孫才智 秦海東 沈華 宋楊 張錚
DOI:10.3760/cma.j.issn.1671-0282.2014.12.009
基金項目:南京市醫(yī)學(xué)科技發(fā)展項目(YKK12074)
作者單位:210006 ?南京,南京醫(yī)科大學(xué)附屬南京醫(yī)院(南京市第一醫(yī)院) 急診科
通信作者:張錚,Email: Zz18351891108@126.com
【摘要】目的探討不同劑量多巴酚丁胺對膿毒癥休克兔急性肺損傷(acute lung injury ,ALI)的保護作用及其可能機制。方法采用盲腸結(jié)扎穿孔聯(lián)合靜脈注射內(nèi)毒素制備膿毒癥休克模型,將70只新西蘭雄性大白兔隨機(隨機數(shù)字法)均分為假手術(shù)組(A組)、ALI模型組(B組)、多巴酚丁胺低劑量組(C組)、多巴酚丁胺中劑量組(D組)和多巴酚丁胺高劑量組(E組),分別于造模后3 h和6 h點處死,留取肺組織標(biāo)本。ELISA法檢測環(huán)磷酸腺苷(cyclic adenosine monophosphate, cAMP)濃度;Western-blot法檢測水通道蛋白5(Aquaporin 5, AQP5)蛋白濃度;計算肺濕/干重(wet to dry weight, W/D)比值;光鏡和電鏡下觀察各組小鼠肺組織病理學(xué)改變,并對肺組織病理損傷進(jìn)行評分。多組間差異比較采用單因素方差分析,組間兩兩比較采用 LSD法。結(jié)果與A組相比,B組cAMP、AQP5蛋白濃度在3 h及6 h明顯降低(3.53±0.43) pmol/mL vs. (21.18±0.62)pmol/mL;(0.44±0.04)pmol/mLvs.(0.99±0.06)pmol/mL; (2.71±0.56)pmol/mL vs.(21.78±0.62)pmol/mL;(0.29±0.05)pmol/mLvs.(0.91±0.06)pmol/mL;P<0.001,W/D比值則明顯升高P<0.00。與B組相比, C組cAMP及AQP5蛋白水平在6 h明顯增加(8.48±0.61)pmol/mLvs.(2.71±0.56)pmol/mL, P<0.001;(0.49±0.04)pmol/mLvs.(0.29±0.05)pmol/mL,(P=0.001),D、E組cAMP及AQP5蛋白水平在3 h、6 h明顯增加(10.86±0.66)pmol/mL vs.(3.53±0.43)pmol/mL;(0.60±0.05)pmol/mLvs.(0.44±0.04)pmol/mL;(13.80±0.49)pmol/mLvs.(2.71±0.56)pmol/mL;(0.64±0.03)pmol/mLvs.(0.29±0.05)pmol/mL;(15.57±0.60)pmol/mLvs.(3.53±0.43)pmol/mL;(0.91±0.05)pmol/mLvs.(0.44±0.04)pmol/mL;(19.30±0.42)pmol/mLvs.(2.71±0.56)pmol/mL;(0.89±0.08)pmol/mLvs.(0.29±0.05)pmol/mL;P<0.01,E組W/D比值明顯降低(P=0.002; P=0.001)。與C 、D組相比, E組cAMP及AQP5水平在3 h、6 h均有所增加(P<0.01)。光鏡及電鏡下,B組肺的病理形態(tài)及超微結(jié)構(gòu)損傷均較重, 肺病理學(xué)評分明顯升高(P<0.01);多巴酚丁胺干預(yù)后,肺的病理形態(tài)及超微結(jié)構(gòu)損傷有所減輕,肺組織病理學(xué)評分明顯降低(P<0.01)。結(jié)論多巴酚丁胺對內(nèi)毒素誘導(dǎo)的急性肺損傷有一定程度保護作用,其機制可能與提高肺組織cAMP水平,上調(diào)AQP5蛋白表達(dá)量有關(guān),且大劑量多巴酚丁胺效果更佳。
【關(guān)鍵詞】急性肺損傷;膿毒癥休克;多巴酚丁胺;水通道蛋白5;環(huán)磷酸腺苷
Effects of dobutamine on acute lung injury in rabbits of septic shock
Sun Caizhi , Qin Haidong, Shen Hua, Song Yang, Zhang Zheng. Department of Emergency, Nanjing Hospital Affiliated to Nanjing Medical University/the First Hospital of Nanjing, Nanjing210006, China
Corresponding Author: Zhang Zheng, Email: Zz18351891108@126.com
【Abstract】ObjectiveTo explore the effect of different doses of dobutamine on acute lung injury (ALI) in rabbits with septic shock and to clarify the possible mechanism. Methods ?The rabbits model of septic shock was made by cecal ligation and puncture combined with intravenous injection of endotoxin, 70 male New Zealand white rabbits were randomly divided into five groups(14 rabbits in each groups): sham operation group (group A),ALI group (group B), dobutamine low-dose group (group C), dobutamine medium-dose group(group D) and dobutamine high-dose group(group E), 7 rabbits from each group were sacrificed 3 h and 6 h after septic shock. The level of cyclic adenosine monophosphate (cAMP) in lung tissue was detected by ELISA .The expression of aquaporin 5 (AQP5) protein was determined by western blotting. The wet to dry weight (W/D) ratio was measured. The pathological and ultrastructural changes of lung tissue were evaluated by optical microscopy and electron microscope, and lung injury score was assessed. The differences among the different groups were analyzed by one-way ANOVA (LSD test). Results ?The level of cAMP and expression of AQP5 protein in lung tissue at 3 h and 6 h were dramatically lower in group B than those in group A (3.53±0.43) pmol/mLvs. (21.18±0.62)pmol/mL;(0.44±0.04) pmol/mLvs.(0.99±0.06)pmol/mL;(2.71±0.56) pmol/mLvs.(21.78±0.62)pmol/mL;(0.29±0.05)pmol/mLvs.(0.91±0.06)pmol/mL; all P<0.001, while the W/D ratio was obviously higher in group B than those in group A (all P<0.001). Compared with group B, the level of cAMP and AQP5 protein expression in lung tissue were significantly increased at 6 h in group C (8.48±0.61)pmol/mLvs.(2.71±0.56)pmol/mL, P<0.01; (0.49±0.04)pmol/mLvs.(0.29±0.05)pmol/mL,P=0.001 and at 3 h and 6 h in group D and E (10.86±0.66)pmol/mLvs.(3.53±0.43)pmol/mL;(0.60±0.05)pmol/mLvs.(0.44±0.04)pmol/mL;(13.80±0.49)pmol/mL vs.(2.71±0.56)pmol/mL;(0.64±0.03)pmol/mLvs.(0.29±0.05)pmol/mL; (15.57±0.60)pmol/mL vs.(3.53±0.43)pmol/mL;(0.91±0.05)pmol/mLvs.(0.44±0.04)pmol/mL; (19.30±0.42)pmol/mL vs.(2.71±0.56)pmol/mL; (0.89±0.08)pmol/mL vs.(0.29±0.05)pmol/mL; all P<0.01, while the W/D ratio in group E was decreased obviously(P=0.002; P=0.001). Compared with group C and D, the level of cAMP and the expression of AQP5 protein at 3 h and 6 h in group E increased significantly(all P<0.01. The pathological and ultrastructural changes of lung tissue were more intensive in group B than those in group A and the lung injury scores were obviously higher (P<0.01). The degree of lung pathological and ultrastructural lesion was ameliorated after administration of dobutanmine. Additionally, histological scores decreased significantly(P<0.01). ConclusionsOur study demonstrated that dobutamine could improve ALI induced by endotoxin, the mechanism of protective effect may involve in increasing the level of cAMP and up-regulating the AQP5 protein expression, and high-dose dobutamine had better effects.
【Key words】Acute lung injury; Septic shock; Dobutamine; Aquaporin 5; Cyclic adenosine monophosphate
在膿毒癥休克的發(fā)展過程中,肺是最易受損的器官之一<sup>[1]</sup>。急性肺損傷(acute lung injury, ALI)/急性呼吸窘迫綜合征(acute respiratory distress syndrome, ARDS)是臨床上的危重癥,病死率可高達(dá)30%~40%<sup>[2]</sup>,其主要病理學(xué)特征是肺泡-毛細(xì)血管屏障受損導(dǎo)致血管外肺水增加和形成肺水腫。已有報道視血管外肺水為膿毒癥患者病死率的獨立預(yù)測指標(biāo)<sup>[3]</sup>,但針對ALI時肺水腫的治療目前并無突破性進(jìn)展。多巴酚丁胺(dobutamine)作為臨床上常用的血管活性藥物之一,近年來在內(nèi)毒素等引起的急性肺損傷肺水腫方面的保護作用已引起廣泛關(guān)注<sup>[4-5]</sup>。本研究旨在觀察不同劑量多巴酚丁胺對膿毒癥休克兔ALI的影響,探討其對肺損傷的可能保護機制。
1材料與方法
1.1動物分組及模型的制備
健康成年雄性新西蘭大白兔70只,體質(zhì)量2.0~2.5 kg,由南京醫(yī)科大學(xué)附屬南京醫(yī)院動物實驗中心提供。兔禁食24 h后稱重,予20%烏拉坦1 g/kg麻醉后,右股靜脈置管補液,左頸動脈插管測動脈壓。參照全竹富等<sup>[6]</sup>改進(jìn)的盲腸結(jié)扎穿孔術(shù)聯(lián)合靜脈注入內(nèi)毒素制備膿毒癥休克模型。術(shù)后待血壓穩(wěn)定15 min后予以內(nèi)毒素(LPS,血清型O55B5,購自美國SIGMA公司)600 μg/kg靜脈推注,時間約15 min。平均動脈壓降至基礎(chǔ)值的60%視為達(dá)到休克標(biāo)準(zhǔn)。休克后隨機(隨機數(shù)字法)分成四組:①ALI模型組(B組,n=14)、多巴酚丁胺低劑量組(C組,n=14)、多巴酚丁胺中劑量組(D組,n=14)、多巴酚丁胺高劑量組(E組,n=14)。四組均以15mL/(kg·h)的速率輸注林格液進(jìn)行液體復(fù)蘇,以平均動脈壓維持至70 mmHg(1 mmHg=0.133 kPa)為準(zhǔn);②C組:液體復(fù)蘇時持續(xù)輸注多巴酚丁胺 2.5 μg/(kg·min);③D組:液體復(fù)蘇時持續(xù)輸注多巴酚丁胺 5 μg/(kg·min);④E組:液體復(fù)蘇時持續(xù)輸注多巴酚丁胺 10 μg/(kg·min)。另設(shè)假手術(shù)組(A組,n=14):動物僅接受盲腸探查術(shù)。各組兔分別于3 h和6 h處死取材。
1.2檢測指標(biāo)及方法
1.2.1肺組織濕/干質(zhì)量(W/D)比值的測定取左肺組織吸干血跡后稱濕質(zhì)量,再置入70 ℃烘箱48 h至恒重后稱干質(zhì)量,計算兩者比值。
1.2.2肺組織環(huán)磷酸腺苷(cyclic adenosine monophosphate, cAMP)含量的測定取右肺下葉小塊組織,常規(guī)勻漿、離心取上清液,采用ELISA法檢測肺組織cAMP水平,操作步驟嚴(yán)格按照試劑盒(cAMP試劑盒購自美國NewEast公司)說明進(jìn)行。
1.2.3Western-blot檢測水通道蛋白5(Aquaporin 5, AQP5)按照試劑盒說明書提取肺組織蛋白,測定蛋白濃度后保存于-70 ℃冰箱待用。將待測樣品行SDS-PAGE凝膠電泳,轉(zhuǎn)膜1 h,封閉1 h,加入多克隆羊抗AQP5(購自美國SantaCruz公司),孵育過夜,加入辣根過氧化酶標(biāo)記的羊抗兔IgG(1∶1500, 購自南京凱基生物科技發(fā)展有限公司),孵育,ECL顯影,曝光。采用Gel-Pro32軟件進(jìn)行灰度分析,目的蛋白與內(nèi)參灰度比值即為目的蛋白的相對含量。
1.2.4肺組織病理學(xué)觀察取右肺上葉小塊組織,光鏡下觀察,并由2名以上病理醫(yī)師以Guo等<sup>[7]</sup>的方法對肺損傷的程度進(jìn)行病理學(xué)評分。再取1 mm × 1 mm × 1 mm大小組織作電鏡檢查。
1.3統(tǒng)計學(xué)方法
采用SPSS 17.0軟件進(jìn)行統(tǒng)計學(xué)處理,各組數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(x±s) 表示,多組間差異比較采用單因素方差分析,組間兩兩比較采用 LSD法,方差不齊則進(jìn)行秩和檢驗,以P<0.05為差異具有統(tǒng)計學(xué)意義。
2結(jié)果
2.1各組肺組織W/D比值的變化
3 h、6 h時間點各組間W/D比值比較,差異具有統(tǒng)計學(xué)意義(F=10.94;F=20.49, P<0.01)。與A組相比, B組W/D比值在3 h和6 h均明顯增加(P<0.05),且6 h W/D比值增加更明顯(P<0.05);與B組相比,3 h C、D 兩組W/D比值無明顯變化(P>0.05), 3 h E組W/D比值顯著降低(P<0.05),6 h D、E兩組W/D比值均明顯降低(P<0.05);與C組比較,E組W/D比值亦明顯降低(P<0.05);D組和E組W/D比值差異無統(tǒng)計學(xué)意義(表1)。
2.2各組肺組織cAMP濃度的變化
3 h、6 h時間點各組間cAMP濃度比較,差異具有統(tǒng)計學(xué)意義(F=1168.49; F=1422.16, P<0.01)。與A組相比,B組cAMP濃度在3 h和6 h均明顯下降(P<0.05);與B組相比,C、D、E組cAMP濃度在3 h和6 h時間點均明顯增加(P<0.05);與C組相比,D、E兩組cAMP濃度增加明顯(P<0.05),且E組較D組cAMP濃度增加更明顯(P<0.05)。
2.3各組肺組織AQP5蛋白量的變化
3 h、6 h時間點各組間AQP5蛋白量比較,差異具有統(tǒng)計學(xué)意義(F=90.19;F=83.32; P<0.01)。與A組相比,B組AQP5蛋白量在3 h明顯減少(P<0.05),隨著損傷時間延長, 6 h時減少更明顯(P<0.05);與B組相比,C組AQP5蛋白表達(dá)量在3 h時無明顯改變,6 h時有所增加(P<0.05),D組和E組AQP5蛋白量在3 h和6 h時表達(dá)均明顯增加(P<0.05);與C組比較,D、E兩組AQP5蛋白量亦有所增加(P<0.05),且E組較D組增加更明顯(P<0.05)(圖1)。
2.3各組肺組織AQP5蛋白量的變化
3 h、6 h時間點各組間AQP5蛋白量比較,差異具有統(tǒng)計學(xué)意義(F=90.19;F=83.32; P<0.01)。與A組相比,B組AQP5蛋白量在3 h明顯減少(P<0.05),隨著損傷時間延長, 6 h時減少更明顯(P<0.05);與B組相比,C組AQP5蛋白表達(dá)量在3 h時無明顯改變,6 h時有所增加(P<0.05),D組和E組AQP5蛋白量在3 h和6 h時表達(dá)均明顯增加(P<0.05);與C組比較,D、E兩組AQP5蛋白量亦有所增加(P<0.05),且E組較D組增加更明顯(P<0.05)(圖1)。
3討論
目前普遍認(rèn)為在ALI/ARDS的發(fā)展過程中,肺水腫起關(guān)鍵作用,且AQP5與肺水腫的形成密不可分。研究發(fā)現(xiàn),在內(nèi)毒素誘導(dǎo)的ALI模型中,多巴酚丁胺可通過上調(diào)AQP5的表達(dá)起減輕肺水腫的作用<sup>[5]</sup>,但不同劑量多巴酚丁胺對AQP5的表達(dá)和肺水腫的影響差異具有統(tǒng)計學(xué)意義無統(tǒng)計學(xué)意義,國內(nèi)外尚未見相關(guān)報道。
本實驗成功復(fù)制兔急性肺損傷模型后,根據(jù)預(yù)實驗結(jié)果及文獻(xiàn)[4-5],分別予多巴酚丁胺2.5、5.0和10 μg/(kg·min)三種劑量,觀察其對ALI的影響。結(jié)果顯示,B組光鏡及電鏡結(jié)果符合ALI 病理改變,肺組織病理評分及W/D比值明顯升高,說明建立的ALI模型有效可行。給予多巴酚丁胺后,肺組織的病理損傷有不同程度的改善,病理學(xué)評分及W/D比值顯著降低,且存在劑量依賴性,提示多巴酚丁胺可減輕肺組織的病理損害,對肺損傷起保護作用。
水通道蛋白是一種調(diào)節(jié)水進(jìn)出細(xì)胞的特異性膜通道蛋白,參與多種病理生理過程。目前所知的水通道蛋白有4種存在于嚙齒類動物的呼吸道,其中AQP5主要表達(dá)在AT I的頂膜<sup>[8-9]</sup>。Ohinata等<sup>[10]</sup>將小鼠AQP5基因敲除后發(fā)現(xiàn)肺泡-毛細(xì)血管屏障對水的滲透能力較野生型小鼠降低約10倍。譚利平等<sup>[11]</sup>在高壓氧所致的大鼠肺損傷模型中發(fā)現(xiàn),隨著高壓氧的暴露時間延長,AQP5表達(dá)量逐漸減少,W/D比值逐漸增加。本實驗通過western-blot檢測肺組織中AQP5的表達(dá)變化,發(fā)現(xiàn)AQP5的表達(dá)量在造模成功后3 h即有明顯下調(diào),肺W/D比值則明顯增加,提示在ALI發(fā)展過程中,AQP5表達(dá)水平下降可能是肺內(nèi)液體在肺泡腔和肺間質(zhì)聚集,形成肺水腫的重要原因。筆者還觀察到,模型組中cAMP水平較正常對照組明顯下降,這與Tang等<sup>[12]</sup>研究結(jié)果相一致。在ALI的發(fā)展過程中,AQP5蛋白水平下調(diào)可能與LPS致傷后機體釋放大量炎癥介質(zhì),激活NF-κB和MAPK信號通路,從而降低AQP5的表達(dá)有關(guān)<sup>[13-15]</sup>,但AQP5表達(dá)量下降是否與其被細(xì)胞吞噬有關(guān),cAMP水平下降是否影響AQP5磷酸化水平還有待進(jìn)一步研究。
β-腎上腺素能受體參與調(diào)節(jié)胞內(nèi)諸如cAMP等第二信使的濃度,在各細(xì)胞和器官中起重要作用<sup>[16]</sup>。通過β-腎上腺素能受體與G蛋白的偶聯(lián)作用,β-腎上腺素能受體激動劑可激活腺苷酸環(huán)化酶,增加胞內(nèi)cAMP的濃度<sup>[17-18]</sup>。作為β-腎上腺素能受體信號傳導(dǎo)通路的第二信使,cAMP可激活蛋白激酶A并使之磷酸化,進(jìn)而對AQP5的表達(dá)進(jìn)行調(diào)節(jié)。近年來的眾多研究均表明,cAMP可上調(diào)AQP5的基因和蛋白的表達(dá)。Yang等<sup>[19]</sup>將肺泡上皮細(xì)胞暴露于cAMP幾小時后發(fā)現(xiàn)AQP5 mRNA和蛋白量均明顯增加。Sidhaye等<sup>[20]</sup>進(jìn)一步研究發(fā)現(xiàn)cAMP對AQP5的表達(dá)具有雙向調(diào)節(jié)的作用,短期(數(shù)分鐘)暴露于cAMP后發(fā)現(xiàn),小鼠肺泡上皮細(xì)胞膜上AQP5含量下降,長期持續(xù)暴露則AQP5含量明顯增加。此外,還有研究發(fā)現(xiàn),PKA誘導(dǎo)AQP5的蘇氨酸256位點磷酸化這一過程是由cAMP信號途徑介導(dǎo)的<sup>[21]</sup>。通過本實驗研究結(jié)果,筆者發(fā)現(xiàn),給予多巴酚丁胺后,cAMP含量和AQP5的蛋白表達(dá)量均呈劑量依賴性增加,W/D比值則明顯降低,提示多巴酚丁胺可能通過提高cAMP的水平上調(diào)AQP5的蛋白表達(dá),從而促進(jìn)肺泡及間質(zhì)內(nèi)液體的清除,減輕肺水腫,改善肺的病理損傷,對肺損傷起一定程度的保護作用。
綜上所述,多巴酚丁胺能有效減輕內(nèi)毒素致兔的急性肺損傷,其機制可能是通過提高胞內(nèi)cAMP水平進(jìn)而促進(jìn)AQP5的蛋白表達(dá)實現(xiàn)的,且其保護作用呈劑量依賴性,從而為臨床上急性肺損傷的治療提供新的思路。
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(收稿日期:2014-07-17)
(本文編輯:何小軍)
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