游泳训练对水浸束缚应激大鼠免疫功能及胃肠粘膜细胞凋亡影响的研究
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摘要
应激(stress)是指机体受到强烈刺激后发生的,以交感神经-肾上腺髓质和垂体-肾上腺皮质功能增强为主要特点的非特异性反应。任何躯体或心理的刺激,只要达到一定的强度,除了引起与刺激因素直接相关的特异性变化外,也可引起一组与刺激因素的性质无直接关系的全身性非特异性反应。强烈的身心应激严重影响人类的身心健康,可导致情感行为异常、免疫功能低下,神经内分泌功能异常等。因此,应激与心身疾病关系的研究在传统生物医学模式向生物-心理-社会医学模式转变的今天已成为医学研究领域的一个重要课题,并受到军事医学的广泛关注。
研究表明,机体受应激影响最严重的两大系统分别是消化系统和免疫系统。一般认为,严重应激反应主要导致机体的细胞免疫功能降低,体液免疫功能增强,形成Thl向Th2漂移。胃肠道不仅是消化、吸收营养物质的场所,而且是体内最大的免疫器官。胃肠道粘膜的免疫屏障功能被各种因素削弱时,外来微生物如细菌、病毒和寄生虫等的入侵,可造成消化道或全身性疾病.胃肠道分泌的sIgA是相对的非炎症物质,其水平的高低决定了机体胃肠道免疫水平的强弱。而自细胞凋亡概念提出后,应激后胃肠粘膜是否存在凋亡?越来越引起了学者们的关注。
现代战争具有高技术、高强度、高杀伤力的特点,在现代战场环境下,参战人员势必受到强烈的心理和生理刺激,产生强烈的应激反应。应激常常累及胃肠道,可引起胃肠道消化、吸收、分泌、运动、免疫、肠粘膜屏障等等功能障碍,其中应激性溃疡、肠粘膜屏障损害和胃肠动力障碍是各种应激引起的重要的病理结果,是伤病员救治的重点和难点。因此,研究应激对机体免疫功能和胃肠粘膜完整性的影响及防治可以深入揭示其发生机制,不仅能提高野战医学救治水平,还可为平时防治应激造成的心理生理损害提供理论指导。
本课题拟以大鼠为实验对象,制备稳定成熟的大鼠应激性胃肠粘膜损害模型——水浸-束缚应激(water-immersion restraint stress ,WRS)模型,探讨应激状态对大鼠整体免疫功能(包括血清TNF-α、IL-6、IL-2)、胃、肠粘膜免疫功能——肠腔内分泌型免疫球蛋白(sIgA)、血清皮质醇,Tunel法检测胃、肠上皮细胞凋亡率,免疫组化法检测凋亡相关蛋白Bax、Bcl-2蛋白表达。同时,设置适当游泳训练组大鼠,比较预先给予合适的心理和生理训练(游泳训练)对提高大鼠胃肠道免疫能力和降低胃肠上皮细胞凋亡的作用,为进一步深入研究大鼠应激反应过程中胃肠功能的变化规律和保护措施的寻找奠定基础,以期在此基础逐步提出现代战争条件下常见的创伤应激后胃肠功能紊乱的防治预案。
【材料与方法】
健康雄性Wistar大鼠48只,经适应性环境后10天,随机分为3组,每组16只。A组:适当游泳训练+水浸束缚应激;B组:适当游泳训练组;C组:无游泳训练+水浸束缚应激。给予A、B两组大鼠在平静水中自由游泳30min,2次/日,连续5天为适当游泳训练。结束游泳训练后,所有大鼠均经禁食24h,禁水1h后,A、C组大鼠在轻度乙醚吸入麻醉下,水浸时间1.5h,制作成为水浸-束缚应激(WRS)模型。
结束水浸-束缚应激试验后,处死大鼠,立即从腹腔主动脉处各取血2ml,低温分离血清,采用ELISA法检测大鼠血清TNF-α和IL-6水平,采用放射免疫法检测大鼠血清IL- 2和皮质酮水平;生化法检测血清超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量;洗涤法获取结肠肠液检测sIgA含量,采用ELISA法进行检测。
采用Tunel法原位检测大鼠胃、肠粘膜上皮细胞凋亡指数,免疫组织化学法检测凋亡相关基因Bax ,Bcl-2表达。
【结果】
1.三组实验大鼠共48只,实验结束后均存活,打开腹腔见均无腹水,腹膜、胃壁、肠壁肉眼观色鲜红、无明显坏死、无淤血扩张;A、B组两组大鼠胃内均未见出血和溃疡灶,C组16只中,有4只发现胃内点状溃疡出血、少量血凝块。三组实验大鼠中分别有3、1、6只见肠管苍白、积气、少量伴有积血;
2.A组(适当游泳训练+水浸束缚应激)和B组(适当游泳训练组)大鼠血清TNF-α、IL-6、IL-2水平明显高于C组(无游泳训练+水浸束缚应激)大鼠(P<0.05);而血清皮质醇水平三组间无显著性差异(P>0.05);
3.A组(适当游泳训练+水浸束缚应激)肠液中sIgA为36.3±3.6mg/L,B组(适当游泳训练组)大鼠肠液中sIgA为39.1±4.0mg/L,均明显高于C组(无游泳训练+水浸束缚应激)大鼠肠液中sIgA水平(27.1±2.8mg/L)(P<0.05);
4.A组(适当游泳训练+水浸束缚应激)和B组(适当游泳训练组)大鼠血清SODA水平明显高于C组(无游泳训练+水浸束缚应激)大鼠(P<0.05),而血清MDA水平则低于C组(P<0.05);
5.三组大鼠胃、肠粘膜均可见程度不等的急性炎症变化,部分标本可见肠壁轻度变薄。腺体无明显萎缩;
6.三组大鼠胃、肠粘膜均可见程度不等的凋亡细胞表达。免疫组化检测Bcl-2、Bax阳性细胞表达示,阳性信号位于细胞浆和核膜,三组大鼠胃、肠粘膜阳性表达率存在差异。统计C组大鼠Tunel凋亡指数AI和Bcl-2、Bax阳性细胞表达率与A、B两组大鼠比较存在显著性差异(P<0.05),AI与Bcl-2表达A值呈正相关,而与Bax表达A值呈负相关。
【结论】
适当游泳训练能提高应激大鼠整体和胃肠道免疫功能,能提高机体清除自由基功能,机体通过Bcl-2上调和Bax下调对抗应激条件下凋亡的发生和发展,适当的心理和生理训练可以降低机体应激时细胞凋亡。
Stress means unspecific response of organism characteres with function reinforcement of sympathetic nerve– adrenal medulla and hypophysis-adrenal gland cortex stimulated fiercenessfully. Stimulated by intensive physical or psychological stimulation factor, organism appears specific response directly associate with the stimulation factor, as well a group of unspecific response that has no direct relationship with that. Whatever the character of stimulation factor, the unspecific response is similar. Though immunological function is reinforced or inhibited by various kinds of neuroendocrine changes caused by stress. Their balance controls the immune state. Persistent stress usually results in immunosuppression due to glucocorticoid and catecholamine. Strong psychophysical stress or longtime chronic psychophysical stress does great harm to human health, which causes emotional abnormalities, excitatory sympathetic neural activity, gastrointestinal injury, low response of immunity and abnormality of neuroendocrine function as people. Stressing incidence has been considered one of the most important contributors to those functional diseases. Modem medical patern is the integrity of biological, psychological and social aspects. So far, no standard animal model has been developed, which limits the research in the efect of psychological factor on stress. Therefore, development of a psychological animal stress model is not only very helpful for management of psychological diseases, but also provides essential information for prevention or relief of stress.
The studies show that gastroenterology system and immune system are the most seriously damaged by stress attack.It is well kown serious stress lead cellare immune function reducing and humoral immunity strengthening,as a result, Th1 turning into Th2. Gastroenterological tube is not only the place where digesting and absorb nourish matter,but also is the biggest immune organ in the body. If the gastroenterological tube immunity is decreased by every damage factor, The microorganism such as bacteria,virus and parasite well invade the body. It will cause gastroenterology system or the whole body pathological chages. The level of gastroenterological tube secretory immunoglobulin A (sIgA) will show the level of gastroenterological immunity.The question of if there is relationship between apoptosis and gastroenterological damage attacked by stress is still well known.It is reseach hot spot.
Modern war notable characteristion is high technique, high intersity and high massacre power. The participater must undertake strong physiological and physical stress under modern battlefield environment. Gastroenterological tube is always attacked by stress, as the results lead to obstaction of digesting, obsorbing,secreting,moving,immunity and mucosal barrier. Stress ulcer, mucosal barrier obstacte and gastrointestinal motility weak are stress leading pathological results,which are difficult point and main point to save the sick and wounded.Soving the difficults can not only announce the principte of disease based of stress bult also improve the level of field medicine cure and prevent.
Wistar Rats were our experiment objects,which were used to prepared water immersion restraint stress (WRS)models.The systematic immunity such as the levels of serum TNF-α、IL-6、IL-2 were tested by ELISA. The level of sIgA and Cortisol(Cor) were studed. The apoptosis index was test by Tunel. The expression of Bcl-2 and Bax was studied by immunohistochemistry. The correlation between this data change and psychological stress was studied.Accoring the research,we will work out the scheme to prevent gastroenterological function disorder base of modern war wound.
Methods
48 Wistar Rats were randomly divided into three groups as A group,B group and C group, the number of every group was 16. A group given suit swimming exercise and water immersion restraint stress . B group given suit swimming exercise nly. C group given water immersion restraint stress only. Suit swimming exercise meas free swimming 30 min 2/d,×5d. After suitswimming exercise, the rats of A and C group were given water immersion restraint stress after light anaesthesia, 90 min.
After WRS experiment, we kill all rats and gain the vein blood and serum after centrifuge. The levels of serum TNF-α、IL-6、IL-2 were tested by ELISA. The level of sIgA and Cortisol(Cor) were studed. The levels of superoxide dismutase(SOD) and molondialdehyde (MDA) were tested by biochemical methods. The apoptosis index was test by Tunel. The expression of Bcl-2 and Bax was studied by immunohistochemistry. The correlation between this data change and psychological stress was studied.
Results
1. After model experiment, all rat were survival. There was not bleed and ulcer in the stomach in either rat of A and B group. Only four from sixteen rats of C group were found bleed and ulcer in the stomach. There three, one and six rat were found pale intestine and small bleed.
2. The levels of serum TNF-α、IL-6、IL-2 of the rats of A and B group were higher than that of C group (P<0.05). There was significantly difference between three group either(P>0.05).
3. The sIgA levels of the rats of A and B group were higher than that of C group (P<0.05).Data was 36.3±3.6mg/L and 39.1±4.0mg/L VS 27.1±2.8mg/L.
4. The level of serum SOD of the rats of A and B group were higher than that of C group (P<0.05). The level of serum MDA of the rats of A and B group were lower than that of C group (P<0.05).
5. Acute inflammation was observed in gastroenterological mucosa in the rats of three group.
6.Tunel showed there was significant difference between three group in the apoptosis index(P<0.05). The expression of Bcl-2 and Bax of A and C group was significant different than that of C group. AI was direct relation with expression of Bcl-2 and inverse relation with expression of Bax.
Conclusions
Suit swimming exercise can improve the rats attacked by stress in their systematic immunity and gastroenterological mucosal immunity. It can improve the rats’elinity ability to radical scavenger. The rats resisted the apoptosis caused by stress by way of promoting expression of bcl-2 and decreasing expression of Bax.Suit physiological and physical exercise can reduce apoptosis caused by stress.
研究表明,机体受应激影响最严重的两大系统分别是消化系统和免疫系统。一般认为,严重应激反应主要导致机体的细胞免疫功能降低,体液免疫功能增强,形成Thl向Th2漂移。胃肠道不仅是消化、吸收营养物质的场所,而且是体内最大的免疫器官。胃肠道粘膜的免疫屏障功能被各种因素削弱时,外来微生物如细菌、病毒和寄生虫等的入侵,可造成消化道或全身性疾病.胃肠道分泌的sIgA是相对的非炎症物质,其水平的高低决定了机体胃肠道免疫水平的强弱。而自细胞凋亡概念提出后,应激后胃肠粘膜是否存在凋亡?越来越引起了学者们的关注。
现代战争具有高技术、高强度、高杀伤力的特点,在现代战场环境下,参战人员势必受到强烈的心理和生理刺激,产生强烈的应激反应。应激常常累及胃肠道,可引起胃肠道消化、吸收、分泌、运动、免疫、肠粘膜屏障等等功能障碍,其中应激性溃疡、肠粘膜屏障损害和胃肠动力障碍是各种应激引起的重要的病理结果,是伤病员救治的重点和难点。因此,研究应激对机体免疫功能和胃肠粘膜完整性的影响及防治可以深入揭示其发生机制,不仅能提高野战医学救治水平,还可为平时防治应激造成的心理生理损害提供理论指导。
本课题拟以大鼠为实验对象,制备稳定成熟的大鼠应激性胃肠粘膜损害模型——水浸-束缚应激(water-immersion restraint stress ,WRS)模型,探讨应激状态对大鼠整体免疫功能(包括血清TNF-α、IL-6、IL-2)、胃、肠粘膜免疫功能——肠腔内分泌型免疫球蛋白(sIgA)、血清皮质醇,Tunel法检测胃、肠上皮细胞凋亡率,免疫组化法检测凋亡相关蛋白Bax、Bcl-2蛋白表达。同时,设置适当游泳训练组大鼠,比较预先给予合适的心理和生理训练(游泳训练)对提高大鼠胃肠道免疫能力和降低胃肠上皮细胞凋亡的作用,为进一步深入研究大鼠应激反应过程中胃肠功能的变化规律和保护措施的寻找奠定基础,以期在此基础逐步提出现代战争条件下常见的创伤应激后胃肠功能紊乱的防治预案。
【材料与方法】
健康雄性Wistar大鼠48只,经适应性环境后10天,随机分为3组,每组16只。A组:适当游泳训练+水浸束缚应激;B组:适当游泳训练组;C组:无游泳训练+水浸束缚应激。给予A、B两组大鼠在平静水中自由游泳30min,2次/日,连续5天为适当游泳训练。结束游泳训练后,所有大鼠均经禁食24h,禁水1h后,A、C组大鼠在轻度乙醚吸入麻醉下,水浸时间1.5h,制作成为水浸-束缚应激(WRS)模型。
结束水浸-束缚应激试验后,处死大鼠,立即从腹腔主动脉处各取血2ml,低温分离血清,采用ELISA法检测大鼠血清TNF-α和IL-6水平,采用放射免疫法检测大鼠血清IL- 2和皮质酮水平;生化法检测血清超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量;洗涤法获取结肠肠液检测sIgA含量,采用ELISA法进行检测。
采用Tunel法原位检测大鼠胃、肠粘膜上皮细胞凋亡指数,免疫组织化学法检测凋亡相关基因Bax ,Bcl-2表达。
【结果】
1.三组实验大鼠共48只,实验结束后均存活,打开腹腔见均无腹水,腹膜、胃壁、肠壁肉眼观色鲜红、无明显坏死、无淤血扩张;A、B组两组大鼠胃内均未见出血和溃疡灶,C组16只中,有4只发现胃内点状溃疡出血、少量血凝块。三组实验大鼠中分别有3、1、6只见肠管苍白、积气、少量伴有积血;
2.A组(适当游泳训练+水浸束缚应激)和B组(适当游泳训练组)大鼠血清TNF-α、IL-6、IL-2水平明显高于C组(无游泳训练+水浸束缚应激)大鼠(P<0.05);而血清皮质醇水平三组间无显著性差异(P>0.05);
3.A组(适当游泳训练+水浸束缚应激)肠液中sIgA为36.3±3.6mg/L,B组(适当游泳训练组)大鼠肠液中sIgA为39.1±4.0mg/L,均明显高于C组(无游泳训练+水浸束缚应激)大鼠肠液中sIgA水平(27.1±2.8mg/L)(P<0.05);
4.A组(适当游泳训练+水浸束缚应激)和B组(适当游泳训练组)大鼠血清SODA水平明显高于C组(无游泳训练+水浸束缚应激)大鼠(P<0.05),而血清MDA水平则低于C组(P<0.05);
5.三组大鼠胃、肠粘膜均可见程度不等的急性炎症变化,部分标本可见肠壁轻度变薄。腺体无明显萎缩;
6.三组大鼠胃、肠粘膜均可见程度不等的凋亡细胞表达。免疫组化检测Bcl-2、Bax阳性细胞表达示,阳性信号位于细胞浆和核膜,三组大鼠胃、肠粘膜阳性表达率存在差异。统计C组大鼠Tunel凋亡指数AI和Bcl-2、Bax阳性细胞表达率与A、B两组大鼠比较存在显著性差异(P<0.05),AI与Bcl-2表达A值呈正相关,而与Bax表达A值呈负相关。
【结论】
适当游泳训练能提高应激大鼠整体和胃肠道免疫功能,能提高机体清除自由基功能,机体通过Bcl-2上调和Bax下调对抗应激条件下凋亡的发生和发展,适当的心理和生理训练可以降低机体应激时细胞凋亡。
Stress means unspecific response of organism characteres with function reinforcement of sympathetic nerve– adrenal medulla and hypophysis-adrenal gland cortex stimulated fiercenessfully. Stimulated by intensive physical or psychological stimulation factor, organism appears specific response directly associate with the stimulation factor, as well a group of unspecific response that has no direct relationship with that. Whatever the character of stimulation factor, the unspecific response is similar. Though immunological function is reinforced or inhibited by various kinds of neuroendocrine changes caused by stress. Their balance controls the immune state. Persistent stress usually results in immunosuppression due to glucocorticoid and catecholamine. Strong psychophysical stress or longtime chronic psychophysical stress does great harm to human health, which causes emotional abnormalities, excitatory sympathetic neural activity, gastrointestinal injury, low response of immunity and abnormality of neuroendocrine function as people. Stressing incidence has been considered one of the most important contributors to those functional diseases. Modem medical patern is the integrity of biological, psychological and social aspects. So far, no standard animal model has been developed, which limits the research in the efect of psychological factor on stress. Therefore, development of a psychological animal stress model is not only very helpful for management of psychological diseases, but also provides essential information for prevention or relief of stress.
The studies show that gastroenterology system and immune system are the most seriously damaged by stress attack.It is well kown serious stress lead cellare immune function reducing and humoral immunity strengthening,as a result, Th1 turning into Th2. Gastroenterological tube is not only the place where digesting and absorb nourish matter,but also is the biggest immune organ in the body. If the gastroenterological tube immunity is decreased by every damage factor, The microorganism such as bacteria,virus and parasite well invade the body. It will cause gastroenterology system or the whole body pathological chages. The level of gastroenterological tube secretory immunoglobulin A (sIgA) will show the level of gastroenterological immunity.The question of if there is relationship between apoptosis and gastroenterological damage attacked by stress is still well known.It is reseach hot spot.
Modern war notable characteristion is high technique, high intersity and high massacre power. The participater must undertake strong physiological and physical stress under modern battlefield environment. Gastroenterological tube is always attacked by stress, as the results lead to obstaction of digesting, obsorbing,secreting,moving,immunity and mucosal barrier. Stress ulcer, mucosal barrier obstacte and gastrointestinal motility weak are stress leading pathological results,which are difficult point and main point to save the sick and wounded.Soving the difficults can not only announce the principte of disease based of stress bult also improve the level of field medicine cure and prevent.
Wistar Rats were our experiment objects,which were used to prepared water immersion restraint stress (WRS)models.The systematic immunity such as the levels of serum TNF-α、IL-6、IL-2 were tested by ELISA. The level of sIgA and Cortisol(Cor) were studed. The apoptosis index was test by Tunel. The expression of Bcl-2 and Bax was studied by immunohistochemistry. The correlation between this data change and psychological stress was studied.Accoring the research,we will work out the scheme to prevent gastroenterological function disorder base of modern war wound.
Methods
48 Wistar Rats were randomly divided into three groups as A group,B group and C group, the number of every group was 16. A group given suit swimming exercise and water immersion restraint stress . B group given suit swimming exercise nly. C group given water immersion restraint stress only. Suit swimming exercise meas free swimming 30 min 2/d,×5d. After suitswimming exercise, the rats of A and C group were given water immersion restraint stress after light anaesthesia, 90 min.
After WRS experiment, we kill all rats and gain the vein blood and serum after centrifuge. The levels of serum TNF-α、IL-6、IL-2 were tested by ELISA. The level of sIgA and Cortisol(Cor) were studed. The levels of superoxide dismutase(SOD) and molondialdehyde (MDA) were tested by biochemical methods. The apoptosis index was test by Tunel. The expression of Bcl-2 and Bax was studied by immunohistochemistry. The correlation between this data change and psychological stress was studied.
Results
1. After model experiment, all rat were survival. There was not bleed and ulcer in the stomach in either rat of A and B group. Only four from sixteen rats of C group were found bleed and ulcer in the stomach. There three, one and six rat were found pale intestine and small bleed.
2. The levels of serum TNF-α、IL-6、IL-2 of the rats of A and B group were higher than that of C group (P<0.05). There was significantly difference between three group either(P>0.05).
3. The sIgA levels of the rats of A and B group were higher than that of C group (P<0.05).Data was 36.3±3.6mg/L and 39.1±4.0mg/L VS 27.1±2.8mg/L.
4. The level of serum SOD of the rats of A and B group were higher than that of C group (P<0.05). The level of serum MDA of the rats of A and B group were lower than that of C group (P<0.05).
5. Acute inflammation was observed in gastroenterological mucosa in the rats of three group.
6.Tunel showed there was significant difference between three group in the apoptosis index(P<0.05). The expression of Bcl-2 and Bax of A and C group was significant different than that of C group. AI was direct relation with expression of Bcl-2 and inverse relation with expression of Bax.
Conclusions
Suit swimming exercise can improve the rats attacked by stress in their systematic immunity and gastroenterological mucosal immunity. It can improve the rats’elinity ability to radical scavenger. The rats resisted the apoptosis caused by stress by way of promoting expression of bcl-2 and decreasing expression of Bax.Suit physiological and physical exercise can reduce apoptosis caused by stress.
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