本文選題：乙酸 + 膿毒癥��； 參考：《第二軍醫(yī)大學(xué)》2017年博士論文

【摘要】：研究背景與目的:膿毒癥是指由感染引起的全身炎癥反應(yīng)綜合征,伴有臟器功能障礙,但其確切分子機(jī)制還不清楚,缺乏有效的治療方法。巨噬細(xì)胞作為膿毒癥促炎因子的主要來源,其代謝過程發(fā)揮著重要作用。巨噬細(xì)胞的糖酵解作為治療靶點(diǎn)越來越受到人們的重視。本文研究了在小鼠盲腸結(jié)扎穿孔致膿毒癥模型中,糖代謝重要中間產(chǎn)物之一乙酸對(duì)小鼠的保護(hù)作用及其作用機(jī)制。研究方法:1.在體觀察乙酸對(duì)膿毒癥小鼠的保護(hù)作用:采用小鼠盲腸結(jié)扎穿孔(CLP)膿毒癥模型,觀察在CLP術(shù)后30分鐘分別腹腔注射乙酸250mg/kg或500mg/kg對(duì)小鼠7天生存率、CLP術(shù)后12小時(shí)肝和肺組織損傷、腹腔細(xì)菌清除率和小鼠血清促炎因子的水平。采用腹腔注射LPS建立小鼠內(nèi)毒素血癥模型,分別在建模前12天連續(xù)給予水中添加乙酸(150mmol/l)或建模前30分鐘后給予乙酸腹腔注射(500mg/kg)預(yù)處理,檢測(cè)小鼠血清促炎因子的水平。2.離體觀察乙酸對(duì)巨噬細(xì)胞的作用:體外分離培養(yǎng)小鼠骨髓來源的巨噬細(xì)胞(BMDM)、小鼠腹腔巨噬細(xì)胞、人外周血單核細(xì)胞(PBMC)、人類THP-1細(xì)胞和人CD14+細(xì)胞,分別用乙酸(5~100mmol/l)預(yù)處理0.5h、1h、2h后用LPS刺激,6h后檢測(cè)小鼠血清促炎因子TNF-α和IL-6的水平。3.乙酸抑炎的機(jī)制探討:首先,通過離體細(xì)胞進(jìn)一步探討乙酸對(duì)巨噬細(xì)胞免疫功能影響的機(jī)制。體外分離培養(yǎng)BMDM,將BMDM和RAW 264.7巨噬細(xì)胞系用乙酸(10mmol/l)預(yù)處理0.5h,在LPS(100ng/ml)刺激后15min、30min和60min收集細(xì)胞,采用Western Blotting法測(cè)定NF-κB p65、JNK、ERK和p38 MAPK磷酸化的改變。并用si RNA敲減GPR41、GPR43兩個(gè)乙酸受體,檢測(cè)小鼠血清促炎因子TNF-α和IL-6的水平。其次觀察乙酸對(duì)糖酵解的影響。體外分離培養(yǎng)BMDM,用乙酸10mmol/l預(yù)處理后0.5h LPS刺激,LPS刺激后6h收集細(xì)胞與上清,測(cè)定BMDMs培養(yǎng)上清中乳酸濃度,分析2-NBDG攝取,檢測(cè)細(xì)胞中糖酵解關(guān)鍵酶GLUT1,PFKFB3,HK2,MCT4m RNA的表達(dá)以及細(xì)胞酸化率(ECAR)和氧耗率(OCR)。隨后通過γ-干擾素(IFN-γ)或粒細(xì)胞-巨噬細(xì)胞集落刺激因子(GM-CSF)預(yù)處理增強(qiáng)BMDM糖酵解后進(jìn)一步觀察乙酸對(duì)巨噬細(xì)胞糖代謝及促炎因子的影響。體外分離培養(yǎng)BMDM,用100ng/ml的IFN-γ或GM-CSF預(yù)孵育3小時(shí),乙酸10mmol/l預(yù)處理后,LPS刺激6h,收集細(xì)胞和上清,ELISA法測(cè)上清TNF-α和IL-6的濃度;比色法測(cè)上清乳酸濃度;Q-PCR法測(cè)細(xì)胞GLUT1、HK2、MCT4、PFKFB3的m RNA表達(dá);流式細(xì)胞儀分析細(xì)胞表面的GLUT1表達(dá);Western Blotting分析測(cè)定NF-κB p65和m TOR磷酸化改變。4.乙酸如何抑制糖酵解:首先離體觀察乙酸預(yù)處理對(duì)HIF-1αm RNA以及蛋白表達(dá)的影響。體外分離培養(yǎng)BMDM,用乙酸(10mmol/l)預(yù)處理0.5h,在LPS(100ng/ml)刺激后15min和30min,收集細(xì)胞,Q-PCR法測(cè)細(xì)胞的HIF-1αm RNA;Western Blotting法測(cè)定HIF-1α蛋白的表達(dá)。隨后探討HIF-1α是不是乙酸作用所必需的,我們?cè)隗w內(nèi)和體外用DMOG,一種HIF-1α脯氨酰羥化酶的競(jìng)爭(zhēng)性抑制劑。將BMDM,先用DMSO或DMOG(0.5mmol/l)孵育2.5h,然后乙酸10mmol/l預(yù)處理0.5h,再用LPS100μg/ml刺激6h后收集上清,ELISA法測(cè)定上清TNF-α和IL-6水平。觀察DMOG是否能反轉(zhuǎn)乙酸降低LPS誘導(dǎo)的促炎因子的產(chǎn)生;Gl UT1、HK2、MCT4和PFKFB3的m RNA表達(dá);巨噬細(xì)胞表面GLUT1表達(dá);培養(yǎng)基中乳酸和葡萄糖的消耗率以及NF-κB的磷酸化改變。又將RAW264.7細(xì)胞,轉(zhuǎn)染HIF-1α特異性載體和空白載體24 h后,觀察過表達(dá)HIF-1α對(duì)乙酸降低LPS誘導(dǎo)的促炎因子的產(chǎn)生作用的影響。最后在體研究HIF-1α是不是乙酸保護(hù)膿毒癥小鼠作用所必需的:將40只野生型(wild type,WT)C57BL/6小鼠隨機(jī)分為假手術(shù)組(sham,n=8),CLP模型組(CLP,n=8),DMOG+CLP組(DMOG+CLP,n=8),乙酸處理組(CLP+A,n=8)和乙酸+DMOG組(DMOG+CLP+A,n=8)五個(gè)組。用DMOG(320mg/kg)腹腔注射2.5h,隨后進(jìn)行CLP術(shù),然后用乙酸(500mg/kg,ip)0.5h,觀察小鼠7天生存率;CLP模型后12小時(shí)收集小鼠肝肺組織、外周血血清、腹腔灌洗液。HE染色觀察各組小鼠肝和肺的病理變化;ELISA法測(cè)外周血血清促炎因子TNF-α、IL-6、IL-1β水平等指標(biāo)。結(jié)果:1.在體觀察乙酸對(duì)膿毒癥小鼠的保護(hù)作用:CLP組小鼠生存率為37.5%,乙酸處理組(250mg/kg、500mg/kg)的生存率分別為75%和85%,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。提示乙酸可明顯改善CLP小鼠的生存率。CLP明顯造成小鼠肝肺組織損傷,可見肝靜脈、中央靜脈和肝竇擴(kuò)張淤血,肝小葉肝細(xì)胞萎縮和壞死,炎性細(xì)胞浸潤(rùn)、變性;肺小靜脈和肺泡壁毛細(xì)血管擴(kuò)張,充血和纖維組織增生;肝功能指標(biāo)AST顯著上升;腹膜細(xì)菌計(jì)數(shù)上升;血清TNF-α、IL-6、IL-1β水平升高。與CLP組相比,乙酸處理組的肝肺組織損傷明顯減輕,肝靜脈、中央靜脈和肝竇淤血,肝細(xì)胞壞死區(qū)域以及炎性細(xì)胞浸潤(rùn)顯著減少;肺小靜脈和肺泡壁毛細(xì)血管管壁重建增加,肺毛細(xì)血管充血和纖維化增生減少。乙酸處理組的AST明顯下降,腹膜細(xì)菌計(jì)數(shù)顯著降低;血漿TNF-α、IL-6、IL-1β水平下降(P0.05)。提示乙酸可改善膿毒癥造成的組織損傷,減少促炎因子的釋放并增強(qiáng)病原體的清除能力。LPS誘導(dǎo)的小鼠內(nèi)毒素血癥模型結(jié)果顯示,LPS能顯著增加小鼠血清的TNF-α和IL-6,口服給藥乙酸組或者腹腔注射給予乙酸組血清的TNF-α和IL-6明顯下降,說明乙酸對(duì)LPS介導(dǎo)的體內(nèi)促炎因子釋放有抑制作用。2.離體觀察乙酸對(duì)巨噬細(xì)胞的作用:在BMDM、小鼠腹腔巨噬細(xì)胞、人外周血單個(gè)核細(xì)胞(PBMC)、人CD14細(xì)胞以及人類的THP-1細(xì)胞中,LPS組的促炎細(xì)胞因子TNF-α和IL-6水平以及m RNA顯著增加,乙酸處理組的TNF-α和IL-6水平以及m RNA較LPS組明顯降低,提示乙酸對(duì)LPS誘導(dǎo)的TNF-α和IL-6釋放和m RNA增加有抑制作用,且此抑制作用呈劑量依賴性和時(shí)間依賴性。在體外證實(shí)了乙酸的抑炎作用。3.乙酸抑炎的機(jī)制探討:在BMDM,LPS組的NF-κB p65的磷酸化顯著增加,而乙酸預(yù)處理導(dǎo)致激活的NF-k B p65顯著減少。LPS組的JNK、ERK和p38 MAPK的磷酸化顯著增加,然而,乙酸預(yù)處理的JNK、ERK和p38 MAPK的磷酸化與LPS組無顯著差異。在BMDM,單獨(dú)敲減GPR43和GPR41兩個(gè)乙酸受體,或同時(shí)敲減GPR43和GPR41的表達(dá),LPS組的TNF-α和IL-6顯著增加,乙酸處理組的TNF-α和IL-6較LPS組明顯降低;與乙酸組相比,無論單獨(dú)敲減GPR43或GPR41的表達(dá),還是同時(shí)敲減GPR43和GPR41的表達(dá),TNF-α和IL-6的釋放均無顯著差異,提示GPR43和GPR41可能不涉及乙酸的抗炎作用。在BMDM,LPS組的血清乳酸水平、2-NBDG攝取略有增加,GLUT1、PFKFB3、HK2、MCT4 m RNA的表達(dá)迅速增加,巨噬細(xì)胞表面GLUT1表達(dá)明顯增加;而乙酸處理組的血清乳酸水平、2-NBDG攝取降低,GLUT1、PFKFB3、HK2、MCT4 m RNA的表達(dá)顯著下降,巨噬細(xì)胞表面GLUT1表達(dá)也顯著降低。這表明,在BMDM中,乙酸抑制LPS誘導(dǎo)的糖酵解率和葡萄糖消耗率增加,提示在LPS刺激后,乙酸抑制糖酵解關(guān)鍵酶的表達(dá)。預(yù)孵育IFN-γ或GM-CSF增加BMDM的糖酵解后,相較LPS組,乙酸組的TNF-α和IL-6釋放減少以及GLUT1,PFKFB3,HK2,MCT4 m RNA的表達(dá)降低;而預(yù)孵育IFN-γ或GM-CSF的乙酸治療組,可見乙酸組的TNF-α和IL-6釋放減少以及GLUT1、PFKFB3、HK2、MCT4 m RNA的表達(dá)降低幾乎被完全反轉(zhuǎn)。表明增加糖酵解能逆轉(zhuǎn)乙酸的抗炎作用。在LPS誘導(dǎo)的內(nèi)毒素血癥模型中,乙酸處理組的小鼠脾巨噬細(xì)胞的2-NBDG攝取顯著減少,說明乙酸在體內(nèi)抑制LPS導(dǎo)致的葡萄糖攝取升高。4.HIF-1α在糖酵解中的作用:在BMDM,LPS組的HIF-1αm RNA和蛋白表達(dá)增加,乙酸組的HIF-1αm RNA和蛋白表達(dá)顯著降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。表明在乙酸在m RNA水平和蛋白水平抑制LPS刺激引起的HIF-1α上調(diào)。用DMOG抑制BMDM的HIF-1α表達(dá),結(jié)果顯示,LPS組的TNF-α和IL-6顯著增加,乙酸處理組的TNF-α和IL-6較LPS組明顯降低,DMOG+乙酸組的TNF-α和IL-6與LPS組無顯著差別。在RAW264.7細(xì)胞,通過轉(zhuǎn)染HIF-1α特異性載體過表達(dá)HIF-1α,結(jié)果顯示,LPS組的TNF-α和IL-6顯著增加,乙酸處理組的TNF-α和IL-6較LPS組明顯降低,而HIF-1α過表達(dá)+乙酸組的TNF-α和IL-6與LPS組無顯著差別。說明HIF-1α是乙酸抑制LPS誘導(dǎo)的促炎因子釋放所必需的。在BMDM,LPS組的GLUT1、PFKFB3、HK2、MCT4 m RNA表達(dá)明顯升高,巨噬細(xì)胞表面GLUT1表達(dá)明顯增加,乳酸產(chǎn)生和葡萄糖水平明顯增加;乙酸組的GLUT1、PFKFB3、HK2、MCT4 m RNA的表達(dá)顯著降低;DMOG+乙酸組的GLUT1、PFKFB3、HK2、MCT4 m RNA的表達(dá)顯著降低,巨噬細(xì)胞表面GLUT1表達(dá)明顯減少,乳酸產(chǎn)生和葡萄糖水平明顯減少,與LPS組無顯著差異。這表明乙酸降低LPS刺激產(chǎn)生的細(xì)胞因子至少部分通過HIF-1α依賴性的糖酵解的調(diào)節(jié)。在BMDM,LPS組的NF-κB p65磷酸化增加,乙酸組的NF-κB p65磷酸化顯著降低;DMOG+乙酸組的NF-κB p65磷酸化增加,與LPS組無顯著差異;證明DMOG能完全逆轉(zhuǎn)乙酸抑制激活NF-κB的作用。最后在體研究HIF-1α是不是乙酸保護(hù)膿毒癥小鼠作用所必需的:用DMOG在體內(nèi)抑制膿毒癥小鼠巨噬細(xì)胞的HIF-1α表達(dá),與CLP組相比,乙酸組小鼠的死亡率顯著降低,肝肺組織損傷明顯減輕,肝靜脈、中央靜脈和肝竇淤血,肝細(xì)胞壞死區(qū)域以及炎性細(xì)胞浸潤(rùn)顯著減少;肺小靜脈和肺泡壁毛細(xì)血管管壁重建增加,肺毛細(xì)血管充血和纖維化增生減少。乙酸處理組的AST明顯下降,腹膜細(xì)菌計(jì)數(shù)顯著降低;血漿IL-6、IL-1β,TNF-α水平下降;而用DMOG預(yù)處理后,乙酸降低死亡率的作用明顯反轉(zhuǎn),乙酸改善膿毒癥造成的組織損傷,減少促炎因子的釋放并增強(qiáng)病原體的清除能力也明顯被反轉(zhuǎn)。說明HIF-1α在乙酸引起的糖酵解改變中起關(guān)鍵作用。結(jié)論:乙酸可通過抑制LPS導(dǎo)致的HIF-1α表達(dá)來抑制LPS導(dǎo)致的糖酵解,進(jìn)而減少促炎因子的釋放,由此緩解膿毒癥導(dǎo)致的臟器功能損害,提高其生存率。
[Abstract]:Background and purpose: sepsis is a systemic inflammatory response syndrome caused by infection, accompanied by organ dysfunction, but its exact molecular mechanism is not clear and effective. Macrophages play an important role in the metabolic process of septic proinflammatory factors. The glycolysis of macrophages is used as a cure. In this paper, the protective effect and mechanism of acetic acid, one of the important intermediate products of sugar metabolism, in mice were studied in the model of cecal ligation and perforation induced sepsis in mice. 1. in vivo observation of the protective effect of acetic acid on sepsis in mice: the use of cecum ligation and perforation (CLP) sepsis in mice The model was to observe the 7 natural survival rate of mice by intraperitoneal injection of acetic acid 250mg/kg or 500mg/kg 30 minutes after CLP, 12 hours of liver and lung tissue injury after CLP, the clearance rate of peritoneal bacteria and the level of serum proinflammatory factors in mice. The mice endotoxemia model was established by intraperitoneal injection of LPS, and the water was added to the water for 12 days before modeling, respectively. Acetic acid (150mmol/l) or intraperitoneal injection of acetic acid (500mg/kg) was given 30 minutes before the modeling to detect the level of serum proinflammatory factors in mice by.2. in vitro. The effect of acetic acid on macrophages was observed in vitro: isolated and cultured macrophages (BMDM), mouse peritoneal macrophages, human peripheral blood mononuclear cells (PBMC), and human THP-1 thin in vitro. The cell and human CD14+ cells were pretreated with acetic acid (5~100mmol/l) for 0.5h, 1H, 2h and LPS stimulation. After 6h, the mechanism of serum proinflammatory factor TNF- alpha and IL-6 level.3. acetic acid inhibition was investigated. First, the mechanism of acetic acid on the immune function of macrophages was further investigated by the isolated cells. In vitro isolation and culture BMDM, BMDM and 2 The 64.7 macrophage system was pretreated with acetic acid (10mmol/l), 0.5h was pretreated with LPS (100ng/ml), 15min, 30min and 60min were collected after the stimulation of LPS (100ng/ml). Western Blotting method was used to determine NF- kappa B p65. The effects of acetic acid on glycolysis were observed. BMDM was isolated and cultured in vitro, 0.5h LPS was pretreated with acetic acid 10mmol/l. After LPS stimulation, 6h collected cell and supernatant, determined the concentration of lactic acid in BMDMs culture supernatant, analyzed 2-NBDG uptake, and detected the expression of the key enzymes, GLUT1, PFKFB3, HK2, MCT4m, and the rate of acidification and oxygen consumption. (OCR). The effect of acetic acid on macrophage glucose metabolism and proinflammatory factors was further observed by preconditioning with interferon gamma (IFN- gamma) or granulocyte macrophage colony stimulating factor (GM-CSF). BMDM was isolated and cultured in vitro, incubated with 100ng/ml IFN- gamma or GM-CSF for 3 hours, and LPS stimulated 6h after acetic acid 10mmol/l preconditioning. To collect cells and supernatants, the concentration of TNF- alpha and IL-6 in the supernatant was measured by ELISA; the concentration of lactic acid in the supernatant was measured by colorimetric method; the m RNA expression of GLUT1, HK2, MCT4, PFKFB3 in cells was measured by Q-PCR; the flow cytometer analyzed the GLUT1 expression of the cell surface; The effects of acetic acid pretreatment on the expression of HIF-1 alpha m RNA and protein were observed. BMDM was isolated and cultured in vitro, 0.5h was pretreated with acetic acid (10mmol/l), 15min and 30min were collected after LPS (100ng/ml) stimulation, and the cells were collected and Q-PCR method was used to determine the expression of alpha protein. In the body and in vitro, we use DMOG in vivo and in vitro, a competitive inhibitor of HIF-1 alpha prolyl hydroxylase. BMDM is incubated with DMSO or DMOG (0.5mmol/l) to incubate 2.5h first, and then acetic 10mmol/l pretreatments 0.5h, then LPS100 um g/ml stimulates 6h to collect the supernatant. The production of pro-inflammatory factors; the expression of M RNA in Gl UT1, HK2, MCT4 and PFKFB3; the expression of GLUT1 on the surface of macrophages; consumption of lactic acid and glucose in the medium and phosphorylation of NF- kappa B. RAW264.7 cells were also transfected to HIF-1 alpha specific carrier and blank carrier 24. The effect of factor production. Finally, in vivo study of whether HIF-1 alpha was necessary for the action of acetic acid to protect sepsis: 40 wild type (WT) C57BL/6 mice were randomly divided into the sham operation group (sham, n=8), CLP model group (CLP, n=8), DMOG+CLP group (DMOG+), acetic acid treatment group and acetic acid group. The five groups were treated with DMOG (320mg/kg) intraperitoneal injection of 2.5h, followed by CLP, and then using acetic acid (500mg/kg, IP) 0.5h to observe the 7 natural survival rate of mice. After CLP model, the liver and lung tissue of mice were collected 12 hours after CLP model, and the pathological changes of liver and lung in each group were observed by.HE staining in the peritoneal lavage solution, and TNF- alpha of peripheral blood serum proinflammatory factor in peripheral blood was measured by ELISA method. IL-1 beta level and other indicators. Results: 1. the protective effect of acetic acid on sepsis in mice was observed in the body of acetic acid: the survival rate of the CLP group was 37.5%, the survival rate of the acetic acid treatment group (250mg/kg, 500mg/kg) was 75% and 85% respectively, the difference was statistically significant (P0.05). It was suggested that the survival rate of the acetic acid can be obviously improved by.CLP in the mice, and the liver and lung tissue damage in mice was obviously caused. The hepatic vein, the central vein and the hepatic sinus dilated and congestion, hepatic lobular liver cell atrophy and necrosis, inflammatory cell infiltration, degeneration, pulmonary venules and alveolar capillary dilatation, hyperemia and fibrous tissue proliferation, liver function index AST increased significantly, peritoneal bacteria count increased; serum TNF- alpha, IL-6, IL-1 beta levels increased. Compared with CLP group, acetic acid The injury of liver and lung tissue in the treatment group was obviously reduced, the hepatic vein, the central vein and the hepatic sinus congestion, the necrosis area of the liver cell and the inflammatory cell infiltration decreased significantly, the reconstruction of the capillary wall of the pulmonary venule and the alveolar wall increased, the pulmonary capillary congestion and the fibrotic hyperplasia decreased. The AST of the acetic acid treatment group was significantly decreased and the count of peritoneal bacteria was significant. Decrease in plasma TNF- alpha, IL-6, IL-1 beta level (P0.05). It suggests that acetic acid can improve tissue damage caused by sepsis, reduce the release of pro-inflammatory factors and enhance the scavenging ability of pathogens,.LPS induced mouse endotoxemia model results show that LPS can significantly increase the TNF- alpha and IL-6 in mice serum, oral Administration of acetic acid group or intraperitoneal injection. TNF- alpha and IL-6 decreased significantly in the serum given to acetic acid, indicating that acetic acid inhibits the release of pro-inflammatory factors mediated by LPS in vivo and the effect of acetic acid on the macrophage is observed in.2. in vitro: in BMDM, mouse peritoneal macrophages, human peripheral blood mononuclear cells (PBMC), human CD14 cells and human THP-1 cells, the proinflammatory cells in group LPS The levels of TNF- alpha and IL-6 and m RNA increased significantly, and the TNF- alpha and IL-6 levels in the acetic acid treatment group and the m RNA were significantly lower than those of the LPS group. It suggested that acetic acid inhibited the TNF- alpha and IL-6 release and increasing of LPS induced IL-6, and the inhibition was dose-dependent and time dependent. The mechanism of inflammation: in BMDM, the phosphorylation of NF- kappa B p65 in group LPS increased significantly, while acetic acid pretreatment led to the activation of NF-k B p65 to significantly reduce the JNK of.LPS group, and the phosphorylation of ERK and p38 significantly increased. However, there was no significant difference between the acetic acid pretreatment and the phosphorylation of the NF-. The TNF- A and IL-6 of the LPS group increased significantly, and the TNF- alpha and IL-6 in the LPS group were significantly lower than those in the LPS group, and there was no significant difference in the expression of GPR43 or GPR41, or at the same time, and the release of GPR43 and GPR41. 1 may not involve the anti-inflammatory effect of acetic acid. In BMDM, the level of serum lactic acid in group LPS, 2-NBDG uptake slightly increased, GLUT1, PFKFB3, HK2, MCT4 m RNA increased rapidly, and the expression of GLUT1 on the surface of macrophages increased significantly, while the serum lactic acid level of the acetic acid treatment group decreased, GLUT1, GLUT1, declining, decreased significantly, The expression of GLUT1 on the surface of macrophages also decreased significantly. This indicated that acetic acid inhibited LPS induced glycolysis and glucose consumption in BMDM, suggesting that acetic acid inhibited the expression of key enzymes in glycolysis after LPS stimulation. After incubating IFN- gamma or GM-CSF to increase the glycolysis of BMDM, the release of TNF- alpha and IL-6 in the acetic acid group decreased and G was less than G. The expression of LUT1, PFKFB3, HK2, and MCT4 m RNA decreased; while the pre incubation of IFN- gamma or GM-CSF in the acetic acid treatment group, the release of TNF- alpha and IL-6 in the acetic acid group was reduced and GLUT1 was seen, and the decrease in the expression was almost completely reversed. It showed that the increase of glycolysis could reverse the anti-inflammatory effect of acetic acid. The 2-NBDG uptake of mouse spleen macrophages in the treatment group was significantly reduced, indicating that the glucose uptake induced by LPS in the body increased the role of.4.HIF-1 alpha in glycolysis: in BMDM, the expression of HIF-1 alpha m RNA and protein in LPS group increased, and the HIF-1 alpha m RNA and protein expression in the acetic acid group decreased significantly (P0.05) was statistically significant (P0.05). At the level of M RNA and the protein level of acetic acid, it inhibited the up regulation of HIF-1 alpha induced by LPS stimulation. The expression of HIF-1 alpha in BMDM was inhibited by DMOG. The results showed that TNF- alpha and IL-6 increased significantly in LPS group, and the TNF- alpha and IL-6 in the acetic acid group were significantly lower than those in the group. The -1 alpha specific vector overexpressed HIF-1 a. The results showed that the TNF- alpha and IL-6 in the LPS group increased significantly, and the TNF- alpha and IL-6 in the acetic acid treatment group were significantly lower than those in the LPS group, but the HIF-1 alpha overexpression and the TNF- alpha and IL-6 in the acetic acid group were not significantly different from those in the LPS group. 1, the expression of PFKFB3, HK2, MCT4 m RNA increased significantly, the expression of GLUT1 on the surface of macrophages increased significantly, the production of lactic acid and glucose increased significantly, and the expression of GLUT1, PFKFB3, HK2, MCT4 m RNA decreased significantly in the acetic acid group. The acid production and glucose level decreased significantly, and there was no significant difference from the LPS group. This indicated that the cytokine produced by the acetic acid reduction of LPS stimulation was at least partly regulated by the HIF-1 alpha dependent glycolysis. In BMDM, the NF- kappa B p65 phosphorylation in the LPS group increased, and the NF- kappa B p65 phosphorylation of the acetic acid group decreased. Addition, there was no significant difference from the LPS group; it was proved that DMOG could completely reverse the inhibitory effect of acetic acid on the activation of NF- kappa B. Finally, it was necessary to study in vivo that HIF-1 alpha was essential for the action of acetic acid to protect sepsis: the expression of HIF-1 a in the macrophages of sepsis mice was inhibited by DMOG in vivo, and the mortality of mice in the acetic acid group was significantly lower than that in the CLP group, and the liver lung group was significantly lower. The liver vein, central vein and hepatic sinus congestion, hepatic necrosis area and inflammatory cell infiltration decreased significantly, the capillary wall reconstruction of pulmonary vein and alveolar wall increased, pulmonary capillary hyperemia and fibrotic hyperplasia decreased. The AST of the acetic acid treatment group decreased significantly, the count of peritoneal bacteria decreased significantly; plasma IL-6, IL-1 The level of beta, TNF- alpha decreased, and the effect of acetic acid on mortality decreased obviously after DMOG pretreatment. Acetic acid improved tissue damage caused by sepsis, reduced the release of pro-inflammatory factors and enhanced the ability to scavenge the pathogen. It indicated that HIF-1 alpha plays a key role in glycolysis induced by acetic acid. Inhibition of the expression of HIF-1 alpha induced by LPS inhibits the glycolysis caused by LPS and reduces the release of proinflammatory factors, thus alleviating the organ dysfunction caused by sepsis and improving its survival rate.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R459.7

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