载脂蛋白A-I及其模拟肽D-4F对气道炎症及气道高反应性的作用的实验研究
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摘要
论文一
载脂蛋白A-I的基因缺失增加小鼠气道反应性,肺部炎症和
胶原沉积的作用及机制的实验研究
1.背景
载脂蛋白A-Ⅰ(apoA-Ⅰ),是高密度脂蛋白(HDL)的重要组成部分,其抗动脉粥样硬化作用已经得到广泛的承认,而且apoA-Ⅰ本身对器官和血管床也有保护作用。最近新的研究证据表明,HDL在肺脏中起着保护作用。与apoA-Ⅰ结合的ABCA-Ⅰ先前已经被证明在维持脂质组成、肺脏结构和呼吸功能中起着重要的作用。最近,在蛋白质组学研究揭示纯合子镰刀细胞贫血病患者的血浆apoA-Ⅰ的水平合并肺动脉高压者总是比没有合并肺动脉高压者低。另外有研究证实,内皮脂酶的基因缺失导致HDL水平升高2倍,并且增加了卵蛋白致敏的BALB/c小鼠的气道高反应性和肺部炎症。尽管这些研究提供了这样一个观点,那就是HDL有助于维持健康的肺脏,但是还没有研究直接证实apoA-Ⅰ或者是apoA-Ⅰ的缺失对肺脏炎症,血管舒张和气道高反应性的作用。
越来越多的证据表明,高水平的HDL并不总是具有抗动脉粥样硬化的作用。实际上,慢性炎症状态和氧化应激已经被证明能将HDL从具有抗炎和抗动脉粥样硬化作用的脂蛋白转换成具有致炎性和致动脉粥样硬化作用的脂蛋白,从而使HDL失去有效的清除LDL,抗动脉粥样硬化和保护血管的功能。哮喘也能增加炎症和氧化应激。这些炎症变化很可能解释为什么成年女性中发作的哮喘和颈内动脉内膜厚度的增加有显著关系。
这些报告为我们提供了一个新的思想,那就是HDL对肺脏具有保护功能。但是目前还没有任何研究表明,载脂蛋白A-I是否直接决定或影响肺部炎症及气道高反应性。
在这个报告中,我们研究了载脂蛋白A-I基因缺失对肺的炎症,血管舒张反应,胶原沉积及气道高反应性的影响。我们的研究结果表明载脂蛋白A-I对肺动脉和气道功能的维持具有保护功能,能有效的防止气道的炎症和胶原沉积。
2方法
2.1对血浆总胆固醇(TC),HDL和致炎性HDL(p-HDL)的测定
总胆固醇是由Wako公司生产的胆固醇氧化酶/酯酶试剂盒测定。HDL是用Richmond公司生产的高密度脂蛋白胆固醇E试剂盒测定。致炎性HDL用MgCl2沉淀后和CuCl2孵化,加入荧光染料H2DCF后测定。
2.2血浆(对氧磷酶)芳香酯酶活性的测定
血浆芳香酯酶活性的测定是用苯乙酸作为底物进行的。稀释后的血浆被加到加到磷酸盐缓冲液体系中(苯基乙酸[100 mmol/L],氯化钙[100 mmol/L],pH值8.0)。苯乙酸的水解率由DU(?)640分光光度计进行测定。芳香酯酶的活性单位相当于每分钟水解的每毫升中的苯乙酸的摩尔数。
2.3肺泡灌洗液中亚硝酸盐+硝酸盐的定量测定
肺泡灌洗液中亚硝酸盐和硝酸盐浓度使用臭氧发光法测定。30ul原液加到包含氯化碘的反应体系中。一氧化氮的信号由一氧化氮分析仪测定。和标准硝酸盐浓度曲线相比而得到原液中硝酸盐和亚硝酸盐的浓度。
2.4血浆中3-NT的斑点杂交
从小鼠体内分离的血浆样品直接点在硝酸纤维素膜上,晾干。膜用牛奶封闭,加一抗3-NT杂交,4℃过夜。第二天洗膜,加二抗,ECL化学发光拍片来测定血浆中3-NT总的含量
2.5 apoA-I基因缺失对血管舒张的影响
本实验全部采用小鼠的面部动脉和肺动脉血管,腹腔注射苯巴比妥后,分离小鼠的双侧的面部动脉,其直径一般在180-250um。血管两侧挂在微玻璃管上并连接60cm H20的压强。所有分离提取的所有血管均不作预处理。先测量最大血管内径(Vmax),然后用U 46619(10-9-10-8mol/L)作预收缩并测值(V con),然后依次用浓度梯度为10-7-10-4mol/L的乙酰胆碱(ACh)扩张血管以测量在依次增加ACh浓度情况下的内皮依赖的血管舒张情况(VACh);按以下公式计算ACh扩张血管的百分值:(VACh-Vcx/n)/(Vmax-Vcon}=血管扩张率,以此血管扩张率表示内皮依赖的血管舒张情况。进而用左旋硝基精氨酸甲酯(L-nitroarginiemethylester, L-NAME)和100umol/L预处理血管30 min,再重复用U 46619和ACh处理血竹以了解内皮一氧化氮合酶(endothelial nitric oxide synthase, eNOS)依赖的血管舒张功能。
2.6脉冲振荡技术测量气道反应性
用Scireq2007小动物肺功能分析仪通过强力脉冲振荡技术来测定呼吸总阻(impedence, Zrs),测定参数为中央气道阻力(resistance, Rn),外周气道阻力(tissue damping,G)和组织弹性回缩力(tissue elastance,H)。
2.7乙酰甲胆碱激发实验
经气道浓度梯度增加应用乙酰甲胆碱(3.125mg/ml-100mg/ml)激发气道高反应性。70ul乙酰甲胆碱在超声雾化器中被雾化,然后经由Scireq2007小动物肺功能分析仪吸气管道输送至小鼠气道。待曲线平稳后进行下一次给药。在不同浓度的乙酰甲胆碱取得曲线,取其峰值,分别取得相应的Rn,G和H值。
2.8肺病理取材制片
0.5毫升中性甲醛经气道灌洗管被缓缓注入肺中进行组织固定。开胸摘取肺,至于10%中性甲醛固定液中固定24h以上。常规石蜡包埋、制备石蜡切片HE染色McLetchies三色染色来评估胶原沉积。
2.9 BALF的分离和分析
收回的肺泡灌洗液2000rpm离心后去除上清液,保存于-80℃。细胞沉淀悬浮于1mlPBS,吸取500ul细胞悬液来制作细胞调整细胞离心涂片,晾干后4%多聚甲醛固定,瑞氏染色,瑞氏染色后行光学显微镜下观察500个细胞,进行细胞分类计数。
2.10免疫荧光测定肺病理切片中4HNE,转化生长因子β-1 (TGFβ-1),3-NT和T-15的表达
肺组织切片进行脱蜡,梯度脱水,封闭,加一抗,加二抗,激光共聚焦显微镜进行免疫荧光测定。
2.11 Western-blot分析黄嘌呤氧化酶(XO),内源性一氧化氮合酶(eNOS)和髓过氧化物酶(MPO)
摘除小鼠肺组织,立即至于-80℃中冻存。研磨裂解肺组织,分离蛋白上清液。测定蛋白含量。SDS-PAGE电泳分离蛋白,转膜,显色,照相。
2.12 BALF的氧化指数的测定
氧化物H2DCF用来测定BALF中的氧化指数。75ul BALF与H2DCF混合(100 u L PBS+25 u L H2DCF和37℃孵育2小时。读板器在2小时后读取荧光值(Ex 485nm;Em 530nm)
3.统计学处理：
所有数据均用X±SEM表示,利用Prisme5统计软件进行统计分析,指标比较采用单因素方差分析,两组间比较采用t检验,P<0.05为差异有显著性。
4.结果：
4.1 ApoA-Ⅰ基因缺失对脂质和氧化应激的作用
和C57BL/6J小鼠相比,apoA-Ⅰ-/-小鼠血浆中总胆固醇和HDL水平降低。尽管apoA-Ⅰ-/-小鼠含有的HDL水平低于C57BL/6J小鼠,但是其HDL的氧化速率要高于C57BL/6J小鼠,这也说明apoA-Ⅰ-/-小鼠血浆中HDL是致炎性的。ApoA-Ⅰ-/-小鼠血浆中PON1的蛋白水平比C57BL/6J小鼠的高。但是,PON1的活性在apoA-Ⅰ-/-小鼠血浆中比C57BL/6J小鼠降低了大约34%。和C57BL/6J小鼠相比,apoA-Ⅰ-/-小鼠血浆中硝酸盐和亚硝酸盐水平下降,血浆中3-NT的水平升高。
4.2 ApoA-Ⅰ基因缺失对血管扩张的作用
分离的面部动脉血管在预增压后对Ach依赖的血管舒张反应实验表明apoA-Ⅰ基因缺失对面部动脉血管没有影响。这些结果和之前的研究结果表明apoA-Ⅰ-/-小鼠对动脉粥样硬化并不易感。然而,肺动脉血管环的舒张反应显示从apoA-Ⅰ-/-小鼠分离的血管环在最高浓度的Ach下舒张功能受损。从apoA-Ⅰ-/-小鼠分离的肺血管环在最高浓度的Ach下实际上表现出收缩反应。相反地,从C57BL/6J小鼠分离的肺血管环在最高浓度的Ach下则表现出舒张反应。ApoA-Ⅰ-/-小鼠肺动脉血管的这些生理反应的变化提示慢性炎症反应和氧化应激损伤了肺部血管的舒张。
4.3 ApoA-Ⅰ基因缺失对气道高反应性的作用
Flexivent动物肺功能仪是对整个支气管呼吸动力参数的测定。在主支气管中,它测定中央气道阻力(RN),在外周支气管中,它测定外周气道阻力(G)和组织弹性回缩力(H)。经MCh后,G(P<0.01)和H(P<0.001)在apoA-Ⅰ-/-小鼠中明显升高,Rn在两组中无明显差别。在不同水平的气道正压通气(PEEP)后,中央气道阻力(RN)在两组中无明显差别。和C57BL/6J小鼠相比,apoA-Ⅰ-/-小鼠外周气道阻力在基线水平(PEEP=0)升高(P<0.01),并且在整个PEEP曲线中,G在apoA-Ⅰ-/-小鼠比在C57BL/6J小鼠中高；在整个PEEP曲线中,组织弹性回缩力H在apoA-Ⅰ-/-小鼠中比在C57BL/6J小鼠中升高(H,P<0.001)。
4.4ApoA-Ⅰ基因缺失对肺病理组织的作用
肺组织病理HE切片显示：与C57BL/6J系小鼠相比,apoA-Ⅰ-/-小鼠肺部血管和支气管周围炎症细胞浸润增多。尽管apoA-Ⅰ-/-小鼠肺部血管和支气管周围炎症细胞比C57BL/6J系小鼠增多2倍以上,但是肺浸润的白细胞数还没有达到OVA致敏的C57BL/6J系小鼠的白细胞数(25-50细胞/PHF)。ApoA-Ⅰ-/-小鼠肺组织切片显示胶原沉积增多。与C57BL/6J系小鼠相比,apoA-Ⅰ-/-小鼠肺组织trichrome染色增强。BALF细胞涂片显示BALF中主要含有肺泡巨噬细胞(>98%)。从apoA-Ⅰ-/-小鼠和C57BL/6J系小鼠的BALF的细胞图片显示apoA-I的基因缺失对支气管淋巴细胞浸润没有明显的作用。
4.5Apo A-I的基因缺失对氧化应激和炎症生物标记物的作用
免疫荧光研究表明apoAⅠ-/-小鼠的肺组织切片比对照组中3-硝基酪氨酸(3-NT)和4-羟基壬烯醛(4HNE)Michael加合物免疫荧光信号明显增强。但是apoA-Ⅰ-/-小鼠的肺组织切片抗T15的自身抗体信号却没有比对照组升高。ApoA-I的基因缺失导致呼吸道中4-HNE衍生的Michael反应的加合物和活性TGF-01表达增加。对照组C57BL/6J系小鼠相比,apoA-Ⅰ-/-小鼠肺组织中XO,MPO和eNOS的表达明显升高。这些数据与硝酸盐和亚硝酸盐的数据相反,apoA-Ⅰ-/-小鼠的BALF中的硝酸盐和亚硝酸盐的水平比C57BL/6J系小鼠BALF硝酸盐和亚硝酸盐的水平下降。然而,与C57BL/6J系小鼠相比,,apoA-Ⅰ-/-小鼠的BALF中含有更多致氧化化合物。
5结论：
1) ApoA-Ⅰ的缺失导致HDL在体内的的保护作用消失,并将HDL转变成致炎性HDL,引起一系列氧化应激反应的加剧,导致脂质过氧化,eNOS解偶联。
2) ApoA-Ⅰ在预防肺部炎症,受损的血管舒张及气道高反应性方面具有重要的保护作用。ApoA-Ⅰ及其相关的抗炎和抗动脉粥样硬化作用的缺失在血管功能和呼吸道生理功能方面具有深刻的和重要的影响。
论文二ApoA-Ⅰ模拟肽,D-4F对apoA-Ⅰ和T-bet双基因敲除小鼠体内氧化应激和炎症状态的改善作用及机制的实验研究
1.背景
高密度脂蛋白(HDL)在胆固醇逆转运和抗动脉粥样硬化和炎症过程中发挥着重要的作用。越来越多的证据表明,高密度脂蛋白水平升高并不总是意味着对动脉的保护。事实上,慢性炎症和氧化应激已被证明可将具有抗炎和抗动脉粥样硬化作用的HDL转换成致炎性和致动脉粥样硬化性颗粒,使它失去对抗低密度脂蛋白(LDL),抗动脉粥样硬化和保护血管的作用。哮喘也增加炎症和氧化应激。在我们之前的研究中,我们发现载脂蛋白A-I在防止气道高反应和肺部炎症中具有重要的保护作用。
D-4F是载脂蛋白A-I的模拟肽,全部由D-氨基酸合成。D-4F有一个疏水螺旋可以结合脂质,这个与载脂蛋白A-I与脂质的结合方式相似。D-4F对氧化的脂质比普通脂质具有更大的结合力,这个作用似乎与其对保护高密度脂蛋白的抗炎能力有关。在小鼠流感的模型中,D-4F被证实可以结合氧化脂质,重塑HDL,这种功能可能与其减少炎症因子的产生有关。
T-bet基因敲除小鼠,以自发地气道高反应性,肺部炎症和气道重塑为特征。之前的研究发现apoA-Ⅰ基因缺失能增加小鼠的气道高反应性。为了研究高密度脂蛋白在气道疾病中的作用,本实验将apoA-Ⅰ-/-小鼠和T-bet-/-小鼠进行杂交,筛选出双基因敲除的T-bet-/-/apoA-Ⅰ-/-小鼠有。为了进一步验证D-4F能够减少气道疾病,实验中加用D-4F治疗双基因敲除的T-bet-/-/apoA-Ⅰ-/-小鼠。研究发现,双基因敲除的T-bet-/-/apoA-Ⅰ-/-小鼠有更高的气道高反应性,肺部炎症和胶原沉积。有趣的是,在T-bet-/-/apoA-Ⅰ-/-小鼠中,D-4F的治疗降低了气道高反应性,减少肺部炎症,胶原沉积。另外,D-4F的治疗也减轻了T-bet-/-/apoA-Ⅰ-/-小鼠肺部的氧化应激状态。
2方法
2.1 T-bet-/-/apoA-Ⅰ-/-小鼠模型的筛选和建立
以C57BL/6J小鼠系的apoA-Ⅰ-/-小鼠和T-bet-/-小鼠均购自美国杰克逊实验室,将其进行杂交,产生T-bet+/-apoA-Ⅰ+/-小鼠。将杂合子小鼠进一步杂交,用PCR法筛选出双基因敲除的T-bet-/-/apoA-Ⅰ-/-小鼠。T-bet-/-/apoA-Ⅰ-/-小鼠与对照组的T-bet-/-小鼠在出生体重上没有明显差别。10周左右时,将雄性T-bet+/-/apoA-Ⅰ+/-小鼠随机分成对两组,一组每天腹腔注射PBS,治疗组注射D-4F,(D-4F,AC-DWFKAFYDKVAEKFKEAF-NH2,3mg/kg体重),治疗6周。所有的动物实验,由威斯康星医学院动物护理和使用委员会(IACUC)批准。
2.2对血浆总胆固醇(TC)和HDL的测定
总胆固醇是由Wako公司生产的胆固醇氧化酶/酯酶试剂盒测定。HDL是用Richmond公司生产的高密度脂蛋白胆固醇E试剂盒测定。
2.3血浆(对氧磷酶)芳香酯酶活性的测定
血浆芳香酯酶活性的测定是用苯乙酸作为底物进行的。稀释后的血浆被加到加到磷酸盐缓冲液体系中(苯基乙酸[100 mmol/L],氯化钙[100 mmol/L],pH值8.0)。苯乙酸的水解率由DU(?)640分光光度计进行测定。芳香酯酶的活性单位相当于每分钟水解的每毫升中的苯乙酸的摩尔数。
2.4肺泡灌洗液中亚硝酸盐+硝酸盐的定量测定
肺泡灌洗液中亚硝酸盐和硝酸盐浓度使用臭氧发光法测定。30ul原液加到包含氯化碘的反应体系中。一氧化氮的信号由一氧化氮分析仪测定。和标准硝酸盐浓度曲线相比而得到原液中硝酸盐和亚硝酸盐的浓度。
2.5脉冲振荡技术测量气道反应性
用Scireq2007小动物肺功能分析仪通过强力脉冲振荡技术来测定呼吸总阻(impedence,Zrs),测定参数为中央气道粘性阻力(resistance,Rn),外周气道粘性阻力(tissue damping,G)和组织弹性回缩力(tissue elastance,H)。
2.6乙酰甲胆碱激发实验
经气道浓度梯度增加应用乙酰甲胆碱(3.125mg/ml～100mg/ml)激发气道高反应性。70ul乙酰甲胆碱在超声雾化器中被雾化,然后经由Scireq2007小动物肺功能分析仪吸气管道输送至小鼠气道。待曲线平稳后进行下一次给药。在不同浓度的乙酰甲胆碱取得曲线,取其峰值,分别取得相应的Rn,G和H值。
2.7肺病理取材制片
常规石蜡包埋、制备石蜡切片、HE染色McLetchies三色染色来评估胶原沉积。
2.8免疫荧光测定肺病理切片中4HNE,转化生长因子β-1(TGFβ-1)
常规脱蜡,梯度脱水,加一抗,加二抗,封闭组织切片
2.9 Western-blot分析黄嘌呤氧化酶(XO)和髓过氧化物酶(MPO)
SDS-PAGE电泳分离蛋白,转膜,显影,照相。
3.统计学处理
所有数据均用X±SEM表示,利用Prisme5统计软件进行统计分析,指标比较采用多因素方差分析,P<0.05为差异有显著性。
4结果
4.1血浆总胆固醇(TC),HDL的水平
注射PBS和D-4F的T-bet-/-/apoA-Ⅰ-/-小鼠血浆中总胆固醇(P<0.001)和高密度脂蛋白(P<0.001)的水平比对照组T-bet-/-/apoA-Ⅰ+/+小鼠明显降低。血浆中总胆固醇和高密度脂蛋白的水平在PBS注射组和D-4F组中无显著性差异。
4.2血浆中PON1的表达和活性
和对照组T-bet-/-/apoA-Ⅰ+/+小鼠相比,PBS组和D-4F组中T-bet-/-/apoA-Ⅰ-/-小鼠PON1的活性明显下降(P<0.001)。在T-bet-/-/apoA-Ⅰ-/-小鼠中,D-4F治疗使PON1的活性有所升高,但没有达到统计学意义。
4.3肺泡灌洗液中硝酸盐和亚硝酸盐水平
肺泡灌洗液中硝酸盐和亚硝酸盐的水平提示体内气道中一氧化氮的产生。和对照组T-bet-/-/apoA-Ⅰ+/+小鼠相比,PBS组和D-4F组中T-bet-/-/apoA-Ⅰ-/-小鼠硝酸盐和亚硝酸盐水平明显下降(P<0.001)。在T-bet-/-/apoA-Ⅰ-/-小鼠中,D-4F治疗使硝酸盐和亚硝酸盐水平的水平有所升高(P<0.05)。
4.4 FIexiVent动物肺功能仪对呼吸力参数的测
乙酰甲胆碱激发实验中,中央气道阻力和外周气道阻力在三组中无明显差异。组织弹性回缩力H(P<0.05)在PBS组中的T-bet-/-/apoA-Ⅰ-/-小鼠比T-bet-/-/apoA-Ⅰ+/+对照组小鼠升高。
4.5免疫荧光测定TGF-β1和4-HNE在肺组织的表达水平
在T-bet-/-/apoA-Ⅰ-/-小鼠中,经D-4F治疗后,TGF-β1 and 4-HNE在肺的含量明显减少。
4.6肺组织病理学
和对照组T.bet-/-/apoA-Ⅰ+/+小鼠相比,PBS组的T-bet-/-/apoA-Ⅰ-/-小鼠肺部炎症明显增加,在气管和血管旁的胶原沉积也增加。然而,经D-4F治疗的T-bet-/-/apoA-Ⅰ-/-小鼠肺中,肺部炎症明显减轻,胶原沉积也减少。
4.7肺组织中XO的表达
与对照组T-bet-/-/apoA-Ⅰ+/+小鼠和PBS组T-bet-/-/apoA-Ⅰ-/-小鼠相比,D-4F治疗的T-bet-/-/apoA-Ⅰ-/-小鼠肺组织中黄嘌呤氧化酶(P<0.05)的蛋白表达明显增加。
5结论
1)总的来说,这些数据显示在哮喘模型T-bet-/-小鼠基础上,apoA-Ⅰ的缺失进一步加重了肺部的炎症和胶原沉积以及气道高反应性。这说明apoA-Ⅰ在哮喘的发病中具有一定的保护作用。这一点也被apoA-Ⅰ模拟物D-4F的治疗进一步证明。经D-4F治疗后,T-bet-/-/apoA-Ⅰ-/-老鼠肺部炎症,胶原沉积及气道反应性减轻。D4F的作用为哮喘的治疗提供了新的策略。
2)哮喘发病还与多种炎性细胞和多种细胞因子等有关,在其复杂的免疫炎症网络中如何实施合理的干预性调节治疗,有待于在今后的实验研究与临床研究结合于一起,以p-HDL中心,从炎症反应和氧化应激及其多因子的综合性探索,以期从根本上达到控制哮喘肺部炎症和高气道反应性的目的。
Part One Genetic Deletion of Apolipoprotein A-ⅠIncreases Airway Hyperresponsiveness, Inflammation and Collagen Deposition in the Lung
1. Background
Although apolipoprotein A-Ⅰ(apoA-Ⅰ), the major anti-atherogenic apolipoprotein of high-density lipoprotein (HDL) is well recognized for protecting the heart against vascular disease, it also protects other vascular beds and organs. Recent studies provide new evidence supporting the notion that HDL plays a protective role in the lung. Earlier ABCA1, which interacts with lipid poor apoA-Ⅰ, was shown to be essential for maintaining normal lipid composition and architecture of the lung as well as respiratory physiology. More recently, proteomic studies revealed that homozygous sickle cell anemia patients with pulmonary arterial hypertension (PAH) consistently had lower apoA-Ⅰlevels than sickle patients without PAH. Interestingly, genetic deletion of endothelial lipase resulted in a nearly 2-fold increase in HDL, which was credited with decreasing airway hyperresponsiveness and pulmonary inflammation in ovalbumin (OVA)-sensitized BALB/c mice. Although these reports provide some support for the idea that HDL helps maintain healthy lungs, no studies have directly determined the effects of apoA-Ⅰ, or lack thereof, on pulmonary inflammation, vasodilatation and airway hyperresponsiveness.
Increasing evidence suggests that elevated levels of HDL are not always atheroprotective. Indeed, chronic states of inflammation and oxidative stress have been shown to convert HDL from an anti-inflammatory and anti-atherogenic lipoprotein into a pro-inflammatory and pro-atherogenic lipoprotein, making it useless for protecting the vessel wall against the effects of atherogenic concentrations of LDL. Asthma also increases inflammation and oxidative stress. Such inflammatory changes may explain why adult-onset asthma is associated with significant increases in carotid artery intimal-medial thickness in women. In this report we examine the effects of genetic deletion of apoA-Ⅰon pulmonary inflammation, vasodilatation, collagen deposition and airway hyperresponsiveness. Our findings suggest apoA-Ⅰplays a critical role in protecting pulmonary artery and airway function as well as preventing inflammation and collagen deposition..
2. Methods
2.1 Plasma Total Cholesterol, HDL Cholesterol and Proinflammatory HDL
Total cholesterol (TC) was quantified using a cholesterol oxidase/esterase kit from Wako Chemical, Inc. (Richmond, VA). HDL cholesterol was quantified using a HDL Cholesterol E kit from Wako Diagnostics. Proinflammatory HDL (p-HDL) was determined using a modified method of a previously published cell-free assay with CuCl2 and H2DCF.
2.2 Plasma PON1 Arylesterase Activity
Arylesterase activity of PON1 was performed on whole plasma using phenyl acetate as the substrate as described. Initial rates of hydrolysis were determined spectrophotometrically at 270 nm on DU(?) 640 spectrophotometer. An aliquot of 20μL of 30X diluted mouse plasma was added to a final reaction volume of 500μL (phenyl acetate [100 mmol/L] and CaCl2 [100 mmol/L] in Tris-HCI [40 mmol/L] buffer, pH 8.0) for 6 min at 25℃. One unit of arylesterase activity equals 1μmole of phenyl acetate hydrolyzed per mL per min.
2.3 Quantification of Plasma Nitrite+Nitrate
Nitrite+nitrate concentrations were determined by ozone chemiluminescence using the NO Analyzer (Model 280i, GE Analytical-Sievers, Boulder, CO) as described. An aliquot of 30μL was injected (plasma was diluted by 1:30) into a sealed glass reaction chamber at 95℃containing VCI3. Nitric oxide chemiluminescence signals were quantified and peak areas compared to the areas of external nitrate standards.
2.4 Estimates of Plasma 3-Nitrotyrosine
An aliquot of plasma from C57BL/6J and apoA-Ⅰ-/- mice was pipetted onto nitrocellulose membranes and allowed to bind. Membranes were blocked with 5% non-fat dry milk dissolved in fresh PBS-Tween (0.1%) and then incubated overnight at 4℃with antibodies for 3-NT (1:5000;Millipore, Billerica, MA). The next day, the membranes were washed and incubated with the appropriate HRP-conjugated secondary antibody for 1 h. Bands of identity were visualized with ECL chemiluminescence following the manufacturer's recommendations. Autoradiograms were scanned with a laser densitometer or a UMax scanner.
2.5 Effects of Genetic Deletion of ApoA-Ⅰon Vasodilatation
Facialis and pulmonary arteries were isolated from C57BL/6J and apoA-Ⅰ-/- mice by microdissection as previously described. Vasodilatation of pressurized (60 cm of H2O) facialis arteries was examined in the absence and presence of L-nitroargininemethylester (L-NAME-200μM, final concentration) as previously described.(14) Changes in pulmonary artery tension in response to acetylcholine (ACh) were recorded on a DMT wire-myograph using protocols similar to that which we have previously described.
2.6 Measurements of Lung Mechanics
Mice were tracheostomized with an 18-gauge cannula and mechanically ventilated in a quasi-sinusoidal fashion with a small animal ventilator at 150 breaths per minute(19) and a tidal volume (VT) of 10 mL·kg-1 body weight. Basal respiratory system impedance (Zrs) was then assessed using the forced oscillation technique (FOT) applied over 2 sec (a Prime-2 perturbation) using the flexiVent system. Each level of PEEP (0,3,6, and 9 cm H2O) was held for 80 sec with the Prime-2 perturbation repeated twice, once at 35 sec and the other at 70 sec. Measurements at each PEEP were Rn, G and H.
2.7 Methacholine challenge
Mice were challenged with 70 L of MCh (Sigma-Aldrich) of increasing concentrations (3.125 to 100 mg/mL). MCh aerosols were generated using an ultrasonic nebulizer and delivered to the inspiratory line of the ventilator using room air. Each aerosol was delivered for 30 sec, during which time ventilation was maintained mechanically by the flexiVent. Then for the next 3 min, each aerosol was delivered for 30 sec, during which time regular ventilation was maintained and measurements(Rn,G and H) were assessed as a curve, whch pick value were picked up for calculation.
2.8 Histology
A 0.5mL aliquot of zinc-formalin was used to inflate the lung prior to removal. The lung was fixed in zinc-formalin, embedded in paraffin, sectioned and then stained with hematoxylin and eosin (H&E) for histology or with McLetchies' trichrome to assess collagen deposition.
2.9 Isolation of BALF and Analysis
BALF was obtained by flushing the lung with PBS, first 1mL followed by 0.5mL. The rinses were combined and the BALF was centrifuged at 2000 rpm for 10 min at 4℃. The supernatant was removed, stored at -80℃. The cell pellet was gently resuspended in 1 mL PBS. An aliquot of 500μL was used to prepare slides using a Cytospin. Differential cell counts were made from slides stained with Diff-Quick. Five hundred cells were counted and identified (magnification, X40) for each mouse by a pathologist who had no prior knowledge of slide identities.
2.10 Immunofluorescence
Immunofluorescence was performed on 5μm sections of paraffin-embedded, PBS-zinc-formalin-fixed lungs. Two sections were present on each slide. The sections were incubated separately with antibodies against either 3-nitrotyrosine anti-T15 autoantibodies as previously described, or with anti-4-HNE Michael's adducts and with TGFβ-1 for co-localization studies. Slides were washed with PBS (3X) and then incubated with the appropriate secondary antibodies. The slides were washed, sealed under cover slips and images captured using a krypton argon laser Nikon Eclipse TE2000U confocal microscope with 10x/0.17 aperture objective. Total magnification was 100 with Ex/Em at 488/580 nm for Alexa 488 and 633/661 nm forTO-PRO-3.
2.11Western Blot Analysis
Proteins were separated by SDS-PAGE (4-20%) and transferred onto nitrocellulose membranes. Membranes were blocked with 5% non-fat dry milk dissolved in fresh PBS-Tween (0.1%) and then incubated overnight at 4℃with antibodies for XO (1:500), MPO (1:100) or eNOS (1:10,000). The next day, the membranes were washed and incubated with the appropriate HRP-conjugated secondary antibody for 1 h. Bands of identity were visualized with ECL chemiluminescence following the manufacturer's recommendations. Autoradiograms were scanned with a laser densitometer or an UMax scanner.
2.12 BALF DCF-detectable Oxidants
Oxidation of H2DCF was used to obtain an index of the levels of oxidants in BALF. An aliquot of BALF (75μL) was mixed with H2DCF (100μL PBS+25μL H2DCF [a 1x10 dilution of 2 mg/mL] in a total volume of 200μL) and this mixture incubated for 2 h at 37℃. Absolute changes in fluorescence (Ex 485 nm;Em 530 nm) were determined at the end of the 2 h incubation period using Spectra Max Gemini EM fluorescence plate reader.
3. Statistical Analysis
Data are presented as mean±SEM. Results were analyzed by Student's t-test, Mann-Whitney test or Fisher's exact test where appropriate. Airway data were analyzed by 2-way ANOVA to determine significance between curves and a Bonferroni post-test to determine significance of points between the curves. All statistical analysis was performed using GraphPad Prism Software (version 4.0).
4. Results
4.1 Effects of ApoA-ⅠDeficiency on Lipids and Oxidative Stress
ApoA-Ⅰ-/- mice had reduced levels of plasma total cholesterol and HDL cholesterol compared with control mice (Figure 1A and 1B). Although apoA-Ⅰ-/-mice contained less HDL than control mice, their HDL oxidized at a faster rate than HDL from control mice, indicating that HDL in apoA-Ⅰ-/- mice was proinflammatory (Figure 1C). PON1 plasma protein in apoA-Ⅰ-/- mice was increased compared with protein levels in C57BL/6J mice (Figure 2A and 2B). However, PON1 activity was decreased (≈34%,p<0.001) compared with controls (Figure 2C). Although the concentration of plasma nitrite+nitrate was decreased in apoA-Ⅰ-/- mice, plasma 3-NT levels were increased compared with the levels in control mice (Figure 3A and 3B, respectively).
4.2 Effects of ApoA-ⅠDeficiency on Vasodilatation.
Studies on ACh-dependent vasodilatation of isolated and pressurized facialis arteries revealed that genetic loss of apoA-Ⅰhad no effect on relaxation responses in these vessels (Figure 4A vs.4B). These observations are consistent with previous studies showing that apoA-Ⅰ-/- mice are not more susceptible to atherosclerosis.(27,28) However, relaxation responses of pulmonary artery rings isolated from apoA-Ⅰ-/- mice were impaired at the highest ACh concentration (Figure 4C). Pulmonary artery rings from apoA-Ⅰ-/-mice actually constricted when treated with the highest ACh concentration. In contrast, the pulmonary artery rings from C57BL/6J mice continued to relax. Such changes in the physiological responses of pulmonary arteries in apoA-Ⅰ-/- mice are consistent with the idea that chronic exposure to inflammation and oxidative stress impair vasodilatation.
4.3 Effects of ApoA-ⅠDeficiency on Airway Hyperresponsiveness
The flexiVent provides quantitative information regarding the mechanical properties of the entire airway tree. In the large airways it assesses Newtonian resistance (RN), while in the smaller airways it is able to determine changes in tissue damping (G) and tissue elastance (H). In both apoA-Ⅰ-/- and C57BL/6J mice, methacholine (MCh) induced dose-dependent increases in airway resistance associated with tissue dampening (G, Figure 5B; p<0.01) and tissue elastance (H, Figure 5C;p<0.001), but not RN (Figure 5A). Effects of increasing PEEP on airway mechanical parameters, RN, G and H, are shown in Figure 5D,4E and 4F, respectively. G was increased in apoA-Ⅰ-/- mice compared with control mice at baseline PEEP=0 (p<0.01) as well as throughout the entire PEEP curve (p<0.001). H was increased in apoA-Ⅰ-/- mice compared with controls throughout the entire PEEP range (p<0.001).
4.4 Effects of apoA-ⅠDeficiency on Histology
H&E sections of lungs harvested from apoA-Ⅰ-/- mice (two lower left images, Figure 6A) contained more inflammatory cells than lungs from C57BL/6J mice (one upper left image, Figure 6A). Although inflammatory cells in perialveolar and perivascular regions of apoA-Ⅰ-/- mice were increased more than two fold compared with C57BL/6J mice (Figure 6B, p<0.025 2-tailed t-test), the number of white blood cell infiltrating the lungs did not achieve the numbers that are achieved in ovalbumin-sensitized C57BL/6J mice, which can approach 25-50 cells PHF. Sections of lungs harvested from apoA-Ⅰ-/- mice also revealed increased collagen deposition (two lower right images, Figure 6A). Trichrome staining is stronger and more extensive in apoA-Ⅰ-/- sections than in sections of lungs from C57BL/6J mice (upper right image, Figure 6A). Cytospins of BALF isolated from apoA-Ⅰ-/- and C57BL/6J mice showed that the BALF contained predominately alveolar macrophages(>98%). Cytospins of BALF isolated from C57BL/6J and apoA-Ⅰ-/- mice reveal that the genetic loss of apoA-Ⅰdid not have significant effect on lymphocyte infiltration in the bronchoalveolar space (Figure 6C).
4.5 Effects of apoA-ⅠDeficiency on Biomarkers of Oxidative Stress and Inflammation
Immunofluorescence studies revealed that lung sections from apoA-Ⅰ-/-mice stained stronger for 3-NT and 4-HNE Michael's adducts than sections from controls (Figure 7A and 7B) but not T15 autoantibodies (Figure 7A). Genetic loss of apoA-Ⅰincreases co-localization of 4-HNE-derived Michael's adducts with active TGFβ-1 in pulmonary airways (Figure 7B). Lungs from apoA-Ⅰ-/- mice express higher levels of XO, MPO and eNOS than lungs from C57BL/6 mice (Figure 8). These data are in contrast to nitrite+nitrate data showing that BALF isolated from apoA-Ⅰ-/- mice contains less nitrite+nitrate than BALF isolated from C57BL/6J mice (Figure 9A). Finally, BALF from apoA-Ⅰ-/- mice increased DCF fluorescence to a greater extent than BALF from C57BL/6J mice (Figure 9B). These data indicate that BALF from apoA-Ⅰ-/- mice contains more pro-oxidant compounds than BALF from C57BL/6J mice.
5. Conclusion
(1) Deletiong of apoA-Ⅰmakes HDL lost its protect fuction and turns it to proinflammatiory HDL, which aggregate a serial oxidatives stress and ucoupled eNOS.
(2) ApoA-Ⅰplays an important protective role in preventing pulmonary inflammation, impaired vasodilatation and increased airway hyperresponsiveness. Loss of apoA-Ⅰand its associated anti-inflammatory and anti-atherogenic properties has a profound and severe negative impact on the lung with respect to vascular function and airway physiology. Paper Two D-4F attenuates oxidative stress and inflammation in T-bet and apoA-Ⅰdouble knock out mice
1 Back ground
High-density lipoprotein (HDL) is important in reverse cholesterol transport and is believed to play important anti-inflammatory and anti-atherogenic roles in vascular physiology. Increasing evidence suggests that elevated levels of HDL are not always atheroprotective. Indeed, chronic states of inflammation and oxidative stress have been shown to convert HDL from an anti-inflammatory and anti-atherogenic particle into a pro-inflammatory and pro-atherogenic particle, making it useless for protecting the vessel wall against the effects of atherogenic concentrations of LDL. Asthma also increases inflammation and oxidative stress. In our previous study, we observed that apoA-Ⅰplays an important protective role in preventing pulmonary inflammation and airway hyperresponsiveness.
D-4F is an apoA-Ⅰmimetic made with all D-amino acids. D-4F is an amphipathic helix with a hydrophobic face that binds lipids in a manner similar to apolipoprotein A-Ⅰ. D-4F appears to protect HDL from becoming proinflammatory by binding oxidized lipids with greater affinity than native lipids. This ability to preferentially bind oxidized lipids and remodel HDL may be the responsible way for decreasing production of inflammatory cytokines in the mouse influenza model.
T-bet knock out mice, which develop airway hyperresponsiveness and are prone to spontaneous asthma, show lung inflammation and airway remodeling. T-bet deficient mice on Balb/c background have demonstrated dysfunctional HDL in lung inflammation and airway remodeling (unpublished abstractions). To examine the role of HDL in airway disease, we crossed T-bet-/- mice and apoA-Ⅰ-/- mice to create double knock out T-bet-/-/ apoA-Ⅰ-/- mice. To determine if D-4F could decrease airway disease we also treated T-bet-/-/apoA-Ⅰ-/- mice with D-4F. We found that T-bet-/-/apoA-Ⅰ-/- mice displayed higher airway hyperresponsiveness, pulmonary inflammation, collagen deposition in perivascular and peri alveolar region compared with T-bet-/-/apoA-Ⅰ+/+ control mice. Intriguingly, D4F reduced airway responsiveness, pulmonary inflammation and collagen deposition in the T-bet-/-/ apoA-Ⅰ-/- mice. Additionally, D4F attenuated oxidative stress in the T-bet-/-/apoA-Ⅰ-/-mice lung.
2 Methods
2.1 Mice
ApoA-Ⅰ-/- mice and T-bet-/- mice on a C57BL/6J background were purchased from Jackson Laboratory. The doubly heterozygous progeny were crossed to generate double knockout T-bet-/-/ apoA-Ⅰ-/- mice. Animal genotype was identified by a PCR-based assay. There were no discernible differences in litter size or appearance of the T-bet-/-/ apoA-Ⅰ-/- mice versus T-bet-/-/ apoA-Ⅰ+/+ control mice. Mice of 10 weeks old were injected with PBS and Apolipoprotein-mimetic peptide (D-4F, AC-DWFKAFYDKVAEKFKEAF-NH2) by daily intraperitoneal (IP) injection (3 mg/kg) for 6 weeks.
2.2 Plasma Total Cholesterol, HDL Cholesterol
Total cholesterol (TC) was quantified using a cholesterol oxidase/esterase kit from Wako Chemical, Inc. (Richmond, VA). HDL cholesterol was quantified using a HDL Cholesterol E kit from Wako Diagnostics. Proinflammatory HDL (p-HDL) was determined using a modified method of a previously published cell-free assay with CuCl2 and H2DCF(14).
2.3 Plasma PON1 Arylesterase Activity
Arylesterase activity of PON1 was performed on whole plasma using phenyl acetate as the substrate as described.(15)
2.4 Quantification of Plasma Nitrite+Nitrate
Nitrite+nitrate concentrations were determined by ozone chemiluminescence using the NO Analyzer (Model 280i, GE Analytical-Sievers, Boulder, CO) as described.(16,17) An aliquot of 30μL was injected (plasma was diluted by 1:30) into a sealed glass reaction chamber at 95℃containing VCl3.(17) Nitric oxide chemiluminescence signals were quantified and peak areas compared to the areas of external nitrate standards.
2.5 Measurements of Lung Mechanics
Each level of PEEP (0,3,6, and 9 cm H2O) was held for 80 sec with the Prime-2 perturbation repeated twice, once at 35 sec and the other at 70 sec. Measurements at each PEEP were Rn,G and H.
2.6 Methacholine challenge
Mice were challenged with 70 L of MCh (Sigma-Aldrich) of increasing concentrations (3.125 to 100 mg/mL) measurements(Rn,G and H) were assessed as a curve, whch pick value were picked up for calculation.
2.7 Histology
A 0.5 mL aliquot of zinc-formalin was used to inflate the lung prior to removal. The lung was fixed in zinc-formalin, embedded in paraffin, sectioned and then stained with hematoxylin and eosin (H&E) for histology or with McLetchies' trichrome to assess collagen deposition.
2.8 Immunofluorescence
4-HNE Michael's adducts and TGFβ-1 expression were detected by immunofluorescence for co-localization studies.Images captured using a krypton argon laser Nikon Eclipse TE2000U confocal microscope (Melville, NY) with 10x/0.17 aperture objective.
2.9 Western Blot Analysis
XO,MPO and eNOS protein levels were determined by western blot analysis. Autoradiograms were scanned with a laser densitometer or an UMax scanner.
3. Statistical Analysis
Data are presented as mean±SEM. Results were analyzed by Student's t-test, Mann-Whitney test or Fisher's exact test where appropriate. Airway data were analyzed by 2-way ANOVA to determine significance between curves and a Bonferroni post-test to determine significance of points between the curves. All statistical analysis was performed using GraphPad Prism Software (version 4.0).
4 Results
4.1 Cholesterol Profiles
The levels of total cholesterol and HDL cholesterol in plasma of T-bet-/-/ apoA-Ⅰ-/- mice treated with either PBS or D-4F were significantly reduced compared with T-bet-/-/7apoA-Ⅰ+/+ control mice (P < 0.001) . No difference in the total cholesterol or HDL cholesterol was observed between T-bet-/-/apoA-Ⅰ-/-mice treated with PBS and D4F.
4.2 Respiratory Mechanics
The effects of MCh on RN and G had no differences between groups. Lung tissue elastance was increased in the T-bet-/-/ apoA-Ⅰ-/- mice treated with PBS compared with T-bet-/-/apoA-Ⅰ-/- control mice (P < 0.05)
4.3 TGF-β1and 4- HNE in Lung Tissue Immunofluorescence
TGF-βand 4-HNE content in lungs is significantly reduced in T-bet-/-/ apoA-Ⅰ-/- mice treated with D-4F.
4.4 Lung histology
4.5 Perivascular and peribronchiolar inflammation and collagen deposition were increased slightly in T-bet-/-/apoA-Ⅰ+/+ control mice; and to a much greater extent in the T-bet-/-/apoA-Ⅰ-/- mice treated with PBS. However D-4F treatments markedly decreased inflammation and collagen deposition in T-bet-/-/ apoA-Ⅰ-/-mice.
4.6 XO Expression
The 145-kDa band corresponding to full length XD expression in the lung was significantly increased in T-bet-/-/apoA-Ⅰ-/- mice treated with D-4F compared with T-bet-/-/apoA-Ⅰ+/+ control mice and T-bet-/-/apoA-Ⅰ-/- mice treated with PBS(P<0.05). There was no difference between T-bef/7apoA-r/+ control mice and T-bet-/-/apoA-Ⅰ-/- mice treated with PBS.
4.7 Nitrate and Nitrite Level in BAL Fluid and Plasma
The nitrate and nitrite concentration in BAL fluids, which indicates the in vivo generation of NO in the airways, from the T-bet-/-/ apoA-Ⅰ-/- mice treated with PBS and D4F was significantly reduced compared with T-bet-/-/ apoA-Ⅰ+/+ control mice. Compared with T-bet-/-/ apoA-Ⅰ-/- mice treated with PBS, T-bet-/-/ apoA-Ⅰ-/- mice treated with D4F had increased nitrate and nitrite levels in the plasma and no significant difference had been achieved.
5 Conclusion
Taken together, these data suggest that genetic loss of apoA-Ⅰon the background of T-bet deficient mice increases pulmonary inflammation and collagen deposition as well as airway hyperresponsiveness. D4F reduced airway responsiveness, pulmonary inflammation and collagen deposition in the T-bet-/-/ apoA-Ⅰ-/- mice lung. D4F plays an important role in the mechanics of asthma.
载脂蛋白A-I的基因缺失增加小鼠气道反应性,肺部炎症和
胶原沉积的作用及机制的实验研究
1.背景
载脂蛋白A-Ⅰ(apoA-Ⅰ),是高密度脂蛋白(HDL)的重要组成部分,其抗动脉粥样硬化作用已经得到广泛的承认,而且apoA-Ⅰ本身对器官和血管床也有保护作用。最近新的研究证据表明,HDL在肺脏中起着保护作用。与apoA-Ⅰ结合的ABCA-Ⅰ先前已经被证明在维持脂质组成、肺脏结构和呼吸功能中起着重要的作用。最近,在蛋白质组学研究揭示纯合子镰刀细胞贫血病患者的血浆apoA-Ⅰ的水平合并肺动脉高压者总是比没有合并肺动脉高压者低。另外有研究证实,内皮脂酶的基因缺失导致HDL水平升高2倍,并且增加了卵蛋白致敏的BALB/c小鼠的气道高反应性和肺部炎症。尽管这些研究提供了这样一个观点,那就是HDL有助于维持健康的肺脏,但是还没有研究直接证实apoA-Ⅰ或者是apoA-Ⅰ的缺失对肺脏炎症,血管舒张和气道高反应性的作用。
越来越多的证据表明,高水平的HDL并不总是具有抗动脉粥样硬化的作用。实际上,慢性炎症状态和氧化应激已经被证明能将HDL从具有抗炎和抗动脉粥样硬化作用的脂蛋白转换成具有致炎性和致动脉粥样硬化作用的脂蛋白,从而使HDL失去有效的清除LDL,抗动脉粥样硬化和保护血管的功能。哮喘也能增加炎症和氧化应激。这些炎症变化很可能解释为什么成年女性中发作的哮喘和颈内动脉内膜厚度的增加有显著关系。
这些报告为我们提供了一个新的思想,那就是HDL对肺脏具有保护功能。但是目前还没有任何研究表明,载脂蛋白A-I是否直接决定或影响肺部炎症及气道高反应性。
在这个报告中,我们研究了载脂蛋白A-I基因缺失对肺的炎症,血管舒张反应,胶原沉积及气道高反应性的影响。我们的研究结果表明载脂蛋白A-I对肺动脉和气道功能的维持具有保护功能,能有效的防止气道的炎症和胶原沉积。
2方法
2.1对血浆总胆固醇(TC),HDL和致炎性HDL(p-HDL)的测定
总胆固醇是由Wako公司生产的胆固醇氧化酶/酯酶试剂盒测定。HDL是用Richmond公司生产的高密度脂蛋白胆固醇E试剂盒测定。致炎性HDL用MgCl2沉淀后和CuCl2孵化,加入荧光染料H2DCF后测定。
2.2血浆(对氧磷酶)芳香酯酶活性的测定
血浆芳香酯酶活性的测定是用苯乙酸作为底物进行的。稀释后的血浆被加到加到磷酸盐缓冲液体系中(苯基乙酸[100 mmol/L],氯化钙[100 mmol/L],pH值8.0)。苯乙酸的水解率由DU(?)640分光光度计进行测定。芳香酯酶的活性单位相当于每分钟水解的每毫升中的苯乙酸的摩尔数。
2.3肺泡灌洗液中亚硝酸盐+硝酸盐的定量测定
肺泡灌洗液中亚硝酸盐和硝酸盐浓度使用臭氧发光法测定。30ul原液加到包含氯化碘的反应体系中。一氧化氮的信号由一氧化氮分析仪测定。和标准硝酸盐浓度曲线相比而得到原液中硝酸盐和亚硝酸盐的浓度。
2.4血浆中3-NT的斑点杂交
从小鼠体内分离的血浆样品直接点在硝酸纤维素膜上,晾干。膜用牛奶封闭,加一抗3-NT杂交,4℃过夜。第二天洗膜,加二抗,ECL化学发光拍片来测定血浆中3-NT总的含量
2.5 apoA-I基因缺失对血管舒张的影响
本实验全部采用小鼠的面部动脉和肺动脉血管,腹腔注射苯巴比妥后,分离小鼠的双侧的面部动脉,其直径一般在180-250um。血管两侧挂在微玻璃管上并连接60cm H20的压强。所有分离提取的所有血管均不作预处理。先测量最大血管内径(Vmax),然后用U 46619(10-9-10-8mol/L)作预收缩并测值(V con),然后依次用浓度梯度为10-7-10-4mol/L的乙酰胆碱(ACh)扩张血管以测量在依次增加ACh浓度情况下的内皮依赖的血管舒张情况(VACh);按以下公式计算ACh扩张血管的百分值:(VACh-Vcx/n)/(Vmax-Vcon}=血管扩张率,以此血管扩张率表示内皮依赖的血管舒张情况。进而用左旋硝基精氨酸甲酯(L-nitroarginiemethylester, L-NAME)和100umol/L预处理血管30 min,再重复用U 46619和ACh处理血竹以了解内皮一氧化氮合酶(endothelial nitric oxide synthase, eNOS)依赖的血管舒张功能。
2.6脉冲振荡技术测量气道反应性
用Scireq2007小动物肺功能分析仪通过强力脉冲振荡技术来测定呼吸总阻(impedence, Zrs),测定参数为中央气道阻力(resistance, Rn),外周气道阻力(tissue damping,G)和组织弹性回缩力(tissue elastance,H)。
2.7乙酰甲胆碱激发实验
经气道浓度梯度增加应用乙酰甲胆碱(3.125mg/ml-100mg/ml)激发气道高反应性。70ul乙酰甲胆碱在超声雾化器中被雾化,然后经由Scireq2007小动物肺功能分析仪吸气管道输送至小鼠气道。待曲线平稳后进行下一次给药。在不同浓度的乙酰甲胆碱取得曲线,取其峰值,分别取得相应的Rn,G和H值。
2.8肺病理取材制片
0.5毫升中性甲醛经气道灌洗管被缓缓注入肺中进行组织固定。开胸摘取肺,至于10%中性甲醛固定液中固定24h以上。常规石蜡包埋、制备石蜡切片HE染色McLetchies三色染色来评估胶原沉积。
2.9 BALF的分离和分析
收回的肺泡灌洗液2000rpm离心后去除上清液,保存于-80℃。细胞沉淀悬浮于1mlPBS,吸取500ul细胞悬液来制作细胞调整细胞离心涂片,晾干后4%多聚甲醛固定,瑞氏染色,瑞氏染色后行光学显微镜下观察500个细胞,进行细胞分类计数。
2.10免疫荧光测定肺病理切片中4HNE,转化生长因子β-1 (TGFβ-1),3-NT和T-15的表达
肺组织切片进行脱蜡,梯度脱水,封闭,加一抗,加二抗,激光共聚焦显微镜进行免疫荧光测定。
2.11 Western-blot分析黄嘌呤氧化酶(XO),内源性一氧化氮合酶(eNOS)和髓过氧化物酶(MPO)
摘除小鼠肺组织,立即至于-80℃中冻存。研磨裂解肺组织,分离蛋白上清液。测定蛋白含量。SDS-PAGE电泳分离蛋白,转膜,显色,照相。
2.12 BALF的氧化指数的测定
氧化物H2DCF用来测定BALF中的氧化指数。75ul BALF与H2DCF混合(100 u L PBS+25 u L H2DCF和37℃孵育2小时。读板器在2小时后读取荧光值(Ex 485nm;Em 530nm)
3.统计学处理：
所有数据均用X±SEM表示,利用Prisme5统计软件进行统计分析,指标比较采用单因素方差分析,两组间比较采用t检验,P<0.05为差异有显著性。
4.结果：
4.1 ApoA-Ⅰ基因缺失对脂质和氧化应激的作用
和C57BL/6J小鼠相比,apoA-Ⅰ-/-小鼠血浆中总胆固醇和HDL水平降低。尽管apoA-Ⅰ-/-小鼠含有的HDL水平低于C57BL/6J小鼠,但是其HDL的氧化速率要高于C57BL/6J小鼠,这也说明apoA-Ⅰ-/-小鼠血浆中HDL是致炎性的。ApoA-Ⅰ-/-小鼠血浆中PON1的蛋白水平比C57BL/6J小鼠的高。但是,PON1的活性在apoA-Ⅰ-/-小鼠血浆中比C57BL/6J小鼠降低了大约34%。和C57BL/6J小鼠相比,apoA-Ⅰ-/-小鼠血浆中硝酸盐和亚硝酸盐水平下降,血浆中3-NT的水平升高。
4.2 ApoA-Ⅰ基因缺失对血管扩张的作用
分离的面部动脉血管在预增压后对Ach依赖的血管舒张反应实验表明apoA-Ⅰ基因缺失对面部动脉血管没有影响。这些结果和之前的研究结果表明apoA-Ⅰ-/-小鼠对动脉粥样硬化并不易感。然而,肺动脉血管环的舒张反应显示从apoA-Ⅰ-/-小鼠分离的血管环在最高浓度的Ach下舒张功能受损。从apoA-Ⅰ-/-小鼠分离的肺血管环在最高浓度的Ach下实际上表现出收缩反应。相反地,从C57BL/6J小鼠分离的肺血管环在最高浓度的Ach下则表现出舒张反应。ApoA-Ⅰ-/-小鼠肺动脉血管的这些生理反应的变化提示慢性炎症反应和氧化应激损伤了肺部血管的舒张。
4.3 ApoA-Ⅰ基因缺失对气道高反应性的作用
Flexivent动物肺功能仪是对整个支气管呼吸动力参数的测定。在主支气管中,它测定中央气道阻力(RN),在外周支气管中,它测定外周气道阻力(G)和组织弹性回缩力(H)。经MCh后,G(P<0.01)和H(P<0.001)在apoA-Ⅰ-/-小鼠中明显升高,Rn在两组中无明显差别。在不同水平的气道正压通气(PEEP)后,中央气道阻力(RN)在两组中无明显差别。和C57BL/6J小鼠相比,apoA-Ⅰ-/-小鼠外周气道阻力在基线水平(PEEP=0)升高(P<0.01),并且在整个PEEP曲线中,G在apoA-Ⅰ-/-小鼠比在C57BL/6J小鼠中高；在整个PEEP曲线中,组织弹性回缩力H在apoA-Ⅰ-/-小鼠中比在C57BL/6J小鼠中升高(H,P<0.001)。
4.4ApoA-Ⅰ基因缺失对肺病理组织的作用
肺组织病理HE切片显示：与C57BL/6J系小鼠相比,apoA-Ⅰ-/-小鼠肺部血管和支气管周围炎症细胞浸润增多。尽管apoA-Ⅰ-/-小鼠肺部血管和支气管周围炎症细胞比C57BL/6J系小鼠增多2倍以上,但是肺浸润的白细胞数还没有达到OVA致敏的C57BL/6J系小鼠的白细胞数(25-50细胞/PHF)。ApoA-Ⅰ-/-小鼠肺组织切片显示胶原沉积增多。与C57BL/6J系小鼠相比,apoA-Ⅰ-/-小鼠肺组织trichrome染色增强。BALF细胞涂片显示BALF中主要含有肺泡巨噬细胞(>98%)。从apoA-Ⅰ-/-小鼠和C57BL/6J系小鼠的BALF的细胞图片显示apoA-I的基因缺失对支气管淋巴细胞浸润没有明显的作用。
4.5Apo A-I的基因缺失对氧化应激和炎症生物标记物的作用
免疫荧光研究表明apoAⅠ-/-小鼠的肺组织切片比对照组中3-硝基酪氨酸(3-NT)和4-羟基壬烯醛(4HNE)Michael加合物免疫荧光信号明显增强。但是apoA-Ⅰ-/-小鼠的肺组织切片抗T15的自身抗体信号却没有比对照组升高。ApoA-I的基因缺失导致呼吸道中4-HNE衍生的Michael反应的加合物和活性TGF-01表达增加。对照组C57BL/6J系小鼠相比,apoA-Ⅰ-/-小鼠肺组织中XO,MPO和eNOS的表达明显升高。这些数据与硝酸盐和亚硝酸盐的数据相反,apoA-Ⅰ-/-小鼠的BALF中的硝酸盐和亚硝酸盐的水平比C57BL/6J系小鼠BALF硝酸盐和亚硝酸盐的水平下降。然而,与C57BL/6J系小鼠相比,,apoA-Ⅰ-/-小鼠的BALF中含有更多致氧化化合物。
5结论：
1) ApoA-Ⅰ的缺失导致HDL在体内的的保护作用消失,并将HDL转变成致炎性HDL,引起一系列氧化应激反应的加剧,导致脂质过氧化,eNOS解偶联。
2) ApoA-Ⅰ在预防肺部炎症,受损的血管舒张及气道高反应性方面具有重要的保护作用。ApoA-Ⅰ及其相关的抗炎和抗动脉粥样硬化作用的缺失在血管功能和呼吸道生理功能方面具有深刻的和重要的影响。
论文二ApoA-Ⅰ模拟肽,D-4F对apoA-Ⅰ和T-bet双基因敲除小鼠体内氧化应激和炎症状态的改善作用及机制的实验研究
1.背景
高密度脂蛋白(HDL)在胆固醇逆转运和抗动脉粥样硬化和炎症过程中发挥着重要的作用。越来越多的证据表明,高密度脂蛋白水平升高并不总是意味着对动脉的保护。事实上,慢性炎症和氧化应激已被证明可将具有抗炎和抗动脉粥样硬化作用的HDL转换成致炎性和致动脉粥样硬化性颗粒,使它失去对抗低密度脂蛋白(LDL),抗动脉粥样硬化和保护血管的作用。哮喘也增加炎症和氧化应激。在我们之前的研究中,我们发现载脂蛋白A-I在防止气道高反应和肺部炎症中具有重要的保护作用。
D-4F是载脂蛋白A-I的模拟肽,全部由D-氨基酸合成。D-4F有一个疏水螺旋可以结合脂质,这个与载脂蛋白A-I与脂质的结合方式相似。D-4F对氧化的脂质比普通脂质具有更大的结合力,这个作用似乎与其对保护高密度脂蛋白的抗炎能力有关。在小鼠流感的模型中,D-4F被证实可以结合氧化脂质,重塑HDL,这种功能可能与其减少炎症因子的产生有关。
T-bet基因敲除小鼠,以自发地气道高反应性,肺部炎症和气道重塑为特征。之前的研究发现apoA-Ⅰ基因缺失能增加小鼠的气道高反应性。为了研究高密度脂蛋白在气道疾病中的作用,本实验将apoA-Ⅰ-/-小鼠和T-bet-/-小鼠进行杂交,筛选出双基因敲除的T-bet-/-/apoA-Ⅰ-/-小鼠有。为了进一步验证D-4F能够减少气道疾病,实验中加用D-4F治疗双基因敲除的T-bet-/-/apoA-Ⅰ-/-小鼠。研究发现,双基因敲除的T-bet-/-/apoA-Ⅰ-/-小鼠有更高的气道高反应性,肺部炎症和胶原沉积。有趣的是,在T-bet-/-/apoA-Ⅰ-/-小鼠中,D-4F的治疗降低了气道高反应性,减少肺部炎症,胶原沉积。另外,D-4F的治疗也减轻了T-bet-/-/apoA-Ⅰ-/-小鼠肺部的氧化应激状态。
2方法
2.1 T-bet-/-/apoA-Ⅰ-/-小鼠模型的筛选和建立
以C57BL/6J小鼠系的apoA-Ⅰ-/-小鼠和T-bet-/-小鼠均购自美国杰克逊实验室,将其进行杂交,产生T-bet+/-apoA-Ⅰ+/-小鼠。将杂合子小鼠进一步杂交,用PCR法筛选出双基因敲除的T-bet-/-/apoA-Ⅰ-/-小鼠。T-bet-/-/apoA-Ⅰ-/-小鼠与对照组的T-bet-/-小鼠在出生体重上没有明显差别。10周左右时,将雄性T-bet+/-/apoA-Ⅰ+/-小鼠随机分成对两组,一组每天腹腔注射PBS,治疗组注射D-4F,(D-4F,AC-DWFKAFYDKVAEKFKEAF-NH2,3mg/kg体重),治疗6周。所有的动物实验,由威斯康星医学院动物护理和使用委员会(IACUC)批准。
2.2对血浆总胆固醇(TC)和HDL的测定
总胆固醇是由Wako公司生产的胆固醇氧化酶/酯酶试剂盒测定。HDL是用Richmond公司生产的高密度脂蛋白胆固醇E试剂盒测定。
2.3血浆(对氧磷酶)芳香酯酶活性的测定
血浆芳香酯酶活性的测定是用苯乙酸作为底物进行的。稀释后的血浆被加到加到磷酸盐缓冲液体系中(苯基乙酸[100 mmol/L],氯化钙[100 mmol/L],pH值8.0)。苯乙酸的水解率由DU(?)640分光光度计进行测定。芳香酯酶的活性单位相当于每分钟水解的每毫升中的苯乙酸的摩尔数。
2.4肺泡灌洗液中亚硝酸盐+硝酸盐的定量测定
肺泡灌洗液中亚硝酸盐和硝酸盐浓度使用臭氧发光法测定。30ul原液加到包含氯化碘的反应体系中。一氧化氮的信号由一氧化氮分析仪测定。和标准硝酸盐浓度曲线相比而得到原液中硝酸盐和亚硝酸盐的浓度。
2.5脉冲振荡技术测量气道反应性
用Scireq2007小动物肺功能分析仪通过强力脉冲振荡技术来测定呼吸总阻(impedence,Zrs),测定参数为中央气道粘性阻力(resistance,Rn),外周气道粘性阻力(tissue damping,G)和组织弹性回缩力(tissue elastance,H)。
2.6乙酰甲胆碱激发实验
经气道浓度梯度增加应用乙酰甲胆碱(3.125mg/ml～100mg/ml)激发气道高反应性。70ul乙酰甲胆碱在超声雾化器中被雾化,然后经由Scireq2007小动物肺功能分析仪吸气管道输送至小鼠气道。待曲线平稳后进行下一次给药。在不同浓度的乙酰甲胆碱取得曲线,取其峰值,分别取得相应的Rn,G和H值。
2.7肺病理取材制片
常规石蜡包埋、制备石蜡切片、HE染色McLetchies三色染色来评估胶原沉积。
2.8免疫荧光测定肺病理切片中4HNE,转化生长因子β-1(TGFβ-1)
常规脱蜡,梯度脱水,加一抗,加二抗,封闭组织切片
2.9 Western-blot分析黄嘌呤氧化酶(XO)和髓过氧化物酶(MPO)
SDS-PAGE电泳分离蛋白,转膜,显影,照相。
3.统计学处理
所有数据均用X±SEM表示,利用Prisme5统计软件进行统计分析,指标比较采用多因素方差分析,P<0.05为差异有显著性。
4结果
4.1血浆总胆固醇(TC),HDL的水平
注射PBS和D-4F的T-bet-/-/apoA-Ⅰ-/-小鼠血浆中总胆固醇(P<0.001)和高密度脂蛋白(P<0.001)的水平比对照组T-bet-/-/apoA-Ⅰ+/+小鼠明显降低。血浆中总胆固醇和高密度脂蛋白的水平在PBS注射组和D-4F组中无显著性差异。
4.2血浆中PON1的表达和活性
和对照组T-bet-/-/apoA-Ⅰ+/+小鼠相比,PBS组和D-4F组中T-bet-/-/apoA-Ⅰ-/-小鼠PON1的活性明显下降(P<0.001)。在T-bet-/-/apoA-Ⅰ-/-小鼠中,D-4F治疗使PON1的活性有所升高,但没有达到统计学意义。
4.3肺泡灌洗液中硝酸盐和亚硝酸盐水平
肺泡灌洗液中硝酸盐和亚硝酸盐的水平提示体内气道中一氧化氮的产生。和对照组T-bet-/-/apoA-Ⅰ+/+小鼠相比,PBS组和D-4F组中T-bet-/-/apoA-Ⅰ-/-小鼠硝酸盐和亚硝酸盐水平明显下降(P<0.001)。在T-bet-/-/apoA-Ⅰ-/-小鼠中,D-4F治疗使硝酸盐和亚硝酸盐水平的水平有所升高(P<0.05)。
4.4 FIexiVent动物肺功能仪对呼吸力参数的测
乙酰甲胆碱激发实验中,中央气道阻力和外周气道阻力在三组中无明显差异。组织弹性回缩力H(P<0.05)在PBS组中的T-bet-/-/apoA-Ⅰ-/-小鼠比T-bet-/-/apoA-Ⅰ+/+对照组小鼠升高。
4.5免疫荧光测定TGF-β1和4-HNE在肺组织的表达水平
在T-bet-/-/apoA-Ⅰ-/-小鼠中,经D-4F治疗后,TGF-β1 and 4-HNE在肺的含量明显减少。
4.6肺组织病理学
和对照组T.bet-/-/apoA-Ⅰ+/+小鼠相比,PBS组的T-bet-/-/apoA-Ⅰ-/-小鼠肺部炎症明显增加,在气管和血管旁的胶原沉积也增加。然而,经D-4F治疗的T-bet-/-/apoA-Ⅰ-/-小鼠肺中,肺部炎症明显减轻,胶原沉积也减少。
4.7肺组织中XO的表达
与对照组T-bet-/-/apoA-Ⅰ+/+小鼠和PBS组T-bet-/-/apoA-Ⅰ-/-小鼠相比,D-4F治疗的T-bet-/-/apoA-Ⅰ-/-小鼠肺组织中黄嘌呤氧化酶(P<0.05)的蛋白表达明显增加。
5结论
1)总的来说,这些数据显示在哮喘模型T-bet-/-小鼠基础上,apoA-Ⅰ的缺失进一步加重了肺部的炎症和胶原沉积以及气道高反应性。这说明apoA-Ⅰ在哮喘的发病中具有一定的保护作用。这一点也被apoA-Ⅰ模拟物D-4F的治疗进一步证明。经D-4F治疗后,T-bet-/-/apoA-Ⅰ-/-老鼠肺部炎症,胶原沉积及气道反应性减轻。D4F的作用为哮喘的治疗提供了新的策略。
2)哮喘发病还与多种炎性细胞和多种细胞因子等有关,在其复杂的免疫炎症网络中如何实施合理的干预性调节治疗,有待于在今后的实验研究与临床研究结合于一起,以p-HDL中心,从炎症反应和氧化应激及其多因子的综合性探索,以期从根本上达到控制哮喘肺部炎症和高气道反应性的目的。
Part One Genetic Deletion of Apolipoprotein A-ⅠIncreases Airway Hyperresponsiveness, Inflammation and Collagen Deposition in the Lung
1. Background
Although apolipoprotein A-Ⅰ(apoA-Ⅰ), the major anti-atherogenic apolipoprotein of high-density lipoprotein (HDL) is well recognized for protecting the heart against vascular disease, it also protects other vascular beds and organs. Recent studies provide new evidence supporting the notion that HDL plays a protective role in the lung. Earlier ABCA1, which interacts with lipid poor apoA-Ⅰ, was shown to be essential for maintaining normal lipid composition and architecture of the lung as well as respiratory physiology. More recently, proteomic studies revealed that homozygous sickle cell anemia patients with pulmonary arterial hypertension (PAH) consistently had lower apoA-Ⅰlevels than sickle patients without PAH. Interestingly, genetic deletion of endothelial lipase resulted in a nearly 2-fold increase in HDL, which was credited with decreasing airway hyperresponsiveness and pulmonary inflammation in ovalbumin (OVA)-sensitized BALB/c mice. Although these reports provide some support for the idea that HDL helps maintain healthy lungs, no studies have directly determined the effects of apoA-Ⅰ, or lack thereof, on pulmonary inflammation, vasodilatation and airway hyperresponsiveness.
Increasing evidence suggests that elevated levels of HDL are not always atheroprotective. Indeed, chronic states of inflammation and oxidative stress have been shown to convert HDL from an anti-inflammatory and anti-atherogenic lipoprotein into a pro-inflammatory and pro-atherogenic lipoprotein, making it useless for protecting the vessel wall against the effects of atherogenic concentrations of LDL. Asthma also increases inflammation and oxidative stress. Such inflammatory changes may explain why adult-onset asthma is associated with significant increases in carotid artery intimal-medial thickness in women. In this report we examine the effects of genetic deletion of apoA-Ⅰon pulmonary inflammation, vasodilatation, collagen deposition and airway hyperresponsiveness. Our findings suggest apoA-Ⅰplays a critical role in protecting pulmonary artery and airway function as well as preventing inflammation and collagen deposition..
2. Methods
2.1 Plasma Total Cholesterol, HDL Cholesterol and Proinflammatory HDL
Total cholesterol (TC) was quantified using a cholesterol oxidase/esterase kit from Wako Chemical, Inc. (Richmond, VA). HDL cholesterol was quantified using a HDL Cholesterol E kit from Wako Diagnostics. Proinflammatory HDL (p-HDL) was determined using a modified method of a previously published cell-free assay with CuCl2 and H2DCF.
2.2 Plasma PON1 Arylesterase Activity
Arylesterase activity of PON1 was performed on whole plasma using phenyl acetate as the substrate as described. Initial rates of hydrolysis were determined spectrophotometrically at 270 nm on DU(?) 640 spectrophotometer. An aliquot of 20μL of 30X diluted mouse plasma was added to a final reaction volume of 500μL (phenyl acetate [100 mmol/L] and CaCl2 [100 mmol/L] in Tris-HCI [40 mmol/L] buffer, pH 8.0) for 6 min at 25℃. One unit of arylesterase activity equals 1μmole of phenyl acetate hydrolyzed per mL per min.
2.3 Quantification of Plasma Nitrite+Nitrate
Nitrite+nitrate concentrations were determined by ozone chemiluminescence using the NO Analyzer (Model 280i, GE Analytical-Sievers, Boulder, CO) as described. An aliquot of 30μL was injected (plasma was diluted by 1:30) into a sealed glass reaction chamber at 95℃containing VCI3. Nitric oxide chemiluminescence signals were quantified and peak areas compared to the areas of external nitrate standards.
2.4 Estimates of Plasma 3-Nitrotyrosine
An aliquot of plasma from C57BL/6J and apoA-Ⅰ-/- mice was pipetted onto nitrocellulose membranes and allowed to bind. Membranes were blocked with 5% non-fat dry milk dissolved in fresh PBS-Tween (0.1%) and then incubated overnight at 4℃with antibodies for 3-NT (1:5000;Millipore, Billerica, MA). The next day, the membranes were washed and incubated with the appropriate HRP-conjugated secondary antibody for 1 h. Bands of identity were visualized with ECL chemiluminescence following the manufacturer's recommendations. Autoradiograms were scanned with a laser densitometer or a UMax scanner.
2.5 Effects of Genetic Deletion of ApoA-Ⅰon Vasodilatation
Facialis and pulmonary arteries were isolated from C57BL/6J and apoA-Ⅰ-/- mice by microdissection as previously described. Vasodilatation of pressurized (60 cm of H2O) facialis arteries was examined in the absence and presence of L-nitroargininemethylester (L-NAME-200μM, final concentration) as previously described.(14) Changes in pulmonary artery tension in response to acetylcholine (ACh) were recorded on a DMT wire-myograph using protocols similar to that which we have previously described.
2.6 Measurements of Lung Mechanics
Mice were tracheostomized with an 18-gauge cannula and mechanically ventilated in a quasi-sinusoidal fashion with a small animal ventilator at 150 breaths per minute(19) and a tidal volume (VT) of 10 mL·kg-1 body weight. Basal respiratory system impedance (Zrs) was then assessed using the forced oscillation technique (FOT) applied over 2 sec (a Prime-2 perturbation) using the flexiVent system. Each level of PEEP (0,3,6, and 9 cm H2O) was held for 80 sec with the Prime-2 perturbation repeated twice, once at 35 sec and the other at 70 sec. Measurements at each PEEP were Rn, G and H.
2.7 Methacholine challenge
Mice were challenged with 70 L of MCh (Sigma-Aldrich) of increasing concentrations (3.125 to 100 mg/mL). MCh aerosols were generated using an ultrasonic nebulizer and delivered to the inspiratory line of the ventilator using room air. Each aerosol was delivered for 30 sec, during which time ventilation was maintained mechanically by the flexiVent. Then for the next 3 min, each aerosol was delivered for 30 sec, during which time regular ventilation was maintained and measurements(Rn,G and H) were assessed as a curve, whch pick value were picked up for calculation.
2.8 Histology
A 0.5mL aliquot of zinc-formalin was used to inflate the lung prior to removal. The lung was fixed in zinc-formalin, embedded in paraffin, sectioned and then stained with hematoxylin and eosin (H&E) for histology or with McLetchies' trichrome to assess collagen deposition.
2.9 Isolation of BALF and Analysis
BALF was obtained by flushing the lung with PBS, first 1mL followed by 0.5mL. The rinses were combined and the BALF was centrifuged at 2000 rpm for 10 min at 4℃. The supernatant was removed, stored at -80℃. The cell pellet was gently resuspended in 1 mL PBS. An aliquot of 500μL was used to prepare slides using a Cytospin. Differential cell counts were made from slides stained with Diff-Quick. Five hundred cells were counted and identified (magnification, X40) for each mouse by a pathologist who had no prior knowledge of slide identities.
2.10 Immunofluorescence
Immunofluorescence was performed on 5μm sections of paraffin-embedded, PBS-zinc-formalin-fixed lungs. Two sections were present on each slide. The sections were incubated separately with antibodies against either 3-nitrotyrosine anti-T15 autoantibodies as previously described, or with anti-4-HNE Michael's adducts and with TGFβ-1 for co-localization studies. Slides were washed with PBS (3X) and then incubated with the appropriate secondary antibodies. The slides were washed, sealed under cover slips and images captured using a krypton argon laser Nikon Eclipse TE2000U confocal microscope with 10x/0.17 aperture objective. Total magnification was 100 with Ex/Em at 488/580 nm for Alexa 488 and 633/661 nm forTO-PRO-3.
2.11Western Blot Analysis
Proteins were separated by SDS-PAGE (4-20%) and transferred onto nitrocellulose membranes. Membranes were blocked with 5% non-fat dry milk dissolved in fresh PBS-Tween (0.1%) and then incubated overnight at 4℃with antibodies for XO (1:500), MPO (1:100) or eNOS (1:10,000). The next day, the membranes were washed and incubated with the appropriate HRP-conjugated secondary antibody for 1 h. Bands of identity were visualized with ECL chemiluminescence following the manufacturer's recommendations. Autoradiograms were scanned with a laser densitometer or an UMax scanner.
2.12 BALF DCF-detectable Oxidants
Oxidation of H2DCF was used to obtain an index of the levels of oxidants in BALF. An aliquot of BALF (75μL) was mixed with H2DCF (100μL PBS+25μL H2DCF [a 1x10 dilution of 2 mg/mL] in a total volume of 200μL) and this mixture incubated for 2 h at 37℃. Absolute changes in fluorescence (Ex 485 nm;Em 530 nm) were determined at the end of the 2 h incubation period using Spectra Max Gemini EM fluorescence plate reader.
3. Statistical Analysis
Data are presented as mean±SEM. Results were analyzed by Student's t-test, Mann-Whitney test or Fisher's exact test where appropriate. Airway data were analyzed by 2-way ANOVA to determine significance between curves and a Bonferroni post-test to determine significance of points between the curves. All statistical analysis was performed using GraphPad Prism Software (version 4.0).
4. Results
4.1 Effects of ApoA-ⅠDeficiency on Lipids and Oxidative Stress
ApoA-Ⅰ-/- mice had reduced levels of plasma total cholesterol and HDL cholesterol compared with control mice (Figure 1A and 1B). Although apoA-Ⅰ-/-mice contained less HDL than control mice, their HDL oxidized at a faster rate than HDL from control mice, indicating that HDL in apoA-Ⅰ-/- mice was proinflammatory (Figure 1C). PON1 plasma protein in apoA-Ⅰ-/- mice was increased compared with protein levels in C57BL/6J mice (Figure 2A and 2B). However, PON1 activity was decreased (≈34%,p<0.001) compared with controls (Figure 2C). Although the concentration of plasma nitrite+nitrate was decreased in apoA-Ⅰ-/- mice, plasma 3-NT levels were increased compared with the levels in control mice (Figure 3A and 3B, respectively).
4.2 Effects of ApoA-ⅠDeficiency on Vasodilatation.
Studies on ACh-dependent vasodilatation of isolated and pressurized facialis arteries revealed that genetic loss of apoA-Ⅰhad no effect on relaxation responses in these vessels (Figure 4A vs.4B). These observations are consistent with previous studies showing that apoA-Ⅰ-/- mice are not more susceptible to atherosclerosis.(27,28) However, relaxation responses of pulmonary artery rings isolated from apoA-Ⅰ-/- mice were impaired at the highest ACh concentration (Figure 4C). Pulmonary artery rings from apoA-Ⅰ-/-mice actually constricted when treated with the highest ACh concentration. In contrast, the pulmonary artery rings from C57BL/6J mice continued to relax. Such changes in the physiological responses of pulmonary arteries in apoA-Ⅰ-/- mice are consistent with the idea that chronic exposure to inflammation and oxidative stress impair vasodilatation.
4.3 Effects of ApoA-ⅠDeficiency on Airway Hyperresponsiveness
The flexiVent provides quantitative information regarding the mechanical properties of the entire airway tree. In the large airways it assesses Newtonian resistance (RN), while in the smaller airways it is able to determine changes in tissue damping (G) and tissue elastance (H). In both apoA-Ⅰ-/- and C57BL/6J mice, methacholine (MCh) induced dose-dependent increases in airway resistance associated with tissue dampening (G, Figure 5B; p<0.01) and tissue elastance (H, Figure 5C;p<0.001), but not RN (Figure 5A). Effects of increasing PEEP on airway mechanical parameters, RN, G and H, are shown in Figure 5D,4E and 4F, respectively. G was increased in apoA-Ⅰ-/- mice compared with control mice at baseline PEEP=0 (p<0.01) as well as throughout the entire PEEP curve (p<0.001). H was increased in apoA-Ⅰ-/- mice compared with controls throughout the entire PEEP range (p<0.001).
4.4 Effects of apoA-ⅠDeficiency on Histology
H&E sections of lungs harvested from apoA-Ⅰ-/- mice (two lower left images, Figure 6A) contained more inflammatory cells than lungs from C57BL/6J mice (one upper left image, Figure 6A). Although inflammatory cells in perialveolar and perivascular regions of apoA-Ⅰ-/- mice were increased more than two fold compared with C57BL/6J mice (Figure 6B, p<0.025 2-tailed t-test), the number of white blood cell infiltrating the lungs did not achieve the numbers that are achieved in ovalbumin-sensitized C57BL/6J mice, which can approach 25-50 cells PHF. Sections of lungs harvested from apoA-Ⅰ-/- mice also revealed increased collagen deposition (two lower right images, Figure 6A). Trichrome staining is stronger and more extensive in apoA-Ⅰ-/- sections than in sections of lungs from C57BL/6J mice (upper right image, Figure 6A). Cytospins of BALF isolated from apoA-Ⅰ-/- and C57BL/6J mice showed that the BALF contained predominately alveolar macrophages(>98%). Cytospins of BALF isolated from C57BL/6J and apoA-Ⅰ-/- mice reveal that the genetic loss of apoA-Ⅰdid not have significant effect on lymphocyte infiltration in the bronchoalveolar space (Figure 6C).
4.5 Effects of apoA-ⅠDeficiency on Biomarkers of Oxidative Stress and Inflammation
Immunofluorescence studies revealed that lung sections from apoA-Ⅰ-/-mice stained stronger for 3-NT and 4-HNE Michael's adducts than sections from controls (Figure 7A and 7B) but not T15 autoantibodies (Figure 7A). Genetic loss of apoA-Ⅰincreases co-localization of 4-HNE-derived Michael's adducts with active TGFβ-1 in pulmonary airways (Figure 7B). Lungs from apoA-Ⅰ-/- mice express higher levels of XO, MPO and eNOS than lungs from C57BL/6 mice (Figure 8). These data are in contrast to nitrite+nitrate data showing that BALF isolated from apoA-Ⅰ-/- mice contains less nitrite+nitrate than BALF isolated from C57BL/6J mice (Figure 9A). Finally, BALF from apoA-Ⅰ-/- mice increased DCF fluorescence to a greater extent than BALF from C57BL/6J mice (Figure 9B). These data indicate that BALF from apoA-Ⅰ-/- mice contains more pro-oxidant compounds than BALF from C57BL/6J mice.
5. Conclusion
(1) Deletiong of apoA-Ⅰmakes HDL lost its protect fuction and turns it to proinflammatiory HDL, which aggregate a serial oxidatives stress and ucoupled eNOS.
(2) ApoA-Ⅰplays an important protective role in preventing pulmonary inflammation, impaired vasodilatation and increased airway hyperresponsiveness. Loss of apoA-Ⅰand its associated anti-inflammatory and anti-atherogenic properties has a profound and severe negative impact on the lung with respect to vascular function and airway physiology. Paper Two D-4F attenuates oxidative stress and inflammation in T-bet and apoA-Ⅰdouble knock out mice
1 Back ground
High-density lipoprotein (HDL) is important in reverse cholesterol transport and is believed to play important anti-inflammatory and anti-atherogenic roles in vascular physiology. Increasing evidence suggests that elevated levels of HDL are not always atheroprotective. Indeed, chronic states of inflammation and oxidative stress have been shown to convert HDL from an anti-inflammatory and anti-atherogenic particle into a pro-inflammatory and pro-atherogenic particle, making it useless for protecting the vessel wall against the effects of atherogenic concentrations of LDL. Asthma also increases inflammation and oxidative stress. In our previous study, we observed that apoA-Ⅰplays an important protective role in preventing pulmonary inflammation and airway hyperresponsiveness.
D-4F is an apoA-Ⅰmimetic made with all D-amino acids. D-4F is an amphipathic helix with a hydrophobic face that binds lipids in a manner similar to apolipoprotein A-Ⅰ. D-4F appears to protect HDL from becoming proinflammatory by binding oxidized lipids with greater affinity than native lipids. This ability to preferentially bind oxidized lipids and remodel HDL may be the responsible way for decreasing production of inflammatory cytokines in the mouse influenza model.
T-bet knock out mice, which develop airway hyperresponsiveness and are prone to spontaneous asthma, show lung inflammation and airway remodeling. T-bet deficient mice on Balb/c background have demonstrated dysfunctional HDL in lung inflammation and airway remodeling (unpublished abstractions). To examine the role of HDL in airway disease, we crossed T-bet-/- mice and apoA-Ⅰ-/- mice to create double knock out T-bet-/-/ apoA-Ⅰ-/- mice. To determine if D-4F could decrease airway disease we also treated T-bet-/-/apoA-Ⅰ-/- mice with D-4F. We found that T-bet-/-/apoA-Ⅰ-/- mice displayed higher airway hyperresponsiveness, pulmonary inflammation, collagen deposition in perivascular and peri alveolar region compared with T-bet-/-/apoA-Ⅰ+/+ control mice. Intriguingly, D4F reduced airway responsiveness, pulmonary inflammation and collagen deposition in the T-bet-/-/ apoA-Ⅰ-/- mice. Additionally, D4F attenuated oxidative stress in the T-bet-/-/apoA-Ⅰ-/-mice lung.
2 Methods
2.1 Mice
ApoA-Ⅰ-/- mice and T-bet-/- mice on a C57BL/6J background were purchased from Jackson Laboratory. The doubly heterozygous progeny were crossed to generate double knockout T-bet-/-/ apoA-Ⅰ-/- mice. Animal genotype was identified by a PCR-based assay. There were no discernible differences in litter size or appearance of the T-bet-/-/ apoA-Ⅰ-/- mice versus T-bet-/-/ apoA-Ⅰ+/+ control mice. Mice of 10 weeks old were injected with PBS and Apolipoprotein-mimetic peptide (D-4F, AC-DWFKAFYDKVAEKFKEAF-NH2) by daily intraperitoneal (IP) injection (3 mg/kg) for 6 weeks.
2.2 Plasma Total Cholesterol, HDL Cholesterol
Total cholesterol (TC) was quantified using a cholesterol oxidase/esterase kit from Wako Chemical, Inc. (Richmond, VA). HDL cholesterol was quantified using a HDL Cholesterol E kit from Wako Diagnostics. Proinflammatory HDL (p-HDL) was determined using a modified method of a previously published cell-free assay with CuCl2 and H2DCF(14).
2.3 Plasma PON1 Arylesterase Activity
Arylesterase activity of PON1 was performed on whole plasma using phenyl acetate as the substrate as described.(15)
2.4 Quantification of Plasma Nitrite+Nitrate
Nitrite+nitrate concentrations were determined by ozone chemiluminescence using the NO Analyzer (Model 280i, GE Analytical-Sievers, Boulder, CO) as described.(16,17) An aliquot of 30μL was injected (plasma was diluted by 1:30) into a sealed glass reaction chamber at 95℃containing VCl3.(17) Nitric oxide chemiluminescence signals were quantified and peak areas compared to the areas of external nitrate standards.
2.5 Measurements of Lung Mechanics
Each level of PEEP (0,3,6, and 9 cm H2O) was held for 80 sec with the Prime-2 perturbation repeated twice, once at 35 sec and the other at 70 sec. Measurements at each PEEP were Rn,G and H.
2.6 Methacholine challenge
Mice were challenged with 70 L of MCh (Sigma-Aldrich) of increasing concentrations (3.125 to 100 mg/mL) measurements(Rn,G and H) were assessed as a curve, whch pick value were picked up for calculation.
2.7 Histology
A 0.5 mL aliquot of zinc-formalin was used to inflate the lung prior to removal. The lung was fixed in zinc-formalin, embedded in paraffin, sectioned and then stained with hematoxylin and eosin (H&E) for histology or with McLetchies' trichrome to assess collagen deposition.
2.8 Immunofluorescence
4-HNE Michael's adducts and TGFβ-1 expression were detected by immunofluorescence for co-localization studies.Images captured using a krypton argon laser Nikon Eclipse TE2000U confocal microscope (Melville, NY) with 10x/0.17 aperture objective.
2.9 Western Blot Analysis
XO,MPO and eNOS protein levels were determined by western blot analysis. Autoradiograms were scanned with a laser densitometer or an UMax scanner.
3. Statistical Analysis
Data are presented as mean±SEM. Results were analyzed by Student's t-test, Mann-Whitney test or Fisher's exact test where appropriate. Airway data were analyzed by 2-way ANOVA to determine significance between curves and a Bonferroni post-test to determine significance of points between the curves. All statistical analysis was performed using GraphPad Prism Software (version 4.0).
4 Results
4.1 Cholesterol Profiles
The levels of total cholesterol and HDL cholesterol in plasma of T-bet-/-/ apoA-Ⅰ-/- mice treated with either PBS or D-4F were significantly reduced compared with T-bet-/-/7apoA-Ⅰ+/+ control mice (P < 0.001) . No difference in the total cholesterol or HDL cholesterol was observed between T-bet-/-/apoA-Ⅰ-/-mice treated with PBS and D4F.
4.2 Respiratory Mechanics
The effects of MCh on RN and G had no differences between groups. Lung tissue elastance was increased in the T-bet-/-/ apoA-Ⅰ-/- mice treated with PBS compared with T-bet-/-/apoA-Ⅰ-/- control mice (P < 0.05)
4.3 TGF-β1and 4- HNE in Lung Tissue Immunofluorescence
TGF-βand 4-HNE content in lungs is significantly reduced in T-bet-/-/ apoA-Ⅰ-/- mice treated with D-4F.
4.4 Lung histology
4.5 Perivascular and peribronchiolar inflammation and collagen deposition were increased slightly in T-bet-/-/apoA-Ⅰ+/+ control mice; and to a much greater extent in the T-bet-/-/apoA-Ⅰ-/- mice treated with PBS. However D-4F treatments markedly decreased inflammation and collagen deposition in T-bet-/-/ apoA-Ⅰ-/-mice.
4.6 XO Expression
The 145-kDa band corresponding to full length XD expression in the lung was significantly increased in T-bet-/-/apoA-Ⅰ-/- mice treated with D-4F compared with T-bet-/-/apoA-Ⅰ+/+ control mice and T-bet-/-/apoA-Ⅰ-/- mice treated with PBS(P<0.05). There was no difference between T-bef/7apoA-r/+ control mice and T-bet-/-/apoA-Ⅰ-/- mice treated with PBS.
4.7 Nitrate and Nitrite Level in BAL Fluid and Plasma
The nitrate and nitrite concentration in BAL fluids, which indicates the in vivo generation of NO in the airways, from the T-bet-/-/ apoA-Ⅰ-/- mice treated with PBS and D4F was significantly reduced compared with T-bet-/-/ apoA-Ⅰ+/+ control mice. Compared with T-bet-/-/ apoA-Ⅰ-/- mice treated with PBS, T-bet-/-/ apoA-Ⅰ-/- mice treated with D4F had increased nitrate and nitrite levels in the plasma and no significant difference had been achieved.
5 Conclusion
Taken together, these data suggest that genetic loss of apoA-Ⅰon the background of T-bet deficient mice increases pulmonary inflammation and collagen deposition as well as airway hyperresponsiveness. D4F reduced airway responsiveness, pulmonary inflammation and collagen deposition in the T-bet-/-/ apoA-Ⅰ-/- mice lung. D4F plays an important role in the mechanics of asthma.
引文
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