急性心理应激对胃肠功能影响的初步研究
|
摘要
目的：研究急性心理应激对胃肠粘膜形态和屏障功能以及结肠运动的影响。
方法：
55只雄性wistar大鼠经过3小时限动束缚后随机分入4个组：A组(3小时即时处死组)14只,B组(束缚后休息3小时处死组)14只,C组(束缚后休息9小时处死组)14只,D组(束缚后休息21小时处死组)13只；另外13只大鼠禁食禁水3小时作为对照；造模其间计数大鼠排便数。在各自时间点解剖大鼠采集标本。肉眼评估胃粘膜、回肠粘膜、结肠粘膜形态；光镜下对胃、回肠、结肠粘膜病理形态进行观察；免疫组化法分析回肠、结肠粘膜紧密连接Occludin蛋白的表达；TUNEL法检测回肠上皮细胞凋亡；检测血浆二胺氧化酶(DAO)活性和D-乳酸(D-lac)浓度。
结果：
束缚成功造成大鼠急性心理应激；急性心理应激导致大鼠胃粘膜广泛糜烂和散在出血；急性心理应激组紧密连接蛋白Occludin蛋白染色变淡,平均光密度略有降低,但与对照组差异不显著；急性心理应激组回肠粘膜上皮细胞凋亡指数明显增高。和对照组比较,急性心理应激各组血浆DAO活性和D-lac浓度未见明显增高；急性心理应激导致大鼠结肠运动增强。
结论：
1本束缚方法制造心理应激模型是成功的。
2本心理应激模型可导致大鼠胃肠粘膜从形态学损害到屏障功能的损害。
3本研究中大鼠肠粘膜屏障功能损害可能以细胞凋亡增加为主要途径,细胞间隙紧密连接破坏为次要途径。
4急性心理应激中大鼠结肠运动增强。
Objective:
To observe gastrointestinal membrane barrier function and colonic dynamics in wistar rats under acute psychological stress.
Methods:
Fifty-five male wistar rats under3hours of restraint stress were randomly divided into4groups, respectively having0hours (14),3hours (14),9hours (14),21hours (13) of recovery. Additional13rats which subjected to3hours of fasting to food and water were treated as control. Feces output were collected and counted after the3hours of stress or fasting. Rats were anesthetized using pentobarbital at respective time point. Blood plasma was collected and stored at-80℃until serving for detection of diamine oxidase(DAO) and D-lactic acid (d-lact). Stomach, ileum and colon were assessed in naked eyes and samples of each were collected for mucosa membrane damage assessments after HE staining. One of the tight junction proteins, occludin, was detected using immunohistochemistry staining in ileum and colon membrane. Apoptosis in ileac membrane were elevated using TUNEL technology.
Results:
After3hours of restraint stress, gastric membrane showed extensive erosion, which recovered during time. Gastric membrane in HE showed congestion, hemorrhage, epithelial cell necrosis after3hours of restraint stress and recovered during time. Ileac and colonic membrane showed similar reaction but much more moderate. Apoptosis increased after the stress significantly. Anti-occludin staining showed decreased optical density in stress rats, but without statistical significance. There was no significant difference between restraint animals and the control in plasma DAO and d-lact. Feacal output significantly increased in the stressed animals.
Conclusions:
(1) The restraint method used in the study successfully induced acute psychological stress of wistar rat.(2) Acute psychological stress resulted in damage of the gastrointestinal membrane from morphology to barrier function.(3) Barrier dysfunction of this model might mainly due to epithelial cell apoptosis increase, accompanying with tight junction damage to some extent.(4) Psychological stress promoted colonic dynamics.
方法：
55只雄性wistar大鼠经过3小时限动束缚后随机分入4个组：A组(3小时即时处死组)14只,B组(束缚后休息3小时处死组)14只,C组(束缚后休息9小时处死组)14只,D组(束缚后休息21小时处死组)13只；另外13只大鼠禁食禁水3小时作为对照；造模其间计数大鼠排便数。在各自时间点解剖大鼠采集标本。肉眼评估胃粘膜、回肠粘膜、结肠粘膜形态；光镜下对胃、回肠、结肠粘膜病理形态进行观察；免疫组化法分析回肠、结肠粘膜紧密连接Occludin蛋白的表达；TUNEL法检测回肠上皮细胞凋亡；检测血浆二胺氧化酶(DAO)活性和D-乳酸(D-lac)浓度。
结果：
束缚成功造成大鼠急性心理应激；急性心理应激导致大鼠胃粘膜广泛糜烂和散在出血；急性心理应激组紧密连接蛋白Occludin蛋白染色变淡,平均光密度略有降低,但与对照组差异不显著；急性心理应激组回肠粘膜上皮细胞凋亡指数明显增高。和对照组比较,急性心理应激各组血浆DAO活性和D-lac浓度未见明显增高；急性心理应激导致大鼠结肠运动增强。
结论：
1本束缚方法制造心理应激模型是成功的。
2本心理应激模型可导致大鼠胃肠粘膜从形态学损害到屏障功能的损害。
3本研究中大鼠肠粘膜屏障功能损害可能以细胞凋亡增加为主要途径,细胞间隙紧密连接破坏为次要途径。
4急性心理应激中大鼠结肠运动增强。
Objective:
To observe gastrointestinal membrane barrier function and colonic dynamics in wistar rats under acute psychological stress.
Methods:
Fifty-five male wistar rats under3hours of restraint stress were randomly divided into4groups, respectively having0hours (14),3hours (14),9hours (14),21hours (13) of recovery. Additional13rats which subjected to3hours of fasting to food and water were treated as control. Feces output were collected and counted after the3hours of stress or fasting. Rats were anesthetized using pentobarbital at respective time point. Blood plasma was collected and stored at-80℃until serving for detection of diamine oxidase(DAO) and D-lactic acid (d-lact). Stomach, ileum and colon were assessed in naked eyes and samples of each were collected for mucosa membrane damage assessments after HE staining. One of the tight junction proteins, occludin, was detected using immunohistochemistry staining in ileum and colon membrane. Apoptosis in ileac membrane were elevated using TUNEL technology.
Results:
After3hours of restraint stress, gastric membrane showed extensive erosion, which recovered during time. Gastric membrane in HE showed congestion, hemorrhage, epithelial cell necrosis after3hours of restraint stress and recovered during time. Ileac and colonic membrane showed similar reaction but much more moderate. Apoptosis increased after the stress significantly. Anti-occludin staining showed decreased optical density in stress rats, but without statistical significance. There was no significant difference between restraint animals and the control in plasma DAO and d-lact. Feacal output significantly increased in the stressed animals.
Conclusions:
(1) The restraint method used in the study successfully induced acute psychological stress of wistar rat.(2) Acute psychological stress resulted in damage of the gastrointestinal membrane from morphology to barrier function.(3) Barrier dysfunction of this model might mainly due to epithelial cell apoptosis increase, accompanying with tight junction damage to some extent.(4) Psychological stress promoted colonic dynamics.
引文
1 Selye H. A syndrome produced by diverse nocuous agents. Nature 1936,138:32.
2 吴琳,李兆申等：急性坏死性胰腺炎早期大鼠肠上皮细胞间紧密连接蛋白occludin表达变化,中华胰腺病杂志2008,8：127-129.
3 崔巍等,肿瘤坏死因子a对爆发性肝衰竭小鼠肠上皮细胞紧密连接蛋白表达的影响,中华内科杂志2007,46：478-481.
4周亚平等,严重烧伤患者肠粘膜通透性变化的研究,中华医学杂志2002,82：1-2.
5 姚咏明,于燕,盛志勇,等.创伤后内毒素血症与脏器损害关系及其防治新方法的系列研究.中国危重病急救医学.1998,10：134-139.
6石慧琳等,慢性束缚应激对大鼠肠粘膜屏障功能的影响.中国行为医学科学2003,12：251-253.
7 J E Mawdsley, D S Rampton. Psychological stress in IBD:new insight into pathogenic and therapeutic implications. Gut 2005;54:1481-1491.
8 Michael T. Bailey, Harald Engler, John F. Sheridan:Stress induces the translocation of cutaneous and gastrointestinal microflora to secondary lymphoid organs of C57BL/6 mice. Journal of Neuroimmunology 2006,171:29-37.
9 E Mazzon, G C Sturniolo, D Puzzolo, N Frisina, W Fries:Effect of stress on the paracellular barrier in the rat ileum. Gut 2002,51:507-513.
10 Jerrold R. Turner. Molecular Basis of Epithelial Barrier Regulation:From Basic Mechanisms to Clinical Application. The American Journal of Pathology 2006,169: 1901-1909.
11 Ikeda H, Suzuki Y, Suzuki M, et al. Apoptosis is a major mode of cell death caused by ischemia and ischemial reperfusion injury to the rat intestinal epithelium. Gut, 1998,42:530-537.
12 齐志伟等,缺血性脑卒中诱发肠屏障改变的实验研究,中国协和医科大学博士研究生学位论文2007年.
13 Masahiro Ochi, et al., Effect of chronic stress on gastric emptying and plasma ghrelin levels in rats. Life Sciences 2008,82:862-868.
14郝悦,黄裕新,王景杰.心理性应激对大鼠胃排空的影响及胃粘膜的损伤.第 四军医大学学报,2002,23：603-605.
15桂先勇,潘国宗,柯美云.胃肠肽在应激所致结肠动力紊乱中的作用.中华医学杂志,1997,77：31-34.
16 Garcia, A., Marti, O., Valles, A., Dal-Zotto, S., Armario, A.,. Recovery of the hypothalamic-pituitary-adrenal response to stress. Effect of stress intensity, stress duration and previous stress exposure. Neuroendocrinology 2000,72:114-125.
17 Whittle BJ, et al. Regulation of gastric mucosal integrity by endogenous nitric oxide:interactions with prostanoids and sensory neuropeptides in the rat. Br J Pharmacol,1990,99:607-611.
18 Chiu CJ, McArdle AH, Brown R, et al. Intestinal mucosal lesion in low flow states. Ⅰ. A morphological, hemodynamic, and metabolic reappraisal. Arch Surg,1970, 101:478-483.
19 Brandt RB, Siegel SA, Waters MG, et al. Spectrophotometric assay for D-(-)-lactate in plasma. Anal Biochem 1980,102:39-46.
20 Smith SM, Eng RHK, Buccini F. Use of D-lactic acid measurement in the diagnosis of bacterial infections. J Infect Dis,1986,154:658-664.
21曹碧茵,施建蓉,童瑶,邹军,心理应激动物模型的研制及其应用,中国行为医学科学2005,14：191-192.
22张红梅,刘晓伟.心理应激的实验研究.Chinese Journal of Behavioral Medical Science 2003,12:590-591.
23 F Barreau, L Ferrier, J Fioramonti, L Bueno. Neonatal maternal deprivation triggers long term alterations in colonic epithelial barrier and mucosal immunity in rats. Gut 2004,53:501-506.
24王韶轩,王金轩,心理应激对小肠细菌和小肠黏膜的影响,中国行为医学科学2008,17：210-211.
25 Kay L. Pals, et al. Effect of running intensity on intestinal permeability. JAppl Physiol 1997,82:571-576.
26 Oktedalen, O., O. C. Lunde, P. K. Opstad, L. Aabakken, and K. Kvernebo. Changes in the gastrointestinal mucosa after long-distance running. Scand. J. Gastroenterol 1992,27:270-274.
27 Mike G. Laukoetter, et al. Regulation of the intestinal epithelial barrier by the apical junctional complex. Current Opinion in Gastroenterology 2006,22:85-89.
28 Wu Z, Nybom P, Magnusson K. Distinct effects of Vibrio cholerae haemagglutinin/protease on the structure and localization of the tight junction-associated proteins occludin and ZO-1. Cell Micriob 2000,2:11-17.
29刘冬妍等,重症肝炎患者肠道屏障功能的变化,中国综合临床2008,24：412-414.
30 Wollin A, Navert H, Bounous G.Effect of intestinal ischemia on diamine oxidase activity in rat intestinal tissue and blood. Gastroenterology 1981,80:349-55.
31 Bounous G, Echave V, Vobecky SJ,et al. Acute necrosis of the intestinal mucosa with high serum levels of diamine oxidase. Dig Dis Sci.1984,29:872-4.
32 Johnston SD, Smye M, Watson RP. Intestinal permeability tests in celiac disease. Clin Lab.2001,47:143-150.
33 PAUL R. SAUNDERS, JAVIER SANTOS, et al:Physical and Psychological Stress in Rats Enhances Colonic Epithelial Permeability via Peripheral CRH. Digestive Diseases and Sciences 2002,47:208-215.
34 C Wallon, P-C Yang, et al:Corticotropin-releasing hormone (CRH) regulates macromolecular permeability via mast cells in normal human colonic biopsies in vitro. Gut 2008,57:50-58.
35 Santos J, Saunders PR, Hanssen NP, Yang PC, Yates D, Groot JA, Perdue MH: Corticotropin-releasing hormone mimics stress-induced colonic epithelial pathophysiology in the rat. Am J Physiol 1999,277:G391-G399
36 Kiliaan AJ, et al. Stress stimulates transepithelial macromolecular uptake in rat jejunum[J]. AM J Physiol,1998,275.G1037-G1044.
37 PAUL R. SAUNDERS, JAVIER SANTOS, et al:Physical and Psychological Stress in Rats Enhances Colonic Epithelial Permeability via Peripheral CRH. Digestive Diseases and Sciences 2002,47:208-215.
38 J Santos, P-C Yang, et al. Role of mast cells in chronic stress induced colonic epithelial barrier dysfunction in the rat. Gut 2001,48:630-636.
39周吕,柯美云主编.神经胃肠病学与动力基础与临床。2005年10月第一版,p108-p112.
2 吴琳,李兆申等：急性坏死性胰腺炎早期大鼠肠上皮细胞间紧密连接蛋白occludin表达变化,中华胰腺病杂志2008,8：127-129.
3 崔巍等,肿瘤坏死因子a对爆发性肝衰竭小鼠肠上皮细胞紧密连接蛋白表达的影响,中华内科杂志2007,46：478-481.
4周亚平等,严重烧伤患者肠粘膜通透性变化的研究,中华医学杂志2002,82：1-2.
5 姚咏明,于燕,盛志勇,等.创伤后内毒素血症与脏器损害关系及其防治新方法的系列研究.中国危重病急救医学.1998,10：134-139.
6石慧琳等,慢性束缚应激对大鼠肠粘膜屏障功能的影响.中国行为医学科学2003,12：251-253.
7 J E Mawdsley, D S Rampton. Psychological stress in IBD:new insight into pathogenic and therapeutic implications. Gut 2005;54:1481-1491.
8 Michael T. Bailey, Harald Engler, John F. Sheridan:Stress induces the translocation of cutaneous and gastrointestinal microflora to secondary lymphoid organs of C57BL/6 mice. Journal of Neuroimmunology 2006,171:29-37.
9 E Mazzon, G C Sturniolo, D Puzzolo, N Frisina, W Fries:Effect of stress on the paracellular barrier in the rat ileum. Gut 2002,51:507-513.
10 Jerrold R. Turner. Molecular Basis of Epithelial Barrier Regulation:From Basic Mechanisms to Clinical Application. The American Journal of Pathology 2006,169: 1901-1909.
11 Ikeda H, Suzuki Y, Suzuki M, et al. Apoptosis is a major mode of cell death caused by ischemia and ischemial reperfusion injury to the rat intestinal epithelium. Gut, 1998,42:530-537.
12 齐志伟等,缺血性脑卒中诱发肠屏障改变的实验研究,中国协和医科大学博士研究生学位论文2007年.
13 Masahiro Ochi, et al., Effect of chronic stress on gastric emptying and plasma ghrelin levels in rats. Life Sciences 2008,82:862-868.
14郝悦,黄裕新,王景杰.心理性应激对大鼠胃排空的影响及胃粘膜的损伤.第 四军医大学学报,2002,23：603-605.
15桂先勇,潘国宗,柯美云.胃肠肽在应激所致结肠动力紊乱中的作用.中华医学杂志,1997,77：31-34.
16 Garcia, A., Marti, O., Valles, A., Dal-Zotto, S., Armario, A.,. Recovery of the hypothalamic-pituitary-adrenal response to stress. Effect of stress intensity, stress duration and previous stress exposure. Neuroendocrinology 2000,72:114-125.
17 Whittle BJ, et al. Regulation of gastric mucosal integrity by endogenous nitric oxide:interactions with prostanoids and sensory neuropeptides in the rat. Br J Pharmacol,1990,99:607-611.
18 Chiu CJ, McArdle AH, Brown R, et al. Intestinal mucosal lesion in low flow states. Ⅰ. A morphological, hemodynamic, and metabolic reappraisal. Arch Surg,1970, 101:478-483.
19 Brandt RB, Siegel SA, Waters MG, et al. Spectrophotometric assay for D-(-)-lactate in plasma. Anal Biochem 1980,102:39-46.
20 Smith SM, Eng RHK, Buccini F. Use of D-lactic acid measurement in the diagnosis of bacterial infections. J Infect Dis,1986,154:658-664.
21曹碧茵,施建蓉,童瑶,邹军,心理应激动物模型的研制及其应用,中国行为医学科学2005,14：191-192.
22张红梅,刘晓伟.心理应激的实验研究.Chinese Journal of Behavioral Medical Science 2003,12:590-591.
23 F Barreau, L Ferrier, J Fioramonti, L Bueno. Neonatal maternal deprivation triggers long term alterations in colonic epithelial barrier and mucosal immunity in rats. Gut 2004,53:501-506.
24王韶轩,王金轩,心理应激对小肠细菌和小肠黏膜的影响,中国行为医学科学2008,17：210-211.
25 Kay L. Pals, et al. Effect of running intensity on intestinal permeability. JAppl Physiol 1997,82:571-576.
26 Oktedalen, O., O. C. Lunde, P. K. Opstad, L. Aabakken, and K. Kvernebo. Changes in the gastrointestinal mucosa after long-distance running. Scand. J. Gastroenterol 1992,27:270-274.
27 Mike G. Laukoetter, et al. Regulation of the intestinal epithelial barrier by the apical junctional complex. Current Opinion in Gastroenterology 2006,22:85-89.
28 Wu Z, Nybom P, Magnusson K. Distinct effects of Vibrio cholerae haemagglutinin/protease on the structure and localization of the tight junction-associated proteins occludin and ZO-1. Cell Micriob 2000,2:11-17.
29刘冬妍等,重症肝炎患者肠道屏障功能的变化,中国综合临床2008,24：412-414.
30 Wollin A, Navert H, Bounous G.Effect of intestinal ischemia on diamine oxidase activity in rat intestinal tissue and blood. Gastroenterology 1981,80:349-55.
31 Bounous G, Echave V, Vobecky SJ,et al. Acute necrosis of the intestinal mucosa with high serum levels of diamine oxidase. Dig Dis Sci.1984,29:872-4.
32 Johnston SD, Smye M, Watson RP. Intestinal permeability tests in celiac disease. Clin Lab.2001,47:143-150.
33 PAUL R. SAUNDERS, JAVIER SANTOS, et al:Physical and Psychological Stress in Rats Enhances Colonic Epithelial Permeability via Peripheral CRH. Digestive Diseases and Sciences 2002,47:208-215.
34 C Wallon, P-C Yang, et al:Corticotropin-releasing hormone (CRH) regulates macromolecular permeability via mast cells in normal human colonic biopsies in vitro. Gut 2008,57:50-58.
35 Santos J, Saunders PR, Hanssen NP, Yang PC, Yates D, Groot JA, Perdue MH: Corticotropin-releasing hormone mimics stress-induced colonic epithelial pathophysiology in the rat. Am J Physiol 1999,277:G391-G399
36 Kiliaan AJ, et al. Stress stimulates transepithelial macromolecular uptake in rat jejunum[J]. AM J Physiol,1998,275.G1037-G1044.
37 PAUL R. SAUNDERS, JAVIER SANTOS, et al:Physical and Psychological Stress in Rats Enhances Colonic Epithelial Permeability via Peripheral CRH. Digestive Diseases and Sciences 2002,47:208-215.
38 J Santos, P-C Yang, et al. Role of mast cells in chronic stress induced colonic epithelial barrier dysfunction in the rat. Gut 2001,48:630-636.
39周吕,柯美云主编.神经胃肠病学与动力基础与临床。2005年10月第一版,p108-p112.