虎杖甙对产兔失血性休克后肺损伤保护作用的研究
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摘要
产后出血是我国乃至全球孕产妇死亡的主要原因,每年估计约有14万妇女死于产后出血。全球超过一半的孕产妇死亡发生在产后24小时内,死因为出血过多。产后出血除导致孕产妇死亡外,还可引起严重的并发症,如急性呼吸窘迫综合征(Acute respiratory distress syndrome, ARDS)、凝血系统疾病和席汉氏综合症。休克和ARDS是围产期妇女入住重症监护中心(Intensive Care Unit, ICU)的主要原因,分别占产科ICU所有疾病的25%和19%。近年研究发现,输血作为失血性休克后的治疗措施之一,本身也可以引起急性肺损伤(Acute lung injury, ALI)即输血相关性急性肺损伤。失血性休克后的ALI,病情发展迅速,容易进一步恶化发展为ARDS,继而进展为多器官功能障碍综合征,导致围产期妇女死亡。如果能够在纠正产后失血性休克的同时进行有效地器官保护,防止ARDS的发生,无疑将显著改善患者的预后,降低孕产妇死亡率。
ALI/ARDS是在严重感染、休克、创伤及烧伤等非心源性疾病过程中,肺毛细血管内皮细胞和肺泡上皮细胞损伤造成弥漫性肺间质及肺泡水肿,导致的急性低氧性呼吸功能不全或衰竭。目前普遍认为肺组织缺血-再灌注损伤、炎性细胞因子的网络作用、肠道细菌／内毒素移位是导致失血性休克后ALI的根本原因,但其确切的发病机制仍有待深入阐明。
肺是失血性休克后缺血再灌注损伤最严重的器官,也是失血性休克后急性肺损伤的重要途径。MDA是脂质过氧化反应的终产物,可以通过MDA含量的检测反映缺血再灌注时体内氧自由基的产生水平,间接了解组织缺血再灌注损伤程度。促炎因子(TNF-α, IL-1,IL-6等)的过度释放与抑炎因子(IL-4、IL-10)的相对表达不足,导致促炎／抗炎的失衡在ALI／ARDS的发病过程起重要作用。TNF-a是机体应激后产生最早、并起到核心作用的炎症介质,主要通过增强中性粒细胞活性,产生活性氧,血小板活化因子等,引起炎症损伤。TNF-α在体内水平的升高,是失血性休克导致ALI的重要因素,而且其升高程度与ARDS的发生有一定的相关性。抗炎因子IL-10,可部分抑制TNF等促炎因子的表达。TNF-α/IL-10升高的水平表示机体炎症反应是向促炎反应还是抗炎反应方向的发展。NF-KB是控制炎性信号在免疫细胞内的信号转导的开关,起着扳机(trigger)样关键性作用。NF-κB的过度激活可引起炎症介质大量合成、释放,导致炎症瀑布效应,加快ALI及ARDS的进程。热休克蛋白-70(Heat shock protein-70,HSP-70)是最保守、最主要和含量最多的一类,应激后生成最为明显,可以提高细胞的应激能力,抵御各种危险因素,起到保护细胞的作用。实验研究证明,HSP-70的高表达可以对磷脂酶A2、内毒素、缺血再灌注等因素引起的急性肺损伤起保护作用。
虎杖甙(Polydatin, PD),又名白藜芦醇甙,是从虎杖的根茎中提取的第4种单体,化学结构已确定为3,4’,5-三羟基3-单-D-葡糖甙。主要功效有祛风利湿,祛痰止咳,清热解毒,活血化瘀。中医临床用于治疗肺热咳嗽,湿热黄疸,疮痈肿毒,关节痹痛,经闭经痛,水火烫伤,跌打损伤等。赵克森等通过大量动物实验表明,虎杖甙能明显提高失血性休克和烧伤性休克动物的存活率,改善微循环,减少白细胞贴壁黏着,还有增加心功能和增加脉压的效应。2006年虎杖甙通过了国家食品药品监督管理局的评审,取得了临床试验的正式批件,成为我国为数不多的Ⅰ类新药的临床试验批件,应用于失血性休克和烧伤性休克的临床研究。目前已有动物实验研究表明,虎杖甙通过降低促炎因子的释放,抑制肺组织磷脂酶A2的活性,减轻内毒性休克后的肺损伤,但未见虎杖甙对失血性休克后肺损伤的研究报道。
基于孕产妇血容量高、血液高凝、肺功能残气量及氧合功能的降低,产后出血导致的失血性休克与非妊娠期失血性休克后的急性肺损伤可能有不同之处。我们前期研究发现在出血未控制条件下,限制性输液联合应用虎杖甙复苏孕兔失血性休克较单纯应用限制性输液能改善微循环,延长动物生存时间,我们推测此药物能改善产兔失血性休克后的肺损伤的严重程度。
[目的]
1建立产兔失血性休克后急性肺损伤模型,通过研究缺血-再灌注损伤、NF-κB介导的炎症反应,多性核中性粒细胞的活化,探讨产兔失血性休克后急性肺损伤的发生机制。
2证实虎杖甙对产兔休克后急性肺损伤的保护作用,通过研究热休克蛋白,NF-κB,ICAM-1等,阐明虎杖甙对休克后急性肺损伤保护作用的的分子机制。
3探讨虎杖甙对产兔休克后肝肾功能的影响,评价药物应用于产兔休克后的安全性,为日后应用于临床研究提供理论依据。
[方法]
1、实验动物40只孕晚期新西兰白兔,年龄2—2.5岁,妊娠年龄25-30天,产后6小时建立动物失血性休克模型。
2、建立产兔失血性休克模型
经过麻醉动物、备皮、股动静脉插管,操作完毕后15分钟后,连续监测平均动脉压(Mean arterial pressure, MAP),记录基础期指标。模型分为2期。①休克期(T0～T60min)：动物自股动脉放血开始时间为TO,右侧股动脉放血,速度2ml/kg/min,约15分钟MAP降至40-45mmHg,维持45分钟,放出的血液经肝素化处理以备回输。②复苏期(T60～T360min)：股动脉放血后第60min(T60),生理盐水与虎杖甙组分别输入4ml／Kg生理盐水与虎杖甙(1mg／ml),20分钟后回输自身血(股动脉失血量的一半)。T360停止实验。拔除动物各管道,缝合股动静脉,缝合皮肤,放回笼内,自由饮水、进食,记录动物存活时间。ALI诊断标准为PaO2/FiO2< 300 mmHg。
3、实验分组
将实验动物编号,随机分为4组,每组10只。①假休克组(Sham shock group:SS)：动物仅接受麻醉、插管监测血压,未接受休克及复苏处理；②休克组(Shock group:SH):动物接受麻醉、插管、休克处理,未予任何复苏；③生理盐水组(Normal saline group, NS):T60,静脉注射生理盐水4ml/Kg,20分钟后回输自身血(股动脉失血量的一半)；④虎杖甙组(Polydatin group, PD):T60,静脉注射4ml／kg虎杖甙(1mg/ml),20分钟后回输自身血(股动脉失血量的一半)。
4、监测指标
4.1持续监测血流动力学指标：MAP
4.2分别于T0(基础期)、T60(放血后60min)、T80(给药后20min)、T360(输血后280min)取股动脉血检测红细胞比容(hematocrit, Hct)、谷丙转氨酶(alanine aminotransferase, ALT)、肌酐(Creatinine, Cr)、PaO2、PaCO2、PaO2/FiO2、pH、乳酸、肿瘤坏死因子-alpha (TNF-α),白介素-10(IL-10)。Hct的测定采用微量血液离心机(金坛市正基仪器有限公司),ALT、Cr采用全自动生化分析仪检测。PaO2、PaCO2、pH的检测采用全自动血气分析仪(Medica Easy Blood Gas Analyzer, Medica Corporaton, U.S.A.)。乳酸试剂盒购于南京建成生物工程研究所。TNF-α、IL-10检测采用酶联免疫吸附方法(ELISA)。TNF-α, IL-10试剂盒购于(R&D Systems, Minneapolis, MN; Biosource International, Camarillo, CA),空气中氧含量为21%,定义FiO2=21%,计算PaO2/FiO2。
4.3肺组织检测：SH组在休克后2-3h死亡,立即取肺组织。其余3组动物在实验6h过程中均存活,于T360min,每组随机取3只动物行安乐死,注射过量戊巴比妥钠,取肺组织。具体如下：左肺下叶组织福尔马林固定、石蜡包埋、切片、HE染色。光镜下观察肺间质水肿及白细胞浸润程度。左肺上叶-80℃冻存,行肺组织髓过氧化物酶(myeloperoxidase, MPO)活性,丙二醛(Malondialdehyde, MDA)含量,细胞间粘附分子-1(Intercellular adhesion molecule-1, ICAM-1)mRNA与热休克蛋白70[Heat Shock Protein(HSP)-70]的表达、NF-κB的表达及其结合力的检测,右肺行湿／干质量比(Wet/dry Weight, WW/DW)检测。MPO、MDA试剂盒分别购于南京建成生物工程研究所。ICAM-1 mRNA的检测采用TRIzol法提取总RNA, SYBR GreenⅠ实时定量逆转录PCR。Trizol试剂盒[美国Molecular Research Center(MRC)公司],第一链cDNA合成试剂盒以及荧光定量试剂盒(RevertAidTM First Strand cDNA Synthesis Kit, MaximaTM SYBR Green/ROX qPCR Master Mix 2 X, Fermentas)。肺组织HSP-70、NF-κB的表达采用Western Blot方法,内对照选择β-actin。肺组织HSP-70单克隆抗体(SPA-antibody, Stressgen Bioreagents Co, Ann Arbor, MI), NF-κB p65(NeoMarkers, Thermo Fisher Scientific, Fremont, CA, USA), P-actin(NeoMarkers, Thermo Fisher Scientific, Fremont, CA, USA)。NF-κB结合力检测采用ELISA方法。NF-κB结合力购于(Cayman Chemical Company, Ann Arbor, MI)。
4.4肝肾组织行病理切片,HE染色
4.5统计出血量及输血量
4.6产兔生存时间。
5统计学分析
所有数据均应用SPSS13.0统计软件分析,每组不同时间点比较采用重复测量方差分析,分组因素的单独效应分析采用完全随机设计资料的方差分析,多重比较采用SNK检验法,数据用均数±标准差(X±SD)表示。生存分析采用Kaplan—Meier法,根据Breslow检验生存时间。生存时间用中位数±标准误(Md±SE)来表示。以P≤0.05(双侧)表示统计学上有显著性差异。
[结果]
1产兔基本特性比较：各实验组产兔基本情况：年龄、体重相互比较,无统计学意义(P>0.05)。
2失血量的比较：SH组、NS组、PD组产兔失血量介于(19.9±1.0～21.0±1.2)ml／Kg,三组比较,统计学无差异(p>0.05)。
3各时间点MAP的比较：SS组未接受放血处理,血压平稳波动在91.9～95.3mmHg。SH组未接受输血输液处理,在休克后2～3小时死亡。基础期,4组产兔的MAP波动在94.2～97.3mmHg,组间差异无差别(P>0.05)。经过股动脉放血,SH、NS、PD组动物在放血后15分钟达到休克状态,维持至放血后60min, MAP波动在40～45 mmHg,三者比较无统计学差异(P>0.05)。NS组静脉注射4ml/Kg生理盐水后,MAP随之上升,10min后达50.9±0.9mmHg,随后MAP逐渐下降,20min后降至46.5±0.8mmHg,回输半量失去的全血后,MAP再次上升,波动在68.5±1.0～73.2±0.7mmHg。PD组静脉注射4ml/Kg虎杖甙(1mg/ml)后,MAP在5～10分钟内迅速上升达70.5±0.9 mmHg,高于生理盐水组(P<0.05),随后MAP缓慢下降,20min后降至58.4±0.8mmHg,高于生理盐水组(P<0.05),回输半量失去的全血后,MAP波动在78.2±0.7～81.7±1.0mmHg,高于生理盐水组(P<0.05)
4各时间点血Hct含量的比较：基础期,4组产兔血Hct比较无统计学意义(P>0.05)。放血后60min,SH、NS、PD组产兔血HCT均明显降低,与基础期相比有统计学差异(P<0.05),考虑与组织间隙液体回流入流,血液稀释有关。静脉注射4ml／Kg生理盐水或虎杖甙后,产兔血Hct继续降低考虑组织间液回流入血以及液体输入有关。SH组在休克后2～3小时死亡。回输半量失去的全血后,NS与PD组Hct略有回升,2组比较无统计学差异(P>0.05)。
5各时间点血清TNF-a的比较：基础期,4组产兔血清TNF-a浓度测不出。放血后60min, SH、NS、PD组产兔血清TNF-a浓度升高,组间比较无统计学差异(P>0.05)。静脉注射4ml/Kg生理盐水或虎杖甙后,产兔血TNF-a继续升高。SH组在休克后2～3小时死亡。回输半量失去的全血治疗后,NS与PD组TNF-a略有下降,其中PD组明显低于NS组,2组比较有统计学差异(P<0.05)。
6各时间点血清IL-10的比较：基础期,4组产兔血清IL-10浓度介于(39.2±16.7)pg/ml至(43.9±17.9)pg/ml,组间比较统计学无差异(P>0.05)。放血后60min,SH、NS、PD组产兔血清IL-10浓度升高,介于(261.2±118.1) pg/ml至(271.6±140.8)pg/ml,组间比较无统计学差异(P>0.05)。静脉注射4ml／Kg生理盐水或虎杖甙后,产兔血IL-10继续升高。SH组在休克后2～3小时死亡。回输半量失去的全血治疗后,NS与PD组IL-10略有下降,其中PD组(247.3±67.4)pg／ml稍高于NS组(219.6±95.9)pg/m1,2组比较无统计学差异(P>0.05)。7不同复苏方式对产兔失血性休克PaO2的影响：基础期,四组产兔动脉血PaO2在为80.8±3.8～84.9±5.7mmHg,统计学比较无差异(P>0.05)。休克后各组动物呼吸频率加快,PaO2明显上升,达91.2±5.1～95.8±7.1mmHg,与基础期比较有统计学差异(P<0.05)。给药后20min,PD组PaO2有所下降,低于基础期状态(P<0.05),低于SS组(P<0.05),与NS组无统计学差异(P>0.05)。输血后280min,PD组PaO2有所回升,高于NS组(P<0.05),与SS组无统计学差异(P>0.05)。
8不同复苏方式对产兔失血性休克PaCO2的影响：基础期,四组产兔动脉血PaCO2为(36.0±3.1-39.7±3.6)mmHg,组间比较无统计学差异(P>0.05)。休克后各组动物呼吸频率加快,PaCO2下降,达30.0±3.6-32.6±4.0mmHg。复苏后,PaCO2缓慢上升。输血后280min,PD组、NS组、SS组PaCO2分别为(35.4±4.1)mmHg, (33.0±5.1)mmHg, (35.0±3.1)mmHg,3组比较无统计学差异(P>0.05)。
9不同复苏方式对产兔失血性休克PaO2／FiO2的影响：四组产兔动脉血PaO2／FiO2在基础期为(384.8±17.9-404.3±27.3)mmHg,组间比较无统计学差异(P>0.05)。休克后各组动物呼吸频率加快,PaO2／FiO2明显上升,达(434.3±24.3～456.2±33.8)mmHg,高于基础期状态(P<0.05)。给药后20min,PD组Pa02/FiO2有所下降,低于基础期状态(P<0.05),与NS、SH组无区别(P>0.05)。输血后280min,PD组Pa02/FiO2有所回升,明显高于NS组(P<0.05),与SS组无差异(P>0.05)。
10各时间点血乳酸浓度的比较：基础期,4组产兔血乳酸浓度在(1.4-1.7)mmol/L之间,差异无统计学意义(P>0.05),休克后,SH、NS、PD组血乳酸浓度明显升高,达(8.6～9.7)mmol/L,三组比较差异无统计学意义(P>0.05),考虑与产兔接受休克打击的程度相当有关。除PD组,给予虎杖甙20min后,血乳酸浓度有所降低,达(7.5±1.6)mmol/L外,NS组与SH组血乳酸继续升高,分别为(10.4±1.7)mmol/L, (13.3±1.4)mmol/L。SH组动物在休克后2-3小时死亡NS、PD组在回输半量失去的全血后,乳酸浓度降低,PD组与NS组分别为(2.3±0.7) mmol/L, (6.2±1.1)mmol/L,均未恢复到基础期值(P<0.05),2组比较统计学有差异(P<0.05)。考虑与PD组MAP稳定,减轻了因休克造成的组织供血、供氧不足。
11各时间点血pH的比较：基础期,4组产兔血pH在7.41～7.43之间,差异无统计学意义(P>0.05),休克后,pH降低,SH、NS、PD组血pH介于7.25～7.27,三组比较差异无统计学意义(P>0.05),考虑与产兔接受休克打击的程度相当有关。给药后20min,PD血pH稍有上升外,其余两组血pH继续降低,PD组明显高于SH组与NS组(P<0.05)。SH组在休克后2-3小时死亡。回输半量失去的全血后,NS、PD组产兔血pH均上升,但未恢复到基础期值(P<0.05)。其中PD组为7.38±0.02,高于NS组7.27±0.03(P<0.05)。考虑与PD组MAP稳定,组织氧供好有关。
12虎杖甙对产兔失血性休克后肺组织MDA含量的影响：与假休克组相比,其余3组产兔肺组织中MDA含量升高,其中PD组明显低于SH与NS组(P<0.05),与SS组无差别(P>0.05)。表明虎杖甙减轻了休克复苏后产兔肺组织MDA含量,减少氧自由基产生,减轻肺组织缺血再灌注损伤。
13肺组织HSP-70表达的比较：产兔肺组织中HSP-70的表达在各组间自高到低排列为PD组4.00±0.20、NS组2.55±0.23、SH组2.32±0.30、SS组1.00±0.00。PD组明显高于其余3组,组间比较有统计学差异(P<0.05)。与生理盐水复苏相比,虎杖甙复苏增强了休克复苏后产兔肺组织中HSP-70的表达。
14肺组织NF-κB核转移的比较：产兔肺组织中NF-κB活性在各组间自高到低排列为SH组2.56±0.57、NS组2.20±0.28、PD组1.21±0.63、SS组1.00±0.00。PD组明显低于SH与NS组(P<0.05),与SS组无差异(P>0.05)。表明休克复苏后产兔肺组织NF-κB活性增强,与生理盐水复苏相比,虎杖甙复苏减轻了休克复苏后产兔肺组织中NF-κB的激活。
15肺组织核NF-κB结合力的比较：产兔肺组织中核NF-κB结合力在各组间自高到低排列为SH组1.72±0.12、NS组1.60±0.15、PD组1.16±0.16、SS组1.00±0.00。PD组明显低于SH与NS组(P<0.05),与SS组无差异(P>0.05)。表明休克复苏后产兔肺组织NF-κB结合力增强,与生理盐水复苏相比,虎杖甙复苏降低了休克复苏后产兔肺组织中NF-κB的结合能力。
16虎杖甙对产兔失血性休克后肺组织ICAM-1 mRNA水平的变化：与假休克组相比,其余3组产兔肺组织中ICAM-1 mRNA含量升高,其中PD组明显低于SH与NS组(P<0.05),与SS组无差别(P>0.05)。表明虎杖甙降低了产兔休克复苏后肺组织ICAM-1 mRNA的含量。
17肺组织MPO活性的比较：产兔肺组织中MPO水平在各组间自高到低排列为SH组(3.80±0.15)单位／克组织、NS组(3.18±0.12)单位／克组织、PD组(2.63±0.12)单位／克组织、SS组(1.32±0.08)单位／克组织,组间比较差异有统计学意义,且PD组明显低于SH与NS组,高于SS组(P<0.05)。表明虎杖甙减轻了休克复苏后产兔肺组织中性粒细胞在肺组织聚集程度的增加。
18肺组织湿干比的比较：产兔肺组织湿干比由大到小分别为SH、NS、PD、SS组,PD组明显小于SH与NS组,大于SS组,统计学有差异(P<0.05),表明虎杖甙可以降低产兔休克后肺组织的含水量。
19肺组织病理损伤的比较：肉眼观：SH组肺组织变重,表面湿润,有散在出血斑,并可见暗红色略凹陷的斑片状肺萎陷区；切面有大量泡沫状液体流出。光镜：SS组肺组织病理HE染色肺组织大致正常。SH组肺间质毛细血管高度扩张及充血,肺间质和肺泡腔内有大量富含蛋白质液体渗出,形成以间质性肺水肿为主的病变。肺间质中还可有不同程度的充血、出血、灶状坏死。肺内发生局灶性肺不张,肺小血管内PMNs积聚。NS组示肺间质水肿,肺泡隔增宽伴中性粒细胞浸润,肺泡内可见大量蛋白质水肿液及中性粒细胞浸润,病理损伤较SH组略有减轻。PD组肺组织病理损伤较SH与NS组均明显减轻,较SS组肺损伤加重。肺组织损伤评分在各组间自高到低排列为SH组(12.0±0.0)、NS组(8.0±1.0)、PD组(3.7±0.6)、SS组(1.3±0.6),组间比较差异有统计学意义,且PD组明显低于SH与NS组,高于SS组(P<0.05)。反映虎杖甙减轻了休克复苏后产兔肺组织中性粒细胞在肺组织损伤程度。
20血清ALT含量的比较基础期,4组产兔血清ALT含量基本相同,介于(19.8±2.0 IU／L至22.8±3.5 IU／L),差异无统计学意义(P>0.05),休克后,SH、NS、PD组血清ALT含量较SS组明显升高,达(149.4±8.6 IU／L至158.2±11.2 IU／L)三组比较差异无统计学意义(P>0.05)。SS组经麻醉、插管等处理后未给予放血处理,各时相ALT浓度各时间点无明显变化,各时间点比较P>0.05。除PD组,给予虎杖甙20min后,血清ALT含量有所降低外,NS组与SH组血清ALT含量继续升高。SH组动物休克后一直未予复苏,在休克后2～3小时死亡。NS、PD组在回输半量失去的全血后,血清ALT含量均有所下降,且尤以PLH组ALT下降幅度大,2组比较统计学有差异(NS vs PD：169.7±6.3IU／L vs 97.5±5.4 IU／L)(P<0.05)。
21血清肌酐含量的比较基础期,4组产兔血清肌酐含量基本相同,介于(82.7±1.7IU／L至84.9±1.8 IU／L)差异无统计学意义(P>0.05),休克后,SH、NS、PD组均较SS组血清肌酐含量明显升高,达(142.7±3.8 IU／L至147.5±4.9 IU／L)三组比较差异无统计学意义(P>0.05)。SS组经麻醉、插管等处理后未给予放血处理,各时间点血清肌酐含量无明显变化,各时间点比较P>0.05。SH组动物休克后一直未予复苏,血清肌酐含量持续升高,在休克后2-3小时死亡。给药后,NS、PD组肌酐含量均继续升高,2组比较无统计学差异(P>0.05)。输血后,NS与PD组血清肌酐含量均下降,2组比较无统计学差异(NS vs PD：164.0±5.2 IU／Lvs 151.0±2.4IU／L,P>0.05)。
22肝组织病理损伤的比较：SS组肝细胞结构大致正常。SH组动物中在休克后2-3h死亡,肝脏大体结构被破坏,中央静脉,肝血窦淤血,出血,肝细胞水肿空泡化,少量炎症细胞浸润。NS组肝细胞水肿,空泡化,大量炎细胞浸润,肝血窦扩张。PD组病理损伤较NS组轻,可见肝细胞轻度水肿并少量炎症细胞浸润。
23肾组织病理损伤的比较：SS组肾脏病理大致正常。SH组动物中在休克2-3h死亡,肾小管上皮细胞水肿,空泡化,肾小球可见出血。NS组与PD组肾组织可见肾小管上皮细胞水肿空泡化。
24生存时间的比较：SS组动物长期存活。SH组动物在休克后2-4小时死亡。NS、PD组12小时分别为(0／7,7／7),差异有统计学意义(P<0.05)、24小时生存率分别为：NS(0／7,2／7),差异无统计学意义(P>0.05)。PD产兔中位生存时间(22.0±1.3)小时明显长于NS组(10.0±0.6)小时(χ2=12.091,P=0.001)。
[结论]
虎杖甙对产兔失血性复苏后的肺损伤存在保护作用,可能与其减轻缺血再灌注损伤,增强肺组织HSP-70的表达,从而抑制肺组织NF-κB的激活,降低血清TNF-α的释放以及肺组织ICAM-1粘附分子的产生,最终减轻PMN在肺脏的扣钾造成组织的损伤。虎杖甙能明显改善产兔失血性休克后的肝功能,但对肾功能无明显改善作用。
Postpartum hemorrhage(PPH) is considered the leading cause of pregnancy-related deaths worldwide, with an estimated 140,000 women dying annually from this complication. Especially in developing coutries, mortality from PPH has remained high despite international efforts to decrease maternal mortality since the launch of the Safe Motherhoold Initiative in 1987. In our country, postpartum hemorrhage is the major reason of maternal death and morbidity.
After hemorrhagic shock induced by postpartum hemorrhage, the systemic inflammatory response was activated and the first organ injured was lung. In the last five years it has gained recognition that transfusion of blood products, a treatment method to postpartum hemorrhage could induce acute lung injury by itself, which is called Transfusion-related acute lung injury. One word, acute lung injury (ALI) is the major reason for peripartum women's entering in Intensive Care Unit. For peripartum women, ALI is readily develop into acute respiratory distress syndrome (ARDS), multiple organ dysfunction syndrome (MODS), which results in death finally.
Even until now, the mechanism of lung injury after shock is still unclear. It is widely accepted that polymorphonuclear is the major cell mediating this process. The unbalanced between over expression of pro-inflammatory mediators and the relatively less expression of anti-inflammatory mediators, over-activated polymorphonuclears, ischemia-reperfusion injury and cellular apoptosis is major mechanism leading to the ALI/ARDS after hemorrhagic shock. The over-activation of nuclear factor-kappa B (NF-κB) could lead to the synthesis and release of inflammatory mediators, promote the development of ALI and ARDS.
Polydatin is extracted from a traditional Chinese herbal medicine, Polygonum cuspidatum,3,4',5 trihydroxystibene-3-monoglucoside. and has a special effect in shock treatment in terms of improvement of heart function and microcirculatory insufficiency.
OBJECTIVE
Because of the beneficial effects of Polydatin in improving microcirculation and prolongation of survival time of pregnant rabbits in hemorrhagic shock. We speculated that it could also improve lung injury after hemorrhagic shock in postpartum rabbit. So, in the present study, we tested the efficacy of Polydatin in improving lung injury in a controlled hemorrhagic shock in postpartum rabbit.
METHODS
40 anesthetized New Zealand white rabbits with 6 hours within postpartum were underwent controlled hemorrhagic shock by bleeding via femoral artery to mean arterial pressure (MAP) of 40-45mmHg. The experiment consisted of two phases, shock phase (T0-T60min) and resuscitation phase (T60-T360min). Animals were randomly divided into four groups (n=10 per group); The first group, SS ("sham shock"), underwent a sham operation in which they were subjected to all the surgical procedures experienced by the hemorrhaged animals but were not hemorrhaged. The second group, SH ("shock"), underwent hemorrhage but without resuscitation. The next two groups, NS, PD, underwent hemorrhage and received a single volume infusion of 4ml/Kg normal saline or Polydatin(1mg/ml) at T60 followed by transfued with half of the heparinized shed blood. At T360, three animals from group SS, NS, PD were euthanized for lung tissue harvest for further test(all the animals in group SH were died during 2-3 hours after hemorrhagic shock and were immediately harvested the lung for further examination); while to the remain animals, the cannulas were then removed after ligation of the femoral artery, femoral vein. The animals were placed in a caged with food and water and observed for survival. Mean arterial pressure (MAP) were continuously monitored, Blood samples for hematocrit(Hct), alanine aminotransferase (ALT), Creatinine (Cr), PaO2, PaCO2, PaO2/FiO2, lactate, pH, Tumor necrosis factor-alpha (TNF-α), interleukin-10(IL-10) were taken at baseline, 60min post bleeding (Postbleeding 60 min),20min post drug (Postdrug 20 min), 280min post transfusion of blood products (Posttransfusion 280min). Lung tissue examination included pathological exmaination, wet weight/dry weight ratio, myeloperoxidase (MPO) assay,Malondialdehyde (MDA), the expression of Intercellular adhesion molecule-1(ICAM-1) mRNA, Heat shock protein-70(HSP-70) and NF-κB. Liver and Kidney underwent pathology examination with HE stained. Statistical Analysis:results are presented as mean±standard deviation (SD) unless otherwise noted, and analyzed by two-way analysis of variance with repeated measures over time. Post hoc analyses were performed with Student-Newman-Keuls multiple comparisons test. The survival time was analyzed by Kaplan-Meier plots and compared by the Breslow test. Analysis was completed with a statistical software package for desktop computers (SPSS13.0). P≤0.05 (two-sided) was considered significant.
RESULTS
Basic status and Blood loss
All groups were comparable in baseline measurements (such as age or body weight) and the volume of blood withdrawl via the femoral artery for induction and maintenance of shock (P>0.05)
Hemodynamic changes
MAP of the rabbits before hemorrhage was between 94.2±97.3 mmHg. After blood withdrawl via femoray artery, MAP decreased markedly over a 15 min period (T0-T15) to a comparable values of 40～45 mmHg in all of the hemorrhaged groups. In the sham shock group SS, MAP remained stable at normotensive levels throughout the experiment. In the group SH, in which the hemorrhagic shock was not resuscitated, MAP remained stable at hypotensive levels and died 2-3 hours after hemorrhage. Immediately after polydatin infusion, the MAP of PD group recovered to (70.5±0.9) mmHg, which was significantly higher than the NS group (50.9±0.9) mmHg and remained at that level for about 10 minutes. After that, MAP was gradually decreased to (58.4±0.8) mmHg, which is also higher than the relative time point of NS group, which was (46.5±0.8) mmHg. After twenty min of drug or normal saline infusion, half of the heparinized blood was reinfused to group NS and PD. In the following time, MAP of group PD increased to (78.2±0.7～81.7±1.0) mmHg, which was significantly higher than that in group NS(68.5±1.0～73.2±0.7) mmHg (P<0.05).
HCT
All groups showed a significant reduction of Hct after hemorrhagic shock and resuscitation, no significant difference was found between group NS and group PD.
Serum TNF-αand IL-10 assay
The blood concentration of TNF-αand IL-10 was increased after hemorrhage and peak at 20min after drug (equal to 280min after hemorrhage) and decreased at 6 hour after hemorrhage. At 6 hour after hemorrhage, the blood concentration of TNF-a was significantly lower in PD group compared with NS group(P<0.05), while no significant differences was found between these two groups.
PaO2, Pa CO2 and PaO2/FiO2
No significant differences was found between PaO2, PaCO2 and PaO2/FiO2 in the baseline between four groups.60 minutes after bleeding, animals respiratory rate increases, PaO2 and PaO2/FiO2 was increased while PaCO2 was decreased.280 minutes after transfusion, PaO2 and PaO2/FiO2 was higher in PD group than NS group, no significant differences was found in PaCO2 between PD, NS and SS group.
Lactate and pH level
There was significant metabolic acidosis as shown by the decrease in pH from 7.41～7.43 to 7.25～7.27 and the increase in lactate increased from 1.4～1.7 to 8.6～9.7 mmol/L. Although, lactate tended to decrease, and pH value tended to increase at 280min after transfusion of blood product in group NS and group PD compared with shock period(P<0.05), all of which had not recovered. However, in PD group, lactate was significantly lower, while pH value was significantly higher than corresponding values in the group NS.
Lung tissue Malondialdehyde (MDA) content
At the end of experiment, the lung tissue MDA content was increased in all the shock groups compared to SS group. The animals in PD group had significantly lower MDA content than those in SH or NS group (P<0.05).
Lung tissue HSP-70 expression
Compared with SS group, HSP-70 expression in lung tissue is up-regulated in the other three groups, and the up-regulation of HSP-70 expression is much evident in PD group than that in other three groups(P<0.05).
Lung tissue NF-κB expression
The down-regulation of NF-κB expression in lung tissue is much evident in PD group than that in SH or NS group (P<0.05). The expression of NF-κB is similar between PD and SS group (P>0.05)
Lung tissue NF-κB activity
Lung tissue NF-κB activity was significantly decreased in PD group than that in SH or NS group (P<0.05). The activity of NF-κB was similar between PD and SS group (P>0.05)
Lung tissue mRNA expression of ICAM-1
Compared with SS group, the mRNA expression of ICAM-1 was significantly higher in other three groups. Compared with NS group, PD inhibited the mRNA expression of ICAM-1 (P<0.05).
Lung MPO activity, wet weight/dry weight ratio, and lung injury score
The lung MPO activity, wet weight/Dry weight ratio and lung injury score was significantly lower in PD group than those in NS or SS group, but was significantly higher than those in SS group (P<0.05).
Serum ALT content & Serum creatinine content
No significant differences was found in serum ALT or creatinine content in the baseline between four groups.60 minutes after bleeding, both ALT and creatinine were increased in SH, NS, PD group.20 minutes after Polydatin administration, ALT was decreased, while AST was still increased. Until blood transfusion, both creatinine and ALT was decreased, while they were still not recovered to the baseline level.ALT in the PD group was significantly lower than that in NS group, while no significant difference was found in creatinine between the two groups.
Liver and Kidney pathology examination
Liver and kidney in the SS group were almost normal. Hemorrhagic shock resulted in severe liver hemorrhagic edema with inflammatory cell inflitration and vacuolar changes of renal tubular epithelial cell. Compared with NS group, Polydatin reduced inflammatory cell infiltration in the hepatic gate-duct area. No significnat differences was found between NS and PD group in the kidney pathological examination, both were shown cell enlargement and vacuolar changes in the renal tubular epithelial cells.
Survival time
Median survival time in PD group (22.0±1.3 hours) was significantly longer than that in NS group(10.0±0.6 hours) (P<0.05).
CONCLUSION
Polydatin has protective effect on hemorrhagic shock induced lung injury in postpartum rabbit, which maybe mediated by alleviates ischemia-reperfusion injury, induces HSP-70 expression, inhibits overactivation of NF-κB and mRNA ICAM-1 expression in lung tissue, finaly decrease lung tissue PMN infiltration and ameliorate lung injury. Polydatin administration could improve liver function while has no obvious effect on kidney function after hemorrhagic shock in postpartum rabbit.
ALI/ARDS是在严重感染、休克、创伤及烧伤等非心源性疾病过程中,肺毛细血管内皮细胞和肺泡上皮细胞损伤造成弥漫性肺间质及肺泡水肿,导致的急性低氧性呼吸功能不全或衰竭。目前普遍认为肺组织缺血-再灌注损伤、炎性细胞因子的网络作用、肠道细菌／内毒素移位是导致失血性休克后ALI的根本原因,但其确切的发病机制仍有待深入阐明。
肺是失血性休克后缺血再灌注损伤最严重的器官,也是失血性休克后急性肺损伤的重要途径。MDA是脂质过氧化反应的终产物,可以通过MDA含量的检测反映缺血再灌注时体内氧自由基的产生水平,间接了解组织缺血再灌注损伤程度。促炎因子(TNF-α, IL-1,IL-6等)的过度释放与抑炎因子(IL-4、IL-10)的相对表达不足,导致促炎／抗炎的失衡在ALI／ARDS的发病过程起重要作用。TNF-a是机体应激后产生最早、并起到核心作用的炎症介质,主要通过增强中性粒细胞活性,产生活性氧,血小板活化因子等,引起炎症损伤。TNF-α在体内水平的升高,是失血性休克导致ALI的重要因素,而且其升高程度与ARDS的发生有一定的相关性。抗炎因子IL-10,可部分抑制TNF等促炎因子的表达。TNF-α/IL-10升高的水平表示机体炎症反应是向促炎反应还是抗炎反应方向的发展。NF-KB是控制炎性信号在免疫细胞内的信号转导的开关,起着扳机(trigger)样关键性作用。NF-κB的过度激活可引起炎症介质大量合成、释放,导致炎症瀑布效应,加快ALI及ARDS的进程。热休克蛋白-70(Heat shock protein-70,HSP-70)是最保守、最主要和含量最多的一类,应激后生成最为明显,可以提高细胞的应激能力,抵御各种危险因素,起到保护细胞的作用。实验研究证明,HSP-70的高表达可以对磷脂酶A2、内毒素、缺血再灌注等因素引起的急性肺损伤起保护作用。
虎杖甙(Polydatin, PD),又名白藜芦醇甙,是从虎杖的根茎中提取的第4种单体,化学结构已确定为3,4’,5-三羟基3-单-D-葡糖甙。主要功效有祛风利湿,祛痰止咳,清热解毒,活血化瘀。中医临床用于治疗肺热咳嗽,湿热黄疸,疮痈肿毒,关节痹痛,经闭经痛,水火烫伤,跌打损伤等。赵克森等通过大量动物实验表明,虎杖甙能明显提高失血性休克和烧伤性休克动物的存活率,改善微循环,减少白细胞贴壁黏着,还有增加心功能和增加脉压的效应。2006年虎杖甙通过了国家食品药品监督管理局的评审,取得了临床试验的正式批件,成为我国为数不多的Ⅰ类新药的临床试验批件,应用于失血性休克和烧伤性休克的临床研究。目前已有动物实验研究表明,虎杖甙通过降低促炎因子的释放,抑制肺组织磷脂酶A2的活性,减轻内毒性休克后的肺损伤,但未见虎杖甙对失血性休克后肺损伤的研究报道。
基于孕产妇血容量高、血液高凝、肺功能残气量及氧合功能的降低,产后出血导致的失血性休克与非妊娠期失血性休克后的急性肺损伤可能有不同之处。我们前期研究发现在出血未控制条件下,限制性输液联合应用虎杖甙复苏孕兔失血性休克较单纯应用限制性输液能改善微循环,延长动物生存时间,我们推测此药物能改善产兔失血性休克后的肺损伤的严重程度。
[目的]
1建立产兔失血性休克后急性肺损伤模型,通过研究缺血-再灌注损伤、NF-κB介导的炎症反应,多性核中性粒细胞的活化,探讨产兔失血性休克后急性肺损伤的发生机制。
2证实虎杖甙对产兔休克后急性肺损伤的保护作用,通过研究热休克蛋白,NF-κB,ICAM-1等,阐明虎杖甙对休克后急性肺损伤保护作用的的分子机制。
3探讨虎杖甙对产兔休克后肝肾功能的影响,评价药物应用于产兔休克后的安全性,为日后应用于临床研究提供理论依据。
[方法]
1、实验动物40只孕晚期新西兰白兔,年龄2—2.5岁,妊娠年龄25-30天,产后6小时建立动物失血性休克模型。
2、建立产兔失血性休克模型
经过麻醉动物、备皮、股动静脉插管,操作完毕后15分钟后,连续监测平均动脉压(Mean arterial pressure, MAP),记录基础期指标。模型分为2期。①休克期(T0～T60min)：动物自股动脉放血开始时间为TO,右侧股动脉放血,速度2ml/kg/min,约15分钟MAP降至40-45mmHg,维持45分钟,放出的血液经肝素化处理以备回输。②复苏期(T60～T360min)：股动脉放血后第60min(T60),生理盐水与虎杖甙组分别输入4ml／Kg生理盐水与虎杖甙(1mg／ml),20分钟后回输自身血(股动脉失血量的一半)。T360停止实验。拔除动物各管道,缝合股动静脉,缝合皮肤,放回笼内,自由饮水、进食,记录动物存活时间。ALI诊断标准为PaO2/FiO2< 300 mmHg。
3、实验分组
将实验动物编号,随机分为4组,每组10只。①假休克组(Sham shock group:SS)：动物仅接受麻醉、插管监测血压,未接受休克及复苏处理；②休克组(Shock group:SH):动物接受麻醉、插管、休克处理,未予任何复苏；③生理盐水组(Normal saline group, NS):T60,静脉注射生理盐水4ml/Kg,20分钟后回输自身血(股动脉失血量的一半)；④虎杖甙组(Polydatin group, PD):T60,静脉注射4ml／kg虎杖甙(1mg/ml),20分钟后回输自身血(股动脉失血量的一半)。
4、监测指标
4.1持续监测血流动力学指标：MAP
4.2分别于T0(基础期)、T60(放血后60min)、T80(给药后20min)、T360(输血后280min)取股动脉血检测红细胞比容(hematocrit, Hct)、谷丙转氨酶(alanine aminotransferase, ALT)、肌酐(Creatinine, Cr)、PaO2、PaCO2、PaO2/FiO2、pH、乳酸、肿瘤坏死因子-alpha (TNF-α),白介素-10(IL-10)。Hct的测定采用微量血液离心机(金坛市正基仪器有限公司),ALT、Cr采用全自动生化分析仪检测。PaO2、PaCO2、pH的检测采用全自动血气分析仪(Medica Easy Blood Gas Analyzer, Medica Corporaton, U.S.A.)。乳酸试剂盒购于南京建成生物工程研究所。TNF-α、IL-10检测采用酶联免疫吸附方法(ELISA)。TNF-α, IL-10试剂盒购于(R&D Systems, Minneapolis, MN; Biosource International, Camarillo, CA),空气中氧含量为21%,定义FiO2=21%,计算PaO2/FiO2。
4.3肺组织检测：SH组在休克后2-3h死亡,立即取肺组织。其余3组动物在实验6h过程中均存活,于T360min,每组随机取3只动物行安乐死,注射过量戊巴比妥钠,取肺组织。具体如下：左肺下叶组织福尔马林固定、石蜡包埋、切片、HE染色。光镜下观察肺间质水肿及白细胞浸润程度。左肺上叶-80℃冻存,行肺组织髓过氧化物酶(myeloperoxidase, MPO)活性,丙二醛(Malondialdehyde, MDA)含量,细胞间粘附分子-1(Intercellular adhesion molecule-1, ICAM-1)mRNA与热休克蛋白70[Heat Shock Protein(HSP)-70]的表达、NF-κB的表达及其结合力的检测,右肺行湿／干质量比(Wet/dry Weight, WW/DW)检测。MPO、MDA试剂盒分别购于南京建成生物工程研究所。ICAM-1 mRNA的检测采用TRIzol法提取总RNA, SYBR GreenⅠ实时定量逆转录PCR。Trizol试剂盒[美国Molecular Research Center(MRC)公司],第一链cDNA合成试剂盒以及荧光定量试剂盒(RevertAidTM First Strand cDNA Synthesis Kit, MaximaTM SYBR Green/ROX qPCR Master Mix 2 X, Fermentas)。肺组织HSP-70、NF-κB的表达采用Western Blot方法,内对照选择β-actin。肺组织HSP-70单克隆抗体(SPA-antibody, Stressgen Bioreagents Co, Ann Arbor, MI), NF-κB p65(NeoMarkers, Thermo Fisher Scientific, Fremont, CA, USA), P-actin(NeoMarkers, Thermo Fisher Scientific, Fremont, CA, USA)。NF-κB结合力检测采用ELISA方法。NF-κB结合力购于(Cayman Chemical Company, Ann Arbor, MI)。
4.4肝肾组织行病理切片,HE染色
4.5统计出血量及输血量
4.6产兔生存时间。
5统计学分析
所有数据均应用SPSS13.0统计软件分析,每组不同时间点比较采用重复测量方差分析,分组因素的单独效应分析采用完全随机设计资料的方差分析,多重比较采用SNK检验法,数据用均数±标准差(X±SD)表示。生存分析采用Kaplan—Meier法,根据Breslow检验生存时间。生存时间用中位数±标准误(Md±SE)来表示。以P≤0.05(双侧)表示统计学上有显著性差异。
[结果]
1产兔基本特性比较：各实验组产兔基本情况：年龄、体重相互比较,无统计学意义(P>0.05)。
2失血量的比较：SH组、NS组、PD组产兔失血量介于(19.9±1.0～21.0±1.2)ml／Kg,三组比较,统计学无差异(p>0.05)。
3各时间点MAP的比较：SS组未接受放血处理,血压平稳波动在91.9～95.3mmHg。SH组未接受输血输液处理,在休克后2～3小时死亡。基础期,4组产兔的MAP波动在94.2～97.3mmHg,组间差异无差别(P>0.05)。经过股动脉放血,SH、NS、PD组动物在放血后15分钟达到休克状态,维持至放血后60min, MAP波动在40～45 mmHg,三者比较无统计学差异(P>0.05)。NS组静脉注射4ml/Kg生理盐水后,MAP随之上升,10min后达50.9±0.9mmHg,随后MAP逐渐下降,20min后降至46.5±0.8mmHg,回输半量失去的全血后,MAP再次上升,波动在68.5±1.0～73.2±0.7mmHg。PD组静脉注射4ml/Kg虎杖甙(1mg/ml)后,MAP在5～10分钟内迅速上升达70.5±0.9 mmHg,高于生理盐水组(P<0.05),随后MAP缓慢下降,20min后降至58.4±0.8mmHg,高于生理盐水组(P<0.05),回输半量失去的全血后,MAP波动在78.2±0.7～81.7±1.0mmHg,高于生理盐水组(P<0.05)
4各时间点血Hct含量的比较：基础期,4组产兔血Hct比较无统计学意义(P>0.05)。放血后60min,SH、NS、PD组产兔血HCT均明显降低,与基础期相比有统计学差异(P<0.05),考虑与组织间隙液体回流入流,血液稀释有关。静脉注射4ml／Kg生理盐水或虎杖甙后,产兔血Hct继续降低考虑组织间液回流入血以及液体输入有关。SH组在休克后2～3小时死亡。回输半量失去的全血后,NS与PD组Hct略有回升,2组比较无统计学差异(P>0.05)。
5各时间点血清TNF-a的比较：基础期,4组产兔血清TNF-a浓度测不出。放血后60min, SH、NS、PD组产兔血清TNF-a浓度升高,组间比较无统计学差异(P>0.05)。静脉注射4ml/Kg生理盐水或虎杖甙后,产兔血TNF-a继续升高。SH组在休克后2～3小时死亡。回输半量失去的全血治疗后,NS与PD组TNF-a略有下降,其中PD组明显低于NS组,2组比较有统计学差异(P<0.05)。
6各时间点血清IL-10的比较：基础期,4组产兔血清IL-10浓度介于(39.2±16.7)pg/ml至(43.9±17.9)pg/ml,组间比较统计学无差异(P>0.05)。放血后60min,SH、NS、PD组产兔血清IL-10浓度升高,介于(261.2±118.1) pg/ml至(271.6±140.8)pg/ml,组间比较无统计学差异(P>0.05)。静脉注射4ml／Kg生理盐水或虎杖甙后,产兔血IL-10继续升高。SH组在休克后2～3小时死亡。回输半量失去的全血治疗后,NS与PD组IL-10略有下降,其中PD组(247.3±67.4)pg／ml稍高于NS组(219.6±95.9)pg/m1,2组比较无统计学差异(P>0.05)。7不同复苏方式对产兔失血性休克PaO2的影响：基础期,四组产兔动脉血PaO2在为80.8±3.8～84.9±5.7mmHg,统计学比较无差异(P>0.05)。休克后各组动物呼吸频率加快,PaO2明显上升,达91.2±5.1～95.8±7.1mmHg,与基础期比较有统计学差异(P<0.05)。给药后20min,PD组PaO2有所下降,低于基础期状态(P<0.05),低于SS组(P<0.05),与NS组无统计学差异(P>0.05)。输血后280min,PD组PaO2有所回升,高于NS组(P<0.05),与SS组无统计学差异(P>0.05)。
8不同复苏方式对产兔失血性休克PaCO2的影响：基础期,四组产兔动脉血PaCO2为(36.0±3.1-39.7±3.6)mmHg,组间比较无统计学差异(P>0.05)。休克后各组动物呼吸频率加快,PaCO2下降,达30.0±3.6-32.6±4.0mmHg。复苏后,PaCO2缓慢上升。输血后280min,PD组、NS组、SS组PaCO2分别为(35.4±4.1)mmHg, (33.0±5.1)mmHg, (35.0±3.1)mmHg,3组比较无统计学差异(P>0.05)。
9不同复苏方式对产兔失血性休克PaO2／FiO2的影响：四组产兔动脉血PaO2／FiO2在基础期为(384.8±17.9-404.3±27.3)mmHg,组间比较无统计学差异(P>0.05)。休克后各组动物呼吸频率加快,PaO2／FiO2明显上升,达(434.3±24.3～456.2±33.8)mmHg,高于基础期状态(P<0.05)。给药后20min,PD组Pa02/FiO2有所下降,低于基础期状态(P<0.05),与NS、SH组无区别(P>0.05)。输血后280min,PD组Pa02/FiO2有所回升,明显高于NS组(P<0.05),与SS组无差异(P>0.05)。
10各时间点血乳酸浓度的比较：基础期,4组产兔血乳酸浓度在(1.4-1.7)mmol/L之间,差异无统计学意义(P>0.05),休克后,SH、NS、PD组血乳酸浓度明显升高,达(8.6～9.7)mmol/L,三组比较差异无统计学意义(P>0.05),考虑与产兔接受休克打击的程度相当有关。除PD组,给予虎杖甙20min后,血乳酸浓度有所降低,达(7.5±1.6)mmol/L外,NS组与SH组血乳酸继续升高,分别为(10.4±1.7)mmol/L, (13.3±1.4)mmol/L。SH组动物在休克后2-3小时死亡NS、PD组在回输半量失去的全血后,乳酸浓度降低,PD组与NS组分别为(2.3±0.7) mmol/L, (6.2±1.1)mmol/L,均未恢复到基础期值(P<0.05),2组比较统计学有差异(P<0.05)。考虑与PD组MAP稳定,减轻了因休克造成的组织供血、供氧不足。
11各时间点血pH的比较：基础期,4组产兔血pH在7.41～7.43之间,差异无统计学意义(P>0.05),休克后,pH降低,SH、NS、PD组血pH介于7.25～7.27,三组比较差异无统计学意义(P>0.05),考虑与产兔接受休克打击的程度相当有关。给药后20min,PD血pH稍有上升外,其余两组血pH继续降低,PD组明显高于SH组与NS组(P<0.05)。SH组在休克后2-3小时死亡。回输半量失去的全血后,NS、PD组产兔血pH均上升,但未恢复到基础期值(P<0.05)。其中PD组为7.38±0.02,高于NS组7.27±0.03(P<0.05)。考虑与PD组MAP稳定,组织氧供好有关。
12虎杖甙对产兔失血性休克后肺组织MDA含量的影响：与假休克组相比,其余3组产兔肺组织中MDA含量升高,其中PD组明显低于SH与NS组(P<0.05),与SS组无差别(P>0.05)。表明虎杖甙减轻了休克复苏后产兔肺组织MDA含量,减少氧自由基产生,减轻肺组织缺血再灌注损伤。
13肺组织HSP-70表达的比较：产兔肺组织中HSP-70的表达在各组间自高到低排列为PD组4.00±0.20、NS组2.55±0.23、SH组2.32±0.30、SS组1.00±0.00。PD组明显高于其余3组,组间比较有统计学差异(P<0.05)。与生理盐水复苏相比,虎杖甙复苏增强了休克复苏后产兔肺组织中HSP-70的表达。
14肺组织NF-κB核转移的比较：产兔肺组织中NF-κB活性在各组间自高到低排列为SH组2.56±0.57、NS组2.20±0.28、PD组1.21±0.63、SS组1.00±0.00。PD组明显低于SH与NS组(P<0.05),与SS组无差异(P>0.05)。表明休克复苏后产兔肺组织NF-κB活性增强,与生理盐水复苏相比,虎杖甙复苏减轻了休克复苏后产兔肺组织中NF-κB的激活。
15肺组织核NF-κB结合力的比较：产兔肺组织中核NF-κB结合力在各组间自高到低排列为SH组1.72±0.12、NS组1.60±0.15、PD组1.16±0.16、SS组1.00±0.00。PD组明显低于SH与NS组(P<0.05),与SS组无差异(P>0.05)。表明休克复苏后产兔肺组织NF-κB结合力增强,与生理盐水复苏相比,虎杖甙复苏降低了休克复苏后产兔肺组织中NF-κB的结合能力。
16虎杖甙对产兔失血性休克后肺组织ICAM-1 mRNA水平的变化：与假休克组相比,其余3组产兔肺组织中ICAM-1 mRNA含量升高,其中PD组明显低于SH与NS组(P<0.05),与SS组无差别(P>0.05)。表明虎杖甙降低了产兔休克复苏后肺组织ICAM-1 mRNA的含量。
17肺组织MPO活性的比较：产兔肺组织中MPO水平在各组间自高到低排列为SH组(3.80±0.15)单位／克组织、NS组(3.18±0.12)单位／克组织、PD组(2.63±0.12)单位／克组织、SS组(1.32±0.08)单位／克组织,组间比较差异有统计学意义,且PD组明显低于SH与NS组,高于SS组(P<0.05)。表明虎杖甙减轻了休克复苏后产兔肺组织中性粒细胞在肺组织聚集程度的增加。
18肺组织湿干比的比较：产兔肺组织湿干比由大到小分别为SH、NS、PD、SS组,PD组明显小于SH与NS组,大于SS组,统计学有差异(P<0.05),表明虎杖甙可以降低产兔休克后肺组织的含水量。
19肺组织病理损伤的比较：肉眼观：SH组肺组织变重,表面湿润,有散在出血斑,并可见暗红色略凹陷的斑片状肺萎陷区；切面有大量泡沫状液体流出。光镜：SS组肺组织病理HE染色肺组织大致正常。SH组肺间质毛细血管高度扩张及充血,肺间质和肺泡腔内有大量富含蛋白质液体渗出,形成以间质性肺水肿为主的病变。肺间质中还可有不同程度的充血、出血、灶状坏死。肺内发生局灶性肺不张,肺小血管内PMNs积聚。NS组示肺间质水肿,肺泡隔增宽伴中性粒细胞浸润,肺泡内可见大量蛋白质水肿液及中性粒细胞浸润,病理损伤较SH组略有减轻。PD组肺组织病理损伤较SH与NS组均明显减轻,较SS组肺损伤加重。肺组织损伤评分在各组间自高到低排列为SH组(12.0±0.0)、NS组(8.0±1.0)、PD组(3.7±0.6)、SS组(1.3±0.6),组间比较差异有统计学意义,且PD组明显低于SH与NS组,高于SS组(P<0.05)。反映虎杖甙减轻了休克复苏后产兔肺组织中性粒细胞在肺组织损伤程度。
20血清ALT含量的比较基础期,4组产兔血清ALT含量基本相同,介于(19.8±2.0 IU／L至22.8±3.5 IU／L),差异无统计学意义(P>0.05),休克后,SH、NS、PD组血清ALT含量较SS组明显升高,达(149.4±8.6 IU／L至158.2±11.2 IU／L)三组比较差异无统计学意义(P>0.05)。SS组经麻醉、插管等处理后未给予放血处理,各时相ALT浓度各时间点无明显变化,各时间点比较P>0.05。除PD组,给予虎杖甙20min后,血清ALT含量有所降低外,NS组与SH组血清ALT含量继续升高。SH组动物休克后一直未予复苏,在休克后2～3小时死亡。NS、PD组在回输半量失去的全血后,血清ALT含量均有所下降,且尤以PLH组ALT下降幅度大,2组比较统计学有差异(NS vs PD：169.7±6.3IU／L vs 97.5±5.4 IU／L)(P<0.05)。
21血清肌酐含量的比较基础期,4组产兔血清肌酐含量基本相同,介于(82.7±1.7IU／L至84.9±1.8 IU／L)差异无统计学意义(P>0.05),休克后,SH、NS、PD组均较SS组血清肌酐含量明显升高,达(142.7±3.8 IU／L至147.5±4.9 IU／L)三组比较差异无统计学意义(P>0.05)。SS组经麻醉、插管等处理后未给予放血处理,各时间点血清肌酐含量无明显变化,各时间点比较P>0.05。SH组动物休克后一直未予复苏,血清肌酐含量持续升高,在休克后2-3小时死亡。给药后,NS、PD组肌酐含量均继续升高,2组比较无统计学差异(P>0.05)。输血后,NS与PD组血清肌酐含量均下降,2组比较无统计学差异(NS vs PD：164.0±5.2 IU／Lvs 151.0±2.4IU／L,P>0.05)。
22肝组织病理损伤的比较：SS组肝细胞结构大致正常。SH组动物中在休克后2-3h死亡,肝脏大体结构被破坏,中央静脉,肝血窦淤血,出血,肝细胞水肿空泡化,少量炎症细胞浸润。NS组肝细胞水肿,空泡化,大量炎细胞浸润,肝血窦扩张。PD组病理损伤较NS组轻,可见肝细胞轻度水肿并少量炎症细胞浸润。
23肾组织病理损伤的比较：SS组肾脏病理大致正常。SH组动物中在休克2-3h死亡,肾小管上皮细胞水肿,空泡化,肾小球可见出血。NS组与PD组肾组织可见肾小管上皮细胞水肿空泡化。
24生存时间的比较：SS组动物长期存活。SH组动物在休克后2-4小时死亡。NS、PD组12小时分别为(0／7,7／7),差异有统计学意义(P<0.05)、24小时生存率分别为：NS(0／7,2／7),差异无统计学意义(P>0.05)。PD产兔中位生存时间(22.0±1.3)小时明显长于NS组(10.0±0.6)小时(χ2=12.091,P=0.001)。
[结论]
虎杖甙对产兔失血性复苏后的肺损伤存在保护作用,可能与其减轻缺血再灌注损伤,增强肺组织HSP-70的表达,从而抑制肺组织NF-κB的激活,降低血清TNF-α的释放以及肺组织ICAM-1粘附分子的产生,最终减轻PMN在肺脏的扣钾造成组织的损伤。虎杖甙能明显改善产兔失血性休克后的肝功能,但对肾功能无明显改善作用。
Postpartum hemorrhage(PPH) is considered the leading cause of pregnancy-related deaths worldwide, with an estimated 140,000 women dying annually from this complication. Especially in developing coutries, mortality from PPH has remained high despite international efforts to decrease maternal mortality since the launch of the Safe Motherhoold Initiative in 1987. In our country, postpartum hemorrhage is the major reason of maternal death and morbidity.
After hemorrhagic shock induced by postpartum hemorrhage, the systemic inflammatory response was activated and the first organ injured was lung. In the last five years it has gained recognition that transfusion of blood products, a treatment method to postpartum hemorrhage could induce acute lung injury by itself, which is called Transfusion-related acute lung injury. One word, acute lung injury (ALI) is the major reason for peripartum women's entering in Intensive Care Unit. For peripartum women, ALI is readily develop into acute respiratory distress syndrome (ARDS), multiple organ dysfunction syndrome (MODS), which results in death finally.
Even until now, the mechanism of lung injury after shock is still unclear. It is widely accepted that polymorphonuclear is the major cell mediating this process. The unbalanced between over expression of pro-inflammatory mediators and the relatively less expression of anti-inflammatory mediators, over-activated polymorphonuclears, ischemia-reperfusion injury and cellular apoptosis is major mechanism leading to the ALI/ARDS after hemorrhagic shock. The over-activation of nuclear factor-kappa B (NF-κB) could lead to the synthesis and release of inflammatory mediators, promote the development of ALI and ARDS.
Polydatin is extracted from a traditional Chinese herbal medicine, Polygonum cuspidatum,3,4',5 trihydroxystibene-3-monoglucoside. and has a special effect in shock treatment in terms of improvement of heart function and microcirculatory insufficiency.
OBJECTIVE
Because of the beneficial effects of Polydatin in improving microcirculation and prolongation of survival time of pregnant rabbits in hemorrhagic shock. We speculated that it could also improve lung injury after hemorrhagic shock in postpartum rabbit. So, in the present study, we tested the efficacy of Polydatin in improving lung injury in a controlled hemorrhagic shock in postpartum rabbit.
METHODS
40 anesthetized New Zealand white rabbits with 6 hours within postpartum were underwent controlled hemorrhagic shock by bleeding via femoral artery to mean arterial pressure (MAP) of 40-45mmHg. The experiment consisted of two phases, shock phase (T0-T60min) and resuscitation phase (T60-T360min). Animals were randomly divided into four groups (n=10 per group); The first group, SS ("sham shock"), underwent a sham operation in which they were subjected to all the surgical procedures experienced by the hemorrhaged animals but were not hemorrhaged. The second group, SH ("shock"), underwent hemorrhage but without resuscitation. The next two groups, NS, PD, underwent hemorrhage and received a single volume infusion of 4ml/Kg normal saline or Polydatin(1mg/ml) at T60 followed by transfued with half of the heparinized shed blood. At T360, three animals from group SS, NS, PD were euthanized for lung tissue harvest for further test(all the animals in group SH were died during 2-3 hours after hemorrhagic shock and were immediately harvested the lung for further examination); while to the remain animals, the cannulas were then removed after ligation of the femoral artery, femoral vein. The animals were placed in a caged with food and water and observed for survival. Mean arterial pressure (MAP) were continuously monitored, Blood samples for hematocrit(Hct), alanine aminotransferase (ALT), Creatinine (Cr), PaO2, PaCO2, PaO2/FiO2, lactate, pH, Tumor necrosis factor-alpha (TNF-α), interleukin-10(IL-10) were taken at baseline, 60min post bleeding (Postbleeding 60 min),20min post drug (Postdrug 20 min), 280min post transfusion of blood products (Posttransfusion 280min). Lung tissue examination included pathological exmaination, wet weight/dry weight ratio, myeloperoxidase (MPO) assay,Malondialdehyde (MDA), the expression of Intercellular adhesion molecule-1(ICAM-1) mRNA, Heat shock protein-70(HSP-70) and NF-κB. Liver and Kidney underwent pathology examination with HE stained. Statistical Analysis:results are presented as mean±standard deviation (SD) unless otherwise noted, and analyzed by two-way analysis of variance with repeated measures over time. Post hoc analyses were performed with Student-Newman-Keuls multiple comparisons test. The survival time was analyzed by Kaplan-Meier plots and compared by the Breslow test. Analysis was completed with a statistical software package for desktop computers (SPSS13.0). P≤0.05 (two-sided) was considered significant.
RESULTS
Basic status and Blood loss
All groups were comparable in baseline measurements (such as age or body weight) and the volume of blood withdrawl via the femoral artery for induction and maintenance of shock (P>0.05)
Hemodynamic changes
MAP of the rabbits before hemorrhage was between 94.2±97.3 mmHg. After blood withdrawl via femoray artery, MAP decreased markedly over a 15 min period (T0-T15) to a comparable values of 40～45 mmHg in all of the hemorrhaged groups. In the sham shock group SS, MAP remained stable at normotensive levels throughout the experiment. In the group SH, in which the hemorrhagic shock was not resuscitated, MAP remained stable at hypotensive levels and died 2-3 hours after hemorrhage. Immediately after polydatin infusion, the MAP of PD group recovered to (70.5±0.9) mmHg, which was significantly higher than the NS group (50.9±0.9) mmHg and remained at that level for about 10 minutes. After that, MAP was gradually decreased to (58.4±0.8) mmHg, which is also higher than the relative time point of NS group, which was (46.5±0.8) mmHg. After twenty min of drug or normal saline infusion, half of the heparinized blood was reinfused to group NS and PD. In the following time, MAP of group PD increased to (78.2±0.7～81.7±1.0) mmHg, which was significantly higher than that in group NS(68.5±1.0～73.2±0.7) mmHg (P<0.05).
HCT
All groups showed a significant reduction of Hct after hemorrhagic shock and resuscitation, no significant difference was found between group NS and group PD.
Serum TNF-αand IL-10 assay
The blood concentration of TNF-αand IL-10 was increased after hemorrhage and peak at 20min after drug (equal to 280min after hemorrhage) and decreased at 6 hour after hemorrhage. At 6 hour after hemorrhage, the blood concentration of TNF-a was significantly lower in PD group compared with NS group(P<0.05), while no significant differences was found between these two groups.
PaO2, Pa CO2 and PaO2/FiO2
No significant differences was found between PaO2, PaCO2 and PaO2/FiO2 in the baseline between four groups.60 minutes after bleeding, animals respiratory rate increases, PaO2 and PaO2/FiO2 was increased while PaCO2 was decreased.280 minutes after transfusion, PaO2 and PaO2/FiO2 was higher in PD group than NS group, no significant differences was found in PaCO2 between PD, NS and SS group.
Lactate and pH level
There was significant metabolic acidosis as shown by the decrease in pH from 7.41～7.43 to 7.25～7.27 and the increase in lactate increased from 1.4～1.7 to 8.6～9.7 mmol/L. Although, lactate tended to decrease, and pH value tended to increase at 280min after transfusion of blood product in group NS and group PD compared with shock period(P<0.05), all of which had not recovered. However, in PD group, lactate was significantly lower, while pH value was significantly higher than corresponding values in the group NS.
Lung tissue Malondialdehyde (MDA) content
At the end of experiment, the lung tissue MDA content was increased in all the shock groups compared to SS group. The animals in PD group had significantly lower MDA content than those in SH or NS group (P<0.05).
Lung tissue HSP-70 expression
Compared with SS group, HSP-70 expression in lung tissue is up-regulated in the other three groups, and the up-regulation of HSP-70 expression is much evident in PD group than that in other three groups(P<0.05).
Lung tissue NF-κB expression
The down-regulation of NF-κB expression in lung tissue is much evident in PD group than that in SH or NS group (P<0.05). The expression of NF-κB is similar between PD and SS group (P>0.05)
Lung tissue NF-κB activity
Lung tissue NF-κB activity was significantly decreased in PD group than that in SH or NS group (P<0.05). The activity of NF-κB was similar between PD and SS group (P>0.05)
Lung tissue mRNA expression of ICAM-1
Compared with SS group, the mRNA expression of ICAM-1 was significantly higher in other three groups. Compared with NS group, PD inhibited the mRNA expression of ICAM-1 (P<0.05).
Lung MPO activity, wet weight/dry weight ratio, and lung injury score
The lung MPO activity, wet weight/Dry weight ratio and lung injury score was significantly lower in PD group than those in NS or SS group, but was significantly higher than those in SS group (P<0.05).
Serum ALT content & Serum creatinine content
No significant differences was found in serum ALT or creatinine content in the baseline between four groups.60 minutes after bleeding, both ALT and creatinine were increased in SH, NS, PD group.20 minutes after Polydatin administration, ALT was decreased, while AST was still increased. Until blood transfusion, both creatinine and ALT was decreased, while they were still not recovered to the baseline level.ALT in the PD group was significantly lower than that in NS group, while no significant difference was found in creatinine between the two groups.
Liver and Kidney pathology examination
Liver and kidney in the SS group were almost normal. Hemorrhagic shock resulted in severe liver hemorrhagic edema with inflammatory cell inflitration and vacuolar changes of renal tubular epithelial cell. Compared with NS group, Polydatin reduced inflammatory cell infiltration in the hepatic gate-duct area. No significnat differences was found between NS and PD group in the kidney pathological examination, both were shown cell enlargement and vacuolar changes in the renal tubular epithelial cells.
Survival time
Median survival time in PD group (22.0±1.3 hours) was significantly longer than that in NS group(10.0±0.6 hours) (P<0.05).
CONCLUSION
Polydatin has protective effect on hemorrhagic shock induced lung injury in postpartum rabbit, which maybe mediated by alleviates ischemia-reperfusion injury, induces HSP-70 expression, inhibits overactivation of NF-κB and mRNA ICAM-1 expression in lung tissue, finaly decrease lung tissue PMN infiltration and ameliorate lung injury. Polydatin administration could improve liver function while has no obvious effect on kidney function after hemorrhagic shock in postpartum rabbit.
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