ASA组各实验动物相对体重随实验时间逐渐上升，与对照组相比无明显差异（P>0.05）。 PQ染毒后实验动物体重下降，明显低于对照组，72h时达到高峰（P<0.05），染毒14d时恢复至与对照组相同水平（P>0.05）。PQ+30mg和75mg5-ASA组动物72小时内相对体重也有下降，明显低于对照组和5-ASA组（P<0.05）；但7d时即可恢复至对照组和5-ASA组水平（P>0.05）。提示5-ASA治疗可以减轻PQ中毒对大鼠体重的影响，侧面反映其可减轻PQ的毒性反应。
4肺系数的比较
5-ASA组动物处理各时间段肺系数无明显变化，与对照组无明显差异（P>0.05）。PQ染毒后肺系数明显上升，至第7d升至高峰，14天时较第7d下降，但28d时再次升高，呈双相升高表现。各时间段肺系数均明显高于对照组和5-ASA组（P<0.05）。PQ+30mg5-ASA组和PQ+75mg5-ASA组动物24h和72h肺系数与对照组和5-ASA组相比均有明显增加，尤以72h时升高明显（P<0.05），但两个治疗组24h和72h肺系数及其后各时间点肺系数均明显低于PQ组（P<0.05），且恢复至与对照组和5-ASA组同一水平的时间均明显短于PQ组（7d vs28d）。但两治疗组之间相比无明显差异（P>0.05）。
综上表明，5-ASA治疗组肺系数明显较中毒组轻，说明5-ASA在一定程度上减轻了PQ中毒引起的充血水肿。
5血清学指标测定结果
5-ASA组各时间点血清SOD、GSH、MDA含量与对照组相比无明显差异（P>0.05），染毒后PQ组血清SOD、GSH活力明显下降，SOD活力在7d前均明显低于对照组和5-ASA组（P<0.05），以72h降低最明显，GSH含量在14d前均明显低于对照组和5-ASA组（P<0.05），以14d时含量最低，MDA含量则在染毒后呈上升趋势，7d前均较对照组和5-ASA组明显升高（P<0.05），其余时间点三个血清学指标均与对照组和5-ASA组相比无明显差异（P>0.05）。而两个治疗组血清学指标变化趋势与PQ组相一致，但程度均低于PQ组，SOD活力仅在72h时下降，与PQ组，对照组和5-ASA组相比均有显著差异（P<0.05），GSH含量仅24h时与对照组和5-ASA组相比有显著差异（P<0.05），24h～14d内均明显高于PQ组（P<0.05）；MDA含量在72h后逐渐下降，PQ+30mg5-ASA组在各时间段与对照组和5-ASA组相比均无显著差异（P>0.05），72h和7d时明显低于PQ组（P<0.05）； PQ+75mg5-ASA组在24h时明显高于对照组和5-ASA组（P<0.05），其在各时间段与PQ组相比均无显著差异（P>0.05）。结果表明PQ中毒早期会出现SOD、GSH含量下降和MDA含量上升，5-ASA治疗使这些变化在持续时间和程度上都有所减轻。
6肺组织病理改变（HE染色）
5-ASA组动物肺组织结构与对照组无明显差异，镜下可见肺泡结构清晰，肺泡壁薄，肺泡间隔无增宽及充血，肺泡腔无炎性细胞浸润及出血现象。PQ染毒后实验动物肺组织出现明显肺泡炎改变，24h可见肺内炎性细胞浸润，肺泡壁毛细血管扩张、瘀血，气管及血管周围有不同程度的水肿，72h、7d时肺间质和肺泡水肿继续进展，7d时达高峰，病变范围超过75%，肺泡间隔充血、增厚，肺泡腔变窄，部分肺泡腔充满粉染均质水肿液，可伴广泛血管内皮细胞损伤，弥漫性肺出血，部分有透明膜形成，肺泡腔内有游离的中性粒细胞和巨噬细胞，14d和28d炎症改变逐渐减轻，炎性细胞浸润以巨噬细胞、中性粒细胞为主，并有少量淋巴细胞，仍有出血及水肿，肺间隔明显增宽，组织增生明显，可见胶原纤维增生。
PQ+30mg5-ASA组和PQ+75mg5-ASA组实验动物肺组织病变较轻，肺泡内充血水肿和炎性细胞浸润程度低于PQ组，病变范围低于60%，后期胶原纤维增生亦较PQ组为轻。
提示：5-ASA的治疗减轻了PQ中毒引起的肺的早期以肺泡炎和晚期以增生和纤维化为主要表现的病理变化。
第二部分5-ASA对PQ引起的肺纤维化的保护作用
目的：TGF-β1/Smads信号途径是肺纤维化发生的关键环节，本部分研究目的旨在探讨作为PPARγ激动剂，5-ASA是否能通过抑制TGF-β1/Smads信号途径，进而减轻PQ中毒引起的肺纤维化。
方法：实验分组与处理同第一部分，留取肺组织测HYP含量，肺组织进行病理形态学观察及VG染色。免疫组化法检测PPARγ、TGF-β1在肺中的表达，蛋白免疫印迹测定P-smad3水平。
结果：
1肺组织HYP含量测定结果
5-ASA组实验动物各时间点肺组织HYP水平与对照组无明显差异（P>0.05）。PQ组于染毒后第7d实验动物肺组织HYP含量开始升高，7d、14d、28d均显著高于对照组和5-ASA组（P<0.05）。给予5-ASA治疗后实验动物肺组织HYP水平升高时间延后至第14d，与对照组和5-ASA组相比14d和28d时肺组织HYP含量增高，但两组动物肺组织HYP水平均明显均低于PQ组。结果表明HYP含量升高发生在PQ中毒后期，5-ASA的治疗使其升高的时间延后，程度减轻。
2Van Gieson胶原纤维染色
Van Gieson胶原纤维染色可见对照组和5-ASA组肺组织仅在大支气管壁、血管周围及肺泡间隔区见少量红色的胶原纤维。PQ染毒早期（24h、72h、7d）实验动物肺组织未见明显胶原纤维增生，但染毒14d以后胶原纤维着色明显增强，血管壁与支气管壁的内皮下与肌层下均有红染的胶原纤维出现，肺泡间隔呈纤维性增厚，部分区域胶原纤维不规则排列。
PQ+30mg5-ASA组和PQ+75mg5-ASA组动物染毒24h至14d时未见明显胶原纤维增生；仅在染毒28d时可见胶原纤维数量多于对照组，但较染毒组明显减轻，有部分肺泡间隔增厚。结果提示5-ASA的治疗减少了胶原纤维的合成。
3PPARγ和TGF-β1的免疫组织化学结果
3.1PPARγ在大鼠肺组织中的表达与分布
免疫组化染色结果表明，PPARγ免疫反应产物主要定位于细胞核，呈棕黄色颗粒状，胞质很少着色，正常组实验动物肺组织内可见PPARγ表达阳性的血管内皮细胞、肺泡上皮细胞和巨噬细胞。给予5-ASA后，实验动物肺组织PPARγ阳性表达明显增强，7天时达高峰（1.61±0.06），与用药时间相一致，较其他组明显升高（P<0.05）。14天后PPARγ表达水平下降，恢复至对照组实验动物水平（P>0.05）。PQ染毒后肺组织内PPARγ表达阳性的血管内皮细胞、肺泡上皮细胞和巨噬细胞均明显减少，尤以PQ染毒7d时最明显（0.44±0.07），14d之前与对照组相比均有显著差异（P<0.05）。至染毒28d时恢复至正常水平。5-ASA治疗组实验动物肺组织PPARγ表达阳性的细胞较对照组减少，但较PQ组多，如7d时染色指数分别为0.59±0.07、0.61±0.06，在14d之前与两组相比均有明显差异（P<0.05）。28d时恢复至正常水平。提示PQ使PPARγ表达下调，而5-ASA使这种作用减弱。
3.2TGF-β1在大鼠肺组织中的表达与分布
免疫组化染色结果表明，TGF-β1表达定位于细胞胞浆，主要表达于支气管上皮细胞、平滑肌细胞、成纤维细胞及散在的炎性细胞。PQ组实验动物染毒后TGF-β1表达明显增强，至14d时达高峰（0.976±0.063），各时间点染色指数均明显高于对照组（P<0.05）。28d时恢复至正常水平。
PQ+30mg5-ASA组和PQ+75mg5-ASA组：TGF-β1表达于染毒后增多，但染色指数介于对照组和PQ组之间，于7d时达高峰（0.557±0.078，0.612±0.056），与前两组相比均有显著差异（P<0.05）。28d时回落至正常。
由上可见5-ASA的治疗抑制了由于PQ中毒引起的肺内TGF-β1的表达上调。
4肺组织中P-smad3的免疫蛋白印迹结果
5-ASA组动物肺组织p-smad3蛋白表达在各时间段无明显变化，与对照组相比无明显差异（P>0.05）。PQ染毒后P-smad3蛋白表达逐渐上升，14d时达高峰（0.688±0.065），28d时下降，但仍高于正常水平，各时间点P-smad3蛋白表达均明显高于对照组（P<0.05）。PQ+30mg5-ASA组和PQ+75mg5-ASA组：P-smad3蛋白表达水平于染毒后升高，但介于对照组和PQ组之间，7d时达高峰（0.349±0.021,0.347±0.027），之后下降，14d前与两组差异均有意义（P<0.05）。28d时回落至正常水平（P>0.05）。结果表明PQ染毒后肺内smad3蛋白磷酸化水平上升，5-ASA的应用抑制了PQ的这种作用。
第三部分5-ASA对WI38VA13细胞PPARγ、TGF-β1、P-Smad3的表达的影响
目的：了解PQ是否对于成纤维细胞的TGF-β1、P-Smad3的分泌产生影响，及应用5-ASA后是否对TGF-β1/Smads信号通路的传导有调控作用。
方法：用MTT法确定试验用PQ浓度，Western Blot方法检测PQ、5-ASA、PQ+5-ASA处理WI38VA13细胞后，在12h、24h和48h时细胞中PPARγ、TGF-β1、P-Smad3含量。
结果：
1PQ对WI38VA13细胞存活率的影响
PQ对WI38VA13细胞的生长有明显的抑制作用。经PQ处理24h后，200、250、300、400、600、800、1000μmol/L PQ处理组WI38VA13细胞存活率均明显低于对照组（P<0.05）。在200-1000μmol/L浓度范围内，随着PQ浓度的升高，WI38VA13细胞的存活率明显降低。
25-ASA对PQ作用下WI38VA13细胞中PPARγ、TGF-β1、P-Smad3的影响
2.15-ASA对PQ作用下WI38VA13细胞PPARγ表达的影响
Western Blot检测结果显示，给予5-ASA处理后，WI38VA13细胞中PPARγ蛋白表达与对照组相比增强，各时间组与对照组相比均有明显差异（P<0.05）。单纯给予PQ处理，WI38VA13细胞中PPARγ蛋白表达与对照组相比明显下降，并随时间延长递减，各时间组与对照组相比均有明显差异（P<0.05）。5-ASA与PQ共同处理组，WI38VA13细胞中PPARγ蛋白与对照组相比表达下降，但介于5-ASA组与PQ组之间，与两组相比均有统计学意义（P<0.05）。提示PQ下调了WI38VA13细胞中PPARγ蛋白的表达，而5-ASA则有上调作用，共同作用下，5-ASA可以部分的逆转PQ对WI38VA13细胞中PPARγ蛋白表达的下调作用。
2.25-ASA对PQ作用下WI38VA13细胞TGF-β1表达的影响
Western Blot检测结果显示，给予5-ASA处理后，WI38VA13细胞中TGF-β1蛋白表达与对照组相比无明显差异（P>0.05），PQ处理后，TGF-β1蛋白表达与对照组相比明显增强，并随时间延长递增，与对照组相比有明显差异（P<0.05）。5-ASA与PQ共同处理组，TGF-β1蛋白表达逐渐增强，数值介于PQ组和对照组之间，与两组相比均有明显差异（P<0.05）。提示PQ激活WI38VA13细胞中TGF-β1蛋白表达，5-ASA可以部分逆转PQ对WI38VA13细胞中TGF-β1蛋白表达的上调作用。
2.35-ASA对PQ作用下WI38VA13细胞P-Smad3表达的影响
Western Blot检测结果显示，给予5-ASA处理后，WI38VA13细胞中Smad3蛋白磷酸化水平与对照组相比无明显差异（P>0.05），PQ处理后，Smad3蛋白磷酸化水平与对照组相比明显增强，并随时间延长递增，与对照组相比有明显差异（P<0.05）。5-ASA与PQ共同处理组，Smad3蛋白磷酸化逐渐增强，数值介于PQ组和对照组之间，与两组相比均有明显差异（P<0.05）。提示PQ作用后，WI38VA13细胞中Smad3蛋白磷酸化水平上调，5-ASA可以部分逆转PQ对WI38VA13细胞中Smad3蛋白磷酸化水平的上调作用。
结论：
1PQ灌胃染毒是建立急性肺损伤和肺纤维化动物模型的可靠方法，为研究PQ中毒机制及药物疗效奠定了基础。
2氧自由基的产生在PQ中毒所致肺损伤中起重要作用，表现为染毒后脂质过氧化产物MDA含量的升高，抗氧化物质SOD、GSH含量的下降。
3TGF-β1/Smads信号转导通路在PQ引起的大鼠肺纤维化中发挥重要作用。
4PQ处理可以引起WI38VA13细胞TGF-β1以及P-Smad3表达增强。
55-ASA有抗自由基及减轻脂质过氧化作用，对PQ引起的肺损伤有一定的保护性抗炎作用。
65-ASA作为PPARγ配体，通过干预TGF-β1/Smads信号通路一定程度上缓解PQ导致的肺纤维化。
75-ASA可以逆转PQ引起的细胞中PPARγ的表达下调，同时使TGF-β1/Smads信号通路受到抑制。
Paraquat dichloride (1,1’-dimethyl-4,4’-bipyridilium dichloride; methylviologen, PQ) is an effective and widely used herbicide in most countries. Theintentional and accidental ingestion of commercial liquid formulations ofparaquat has caused a large number of human fatalities. It has become one ofthe most important medical and social problems which can not be ignored inour country.
The toxicity of PQ was first described by Clark. It can cause injury tomany organs, including liver, kidney, thymus and lung. Most importantly, lungtissue is the major targeted organ for paraquat toxicity in rat and man, becauseit has active polyamine uptake transport systems that concentrate paraquatrapidly into the type II epithelial cells of the alveoli. The toxic effects ofparaquat on the lungs result in pulmonary edema, hypoxia, respiratory failure,and pulmonary fibrosis. Survivors of paraquat poisoning may be left with arestrictive type of long-term pulmonary dysfunction. Because of themechanism is complex and not fully understood, so the treatment of paraquatpoisoning is difficulty. So the mechanism of paraquat toxicity and treatment,especially for the study of lung injury caused by PQ, is hotspot and difficultyin the field of poisoning.
Transforming growth factor-β1(TGF-β1) is a key growth factor thatinitiates tissue repair and its sustained production underlies the developmentof tissue fibrosis. TGF-β signals are mediated by a family of Smad proteins.Inrecent years many studies have demonstrated that PPAR agonists could inhibitthe TGF-β signaling pathway, thereby inhibiting chronic fibrosis in majororgans. The effect of PPARγ in PQ-induced lung injure is seldom reported.Over the past decade,5-ASA has been first-line therapy for mild to moderateactive IBD. Recent researchs have demonstrated that, as a ligand for PPARγ, 5-ASA was found to reduce TGF-β1signalling as judged by the reduction inTGF-β specific reporter gene activity.
In this study, we aimed to investigate whether PPARγ played a regulatoryrole in paraquat induced acute lung injury and pulmonary fibrosis both in vitroand in vivo.
PartⅠThe protective anti-inflammatory effects of5-ASA on lungtissue of rats with PQ poisoning.
Objective: To explor whether5-ASA has therapeutic effect to PQ-induced lung injure of rat.
Methods:125adult healthy Wister male rats were randomly distribu-tioned to five groups.(1) Control group, n=25: animals were intragastricadministered with distilled water;(2)5-ASA group, n=25: animals were intra-gastric administered with5-ASA(75mg/kg);(3) PQ group, n=25: animalswere intragastric administered with PQ(80mg/kg);(4) PQ+30mg5-ASA group,n=25, animals were intragastric administered with PQ(80mg/kg)+5-ASA(30mg/kg);(5) PQ+75mg5-ASA group, n=25, animals were intragastric adminis-tered with PQ(80mg/kg)+5-ASA(75mg/kg). At24h,72h,7d,14d,28day ratswere sacrificed, venous blood were collected to detect SOD, GSH andMDA.Weight for lung coefficient. Optical microscope was performed toexamine pathological changes in lung tissues with HE.
Results:
1The general condition of each group
Intoxication manifestations occurred in rats only2hours after PQ ad-ministered, and were especially severe during the first three days. Intoxicationinvolves a combination of signs and symptoms including lethargy, hypoxia,dyspnea, tachyeardia, hyperpnea, hyperexcitability, convulsions, unhairing,nose and/or eyes bleeding, anorexia, diarrhea, oliguria, hematuria and markedlosses in body weight. In5-ASA treatment group, intoxication manifestationswere lighter than PQ group.
2Each group’s gross pathology changes
In PQ group, double lung’s volume increased significantly at the early stage(24h,72h,7d). Hyperemia, edema, hemorrhage could be observed.Thereis hemorrhagic exudate in rat pleural. Gastrointestinal obvious flatulence,hyperemia, mucous membrane can be seen ulcers; liver congestion, swelling,bilateral renal enlargement, the color is purple, film tension, even visiblebleeding. In the late stage(14d,28d), double lung’s surface uneven, oldhaemorrhage dot could be seen.In PQ+30mg5-ASA group and PQ+75mg5-ASA group, each organ manifestations were lighter than PQ group. Doublelung volume increases, congestion and edema, but no obvious bleeding inearly period. Lung surface is smooth，there is a bit flaky white on it, less thanthat of PQ group.
The PQ-induced pathological changes could be improved by5-ASA.There was no significant difference between the two treatment doses.
3Comparison of the relative weight
Relative body weight gradually increases with time in5-ASA group. Ithas no change compared with the Control group(P>0.05). After the PQexposure, the relative weight decreased to minimum at72h(P<0.05), thengradually increased to the control level at14d(P>0.05). In PQ+30mg5-ASAgroup and PQ+75mg5-ASA group, relative body weight decreased to aminimum at72h. It had statistical significance when compared with controlgroup and5-ASA group at72h(P <0.05).So the PQ-induced relative bodyweight decrease could be improved by5-ASA.
4Comparison of the lung coefficient
The lung coefficient did not change at all time points in5-ASA group. Ithad no statistical significance compared with control group(P>0.05).In PQgroup, the lung coefficient increased significantly after exposure, it rosed tothe highest position at7d, then decreased at14d, increased again at28d. Eachdata had statistical significance compared with control group(P<0.05). InPQ+30mg5-ASA group and PQ+75mg5-ASA group, The lung coefficientincreased after exposure. It had statistical significance when compared withcontrol group and5-ASA group at24h,72h(P<0.05). But it was significantlylower than PQ group at all time points(P<0.05). There was no difference between two treatment group(P>0.05).
The lung coefficients of5-ASA treatment group were lower than that ofthe PQ group. Which indicated that5-ASA reduced congestion and edemacaused by PQ poisoning.
5Serum measurement
In5-ASA group, the activities of serum SOD, GSH, MDA have nosignificantly difference compared with the control group(P>0.05). In PQgroup, the activities of serum SOD and GSH significantly decreased afterexposure. SOD down to the lowest point at72h, then began to increase. It hadstatistical significance when compared with control group and5-ASA group at24h,72h and7d(P<0.05). GSH were significantly lower than control groupand5-ASA group. The lowest point was at14d(P<0.05). The content of MDAin serum increased after exposure. It was much higher than control group and5-ASA group before7d(P<0.05). These three serum index had no differencecompared with control group and5-ASA group at other time points(P>0.05).The three serum index had a similar changing trend to PQ group in the twotreatment groups. But the level was lower than that of PQ group.SOD onlydecreased at72h(P<0.05).GSH had significantly difference with control and5-ASA groups only at24h(P<0.05). it was much higher than PQ group from24h to14d. The content of MDA in serum of PQ+30mg5-ASA group has nochange compared with the control group and5-ASA group at eachtime(P>0.05), it was lower than PQ group at72h and7d(P<0.05).There wasdifference between PQ+75mg5-ASA group and control group at24h(P<0.05).There was no difference between PQ+75mg5-ASA group and PQ group ateach time. The reduction of SOD and GSH activity and the increased of MDAoccurred in the early. Therapy with5-ASA reduced these changes in bothduration and extent.
6Histological changes: HE staining
The lung structure of5-ASA group had no significantly difference withthat of control group. The normal alveolar wall was thin and clear, with noinfiltration of inflammatory cells in the lumen of alveoluses or oceasionally only a few erythrocytes. In PQ group, there were alveolitis occurred in lungafter exposure.We can see inflammatory cell infiltration, telangiectasis inalveolar, pulmonary congestion, edema around the trachea and vessels at24h.The edema continued to increase, reached a peak at7d. Alveolar septalhyperemia, thickening, alveolar lumen narrowed, diffuse pulmonaryhemorrhage, hyaline membrane formation could be seen in72h and7d; At thelater stage, we also observed proliferation of collagen fibers, fibrousthickening of the alveolar wall and fibroblastial hyperplasia in differentdegrees.
PQ+30mg5-ASA group and PQ+75mg5-ASA group:5-ASA reducedthe pulmonary pathological changes caused by PQ.
PartⅡ Protective effect of5-ASA on PQ induced pulmonary fibrosis
Objective: TGF-β1/Smads Signaling pathway is a key link of pulmonaryfibrosis
To explore as a PPARγ agonist, whether5-ASA can inhibitTGF-β1/Smad3signal pathway, then reduce pulmonary fibrosis induced by PQpoisoning.
Methods: The experimental group and treatment same with the part I.Lung homogenate to detect HYP. Optical microscope was performed toexamine pathological changes in lung tissues with VG staining. To detect theexpress of PPARγ, TGF-β1in lung tissues by immunohistochemical staining.To detect the express of P-smad3by Western Blot.
Results:
1the content of HYP in lung tissue
The content of HYP in lung tissue of5-ASA group had no changecompared with that of the control group(P>0.05). In PQ group, HYP had asignificantly increased after the7thday. It was much higher than control groupand5-ASA group at7d,14d and28d(P<0.05). The lever of HYP were higherthan control group in the two5-ASA treatment groups at14d and28d(P<0.05),but they were also lower than PQ group(P<0.05).
2Van Gieson stainingSurrounding blood vessels and alveolar septal area,we could see little redcollagen fibers in bronchial wall in control group and5-ASA group. In PQgroup, collagen fiber hyperplasia was not obvious in24h,72h and7d. Afterthe14thday, fibrous thickening occurred in bronchia walls and the alveolusinterspacea, in some areas with an irregular collocation of collagen fibres.PQ+30mg5-ASA group and PQ+75mg5-ASA group: Treated with5-ASA, collagen fiber hyperplasia was not obvious in24h to14d. Collagenfiber was higher than that of control group, but less than PQ group at28d.Suggested that treated with5-ASA could reduce collagen synthesis.
3immunohistochemical staining
3.1The distribution and expression of PPARγ in lung tissues of ratIn control group, there was strong expression of PPARγ in vascularendothelial cells, Alveolar epithelial cells and macrophages, The nuclearstaining is deep brown. Single treated with5-ASA the positive expression ofPPARγ is stronger than other group before the7thday(P<0.05). After treatedwith PQ, the cell which positive express PPARγ is significantly less thancontrol group before the14thday(P<0.05). In PQ+30mg5-ASA group andPQ+75mg5-ASA group, the cell which positive express PPARγ is less thancontrol group but more than PQ group before the14thday(P<0.05).
3.2The distribution and expression of TGF-β1in lung tissues of ratTGF-β1is stain at cytoplasm. It was main expressed in bronchialepithelial cells, smooth muscle cells, fibroblasts and scattered inflammatorycells. The positive expression of TGF-β1in PQ group was much stronger thanthat of control group before the14thday(P<0.05).PQ+30mg5-ASA group and PQ+75mg5-ASA group: The cells whichpositive express TGF-β1were more than Control group but less than PQ groupbefore the14thday(P<0.05).
4Western blot detect the expression of P-smad3in lung tissues of ratThe5-ASA group has no change compared with the control group(P>0.05). The expression of P-smad3was significantly increased in comparedPQ group with the control group at all the time(P<0.05). In PQ+30mg5-ASA group and PQ+75mg5-ASA group: The expression of P-smad3wassignificantly increased compared with the control group before the14thday(P<0.05). But it was decreased compared with the PQ group at all thetime(P<0.05).
PartⅢ Effect of5-ASA on PPARγ、TGF-β1、P-Smad3in WI38VA13cellin vitro
Objective: To explore whether PQ could regulate TGF-β, P-Smad3inWI38VA13cell and whether5-ASA could regulate TGF-β/Smads signalpathway.
Method: Cell survival rates was tested by MTT assay. PPARγ, TGF-β1and P-Smad3in WI38VA13cell were detected at protein lever by WesternBlot, the cell respectively treated with PQ,5-ASA and PQ+5-ASA.
Results:
1Effects of PQ on the survival rates in WI38VA13cells in vitro
MTT assay showed that PQ had a potent inhibitory effect on the growthof WI38VA13cells in vitro (P<0.05). The cell survival rates in PQ treatedgroups displayed a significant decrease in a dose-dependent manner rangingfrom200to1000μmol/L.
2Effects of5-ASA on PPARγ, TGF-β1, P-Smad3in PQ-treated WI38-VA13cells in vitro
2.1Effects of5-ASA on PPARγ in PQ-treated WI38VA13cells in vitro
Western Blot results showed that the expresion of PPARγ protein wassignificantly increased in5-ASA treatment group as compared to the controlgroup in WI38VA13cells (P<0.05). But the PPARγ were significantlydecreased in PQ group and PQ+5-ASA group compared with the controlgroup(P<0.05).However, that in PQ+5-ASA group was higher than PQ group(P<0.05).
2.2Effects of5-ASA on TGF-β1in PQ-treated WI38VA13cells in vitro
Western Blot results showed that the expresion of TGF-β1protein has nosignificant difference in5-ASA treatment group compared with control groupin WI38VA13cells (P>0.05). But the TGF-β1were significantly increased in PQ group and PQ+5-ASA group as compared to the control group(P<0.05).However, that in PQ+5-ASA group was less than PQ group (P<0.05).
2.3Effects of5-ASA on P-Smad3in PQ-treated WI38VA13cells in vitroWestern Blot results showed that the expresion of P-Smad3protein hasno significant difference in5-ASA treatment group compared with controlgroup in WI38VA13cells (P>0.05). But the P-Smad3were significantlyincreased in PQ group and PQ+5-ASA group as compared to the controlgroup(P<0.05). However, that in PQ+5-ASA group was less than PQ group(P<0.05).
Conclusions:
1Intragastric administered with PQ is a reliable method to establish acutelung injury and pulmonary fibrosis animal model. It lay the foundation forthe research of PQ poisoning mechanism and drug therapy.
2The production of oxygen free radicals plays an important role inPQ-induced lung injury. It can induce the increase of MDA while decreasethe content of SOD and GSH.
3TGF-β1/Smads signal transduction pathway plays an important role in PQinduced pulmonary fibrosis in rat.
4Treatment with PQ can induce the expression of TGF-β1and P-Smad3inWI38VA13cell.
55-ASA has the effects of anti-free radical and resisting lipid peroxidation,which can inhibit PQ induced lung injury.
6As PPARγ ligand,5-ASA inhibits PQ induced pulmonary fibrosis incertain degree through regulate TGF-β1/Smads signaling pathway.
75-ASA can up-regulate the expression of PPARγ which was inhibited byPQ, while inhibites the TGF-β1/Smads signaling pathway at the same time.
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