【作者】 张楠；

【导师】 唐其柱；

【作者基本信息】 武汉大学 ， 心血管内科， 2019， 硕士

【摘要】 研究背景:脓毒症是由宿主对感染的反应失调引起的,威胁患者生命的全身炎症反应综合征,伴随着严重的并发症,常导致多器官损伤。脓毒症心肌病是脓毒症患者最严重的并发症之一,也是导致患者死亡的关键病理过程,寻找和探索脓毒症心肌病的治疗方案和干预靶点具有重要的科学意义。近年来研究发现,自噬与脓毒症心肌病关系密切。一些研究表明早期自噬激活拮抗脓毒症心肌病的炎症反应,晚期自噬损伤导致心肌细胞凋亡,心脏收缩功能障碍;另一些研究表明自噬激活加剧脓毒症心肌病。目前自噬在脓毒症心肌病中的作用尚未完全阐明。自噬相关基因(autophagy associated gene,Atg)家族参与自噬的形成与调控,Atg家族的关键成员Atg7是自噬研究的一个热点。Atg7具有E1样泛素化激活酶活性,可促进LC3Ⅰ向LC3Ⅱ的转变和促进E3样泛素酶Atg12-Atg5复合体的形成。Atg7在自噬囊泡形成中发挥至关重要的作用。目前Atg7是否可以通过调控自噬或者其他非自噬途径参与脓毒症心肌病的发生发展,国内外尚未见报道,值得我们深入研究。研究内容:本课题主要探讨(1)Atg7在LPS诱导的脓毒症心肌病小鼠心脏组织和心肌细胞中的表达变化;(2)小鼠心肌细胞中特异性Atg7过表达或缺失后对小鼠生存率和心功能的影响和作用;(3)Atg7参与调控小鼠脓毒症心肌病的潜在作用机制;(4)Atg7调控的非自噬途径对脓毒症心肌病的作用。研究方案:第一部分:(1)检测Atg7和LC3Ⅰ/Ⅱ在LPS作用的野生型(C57)小鼠及原代大鼠心肌细胞(NRCM)中不同时间点的表达变化。动物实验:腹腔注射5mg/kg LPS建立模型。实验动物随机分为对照组(CON),LPS刺激3h组、6h组、12h组、24h组和48h组;细胞实验:分离培养NRCM并给予1μg/mL LPS建立细胞模型。实验分为对照组(CON),LPS刺激3h组、6h组、12h组、24h组和48h组,在相应时间点获取组织和细胞蛋白,Western-blot检测目标蛋白的表达。(2)探究小鼠心肌细胞中特异性Atg7过表达或缺失后对小鼠生存率和心功能的影响和作用。动物实验:选取(8-10周龄,24.5-25.5g)雄性C57小鼠,心肌细胞特异性Atg7敲除小鼠(CKO)及心肌细胞特异性Atg7过表达小鼠(TG),给予30mg/kg LPS观察小鼠死亡率;给予5mg/kg LPS后9-12h检测小鼠心功能;收集心脏组织,HE染色观察组织形态结构变化;电镜观察自噬体和溶酶体的表达;免疫荧光观察LC3和LAMP1的共定位;TUNEL检测心肌细胞凋亡;提取心脏总蛋白和RNA检测自噬、凋亡和AMPKα/mTORC1信号通路的改变。第二部分:(1)Atg7在LPS脓毒症心肌病中的潜在作用机制探究。动物实验:TG 小鼠给予二甲双胍(Metformin,Met,100mg/kg/d)1 周后获得(TG+Met)小鼠,用TG小鼠与AMPKα敲除小鼠杂交获取Atg7过表达且AMPKα敲除的小鼠(TG+AMPKα-KO),给予30mg/kg LPS观测小鼠死亡率;腹腔注射5mg/kg LPS构建脓毒症心肌病模型,LPS注射后9-12h超声监测小鼠心功能,收集小鼠心脏组织,Western-blot检测AMPKα2/mTORC1信号通路、溶酶体和凋亡相关蛋白的表达。(2)Atg7调控的非自噬途径对脓毒症心肌病的作用研究。收集5mg/kg LPS处理过的小鼠心脏组织,western-blot检测并定量Atg7沉默或过表达时,Nrf2相关的非自噬途径相关蛋白的表达。细胞实验:提取并培养NRCM,用lip6000转染Nrf2-siRNA沉默心肌细胞中Nrf2的同时用Atg7腺相关病毒(Ad-Atg7-KO)沉默Atg7,之后再用LPS刺激9-12h收集细胞,利用SOD及GPX检测试剂盒检测氧化应激水平的变化,进一步验证Atg7对Nrf2的表达调控。研究结果:第一部分:(1)LPS刺激使心脏组织及心肌细胞中的Atg7和LC3Ⅱ/LC3Ⅰ的表达升高,在本实验观察时间节点内Atg7的表达和LC3Ⅱ/LC3Ⅰ的比值较CON组均显著增加(P<0.05),且在LPS刺激12h时变化最明显。(2)LPS作用诱导实验组小鼠心功能障碍,与C57+LPS组相比,TG+LPS组小鼠的心功能显著恶化,而CKO+LPS小鼠的心功能有所改善。高浓度LPS使小鼠死亡率增加,TG+LPS组小鼠24h死亡率达100%,CKO+LPS组和C57+LPS组小鼠死亡率无显著差异。在组织水平上,TG+LPS组较C57+LPS组心肌排列显著紊乱,炎性细胞浸润显著增加,心肌细胞中空泡数量和心肌细胞凋亡比例显著增加,而CKO+LPS组和C57+LPS组之间无显著差异;透射电子显微镜显示C57+LPS组自噬小体和溶酶体较少且同步增加,TG+LPS组自噬小体明显增多而溶酶体较少,CKO+LPS组自噬小体显著减少而溶酶体增多;免疫荧光检测进一步证实了 LPS促进心脏组织中自噬流的增强,和C57+LPS组相比,TG+LPS组小鼠心脏组织中LC3Ⅱ显著聚集自噬小体增加,LAMP1的表达减少,自噬流并未增强;CKO+LPS组小鼠心脏组织中自噬小体减少,LAMP1的表达增加,自噬流增强;TUNEL染色显示LPS促进心肌细胞凋亡,且TG+LPS组较C57+LPS组心肌细胞凋亡阳性率明显增加,而CKO+LPS组心肌细胞凋亡较少;相关信号通路检测发现:相较C57+LPS组,TG+LPS组小鼠心肌组织中Atg7表达增加,LC3Ⅱ/Ⅰ比值增加,LAMP1及LAMP2表达减少,Bax表达增加,p-AMPKα表达减少,mTORC1磷酸化增加;而相较C57+LPS组,CKO+LPS组小鼠心肌组织中Atg7表达减少,LC3Ⅱ/Ⅰ比值降低,LAMP1及LAMP2表达增加,Bax表达减少,p-AMPKα增加,mTORC1磷酸化减少。第二部分:(1)LPS刺激下,相比TG组,TG+AMPKα-KO组小鼠心功能显著恶化,而TG+Met组小鼠心功能明显改善。相关信号通路检测发现:LPS刺激下,和TG组相比较,TG+Met组小鼠心肌组织中p-AMPKα/T-AMPKα增加,p-mTORC1/T-mTORC1减少,LAMP1表达增加,促凋亡蛋白Bax表达减少;而相比于TG+LPS组,TG+AMPKα-KO+LPS组小鼠心肌组织中p-mTORC1/T-mTORC1的比值增加,Bax表达明显增加,提示AMPKα敲除,AMPKα/mTORC1阻断,自噬受阻,凋亡增加(P均<0.05)。(2)Nrf2相关的非自噬途径相关蛋白western blot结果发现,和C57+CON组相比较,CKO+CON组,P62聚集,SOD1和SOD2表达增加;C57+LPS组Nrf2,HO-1,SOD1及SOD2表达显著降低;而和C57+LPS组相比较,CKO+LPS组小鼠心肌组织中,P62表达增加,Nrf2,HO-1,SOD1,SOD2的表达显著增加(P均<0.05);和 C57+CON 组相比较,TG+CON 组,Nrf2,HO-1 及 SOD1显著降低;C57+LPS组,Nrf2,HO-1,SOD1表达显著降低;而和C57+LPS组相比较,TG+LPS组小鼠心肌组织中,这些蛋白分子的表达无显著差异。此外,小鼠心脏组织SOD和GPx活性检测结果发现,和CON相比,LPS使CON组和TG组SOD及GPX的表达显著减少;与CON+LPS组比较,TG+LPS组SOD及GPX的表达显著减少;与TG+LPS组比较,KO+LPS组SOD及GPX的表达显著增多。NRCM中检测结果发现,和CON相比,LPS使CON组,si-Nrf2组,CKO+si-Nrf2组SOD及GPX的表达显著减少;与CON+LPS组比较,si-Nrf2+LPS组,CKO+si-Nrf2+LPS组SOD及GPX的表达显著减少。研究结论:1.心肌细胞特异性敲除Atg7减轻LPS所致的心功能恶化,改善脓毒症心肌病引起的氧化应激及凋亡;心肌细胞特异性过表达Atg7加重LPS所致的心功能恶化,加重脓毒症心肌病诱导的氧化应激及凋亡。2.Atg7特异性过表达不能改善LPS诱导的小鼠心功能障碍和减轻心肌细胞凋亡,机制可能是在脓毒症心肌病心脏中AMPKα2活性下调,mTROC1活性上调,抑制了溶酶体途径活性,自噬流并未增强,自噬小体在心肌细胞中的聚积反而促进了心肌细胞凋亡和心功能恶化。3.Atg7特异性敲除保护LPS诱导的脓毒症心肌病,其机制为Atg7依赖的自噬缺失导致P62在心肌细胞中聚积,P62降解细胞中的Keap 1促进Nrf2的释放,增强心肌细胞抗氧化应激的能力,从而保护LPS诱导的心肌细胞损伤。4.在LPS诱导的脓毒症心肌病中Atg7可作为其治疗的潜在靶点。更多还原

【Abstract】 Background:Sepsis is a life-threatening systemic inflammatory response syndrome,followed with severe complications and multiple organ damage,especially the heart.Septic cardiomyopathy,one of the most severe complications of sepsis,is a key pathological process leading to death.Therefore,It is of great scientific significance to find and explore treatment options and intervention targets for sepsis cardiomyopathy.In recent years,studies have found that there is a close relationship between autophagy and sepsis cardiomyopathy.Some evidences have shown that early autophagy can antagonize the inflammatory response of sepsis cardiomyopathy,but late autophagy damage leads to cardiomyocyte apoptosis and systolic dysfunction;others have shown that autophagy may be able to aggravates sepsis cardiomyopathy.The role of autophagy in sepsis cardiomyopathy has not yet been fully elucidated.The autophagy associated gene(autophagy associated gene,Atg)family is involved in the formation and regulation of autophagy.Among the all,Atg7,a key member of the Atg family,is a hotspot in autophagy research.Atg7 has El-like ubiquitination activating enzyme activity,which promotes the conversion of LC3I to LC3II and the formation of the E3-like ubiquitinase Atg12-Atg5 complex,which means Atg7 plays a crucial role in the formation of autophagic vesicles.So far,whether Atg7 can participate in the development of sepsis cardiomyopathy by regulating autophagy or other non-autophagy pathways has not been reported at home and abroad yet,thus it is worthy of further study.Objective:This study focused on(1)the expression changes of Atg7 in the heart and cardiomyocytes of LPS-induced septic cardiomyopathy mice;(2)the effects of specific Atg7’s overexpression or deletion on the survival rate and cardiac function of mice;(3)the potential mechanism of the regulation in septic cardiomyopathy by Atg7;(4)The effect of Atg7-regulated non-autophagy pathway on septic cardiomyopathy.Methods:Part 1:(1)To detect the expression changes of Atg7 and LC3I/II in wild type(C57)mice and primary rat cardiomyocytes(NRCM)under the stimulation of LPS.Animal experiments:The model was established by intraperitoneal injection of 5 mg/kg LPS.The experimental animals were randomly divided into control group(CON),LPS stimulated 3h group,6h group,12h group,24h group and 48h group;Cell experiment:Separated NRCM added with 1μg/mL LPS to establish a cell model.The experiment was divided into control group and LPS stimulated 3h group,6h group,12h group,24h group and 48h group.Tissue and cell proteins were obtained at corresponding time points,and the expression of target protein was detected by Western-blot.(2)To detect the effects of specific Atg7’s overexpression or deletion on the survival rate and cardiac function of mice.Animal experiments:Male C57 mice(8-10 weeks old,24.5-25.5 g),cardiac-specific Atg7 knockout mice(Atg7-CKO)and cardiac-specific Atg7 overexpressing mice(Atg7-TG)were administered.The mortality of mice was observed with 30 mg/kg LPS injected;the cardiac function of mice was detected after 9-12 h with5 mg/kg LPS injected;the tissue morphology changes were observed by HE staining,and the expression of autophagosomes and lysosomes was observed by electron microscopy.Colocalization of LC3 and LAMP1 was observed,TUNEL was used to detect cardiomyocyte apoptosis;total cardiac protein and RNA were extracted to detect autophagy,apoptosis and AMPKα/mTORC1 signaling pathway changes.Part 2:(1)The potential mechanism of Atg7 in LPS sepsis cardiomyopathy.Animal experiments:Atg7-TG mice were given(TG+Met)mice 1 week after metformin(Metformin,Met,100 mg/kg/d),and TG mice were hybridized with AMPKa knockout mice to obtain Atg7 overexpression and AMPKα knockdown;5 mg/kg LPS was intraperitoneally injected to construct a model of sepsis cardiomyopathy,and the heart function of mice was monitored by ultrasound 9-12 h after LPS injection.Mouse heart tissues were collected,and AMPKα2/mTORC1 signaling pathway,lysosome and apoptosis-related proteins were detected by Western-blot.(2)Detected the role of Atg7-regulated non-autophagy pathway in sepsis cardiomyopathy.The heart tissue of 5 mg/kg LPS-treated mice was collected,and the expression of Nrf2 pathway-related proteins was detected by western-blot when Atg7 was silenced or overexpressed.Cell experiment:NRCM was extracted and cultured,Nrf2-siRNA was transfected with lip6000 to silence Nrf2 in cardiomyocytes,and Atg7 was silenced with Ad-Atg7-KO,and then cell were collected after LPS stimulation for 9-12 h.And the oxidative stress level were detected,then the regulation of Nrf2 expression by Atg7 was further verified..Results:Part 1:(1)With the stimulation of LPS the expression of Atg7 and LC3Ⅱ/LC3Ⅰ in cardiac tissue and cardiomyocytes increased,The expression level was the highest at 12h.(2)Compared with the C57+LPS group,the cardiac function of the TG+LPS group was significantly worsened,while the cardiac function of the CKO+LPS mice was improved.High concentration of LPS increased the mortality of mice,and the mortality of mice in TG+LPS group was 100%within 24h.There was no significant difference in mortality between CKO+LPS group and C57+LPS group.At the tissue level,the TG+LPS group had more disordered myocardial alignment than C57+LPS group,the inflammatory cell infiltration,myocardial cell vacuoles and cardiomyocyte apoptosis were significantly increased,but in the.There was no significant difference between CKO+LPS group and the C57+LPS group.Transmission electron microscopy showed that autophagosomes and lysosomes in C57+LPS group were less and increased synchronously.Autophagosomes in TG+LPS group increased significantly but lysosomes were much less.By contrast,in CKO+LPS group The corpuscles are significantly reduced while the lysosomes are increased;Immunofluorescence assay further confirmed that LPS promoted the enhancement of autophagic flow in cardiac tissue.In the TG+LPS group,LC3II was significantly aggregated in mouse heart tissue,the autophagosomes and the expression of lysosomal-associated protein LAMP1 enhanced,but the autophagic flow did not;However,a reverse trend found in CKO+LPS group;TUNEL staining showed that LPS promoted cardiomyocyte apoptosis,and the positive rate of cardiomyocyte apoptosis was significantly increased in TG+LPS group compared with C57+LPS group,while cardiomyocyte apoptosis was less in CKO+LPS group.The detection of related signal pathways showed that the expression of Atg7 and Bax,the ratio of LC3II/I and the degree of phosphorylation of mTORCl in the myocardial tissue of the TG+Met group was increased,while the expression of LAMP 1,LAMP2 and p-AMPKa was decreased.However,in C57+LPS group,a reverse trend can be seen.Part 2:With LPS stimulation,the cardiac function of the TG+AMPKα-KO group was significantly worse than that of the TG group,while the cardiac function of the TG+Met group was significantly improved.Compared with the TG group,the expression of Atg7 and Bax in the myocardial tissue of the TG+Met group,the ratio of LC3Ⅱ/Ⅰ and the degree of phosphorylation of mTORC1 was decreased,while the expression of LAMP1,LAMP2 and p-AMPKα was increased.However,a reverse trend can also be seen in the TG+AMPKα-KO group.The western blot analysis of NRF2 pathway-related proteins revealed that the expression of Nrf2,HO-1,SOD 1,SOD2 and P62 was significantly increased in CK+LPS group compared with C57+LPS group,but the expression of these protein molecules in TG+LPS group has no significant difference.In addition,the results of SOD and GPx activity in mouse heart tissue showed that compared with CON,LPS significantly decreased the expression of SOD and GPX in CON and TG groups.Compared with CON+LPS group,the expression of SOD and GPX in TG+LPS group Significantly reduced;the expression of SOD and GPX in the KO+LPS group was significantly increased compared with the TG+LPS group.The results of NRCM showed that compared with CON,LPS significantly decreased the expression of SOD and GPX in the CON group,the si-Nrf2 group,and the CKO+si-Nrf2 group.Compared with the CON+LPS group,the expression of SOD and GPX in the si-Nrf2+LPS group and the CKO+si-Nrf2+LPS group was significantly reduced.Conclusion:1.Cardiac tissue-specific knockout of Atg7 attenuates LPS-induced cardiac function deterioration,improves the inflammation,oxidative stress and apoptosis caused by LPS;Cardiac tissue-specific overexpression of Atg7 aggravates the deterioration of cardiac function caused by LPS,aggravates inflammatory response,oxidative stress and cardiomyocyte apoptosis induced by septic cardiomyopathy.2.Atg7-specific overexpression cannot improve LPS-induced cardiac dysfunction and decrease myocardial cell apoptosis.The mechanism may be that AMPKα2 activity is down-regulated in septic heart,mTROCl activity is up-regulated to inhibit lysosomal pathway activity,autophagic flow Not enhanced,the accumulation of autophagosomes in cardiomyocytes promoted cardiomyocyte apoptosis and deterioration of cardiac function.3.Atg7-specific overexpression protects LPS-induced sepsis cardiomyopathy.The mechanism is that Atg7-dependent autophagy loss leads to the accumulation of P62 in cardiomyocytes.Keapl in P62-degraded cells promotes the release of Nrf2 and enhances the anti oxidation of cardiomyocytes,thereby protecting cardiomyocyte from damage by LPS-induced sepsis cardiomyopathy.4.Atg7 may be a potential therapeutic target in LPS-induced septic cardiomyopathy.更多还原