西沙比利、乳果糖及反义tlr_4表达质粒对肝硬化大鼠肠细菌转位的影响
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摘要
背景和目的:小肠细菌转位(intestinal bacterial translocation, IBT)是肝硬化伴发自发性腹膜炎(spontaneous bacterial peritonitis, SBP)的主要原因之一。但有关肝硬化肠细菌转位的机理尚不清楚,可能涉及多种机制,其中肠转运障碍,肠细菌过度生长(intestinal bacterial overgrowth, IBO)及肠通透性增加是造成IBT的最主要原因。诺氟沙星已广泛应用于肝硬化SBP的预防,但耐药菌株产生并发生IBT是最为严重的后果。普萘洛尔及其他益生菌制剂对肝硬化IBT的作用也有报道。本研究旨在:(1)了解实验性肝硬化大鼠IBT的发生率(2)探讨肝硬化IBT发生的机制(3)评价西沙比利、乳果糖及反义tlr4表达质粒对肝硬化大鼠IBT及肠粘膜形态的影响。
方法:肝硬化大鼠动物模型制作采用CCl4皮下诱导法,共12周。实验动物共分7组,分别为正常组(25只),肝硬化组(25只),肝硬化对照组(20只),西纱比利组(20只),乳果糖组(20只),诺氟沙星组(20只)及反义tlr4表达质粒组(20只)。所有动物均评价下列参数,即IBT、IBO、血清内毒素、小肠转运时间、小肠通透性、回肠粘膜病理及电镜以及肠、肝Toll样受体4(Toll-like receptor 4, TLR4)。
IBT定义为肝、脾、肠系膜淋巴结、血和/或腹水细菌培养阳性者。IBO定义为空肠细菌数超过正常大鼠的细菌数+2SD。内毒素测定采用鲎试验偶氮基质显色法。肠转运时间采用51Cr方法,以几何中心比值为代表。肠通透性测定采用99Tc-DTPA尿排泄率。回肠病理评价指标为绒毛高度,绒毛高/宽比值,绒毛密度及炎症指数。电镜观察指标为微绒毛、紧密连接及细胞间隙。TLR4受体表达采用RT-PCR及免疫组化。
结果:应用51Cr测定小肠转运时间进行了各种参数的比较,结果采用几何中心或几何中心比,较其他参数(小肠放射性分布,最远端放射性)更能客观地反应小肠转运时间,60?较30?更能拉开各组的差异。
在99Tc-DTPA测定小肠通透性方面,24h尿排泄率不受胃排空及肠转运时间的影响。肝硬化大鼠小肠通透性明显高于正常大鼠及肝纤维化大鼠,而肝纤维化大鼠无肠通透性的改变。
肝硬化大鼠IBT及IBO的发生率分别为48%和80%。而正常大鼠则无IBT发生。肝硬化大鼠99Tc-DTPA尿排泄率(16.1±7.6%)明显高于正常大鼠(1.62±0.8%,P<0.01)。伴IBT的肝硬化大鼠99Tc-DTPA尿排泄率为22.2±7.8%,明显高于无IBT的肝硬化大鼠(10.5±2.9%,P<0.01)。肝硬化大鼠的小肠转运时间(几
何中心比)为0.31±0.06,这较正常大鼠(0.49±0.08)明显减慢。伴IBT的几何中心比(0.24±0.06)又较无IBT的(0.38±0.11)更慢。与无IBO的肝硬化大鼠比较,伴IBO者IBT发生率更高,血清内毒素浓度更高,小肠转运时间更慢,肠通透性更大。肝硬化大鼠IBT菌株与IBO菌株基本一致。经西沙比利、乳果糖及诺氟沙星治疗后,各参数包括IBT、IBO、小肠通透性及小肠转运时间、血清内毒素均有改善。正常大鼠小肠tlr4很少,肝硬化大鼠肠tlr4上调。肝硬化大鼠肝脏的tlr4受体有下调现象,经西沙比利、乳果糖及诺氟沙星处理后,小肠tlr4明显下调,而肝脏tlr4下调不明显。
肝硬化大鼠肠粘膜绒毛有不同程度的损伤,包括绒毛断裂、缩短、高/宽比值减少、绒毛密度减少,炎症细胞浸润,经西沙比利、乳果糖及诺氟沙星处理后病理无明显改善。电镜显示肝硬化组的微绒毛缺失、斜切,细胞间隙扩大,紧密连接消失,经乳果糖、西沙比利及诺氟沙星处理后,肠超微结构有所改善。
反义tlr4表达质粒处理后的肝硬化大鼠的IBT发生率明显高于肝硬化大鼠,但肠转运时间及通透性均无明显改变。小肠病理也无明显改变。肠tlr4表达无明显下调,肝tlr4则表达明显抑制。
结论:
一、肝硬化大鼠IBT发生率明显增加,这可能与肠转运时间延长,IBO及小肠通透性增加有关。
二、肝硬化大鼠小肠粘膜有损伤,表现为绒毛断裂、减少,炎症细胞侵润,电镜下紧密连接缺失,细胞间隙扩大。
三、肝硬化大鼠肠tlr4表达增加,肝tlr4下调,这可能是对肠及血中内毒素的适应性反应,经西沙比利、乳果糖及诺氟沙星处理后,肠tlr4表达有下调,但肝tlr4表达下调不明显。
四、西沙比利、乳果糖及诺氟沙星能有效地防止IBT的发生,改善肠转运时间及肠通透性,降低内毒素水平,提示肠内毒素在致IBT、小肠转运时间延长及肠通透性增加方面起一定作用。
五、反义tlr4表达质粒增加IBT发生,但对其他参数无影响。肝tlr4明显抑制,肠tlr4表达无明显影响,提示外周tlr4在防止IBT中起着重要作用。
Background and aims:Intestinal bacterial translocation (IBT) was one of the factors leading to spontaneous bacterial peritonitis (SBP) in liver cirrhosis. However, the major mechanisms concerning bacterial translocation in cirrhortics had not been completely clarified, although it had been showed that factors such as prolonged intestinal transit, increased intestinal permeability and intestinal bacterial overgrowth (IBO) were involved in development of IBT. Norfloxacin had been widely used in prevention of SBP in liver cirrhosis, but the development of resistant-species could lead to more severe IBT and SBP. It has been also reported that propranolol and Arobiotics may be helpful to IBT.Aims of the study were to: (1) understand in the incidence of IBT in experimental cirrhotic rats (2) explore the mechanisms of IBT in cirrhostics (3) evaluate effect of cisapride, lactulose and anti-sense tlr4 plasmid on IBT and intestinal mucosa of cirrhotic rats.
Material and methods: Cirrhotic rats were induced by injection of 50% CCl4 solution subcutaneously for 12 weeks. Rats were divided into 7 groups, that is normal controls (n=25), cirrhotics (n=25), cirrhotic controls (n=20), cisapride group (n=20), lactulose group (n=20), norfloxacin (n=20) and antisense tlr4 plasmid group (n=20). All animals were assessed with following variables including IBT, IBO, serum endotoxin level, intestinal transit, intestinal permeability, pathological change and ultrastructure of ileum and tlr4 expression of intestine and liver.
IBT was defined as positive culture of bacteria in the liver, spleen, mesenteric lymph node (MLN), blood and/or ascites. IBO was regarded to be bacteria number in jejunum more than mean value, plus 2 SD of normal rats. Serum endotoxin was determined by limulus ameobatic chromogenic assay. Intestinal transit was measured by 51Cr as geometric center ratio in intestine. Intestinal permeability was evaluated by urinary excretion of 99Tc-DTPA. Index of pathological change of ileum mucosa included villus height, villus height/width ratio, villi density and inflammatory index.
Index of electronic microscopy included microvilli, tight junction and cell space. Expression of tlr4 in tissue were determined by RT-PCR and immune-biochemistry.
Results: By comparing several variables obtained from measurement of intestinal transit with 51Cr, geometric center ratio or geometric center were showed better index than other two variables (intestinal distribution of 51Cr and leading edge ratio activity of intestine), 60 min in measurement interval was better than 30 min.
24h urinary excretion of 99Tc-DTPA was not influenced by gastric emptying and intestinal transit. Intestinal permeability in cirrhotic rats was higher than normal and fibrotic rats, which was showed normal intestinal permeability.
IBT and IBO were found 48% and 80% respectively in cirrhotic rats and none in control rats. Urinary excretion of 99Tc-DTPA in cirrhotic rats (16.1±7.6%) was higher than normal rats (1.62±0.8%), and in cirrhotic rats with BT (22.2±7.8%) was greater than those without BT (10.5±2.9%). Intestinal transit (geometric center ratio) was significantly delayed in cirrhotic rats (0.31±0.06) and further more delayed in cirrhotic rats with BT (0.24±0.06) than those without BT (0.38±0.11). Cirrhotic rats with IBO had significantly higher rates of bacterial and endotoxin translocation, slower intestinal transit and higher intestinal permeability than those without IBO. The same organism was always found at the same time both in IBT and IBO. All of the tested variables including IBO, IBT, intestinal permeability, intestinal transit and serum endotoxin were improved after treatment of cisapride, lactulose and norfloxacin. Tlr4 was rarely expressed in the intestine of normal rats but up-regulated in the intestine and down-regulated in the liver of cirrhotic rats. Tlr4 expression was greatly down-regulated in the liver after treatment. In cirrhotic rats, intestinal villi were injured including ruptured and shortened villi, decreased height/width ra
方法:肝硬化大鼠动物模型制作采用CCl4皮下诱导法,共12周。实验动物共分7组,分别为正常组(25只),肝硬化组(25只),肝硬化对照组(20只),西纱比利组(20只),乳果糖组(20只),诺氟沙星组(20只)及反义tlr4表达质粒组(20只)。所有动物均评价下列参数,即IBT、IBO、血清内毒素、小肠转运时间、小肠通透性、回肠粘膜病理及电镜以及肠、肝Toll样受体4(Toll-like receptor 4, TLR4)。
IBT定义为肝、脾、肠系膜淋巴结、血和/或腹水细菌培养阳性者。IBO定义为空肠细菌数超过正常大鼠的细菌数+2SD。内毒素测定采用鲎试验偶氮基质显色法。肠转运时间采用51Cr方法,以几何中心比值为代表。肠通透性测定采用99Tc-DTPA尿排泄率。回肠病理评价指标为绒毛高度,绒毛高/宽比值,绒毛密度及炎症指数。电镜观察指标为微绒毛、紧密连接及细胞间隙。TLR4受体表达采用RT-PCR及免疫组化。
结果:应用51Cr测定小肠转运时间进行了各种参数的比较,结果采用几何中心或几何中心比,较其他参数(小肠放射性分布,最远端放射性)更能客观地反应小肠转运时间,60?较30?更能拉开各组的差异。
在99Tc-DTPA测定小肠通透性方面,24h尿排泄率不受胃排空及肠转运时间的影响。肝硬化大鼠小肠通透性明显高于正常大鼠及肝纤维化大鼠,而肝纤维化大鼠无肠通透性的改变。
肝硬化大鼠IBT及IBO的发生率分别为48%和80%。而正常大鼠则无IBT发生。肝硬化大鼠99Tc-DTPA尿排泄率(16.1±7.6%)明显高于正常大鼠(1.62±0.8%,P<0.01)。伴IBT的肝硬化大鼠99Tc-DTPA尿排泄率为22.2±7.8%,明显高于无IBT的肝硬化大鼠(10.5±2.9%,P<0.01)。肝硬化大鼠的小肠转运时间(几
何中心比)为0.31±0.06,这较正常大鼠(0.49±0.08)明显减慢。伴IBT的几何中心比(0.24±0.06)又较无IBT的(0.38±0.11)更慢。与无IBO的肝硬化大鼠比较,伴IBO者IBT发生率更高,血清内毒素浓度更高,小肠转运时间更慢,肠通透性更大。肝硬化大鼠IBT菌株与IBO菌株基本一致。经西沙比利、乳果糖及诺氟沙星治疗后,各参数包括IBT、IBO、小肠通透性及小肠转运时间、血清内毒素均有改善。正常大鼠小肠tlr4很少,肝硬化大鼠肠tlr4上调。肝硬化大鼠肝脏的tlr4受体有下调现象,经西沙比利、乳果糖及诺氟沙星处理后,小肠tlr4明显下调,而肝脏tlr4下调不明显。
肝硬化大鼠肠粘膜绒毛有不同程度的损伤,包括绒毛断裂、缩短、高/宽比值减少、绒毛密度减少,炎症细胞浸润,经西沙比利、乳果糖及诺氟沙星处理后病理无明显改善。电镜显示肝硬化组的微绒毛缺失、斜切,细胞间隙扩大,紧密连接消失,经乳果糖、西沙比利及诺氟沙星处理后,肠超微结构有所改善。
反义tlr4表达质粒处理后的肝硬化大鼠的IBT发生率明显高于肝硬化大鼠,但肠转运时间及通透性均无明显改变。小肠病理也无明显改变。肠tlr4表达无明显下调,肝tlr4则表达明显抑制。
结论:
一、肝硬化大鼠IBT发生率明显增加,这可能与肠转运时间延长,IBO及小肠通透性增加有关。
二、肝硬化大鼠小肠粘膜有损伤,表现为绒毛断裂、减少,炎症细胞侵润,电镜下紧密连接缺失,细胞间隙扩大。
三、肝硬化大鼠肠tlr4表达增加,肝tlr4下调,这可能是对肠及血中内毒素的适应性反应,经西沙比利、乳果糖及诺氟沙星处理后,肠tlr4表达有下调,但肝tlr4表达下调不明显。
四、西沙比利、乳果糖及诺氟沙星能有效地防止IBT的发生,改善肠转运时间及肠通透性,降低内毒素水平,提示肠内毒素在致IBT、小肠转运时间延长及肠通透性增加方面起一定作用。
五、反义tlr4表达质粒增加IBT发生,但对其他参数无影响。肝tlr4明显抑制,肠tlr4表达无明显影响,提示外周tlr4在防止IBT中起着重要作用。
Background and aims:Intestinal bacterial translocation (IBT) was one of the factors leading to spontaneous bacterial peritonitis (SBP) in liver cirrhosis. However, the major mechanisms concerning bacterial translocation in cirrhortics had not been completely clarified, although it had been showed that factors such as prolonged intestinal transit, increased intestinal permeability and intestinal bacterial overgrowth (IBO) were involved in development of IBT. Norfloxacin had been widely used in prevention of SBP in liver cirrhosis, but the development of resistant-species could lead to more severe IBT and SBP. It has been also reported that propranolol and Arobiotics may be helpful to IBT.Aims of the study were to: (1) understand in the incidence of IBT in experimental cirrhotic rats (2) explore the mechanisms of IBT in cirrhostics (3) evaluate effect of cisapride, lactulose and anti-sense tlr4 plasmid on IBT and intestinal mucosa of cirrhotic rats.
Material and methods: Cirrhotic rats were induced by injection of 50% CCl4 solution subcutaneously for 12 weeks. Rats were divided into 7 groups, that is normal controls (n=25), cirrhotics (n=25), cirrhotic controls (n=20), cisapride group (n=20), lactulose group (n=20), norfloxacin (n=20) and antisense tlr4 plasmid group (n=20). All animals were assessed with following variables including IBT, IBO, serum endotoxin level, intestinal transit, intestinal permeability, pathological change and ultrastructure of ileum and tlr4 expression of intestine and liver.
IBT was defined as positive culture of bacteria in the liver, spleen, mesenteric lymph node (MLN), blood and/or ascites. IBO was regarded to be bacteria number in jejunum more than mean value, plus 2 SD of normal rats. Serum endotoxin was determined by limulus ameobatic chromogenic assay. Intestinal transit was measured by 51Cr as geometric center ratio in intestine. Intestinal permeability was evaluated by urinary excretion of 99Tc-DTPA. Index of pathological change of ileum mucosa included villus height, villus height/width ratio, villi density and inflammatory index.
Index of electronic microscopy included microvilli, tight junction and cell space. Expression of tlr4 in tissue were determined by RT-PCR and immune-biochemistry.
Results: By comparing several variables obtained from measurement of intestinal transit with 51Cr, geometric center ratio or geometric center were showed better index than other two variables (intestinal distribution of 51Cr and leading edge ratio activity of intestine), 60 min in measurement interval was better than 30 min.
24h urinary excretion of 99Tc-DTPA was not influenced by gastric emptying and intestinal transit. Intestinal permeability in cirrhotic rats was higher than normal and fibrotic rats, which was showed normal intestinal permeability.
IBT and IBO were found 48% and 80% respectively in cirrhotic rats and none in control rats. Urinary excretion of 99Tc-DTPA in cirrhotic rats (16.1±7.6%) was higher than normal rats (1.62±0.8%), and in cirrhotic rats with BT (22.2±7.8%) was greater than those without BT (10.5±2.9%). Intestinal transit (geometric center ratio) was significantly delayed in cirrhotic rats (0.31±0.06) and further more delayed in cirrhotic rats with BT (0.24±0.06) than those without BT (0.38±0.11). Cirrhotic rats with IBO had significantly higher rates of bacterial and endotoxin translocation, slower intestinal transit and higher intestinal permeability than those without IBO. The same organism was always found at the same time both in IBT and IBO. All of the tested variables including IBO, IBT, intestinal permeability, intestinal transit and serum endotoxin were improved after treatment of cisapride, lactulose and norfloxacin. Tlr4 was rarely expressed in the intestine of normal rats but up-regulated in the intestine and down-regulated in the liver of cirrhotic rats. Tlr4 expression was greatly down-regulated in the liver after treatment. In cirrhotic rats, intestinal villi were injured including ruptured and shortened villi, decreased height/width ra
引文
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3.张顺财,王唯,任卫英,等。西沙比利对肝硬化大鼠肠细菌及内毒素转位的影响。中华肝脏病杂志,2003; 11: 539-41
4. Zhang S, Wang W, Ren W et al. Effects of lactulose on intestinal endotoxin and bacterial translocation in cirrhotic rats.Chin Med J (Engl). 2003; 116: 767-71
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6. Kuebler JF, Toth B, Rue LW et al. Differential alterations in intestinal permeability after trauma-hemorrhage. J Surg Res, 2003; 15: 198-204
7. Di Leo V, Venturi C, Baragiotta A et al. Gastroduodenal and intestinal permeability in primary biliary cirrhosis. Eur J Gastroenterol Hepatol. 2003;15: 967-73
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2. Pascual S, Such J, Esteban A et al. Intestinal permeability is increased in patients with advanced cirrhosis. Hepatogastroenterology. 2003; 50: 1482-6
3.张顺财,王唯,任卫英,等。西沙比利对肝硬化大鼠肠细菌及内毒素转位的影响。中华肝脏病杂志,2003; 11: 539-41
4. Zhang S, Wang W, Ren W et al. Effects of lactulose on intestinal endotoxin and bacterial translocation in cirrhotic rats.Chin Med J (Engl). 2003; 116: 767-71
5. Hashimoto N, Ohyanagi H. Effect of acute portal hypertension on gut mucosa. Hepatogastroenterology. 2002; 49: 1567-70
6. Kuebler JF, Toth B, Rue LW et al. Differential alterations in intestinal permeability after trauma-hemorrhage. J Surg Res, 2003; 15: 198-204
7. Di Leo V, Venturi C, Baragiotta A et al. Gastroduodenal and intestinal permeability in primary biliary cirrhosis. Eur J Gastroenterol Hepatol. 2003;15: 967-73
8. Zhang SC, Wang W, Ren WY et al. Effect of cisapride on intestinal bacterial and endotoxin translocation in cirrhosis. World J Gastroenterol, 2003; 9: 534-8
9. Gotloib L, Wajsbrot V, Cuperman Y et al. Acute oxidative stress induces peritoneal hyperpermeability, mesothelial loss,and fibrosis. J Lab Clin Med, 2004;143:31-40
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