二次打击大鼠部分体液因子变化与肠系膜淋巴管结扎的影响

Effect of mesenteric lymphatic duct ligation on humoral factors in two-hit rats

目的:观察结扎肠系膜淋巴管对二次打击大鼠部分体液因子的影响,探讨肠淋巴途径在二次打击发病学中的意义。方法:实验于2002-03/10在河北北方学院病理生理学教研室及附属第一医学检验科完成。①实验分组:选择雄性Wistar大鼠45只,按随机数字表法分为结扎组、未结扎组、假手术组,每组15只。②实验方法:所有大鼠行腹部手术,结扎组行肠系膜淋巴管结扎术;未结扎组与假手术组仅在肠系膜淋巴管下穿线,不结扎。结扎组、未结扎组大鼠手术创伤后,以失血-脂多糖方法复制二次打击模型。③实验评估:手术创伤后24h进行。采用酶联免疫吸附法测定实验前后血清肿瘤坏死因子α、白细胞介素6含量;应用黄嘌呤氧化酶法测定超氧化物歧化酶活性、改良TBA微量法测定丙二醛水平;以硝酸还原酶法、化学显色法分别检测NO2-/NO3-浓度及一氧化氮合酶、诱导型一氧化氮合酶活性;以免疫比浊法经全自动生化分析仪检测血清免疫球蛋白IgG、IgM、IgA及补体C3、C4含量。结果:①3组大鼠存活率:实验24h期间,假手术组全部存活(15/15),未结扎组53.3%(8/15)存活,结扎组73.3%(11/15)存活,3组动物24h存活率差异有显著性意义(χ2=8.904,P<0.05)。②3组血清肿瘤坏死因子α、白细胞介素6水平比较:创伤含量高于实验前、假手术组、结扎组,差异有显著性意义(t=3.08,3.12,2.73,P<0.01;t=8.35,6.76,3.84,P<0.01)。③3组血清超氧化物歧化酶活性、丙二醛水平比较:创伤后24h,未结扎组超氧化物歧化酶活性显著低于实验前及假手术组、结扎组,差异有显著性意义(t=1.92,2.01,2.22,P<0.05);未结扎组与结扎组大鼠血清丙二醛含量均显著高于实验前及假手术组(t=3.64,2.67,1.42,P<0.05)。④3组血清一氧化氮及其合酶比较:创伤后24h,未结扎组大鼠血清NO2-/NO3-水平、一氧化氮合酶、诱导型一氧化氮合酶活性均显著高于假手术组,差异有显著性意义(t=3.54,P<0.05;t=2.05,P<0.05;t=2.57,P<0.01);结扎组大鼠血清NO2-/NO3-水平和一氧化氮合酶活性高于假手术组(t=2.10,P<0.05),NO2-/NO3-水平、诱导型一氧化氮合酶活性显著低于未结扎组(t=1.74,P<0.05;t=1.92,P<0.05)。⑤3组血清免疫球蛋白及补体水平比较:创伤后24h,未结扎组大鼠血清IgG、IgA、IgM、C3、C4均高于假手术组及结扎组,差异有显著性意义(t=2.98,P<0.01;t=2.64,2.11,2.54,2.23,P<0.05;t=2.92,2.28,P<0.01;t=1.73,2.02,1.72,P<0.05)。结论:肠系膜淋巴管结扎抑制了二次打击大鼠体液因子变化表现出的免疫反应以及过度炎症反应,肠系膜淋巴途径是二次打击过程中重要的发病环节。

AIM： To study the effect of mesenteric lymphatic duct ligation on part humoral factors in rats subjected to two-hit, so as to explore the significance of mesenteric lymphatic approach in pathogenesis after two-hit. METHODS： The experiment was conducted in Department of Pathophysiology and Clinical Laboratory, First Affiliated Hospital of Hebei North University from March to October 2002. ① Forty-five male Wistar rats were selected and randomly divided into mesenteric lymphatic duct ligation group, non-ligation group and sham operation group with 15 rats in each group. ②All rats received an abdominal operation, then the mesenteric lymphatic duct in ligation group was ligated, whereas the sham operation and non-ligation groups were only threaded with cotton below the mesenteric lymphatic duct without ligation. The two-hit model was established in ligation and non-ligation groups by hemorrhage and lipopolysaccharide （LPS） methods.③After 24 hours, all rats were anesthetized again and their left common carotid arteries were cannulated to facilitate blood withdrawal for serum sample. The contents of tumor necrosis factor-α （TNFα） and interleukin-6 （IL-6） in serum were determined before and after experiment by ELISA; the activity of superoxide dismutase （SOD） and content of malondialdehyde （MDA） were determined by xanthinoxidanse method and modified TBA microdetermination. The content of NO2^-/NO3^-, and activity of nitric oxide synthase （NOS） and inducible NOS （iNOS） were determined by nitrate reductase method, and chemical chromogenic reaction, respectively; the immunoglobulin G （IgG）, IgA, IgM and complement 3 （C3） and CA were determined by immunoprecipitation method before and after experiment. RESULTS：①Survival rate of rats： In the course of two-hit for 24 hours, the survival rates of rats in sham operation group, non-ligation group and ligation group were 100% （15/15）, 53.3% （8/15）, and 73.3% （11/15）, respectively; the differences were significantly （x^2=8.904,P 〈 0.05）. ②Twenty-four hours after operation, the contents of TNFα and IL-6 in non-ligation were obviously higher than that of pre-experiment, sham operation group and ligation group （t =3.08, 3.12, 2.73, P〈 0.01; t =8.35, 6.76, 3.84, P〈 0.01）. ③Twenty-four hours after operation, the activity of SOD in non-ligation g roup was remarkably lower than that of pre-experiment, sham operation group and ligation group （t =1.92, 2.01, 2.22, P 〈 0.05）, while the content of MDA was markedly higher than that of pre-experiment and sham opeartion group （t =3.64, 2.67, 1.42, P 〈 0.05）. ④The content of NO2^-/NO3^-, and activity of NOS and iNOS in serum in non-tigation group were obviously higher than sham operation group 24 hours after operation （t =3.54, P 〈 0.05; t =2.05, P 〈 0.05; t =2.57, P 〈 0.01）. The NO2^-/NO3^- content and NOS activity in ligation group were higher than that of sham operation group （t =2.10, P 〈 0.05）, and NO2^-/NO3^- content and iNOS activity were lower than non-ligation group （t =1.74, P〈 0.05; t =1.92, P 〈 0.05）. ⑤Twenty-four hours after operation, the serum IgG, IgA, IgM, C3 and C4 of non-ligation group were all significantly higher than those of sham operation group and ligation group （t =2.98, P 〈 0.01; t =2.64, 2.11, 2.54, 2.23, P 〈 0.05; t =2.92, 2.28, P 〈 0.01; t =1.73, 2.02, 1.72, P 〈 0.05）. CONCLUSION： Mesenteric lymphatic duct ligation inhibits the immune response and excessive inflammatory response resulting from humoral factor changes in rats subjected to two-hit. Mesenteric lymphatic approach is significant in morbidity after two-hit.