【作者】 吴兰；

【导师】 张文胜；

【作者基本信息】 四川大学 ， 麻醉学， 2021， 博士

【摘要】 目的:机械通气作为麻醉维持和危重患者抢救中不可或缺的手段,在现代医学中的应用越来越广泛。由于呼吸机模式或参数选择的不合理及与患者基础疾病的交互作用,可能会带来新的损伤——机械通气相关性肺损伤^[1]（ventilator induced lung injury,VILI）。1998年以后逐渐证实炎症在此种损伤中有重要的核心意义。目前,针对机械通气相关性肺损伤,主要是进行策略性预防,缺乏有效的药物治疗手段;研究药物的大部分靶点都集中在信号转导、抑制炎症和线粒体的功能上。阿托伐他汀（atorvastatin,ATV）是一种在临床应用多年的降脂药物,由于发现其具有改善病人的远期生活质量、改善神经系统炎症等作用,其降低血浆胆固醇以外的作用效果被广泛研究。有研究发现,阿托伐他汀在肺部具有抗炎和增强肺功能的作用,但是目前未见关于阿托伐他汀对于VILI的作用及其机制研究。随着代谢组学技术的发展,发现脂质代谢通路产物在炎症发生发展中起重要作用。鞘脂代谢通路作为脂质代谢中的一大类,其代谢产物是构成细胞膜的重要分子,参与细胞的生长、分化、衰老和炎症等一系列重要的信号转导过程。鞘氨醇激酶（sphingosine kinase,SphK）为鞘脂代谢通路中的关键酶,主要作用为催化鞘氨醇生成鞘氨醇-1-磷酸（sphingosine 1-phosphate,S1P）,并分为两种同工酶:鞘氨醇激酶1（SphK1）和鞘氨醇激酶2（SphK2）,前者主要分布在细胞质,后者主要分布在细胞核。SphK1在不同模型上通过多种机制发挥重要的抗炎作用。SphK-S1P轴被认为是许多疾病潜在治疗靶点,S1P可以改善脓毒症、肺动脉高压、肺纤维化、哮喘、支气管肺发育不良;同时有研究发现,他汀类药物可以诱导S1P1受体,改善脓毒症急性肺损伤。综上,我们提出假说:阿托伐他汀能减低机械通气相关性肺损伤,主要通过SphK1介导,一方面影响丝裂原活化蛋白激酶（mitogen-activated protein kinase,MAPK）通路相应蛋白的失活降低炎症反应;另一方面通过增强内皮细胞连接的完整性发挥作用。本研究采用高潮气量机械通气损伤小鼠模型模拟临床VILI,结合动物、组织、细胞实验、代谢组学和基因敲除小鼠,首先明确阿托伐他汀对机械通气的肺保护作用,并通过脂质代谢组学筛选出差异代谢物,进一步通过敲基因老鼠明确鞘氨醇1是否与阿托伐他汀对机械通气的肺保护作用有关,最后探索其参与阿托伐他汀对机械通气的肺保护作用是否与MAPK信号通路在此过程中表达的变化以及维持内皮细胞连接完整性有关。材料和方法:选用8-12周野生型C57BL/6小鼠,戊巴比妥钠腹腔内注射麻醉,并按照是否机械通气和给予实验药物,分为4组。气管切开前14 h,腹腔内注射阿托伐他汀10 mg/kg或者等容积二甲亚砜,机械通气参数采用40 ml/Kg,频率65次/分,通气4 h后取材,包括肺组织、血液和肺泡灌洗液,分别行血气分析、病理、免疫组化、细胞因子测定、Western blot（WB）、血浆和肺泡灌洗液代谢组学测定,从而明确阿托伐他汀在VILI中的抗炎作用及其可能的差异脂质代谢物。接着选用SphK1基因敲除小鼠,实验分组同野生型小鼠一致,通过行肺组织病理、肺泡灌洗液细胞因子测定,评价在基因敲除小鼠中阿托伐他汀的抗炎作用;同时原代培养野生型及SphK1基因敲除小鼠肺微血管内皮细胞（pulmonary microvascular endothelial cells,PMVECs）,并采用流式细胞分选的办法,准确分选目标细胞,利用Western blot和RT-PCR验证敲基因小鼠的PMVECs中SphK1基因及蛋白表达情况;采用细胞周期牵张,体外模拟VILI,Western blot检测下游信号通路改变,并采用MAPK通路的三种抑制剂预处理周期性牵张的PMVECs,进一步验证细胞因子的变化。最后通过Western blot检测SphK1/2的变化,免疫荧光技术检测细胞间的连接状态,测量PMVECs细胞的通透性,探究了阿托伐他汀通过鞘氨醇激酶1增强细胞间连接的作用。结果:（1）野生型（wild type,WT）小鼠高潮气量机械通气和阿托伐他汀预处理后的肺损伤:高潮气量机械通气（high tidal volume,HT）与自主呼吸（spontaneous breath,SB）比较,肺组织肺湿干比增加;支气管肺泡灌洗液（bronchoalveolar lavage fluid,BALF）中的总蛋白含量（HT vs SB,0.54±0.025 vs 0.23±0.02mg/ml;P<0.01,n=5）和细胞数（HT vs SB,2.26±0.025×10⁵ vs 1.35±0.67×10⁵/m L;P<0.01,n=5）显著增高;肺组织HE染色显示小鼠肺组织呈现明显的急性肺损伤性改变。阿托伐他汀预处理后,肺组织肺湿干比下降;降低了蛋白和细胞渗漏BALF中肺细胞总数（HT+ATV,0.37±0.026 mg/ml and 0.93±0.23×10⁵/m L;P<0.01,n=5）;肺HE染色显示肺损伤评分下降（P<0.05,n=5）,Tunel和Western Blot测定caspase3提示凋亡减少;提示阿托伐他汀对高通气造成的肺损伤有保护作用。（2）野生型小鼠高潮气量机械通气和阿托伐他汀预处理后的炎症反应:高潮气量机械通气后,小鼠支气管肺泡灌洗液中的炎性细胞因子:IL-6、KC、MIP-1α、MIP-2、MCP-1均增高;阿托伐他汀预处理后,显著抑制了IL-6、KC、MIP-2的水平,提示阿托伐他汀预处理可以减轻高通气造成的炎性反应。（3）广泛靶向脂质代谢组学:肺泡灌洗液代谢产物中,鞘磷脂呈现SB组低于HT,HT+ATV低于HT但高于SB组。（4）SphK1基因敲除（SK1 knockout,SK1KO）小鼠行机械通气和阿托伐他汀预处理后的肺损伤:SphK1基因敲除后,行高潮气量机械通气,支气管肺泡灌洗液中的蛋白总含量（WT HT vs SK1KO HT;0.54±0.03 vs 0.80±0.07mg/ml;P<0.01,n=5）以及细胞计数（WT HT vs SK1KO HT;2.26±0.025×10⁵/ml vs 3.47±0.21×10⁵/ml;P<0.01,n=5）显著增加;SphK1基因敲除老鼠肺组织HE染色显示炎性细胞浸润和肺间质水肿较野生型增多。阿托伐他汀预处理高潮气量组,SphK1基因敲除组支气管肺泡灌洗液中的蛋白含量（SK1KO HT/ATV vs WT HT/ATV;0.52±0.07mg/ml vs 0.37±0.03mg/m L,P<0.05,n=5）和细胞（SK1KO HT/ATV vs WT HT/ATV;2.93±0.45×10⁵/ml vs 0.93±0.23×10⁵/ml,P<0.05,n=5）明显较野生型上升;肺HE染色显示,肺组织的炎性浸润较野生型组明显。SphK1基因敲除并行高潮气量通气,阿托伐他汀预处理组比未给药组,肺HE染色显示肺组织结构未见明显改善。（5）SphK1基因敲除小鼠行机械通气和阿托伐他汀预处理后的炎症反应:SphK1基因敲除后,行高潮气量机械通气,小鼠支气管肺泡灌洗液中的炎性细胞因子:IL-6 KC、MIP-1α、MIP-2、MCP-1较自主呼吸组均增高;同时,基因敲除组与野生型组同时行高潮气量通气,除了IL-1β外,其余测量的五种细胞因子均为基因敲除组高于野生型组。在SphK1基因敲除的老鼠,进行高潮气量通气,无论有无阿托伐他汀预处理,其炎症因子的水平均无明显变化。（6）细胞牵张实验后细胞因子测量的结果:在野生型的内皮细胞中,行机械牵张,其细胞因子的水平较对照组明显增加。阿托伐他汀预处理,显著抑制了细胞牵张后IL-6、KC、MIP-1α、MIP-2、MCP的水平,提示阿托伐他汀能减轻细胞牵张引起的炎性反应。与野生型细胞类似,在SphK1基因敲除的内皮细胞中,行机械牵张,炎性细胞因子的水平较静息状态增加,SphK1基因敲除后,逆转了阿托伐他汀引起的炎性因子水平的下降,包括IL-6、KC、MCP-1,但是没有逆转MIP-1α、MIP-2的水平。（7）肺微血管内皮细胞给予细胞牵张和MAPK抑制剂处理后:a)静息状态下野生型肺微血管内皮细胞,MAPK主要以非磷酸化的状态存在,活性低;当给予18%的机械牵张刺激,EKR的磷酸化不显著,而P38和JNK明显活化。阿托伐他汀预处理显著抑制JNK,而但不抑制ERK和P38磷酸化。b)静息状态下的SphK1基因敲除肺微血管内皮细胞,ERK以磷酸化的状态存在,活性高,JNK和P38以非磷酸化状态存在;当给予18%的机械牵张刺激,EKR变化不显著,而P38和JNK明显活化。阿托伐他汀预处理抑制JNK磷酸化,但其磷酸化程度仍高于野生型肺微血管内皮细胞。c)与野生型内皮细胞相比,SphK1基因敲除内皮细胞在进行机械牵张后会产生大量IL-6和KC。SphK1敲除的内皮细胞中IL-6生成被JNK抑制剂抑制,并被SB203580（P38抑制剂）部分抑制,但不被PD58059（ERK抑制剂）抑制;KC的趋势与IL-6类似。（8）肺微血管内皮细胞间连接功能的检测提示:免疫荧光染色显示VE-钙粘蛋白主要位于肺血管内皮细胞胞浆内,野生型内皮细胞间有少量间隙。凝血酶处理增加了内皮细胞之间的间隙的大小和数量。阿托伐他汀治疗导致VE-钙粘蛋白转移到细胞膜,增强内皮连接完整性;然而,阿托伐他汀介导的内皮细胞VE-钙粘蛋白连接完整性增强效应在SphK1基因敲除的内皮细胞中显著降低。与对照组相比,凝血酶刺激引起的FITC-葡聚糖通透性显著增加;凝血酶介导的FITC-葡聚糖渗透性通过阿托伐他汀预处理而减弱。与野生型内皮细胞相比,凝血酶攻击的SphK1基因敲除的内皮细胞具有显著更高的FITC-葡聚糖渗透性;阿托伐他汀并没有显著逆转这种渗透性的增加。结论:小鼠机械通气相关性肺损伤模型中,阿托伐他汀预处理可减轻高潮气量通气所致的急性肺损伤,并发挥抗炎效应;进一步肺泡灌洗液脂质代谢组学测量结果显示鞘磷脂为SB、HT、HT+ATV组唯一差异代谢产物,提示阿托伐他汀可以影响鞘脂信号通路,是一条主要的差异性代谢通路;SphK1基因敲除加重了VILI的肺损伤,阿托伐他汀介导的小鼠VILI模型的肺保护效应被部分消除,提示SphK1部分介导了阿托伐他汀抗炎作用;SphK1在机械牵张刺激介导的MAPK活化中起重要作用,SphK1同时介导阿托伐他汀增强内皮连接的完整性,提示SphK1可能通过介导MAPK失活以及增强内皮连接的完整性发挥参与阿托伐他汀的肺保护效应。更多还原

【Abstract】 Objective:Mechanical ventilation has become an indispensable treatment to rescue critically ill patients and maintain general anesthesia.Due to the pathophysiological changes of disease and the inappropriate ventilator parameters,ventilator induced lung injury(VILI)can be caused.The importance of biotrauma has been gradually attracted our attention since 1998 and the important role of inflammation have been confirmed in many studies subsequently.At present,strategic prevention of VILI is almost the only choice,due to the lack of effective drug therapy.Most studies focus on signal transduction,inhibition of inflammation and mitochondrial function.Atorvastatin(ATV)is a 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMG-Co A reductase)inhibitor and inhibits cholesterol synthesis.Recently,atorvastatin also showed anti-inflammatory effect in acute lung injury,ameliorating pulmonary gas-blood exchanging function.However,there are no studies on the effect and mechanism of atorvastatin on VILI.In recent years,it has been found that lipid metabolic pathway plays an important role in the occurrence and development of inflammation.Sphingolipid metabolism is a large class of lipid metabolism,and its metabolites are important molecules that make up the cell membrane and participate in a series of important signal transduction processes such as cell growth,differentiation,inflammation and death.Sphingosine kinase(SphK)catalyzes sphingosine to sphingosine-1-phosphate(sphingosine1-phosphate,S1P),which is divided into two isoenzymes:sphingosine kinase 1(SphK1)and sphingosine kinase 2(SphK2).The former is mainly distributed in the cytoplasm and the latter is mainly distributed in the nucleus.SphK1 plays an important role in anti-inflammation with multiple mechanisms on different models.SphK-S1P axis is currently considered as a potential therapeutic target for many diseases.S1P can regulate sepsis,pulmonary hypertension,pulmonary fibrosis,asthma,bronchopulmonary dysplasia and VILI.Meanwhile,some studies have found that statins can induce S1P1 receptors and improve acute lung injury in sepsis.Therefore,we propose the hypothesis that atorvastatin can reduce the inflammatory response of VILI,enhance the integrity of endothelial cell junctions,and partially alleviate acute lung injury through sphingosine kinase 1 to affect the expression of corresponding proteins in mitogen activated protein kinase(mitogen-activated protein kinase,MAPK)pathway.In this study,the high tidal volume mechanical ventilation injury model was used to simulate clinical VILI,combined with metabonomics and gene knockout mice,to explore the anti-inflammatory effect of atorvastatin in VILI through sphingolipid metabolism and signal pathway,and to explore the changes of MAPK expression in this process.Besides to explore the important role of sphingosine kinase 1 mediated by atorvastatin in maintaining the integrity of endothelial cell junction.Materials and Methods:Adult C57BL/6,8-12 weeks,mice were anesthetized with sodium pentobarbital(60 mg/Kg),then tracheal intubation and mechanical ventilation were performed.Mice were randomly divided into 4 groups according to mechanical ventilation or not,given atorvastatin or not.14 hours before endotracheal intubation,10 mg/kg atorvastatin or DMSO was given.Ventilation was performed with parameters:tidal volume of 40 ml/Kg,65 per/min.Blood gas,lung tissue wet/dry ratio,lung pathology,caspase-3,total proteins and cell counts in bronchoalveolar lavage fluid(BALF),and inflammatory cytokines(IL-6,KC,MIP-1α,MIP-2,MCP-1,IL-1β)levels were tested.At the meanwhile,the bronchoalveolar lavage fluids were tested by metabonomics to determine the pathway which influence the effect of atorvastatin.Furthermore,we selected SphK1 knockout mice,the experimental group was the same as the wild type and evaluated the anti-inflammatory effect of atorvastatin in gene knockout mice by lung histopathology and cytokines in bronchoalveolar lavage fluid.At the same time,the pulmonary microvascular endothelial cells(pulmonary microvascular endothelial cells,PMVECs)of wild type and SphK1 knockout mice were cultured after the target cells were sorted accurately by flow cytometry.Western-blot and RT-PCR were used to determine the expression of SphK1 gene and protein in PMVECs of knockout mice.PMVECs were cyclic stretched.Western-blot in vitro to detect the changes of downstream signal pathways,and three inhibitors of MAPK pathway were pretreated.Finally,the state of intercellular junction was detected by immunofluorescence technique,and the permeability of PMVECs cells was measured by transwell to explore the effect of atorvastatin on enhancing intercellular junction through sphingolipid pathway.Results:(1)Lung injury in wild type(WT)moues with the pretreatment of atorvastatin and high vital tidal ventilation:HE staining of lung tissue in mice subjected to high tidal volume(HT)ventilation showed obvious acute lung injury changes.The lung wet/dry weight ratio increased in the HT group.Meanwhile,the protein amount and cell count in bronchoalveolar lavage fluid(BALF)of HT group were significance higher than spontaneous breath(SB)group(HT vs SB,0.54±0.025 vs 0.23±0.02mg/ml,P<0.01,n=5);(HT vs SB,2.26±0.025×10~5 vs 1.35±0.67×10~5/m L,P<0.01,n=5)which indicated that the lung injury model was successfully established.After intraperitoneal injection of atorvastatin,HE staining showed a significant improvement in lung pathological damage.TUNEL test and caspase-3indicated atorvastatin can decrease the damage caused by HT.In addition,compared with the HT group,the mice in the HT+ATV group lower lung wet/dry weight ratio.The protein amount and cell count in BALF decreased in HT+ATV group(HT+ATV,0.37±0.026 mg/ml and 0.93±0.23×10~5/m L;P<0.01,n=5).(2)Cytokine level evaluated in wild type moues with the pretreatment of atorvastatin and high vital tidal ventilation:the levels of cytokine IL-6,KC,MIP-1α,MIP-2,MCP-1 in BALF were significantly increased after high tidal volume ventilation,but IL-1β.Pretreatment with atorvastatin reduced the levels of IL-6,KC,MCP-1,while the IL-1βremains stable,indicating that atorvastatin pretreatment could reduce the inflammation of lung tissue in mice.(3)The only differential metabolite is sphingomyelin in SB,HT and HT+ATV,which indicate the sphingolipid pathway may the main mechanism.(4)Lung injury evaluated in SphK1 knockout(SK1KO)moues with the pretreatment of atorvastatin and high vital tidal ventilation:HE staining of lung tissue in SphK1 knockout mice subjected to high tidal volume ventilation showed obvious acute lung injury changes.Meanwhile,the protein amount and cell count in BALF also increased(WT HT vs SK1KO HT;0.54±0.03 vs 0.80±0.07mg/ml;P<0.01,n=5);(WT HT vs SK1KO HT;2.26±0.025×105/ml vs 3.47±0.21×10~5/ml;P<0.01,n=5.In pretreatment atorvastatin and high tidal group,the wet/dry ratio was higher in SphK1 knockout mice.Compare with vehicle group,pretreatment of atorvastatin decreased the wet/dry ratio,protein amount in BALF of SphK1knockout mice subjected to high tidal volume ventilation(SK1KO HT/ATV vs WT HT/ATV;0.52±0.07mg/ml vs 0.37±0.03mg/m L,P<0.05,n=5)(SK1KO HT/ATV vs WT HT/ATV;2.93±0.45×10~5/ml vs 0.93±0.23×10~5/ml,P<0.05,n=5)(5)Cytokine level evaluated in SphK1 knockout moues with the pretreatment of atorvastatin and high vital tidal ventilation:the levels of cytokine IL-6,KC,MIP-1α,MIP-2,MCP-1 were increased after high tidal volume ventilation compare to spontaneous breathing in SphK1 knockout mice.The levels of cytokine IL-6,KC,MIP-1α,MIP-2,MCP-1 were higher in SphK1 knockout group than wildtype in high tidal with pretreatment of atorvastatin.However,the levels of cytokine tested did not change with or without atorvastatin in SphK1 knockout and high tidal volume ventilation.(6)After cyclic stretching,the cytokine levels in PMVECs were significantly higher than control group no matter in wildtype nor SphK1 knockout group.Atorvastatin pretreatment significantly inhibited the release of IL-6,KC,MIP-1α,MIP-2 and MCP after cell stretching in wildtype.After SphK1 knockout,the decrease in the levels of cytokines induced by atorvastatin,including IL-6,KC and MCP-1 were reversed,but the levels of MIP-1αand MIP-2 were not reversed.(7)The MAPK pathway was involved in cyclic stretching of PMVECs.a)MAPK existed mainly in the state of non-phosphorylation and its activity was low in static status in wildtype PMVECs.The phosphorylation of EKR was not,while P38and JNK were significantly activated after 18%cyclic stretch.Atorvastatin pretreatment significantly inhibited the phosphorylation JNK,but did not inhibit ERK and P38 phosphorylation.b)P38 and JNK existed mainly in the state of non-phosphorylation and its activity was low in static status in SphK1 knockout PMVECs,but ERK.The phosphorylation of EKR was not,while P38 and JNK were significantly activated after 18%cyclic stretch.Atorvastatin pretreatment significantly inhibited the phosphorylation of JNK.c)Compared with wildtype PMVECs,SphK1 knockout PMVECs produced a large amount of IL-6 and KC after18%cyclic stretch.IL-6 production in SphK1 knockout PMVECs was inhibited by JNK inhibitor and partially inhibited by SB203580(P38 inhibitor),but not by PD58059(ERK inhibitor).The trend of KC is similar to that of IL-6.(8)Immunostaining showed that VE-cadherin mostly located in the cytosolic plasma and a few small gaps existed between wildtype PMVECs.Thrombin challenge increased both the size and the numbers of gaps between PMVECs.Atorvastatin treatment caused VE-cadherin translocating to cellular membrane accompanying enhancement of endothelial junction integrity at both control and thrombin challenge condition;However,the atorvastatin-mediated EC VE-cadherin junction integrity enhancement effect significantly decreased in SphK1 knockout PMVECs.Thrombin challenge caused FITC-dextran permeability significantly increased at wildtype PMVECs compared to control.Additionally,the thrombin-mediated FITC-dextran permeability was attenuated by pretreatment by atorvastatin.Compared to wildtype PMVECs,thrombin challenged SphK1 knockout PMVECs had significantly higher FITC-dextran permeability.Moreover,this permeability increase was not significantly reversed by atorvastatin.Conclusion:Pretreatment with the atorvastatin can greatly alleviate acute lung injury caused by high tidal volume ventilation via reducing inflammation.The only differential metabolite in BALF compared during SB,HT,HT+ATV group,sphingomyelin,indicates the sphingolipid pathway may the main mechanism.Atorvastatin has been shown to reduce VILI in vivo and in vitro.SphK1 knockout enhances inflammatory response on VILI model.Atorvastatin anti-inflammatory effect may partially be mediated by Sphk1.SphK1 plays an important role in the activation of MAPK mediated by cyclic stretch.Another important contribution of SphK1 in atorvastatin-mediated anti-inflammatory effects is the enhancement of endothelial junction integrity.更多还原