【作者】 刘明东；

【导师】 邹晓平；

【作者基本信息】 南京中医药大学 ， 中西医结合临床， 2010， 博士

【摘要】 研究背景急性胰腺炎是以胰腺间质水肿、出血、腺泡细胞空泡变性、坏死及炎症细胞浸润为病理特征、病变程度不一、结局难以预料的急性炎症性疾病。急性胰腺炎常伴有全身炎症反应,约20%发展为重症急性胰腺炎(severe acute pancreatitis, SAP),其病死率可高达30%-50%。随着国人生活习惯和饮食结构的变化,我国急性胰腺炎(包括重症胰腺炎)的发病呈现增高的趋势。因此,SAP是目前我国常见的、严重威胁人类健康的重大疾病。急性胰腺炎相关性肺损伤(acute pancreatitis-associated lung Injury, APALI)是急性重症胰腺炎最早出现、最常见、也是最为严重的全身并发症。急性胰腺炎相关性肺损伤发病机制复杂,近年来很多实验性研究集中在炎性细胞和炎性介质激活途径上,一般认为肺损伤是全身炎症反应在肺部的局部表现,而肺脏由于其组织特异性易于发生炎性损伤。积累的大量证据表明胰腺实质中活化的单核巨噬细胞、多核粒细胞释放的TNF-α、LI-1和IL-6等多种促炎细胞因子不仅有助于启动急性胰腺炎局部炎症的形成和发展,而且也是导致包括肺损伤在内全身炎症反应的重要细胞因子,促炎因子的过表达不仅加重了急性胰腺炎的损伤程度,同时也加重了胰腺炎相关性肺损伤的的严重程度。所以降低重症胰腺炎肺组织中炎症因子的水平一直是人们研究如何治疗急性胰腺炎相关性肺损伤的方向。大量的动物实验证明,降低全身或肺组织中炎性细胞因子的水平对急性胰腺炎相关性肺损伤具有治疗作用。但是在急性胰腺炎相关性肺损伤的发病过程中引起肺组织中炎性细胞因子水平增加的原因和机制目前尚不清楚,只有在发病机制上找到原因才能给我们对急性胰腺炎相关性肺损伤的治疗带来理论指导,从而大大降低急性重症胰腺炎的死亡率。最近,JNK信号通路在急性胰腺炎发病中所起的作用越来越受到人们的关注。研究显示,在急性胰腺炎的动物模型中,JNK信号通路被激活,JNK信号通路的激活与炎症因子密切相关,JNK信号通路被激活是胰腺损伤的早期事件,运用JNK通路特异性阻断剂SP600125,可以通过抑制JNK信号通路的激活来下调TNF-α、ICAM-1、IL-1β等促炎症因子的表达,明显减轻胰腺的病理损害,对急性胰腺炎具有治疗作用。由此可见JNK信号通路在急性胰腺炎的发病过程中起着重要的作用。同时人们发现JNK信号通路的激活在肺损伤的发病过程中起着重要的作用,体外动物实验表明,JNK信号通路的激活在镍、脂多糖(lipopolysaccharide, LPS)诱导的急性肺损伤中起着重要的作用,而且应用特异性JNK信号通路阻断剂对肺损伤的治疗,特别是脂多糖(LPS)诱导的急性肺损伤(一种被认为与急性胰腺炎相关性肺损伤有相同的发病途径)的治疗均取得了一定的效果。但到目前为止,JNK信号通路在急性胰腺炎相关性肺损伤中的作用机制尚不清楚。本实验分为两个部分：第一部分JNK通路在急性胰腺炎相关性肺损伤的作用及其机制；第二部分：丹参酮ⅡA对急症胰腺炎相关性肺损伤的干预作用。第一部分：JNK通路在急性胰腺炎相关性肺损伤的作用及其机制目的本研究旨在观察JNK信号通路在牛磺胆酸钠诱导的大鼠急性出血坏死性胰腺炎肺组织中的激活情况,并探讨JNK信号通路激活与肺组织损伤程度的相关性；同时应用特异性JNK通路阻断剂SP600125,进一步证实JNK信号通路激活在急性胰腺炎相关性肺损伤发病过程中所起的作用。方法SD大鼠72只,分为3组,每组24只。S组：假手术对照组,24只大鼠,开腹后翻动大鼠十二指肠并轻触胰腺数次,缝合腹部；SAP组：以1ml／kg的剂量逆行胰胆管注射灭菌5%牛磺胆酸钠,诱导大鼠急性出血坏死性胰腺炎模型,这种模型类似于人类的重症胰腺炎；SP600125干预组：在急性出血坏死性胰腺炎大鼠模型诱导前30分钟,以15mg／kg的剂量通过腹腔注射SP600125；每组分3小时、6小时、12小时和24小时4个时间点,各时间点大鼠数目为6只。按计划于各时间点处死大鼠,收集血清及胰腺和肺组织。肺水肿严重程度以肺组织湿干重比表示；以碘一淀粉比色法测定血清淀粉酶,ELISA方法测定肺组织TNF-α水平和工L-1β水平,H.E染色用于胰腺和肺组织损伤的病理学评价,采用分光光度计测定肺组织中髓过氧化酶(MPO)、免疫组化方法测定肺组织中ICAM-1表达,用Westernblot方法对肺组织中JNK和PJNK进行蛋白定量。结果假手术对照组3hr、6hr、12hr、24hr血清淀粉酶分别为501±85、512±91、518±94和532±86,胰腺病理损伤评分为0；肺脏病理损伤评分为0,肺湿干重比为2.29±0.10、2.22±0.23、2.17±0.11和2.31±0.13；肺脏MPO活性为1.33±0.56、1.56±0.43、1.51±0.23和1.47±0.48；肺组织IL-1β含量为80.3±21.2、92.3±23.5、87.3±19.4和89.7±22.1；肺组织TNF-α含量为70.6±21.2、67.6±19.8、68.4±19.5和74.5±22.6；肺组织ICAM-1免疫组化阳性积分S组0.11±0.01、0.17±0.02、0.14±0.02和0.16±0.09；PJNK相对光密度值为0.65±0.12。SAP组3hr、6hr、12hr、24hr血清淀粉酶分别为2506±215、3701±264、4101±411和3212±295；胰腺病理损伤评分8.24±0.63、10.65±1.02、14.00±0.96和14.09±0.75；肺脏病理损伤评分为3.0±0.4、4.7±0.7、7.5±0.3和6.5±0.7；肺湿干重比为3.52±0.42、3.96±0.15、4.53±0.08和4.26±0.12；肺脏MPO活性为5.64±0.76、6.44±0.77、7.19±0.83和7.36±0.71；肺组织IL-1β含量211.2±19.3、315.4±34.2、399.2±43.2和353.4±38.3；肺组织TNF-α含量为348.5±42.8、433.5±63.4、394.3±56.3和365.3±39.9；肺组织ICAM-1免疫组化阳性积分为0.8±0.09、104±0.11、1.44±0.12和1．22±0.13；PJNK相对光密度值为1.03±0.22、1.24±0.17、1.38±0.21、1.28±0.24。与假手术对照组相比,SAP组在各个时间点的血清淀粉酶、胰腺病理损伤评分和肺脏病理评分均显著增高(P<0.05),我们成功的建立了重症胰腺炎相关性肺损伤的动物模型,SAP组个时间点的肺湿干重比、MPO活性、IL-1β含量、TNF-α含量、ICAM-1表达均明显增高,JNK蛋白总量无变化,但各时间点的PJNK蛋白均显著高于假手术对照组(P<0.05)。应用特异性JNK信号通路阻断剂SP600125后,检测肺组织3hr、6hr、12hr和24hr肺脏病理损伤评分为2.1±0.3、3.2±0.2、6.0±0.7和4.0±0.3；肺湿干重比为SP600125组3.08±0.09、3.54±0.22、3.35±0.16和3.01±0.38；肺脏MPO活性为3.36±0.36、4.27±0.52、5.16±0.26和5.46±0.61(单位：u／g)；肺组织IL-1β含量为154.3±11.2、221.4±21.5、289.4±23.4和267.7±15.1(单位：pg／mg)肺组织TNF-α含量为243.8±32.3、317.6±22.8、272.3±20.5和225.5±32.6(单位：pg／mg)；肺组织ICAM-1免疫组化阳性积分为0.59±0.18、0.77±0.20、0.84±0.23、0.93±0.31；PJNK相对光密度值为0.69±0.14、0.58±0.22、0.85±0.17、0.18±0.05。与SAP组相比,SP600125组各个时间点的肺湿干重比、MPO活性、IL-1β含量、TNF-α含量、ICAM-1表达均明显降低,JNK蛋白无明显变化,但PJNK蛋白显著降低,同时肺脏病理损伤评分明显改善(P<0.05)。通过相关性比较我们发现,肺组织PJNK表达与肺病理损伤评分存在显著相关性(P<0.05)。结论：1、利用5%牛磺胆酸钠逆行大鼠胰胆管注射可建立良好的APALI动物模型,为研究APALI的机制及探讨防治策略提供了试验平台。2、JNK通路激活是重症胰腺炎相关性肺损伤发病的早期事件,其激活可能参与了APALI的发生和发展。3、应用JNK通路阻断剂对急性胰腺炎相关性肺损伤有治疗作用,第二部分：丹参酮ⅡA对急症胰腺炎相关性肺损伤的治疗作用目的评价传统中药丹参酮的有效单体丹参酮ⅡA对大鼠急性胰腺炎相关性肺损伤是否具有治疗作用,并初步探讨其治疗机理。方法SD大鼠72分为3组,每组24只。S组：假手术对照组,24只大鼠,开腹后翻动大鼠十二指肠并轻触胰腺数次,缝合腹部；SAP组：以lml／kg的剂量逆行胰胆管注射灭菌5%牛磺胆酸钠,诱导大鼠急性出血坏死性胰腺炎模型,这种模型类似于人类的重症胰腺炎；丹参酮ⅡA组：在急性出血坏死性胰腺炎大鼠模型诱导前30分钟,以20mg／kg的剂量通过腹腔注射丹参酮ⅡA。每组分3小时、6小时、12小时和24小时4个时间点,各时间点大鼠数目为6只。按计划于各时间点处死大鼠,收集血清及胰腺和肺组织。肺水肿严重程度以肺组织湿干重比表示；以碘一淀粉比色法测定血清淀粉酶,ELISA方法测定肺组织TNF-α水平和IL-1β水平,H.E染色用于胰腺和肺组织损伤的病理学评价,采用分光光度计测定肺组织中髓过氧化酶(MPO)、免疫组化方法测定肺组织中ICAM-1表达,用Westernblot方法对肺组织中JNK和PJNK进行蛋白定量。结果S组肺湿干重比为、2.12±0.23、2.15±0.11和2.33±0.23；肺脏MPO活性为1.23±0.26、1.66±0.33、1.54±0.21和1.42±0.28；肺组织IL-1β含量为82.3±11.2、95.3±21.5、82.3±18.4和92.7±21.1；肺组织TNF-α含量为72.6±11.2、69.6±13.8、69.4±12.5和77.5±21.6；肺组织ICAM-1免疫组化阳性积分S组0.11±0.01、0.17±0.02、0.14±0.02和0.16±0.09；SAP组肺脏病理损伤评分为3.1±0.3、4.6±0.6、7.6±0.3和6.6±0.7；肺湿干重比为3.62±0.43、3.98±0.35、4.54±0.28和4.27±0.12；肺脏MPO活性为5.66±0.66、6.47±0.73、7.21±0.84和7.41±0.72；肺组织IL-1β含量212.2±18.3、319.4±32.2、402.2±41.2和357.4±34.3；肺组织TNF-α含量为351.5±41.8、443.5±53.4、397.3±51.3和365.3±37.9；肺组织ICAM-I免疫组化阳性积分为0.81±0.09、1.14±0.11、1.45±0.12和1.26±0.13。应用丹参酮ⅡA干预后,我们检测肺组织中3hr、6hr、12hr和24hr时间点各项指标的结果分别为：肺湿干重比为3.17±0.29、3.24±0.22、3.49±0.26和3.21±0.38；肺组织MPO活性为4.03±0.39、4.75±0.55、5.66±1.26和5.45±0.719(单位：u／g)；肺组织IL-1β含量为174.3±15.2、211.4±17.5、297.7±21.4和277.9±15.6(单位：pg/mg);肺组织TNF-α含量为253.8±27.3、337.6±22.3、312.3±22.5和311.5±32.6(单位：pg／mg)；肺组织ICAM-1免疫组化阳性积分为0.63±0.15、0.82±0.19、1.01±0.23和0.96±0.31；肺脏病理损伤评分为2.2±0.3、3.6±0.2、6.3±0.7和5.1±0.3；PJNK相对光密度值为1.15±0.08、1.05±0.23、1.34±0.18、0.74±0.16。与SAP组相比,PJNK蛋白显著减少(P<0.05),肺组织湿干重比、肺组织中MPO活性、细胞因子IL-1β和TNF-α的含量、肺组织中ICAM-1的表达均显著性减少(P<0.05),同时肺脏病理损伤评分显著改善(P<0.05)。结论1、丹参酮ⅡA对实验动物的APALI具有治疗作用2、抑制JNK信号通路可能是丹参酮ⅡA治疗作用的重要机制更多还原

【Abstract】 BackgroundAcute pancreatitis is a kind of disease characterized by pancreatic interstitial edema, bleeding, acinous cell vacuolar degeneration, necrosis and inflammatory cells infiltration. Most acute pancreatitis accompany with whole body inflammatory reaction, about 20%patients develop to severe acute pancreatitis (SAP), which is 30%-50%mortality rate. With the changing of life habits and food structure, acute pancreatitis (including SAP) becomes more and more common. Therefore SAP has become a frequent and severe threaten disease to people.Acute pancreatitis-associated lung Injury (APALI) is the most frequent, earliest and severest complication of SAP. Pathogenesy of APALI is still not clear, recently most studying focused on the inflammatory cells and medium. In common APALI is considered to be a part of whole body inflammatory reaction, and because of the tissues specificity of lung it’s easy to get hurt from inflammation. Accumulative evidences indicate that TNF-α, IL-1, IL-6 secreted by active mononuclear macrophage, polynuclear granulocytes in pancreas are very important factors to induce pancreatitis and APALI. The overexpression of pro inflammatory factor can aggravate experimental pancreatitis and APALI. So to decrease the inflammatory factors in lung is a hot point that people study how to treat APALI. Abundant animal experiments show that low the level of inflammatory factor in lung or whole body can treat APALI. But the real mechanism of increasing of inflammatory factor in lung during pancreatitis is still unknown. Only finding the real mechanism it can guide the treatment of APALI and decrease the mortality rate of SAP.Recently, JNK path has got more and more people’s attention in the developing of acute pancreatitis. In acute pancreatitis animal models JNK path is activated and induce inflammatory factors release. JNK path is activated, which is an early incidence in the developing of acute pancreatitis. Using specific inhibitor of JNK path (SP600125) can decrease the expression of TNF-α, ICAM-1, IL-1βand so on, it can significantly improve the impairing of pancreas and treat the acute pancreatitis. The evidences show that JNK path plays a very important role during the development of acute pancreatitis. At the same time people have found the connection between APALI and acute pancreatitis. In vitro, studying show that JNK path plays an important role in the APALI animal models induced by nickel or LPS, and JNK path specific inhibitor can be used to treat APALI especially animal models induced by LPS. But it’s still not clear the mechanism of JNK path in acute pancreatitis.The studying is divided into 2 parts. Part one is to observe the activation of JNK path in the lung of rat acute hemorrhagic necrotizing pancreatitis induced by bile salts and investigate the correlation between JNK path and APALI. At the same time SP600125 is used to confirm the relationship between JNK path and APALI. Part two is to investigate if tanshinone can treat APALI and the mechanism of it.Part one The mechanisms of JNK pathway in acute pancreatitis associated lung injury in rat AimThe aim of studying is to detect the mechanism of JNK path in the developing of acute pancreatitis, and primarily research the therapeutical effect of tanshinone in APALI.Methods72 SD rats were individed into 3 groups. Group S:sham operated group,24 rats, after opening abdomen duodenum and pancreas were lightly touched several times, than abdomen was closed again. Group SAP:To 1ml/kg dose retrograde cholangiopancreatography sterile injection of 5%sodium taurocholate, induced SD rat model of acute hemorrhagic necrotizing pancreatitis. Group SP600125:Acute hemorrhagic necrotizing pancreatitis in rats 1 hour before induction to the dose of 35mg/kg by intraperitoneal injection of SP600125. Every group was divided into 3h,6h,12h and 24h stages. We killed the rats at different stages and collected serum, pancreas and lung. The severity of pulmonary edema was expressed by wet/dry. TNF-a, IL-1βin lung and serum amylase were detected in serum. HE staining was used to value the injure of pancreas and lung. Spectrophotometer was used to detected myeloperoxidase in lung tissue and expression of ICAM-1 was tested by immunohistochemistry in lung. Western-blot was used to detect the expression of JNK and PJNK in lung tissue.ResultsIn Group S at 3hr,6hr,12hr and 24hr four different steps serum amylase were 501±85、512±91、518±94 and 532±86; pancreas pathology score was 0; lung pathology score was 0; lung wet/dry ratio were 2.29±0.10、2.22±0.23、2.17±0.11 and 2.31±0.13; the activity of MPO in lung was 1.33±0.56、1.56±0.43、1.51±0.2andl.47±0.48,IL-1βin lung were 80.3±21.2、92.3±23.5、87.3±19.4 and 89.7±22.1; TNF-αin lung were70.6±21.2、67.6±19.8、68.4±19.5 and 74.5±22.6; ICAM-1 score in lung were 0.11±0.01、0.17±0.02、0.14±0.02 and 0.16±0.09 the relative optical density of PJNK was 0.65±0.12. In Group SAP at 3hr,6hr,12hr and 24hr four different steps serum amylase were 2506±215、3701±264、4101±411 and 3212±295; pancreas pathology score was 8.24±0.63、10.65±1.02、14.00±0.96 and 14.09±0.75; lung pathology score was 3.0±0.4、4.7±0.7、7.5±0.3 and 6.5±0.7; lung wet/dry ratio were 3.52±0.42、3.96±0.15、4.53±0.08 and 4.26±0.12; the activity of MPO in lung was 5.64±0.76、6.44±0.77、7.19±0.83 and 7.36±0.71IL-1βin lung were 211.2±19.3、315.4±34.2、399.2±43.2 and 353.4±38.3; TNF-αin lung were 348.5±42.8、433.5±63.4、394.3±56.3 and 365.3±39.9; ICAM-1 score in lung were 0.81±0.22、1.04±0.27、1.44±0.32 and 1.22±0.26; the relative optical density of PJNK was 1.03±0.22、1.24±0.17、1.38±0.21 and 1.28±0.24. In SAP group serum amylase, pancreas pathology score and lung pathology score were significantly higher than those in control group (P<0.05). We successfully set up the sever acute pancreatitis model in rats. Though JNK protein didn’t increased in 4 time steps, PJNK in SAP group significantly increased. In Group SP600125 at 3hr,6hr,12hr and 24hr four different lung pathology score were 2.1±0.3、3.2±0.2、6.0±0.7 and 4.0±0.3; lung wet/dry ratio were 3.08±0.09、3.54±0.22、3.35±0.16 and 3.01±0.38; the activity of MPO in lung was 3.36±0.36、4.27±0.52、5.16±0.26and 5.46±0.61、IL-1βin lung were 154.3±11.2、221.4±21.5、289.4±23.4 and 267.7±15.1; TNF-a in lung were 243.8±32.3、317.6±22.8、272.3±20.5 and 225.5±32.6;ICAM-1 score in lung were 0.59±0.18、0.77±0.20、0.84±0.23、0.93±0.31; the relative optical density of PJNK were 0.69±0.14、0.58±0.22、0.85±0.17、0.18±0.05. To compare with SAP group, lung dry/wet ratio, MPO activity, IL-1β, TGF-αand ICAM-1 in SP60025 group significantly decreased. Though JNK protein didn’t changed, PJNK protein significantly decreased and lung pathology score improved greatly (P<0.05). We found a significant correlation between the expression of PJNK and lung pathology score (P<0.05). Conclusion:We successful established acute pancreatitis associated lung injure in rat by retrograde pancreatic duct injection of 5%sodium taurocholate.JNK is a very important signal pathway in acute pancreatitis associated lung injure in rat. The activation of JNK is an early incident in the development of SAP. SP600125 can inhibit the activation of JNK, SP600125 can treat the acute pancreatitis associated lung injure and decrease the expression of inflammatory factor.Part two:Mechanisms of and effect of tashinone-ⅡA in acute pancreatitis associated lung injury in ratsAimThe aim of this part to investigate if tanshinone can treat APALI and the mechanism of it.Methods72 SD rats were individed into 3 groups. Group S:sham operated group,24 rats, after opening abdomen duodenum and pancreas were lightly touched several times, than abdomen was closed again. Group SAP:To lml/kg dose retrograde cholangiopancreatography sterile injection of 5%sodium taurocholate, induced SD rat model of acute hemorrhagic necrotizing pancreatitis.. Group tanshinoneⅡA:Acute hemorrhagic necrotizing pancreatitis in rats 30 minutes before induction to the dose of 20mg/kg by intraperitoneal injection of tanshinone IIA. Every group was divided into 3h,6h,12h and 24h stages. We killed the rats at different stages and collected serum, pancreas and lung. The severity of pulmonary edema was expressed by wet/dry. TNF-a, IL-1βin lung and serum amylase were detected in serum. HE staining was used to value the injury of pancreas and lung. Spectrophotometer was used to detected myeloperoxidase in lung tissue and expression of ICAM-1 was tested by immunohistochemistry in lung. Western-blot was used to detect the expression of JNK and PJNK in lung tissue.In Group tanshinone IIA at 3hr,6hr,12hr and 24hr four different steps lung wet/dry ratio were 3.17±0.29、3.24±0.22、3.49±0.26 and 3.21±0.38; the activity of MPO in lung was 20.13±0.39、19.65±1.26 and 24.84±0.71; TNF-αin lung were 253.8±27.3、337.6±22.3、312.3±22.5 and 311.5±32.6; ICAM-1 score in lung were0.63±0.15、0.82±0.19、1.01±0.23 and 0.96±0.31; lung pathology score were 2.3±0.3、3.5±0.2、6.3±0.7 and 5.1±0.3; the relative optical density of PJNK were 1.15±0.08、1.05±0.23、1.34±0.18、0.74±0.16. To compare with SAP group, the expression of PJNK, lung wet/dry ratio, MPO activity, IL-1β, TGF-βand ICAM-1 in lung were significantly decreased, and lung pathology score was also improved (P<0.05).Conclusion:Tanshinone IIA can block the expression of PJNK in lung of SAP rat, which could decrease injure of lung in SAP rat.更多还原