【作者】 王浩；

【导师】 肖献忠；

【作者基本信息】 中南大学 ， 基础医学， 2022， 博士

【摘要】 背景及目的:脓毒症是机体对感染的免疫反应失调所致的危及生命的器官功能障碍。脓毒症患者失调的免疫反应以失控性炎症反应和免疫抑制为主要特征,这些失调的免疫反应导致细胞结构损伤和功能障碍,继而引起多器官功能障碍综合征,其中急性肾损伤(acute kidney injury,AKI)是脓毒症多器官功能障碍中最常见、最严重的并发症之一。脓毒症时,肾小管管周和肾小球的广泛微血管血流障碍所致的细胞缺氧以及各种炎症介质释放所致的细胞代谢、能量合成、线粒体功能障碍是急性肾损伤的主要发病机制。细胞缺氧和代谢改变继而导致许多生物过程发生紊乱,比如细胞死亡、自噬、线粒体功能障碍、线粒体自噬以及活性氧增多。自噬是一种进化上非常保守的由溶酶体水解酶介导的多步骤降解途径,它利用溶酶体水解酶类降解细胞内受损的细胞器、蛋白质和其他大分子物质,主要由腺苷单磷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)、去乙酰化酶sirtuins(SIRTs)和雷帕霉素的哺乳动物靶点蛋白(mammalian target of rapamycin,m TOR)等正、负调控因子介导。已有的研究显示,自噬参与脓毒症急性肾损伤的发生发展,对急性肾损伤具有保护作用。但是,脓毒症急性肾损伤时自噬调控的机制尚不十分清楚。有氧糖酵解(aerobic glycolysis)是指当氧气足够的情况下,细胞内糖酵解途径被激活,其代谢产物乳酸的含量明显增高。许多研究表明有氧糖酵解在脓毒症的发生发展中发挥重要的作用。血清乳酸水平的测量通常被纳入危重病的临床管理中,用来评价脓毒症患者的疾病活动度、治疗反应和预后。脓毒症的高乳酸血症主要来自活化的免疫细胞,调控固有免疫细胞和适应性免疫细胞的免疫功能。使用有氧糖酵解抑制剂2脱氧葡萄糖(2-deoxyglucose,2-DG)进行干预,能显著降低脓毒症小鼠血清乳酸水平,改善多器官功能障碍,提高生存率。我们前期的研究显示2-DG抑制有氧糖酵解,能够保护脓毒症小鼠的多器官损伤,包括急性肾损伤。有氧糖酵解是否参与脓毒症急性肾损伤的发生目前并不十分清楚,有氧糖酵解抑制剂保护脓毒症急性肾损伤的机制有待进一步研究阐明。探究脓毒症时肾脏组织的糖代谢是否向有氧糖酵解转变以及该变化对于急性肾损伤产生怎样的影响,对于脓毒症急性肾损伤的发病机制及其防治具有重要意义。本论文采用盲肠结扎穿孔术(cecal ligation and puncture,CLP)复制脓毒症小鼠模型,用有氧糖酵解抑制剂2-脱氧葡萄糖2-DG或者自噬抑制剂3-MA进行干预,观察小鼠肾功能、肾组织病理损害、细胞凋亡、自噬标志物和自噬调控因子SIRT3、AMPK表达的变化,从整体水平探讨有氧糖酵解对脓毒症及其急性肾损伤的影响及其机制;随后采用脂多糖(lipopolysaccharide,LPS)刺激肾小管上皮细胞制备脓毒症细胞模型,并进行2-DG或者3-MA干预,观察细胞凋亡、自噬标志物和自噬调控因子SIRT3、AMPK表达的变化,从细胞水平探究有氧糖酵解对LPS诱导的肾小管上皮细胞凋亡的影响,并进一步从SIRT3/AMPK通路探究2-DG是否通过干预自噬影响脓毒症急性肾损伤的发生。研究方法:(1)采用CLP术复制脓毒症小鼠模型,用商业化试剂盒检测脓毒症小鼠血清乳酸、血清尿素氮(blood urea nitrogen,BUN)、肌酐和肾损伤分子-1(kidney injury molecule-1,KIM-1)水平;采用H&E染色观察肾组织的病理学改变;采用TUNEL染色检测肾组织细胞的凋亡情况;采用Realtime-PCR检测脓毒症小鼠肾组织中糖酵解相关基因丙酮酸脱氢酶激酶1(pyruvate dehydrogenase kinase 1,PDK1),乳酸脱氢酶A(lactate dehydrogenase A,LDHA)和丙酮酸激酶M2(pyruvate kinase M2,PKM2)的m RNA表达水平;采用Western Blot或者细胞免疫荧光检测自噬标志物LC3-I、LC3-II和p62以及自噬调控因子SIRT3、AMPK、p-AMPK的水平。(2)CLP前3小时腹腔注射2g/kg2-DG,采用商业化试剂盒检测小鼠血清乳酸、血清尿素氮、肌酐和KIM-1水平;采用H&E染色观察肾组织的病理学改变;采用TUNEL染色检测肾组织的凋亡情况;采用Realtime-PCR检测脓毒症小鼠肾组织中糖酵解相关基因PDK1、LDHA和PKM2的m RNA表达水平;采用Western Blot法检测自噬标志物LC3-I、LC3-II和p62以及自噬调控因子SIRT3、AMPK、p-AMPK的水平,观察有氧糖酵解的抑制对脓毒症小鼠肾功能、肾组织病理损害、细胞凋亡、自噬标志物和自噬调控因子的影响。(3)用1μg/ml LPS刺激HK-2细胞,采用流式细胞术检测HK-2细胞的凋亡情况;采用Realtime-PCR检测糖酵解相关基因PDK1、LDHA和PKM2的m RNA表达水平;采用Western Blot法检测自噬标志物LC3-I、LC3-II和p62以及自噬调控因子SIRT3、AMPK、p-AMPK的水平;并观察2-DG对这些指标的影响。(4)采用30 mg/kg自噬抑制剂3-MA预处理脓毒症小鼠或者LPS刺激的HK-2细胞,观察2-DG对脓毒症小鼠肾功能、肾组织病理损害以及肾组织和HK-2细胞的细胞凋亡、自噬标志物和自噬调控因子的影响发生的变化。(5)采用乳酸预处理LPS刺激的HK-2细胞,采用Western Blot法检测自噬标志物LC3-I、LC3-II和p62以及自噬调控因子SIRT3、AMPK、p-AMPK的水平。研究结果:(1)有氧糖酵解在脓毒症小鼠肾组织中增加,2-DG预处理减轻脓毒症急性肾损伤。脓毒症小鼠血清的乳酸、尿素氮、肌酐和KIM-1的水平在CLP后24小时显著升高,小鼠肾组织中糖酵解相关基因PDK1,LDHA和PKM2的m RNA表达水平增高;而有氧糖酵解抑制剂2-DG预处理明显降低脓毒症小鼠血清BUN、肌酐和KIM-1的水平,减轻肾组织的病理学损伤。(2)自噬在脓毒症小鼠肾组织中增强,2-DG预处理进一步增强肾组织中的自噬。脓毒症小鼠肾组织中的自噬标志物LC3-I表达减少,而LC3-II表达明显增加,LC3-II/I比值显著增加,p62的蛋白质表达在CLP后降低;而2-DG预处理后,LC3-II/I的比值增加和p62的表达下调更加明显,肾小管上皮细胞的凋亡明显减少。(3)有氧糖酵解和自噬在LPS刺激的人肾小管上皮细胞HK-2中增加,2-DG预处理进一步增强LPS刺激下HK-2的自噬,降低LPS诱导的SIRT3、p-AMPK水平,抑制HK-2的细胞凋亡。当LPS刺激人肾小管上皮细胞HK-2时,培养上清的乳酸水平增加,LDHA和PKM2的m RNA水平明显升高,LC3-II/I比值升高,p62蛋白质表达降低,SIRT3、p-AMPK蛋白质水平明显升高,肾小管上皮细胞的凋亡明显增加。而2-DG预处理降低LPS诱导的LDHA和PKM2m RNA水平和培养上清液的乳酸水平以及SIRT3、p-AMPK蛋白质水平,并进一步增高LC3-II/I比值,降低p62表达,减少肾小管上皮的细胞凋亡。(4)自噬抑制剂3-MA预处理减弱2-DG对脓毒症小鼠和LPS刺激下的HK-2的保护作用。自噬抑制剂3-MA预处理后,2-DG对脓毒症小鼠血清BUN、SCR和KIM-1水平、肾组织细胞凋亡以及LPS诱导的HK-2细胞凋亡的抑制作用明显减弱。(5)乳酸预处理抑制LPS诱导的自噬,逆转2-DG对HK-2细胞自噬的增强作用。乳酸预处理降低LPS刺激下的HK-2细胞LC3-II/I比值以及SIRT3和p-AMPK的表达,增加p62的表达,并部分地消除2-DG对LC3II/I比值、SIRT3和p-AMPK表达的增加和对p62的降低,从而逆转2-DG对HK-2细胞凋亡的抑制。研究结论:(1)脓毒症小鼠肾组织的有氧糖酵解增强,参与急性肾损伤发生发展;同时自噬增强,发挥保护作用。(2)有氧糖酵解抑制剂2-DG通过增强自噬减少肾小管上皮细胞凋亡,减轻脓毒症急性肾损伤,发挥保护作用。(3)有氧糖酵解抑制剂2-DG通过SIRT3/AMPK通路增强自噬减轻脓毒症急性肾损伤,提示有氧糖酵解可能是脓毒症时急性肾损伤的潜在治疗靶点。图26幅,表1个,参考文献117篇更多还原

【Abstract】 Background and Objective:The immune response dysfunction in patients with sepsis is characterized by uncontrolled inflammatory response and immunosuppression.These dysfunctional immune responses lead to cell dysfunction and then organ failure.Acute kidney injury(AKI)is one of the most common and serious complications in sepsis.The main pathogenesis of acute kidney injury in sepsis is cell hypoxia,which is caused by extensive microvascular blood flow disturbance around renal tubules and glomeruli;and cell metabolism,energy synthesis and mitochondrial dysfunction caused by various inflammatory mediators released.Hypoxia and metabolic disturbance lead to many biological processes’ disturbance.Many cell death pathways,such as necrosis,apoptosis,autophagy,programmed necrosis,netosis and pyrosis,are directly activated by uncontrolled inflammatory response or pathogens in sepsis.Autophagy is an evolutionarily conserved multi-step degradation pathway mediated by lysosomal hydrolases,which degrades damaged organelles,proteins and other macromolecules in cells.It is mainly mediated by positive or negative regulatory factors such as AMP activated protein kinase(AMPK),sirtuins(sirts)and mammalian target of rapamycin,MTOR and others.Many studies have shown that autophagy is involved in the occurrence and development of acute kidney injury in sepsis,and has a protective effect on acute kidney injury.The mechanism of autophagy regulation in septic acute kidney injury still remains unclear.Aerobic glycolysis is that when oxygen is enough,the glycolysis pathway in cells is activated,and the lactate is obviously increased,plays an important role in the development of sepsis.The measurement of serum lactate level is usually included in the clinical management of critical diseases,and is often used to evaluate the disease activity,treatment response and prognosis of sepsis patients.The hyperlactation of sepsis mainly comes from activated immune cells,regulates the immune function of innate immune cells and adaptive immune cells.The intervention of 2-deoxyglucose(2-DG)as aerobic glycolysis inhibitor can significantly reduce the serum lactoic acid level,improve the function of multiple organs and improve survival rate.Our previous studies have shown that 2-DG inhibits aerobic glycolysis and protects multiple organ damage in sepsis mice,including acute renal injury.Why inhibition of aerobic glycolysis can protect acute renal injury in sepsis needs further study.It is not clear whether aerobic glycolysis is involved in the occurrence of acute renal injury in sepsis.It is of great significance to explore whether the glucose metabolism of kidney tissue transverse to aerobic glycolysis and how the change affects acute renal injury in sepsis.In this study,the sepsis mouse model was established by cecal ligation and puncture(CLP),and the changes of renal function,renal pathological damage,apoptosis,the expression of autophagy markers and autophagy regulatory factors SIRT3 and AMPK in mice by intervention of 2-deoxyglucose or 3-MA was determined in order to explore the effect and mechanism of aerobic glycolysis on sepsis and acute kidney injury.Renal tubular epithelial cells were stimulated by lipopolysaccharide(LPS)to establish sepsis cell model,and 2-DG or3-MA intervention was performed to observe apoptosis,the expression of autophagy markers and autophagy regulatory factors SIRT3 and AMPK,and to study the effect of aerobic glycolysis on renal tubular epithelial cell apoptosis at the cellular level and explore whether 2-DG interfere with autophagy via SIRT3/AMPK pathway in sepsis-induced acute kidney injury.Methods:(1)sepsis mouse model was established by CLP.The levels of serum lactic acid,serum urea nitrogen(BUN),creatinine and kidney injury molecule-1(KIM-1)were detected by commercial kit.The pathological changes of renal tissue were observed by H & E staining.TUNEL staining was used to detect the apoptosis of renal cells.Realtime-PCR was used to detect the m RNA expression levels of glycolysis related genes pyruvate dehydrogenase kinase 1(PDK1),lactate dehydrogenase A(LDHA)and pyruvate kinase M2(PKM2)in the kidney of septic mice.The levels of autophagy markers LC3-I,LC3-II and p62 and autophagy regulators SIRT3,AMPK and p-AMPK were detected by Western blot or immunofluorescence.(2)2 g / kg 2-DG was injected intraperitoneally 3 hours before CLP,and the levels of serum lactic acid,serum urea nitrogen,creatinine and KIM-1 were detected by commercial kits.The pathological changes of renal tissue were observed by H & E staining.TUNEL staining was used to detect the apoptosis of renal tissue.The m RNA levels of PDK1,LDHA and PKM2 were detected by real-time PCR.Western blot was used to detect the levels of autophagy markers LC3-I,LC3-II and p62,as well as autophagy regulatory factors SIRT3,AMPK and p-AMPK.The effects of inhibition of aerobic glycolysis on renal function,renal pathological damage,apoptosis,autophagy markers and autophagy regulatory factors in septic mice were observed.(3)The HK-2 cells are stimulated by 1μg/ml LPS and the apoptosis of HK-2 cells was detected by flow cytometry.The m RNA expression levels of PDK1,LDHA and PKM2 were detected by real-time PCR.Western blot was used to detect the levels of autophagy markers LC3-I,LC3-II and p62,and the expression of autophagy regulators SIRT3,AMPK and p-AMPK.(4)The septic mice or HK-2 cells stimulated by LPS were pretreated with autophagy inhibitor 3-MA(30 mg / kg)in order to observe wherther 3-MA regulate the effects of 2-DG on renal function and pathological damage,apoptosis,the expression of autophagy markers and autophagy regulatory factors.(5)The HK-2 cells stimulated by LPS were pretreated with lactic acid.The levels of autophagy markers LC3-I,LC3-II and p62 and autophagy regulators SIRT3,AMPK and p-AMPK were detected by Western blot.Results:(1)Aerobic glycolysis was increased in the renal tissue of septic mice,and 2-DG pretreatment reduced the acute renal injury of septic mice.The levels of lactic acid,bun,creatinine and KIM-1 in serum of sepsis mice were significantly increased at 24 hours after CLP,and the m RNA expression levels of glycolysis related genes PDK1,LDHA and PKM2 in renal tissue were increased.Aerobic glycolysis inhibitor 2-DG reduced the levels of serum BUN,creatinine and KIM-1 in septic mice,and alleviated the pathological damage of renal tissue.(2)Autophagy was enhanced in renal tissue of septic mice,and 2-DG pretreatment further enhanced autophagy in renal tissue.The expression of autophagy marker LC3-I was decreased,but the expression of LC3-II was significantly increased,the ratio of LC3-II / I was significantly increased,and the expression of p62 was decreased after CLP;After 2-DG pretreatment,the ratio of LC3-II / I increased and the expression of p62 decreased more significantly,and the apoptosis of renal tubular epithelial cells decreased significantly.(3)Aerobic glycolysis and autophagy were increased in human renal tubular epithelial cells HK-2 stimulated by LPS.2-DG pretreatment further enhanced the autophagy of HK-2 induced by LPS,reduced the levels of SIRT3 and p-AMPK induced by LPS,and inhibited the apoptosis of HK-2.When HK-2 cells were stimulated with LPS,the levels of lactate,LDHA and PKM2 m RNA,LC3-II / I ratio,p62 expression and apoptosis of HK-2 cells were significantly increased.However,2-DG treatment decreased the levels of LDHA and PKM2 m RNA and the levels of lactate in the supernatant induced by LPS,and further increased LC3-II /I ratio,decreased the expression of p62 and reduced the apoptosis of renal tubular epithelial cells.When HK-2 cells were stimulated by LPS,the levels of SIRT3 and p-AMPK in HK-2 cells were significantly increased,while 2-DG treatment decreased the increase of SIRT3 and p-AMPK induced by LPS.(4)Pretreatment with autophagy inhibitor 3-m A attenuated the protective effect of 2-DG on septic mice and HK-2 under stimulation with LPS.After pretreatment with autophagy inhibitor 3-MA,the inhibition of2-DG on serum BUN,SCR and KIM-1 levels,renal cell apoptosis and LPS induced HK-2 cell apoptosis in septic mice were attenuated signifiacantly.(5)Lactate pretreatment inhibited LPS-induced autophagy and reversed the enhancement effect of 2-DG on autophagy.Lactic acid pretreatment decreased LPS-induced the upregulation of LC3-II / I ratio,SIRT3 and p-AMPK,increased p62 expression,and partially eliminated the effect of 2-DG on the increase of LC3-II / I ratio,SIRT3 and p-AMPK and the decrease of p62 expression,thus finally reversed the inhibition of 2-DG on apoptosis.更多还原