【摘要】：第一部分亞硝基谷胱甘肽對(duì)內(nèi)毒素血癥大鼠系統(tǒng)性及腸道炎癥反應(yīng)的影響背景：膿毒癥是針對(duì)感染引起的失控宿主反應(yīng)造成的多器官功能障礙,一直是重癥患者中發(fā)病率與死亡率均高的疾病,早期的過(guò)度全身炎性反應(yīng)以及腸道炎癥反應(yīng)與膿毒癥的進(jìn)展密切相關(guān),其中腸道炎癥反應(yīng)能夠引起腸道組織損傷,使腸內(nèi)細(xì)菌以及毒素等有害物質(zhì)進(jìn)入系統(tǒng)循環(huán),加重全身炎癥反應(yīng),使膿毒癥進(jìn)一步惡化。近年來(lái),亞硝基谷胱甘肽(S-nitrosoglutathione, GSNO)被發(fā)現(xiàn)在多種疾病模型中具有顯著的抗炎作用。本研究中,我們檢測(cè)了給予GSNO對(duì)內(nèi)毒素血癥大鼠系統(tǒng)性炎癥反應(yīng)及腸道炎癥反應(yīng)和組織損傷的影響。方法：雄性SD大鼠被隨機(jī)分配到四組：生理鹽水對(duì)照組,GSNO對(duì)照組,內(nèi)毒素血癥組及GSNO處理組。麻醉暴露大鼠股靜脈后,由股靜脈注射脂多糖(LPS, 10mg/kg)或生理鹽水建立內(nèi)毒素血癥模型或?qū)φ战M模型,生理鹽水對(duì)照組大鼠于生理鹽水注射15分鐘后再次給予注射生理鹽水,GSNO對(duì)照組大鼠于生理鹽水注射后15分鐘注射GSNO (1mg/kg),內(nèi)毒素血癥組大鼠于LPS注射15分鐘后給予注射生理鹽水,GSNO處理組大鼠于LPS注射15分鐘后給予注射GSNO。LPS或生理鹽水注射后3小時(shí)處死大鼠,獲得血液及末段回腸組織標(biāo)本。檢測(cè)血漿中腫瘤壞死因子(TNF)及白介素-1β (IL-1β)的含量來(lái)評(píng)估大鼠系統(tǒng)性炎癥反應(yīng),檢測(cè)末段回腸組織中TNF及IL-1β的含量來(lái)評(píng)估腸道的炎癥反應(yīng),并對(duì)回腸組織進(jìn)行切片和HE染色,對(duì)腸道組織損傷情況進(jìn)行評(píng)級(jí)。結(jié)果：生理鹽水對(duì)照組與GSNO對(duì)照組各項(xiàng)指標(biāo)無(wú)顯著差異,相對(duì)于生理鹽水對(duì)照組,內(nèi)毒素血癥組大鼠在血漿以及回腸組織中均可見(jiàn)顯著增高的TNF和IL-1p含量[血漿：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；組織：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處理組大鼠較內(nèi)毒素血癥大鼠血漿以及腸道組織中TNF和IL-1β含量顯著降低[血漿：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；組織：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]。在回腸組織切片觀察中也可以看到內(nèi)毒素大鼠較生理鹽水對(duì)照組大鼠有明顯損傷,而GSNO處理可以減輕這種損傷,相應(yīng)地,在對(duì)組織損傷評(píng)級(jí)中,內(nèi)毒素血癥組大鼠評(píng)級(jí)顯著高于生理鹽水對(duì)照組(3.33±0.19比0.22±0.19,P0.01),而GSNO處理組大鼠較內(nèi)毒素血癥大鼠評(píng)級(jí)明顯降低(1.56±0.69比3.33±0.19,P0.05)。結(jié)論：GSNO可以降低內(nèi)毒素血癥大鼠的系統(tǒng)性及腸道的炎癥反應(yīng),并減輕腸道組織損傷。第二部分亞硝基谷胱甘肽對(duì)內(nèi)毒素血癥大鼠腸道上皮屏障損傷的影響背景：膿毒癥時(shí)腸道屏障功能損傷是造成多器官功能障礙和不良結(jié)局的重要因素。近年來(lái),腸道膠質(zhì)細(xì)胞來(lái)源的GSNO被認(rèn)為是調(diào)控腸道屏障完整性的重要介質(zhì)。本研究中,我們檢測(cè)了給予GSNO對(duì)內(nèi)毒素血癥大鼠腸道上皮通透性,緊密連接的結(jié)構(gòu)以及蛋白表達(dá)變化的影響。方法：分組同第一部分。LPS或生理鹽水注射后3小時(shí)處死大鼠,獲得末段回腸組織標(biāo)本,進(jìn)行以下檢測(cè)：(1)腸道上皮緊密連接的超微結(jié)構(gòu),使用透射電子顯微鏡觀察；(2)腸道上皮緊密連接蛋白ZO-1的分布,使用激光掃描共聚焦顯微鏡觀察；(3)緊密連接蛋白ZO-1的表達(dá),使用western-blot來(lái)檢測(cè)。在LPS或生理鹽水注射后3小時(shí)麻醉開(kāi)腹,結(jié)扎10cm小腸,于腸腔內(nèi)注射1 ml異硫氰酸熒光素標(biāo)記的右旋糖酐(FITC-Detran)溶液,30分鐘后處死大鼠,留取血液標(biāo)本,進(jìn)行小腸通透性檢測(cè),用血漿中FITC-Detran的含量來(lái)評(píng)估腸道上皮通透性和腸道上皮屏障功能情況。結(jié)果：相對(duì)于生理鹽水對(duì)照組,GSNO對(duì)照組大鼠各項(xiàng)指標(biāo)并無(wú)明顯變化,內(nèi)毒素血癥組大鼠血漿中FITC-Detran含量較生理鹽水對(duì)照組顯著增加(FITC-Detran: 4.29±0.71比1.71±0.35,P0.01),GSNO處理組大鼠較內(nèi)毒素血癥大鼠血漿中FITC-Detran含量明顯降低(FITC-Detran: 2.93±0.65比4.29±0.71,P0.05),而且在內(nèi)毒素血癥組大鼠電鏡標(biāo)本中可以看到腸道上皮緊密連接結(jié)構(gòu)顯著破壞,在激光共聚焦顯微鏡下可以看到緊密連接蛋白ZO-1熒光沾染不連續(xù),western-blot結(jié)果也顯示出ZO-1的條帶相對(duì)密度明顯降低(0.59±0.11比1.00±0.00,P0.05),GSNO處理組的大鼠較內(nèi)毒素血癥大鼠腸道上皮細(xì)胞間緊密連接結(jié)構(gòu)的損傷減輕,緊密連接蛋白ZO-1表達(dá)的減少被抑制(ZO-1條帶相對(duì)密度：0.85+0.11比0.59±0.11,P0.05)。結(jié)論：GSNO可以降低內(nèi)毒素血癥大鼠的腸道通透性,改善其腸道上皮緊密連接結(jié)構(gòu)和蛋白表達(dá),保護(hù)腸道上皮屏障完整性及功能。第三部分亞硝基谷胱甘肽保護(hù)內(nèi)毒素血癥大鼠腸道上皮屏障的機(jī)制背景：腸道上皮緊密連接的損傷與促炎性細(xì)胞因子、轉(zhuǎn)錄因子κB (NF-κB)活性以及肌球蛋白輕鏈激酶(myosin light chain kinase, MLCK)的水平密切相關(guān)。有研究指出,炎性細(xì)胞因子可以通過(guò)激活腸上皮細(xì)胞NF-κB的活性,增加MLCK的表達(dá)破壞上皮細(xì)胞間的緊密連接,前面我們已指出GSNO可以減輕內(nèi)毒素血癥大鼠的腸道炎癥,保護(hù)腸道屏障完整性,本部分中,我們給予GSNO干預(yù)內(nèi)毒素血癥大鼠,檢測(cè)腸道組織細(xì)胞中MLCK和細(xì)胞核中NF-κB p65的水平。方法：分組同第一部分。LPS或生理鹽水注射后3小時(shí)處死大鼠,獲得血液及末段回腸組織標(biāo)本。將回腸組織標(biāo)本進(jìn)行勻漿并提取細(xì)胞蛋白或核蛋白,使用western-blot方法檢測(cè)腸道組織細(xì)胞中MLCK和細(xì)胞核中NF-κB p65的水平。結(jié)果：相對(duì)于生理鹽水對(duì)照組,GSNO對(duì)照組大鼠腸道組織細(xì)胞中MLCK和細(xì)胞核中NF-κB p6的水平并無(wú)顯著差異。內(nèi)毒素血癥組大鼠較生理鹽水對(duì)照組腸道組織細(xì)胞中MLCK的表達(dá)顯著增多(MLCK條帶相對(duì)密度：2.08±0.32比1.00±0.00,P0.01),細(xì)胞核中NF-κB p65的含量也顯著增高(NF-κB p65條帶相對(duì)密度：1.83+0.09比1.00±0.00,P0.01),GSNO處理組較內(nèi)毒素血癥大鼠腸道組織細(xì)胞中MLCK以及核中NF-κB p65的含量明顯降低(MLCK條帶相對(duì)密度：1.52±0.09比2.08+0.32,P0.05；NF-κB p65條帶相對(duì)密度：1.26±0.14比1.83+0.09,P0.01)。結(jié)論：GSNO對(duì)內(nèi)毒素血癥大鼠腸道炎癥以及上皮緊密連接的保護(hù)作用可能與抑制NF-κB的激活有關(guān),GSNO可以通過(guò)抑制NF-κB/MLCK通路保護(hù)腸道上皮細(xì)胞緊密連接的結(jié)構(gòu)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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R459.7

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