硫化氢在肠淋巴液引流减轻失血性休克大鼠多器官损伤中的作用与机制

发布时间：2018-03-30 23:15

本文选题：失血性休克　切入点：肠淋巴液　出处：《河北北方学院》2013年硕士论文

【摘要】：大量研究表明,肠淋巴管结扎或肠淋巴液引流可减轻失血性休克后多器官的功能障碍与结构损伤,经淋巴途径的肠源性感染是重症休克后器官损伤的重要因素；气体信号分子硫化氢(hydrogen sulfide, H2S)加剧了失血性休克后的炎症反应,抑制H2S合成可降低器官损伤的程度。但在休克肠淋巴液介导器官损伤的这一过程中,H2S是否发挥作用,其具体作用机制如何?尚不清楚。为此,本研究应用胱硫醚-γ-裂解酶(cystathionine-y-lyase, CSE)抑制剂D,L-炔丙基甘氨酸(D, L-propargylglycine, PPG)和H2S供体硫氢化钠(sodium hydrosulfide hydrate, NaHS)作用于行肠淋巴液引流的大鼠,观察H2S对肠淋巴液引流改善器官功能与结构以及Toll样受体4(Toll-like receptor4, TLR4)、白介素10(interleukin10, IL-10)、IL-12、肿瘤坏死因子α(tumor necrosis factor a, TNFa)含量的影响,探讨H2S在肠淋巴液引流减轻休克大鼠器官损伤中的作用与机制。雄性Wistar大鼠30只随机均分为：假手术组(Sham)、休克组(Shock)、休克+引流组(Shock+drainage)、休克+引流+PPG组(Shock+drainage+PPG)、休克+引流+NaHS组(Shock+drainage+NaHS)。放血前30min,休克+引流十PPG组、休克+引流+NaHS组腹腔分别注射PPG (45ml.kg-1)、NaHS (28μmol.kg-1);其它三组大鼠腹腔注射等量的生理盐水(1ml.kg-1)。所有大鼠经戊巴比妥钠肌肉注射麻醉后,行股部手术,右股静脉注射肝素钠全身抗凝,右股动脉插管连接生物信号采集系统,连续监测平均动脉血压(mean artery pressure, MAP);左股侧动脉插管后连接注射器固定于抽注机上备放血用；腹部手术,剥离肠系膜淋巴管。待所有手术完成、稳定30min后,休克组、休克+引流组、休克+引流+PPG组、休克+引流+NaHS组大鼠经左股动脉匀速放血,10min内将MAP降至40mmHg,复制失血性休克模型。维持低血压60min后,将放出的全血与林格氏液按1：1混匀,经右侧股静脉输液复苏,30min完成；观察至输液结束后3h。假手术组进行相同的手术操作,但不放血、不输液。休克+引流组、休克+引流+PPG组、休克+引流+NaHS组在输液结束后,行肠系膜淋巴管插管,引流休克肠淋巴液至休克3h。各组大鼠在相当于输液结束后3h这一时间点,经腹主动脉取血,制备血浆,检测反映心肌细胞损伤的指标肌酸激酶同工酶(creatine phosphokinase isoenzyme, CK-MB)与乳酸脱氢酶-1(lactic acid dehydrogenase-1, LDH-1)、反映肝功能的指标天门冬氨酸氨基转移酶(aspartate aminotransferase, AST)、丙氨酸氨基转移酶(alanine aminotransferase, ALT)与总胆汁酸(total bile acid, TBA)、反映肾功能的指标尿素(Urea)、肌酐(Cre),以及总蛋白、H2S含量。取肺左叶,制备支气管肺泡灌洗液(bronchoalveolar lavage fluid, BALF),进行细胞计数以及蛋白检测,计算肺通透性指数(lung permeability index, LPI),同时取右肺上叶,测定肺干／湿(D/W)比。留取固定位置的肺、心肌、肝、肾组织,部分经多聚甲醛固定用于观察组织形态学变化；另一部分制备组织匀浆用于检测H2S含量、CSE活性以及TLR4、IL-10、IL-12、TNFα的含量。结果发现,肠淋巴引流可提高失血性休克大鼠液体复苏后一些时间点的MAP,尽管PPG没有明显的作用,但NaHS降低了肠淋巴引流对MAP的提升作用。组织形态学观察表明,假手术组大鼠肺、心肌、肝、肾组织结构基本正常,休克组大鼠肺、心肌、肝组织出现了形态学损伤,休克+引流组与休克+引流+PPG组大鼠肺、心肌、肝、肾的组织损伤较轻,NaHS加重了各器官的损伤程度。休克组大鼠BALF中细胞总数、LPI显著高于假手术组,休克+引流组BALF中细胞总数、LPI显著低于休克组,休克+引流+PPG组BALF中细胞总数、LPI显著低于休克+引流组,休克+引流+NaHS组BALF细胞总数、LPI显著高于休克+引流组；而大鼠肺组织的D/W比出现了相反的变化。同时可见,休克组大鼠血浆AST、ALT、TBA、LDH-1、CK-MB、Urea、Cre均显著高于假手术组,休克+引流组大鼠血浆的AST、ALT、TBA、 Urea、Cre含量显著低于休克组,休克+引流+PPG组血浆AST、ALT、TBA、Cre水平显著低于休克+引流组,休克+引流+NaHS组血浆AST、ALT、Urea、Cre含量显著高于休克+引流组。同时,研究发现,休克组血浆以及肺、肝、肾、心肌组织的H2S含量均显著升高,肠淋巴液引流显著降低了休克大鼠血浆以及肺、肝、肾、心肌组织H2S的含量,PPG进一步降低了休克+引流组大鼠这些组织H2S的含量,H2S供体NaHS则提高了休克+引流组大鼠的血浆以及肺、肝、肾、心肌组织H2S的含量。进一步的研究结果显示,休克组大鼠肺、肝、肾、心肌组织的TLR4、IL-10、IL-12、TNFα含量均显著升高,肠淋巴液引流则降低了肝匀浆IL-10、肺、肝匀浆IL-12含量以及肺、肝、肾、心肌组织的TNFα含量；休克+引流组大鼠给予CSE抑制剂PPG后,进一步降低了各器官组织的TLR4含量,肺、肾、心肌IL-10含量、肺组织IL-12含量、肺、肝、肾、心肌的TNFα含量均显著下降；H2S供体NaHS在增加休克+引流组大鼠肺、肝、肾、心肌的TLR4含量的同时,增加了肺、肝、肾组织IL-10、IL.12以及肺、肝、肾、心肌的TNFα含量。研究结果表明,H2S在休克肠淋巴液引流减轻肺、肝、心、肾等重要器官的功能障碍及结构损伤中起负面作用,其作用机制与TLR4引起的炎症反应加重有关。研究结果补充完善了“多器官损伤的肠淋巴途径学说”,为以淋巴为靶点,针对H2S的调节,进一步应用于危重病的临床防治奠定了实验基础。
[Abstract]:A large number of studies show that intestinal lymph duct ligation or lymph drainage can reduce the structural damage and multiple organ dysfunction after hemorrhagic shock, the intestinal lymph infection is an important factor in organ injury after severe hemorrhagic shock; HYDROGENSULFIDE (hydrogen sulfide H2S) exacerbated inflammation after hemorrhagic shock. Inhibition of H2S synthesis can reduce the extent of organ damage. But in shock lymph mediated the process of organ damage, whether H2S play a role in how the specific mechanism is not clear.?
Therefore, the research and application of cystathionine gamma lyase (cystathionine-y-lyase, CSE) inhibitor D, L- propargylglycine (D, L-propargylglycine, PPG) and H2S donor sodium hydrosulfide (sodium hydrosulfide, hydrate, NaHS) on mesenteric lymph drainage in rats, the effects of H2S on intestinal lymph drainage to improve organ function with the structure and Toll like receptor 4 (Toll-like, receptor4, TLR4), interleukin 10 (Interleukin10, IL-10), IL-12, tumor necrosis factor alpha (tumor necrosis factor A, TNFa) on the content of H2S in intestinal lymph drainage to reduce the role and mechanism of organ injury in shock rats.
30 male Wistar rats were randomly divided into sham operation group (Sham), shock group (Shock), the shock + drainage group (Shock+drainage), the shock + drainage group +PPG (Shock+drainage+PPG), the shock + drainage group +NaHS (Shock+drainage+NaHS). Blood 30min before the shock + drainage group ten PPG, the shock + drainage +NaHS intraperitoneal injection of PPG group respectively (45ml.kg-1), NaHS (28 mol.kg-1); the other three groups of normal saline was injected to rats (1ml.kg-1). All rats were anesthetized with pentobarbital sodium after intramuscular injection, femoral surgery, right femoral vein injection of heparin sodium anticoagulation, the right femoral artery cannula connected with biological the signal acquisition system, continuous monitoring of mean arterial pressure (mean, artery pressure, MAP); the left femoral artery after intubation side connected to the injector is fixed on the pump injection machine bloodletting; abdominal surgery, dissection of mesenteric lymph duct. After all the operation is complete, stable 30min, shock group, Hugh G + drainage group, the shock + drainage group +PPG, the shock + drainage group +NaHS rats via left femoral artery at MAP to 40mmHg 10min of the blood, the rat model of hemorrhagic shock. Maintain hypotension after 60min, will release the whole blood and Ringer's solution according to 1:1 mix, the right femoral vein infusion recovery. 30min; to observe 3h. infusion after the sham operation group were same operation, but no bleeding, no infusion. The shock + drainage group, the shock + drainage group +PPG, the shock + drainage in group +NaHS after infusion for mesenteric lymph duct intubation, drainage of shock lymph to shock 3h.
The rats in the equivalent of 3H infusion after this point in time, abdominal aortic blood plasma preparation, detection index of creatine kinase myocardial cell injury (creatine phosphokinase isoenzyme CK-MB -1 (lactic) and lactate dehydrogenase acid dehydrogenase-1, LDH-1), index of aspartate amino liver metastasis enzyme (aspartate aminotransferase, AST), alanine aminotransferase (alanine, aminotransferase, ALT) and total bile acid (total bile, acid, TBA), renal function indexes of urea (Urea), creatinine (Cre), and the total protein content of H2S. The left lung lobe preparation, bronchoalveolar lavage fluid (bronchoalveolar lavage fluid, BALF), detect cell count and protein, calculate the lung permeability index (lung permeability, index, LPI), at the same time, the upper lobe of the right lung, lung wet / dry (D/W) ratio. Take a fixed position The lung, myocardium, liver and kidney tissue were partly fixed by paraformaldehyde to observe histomorphology. The other part was prepared tissue homogenate to detect H2S content, CSE activity and TLR4, IL-10, IL-12 and TNF alpha content.
The results showed that the intestinal lymph drainage can improve the MAP some time points of hemorrhagic shock rats after resuscitation, while PPG had no obvious effect, but NaHS reduced the intestinal lymph drainage to increase MAP. It shows that the histomorphology of the sham rats myocardium, liver, lung, kidney tissue structure was normal, shock rats lungs, myocardium, liver tissue morphological damage, the shock + drainage group and shock + drainage group +PPG rat lungs, myocardium, liver, renal tissue damage was lower, NaHS increased the damage degree of various organs. The total number of cells in BALF shock group rats, LPI was significantly higher than that of sham operation group the total number of cells, the shock + drainage group BALF, LPI group was significantly lower than the shock, the shock + drainage group +PPG the total number of cells in BALF, LPI was significantly lower than the shock + drainage group, the total number of BALF cells in +NaHS shock + drainage group, LPI was significantly higher than that of the shock + drainage group; while the rat lung tissue than D/W Now the opposite change. At the same time visible, plasma AST, rats in group TBA, shock ALT, LDH-1, CK-MB, Urea, Cre were significantly higher than those in sham group, shock + drainage group rat plasma AST, ALT, TBA, Urea, Cre were significantly lower than in the shock group, shock + drainage of plasma AST, +PPG group ALT, TBA, Cre level was significantly lower than the shock + drainage group, the shock + drainage group +NaHS plasma AST, ALT, Urea, Cre were significantly higher than those in shock + drainage group.
At the same time, the study found that shock plasma and lung, liver, kidney, myocardial H2S content increased significantly and the intestinal lymph drainage significantly decreased the plasma shock rats, lung, liver, kidney, myocardial H2S content, PPG further decreased the content of H2S of the drainage group shock +. H2S donor NaHS can improve the shock + drainage group rat plasma and lung, liver, kidney, myocardial H2S content. Further research results show that the shock group rat lung, liver, kidney, myocardium TLR4, IL-10, IL-12, TNF were all significantly increased, decreased intestinal lymph drainage the liver homogenate IL-10, lung, liver IL-12 content and lung, liver, kidney, TNF alpha in myocardial tissue; the shock + drainage group rats were treated with CSE inhibitor PPG, to further reduce the content of TLR4, the organs and tissues of lung, kidney, myocardial IL-10 content, IL-12 content, lung and liver. Kidney, myocardium The level of TNF alpha decreased significantly. H2S donor NaHS increased the TLR4 content of lung, liver, kidney and myocardium in shock + draining group, and increased TNF, IL-10 and IL.12 in lung, liver and kidney as well as TNF, alpha in lung, liver, kidney and myocardium.
The results show that the H2S in shock lymph drainage reduce the lung, liver, heart, dysfunction and structural damage play a negative role in kidney and other important organs in the inflammatory reaction and its mechanism caused by TLR4 increases. The results of the multiple organ injury of intestinal lymphatic pathway theory ", as for the lymph the target, the regulation of H2S, and further applied to clinical prevention and treatment of critically ill lay the foundation.

【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R459.7

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