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P物质在急性胰腺炎相关肺损伤中的作用
The Effect of Substance P on Acute Pancreatitis Associated with Lung Injury in Rats
【作者】 刘迎迎;
【导师】 冯志杰;
【作者基本信息】 河北医科大学 , 内科学, 2008, 硕士
【摘要】 急性胰腺炎(acute pancreatitis, AP)是临床上常见的急腹症之一,特别是重症急性胰腺炎(severe acute pancreatitis , SAP)并发症多,预后凶险,病死率可高达30%~40%。急性肺损伤及其终末阶段急性呼吸窘迫综合征(acute respiratory distress syndrome, ARDS)是AP最常见而严重的并发症,为临床SAP病人死亡的重要原因之一。神经激肽P物质是神经源性炎症反应中的重要介质,在免疫反应中亦起着重要作用。近年来,P物质在AP与其相关性肺损伤中的作用已被人们认识,并逐渐引起重视。已知P物质是一种舒血管物质,能增加血管通透性,导致血浆渗出与水肿。P物质能够诱导白细胞黏附与浸润,还能调节多种细胞因子的产生并与T细胞增殖,B淋巴细胞产生抗体及单核巨噬细胞与肥大细胞释放炎性介质,多形核白细胞呼吸爆发等功能作用相关。P物质主要是通过与相应神经激肽受体(neurokinin-1 receptors, NK-1R)结合,介导G蛋白偶联的第二信号途径而发挥作用的。研究发现,在AP过程中,P物质释放明显增加,其相应NK-1R表达亦明显增加。反映肺组织血浆、炎细胞渗出程度的肺湿/干重率明显增加,作为反映中性粒细胞聚集程度指标的髓过氧化物酶(myeloperoxidase, MPO)活力亦明显增强。Lau等发现在雨蛙肽诱导的小鼠急性胰腺炎模型中通过腹腔给予P物质受体拮抗剂CP-96,345可以降低血清淀粉酶水平,减轻胰腺与肺组织的水肿、出血、炎细胞浸润等病理损伤,降低MPO活性,降低微血管通透性,从而为AP相关性肺损伤的治疗提供一新的途径。目的:本研究通过建立大鼠AP模型,并施予P物质受体拮抗剂L-703,606干预,观察肺组织的湿/干重比率,MPO活性,病理改变及P物质与NK-1R的表达情况,探讨其在AP炎症反应中的作用,探讨L-703,606对实验性AP的治疗作用及机制。方法:1动物模型的制备:选取雄性大鼠,以10%水合氯醛腹腔注射麻醉,取上腹正中作切口进入腹腔,用4号加工钝头头皮静脉针头于十二指肠乳头附近逆行插入胰胆管,以0.2 ml/min速度注入5%牛磺胆酸钠(0.1 ml/100g),推药后捏闭胰胆管入十二指肠处,观察3 min,剪除除结扎线,关腹。2实验分组:将雄性Sprague-Dawley大鼠54只,随机分为假手术组(SO组, n=18)、急性胰腺炎组(AP组, n=18)、急性胰腺炎治疗组(AP+ L-703,606组, n=18),后又以不同时间点3 h、6 h、12 h分为三个亚组,每个亚组为6只大鼠。AP+ L-703,606组于不同时间点造模前15 min经尾静脉预防性注射L-703,606(250 nmol /kg)。3肺组织湿/干重率测定:取左肺叶,以吸水纸吸干表面液体,称取湿重,置于60°C烤箱烤72 h至恒重,称取干重,计算湿/干重比率。4生化指标测定:取血清样本,采用全自动血清生化分析仪检测血清淀粉酶、脂肪酶,由河北医科大学第二医院生化室协助测定。5胰腺、肺组织学检查:采用石蜡切片,HE染色。6 MPO活力测定:取肺组织制成组织匀浆,用化学比色法测定,具体操作按南京建成生物工程研究所提供的测试盒说明书进行。7免疫组织化学染色:脱蜡至水,抗原热修复后,用3%过氧化氢孵育15 min。封闭一抗4°C过夜,阴性对照用PBS代替一抗。先后滴加二抗、三抗各15 min,后用3,3-二氨基苯联胺(DAB)显色,复染后封片。以细胞浆内呈现黄褐色颗粒为阳性表达。8 western blot: 100 mg肺组织经超声细胞粉碎机彻底粉碎,加入1 ml细胞裂解液,充分裂解,提取NK-1R膜蛋白(具体操作按说明书),取20μg与上样缓冲液充分混合,置于SDS变性聚丙烯酰胺凝胶电泳,转移至NC膜,以5%的脱脂奶粉封闭,一抗(1:250) 4°C孵育过夜,以相应的二抗室温下孵育2 h,抗体检测按Santa Cruz公司试剂盒说明书进行。结果:1大鼠胰腺的病理学变化:肉眼和光镜所见SO组胰腺及周围组织结构始终大致正常;AP组和AP+L-703,606组随着时间的推移,胰腺及周围组织水肿、出血和坏死面积逐渐扩大,程度逐渐加重,腹水也增多。2大鼠肺组织的病理学变化:肉眼和光镜所见SO组肺组织结构始终大致正常;AP组随着时间的推移,肺组织水肿、充血、出血,肺萎陷与不张和炎细胞聚集面积逐渐扩大,程度逐渐加重;而AP+ L-703,606组较AP组的组织结构损伤相对较轻。3血清淀粉酶的变化:在各时间点,SO组无明显变化,AP组及AP+ L-703,606组与SO组比较均明显升高(P<0.05)。4血清脂肪酶的变化:在各时间点,SO组无明显变化,AP组及AP+ L-703,606组与SO组比较均明显升高(P<0.05)。AP+ L-703,606组与AP组比较,12h时间点的脂肪酶升高(P<0.05)。5肺组织湿/干重比率:在各时点,SO组肺组织湿/干重比率在不同时间点无明显变化,与SO组比较,AP组与AP+ L-703,606组在各时点均明显升高(P<0.05),与AP组比较,AP+ L-703,606组在各时间点肺组织湿/干重比率明显降低(P<0.05)。6 MPO活力的变化:SO组MPO活力在各时间点无明显差异,与SO组比较,AP组与AP+ L-703,606组MPO活力在各时点明显增强(P<0.05)。与AP组比较,AP+ L-706, 303,在各时间点MPO活力明显降低(P<0.05)。7 P物质在肺组织中的表达:SO组肺组织有弱的P物质表达,AP组与AP+ L-703,606组,在血管内皮细胞,肺泡上皮细胞,炎性细胞及肺间质均有明显增强的P物质表达,免疫组化评分显示AP+L-703,606组表达强度低于AP组(P<0.05)。8 NK-1R在肺组织中的表达:密度扫描分析示SO组肺组织有弱的NK-1R表达,AP组与AP+ L-703,606组NK-1R表达明显增强(P<0.05),AP+ L-703,606组与AP组比较NK-1R表达无明显改变。结论:1逆行胆胰管内注射5%牛磺胆酸钠,通过检测血清淀粉酶、脂肪酶升高,胰腺组织HE染色观察到胰腺及周围组织水肿、出血和坏死,肺组织水肿、充血、出血、萎陷、不张与炎细胞浸润等表现,证实大鼠AP相关性肺损伤模型制备成功。2在AP大鼠肺组织中,P物质与其受体NK-1R的表达均明显高于对照组,表明P物质介导的信号转导途径在AP相关性肺损伤的发病机制中可能起作用。3 L-703,606是强有力的NK-1R拮抗剂,能降低P物质表达,降低肺组织湿/干重比率与MPO活力,改善肺组织病理损伤,认为L-703,606对大鼠AP相关肺损伤起到有效的治疗作用。
【Abstract】 Acute pancreatitis (AP) is a a frequent acute abdomen in clinic. Especially severe acute pancreatitis (SAP) has multiple complications and a hazardous prognosis. It results in mortality rates ranging from 30 to 40%. Acute lung injury as well as acute respiratory distress syndrome (ARDS) as its late stage is the most common and severe complication in AP. It is also one of the important death reasons in clinical patients with SAP.Substance P as one of neurokinins is a important mediator in neurogenic inflammation. It plays a key role in immunereaction, too. For the past few years, the effect of substance P in AP and associated acute lung injury has been recognized and gradually evaluated. As far as I know, substance P is a kind of substance which can relax vessel. It can increase microvascular permeability, lead to plasm infiltration and oedema. Substance P can induce the adhesion and infiltration of neutrophils. it correlates to the effects of regulating the production of multiple cytokines, T lymphocyte proliferation, B lymphocyte producting antibody, mononuclear macrophage and mast cell realeasing inflammatory media, polymorphonnuclear leucocytes respiratory bursting. Substance P plays a role mainly via G-protein link-coupled the second signal pathway by binding to accord neurokinin-1 receptor(NK-1R). On the basis of current research findings, the production of substance P and the expression of NK-1R obviously increase in AP. The ratio of wet/dry weight which reflects the exudation degree plasm and inflammatory cell also increases. the myeloperoxidase(MPO) activity as the index which reflects the accumulated degree of nutrophils raises, too. H.Y. Lau et al found CP-96,345--the receptor antagonist of substance P which is administrated by intraperitoneal injection in AP mice was induced by intraperitoneal injection of caerulein could degrade the level of amylase, relieve the pathology damage such as oedema, hemorrhage, inflammatory cell infiltration, depress the activity of MPO, degrade the microvessel permeability. Thereby it provides a new pathway for the therapy of acute pancreatitis.Objective:To investigate pulmonary wet/dry weight ratio, MPO activity, pathology, expression of substance P and o NK-1R and their possible roles in inflammatory reaction of AP performed by establishing AP model and admistrating L-703,606 therapy, to explore the protective effect and mechanism of L-703,606 on experimental AP in rats.Method:1 Animal model: The experiment was performed in Sprague-Dawley rats under anesthesia injected intra-peritoneally with 10% chloral hydrate. Then, the abdomen median incision was performed. The pancreatic gland was exposed, bile duct was injected antidromicly by microinjector with 5% Sodium Cholate (0.1 ml/100g) at the speed of 0.2 ml/min. Then the bile duct entering duodenum was pinched. 3 minutes later, the ligature was cut off and abdomen was sutured.2 Animal groups: All Sprague-Dawley rats (n=54) were divided into three groups at random: sham-operation group (n=18), AP group (n=18), and AP+ L-703,606 group (n=13). Then every group was divided into 3 subgroup (n=6) at different time points: 3 h、6 h、12 h. L-703,606(250 nmol/kg) were administrated prophylacticly via vena caudalis injection in AP+ L-703,606 group 15 min before making the animal models.3 The wet/dry weight ratio : the surface fluid of the left lobe of lung obtained was blotted with absorbent paper, then it was weighed and put into baker of 60°C for 72 h, it was weighed again, then the wet/dry weight rate was caculated .4 Mensuration of biochemical parameters: The levels of serum amylase and limpse were determined respectively by automatic biochemical analyzer.5 Pancreatic gland and Pulmonary histological analysis: Paraffin section was stained by hematoxylin-eosin (HE).6 Mensuration of MPO activity: The homogenate of lung tissue MPO, according to the direction of MPO kit that was offered by Nanjin Jiancheng Bioengineering Institute.7 Immunohistochemical staining of substance P: After dehydration and antigen heat plerosis, the sections were incubated with 3% hydrogen peroxide for 15 min. The sections were dripped with goat serum for 11 min, then incubated with first antibody at 4°C overnight. In negative control group, PBS replaced first antibody. Dripping successively the second antibody and the third antibody, which were kept 15 min respectively. The results were visualized by using 3,3-diaminobenzidine (DAB) as chromogen. At last, the sections were redyed with hematoxylin.8 Western blotting: a piece of lung tissue about 100 mg was homogenized thoroughly by ultrasonic disintegrator, then 1ml lysis buffer was added to it for extraction of the protein. (the process according to the instruction of kit ) the protein about 20μg was mixed with loading buffer, then it was performed SDS denaturing polycrylamide gel electrophoresis, Successively it was thansferred to NC film. then it was incubated with first antibody(1 : 250) at 4°C overnight, successively the second antibody for 2 h. the assay of antibody according to the instruction of kit that was offered by Santa Crua company .Results:1 Histological findings: It was seen by naked eyes and light microscope that the pancreatic gland and its surrounding tissue of sham-operation group was approximatly normal; areas and degrees of dropsy, hemorrhage and necrosis of pancreatic gland and its surrounding tissue were being more and more severe, and the seroperitoneum was growing in AP group and AP+L-703,606 group following the lapse of time.2 Histological findings: It was seen by naked eyes and light microscope that the lung tissue of sham-operation group was approximatly normal; areas and degrees of dropsy, congestion, hemorrhage, detelectasis, atelectasis, and flammatory cells infiltration of lung tissue were becoming more and more severe following the lapse of time in AP group ; compared with AP group, the destruction of tissue in AP+ L-703,606 group was ligther.3 Serum amylase: There was no significant difference in sham-operation group at different time points. Compared with sham-operation group, the levels of serum amylase were increased in AP group and AP+ L-703,606 group (P<0.05).4 Serum lipase: There was no significant difference in sham-operation group at different time points. Compared with sham-operation group, the levels of serum lipase were increased in AP group and AP+ L-703,606 group (P<0.05). Compared with AP group,the levels of serum lipase were increased at 12 h in AP+ L-703,606 group (P<0.05).5 The wet/dry weight ratio: There was no significant difference in sham-operation group at different time points. Compared with sham-operation group, the wet/dry weight rate were increased in AP group and AP+ L-703,606 group (P<0.05). Compared with AP group, the wet/dry weight ratio in AP+ L-703,606 group were declined at different time point (P<0.05).6 The activity of MPO: There was no significant difference in sham-operation group at different time points. Compared with sham-operation group, the activity of MPO were increased in AP group and AP+ L-703,606 group (P<0.05). Compared with AP group, the activity of MPO in AP+ L-703,606 group was obviously decreased in AP group at different time points (P<0.05).7 The expression of substance P: In sham-operation group, substance P was hardly any expression. We found an overexpression of substance P in the vascular endothelial cell , alveolar epithelial cell, inflammatory cells,and pulmonary interstitium in AP group and L-703,606 group. Compared with AP group , It showed that the expression of substance P was decreased in AP+ L-703,606 group by immunohistochemistry score (P<0.05).8 The expression of NK-1R: There were no significant differences in sham-operation group at different time points. Compared with sham-operation group, It showed that the expression of NK-1R were increased at all time points by Optical density analysis in AP group and AP+ L-703,606 group(P<0.05). Compared with AP group, the expression of NK-1R had no significant differences in AP+ L-703,606 group.Conclusions:1 On the basis of increasing of serum amylase and lypase and those histological findings of areas and degrees of oedema, hemorrhage and necrosis of pancreas and periph-tissues and the degrees of congestion, hemorrage, detelectasis, atelectasis, and flammatory cells infiltration of lung tissue from this study, we concluded that AP associated lung injury model group has been successfully manufactured by bile duct injecting antidromic with 5% Sodium Cholate.2 Compared with control group the expression of substance P and NK-1R were conspicuously increased in the lung tissue of pancreatitic rats, it is concluded that the second signal pathway via substance P possiblely plays an important role in the pathogenesis of AP.3 L-703,606 as one of the drastic antagon of substance P can decrease the expression of substance P, ameliorate the changes of pathobiology, downgrade the activity of MPO. L-703,606 plays an important role in therapeutic effectiveness in AP.
【Key words】 acute pancreatitis; lung injury; substance P; neurokinin-1 receptor; myeloperoxidase;
- 【网络出版投稿人】 河北医科大学 【网络出版年期】2009年 01期
- 【分类号】R576;R563.8
- 【被引频次】1
- 【下载频次】103