摘要
目的急性肺损伤（ALI）以失控性炎症反应、通透性肺水肿、肺泡内液体聚集及呼吸衰竭为病理生理特征。血管紧张素II（Ang II）是肾素-血管紧张素系统（RAS）的主要效应物，具有维持体内水盐平衡和细胞外液容量的生理作用。Ang II在ALI失控性炎症反应中扮演重要作用，但是在ALI肺水肿形成及肺泡内液体聚集中作用尚未阐明。肺泡上皮钠通道（ENaC）是肺泡内钠水重吸收的关键限速步骤，调控肺泡内液体容量平衡。来源于肾及结肠等多个组织器官的研究均提示Ang II能调控ENaC表达及功能活性，尚不清楚Ang II在肺组织中对ENaC的作用。故本研究通过气道内滴入脂多糖诱导ALI动物模型及微量泵泵入不同浓度Ang II两种方式，探讨内源性生成及外源性给予AngII对肺泡液体清除（AFC）及肺泡上皮钠通道（ENaC）表达的影响，并用A549细胞进一步证实Ang II对ENaC的表达调控作用。
方法内源性Ang II实验部分：SD大鼠用戊巴比妥腹腔注射麻醉后，行气管插管，通过气管导管向气管内注入LPS（1mg/kg）诱导ALI。空白对照组大鼠则给予等量的生理盐水。血管紧张素II1型（AT1）受体阻滞剂ZD7155（10mg/kg）在LPS滴入前30min于腹腔内注入。SD大鼠均杀死后抽取血液及取出完整肺组织，右肺用于AFC测量，左肺用于测量肺组织水含量及支气管肺泡灌洗液分析，并采用HE染色观察病理改变，用免疫组织化学分析ENaC在肺组织内的表达变化。用ELISA方法分析肺组织和血浆中内Ang II浓度。肺组织内的ENaC蛋白表达用蛋白免疫印迹法（Western blotting）测定。外源性Ang II实验部分：SD大鼠以戊巴比妥腹腔注射麻醉后，行颈静脉置管术，通过微量泵泵入不同浓度（1，10及100μg/kg·min）的外源性Ang II。空白对照组大鼠则泵入等量的生理盐水。血管紧张素II1型（AT1）受体阻滞剂ZD7155（10mg/kg）及cAMP降解抑制剂罗利普兰（1mg/kg）在Ang II泵入前30min于腹腔内注入。SD大鼠均杀死后抽取血液及取出完整肺组织，右肺用于AFC测量，左肺用于测量肺组织水含量及支气管肺泡灌洗液分析，并采用HE染色观察病理改变，用免疫组织化学分析ENaC在肺组织内的表达变化。用ELISA方法分析肺组织中TNF-α、IL-β浓度。采用RIA法测定肺组织内环磷酸腺苷（cAMP）水平。肺组织内的ENaC mRNA及蛋白表达分别用逆转录聚合酶链反应（RT-PCR）和蛋白免疫印迹法（Western blot）测定。并进一步给予A549细胞含有不同浓度（10-7M，10-5M）的Ang II培养基培养，用Western blotting分析不同浓度Ang II对A549细胞ENaC蛋白表达的作用。
结果内源性Ang II研究中：气道内滴入LPS诱导肺组织及血浆中Ang II水平显著升高，并伴随AFC降低。LPS的作用时间与Ang II升高和AFC的降低程度相关(血浆：ALI2h组2.53±0.4μg/l，ALI4h组3.78±1.15μg/l，ALI6h组4.91±1.1μg/l；肺组织：ALI2h组190.2±38.23μg/l，ALI4h组305±72.99μg/l，ALI6h组580.4±129.16μg/l)。预先给予ZD7155作用后，能显著抑制ALI诱导的AFC降低作用(7.8±1.61%vs4.4±1.14%)，同时逆转ALI诱导的对β及γ-ENaC蛋白表达的下调作用，但却进一步下调α-ENaC蛋白表达(P<0.05)。ZD7155还显著改善ALI诱导的间质水肿及炎症细胞浸润的病理改变。外源性AngII研究中：给予外源性高浓度（10及100μg/kg·min）Ang II显著抑制AFC并呈剂量依赖效应(P<0.05)。Ang II泵入导致对α-ENaC和β及γ-ENaC表达的非协同调节作用，上调α-ENaC和下调β及γ-ENaC表达。给予ZD7155干预后，能显著拮抗外源性Ang II对AFC(10.8±1.483%vs6.2±2.683%)及ENaC表达的调控作用(P<0.05)。在A549细胞研究中，也显示Ang II对ENaC表达的非协同调节作用，并呈浓度依赖的方式调控ENaC表达(P<0.05)。外源性Ang II显著抑制肺组织内cAMP水平，给予罗利普兰后则缓解Ang II对AFC的抑制作用(9.8±3.033%vs6.2±2.683%)。
结论内外源性Ang II都能够通过激活AT1受体抑制肺泡液体清除，导致肺水含量增加，诱导肺水肿形成。
Ang II对ENaC三个亚基的表达呈非协同的调节作用，可能导致了ENaC中HSC与NSC通道比例的失调，从而影响了肺泡内液体的清除。
AT1受体阻滞剂及cAMP降解抑制均能够对Ang II诱导的肺泡液体清除降低具有保护作用。
Objective Acute lung injury (ALI) is characterized by uncontrolledinflammatory reponse, increased permeablility pulmonary edema, alveolarfilling and respiratory failure. Angiotensin II (Ang II) is the main effector ofrennin-angiotensin system (RAS), and exerts its effect in maintainingsodium balance and extracellular fluid volume. Ang II has beendemonstrated a pro-inflammatory effect in acute lung injury, however,studies of the effect of Ang II on the formation of pulmonary edema andalveolar filling remains unclear. Epithelial sodium channel (ENaC) is therate-limiting step for sodium absorption in alveoli, which in turn regulatesthe alveolar fluid volume. There is mounting evidence form renal andcolonic studies demonstrating that Ang II regulates ENaC expression andits electrogenous activity. However, the effect of Ang II on ENaCexpression in lung is still unknown. Therefore, in this study was verified theregulation of alveolar fluid clearance (AFC) and the expression of epithelialsodium channel (ENaC) by endogenous and exogenous Ang II. The effect ofAng II on ENaC expression was still verified in A549cells.
Methods In study on endogenous Ang II effects, SD rats wereanesthetized and intratracheally injected with1mg/kg lipopolysaccharide (LPS), whereas control rats received only saline vehicle. Angiotensin type1(AT1) receptor antagonist ZD7155(10mg/kg) was injected intraperitoneally30min before administration of LPS. Then lungs were isolated en bloc. Theleft lungs were separated to measure lung water volume and bronchoalveolarlavage fluid (BALF). The right lungs were prepared to assess alveolar fluidclearance. The lungs were processed histological analysis andimmunological studies with HE stain and immunocytochemistry. Ang IIlevels in plasma and lung tissue were determined by ELISA. The proteinexpression of ENaC was detected by Western blot. In study on exogenousAng II effects, SD rats were anesthetized and were given Ang II withincreasing doses (1,10and100μg/kg-1·min-1) via osmotic minipumps,whereas control rats received only saline vehicle. AT1receptor antagonistZD7155(10mg/kg) and inhibitor of cAMP degeneration rolipram (1mg/kg)were injected intraperitoneally30min before administration of Ang II. Thenlungs were isolated en bloc. The left lungs were separated to measure lungwater volume and bronchoalveolar lavage fluid (BALF). The right lungswere prepared to assess alveolar fluid clearance. The lungs were processedhistological analysis and immunological studies with HE stain andimmunocytochemistry. TNF-α, IL-1β and cAMP levels in lung tissue weredetermined by ELISA and RIA, respectively. The mRNA and proteinexpression of ENaC were detected by RT-PCR and Western blot. A549cellswere treated with different doses (10-7M and10-5M) of Ang II and the change in protein level caused by Ang II was determined by Western blot.
Results In study on endogenous Ang II effects, ALI induced by LPScaused an increase in Ang II levels in plasma and lung tissue, but adecrease in alveolar fluid clearance. The time-course of the Ang II levelsparalleled that of the alveolar fluid clearance (In plasma: ALI2h2.53±0.4μg/l, ALI4h3.78±1.15μg/l, ALI6h4.91±1.1μg/l; In lung: ALI2h190.2±38.23μg/l, ALI4h305±72.99μg/l, ALI6h580.4±129.16μg/l).Pretreatment with ZD7155prevented ALI-induced reduction of alveolarfluid clearance (7.8±1.61%vs4.4±1.14%). ZD7155also reversed theALI-induced reduction of the abundance of β and γ-ENaC, whereas furtherdecreased the abundance of α-ENaC (P<0.05), and ameliorated interstitialedema and inflammatory cell infiltration induced by ALI. In study onexogenous Ang II effects, Exposure to higher doses of Ang II reduced AFCin a dose-dependent manner and resulted in a non-coordinate regulation ofα-ENaC versus the regulation of β-and γ-ENaC, however Ang II type1(AT1) receptor antagonist ZD7155prevented the Ang II-induced inhibitionof fluid clearance (10.8±1.483%vs6.2±2.683%) and dysregulation ofENaC expression (P<0.05). The similar non-coordinate regulation ofα-ENaC versusβ-and γ-ENaC by Ang II was still observed in A549cells,and Ang II dysregulated ENaC expression in a concentration-dependentmanner (P<0.05). In addition, exposure to inhibitor of cAMP degradationrolipram blunted the Ang II-induced inhibition of fluid clearance (9.8± 3.033%vs6.2±2.683%).
Conclusions Endogenous and exogenous Ang II promotes pulmonaryedema and alveolar filling by inhibition of alveolar fluid clearance throughactivation of AT1receptor. Ang II regulates α-ENaC expresson versus β-and γ-ENaC expression in a non-coordinate manner, which may lead tochanges in proportion of HSC channels and NSC channels, and results inreduction of AFC. AT1receptor antagonist and inhibitor of cAMPdegeneration can protect from Ang II-induced reduction of AFC.
引文
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