摘要
目的：内毒素是一种广泛分布于革兰阴性菌及其它微生物如衣原体、立克次体、螺旋体等的细胞壁成分中的脂多糖，当这些微生物死亡后或处于高速增殖期时，就会释放大量内毒素，当血液中内毒素的含量超过0.1EU/ml时，即可引起内毒素血症，并导致肝脏持续性损害，从而形成肝病内毒素血症。血液中长期反复内毒素升高可引起细胞因子级联反应，加速肝病的发展，从而诱发致死性感染性休克、多器官功能衰竭、弥漫性血管内凝血等病死率极高的临床并发症，严重影响患者的预后。肝病内毒素血症的发生率很高，尤其是肝硬化患者，常常伴有肠源性内毒素血症，其发生率为84.3%。肝硬化失代偿期患者，肠道菌群紊乱、比例失调是引起内毒素血症的主要原因之一。造成内毒素血症患者肝功能受损的细胞因子，主要由巨噬细胞释放，包括肿瘤坏死因子(TNF)、白介-1(IL-1α)、白介-6(IL-6)、集落刺激因子、生长因子和趋化因子等。本课题旨在初步探讨在常规保肝治疗的基础上辅助应用双歧三联活菌制剂治疗肝硬化内毒素血症的疗效，并观察治疗前后血浆中内毒素以及IL-1α、IL-6、TNFα水平的变化，为临床内毒素血症治疗提供可靠的具有指导意义的临床实验理论依
据。
方法：随机选择典型肝硬化失代偿期患者60例，男性50例，年龄在39岁～71岁，女性10例，年龄在50岁～69岁。排除合并感染、肝癌及其他重要器官衰竭的患者，排除在治疗中应用抗生素与其他影响肠道菌群制剂的患者。将60例患者随机分为两组，一组为常规治疗组（对照组，control group）给予肝硬化常规治疗：低盐饮食、保肝、利尿、对症支持治疗；一组为双歧三联活菌制剂治疗组(实验组，BTV group)，在常规治疗的基础上加服双歧三联活菌胶囊，每次两粒，日服三次，疗程均为10天。对照组和实验组均在用药前和用药10天后及时抽取空腹静脉血，分离血浆，应用鲎试剂动态比浊法准确定量测定内毒素含量，全部过程在无菌条件下完成；应用酶联免疫技术测定IL-1α、IL-6、TNFα的含量。实验前、后应用全自动生化仪检测有关生化指标（ALT 、AST、BUN 、Cr）。
结果：（1）.治疗前两组间各项指标的比较：实验组治疗前血浆中内毒素、IL-1α、IL-6、TNFα、ALT、AST、BUN和Cr的值分别为0.297EU/ml、13.39pg/ml、12.94pg/ml、8.96pg/ml、48.30mg/ml、61.85mg/ml、5.59mg/ml和60.63mg/ml，对照组治疗前以上指标的含量分别为0.255EU/ml、11.86pg/ml、11.45pg/ml、10.94pg/ml、50.30mg/ml、54.78mg/ml、5.29mg/ml和59.59mg/ml，各项指标的含量在治疗前两组间比较均无显著差异，经t检验，t（P）值分别为0.740（0.462）、1.928（0.059）、0.614（0.541）、-1.325（0.190）、-0.538
（0.593）、1.446（0.153）、0.696（0.489）和0.957（0.343），二者间差异无统计学意义；（2）.实验组治疗前后各项指标的比较：实验组用药后测定血浆中内毒素、IL-1α、IL-6、TNFα、ALT、AST、BUN和Cr分别为0.132EU/ml、8.32pg/ml、4.96pg/ml、4.43pg/ml、38.78 mg/ml、47.87mg/ml、4.97mg/ml和55.45mg/ml与治疗前比较，各项均有明显降低，经t检验，t（P）值分别为5.067（0.000）、5.251（0.000）、3.842（0.001）、6.122（0.000）、8.550（0.000）、8.248（0.000）、1.675（0.105）和7.697（0.000），P值除BUN这一指标外均小于0.05，差异有统计学意义；（3）.对照组治疗前后各项指标的比较：在对照组治疗前血浆中内毒素含量为0.255 EU/ml，而治疗后其值为0.326 EU/ml，与治疗前比较，略有上升趋势，经t检验，t=-1.416，P=0.167，差异无没有统计学意义；对照组治疗后血浆中IL-1α、IL-6、TNFα的含量分别为11.84 pg/ml、12.37pg/ml、11.89pg/ml，与治疗前各项指标的含量相比均无明显变化，经配对t检验，t（P）值分别为0.021（0.983）、-0.945（0.353）、-0.927（0.362），差异没有统计学意义；对照组治疗后血浆中ALT值为44.56mg/ml，与治疗前相比有明显变化，经配对t检验，t值为2.231，P值为0.034，小于0.05，差异有统计学意义；而对照组治疗后血浆中AST的值为46.62mg/ml，与治疗前相比略有变化，经配对t检验，t值为1.143，P值为0.262，大于0.05，差异无统计学意义。（4）.治疗后两组间各项指标的比较：治疗后实验组血浆中内毒素、IL-1α、IL-6、TNFα的检测值均比对照组低，经t检验，
P值均小于0.05，差异有统计学意义；而治疗后实验组血浆中ALT、AST与对照组比较没有明显差别，经t检验，P值均大于0.05，差异没有统计学意义。
结论:研究证实在常规保肝治疗的基础上辅助应用双歧三联活菌制剂，与单纯常规保肝治疗比较，可以明显降低血浆中内毒素的水平，减少生物活性介质IL-1α、IL-6和TNFα的释放，改善肝功能，使临床症状有所好转，具有重要的临床实用意义
Objective: Endotoxin is a kind of lipopolysaccharide in the cell wall layer of gram negative bacterium and other microorganism including chlamydia, rickettsia, spirochete or something else. When they died or propagated actively amount of Endotoxin will be released. And when the plasma Endotoxin level was beyond 0.1EU/ml it is called endotoxemia which can lead to liver injury increasingly in patients undergoing liver disease. High level endotoxin in plasma may up-regulate some cytokines that aggravate liver disease. With the development of endotoxemia the patients undergoing liver disease will have a bad prognosis because serious septic shock, multiple organ failure (MOF), disseminated intravascular coagulation (DIC) or other serious complications. Endotoxemia is a frequent finding in liver disease patients especially liver cirrhosis patients, the incidence of which is high as 84.3%. Generally, endotoxemia in liver cirrhosis patients were intestinal
endotoxemia (IETM). It is one of the main reasons of endotoxemia that intestinal flora of the liver cirrhosis patients in the uncompensated period were disturbance. The cytokines including tumor necrosis factor alpha, interleukin-1 alpha, interleukin-6, interferon, colony stimulating factor, transforming growth foctor, chemotactic factor and other cytokines were secreted by mononuclear phagocye, which can injury the liver function of patients with endotoxemia. The study is conducted to investigate the clinical significance of Bifid Triple Viable that was used to treat endotoxemia of Liver cirrhosis on the base of general therapy, and to observe the change of endotoxin and some cytokines (IL-1α, IL-6, TNFα) in plasma during the treatment, and also to do some useful work which are helpful to the theory for clinic using.
Methods : 60 hospitalizing patients with liver cirrhosis in the uncompensated period (50 male patients, aged 39-71 years; 10 female patients, aged 50-69 years) were included in present study. The patients of the study excluded those who was companied with serious infection, hepatoma or major organ failure, and also excluded those who are applying antibiotic or other drugs that can affect whose intestines flora. The patients satisfied the conditions were separated into two groups at random. One is control group (n=30) treated with general therapy, another is BTV group (n=30) treated with Bifid Triple Viable on the base of
general therapy. In BTV group the Bifid Triple Viable capsule was took orally continuously for 10 days in the dose of two capsules one time, 3 times one day. The fasting venous blood of patients in both of the groups was phlebotomized before therapy and after 10 days therapy. The plasma was separated to detect endotoxin accurately by dynamic limulus lysate nephometric assay, and to detect the level of IL-1α, IL-6 and TNFα by enzyme-linked immunosorbent assay (ELISA). Before and after the therapy ALT, AST, BUN and Cr were detected by biochemistry method.
Results : (1) The compare of all the items before therapy: The endotoxin, IL-1α, IL-6, TNFα, ALT, AST, BUN and Cr in BTV group were 0.297 EU/ml, 13.39 pg/ml, 12.94 pg/ml, 8.96 pg/ml, 48.30 mg/ml, 61.85 mg/ml, 5.59 mg/ml and 60.63 mg/ml respectively. That in control group were as follows: endotoxin (0.255 EU/ml), IL-1α (11.86 pg/ml), IL-6 (11.45 pg/ml), TNFα (10.94 pg/ml), ALT (50.30 mg/ml), AST (54.78 mg/ml), BUN (5.29 mg/ml) and Cr (59.59 mg/ml) respectively. There were no significant differences when the items in control group compared correspondingly with that in BTV group (p>0.05). (2) The compare of all the items before and after therapy in BTV group: The endotoxin, IL-1α, IL-6, TNFα, ALT, AST, BUN and Cr in BTV group after therapy were 0.132 EU/ml, 8.32 pg/ml, 4.96 pg/ml, 4.43 pg/ml,
38.78 mg/ml, 47.87 mg/ml, 4.97 mg/ml and 55.45 mg/ml respectively. Compared correspondingly with that before therapy the differences were significant (p<0.05) except BUN (p>0.05). (3) The compare of all the items before and after therapy in control group: The
引文
1 中华医学会.内毒素血症诊断与治疗[M].中华医学会继续教育部出版.2000，8~9.
2 赵龙凤, 韩德五. 肝病患者内毒素血症的临床意义[J]. 世界华人消化杂志. 1999, 7(5): 391~393
3 Mathison JC, Ulevitch RJ. Clearance, tissue distribution and cellular localization of intervenously injected LPS in rabbits. J Immunol, 1979, 123: 2133~2143
4 Fox ES, Thomas P, Broitman SA. The macrophage 80 Kda endotoxin binding protein is not present on rat kuptter cells. Gastroenterology, 1990, 98: 586~591
Lin RS, Lee SD, Tsai YT et al. Endotoxemia in patients
5 with chronic liver diseases:relationship to severity of liver diseases, presence of esophageal varices, and hyper -dynamic circulation. J Hepatol. 1995, 22:165~172
6 黄自平. 肠源性内毒素血症在肝硬化失代偿中的作用及其处理.胃肠病学和肝病学杂志. 1997, 6(1) 9~22
7 Wang XD, Soltesz V, Molin G, et al. The role of oral administration of oatmeal fermented by Lactobacillus reuteri R21C on bacterial translocation after liver failure induced by subtotal liver resection in the rat, Scand J Gastro-enterol. 1995, 30: 180~185
8 陆付耳, 李鸣真, 叶望云等. 论内毒素血症的治疗对策[J]. 中国危重病急救医学. 2000, 12(10): 579~580
9 Liehr H, Englisch G, Rasenack U et al. Lactulose -adrug, with antiendotoxin effect. Hepato-Gastroentrology 1980, 27; 356
10 Takeyama N, Takagi D Matsuo N, et al. Altered hepatic fatty acid metabolism in endotoxicosis: effect of L-Carnitine on Survival. Am Jphysiol. 1989,256: E31
11 吴锐, 伍津津. 中医药治疗内毒素血症进展及对策[J].实用中西医结合杂志. 1994, 7(12): 749~751
12 邓文龙. 中药的抗内毒素作用研究进展[J]. 中药药理与临床. 1988, 4(3)：58
13 Schafer C, Greiner B, Landig J et al. Decreased endotoxinbinding capacity of whole blood in patients with alcoholic liver disease. J Hepatology. 1997, 26: 567~573
14 华静, 李继法, 曾民德, 等. 双歧三联活菌对肝硬化患者肠道菌群的调节作用及其对内毒素血症的影响[J]. 中华消化杂志, 2000, 20(2): 136～138
15 Tsan MF, Clark RN, Goyert SM et al. Induction of TNF -alpha and MnSOD by endotoxin: Role of membrane cd14 and toll-like receptor-4. Am J Physiol Cell Physion，2001, 80(6): C1422～1430
16 Guidet B, Staikowsky F, Offenstadt G. interleukins and TNF in septic shock. Rev Prat, 1993, 43: 13~17.
17 Chesta J, Deflippic C. Abnormalities in proximal small bowel motility in patients with cirrhosis. Hepatology. 1993, 17: 828
18 Luster MI, Germolec DR, Yoshida T et al. Endotoxin induced cytokine gene expression and excretion in the liver.Hepatology. 1994, 19: 480~488
19 Lee FY, wang SS, Yang MC, et al. Role of endotoxaemia in hyperdynamic circulation in rats with extrahepatic or intrahepatic portal hypertension. J Gastro -enterol Hepatol. 1996, 11: 152~158
20 华静, 李继强, 曾民德. 肝硬化患者肠道菌群的研究. 中华肝脏病杂志. 1998, 6: 79~81
21 Bernet MF, Brassart D, Neeser JR, et al. Lactobacillus acidophilus LA1 binds to cultured human and cell invasion by enterovirulent bacteria. Gut. 1994, 35: 483~489
Hoofnagle JH, Carthers RL, Shapiro e, Ellis LM, Bell
22 MV. Fulminant hepatic failure: Summary of workshop, Hemepatologyem, 1995, 21: 240~247