【作者】 刘伟；

【导师】 余剑波；

【作者基本信息】 天津医科大学 ， 麻醉学（专业学位）， 2016， 硕士

【摘要】 急性呼吸窘迫综合征(ARDS)病因复杂,病情发展迅速,死亡率高,是临床常见的急危重症[1]。肺泡巨噬细胞作为机体呼吸道防御外界细菌病毒等有害物质的第一道防线,在ARDS的发生、发展及转归中发挥重要作用[2]。当有毒物质或致敏颗粒进入呼吸道后,可以通过激活肺泡巨噬细胞生成大量氧自由基、分泌大量促炎因子,募集并激活中性粒细胞等多种途径导致肺损伤[3]。研究表明,线粒体是活性氧(ROS)产生的主要场所[4]。ARDS时肺泡巨噬细胞线粒体受损,正常氧化磷酸化途径受抑制,ROS生成增多,并且内源性自由基清除剂的活性降低,导致线粒体大量氧自由基堆积。同时伴随线粒体膜电位下降,ATP生成减少,造成细胞氧化应激损伤[5]。线粒体的功能与形态结构密切相关,当细胞遭受氧化应激损伤时线粒体融合分裂平衡被打破,即分裂增加,融合减少[6,7]。众所周知,一氧化碳(CO)是一种污染大气的有害气体,早在1857年Claude Bernard首次记录CO与血红蛋白结合后导致血红蛋白和氧结合的能力减弱甚至丧失,严重者可引起窒息。然而,近年来研究发现CO在某些病理状态动物模型中起着重要作用,目前的研究大多集中在CO的抗炎、抗氧化应激、抗细胞凋亡及细胞保护方面,即CO可减轻应激所致的细胞或组织损伤[8-11]。研究表明,CO可通过调控线粒体细胞色素氧化酶活性减轻异氟烷所致大鼠大脑细胞的氧化应激[9]。CO是否可通过影响线粒体的功能和形态起到减轻肺脂多糖(LPS)致肺泡巨噬细胞损伤的作用尚有待研究。目的探讨CO对LPS致大鼠肺泡巨噬细胞系NR8383损伤的影响及作用机制。方法本研究分为两部分。第一部分:使用不同浓度LPS和CORM-2处理NR8383,确定LPS致细胞损伤模型的浓度和CORM-2的安全工作浓度。第二部分:确定LPS及CORM-2浓度后将细胞按照随机分为8组(n=10)C组、CORM-2组、ZnPP组、LPS组、LPS+CORM-2组、LPS+iCORM-2组、LPS+ZnPP组、LPS+NaOH组。各组处理如下:CORM-2组给予CORM-2 100μM处理细胞,ZnPP组给予Zn PP 10μM处理细胞,LPS组向培养基中加入LPS 10μg/ml,LPS+CORM-2组使用CORM-2 100μM预处理1h后加入LPS 10μg/ml,LPS+iCORM-2组使用iCORM-2 100μM预处理1h后加入LPS 10μg/ml,LPS+ZnPP组使用ZnPP 10μM预处理细胞0.5h后加入LPS 10μg/ml,LPS+NaOH组使用ZnPP溶剂0.2M NaOH预处理0.5h后向培养基中加入LPS 10μg/ml,C组加入等量PBS液。各组细胞处理完毕后继续孵育24h收集各组细胞上清与沉淀用于后续指标检测。用MTT法检测细胞活力,DCFH-DA探针检测细胞ROS含量,试剂盒检测细胞SOD活力,流式细胞仪检测线粒体膜电位和细胞凋亡率,ATP酶含量试剂盒检测ATP含量,RT-PCR检测HO-1、Drp1、Fis1、Mnf1/2、OPA1 mRNA含量,Western bolt法检测HO-1、Drp1、Fis1、Mnf1/2、OPA1的表达。结果第一部分确定LPS致肺泡巨噬细胞损伤模型的浓度为10μg/ml,CORM-2的安全工作浓度为100μM。第二部分中与C组相比,CORM-2组、ZnPP组各检测指标无统计学意义(P>0.05);LPS组、LPS+CORM-2组、LPS+iCORM-2组、LPS+ZnPP组、LPS+NaOH组细胞活力、SOD酶活性、线粒体膜电位、ATP含量、Mnf1/2、OPA1 mRNA含量及表达降低(P<0.05),ROS含量、细胞凋亡率、HO-1、Drp1、Fis1 mRNA含量及表达增加(P<0.05)。与LPS组相比,LPS+iCORM-2组、LPS+NaOH组各检测指标无统计学意义(P>0.05);LPS+CORM-2组细胞活力、SOD酶活性、线粒体膜电位、ATP含量、HO-1、Mnf1/2、OPA1 mRNA含量及表达增加(P<0.05),ROS含量、细胞凋亡率、Drp1、Fis1 mRNA含量及表达减少(P<0.05);LPS+ZnPP组细胞活力、SOD酶活性、线粒体膜电位、ATP含量、HO-1、Mnf1/2、OPA1 mRNA含量及表达降低(P<0.05),ROS含量、细胞凋亡率、Drp1、Fis1 mRNA含量及表达增加(P<0.05)。结论CO通过HO-1调节线粒体融合分裂蛋白的表达及线粒体功能从而减轻LPS致NR8383氧化应激损伤。更多还原

【Abstract】 Acute respiratory distress syndrome(ARDS) caused by complex etiology is a clinical common critical disease, which develop rapid and have high mortality. The alveolar macrophages, as the main residence of phagocytes, constitute the lung respiratory pathogen’s first line of defense, which play an important role in the occurrence, development and outcome in ARDS. When toxic substances or sensitized particles entered respiratory tract, alveolar macrophages can be activated as well as neutrophils. Activated cells producing a large number of oxygen free radicals and secretion of pro-inflammatory factor may lead to cell apoptosis and lung damage. Studies have shown that mitochondrial is main venue of reactive oxygen species(ROS). Mitochondria suffer oxidant stress which contain normal oxidative phosphorylation pathway inhibition and decreased the activity of endogenous free radical scavenger resulted in a large number of ROS generated, mitochondrial membrane potential decreased and ATP production decreased. The above mitochondrial changes cause cell oxidative stress damage in ARDS. Mitochondrial function is closely related to the morphological structure. when cells suffer oxidative stress damage the balance between mitochondria fusion and fission is broken, that is fission more, fusion less.Carbon monoxide(CO) is known as a kind of pollution and harmful gas in the atmosphere. Claude Bernard recorded CO can be combined with hemoglobin, which could weaken even deprive the binding force of O2 and hemoglobin eventually lead to asphyxia in 1857. However, researches have found that CO played an important role in many pathological state in animal models. The researches is mainly around the CO ability of anti-inflammatory role, resistance to oxidative stress and apoptosis and cell protection ways in recent years. These functions are all under stress state in cells. Studies have demonstrated that CO can reduce oxidative stress caused by isoflurane in rat brain cells through the regulation of mitochondrial cytochrome oxidase activity. whether the carbon monoxide play a role against LPS caused alveolar macrophages damage by influencing the morphology and function of mitochondria remain to be studied.Objective To explore effects of CO on LPS induced damage in rat alveolar macrophage NR8383 and possible mechanisms.Methods This study was divided into two parts. Part 1: Different concentrations of LPS and CO releasing molecule-2(CORM-2) were added into NR8383 to determine the concentration of LPS induced NR8383 damage model and CORM-2 working concentration. Part 2: Cells were randomly divided into 8 groups(n=10).Treatments of every group were as follows: CORM-2 100μM was added into CORM-2 group. ZnPP 10μM was added into ZnPP group. LPS 10μ/ml was into LPS group. LPS+CORM-2 group cells were pretreated with CORM-2 100μM for 1h, then cultured with LPS 10μ/ml. LPS+iCORM-2 group treatments was same with LPS+CORM-2 group except for replacing CORM-2 with iCORM-2. LPS+ZnPP group cells were pretreated with ZnPP 10μM for 0.5h, then cultured with LPS10μ/ml. LPS+NaOH group treatments was same with LPS+ ZnPP group except for replacing ZnPP with NaOH. The amount of PBS was added into group C. Cell precipitation and supernatant was collected after treatments 24 h for targets detection. Proliferation was measured by MTT assay, ROS was detected by DCFH- DA probe, SOD activity was tested by SOD kit, apoptosis and mitochondrial membrane potential were detected with flow cytometer, content of ATP was tested by ATP content kit, HO-1, Drp1, Fis1, Mnf1/2 and OPA1 mRNA was measured by RT-PCR, HO-1, Drp1, Fis1, Mnf1/2 and OPA1 expression was determined by Western blot.Results Part1: LPS induced NR8383 damage model was successfully built with LPS concentration 10μ/ml. CORM-2 safe working concentration was 100μM. Part2: LPS induced NR8383 damage, however this damage condition can be alleviated by pretreatment CO. Furthermore, cytoprotection of CO is associated with HO-1 to regulate mitochondrial fusion and fission protein expression and function.Conclusion CO can alleviate LPS-induced damage in rat alveolar macrophages through HO-1 to regulate mitochondrial fusion and fission protein expression and function.更多还原