亚硝基谷胱甘肽减轻内毒素血症大鼠炎症反应和肠道上皮屏障损伤
发布时间：2018-09-06 17:34
【摘要】：第一部分亚硝基谷胱甘肽对内毒素血症大鼠系统性及肠道炎症反应的影响背景：脓毒症是针对感染引起的失控宿主反应造成的多器官功能障碍,一直是重症患者中发病率与死亡率均高的疾病,早期的过度全身炎性反应以及肠道炎症反应与脓毒症的进展密切相关,其中肠道炎症反应能够引起肠道组织损伤,使肠内细菌以及毒素等有害物质进入系统循环,加重全身炎症反应,使脓毒症进一步恶化。近年来,亚硝基谷胱甘肽(S-nitrosoglutathione, GSNO)被发现在多种疾病模型中具有显著的抗炎作用。本研究中,我们检测了给予GSNO对内毒素血症大鼠系统性炎症反应及肠道炎症反应和组织损伤的影响。方法：雄性SD大鼠被随机分配到四组：生理盐水对照组,GSNO对照组,内毒素血症组及GSNO处理组。麻醉暴露大鼠股静脉后,由股静脉注射脂多糖(LPS, 10mg/kg)或生理盐水建立内毒素血症模型或对照组模型,生理盐水对照组大鼠于生理盐水注射15分钟后再次给予注射生理盐水,GSNO对照组大鼠于生理盐水注射后15分钟注射GSNO (1mg/kg),内毒素血症组大鼠于LPS注射15分钟后给予注射生理盐水,GSNO处理组大鼠于LPS注射15分钟后给予注射GSNO。LPS或生理盐水注射后3小时处死大鼠,获得血液及末段回肠组织标本。检测血浆中肿瘤坏死因子(TNF)及白介素-1β (IL-1β)的含量来评估大鼠系统性炎症反应,检测末段回肠组织中TNF及IL-1β的含量来评估肠道的炎症反应,并对回肠组织进行切片和HE染色,对肠道组织损伤情况进行评级。结果：生理盐水对照组与GSNO对照组各项指标无显著差异,相对于生理盐水对照组,内毒素血症组大鼠在血浆以及回肠组织中均可见显著增高的TNF和IL-1p含量[血浆：TNF (pg/ml):119.30±50.91比6.07±0.37, IL-1β (pg/ml):1456.00±487.90比28.80±13.35,均P0.01；组织：TNF (pg/mg pro):8.19±3.62比0.55±0.15,IL-1β (pg/mg pro):139.30±433.12比27.71+9.39,均P0.01],而GSNO处理组大鼠较内毒素血症大鼠血浆以及肠道组织中TNF和IL-1β含量显著降低[血浆：TNF(pg/ml)：60.34±16.86比119.30±50.91,P0.05；IL-1β(pg/ml)：373.30±226.90比1456.00±487.90,P0.01；组织：TNF(pg/mg pro):4.12±1.42比8.19±3.62,IL-1β(pg/mg pro):74.91±27.98比139.30±33.12,均P0.05]。在回肠组织切片观察中也可以看到内毒素大鼠较生理盐水对照组大鼠有明显损伤,而GSNO处理可以减轻这种损伤,相应地,在对组织损伤评级中,内毒素血症组大鼠评级显著高于生理盐水对照组(3.33±0.19比0.22±0.19,P0.01),而GSNO处理组大鼠较内毒素血症大鼠评级明显降低(1.56±0.69比3.33±0.19,P0.05)。结论：GSNO可以降低内毒素血症大鼠的系统性及肠道的炎症反应,并减轻肠道组织损伤。第二部分亚硝基谷胱甘肽对内毒素血症大鼠肠道上皮屏障损伤的影响背景：脓毒症时肠道屏障功能损伤是造成多器官功能障碍和不良结局的重要因素。近年来,肠道胶质细胞来源的GSNO被认为是调控肠道屏障完整性的重要介质。本研究中,我们检测了给予GSNO对内毒素血症大鼠肠道上皮通透性,紧密连接的结构以及蛋白表达变化的影响。方法：分组同第一部分。LPS或生理盐水注射后3小时处死大鼠,获得末段回肠组织标本,进行以下检测：(1)肠道上皮紧密连接的超微结构,使用透射电子显微镜观察；(2)肠道上皮紧密连接蛋白ZO-1的分布,使用激光扫描共聚焦显微镜观察；(3)紧密连接蛋白ZO-1的表达,使用western-blot来检测。在LPS或生理盐水注射后3小时麻醉开腹,结扎10cm小肠,于肠腔内注射1 ml异硫氰酸荧光素标记的右旋糖酐(FITC-Detran)溶液,30分钟后处死大鼠,留取血液标本,进行小肠通透性检测,用血浆中FITC-Detran的含量来评估肠道上皮通透性和肠道上皮屏障功能情况。结果：相对于生理盐水对照组,GSNO对照组大鼠各项指标并无明显变化,内毒素血症组大鼠血浆中FITC-Detran含量较生理盐水对照组显著增加(FITC-Detran: 4.29±0.71比1.71±0.35,P0.01),GSNO处理组大鼠较内毒素血症大鼠血浆中FITC-Detran含量明显降低(FITC-Detran: 2.93±0.65比4.29±0.71,P0.05),而且在内毒素血症组大鼠电镜标本中可以看到肠道上皮紧密连接结构显著破坏,在激光共聚焦显微镜下可以看到紧密连接蛋白ZO-1荧光沾染不连续,western-blot结果也显示出ZO-1的条带相对密度明显降低(0.59±0.11比1.00±0.00,P0.05),GSNO处理组的大鼠较内毒素血症大鼠肠道上皮细胞间紧密连接结构的损伤减轻,紧密连接蛋白ZO-1表达的减少被抑制(ZO-1条带相对密度：0.85+0.11比0.59±0.11,P0.05)。结论：GSNO可以降低内毒素血症大鼠的肠道通透性,改善其肠道上皮紧密连接结构和蛋白表达,保护肠道上皮屏障完整性及功能。第三部分亚硝基谷胱甘肽保护内毒素血症大鼠肠道上皮屏障的机制背景：肠道上皮紧密连接的损伤与促炎性细胞因子、转录因子κB (NF-κB)活性以及肌球蛋白轻链激酶(myosin light chain kinase, MLCK)的水平密切相关。有研究指出,炎性细胞因子可以通过激活肠上皮细胞NF-κB的活性,增加MLCK的表达破坏上皮细胞间的紧密连接,前面我们已指出GSNO可以减轻内毒素血症大鼠的肠道炎症,保护肠道屏障完整性,本部分中,我们给予GSNO干预内毒素血症大鼠,检测肠道组织细胞中MLCK和细胞核中NF-κB p65的水平。方法：分组同第一部分。LPS或生理盐水注射后3小时处死大鼠,获得血液及末段回肠组织标本。将回肠组织标本进行匀浆并提取细胞蛋白或核蛋白,使用western-blot方法检测肠道组织细胞中MLCK和细胞核中NF-κB p65的水平。结果：相对于生理盐水对照组,GSNO对照组大鼠肠道组织细胞中MLCK和细胞核中NF-κB p6的水平并无显著差异。内毒素血症组大鼠较生理盐水对照组肠道组织细胞中MLCK的表达显著增多(MLCK条带相对密度：2.08±0.32比1.00±0.00,P0.01),细胞核中NF-κB p65的含量也显著增高(NF-κB p65条带相对密度：1.83+0.09比1.00±0.00,P0.01),GSNO处理组较内毒素血症大鼠肠道组织细胞中MLCK以及核中NF-κB p65的含量明显降低(MLCK条带相对密度：1.52±0.09比2.08+0.32,P0.05；NF-κB p65条带相对密度：1.26±0.14比1.83+0.09,P0.01)。结论：GSNO对内毒素血症大鼠肠道炎症以及上皮紧密连接的保护作用可能与抑制NF-κB的激活有关,GSNO可以通过抑制NF-κB/MLCK通路保护肠道上皮细胞紧密连接的结构和功能。
[Abstract]:Part I Effects of nitroso-glutathione on systemic and intestinal inflammation in endotoxemia rats Background: Sepsis is a multiple organ dysfunction caused by uncontrolled host response to infection. It has always been a disease with high morbidity and mortality in severe patients, early excessive systemic inflammatory response and intestinal inflammation. In recent years, S-nitrosoglutathione (GSNO) has been found in many disease models. Methods: Male SD rats were randomly divided into four groups: normal saline control group, GSNO control group, endotoxemia group and GSNO treatment group. After femoral vein injection of lipopolysaccharide (LPS, 10mg/kg) or normal saline into femoral vein, endotoxemia model or control group was established. Rats in normal saline control group were injected with normal saline 15 minutes later. Rats in GSNO control group were injected with GSNO (1mg/kg) 15 minutes after injection of normal saline. Rats were injected with normal saline 15 minutes after LPS injection. Rats in the GSNO treatment group were sacrificed 3 hours after LPS injection and GSNO. LPS or normal saline injection. Blood and terminal ileal tissue samples were obtained. The levels of tumor necrosis factor (TNF) and interleukin-1 beta (IL-1 beta) in plasma were measured to evaluate systemic inflammation in rats. The levels of TNF and IL-1 beta in the terminal ileum were measured to evaluate the intestinal inflammation, and the ileum was sliced and stained with HE. The intestinal injury was graded. Results: There was no significant difference between the normal saline control group and the GSNO control group in the indexes. Compared with the normal saline control group, the rats in the endotoxemia group had no significant difference. Increased levels of TNF and IL-1p were observed in plasma and ileum tissues [plasma: TNF (pg/ml): 119.30 (+50.91) vs 6.07 (+0.37), IL-1 beta (pg/ml): 1456.00 (+487.90) vs 28.80 (+13.35), all P 0.01; tissue: TNF (pg/mg pro): 8.19 (+3.62) vs 0.55 (+0.15), IL-1 beta (pg/pro): 139.30 (+433.12) vs. 7.71 (+9.39), P 0.01], and NO treatment, respectively. The levels of TNF and IL-1 beta in plasma and intestinal tissues of rats in group A were significantly lower than those in endotoxemia rats [plasma: TNF (pg/ml): 60.34 + 16.86 vs 119.30 + 50.91, P 0.05; IL-1 beta (pg/ml): 373.30 + 226.90 vs 1456.00 + 487.90, P 0.01; tissue: TNF (pg/pro): 4.12 + 1.42 vs 8.19 + 3.62, IL-1 beta (pg/pro): 74.91 + 27.98 vs 139.30 + 33.33 12, all P 0.05). In the ileum tissue section observation, the endotoxin rats were more obviously injured than the normal saline control group, but GSNO treatment could alleviate the injury. Correspondingly, in the tissue injury rating, the endotoxemia group rats were significantly higher than the normal saline control group (3.33 + 0.19 vs 0.22 + 0.19, P 0.01), while the GSNO treatment could alleviate the injury. Conclusion: GSNO can reduce the systemic and intestinal inflammation in endotoxemia rats and alleviate the intestinal tissue damage. Part II The background of the effect of nitroso glutathione on intestinal epithelial barrier damage in endotoxemia rats. In recent years, gut glial cell-derived GSNO has been considered as an important mediator in regulating intestinal barrier integrity. In this study, we examined the effects of GSNO on intestinal epithelial permeability and tight junction in endotoxemia rats. Methods: The rats were sacrificed 3 hours after LPS or saline injection, and the final ileal tissue samples were obtained for the following detection: (1) ultrastructure of tight junction of intestinal epithelium was observed by transmission electron microscopy; (2) distribution of tight junction protein ZO-1 in intestinal epithelium by stimulation. The expression of tight junction protein ZO-1 was detected by Western-blot.The rats were sacrificed 30 minutes later and the blood samples were taken. The intestinal permeability and intestinal epithelial barrier function were assessed by plasma FITC-Detran. Results: Compared with normal saline control group, there was no significant change in the indexes of GSNO control group, and the plasma FITC-Detran content in endotoxemia group was significantly higher than that in normal saline control group (FITC-Detran). - Detran: 4.29 + 0.71 vs. 1.71 + 0.35, P 0.01). Compared with endotoxemia rats, the plasma FITC-Detran content of GSNO treated rats was significantly decreased (FITC-Detran: 2.93 + 0.65 vs. 4.29 + 0.71, P 0.05). In endotoxemia group, the tight junction of intestinal epithelium was significantly destroyed, and under laser confocal microscopy. The fluorescence staining of tight junction protein ZO-1 was discontinuous, and the relative density of ZO-1 was significantly decreased by Western-blot (0.59.11 vs. 1.00.00, P 0.05). Compared with endotoxemia rats, the damage of tight junction between intestinal epithelial cells in GSNO treatment group was lessened, and the expression of tight junction protein ZO-1 was inhibited. CONCLUSION: GSNO can reduce intestinal permeability, improve the tight junction structure and protein expression of intestinal epithelium, and protect the integrity and function of intestinal epithelial barrier in endotoxemia rats. Part III Nitroso glutathione protects intestinal epithelial barrier in endotoxemia rats. Background: Intestinal tight junction injury is closely related to pro-inflammatory cytokines, transcription factor-kappa B (NF-kappa B) activity and myosin light chain kinase (MLCK) levels. In this part, we give GSNO to intervene endotoxemia rats to detect the levels of MLCK in intestinal tissue cells and NF-kappa B p65 in nucleus. Methods: Grouping the same as the first part, LPS or raw. The rats were sacrificed 3 hours after injection of normal saline to obtain blood and terminal ileum tissue specimens. The ileum tissue specimens were homogenized and the protein or nuclear protein was extracted. The levels of MLCK and NF-kappa B p65 in the nucleus of the intestinal tissue cells were detected by Western blot. Results: Compared with the normal saline control group, the intestine of rats in GSNO control group was detected. There was no significant difference between the levels of MLCK and nuclear NF-kappa B P6 in the tracheal tissue cells. Compared with the normal saline group, the expression of MLCK in the intestinal tissue cells of endotoxemia group increased significantly (relative density of MLCK bands: 2.08 + 0.32 vs. 1.00 + 0.00, P 0.01), and the content of NF-kappa B p65 in the nucleus increased significantly (relatively dense bands of NF-kappa B p65). Degree: 1.83 + 0.09 vs. 1.00 + 0.00, P 0.01). Compared with endotoxemia rats, the contents of MLCK and NF-kappa B p65 in intestinal tissue cells and nucleus of GSNO treatment group were significantly decreased (MLCK band relative density: 1.52 + 0.09 vs. 2.08 + 0.32, P 0.05; NF-kappa B p65 band relative density: 1.26 + 0.14 vs. 1.83 + 0.09, P 0.01). The protective effects of GSNO on inflammation and tight junction may be related to the inhibition of NF-kappa B activation. GSNO can protect the structure and function of tight junction of intestinal epithelial cells by inhibiting NF-kappa B/MLCK pathway.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R459.7
[Abstract]:Part I Effects of nitroso-glutathione on systemic and intestinal inflammation in endotoxemia rats Background: Sepsis is a multiple organ dysfunction caused by uncontrolled host response to infection. It has always been a disease with high morbidity and mortality in severe patients, early excessive systemic inflammatory response and intestinal inflammation. In recent years, S-nitrosoglutathione (GSNO) has been found in many disease models. Methods: Male SD rats were randomly divided into four groups: normal saline control group, GSNO control group, endotoxemia group and GSNO treatment group. After femoral vein injection of lipopolysaccharide (LPS, 10mg/kg) or normal saline into femoral vein, endotoxemia model or control group was established. Rats in normal saline control group were injected with normal saline 15 minutes later. Rats in GSNO control group were injected with GSNO (1mg/kg) 15 minutes after injection of normal saline. Rats were injected with normal saline 15 minutes after LPS injection. Rats in the GSNO treatment group were sacrificed 3 hours after LPS injection and GSNO. LPS or normal saline injection. Blood and terminal ileal tissue samples were obtained. The levels of tumor necrosis factor (TNF) and interleukin-1 beta (IL-1 beta) in plasma were measured to evaluate systemic inflammation in rats. The levels of TNF and IL-1 beta in the terminal ileum were measured to evaluate the intestinal inflammation, and the ileum was sliced and stained with HE. The intestinal injury was graded. Results: There was no significant difference between the normal saline control group and the GSNO control group in the indexes. Compared with the normal saline control group, the rats in the endotoxemia group had no significant difference. Increased levels of TNF and IL-1p were observed in plasma and ileum tissues [plasma: TNF (pg/ml): 119.30 (+50.91) vs 6.07 (+0.37), IL-1 beta (pg/ml): 1456.00 (+487.90) vs 28.80 (+13.35), all P 0.01; tissue: TNF (pg/mg pro): 8.19 (+3.62) vs 0.55 (+0.15), IL-1 beta (pg/pro): 139.30 (+433.12) vs. 7.71 (+9.39), P 0.01], and NO treatment, respectively. The levels of TNF and IL-1 beta in plasma and intestinal tissues of rats in group A were significantly lower than those in endotoxemia rats [plasma: TNF (pg/ml): 60.34 + 16.86 vs 119.30 + 50.91, P 0.05; IL-1 beta (pg/ml): 373.30 + 226.90 vs 1456.00 + 487.90, P 0.01; tissue: TNF (pg/pro): 4.12 + 1.42 vs 8.19 + 3.62, IL-1 beta (pg/pro): 74.91 + 27.98 vs 139.30 + 33.33 12, all P 0.05). In the ileum tissue section observation, the endotoxin rats were more obviously injured than the normal saline control group, but GSNO treatment could alleviate the injury. Correspondingly, in the tissue injury rating, the endotoxemia group rats were significantly higher than the normal saline control group (3.33 + 0.19 vs 0.22 + 0.19, P 0.01), while the GSNO treatment could alleviate the injury. Conclusion: GSNO can reduce the systemic and intestinal inflammation in endotoxemia rats and alleviate the intestinal tissue damage. Part II The background of the effect of nitroso glutathione on intestinal epithelial barrier damage in endotoxemia rats. In recent years, gut glial cell-derived GSNO has been considered as an important mediator in regulating intestinal barrier integrity. In this study, we examined the effects of GSNO on intestinal epithelial permeability and tight junction in endotoxemia rats. Methods: The rats were sacrificed 3 hours after LPS or saline injection, and the final ileal tissue samples were obtained for the following detection: (1) ultrastructure of tight junction of intestinal epithelium was observed by transmission electron microscopy; (2) distribution of tight junction protein ZO-1 in intestinal epithelium by stimulation. The expression of tight junction protein ZO-1 was detected by Western-blot.The rats were sacrificed 30 minutes later and the blood samples were taken. The intestinal permeability and intestinal epithelial barrier function were assessed by plasma FITC-Detran. Results: Compared with normal saline control group, there was no significant change in the indexes of GSNO control group, and the plasma FITC-Detran content in endotoxemia group was significantly higher than that in normal saline control group (FITC-Detran). - Detran: 4.29 + 0.71 vs. 1.71 + 0.35, P 0.01). Compared with endotoxemia rats, the plasma FITC-Detran content of GSNO treated rats was significantly decreased (FITC-Detran: 2.93 + 0.65 vs. 4.29 + 0.71, P 0.05). In endotoxemia group, the tight junction of intestinal epithelium was significantly destroyed, and under laser confocal microscopy. The fluorescence staining of tight junction protein ZO-1 was discontinuous, and the relative density of ZO-1 was significantly decreased by Western-blot (0.59.11 vs. 1.00.00, P 0.05). Compared with endotoxemia rats, the damage of tight junction between intestinal epithelial cells in GSNO treatment group was lessened, and the expression of tight junction protein ZO-1 was inhibited. CONCLUSION: GSNO can reduce intestinal permeability, improve the tight junction structure and protein expression of intestinal epithelium, and protect the integrity and function of intestinal epithelial barrier in endotoxemia rats. Part III Nitroso glutathione protects intestinal epithelial barrier in endotoxemia rats. Background: Intestinal tight junction injury is closely related to pro-inflammatory cytokines, transcription factor-kappa B (NF-kappa B) activity and myosin light chain kinase (MLCK) levels. In this part, we give GSNO to intervene endotoxemia rats to detect the levels of MLCK in intestinal tissue cells and NF-kappa B p65 in nucleus. Methods: Grouping the same as the first part, LPS or raw. The rats were sacrificed 3 hours after injection of normal saline to obtain blood and terminal ileum tissue specimens. The ileum tissue specimens were homogenized and the protein or nuclear protein was extracted. The levels of MLCK and NF-kappa B p65 in the nucleus of the intestinal tissue cells were detected by Western blot. Results: Compared with the normal saline control group, the intestine of rats in GSNO control group was detected. There was no significant difference between the levels of MLCK and nuclear NF-kappa B P6 in the tracheal tissue cells. Compared with the normal saline group, the expression of MLCK in the intestinal tissue cells of endotoxemia group increased significantly (relative density of MLCK bands: 2.08 + 0.32 vs. 1.00 + 0.00, P 0.01), and the content of NF-kappa B p65 in the nucleus increased significantly (relatively dense bands of NF-kappa B p65). Degree: 1.83 + 0.09 vs. 1.00 + 0.00, P 0.01). Compared with endotoxemia rats, the contents of MLCK and NF-kappa B p65 in intestinal tissue cells and nucleus of GSNO treatment group were significantly decreased (MLCK band relative density: 1.52 + 0.09 vs. 2.08 + 0.32, P 0.05; NF-kappa B p65 band relative density: 1.26 + 0.14 vs. 1.83 + 0.09, P 0.01). The protective effects of GSNO on inflammation and tight junction may be related to the inhibition of NF-kappa B activation. GSNO can protect the structure and function of tight junction of intestinal epithelial cells by inhibiting NF-kappa B/MLCK pathway.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R459.7
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