特布他林对急性肺损伤大鼠肺泡液体清除功能影响的实验研究

目的 :测定不同 p H值条件下酸性溶液对肺泡上皮屏障的损伤 ;观察特布他林对急性肺损伤大鼠肺泡液体清除功能的影响。方法 :向大鼠肺内滴注等渗酸溶液 ,机械通气 1小时后测定通过肺泡上皮屏障的双向蛋白流和肺泡液体清除率。结果 :随滴注液 p H值的降低 ,通过肺泡上皮屏障的双向蛋白流明显增加。当滴注液的 p H值由 7.0降到 1.5时 ,渗入血浆的肺泡蛋白示踪剂由 (5± 4) %增至 (33± 2 ) % ,进入肺泡腔内的血浆蛋白示踪剂由 (5± 1) %逐渐增至 (2 5± 2 ) %。当滴注液 p H≥ 3.5时 ,特布他林组 (1× 10 - 4 m ol/ L)的肺泡液体清除率较对照组高 2 1%～ 36 % (P<0 .0 5 ) ,但其作用随滴注液 p H值降低而减小 ;当 p H值≤ 2 .5时 ,特布他林对大鼠的肺泡液体清除功能无明显作用。结论 :酸溶液可损伤肺泡上皮 ,增加肺泡上皮屏障对蛋白质的通透性 ,降低肺泡上皮对液体的清除 ;特布他林通过激活肺泡上皮钠通道 ,增加肺泡液体的清除 ,进而改善急性肺损伤的病理生理变化 ,但其作用随气道 p H值降低而逐渐减小     

GABA receptor ameliorates ventilator-induced lung injury in rats by improving alveolar fluid clearance

ABSTRACT: INTRODUCTION: Mechanical ventilators are increasingly used in critical care units. However, they can cause lung injury, including pulmonary edema. Our previous studies indicated that γ-aminobutyric acid (GABA) receptors are involved in alveolar-fluid homeostasis. The present study investigated the role of GABA receptors in ventilator-induced lung injury. METHODS: Adult female Sprague-Dawley rats were subjected to high-tidal-volume ventilation of 40 ml/kg body weight for 1 hour, and lung injuries were assessed. RESULTS: High-tidal-volume ventilation resulted in lung injury, as indicated by an increase in total protein in bronchoalveolar fluid, wet-to-dry ratio (indication of pulmonary edema), and Evans Blue dye extravasation (indication of vascular damage). Intratracheal administration of GABA before ventilation significantly reduced the wet-to-dry ratio. Further, histopathologic analysis indicated that GABA reduced ventilator-induced lung injury and apoptosis. GABA-mediated reduction was effectively blocked by the GABAA-receptor antagonist, bicuculline. The GABA-mediated effect was not due to the vascular damage, because no differences in Evans Blue dye extravasation were noted. However, the decrease in alveolar fluid clearance by high-tidal-volume ventilation was partly prevented by GABA, which was blocked by bicuculline. CONCLUSIONS: These results suggest that GABA reduces pulmonary edema induced by high-tidal-volume ventilation via its effects on alveolar fluid clearance and apoptosis.     

特普他林对油酸性肺损伤鼠肺泡液体清除功能的影响

目的：观察特普他林对油酸致伤大鼠肺损伤后肺泡上皮液体清除能力的影响。方法：采用大鼠油酸型肺损伤模型。大鼠伤后２４小时经气管滴注特普他林（１×１０－ ４ ｍ ｏｌ／Ｌ）溶液５ ｍ ｌ／ｋｇ;１ 小时后检测肺泡内液体清除率（ＡＬＣ）、总肺水量（ＴＬＷ）、肺血管外肺水量（ＥＶＬＷ）和动脉血气参数。结果：大鼠伤后２４ 小时ＡＬＣ降低４９．２％ ,ＴＬＷ 和ＥＶＬＷ 明显增加〔分别由（３．１４±０．１４）ｍ ｌ／ｇ 和（２．２５±０．１１）ｍ ｌ／ｇ 增至（４．０４±０．１３）ｍ ｌ／ｇ和（４．３２±０．１１）ｍ ｌ／ｇ〕,严重的低氧血症〔ＰａＯ２ 为（６．５５±０．２３）ｋＰａ（１ ｋＰａ＝ ７．５ ｍ ｍ Ｈｇ）〕。特普他林组ＡＬＣ较油酸致伤组增高６３．７％ ,ＴＬＷ 和ＥＶＬＷ 显著减少〔分别为（３．３９±０．１６）ｍ ｌ／ｇ 和（２．９４±０．１２）ｍ ｌ／ｇ〕。钠转运特异性抑制剂 阿咪洛利或哇巴因能部分抑制特普他林刺激肺泡内液体清除的作用。结论：特普他林通过上调钠主动转运机能,促进肺水肿液的吸收,从而改善换气功能,对急性肺损伤后肺水肿有一定的治疗作用     

肺泡内液体清除与肺水肿的研究进展

肺水肿是急性肺损伤(ALI)病理进程中的中心环节,与肺泡毛细血管通透性增加和肺泡内液体清除减少有关。以往对肺泡毛细血管通透性导致的肺水肿研究较多,而对肺泡内液体清除在肺水肿形成中的作用关注不足。肺泡内液体清除受水通道蛋白、钠离子通道、钠钾ATP酶的影响。认识肺泡内液体清除机制有望能为临床医师治疗ALI提供参考。     

Cerebrospinal fluid sodium increases in migraine

BACKGROUND: Pharmaceuticals with calcium- or sodium-channel-blocking activity have proven useful for migraine prophylaxis, and calcium channel, sodium transporter, and sodium channel gene mutations have been found in familial hemiplegic migraine. However, it is not known whether calcium or sodium homeostasis is altered in migraine. OBJECTIVE: To compare levels of sodium, calcium, potassium, and magnesium in cerebrospinal fluid (CSF) and blood plasma between migraineurs and controls. METHODS: We recruited 20 migraineurs without aura and 11 controls prospectively, and studied migraineurs in sick (MH(+)) and well (MH(-)) states. We collected lumbar CSF and venous blood plasma, quantified elements with ion-selective electrodes or colorimetry, and determined osmolality by depression of freezing point. We compared levels of Na(+), Ca(2+), K(+), and Mg among and also within subjects who were studied in both MH(+) and MH(-) states. RESULTS: Mean CSF Na(+) levels were increased by 3 mmol/L in MH(+) compared with MH(-) and by 4 mmol/L compared to controls (P < 0.005). In 4 subjects who were sampled in both MH(+) and MH(-) states, mean CSF Na(+) concentration increased by 2 mmol/L in the MH(+) state compared with the MH(-) state (P < 0.05). Simultaneous plasma Na(+) levels did not differ among the 3 clinical groups, nor did osmolality, total Ca and Ca(2+), K(+), and total Mg levels in CSF. CONCLUSIONS: Compared to both controls and the MH(-) state, CSF Na(+) concentration increased in MH(+) independently from other clinical or pharmacological fluctuations, CSF concentrations of Ca(2+), Mg, and K(+), and blood plasma Na(+) levels. These results implicate a deviation of Na(+) homeostasis in migraine. The modestly elevated extracellular Na(+) in MH(+) may cause the neural changes that underlie clinical features of migraine.     

Bench-to-bedside review: The role of the alveolar epithelium in the resolution of pulmonary edema in acute lung injury

Clearance of pulmonary edema fluid is accomplished by active ion transport, predominantly by the alveolar epithelium. Various ion pumps and channels on the surface of the alveolar epithelial cell generate an osmotic gradient across the epithelium, which in turn drives the movement of water out of the airspaces. Here, the mechanisms of alveolar ion and fluid clearance are reviewed. In addition, many factors that regulate the rate of edema clearance, such as catecholamines, steroids, cytokines, and growth factors, are discussed. Finally, we address the changes to the alveolar epithelium and its transport processes during acute lung injury (ALI). Since relevant clinical outcomes correlate with rates of edema clearance in ALI, therapies based on our understanding of the mechanisms and regulation of fluid transport may be developed.     

Aldosterone alleviates lipopolysaccharide-induced acute lung injury by regulating epithelial sodium channel through PI3K/Akt/SGK1 signaling pathway

Reduced alveolar fluid clearance (AFC) is a major pathological feature of acute lung injury (ALI). Epithelial sodium channel (ENaC) plays a key role in regulating the transport of Na⁺ and clearing alveolar edema fluid effectively. ENaC has been reported to be regulated by aldosterone in the distal collecting tube of the kidney. We hypothesized whether aldosterone regulated ENaC in alveolar epithelium and correspondingly played a role in ALI. In this study we found that the expression of aldosterone synthesis encoding gene, CYP11B2, and ENaC were decreased in the lung tissue of LPS-induced ALI mice. Furthermore, aldosterone alleviated ALI by increasing the expression of ENaC-α and relieving pulmonary edema. Besides, we found that aldosterone upregulated ENaC-α through PI3K/Akt/SGK1 pathway. In conclusion, our study demonstrated that aldosterone attenuated pulmonary edema by upregulating ENaC-α through the PI3K/Akt/SGK1 pathway in LPS-induced ALI, indicating that aldosterone might be a promising adjuvant drug for ALI treatment.     

丹参酮ⅡA磺酸钠对海水浸泡人肺上皮A549细胞水通道蛋白5的影响

目的 探讨水通道蛋白5(AQP5)在海水浸泡致细胞损伤中的变化,同时了解丹参酮Ⅱ A可能的作用机制.方法 体外传代培养肺腺癌细胞株A549细胞,接种于培养皿中,按不同海水含量分为空白对照组及15%、25%、50%、75%、100%海水组;以25%海水浸泡不同时间分为空白对照组及海水1、4、8 h组;按给予不同剂量丹参酮ⅡA干预分为空白对照组、25%海水组及25、50、75、100μg/ml丹参酮ⅡA干预4 h组.用蛋白质免疫印迹法(Western blotting)检测AQP5蛋白表达;用免疫组化法检测AQP5阳性表达.结果 Western blotting结果显示,25%与50%海水组8 h时A549细胞AQP5蛋白表达均较空白对照组明显增高(1.053±0.231、1.116±0.316比0.101±0.081,均P＜0.05);海水1 h组AQP5表达较空白对照组稍有增加(0.306±0.125比0.288±0.098,P＞0.05),4 h组(1.423±0.377)明显增加(P＜0.01),8 h组AQP5表达(1.507±0.461)较4 h组略有增加,但差异无统计学意义;25μg/ml与50 μg/ml丹参酮Ⅱ A组4 h时AQP5蛋白表达较25%海水组明显减少(0.580±0.186、0.499±0.172比1.013±0.287,均P＜0.05).免疫组化显示,25%海水4 h组AQP5阳性表达较空白对照组明显增多(7.21±0.78比0.41±0.07,P＜0.01),染色变深;25μg/ml丹参酮ⅡA干预4 h组AQP5阳性表达(3.02±0.23)较25%海水4 h组明显减少(P＜0.05).结论 丹参酮Ⅱ A在25μg/ml浓度时毒副作用最小,对海水浸泡A549细胞的保护作用最佳,其机制可能与抑制AQP5的过度表达有关.

Abstract：

Objective To explore the effects of tanshinone Ⅱ A on the activity of aquaporin-5 (AQP5)in human alveolar epithelial cells (A549) after seawater exposure and its possible mechanism. Methods Routinely cultured A549 cells were divided into different groups according to different content of seawater:blank control group, 15%, 25%, 50%, 75%, 100% seawater groups; they were divided into different groups according to the duration of exposure to 25 % seawater : blank control group, 1, 4, 8 hours groups ;they were also divided into different groups according to concentration of tanshinone ⅡA and exposed to seawater for 4 hours: blank control group, 25% seawater group, 25, 50, 75, 100 μg/ml tanshinone ⅡA intervention groups. The expressions of AQP5 were respectively assayed by Western blotting and immunohistochemistry. Results The results of Western blotting showed that the expressions of AQP5 were remarkably higher at 8 hours of exposure to seawater in 25% and 50% seawater groups than those in blank control group (1. 053±0. 231, 1. 116±0. 316 vs. 0. 101±0. 081, both P＜0. 05); the expression of AQP5 in 1-hour group showed a slight increase compared with blank control group (0. 306±0. 125 vs. 0. 288±0. 098,P＞0. 05), that in 4-hour group was increased significantly (1. 423±0. 377, P＜0. 01), and in 8-hour group (1. 507± 0. 461 ) it was slightly higher than that in 4-hour group without statistical significance. The AQP5 expression was significantly lower in tanshinone ⅡA 25 μg/ml and 50μg/ml intervention groups than that in 25% seawater group (0. 580 ± 0. 186, 0. 499 ± 0. 172 vs. 1.013 ± 0. 287, both P ＜ 0. 05). Immunohistochemistry showed that the expression of AQP5 was markedly up-regulated after A549 cells were stimulated with 25% seawater for 4 hours as compared with blank control group (7.21±0. 78 vs. 0. 41 ±0.07, P＜0.01), but intervention of tanshinone ⅡA significantly inhibited the up-regulation of AQP5 expression (3.02±0.23) induced by 25% seawater (P＜0.05). Conclusion The experimental results showed that tanshinone ⅡA is innocuous to A549 at a dosage of 25 μg/ml, and it can decrease the overexpression of AQP5 induced by seawater.     

细胞因子和水钠通道蛋白在急性肾损伤致肺损伤中的作用

目的 通过双肾动静脉夹闭建立急性缺血性肾损伤大鼠模型,观察大鼠肺病理生理的变化,观察上皮细胞钠通道蛋白(α-ENaC)和水通道蛋白1(AQP1)在急性肾损伤所致肺损伤中的作用.方法 健康雄性Wistar大鼠60只.体质量300～ 320 g,随机(随机数字法)分成健康对照组(A组),急性肾损伤组(B组),每组30只.造模后,处死大鼠,苏木素伊红(HE)染色检查肺组织病理变化,计算肺W/D比值,支气管肺泡灌洗液(BALF)中蛋白质量浓度.检测肺组织中水通道蛋白1、肺上皮钠通道蛋白的质量浓度.测定血清及BALF中IL-6与TNF-α的质量浓度.结果 B组大鼠在实验后6h动脉血pH值开始下降,酸中毒逐渐加重,与A组比较差异具有统计学意义(P＜0.05).B组与A组的氧分压各时间点之间相比差异无统计学意义(P＞0.05).与A组相比,B组在实验后2h肺泡灌洗液中蛋白水平、肺W/D值开始明显增加,差异具有统计学意义(P＜0.05).B组实验后8h肺泡上皮肿胀,肺泡壁增宽,肺泡间质水肿明显,肺泡内可见炎症细胞、红细胞和蛋白渗出,表现出急性肺损伤的病理改变.B组在实验后2h血清及肺泡灌洗液中TNF-、IL-6的质量浓度开始增加,肺组织中AQP1、α-ENaC表达开始逐渐减少,与A组比较,差异具有统计学意义(P＜0.05).结论 急性肾损伤早期肺泡上皮-内皮屏障功能已经受到了影响,急性肺损伤已经发生.急性肾损伤后早期体内TNF-α、IL-6含量明显增加,肺表达AQP1及α-ENaC的减少,可能是急性肾损伤早期引起肺损伤的原因之一.     

重组人超氧化物歧化酶加强一氧化氮吸入治疗幼鼠急性胎粪肺损伤的实验研究

目的　探讨联合应用重组人超氧化物歧化酶 (recombinanthumansuperoxidedismatase ,rhSOD)和一氧化氮吸入 (inhalednitricoxide,iNO)对胎粪诱导幼鼠急性肺损伤的保护作用及可能机制。方法　 32只雄性SD幼年大鼠 ,通过气管置管注入 2 0 %胎粪 1ml/kg建立幼鼠胎粪性肺损伤模型 ,随机分为 :( 1)对照组 (Control,C) :暴露于空气中 ;( 2 )NO吸入组 (iNO) :暴露于 2 0× 10 - 6 NO中 ;( 3)rhSOD组 (SOD) ) :rhSOD 2 0mg/kg·ml气管内注入并暴露于空气中 ;( 4 )联合应用 2 0× 10 - 6 NO和 2 0mg/kgrhSOD组 (iNO/SOD) ,暴露 2 4h后观察支气管肺泡灌洗液 (bronchoalveolarlavagefluid ,BALF)细胞数、肺组织匀浆髓过氧化物酶 (myeloperoxidase ,MPO)活性、丙二醛 (malonyldialdehyde,MDA)、一氧化氮 (nitricoxide,NO)含量和肺损伤病理改变。结果　与对照组比较 ,iNO组、SOD组BALF细胞数、MPO活性、肺损伤病理评分下降(P <0 0 5或 0 0 1)、iNO/SOD组较iNO组BALF细胞数、MPO活性进一步降低 (均P <0 0 5 ) ;但肺损伤病理评分差异无显著牲 ;与对照组比较 ,iNO组肺组织MDA、NO含量略有下降 ,但差异无意义 (均P >0 0 5 ) ,SOD组、iNO/SOD组肺组织MDA、NO含量显著下降 (P <0 0 5或 0 0 1) ,且iNO/SOD组显著低于iNO     

Tumor necrosis factor-α small interfering RNA alveolar epithelial cell-targeting nanoparticles reduce lung injury in C57BL/6J mice with sepsis

BackgroundThe role of tumor necrosis factor (TNF)-α small interfering (si)RNA alveolar epithelial cell (AEC)-targeting nanoparticles in lung injury is unclear.MethodsSixty C57BL/6J mice with sepsis were divided into normal, control, sham, 25 mg/kg, 50 mg/kg, and 100 mg/kg siRNA AEC-targeting nanoparticles groups (n = 10 per group). The wet:dry lung weight ratio, and hematoxylin and eosin staining, western blotting, and enzyme-linked immunosorbent assays for inflammatory factors were conducted to compare differences among groups.ResultsThe wet:dry ratio was significantly lower in control and sham groups than other groups. TNF-α siRNA AEC-targeting nanoparticles significantly reduced the number of eosinophils, with significantly lower numbers in the 50 mg/kg group than in 25 mg/kg and 100 mg/kg groups. The nanoparticles also significantly reduced the expression of TNF-α, B-cell lymphoma-2, caspase 3, interleukin (IL)-1β, and IL-6, with TNF-α expression being significantly lower in the 50 mg/kg group than in 25 mg/kg and 100 mg/kg groups.ConclusionTNF-α siRNA AEC-targeting nanoparticles appear to be effective at improving lung injury-related sepsis, and 50 mg/kg may be a preferred dose option for administration.     

裸鼠模型的人钠/碘转运体基因转染人大细胞肺癌介导放射性核素显像

目的在动物体内实验观察人钠/碘转运体(hNIS)基因转染人大细胞肺癌介导放射性核素显像是否可行。方法①利用重组质粒以脂质体转染法将hNIS基因转染入人大细胞肺癌H460细胞系中,获得稳定表达hNIS的细胞株(hNIS-H460)。②用hNIS-H460细胞株建立大细胞肺癌荷瘤裸鼠模型,进行放射性核素99mTcO4-显像和131I显像。结果①体外实验表明hNIS-H460细胞株可以摄取碘。②裸鼠大细胞肺癌(hNIS-H460)移植瘤的99mTcO4-和131I显像结果较清晰。结论转染后的hNIS-H460细胞具有一定的摄碘能力。裸鼠大细胞肺癌(hNIS-H460)移植瘤可以进行99mTcO4-和131I显像;99mTcO4-显像的质量优于131I显像。     

人肺腺癌细胞总RNA电穿孔法转染的树突状细胞疫苗的体外抗肿瘤效应研究

目的探讨人肺腺癌细胞A549总RNA电穿孔法转染的树突状细胞(dendritic cell,DC)的特征及其特异性抗肿瘤免疫反应的能力。方法用重组人粒细胞巨噬细胞集落刺激因子(rhGM-CSF)和重组人白细胞介素4(rhIL-4)从血细胞分离机分离出的外周血采集物中诱导DCs产生,同时从人肺腺癌细胞A549中提取总RNA用以转染DCs,并用转染后的DCs与自体T细胞共同培养,诱导成为细胞毒性T淋巴细胞(cytotoxic T lymphocytes,CTL)。实验分未转染DCs组,转染PBS的DCs组,转染RNA的DCs组,以流式细胞术(FCM)鉴定DCs、T细胞表型、RT-PCR证明总RNA转染效率,MTT检测T细胞增殖能力及乳酸脱氢酶(LDH)释放法测定杀伤肿瘤活性,酶联免疫吸附法(ELISA)检测IL-12、IFN-γ分泌水平并比较各组差异。结果 1)RT-PCR证明电穿孔法可成功使A549的总RNA转染入DCs并可使DCs表达肿瘤抗原。2)RNA转染组的DCs表面标志物CD40、CD80、CCR-7、CD83、HLA-DR都呈强阳性表达,表达率明显高于PBS转染组DCs和未转染组,差异有统计学意义(P<0.05);3)流式结果显示RNA转染的DCs刺激后的T细胞表面标志物CD8比例明显上升;4)RNA转染组IL-12、IFN-γ分泌水平明显高于PBS转染组和未转染组(P<0.05);5)RNA转染组DCs可以有效刺激T淋巴细胞增殖,并且诱导肿瘤特异性CTLs产生,对A549细胞产生特异性杀伤作用。而PBS转染组与未转染组则无明显作用(P<0.05)。结论利用电转染法可成功将A549的总RNA转入DCs内并使其表达肿瘤抗原,在体外能诱导肿瘤特异性免疫反应。     

肺保护性通气对肺内外源性急性呼吸窘迫综合征外周血和肺泡灌洗液中炎性介质影响的比较

目的　观察在肺保护性通气条件下急性呼吸窘迫综合征 (ARDS)模型犬氧合指数以及外周血和肺不同部位 (肺上区、肺下区腹侧和肺下区背侧 )支气管肺泡灌洗液 (BAL F)中炎性介质的变化。方法　健康雄性杂种犬 2 4只 ,随机分为肺内源性 ARDS(ARDSp)实验组、ARDSp 对照组、肺外源性 ARDS(ARDSexp)实验组和 ARDSexp对照组 ,每组 6只。采用静脉注射油酸形成 ARDSexp模型 ,应用十六烷磺基丁二酸钠盐气管内吸入形成 ARDSp模型。实验组肺损伤后进行肺保护性通气〔潮气量 8ml/ kg,呼气末正压(PEEP) 10 cm H2 O(1cm H2 O=0 .0 98k Pa)〕;对照组则继续进行大潮气量通气。动态观察肺保护性通气条件下 ARDS模型犬外周血和肺不同部位 (如肺尖叶、肺心叶和肺膈叶 ) BAL F中的炎性介质 ,如肿瘤坏死因子α(TNFα)、白细胞介素 (IL 1β,IL 6 )的变化。结果　肺损伤后 ARDS模型犬氧合指数均显著恶化 ,外周血中炎性介质明显升高 (P均 <0 .0 5 ) ,ARDSp模型犬肺尖叶和心叶 BAL F中炎性介质水平明显高于 ARDSexp模型犬 (P均 <0 .0 5 )。应用肺保护性通气治疗后 ,实验组犬氧合指数有不同程度改善 ,炎性介质水平有不同程度下降 ;但 ARDSp实验组的治疗效果不如 ARDSexp实验组。结论　 ARDSexp和 ARDSp的肺不同部位炎性介质释放和氧合