急性肺损伤大鼠PAI-1的表达与一氧化氮吸入干预的实验研究
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摘要
第一部分急性肺损伤大鼠PAI-1与相关因子的表达及其意义
背景:
急性肺损伤(ALI)和急性呼吸窘迫综合征(ARDS)是心源性以外的各种肺内外致病因素导致的急性进行性缺氧性呼吸衰竭,病死率高。研究表明炎症是ALI发病的中心环节,炎症和肺组织重塑及胶原的沉积是同时进行的。纤溶酶原激活物抑制剂-1(PAI-1)是纤溶酶原激活剂主要的抑制物,其基因表达水平与肺中胶原沉积密切相关。PAI-1还通过作用于基质金属酶、肝细胞生长因子、细胞迁移等影响肺损伤修复。
目的:
探索急性肺损伤大鼠PAI-1表达的变化规律,以便寻找合适的干预手段和时机;初步探讨PAI-1相关因子的表达情况及其在急性肺损伤中的意义。
方法:
4～5周清洁级SD雄性大鼠(180～200g)随机分7组:生理盐水组作为基线(B-A),内毒素(LPS)组分别于造模2h、4h、8h、16h、24h、48h处死,即为LPS-A 2h、LPS-A 4h、LPS-A 8h、LPS-A 16h、LPS-A 24h、LPS-A 48h组,每组大鼠8～10只。采用腹腔注射LPS 0.1mg/kg 16h后气管滴入LPS 1mg/kg的二次打击方法诱导大鼠急性肺损伤模型,生理盐水组相应予等量生理盐水处理。测定各时点的血气分析、肺组织病理评分、支气管肺泡灌洗液(BALF)白细胞(WBC)计数和总蛋白(TP)、肺组织匀浆髓过氧化物酶(MPO)活性和丙二醛(MDA)含量、肺湿干重比(B-A、LPS-A 4h、LPS-A 24h组另取6只)。酶联免疫吸附试验(ELISA)测定血浆和BALF中PAI-1水平,实时荧光定量PCR测定肺组织PAI-1及尿激酶型纤溶酶原激活剂(uPA)、基质金属蛋白酶-9(MMP-9)、肝细胞生长因子(HGF)mRNA表达水平,免疫组化法测定肺组织PAI-1及uPA、MMP-9、HGF蛋白表达水平,改良的MSB染色法进行肺纤维素染色。
结果:
1.急性肺损伤模型的建立:LPS二次打击2h后动脉血氧分压(PaO_2)下降,4h、8h达谷底,直至48h均低于基线水平。肺部病理显示二次打击2h即出现急性肺损伤表现,4h病变加重弥漫,高峰持续至24～48h,表现有炎性细胞浸润、肺泡内和间质出血,肺水肿、肺实变不张等。BALF WBC计数和肺组织MPO活性,BALF TP均在2h上升高于基线水平,渐达高峰持续至24～48h,与病理评分变化一致。肺组织MDA含量在4h上升,渐达高峰持续至24～48h(P＜0.05)。肺湿干重比4h有上升趋势,24h有进一步加重的趋势,统计学上无显著差异(P＞0.05)。
2.肺组织PAI-1的表达情况:LPS二次打击后2h血浆PAI-1浓度有上升趋势,8h～16h高于基线水平;而BALF PAI-1水平和肺组织PAI-1蛋白表达水平在二次打击2h直至48h均高于基线水平(P＜0.05),并有进行性加重的趋势;PAI-1mRNA表达在二次打击2h时开始增高,高峰持续于2～16h(P＜0.05),48h恢复至基线水平。血浆与BALF PAI-1浓度、肺组织PAI-1 mRNA和蛋白表达水平均相互呈正相关,其中血浆、BALF PAI-1水平、肺组织PAI-1蛋白的表达与肺病理评分呈正相关(P＜0.05)。
3.肺组织PAI-1相关因子的表达情况:
①LPS二次打击后uPA mRNA表达水平很快增高,2h达高峰(P＜0.05),但4h即降至基线水平;uPA蛋白表达水平有2h上升、4h下降的趋势(P＞0.05),8h后uPA蛋白表达水平均低于2h,24h时最低,低于基线水平(P＜0.05)。
②PAI-1/uPAmRNA在LPS二次打击2h时较基线水平增高,8h～16h达高峰,直至48h仍高于基线水平(P＜0.05),肺组织PAI-1/uPA蛋白比值在LPS二次打击后4h时后高于基线水平,8h及之后各时点高于4h(P＜0.05)。纤维素染色显示肺纤维蛋白沉积的变化趋势与肺组织PAI-1蛋白和PAI-1/uPA蛋白比值的变化趋势基本一致。PAI-1 mRNA/uPA mRNA和PAI-1/uPA蛋白比值与肺病理评分呈正相关(P＜0.01)。
③MMP-9 mRNA在LPS二次打击2h有升高趋势,之后维持在较高水平,48h有下降趋势(P＞0.05);LPS二次打击后4h时MMP-9蛋白表达水平较基线水平增高,持续至24h(P＜0.05),48h降至基线水平。
④HGF mRNA在LPS二次打击后2h、4h有进行性上升趋势,8h时较基线水平增高(P＜0.05),24h下降至基线水平;LPS二次打击后4h时HGF蛋白较基线水平增高,持续至24h(P＜0.05),48h降至基线水平(P＞0.05)。
结论:
1.PAI-1在内毒素诱导的大鼠急性肺损伤早期即表达增高,且持续时间相对长,与uPA之间比例失调导致纤溶失衡;PAI-1高表达和纤溶失衡与急性肺损伤发生发展及异常修复密切相关;BALF PAI-1浓度能较好地反映肺组织PAI-1蛋白的表达,预示急性肺损伤的严重性。
2.MMP-9和HGF在急性肺损伤中表达先增高后降低,下降时间早于PAI-1,可能影响急性肺损伤的细胞外基质清除和肺修复过程。
第二部分一氧化氮和高氧吸入对大鼠急性肺损伤PAI-1表达的影响及其作用
背景:
急性肺损伤时由炎症促发的凝血纤溶功能紊乱是肺泡和肺微血管纤维蛋白沉积的主要原因。纤溶和凝血紊乱影响ALI/ARDS的发生发展和预后,成为治疗ALI/ARDS的新目标。PAI-1增高与ALI/ARDS肺局部纤维蛋白沉积密切相关,因此抑制PAI-1基因的表达可作为急性肺损伤的新的干预点。吸入一氧化氮(iNO)在临床上作为严重ARDS的急救手段,往往在ARDS的晚期机械通气等方法难以控制时使用。有研究表明早期iNO可以预防内毒素引起的血管内皮细胞功能失调。也有资料显示一氧化氮(NO)可抑制PAI-1的表达。我们将采用早期iNO,探讨治疗剂量NO干预对急性肺损伤PAI-1表达的影响。严重ARDS的病人往往高浓度氧疗。研究表明长时间高氧暴露的小鼠过度表达PAI-1。有动物实验提示iNO可降低长期高氧暴露下新生大鼠的病死率。iNO对高氧治疗下急性肺损伤的PAI-1表达的影响尚不明了。体内内皮细胞、上皮细胞、血小板、中性粒细胞、巨噬细胞、神经组织在一定刺激下由NOS合成酶(NOS)合成NO。外源性的NO势必对内源性NO系统产生影响,这种影响对急性肺损伤PAI-1的表达的意义值得进一步探讨。
目的:
探讨吸入一氧化氮和高氧对内毒素(LPS)二次打击诱导大鼠急性肺损伤PAI-1表达的影响及其意义;初步探讨吸入一氧化氮和高氧对急性肺损伤大鼠PAI-1相关因子表达的影响;探讨吸入一氧化氮和高氧后急性肺损伤大鼠内源性NO系统的变化及其与PAI-1表达的关系。
方法:
4～5周清洁级SD雄性大鼠(180～200g)随机分为生理盐水对照组简称C,内毒素造模组简称LPS。采用腹腔注射LPS 0.1mg/kg 16h后气管滴入LPS 1mg/kg的二次打击方法诱导大鼠急性肺损伤模型,生理盐水组相应予等量生理盐水处理。造模后C组和LPS组分别随机给予吸入空气(A)、20ppmNO(NO)、95%氧气(O)、20ppmNO加95%氧气(ONO)4种气体,不同气体干预4h、24h及干预24h观察至造模48h。C组每个时点6～8只大鼠,LPS组每个时点8～10只。实时荧光定量PCR测定肺组织PAI-1及uPA、MMP-9、HGF mRNA表达水平,免疫组化法测定肺组织PAI-1及uPA、MMP-9、HGF蛋白表达水平;改良MSB染色法进行肺纤维素染色,HE染色肺病理评分;测定肺组织总一氧化氮合酶(tNOS)、构成型一氧化氮合酶(cNOS)和诱导型一氧化氮合酶(iNOS)活性以及NO含量。
结果:
1.一氧化氮吸入与高氧对急性肺损伤PAI-1及相关因子表达的影响:
①肺组织PAI-1 mRNA表达在造模4h、24h时,LPS-A组和LPS-O组较相应C组增高,iNO干预的LPS-NO组和LPS-ONO组较LPS-A和LPS-O组降低;造模48h时,LPS-A组表达降至正常对照水平,而高氧干预的LPS-O组表达仍持续增高,高于C-O组,iNO干预的LPS-ONO组较LPS-O组降低(P＜0.05)。
肺组织PAI-1蛋白在造模4h时,iNO干预的LPS-NO组表达低,与对照组无显著差异,并低于其他气体干预的LPS组(P＜0.05);造模24h时,所有LPS组较相应C组表达增高(P＜0.05),iNO干预的LPS-NO组和LPS-ONO组较LPS-A组和LPS-O组有下降趋势(P＞0.05);造模48h时,iNO干预的LPS-NO组和LPS-ONO组降至正常对照水平(P＞0.05),LPS-NO组较其他气体干预的LPS组降低,LPS-ONO组较LPS-O组降低(P＜0.05)。
②肺组织uPA mRNA表达在造模4h、24h、48h各时点,所有LPS组与相应C组比较差异无统计学意义(P＞0.05)。
肺组织uPA蛋白表达在造模24h时LPS-A组和LPS-O组分别低于相应C组(P＜0.05);LPS-NO组和LPS-ONO组较LPS-A组和LPS-O组升高(P＜0.05)。
③肺组织PAI-1 mRNA/uPA mRNA在造模4h时,所有LPS组大鼠较相应C组增高;造模24h、48h时,仅LPS-A组和LPS-O组高于相应C组(P＜0.05),iNO干预的LPS-NO组和LPS-ONO组已降至正常对照水平,其中造模48h时LPS-NO组和LPS-ONO组较LPS-A组和LPS-O组降低(P＜0.05)。
肺组织PAI-1/uPA蛋白比值在造模4h时,iNO干预的LPS-NO组比值低,与对照组比较无显著差异(P＞0.05),其他气体干预的LPS组则高于相应C组(P＜0.05),LPS-NO组较LPS-A组,LPS-ONO组较LPS-O组有降低趋势,但差异无统计学意义(P＞0.05);造模24h时,所有LPS组均高于相应C组(P＜0.05),造模48h时,只有LPS-NO组降至正常对照水平;在造模24h和48h时iNO干预的LPS-NO组和LPS-ONO组较LPS-A组和LPS-O组降低(P＜0.05)。
④肺组织MMP-9mRNA在造模24h时,所有LPS组高于相应C组(P＜0.05);不同气体干预的LPS组肺组织PAI-1mRNA表达差异无统计学意义(P＞0.05)。
肺组织MMP-9蛋白在造模4h和24h时,所有LPS组较相应C组有增高趋势;造模48h时,LPS-O组高于C-O组(P＜0.05),其他LPS组均在正常对照水平。
⑤肺组织HGFmRNA表达在造模4h和24h时,所有LPS组较相应C组有增高趋势(P＞0.05),其中LPS-NO组高于C-NO组(P＜0.05);造模48h时,所有LPS组HGF mRNA表达下降至正常对照水平。
肺组织HGF蛋白表达在造模4h和24h时,所有LPS组较相应C组增高(P＜0.05),造模48h时,LPS组大鼠HGF蛋白表达均降至正常对照水平。
2.iNO与高氧对急性肺损伤内源性NO系统的影响及与PAI-1表达的关系:
①肺组织iNOS活性在造模4h、24h和48h,所有LPS组较相应C组均有增高趋势,其中在4h、24h时LPS-A组和LPS-O组以及48h时LPS-A组较相应C组增高,差异有统计学意义(P＜0.05);一氧化氮干预后iNOS活性有下降趋势,在造模24h时LPS-NO和LPS-ONO组低于LPS-A组(P＜0.05)。
②肺组织cNOS活性在造模4h和24h时,LPS-A组和LPS-O组大鼠较相应C组有下降趋势;其中造模24h时,LPS-A组较C-A组下降,差异有统计学意义(P＜0.05),LPS-A组较其他气体干预下的LPS组肺组织cNOS活性降低(P＜0.05);造模48h时,LPS-NO组肺组织cNOS活性高于LPS-A组(P＜0.05)。
③肺组织tNOS活性在造模4h时LPS组与相应C组大鼠比较差异无统计学意义;在造模24h和48h时LPS组肺组织tNOS活性较相应C组有增高趋势(P＞0.05)。
④肺组织NO含量以NO_2~-和NO_3~-之和表示。NO含量在造模4h、48h时所有LPS组高于相应C组(P＜0.05);在造模24h时LPS-NO组NO含量下降至正常对照水平,并较其LPS-A组下降(P＜0.05)。其他气体干预的LPS组仍高于相应C组(P＜0.05)。
⑤在造模4h、24h、48h时,iNOS活性与PAI-1 mRNA和蛋白表达均呈正相关(P＜0.05),而cNOS活性与PAI-1 mRNA和蛋白表达均呈负相关(P＜0.05)。在各时点,肺组织NO含量与PAI-1 mRNA和蛋白表达均呈正相关(P＜0.05)。
3.一氧化氮吸入与高氧对肺组织病理学的影响:
肺病理评分在造模4h、24h、48h各时点,所有LPS组均大于相应C组(P＜0.05)。LPS-NO组、LPS-ONO较LPS-A和LPS-O组肺病理评分有下降趋势(P＞0.05),其中24h时,LPS-NO组较LPS-A组下降差异有统计学意义(P＜0.05)。
肺组织纤维素染色C组肺组织未见明显纤维蛋白沉着,LPS组可见肺泡腔,小血管腔及间质有纤维蛋白沉积,LPS-NO组和LPS-ONO组较LPS-A组和LPS-O组有所减轻。
结论:
1.早期20ppmNO吸入可抑制内毒素所致大鼠急性肺损伤PAI-1的高表达,纠正高氧暴露后PAI-1高表达延长,缓解纤溶失衡,减轻肺损伤,同时也为临床急性肺损伤早期iNO干预治疗提供实验依据。
2.iNO降低急性肺损伤肺组织iNOS活性和NO产量,这种作用与PAI-1mRNA和蛋白表达下调密切相关。
PartⅠExpression and Role of PAI-1 and Correlation Factors in a Rat Model of Acute Lung injury
Backgroud:
Acute lung injury(ALI) and acute respiratory distress syndrome(ARDS) are common,life-threatening causes of acute hypoxemic respiratory failure that is associated with both intrapulmonary and extrapulmonary disorders and with no left atrial hypertension.It is well established that acute lung injury and acute respiratory distress syndrome are characterized by local and intense inflammatory responses. Fibroproliferation,with deposition of collagen is an early response to lung injury and an important therapeutic target as well as anti-inflammation.Plasminogen activator inhibitor(PAI)-1 is the key inhibitor of the plasminogen activation system(PAS). The level of PAI-1 gene expression correlates closely with the amount of collagen deposition in lung tissues.PAI-1 blocking MMPs may prevent the extracellular matrix degradation.Enhanced plasminogen activation leads to increased levels of active hepatocyte growth factor(HGF) within the alveolar spaces and contributes to reducing the extent of subsequent fibrosis.Inhibition of the plasminogen system by increased expression of PAI-1 in lung injury leads to abnormal accumulation of fibrin and play an essential role in lung remodeling.
Objectives:
The objectives of the experimental study are 1) to observe changes of PAI-1 expression in a rat model of acute lung injury so as to search suitable therapeutic opportunity;2) to explore expression and role of the factors related to PAI-1 in acute lung injury.
Methods:
Male SD rats aged 4～5weeks(clean conventional rats,180～200g) were primed by lipopolysaccharide(LPS)(0.1mg/kg) via intraperitoneal challege and then followed by a second dose of LPS(1mg/kg) that were given intratracheally 16h later,wheras control groups of rats were given normal saline.LPS-treated rats were sacrificed at 2h、4h、8h、16h、24h、48h and saline-treated rats at 2～4h(marked as baseline)(n=8～10).Measuring of blood gas analysis,histopathological lung injury score,total proteins(TP) and white blood cell(WBC) count in bronchoalveolar lavage fluid(BALF),pulmonary myeloperoxidase(MPO), pulmonary malondialdehyde(MDA) and wet-to-dry lung weight(another six rats sacrificed at baseline,at 4h and 24h respectively) were measured.PAI-1 antigen levels in both plasma and BALF were also measured.Meanwhile,expression of PAI-1, uPA,MMP-9 and HGF mRNA of the lung tissue were evaluated by real-time polymerise chain reaction;PAI-1,uPA,MMP-9 and HGF proteins were determined by immunohistochemistry.Modified MSB stains were performed to evaluate fibrin of the lung tissue.
Results:
1.Establishing acute lung injury in a rat model:At 2～48h time points,arterial partial pressure of oxygen(PaO_2)were decreased when compared to baseline(P＜0.05),and were lower than 60mmHg during 4～8h.In LPS-treated rats,the histopathological lung injury score values at 2h were significantly higherthan that of baseline and the score values in time points after 2h were higher than that of LPS group at 2h(P＜0.05).They were manifested by widerspread neutrophil infiltration, edema,hemorrhage and alveolar atelectasis in the lungs.During 2h～48h after second hit of LPS,TP concentration and WBC counts in BALF,pulmonary MPO were all higher than baseline(P＜0.05) with a trend of progressing.At 4h～48h time points, pulmonary MDA were increased compared with baseline(P＜0.05) with a trend of progressing.At 4h,24h time points,wet-to-dry lung weight showed a trend of increasing.
2.Expression of PAI-1 in the lung tissue:After 2h with two LPS attacks,PAI-1 antigen levels in plasma showed a trend of increasing and PAI-1 levels were increased significantly at 8～16h time points(P＜0.05).PAI-1 antigen levels in BALF and PAI-1 proteins were increased compared with baseline during 2～48h(P＜0.05)with a trend of progressing.Expression of PAI-1 mRNA in lung were increased gradually in LPS groups during 2h～24h(P＜0.05) and decreased at 48h.PAI-1 antigen levels in plasma and BALF,expression of PAI-1 mRNA and proteins had a significant correlation with each other;PAI-1 antigen levels in plasma and BALF,PAI-1 proteins had significant correlations with histopathological lung injury scores(P＜0.05).
3.Expression of the factors related to PAI-1 in the lung tissue:
①Expression of uPA mRNA was increased rapidly after second LPS hit,and at 2h was higher than baseline(P＜0.05).But then uPA mRNA had no significant change during 4～48 time points compared with baseline(P＞0.05).uPA proteins showed a increasing trend at 2h compared with baseline and were decreased at 4h. After 8h with two LPS attacks,uPA proteins were lower than that at 2h and at 24h lower than baseline(P＜0.05).
②PAI-1/uPAmRNA was increased at 2～48h time points in contrast to baseline and PAI-1/uPAmRNA during 8～16h was higher than that at 2h(P＜0.05). PAI-1/uPA ratio was increased at 4h compared with baseline and was significant higher after 8h than that at 4h(P＜0.05).Fibrin deposition in the lung tissure showed the semblable trend with PAI-1 protein and PAI-1/uPA.PAI-1 mRNA/uPA mRNA and PAI-1/uPA had significant correlations with histopathological lung injury scores(P＜0.01 ).
③Expression of MMP-9 mRNA tended to be increased compared with baseline at 2～24h time points and tended to be decresed at 48h in contrast to at 24h(P＞0.05).At 4～24h time points,MMP-9 proteins were higher than baseline(P＜0.05).
④Expression of HGF mRNA tended to be increased at 2～4h time points compared with baseline(P＞0.05) and was higher than baseline at 8～24h(P＜0.05).At 4～24h time points,HGF proteins were higher than baseline(P＜0.05).
Conclusions:
1.Expression of PAI-1 is early elevated in rats with two hits of LPS induced acute lung injury.The prolonged time course of the increase in PAI-1 levels causes imbalance between PAI-1 and uPA and results in depression of the plasminogen activation system in the lung tissue.PAI-1 plays a pivotal role in lung injury and repair and it is important in the progression of ALI.The elevated levels of PAI-1 antigen in BALF corresponding to PAI-1 proteins in the lung tissue predicts severity of ALI.
2.Expression of MMP-9 and HGF are increased at first and then decreased.They are decreased earlier than PAI-1 and it may contribute to abnormal deposition of extracellular matrix and lung remodeling.
PartⅡEffects of Inhaled Nitric Oxide and Hyperoxia on Expression of PAI-1 in a Rat Model of Acute Lung Injury
Backgroud:
Fibrin deposition in the alveolar and lung microvasculature likely results from disordered coagulation and fibrinolysis,triggered by inflammation in ALI.The degree of alterations in coagulation and fibrinolysis may be an important pathogenetic and prognostic determinant of mortality in ALI/ARDS.Modulation of fibrin deposition in the lung through targeting modulation of fibrinolysis may be an important therapeutic target in ALI/ARDS.Locally increased amplification of plasminogrn activator inhiitor-1(PAI-1) is largely responsible for the fibrinolytic defect in the acutely injuried lung.A variety of strategies are being explored to develop inhibitors of PAI-1. Inhibition of PAI-1 has been reported with nitric oxide.Inhaled nitric oxide(iNO) has been demonstrated to improve oxygen by selective pulmonary vasolation to alter ventilation-perfusion mismatching in refractory ARDS.It was reported that iNO prevents pulmonary vascular endothelial cell dysfunction in early-stage of acute lung injury.It's unknown that effects of early intervention with inhaled nitric oxide on expression of PAI-1 in ALI.iNO is usually used in combination with high oxygen supply in critical conditions.An animal study showed that lungs of mice exposed to hyperoxia overproduced plasminogen activator inhibitor-1(PAI-1).Whether iNO together with high oxygen supply would have effects on expression of PAI-1 in ALI remains unknown.NO is synthesized from the amino acid L-arginine by NO synthase (NOS) and released by various cells after chemical and mechanical activation. Exogenous NO may influence endogenous NO system.The relation between exogenous NO and endogenous NO system and the effect on expression of PAI-1 in ALI are still unclear.
Objectives:
The objectives of the experimental study are 1) to explore the effects of inhaled nitric oxide and/or oxygen of high concentration on expression of PAI-1 in early-stage of experimental acute lung injury in a rat model;2) to explore expression and role of correlation factors of PAI-1 with above-mentioned intervention;3) to explore the effects of inhaled nitric oxide and/or oxygen of high concentration on endogenous NO system in experimental acute lung injury.
Methods:
Male SD rats aged 4～5weeks(clean conventional rats,180～200g) received lipopolysaccharide(LPS)(0.1mg/kg ) with intraperitoneal challege followed by a second LPS(1mg/kg) attack via intratracheal injection 16h later,wheras control rats received normal saline.LPS-treated(LPS) rats(n=8～10 per subgroup) and control(C) rats(n=6～8 per subgroup) were randomly allocated to subgroups exposed to:air(A),95%oxygen(O),20ppm iNO(NO),95%oxygen and 20ppm iNO(ONO).They were sacrificed at 4h、24h、48h(NO and/or 95%oxygen exposure for 24h).Expression of PAI-1,uPA,MMP-9 and HGF mRNA of the lung tissue were evaluated by real-time polymerise chain reaction;PAI-1,uPA,MMP-9 and HGF proteins were determined by immunohistochemistry.NO production in the lung tissues and pulmonary NOS activity including total NOS(tNOS),inducible NOS(iNOS) and constitutive NOS(cNOS) were measured.Meanwhile, histopathological lung injury scores were evaluated and modified MSB stains were performed to evaluate fibrin of the lung tissues.
Results:
1.Altered expression of PAI-1 and related factors:
①PAI-1 in lung tissure:At 4 and 24h time points,expression of PAI-1 mRNA in LPS-A subgroup and LPS-O subgroup were elevated in contrast to control;with intervention of iNO,PAI-1 mRNA levels in LPS-NO subgroup and LPS-ONO subgroup were decreased compared with other LPS subgroups.At 48h,only in LPS-O subgroup PAI-1 mRNA levels were higher than control and with iNO that in LPS-ONO subgroup was lower than LPS-O subgroup(P＜0.05).
At 4h,expression of PAI-1 proteins in LPS-NO subgroup were lower than other LPS subgroups and there were no statistical difference between LPS-NO subgroup and control.At 24h,All LPS subgroups had higher PAI-1 protein levels than corresponding control subgroups(P＜0.05).It tended to be decreased in LPS-NO and LPS-ONO subgroups in contrast to other LPS subgroups(P＞0.05).At 48h,with iNO,PAI-1 protein levels in LPS-NO and LPS-ONO were decreased compared with corresponding LPS subgroups(P＜0.05).
②uPA in lung tissure:At 4h,24h and 48 time points,there were no significant difference in uPA mRNA levels between LPS subgroups and corresponding control.
At 24h,uPA protein levels in LPS-A subgroup and LPS-O subgroup were lower than correspongding control and that in LPS subgroups with iNO were increased in contrast to LPS subgroups without iNO(P＜0.05).
③PAI-1/uPA in lung tissue:At 4h,PAI-1 mRNA/uPA mRNA in all LPS subgroups was higher than correponding controls.At 24h and 48h,only in LPS-A and LPS-O subgroups the ratios were higher than correponding controls.At 48h,the ratios in LPS subgroups with iNO were decreased in contrast to LPS subgroups without iNO (P＜0.05).
At 4h,PAI-1/uPA in LPS-NO and LPS-ONO subgroups tended to be decreased compared with LPS-A and LPS-O subgroups respectively.At 24h,the ratios in all LPS subgroups were higher than corresponding controls.At 48h the radios in LPS-NO subgroup were decreased and there were no difference between LPS-NO subgroup and control.At 24h and 48h,the ratios in LPS subgroups with iNO were decreased in contrast to LPS subgroups without iNO(P＜0.05).
④MMP-9 in lung tissue:At 24h,MMP-9 mRNA levels in all LPS subgroups were elevated in contrast to corresponding controls(P＜0.05).There were no difference among all LPS subgroups(P＞0.05).
At 4h and 24h,MMP-9 protein levels in all LPS subgroups tended to be elevated in contrast to corresponding controls(P＞0.05).At 48h,only LPS-O subgroup was higher than control(P＜0.05).
⑤HGF in lung tissue:At 4h and 24h,HGF mRNA levels in all LPS subgroups appeared increasing trend compared with corresponding controls(P＞0.05),and LPS-NO subgroup were marked higher than control(P＜0.05).At 48h,there were no difference in HGF mRNA levels between all LPS subgroups and corresponding controls(P＞0.05).
At 4h and 24h,HGF protein levels in all LPS subgroups were higher than corresponding controls(P＞0.05),and at 48h,there were no difference between all LPS subgroups and corresponding controls(P＞0.05).
2.Altered endogenous NO system consistent with expression of PAI-1:
①iNOS activity in lung tissue:At 4h,24h and 48h,iNOS activity in all LPS subgroups showed a trend of increasing in contrast to corresponding controls,while LPS-A subgroups at all time points and LPS-O subgroup at 4h,24h iNOS activity was higher than corresponding controls,iNOS activity in LPS subgroups with iNO tended to decrease in contrast to LPS subgroups without iNO,and at 24h,LPS-NO and LPS-ONO subgroups were lower than LPS-A subgroup(P＜0.05).
②cNOS activity in lung tissue:At 24h,cNOS activity in LPS-A subgroup was significantly lower than control and other LPS subgroups.And at 48h,cNOS activity in LPS-NO subgroup was increased in contrast to LPS-A subgroup(P＜0.05).
③tNOS activity in lung tissue:At 24h and 48h,tNOS activity in all LPS subgroups showed a trend of increasing compared with corresponding controls(P＞0.05).
④NO productions in lung tissue:At 4h and 48h,NO productions in all LPS subgroups were increased compared with corresponding controls.At 24h,NO productions in LPS-NO subgroup were decreased in contrast to LPS-A subgroup(P＜0.05);There was no difference between LPS-NO subgroup and control(P＞0.05).
⑤Correlations between NO system and PAI-1:At 4h,24h and 48h,iNOS activity had significant correlation with expression of PAI-1 mRNA and protein in lung tissue;cNOS activity had inverse correlation with expression of PAI-1 mRNA and protein in lung tissue;NO production had significant correlation with expression of PAI-1 mRNA and protein in lung tissue(P＜0.05).
3.Histopathologic changes in lung tissue:At 4h,24h and 48h,the histopathologic lung injury scores in all LPS subgroups were increased in contrast to corresponding controls.LPS subgroups with iNO appeared a decreasing trend in contrast to LPS subgroups without iNO and at 24h,the scores in LPS-NO subgroup were lower than LPS-A subgroup(P＜0.05).
Fibrin deposition evaluated by modified MSB stains in LPS subgroups was found in alveolar space,lumen of blood vessel and mesenchymal;LPS subgroups with iNO appeared a decreasing trend in contrast to LPS subgroups without iNO.
Conclusions:
1.Inhaled nitric oxide of 20ppm can suppress elevated expression of PAI-1 in rats with two hits of LPS induced acute lung injury.Exposure to high concentration oxygen prolongs the time course of the increase in PAI-1 mRNA and protein in ALI, while iNO can reduce it.iNO can improve imbalance of plasminogen activation system and alleviate lung injury.
2.Inhaled nitric oxide down-regulates intrapulmonary iNOS activity as well as endogenous nitric oxide productions in rats with two hits of LPS induced acute lung injury.These changes also have a closed correlation with down-regulation of PAI-1 mRNA and protein.
背景:
急性肺损伤(ALI)和急性呼吸窘迫综合征(ARDS)是心源性以外的各种肺内外致病因素导致的急性进行性缺氧性呼吸衰竭,病死率高。研究表明炎症是ALI发病的中心环节,炎症和肺组织重塑及胶原的沉积是同时进行的。纤溶酶原激活物抑制剂-1(PAI-1)是纤溶酶原激活剂主要的抑制物,其基因表达水平与肺中胶原沉积密切相关。PAI-1还通过作用于基质金属酶、肝细胞生长因子、细胞迁移等影响肺损伤修复。
目的:
探索急性肺损伤大鼠PAI-1表达的变化规律,以便寻找合适的干预手段和时机;初步探讨PAI-1相关因子的表达情况及其在急性肺损伤中的意义。
方法:
4～5周清洁级SD雄性大鼠(180～200g)随机分7组:生理盐水组作为基线(B-A),内毒素(LPS)组分别于造模2h、4h、8h、16h、24h、48h处死,即为LPS-A 2h、LPS-A 4h、LPS-A 8h、LPS-A 16h、LPS-A 24h、LPS-A 48h组,每组大鼠8～10只。采用腹腔注射LPS 0.1mg/kg 16h后气管滴入LPS 1mg/kg的二次打击方法诱导大鼠急性肺损伤模型,生理盐水组相应予等量生理盐水处理。测定各时点的血气分析、肺组织病理评分、支气管肺泡灌洗液(BALF)白细胞(WBC)计数和总蛋白(TP)、肺组织匀浆髓过氧化物酶(MPO)活性和丙二醛(MDA)含量、肺湿干重比(B-A、LPS-A 4h、LPS-A 24h组另取6只)。酶联免疫吸附试验(ELISA)测定血浆和BALF中PAI-1水平,实时荧光定量PCR测定肺组织PAI-1及尿激酶型纤溶酶原激活剂(uPA)、基质金属蛋白酶-9(MMP-9)、肝细胞生长因子(HGF)mRNA表达水平,免疫组化法测定肺组织PAI-1及uPA、MMP-9、HGF蛋白表达水平,改良的MSB染色法进行肺纤维素染色。
结果:
1.急性肺损伤模型的建立:LPS二次打击2h后动脉血氧分压(PaO_2)下降,4h、8h达谷底,直至48h均低于基线水平。肺部病理显示二次打击2h即出现急性肺损伤表现,4h病变加重弥漫,高峰持续至24～48h,表现有炎性细胞浸润、肺泡内和间质出血,肺水肿、肺实变不张等。BALF WBC计数和肺组织MPO活性,BALF TP均在2h上升高于基线水平,渐达高峰持续至24～48h,与病理评分变化一致。肺组织MDA含量在4h上升,渐达高峰持续至24～48h(P＜0.05)。肺湿干重比4h有上升趋势,24h有进一步加重的趋势,统计学上无显著差异(P＞0.05)。
2.肺组织PAI-1的表达情况:LPS二次打击后2h血浆PAI-1浓度有上升趋势,8h～16h高于基线水平;而BALF PAI-1水平和肺组织PAI-1蛋白表达水平在二次打击2h直至48h均高于基线水平(P＜0.05),并有进行性加重的趋势;PAI-1mRNA表达在二次打击2h时开始增高,高峰持续于2～16h(P＜0.05),48h恢复至基线水平。血浆与BALF PAI-1浓度、肺组织PAI-1 mRNA和蛋白表达水平均相互呈正相关,其中血浆、BALF PAI-1水平、肺组织PAI-1蛋白的表达与肺病理评分呈正相关(P＜0.05)。
3.肺组织PAI-1相关因子的表达情况:
①LPS二次打击后uPA mRNA表达水平很快增高,2h达高峰(P＜0.05),但4h即降至基线水平;uPA蛋白表达水平有2h上升、4h下降的趋势(P＞0.05),8h后uPA蛋白表达水平均低于2h,24h时最低,低于基线水平(P＜0.05)。
②PAI-1/uPAmRNA在LPS二次打击2h时较基线水平增高,8h～16h达高峰,直至48h仍高于基线水平(P＜0.05),肺组织PAI-1/uPA蛋白比值在LPS二次打击后4h时后高于基线水平,8h及之后各时点高于4h(P＜0.05)。纤维素染色显示肺纤维蛋白沉积的变化趋势与肺组织PAI-1蛋白和PAI-1/uPA蛋白比值的变化趋势基本一致。PAI-1 mRNA/uPA mRNA和PAI-1/uPA蛋白比值与肺病理评分呈正相关(P＜0.01)。
③MMP-9 mRNA在LPS二次打击2h有升高趋势,之后维持在较高水平,48h有下降趋势(P＞0.05);LPS二次打击后4h时MMP-9蛋白表达水平较基线水平增高,持续至24h(P＜0.05),48h降至基线水平。
④HGF mRNA在LPS二次打击后2h、4h有进行性上升趋势,8h时较基线水平增高(P＜0.05),24h下降至基线水平;LPS二次打击后4h时HGF蛋白较基线水平增高,持续至24h(P＜0.05),48h降至基线水平(P＞0.05)。
结论:
1.PAI-1在内毒素诱导的大鼠急性肺损伤早期即表达增高,且持续时间相对长,与uPA之间比例失调导致纤溶失衡;PAI-1高表达和纤溶失衡与急性肺损伤发生发展及异常修复密切相关;BALF PAI-1浓度能较好地反映肺组织PAI-1蛋白的表达,预示急性肺损伤的严重性。
2.MMP-9和HGF在急性肺损伤中表达先增高后降低,下降时间早于PAI-1,可能影响急性肺损伤的细胞外基质清除和肺修复过程。
第二部分一氧化氮和高氧吸入对大鼠急性肺损伤PAI-1表达的影响及其作用
背景:
急性肺损伤时由炎症促发的凝血纤溶功能紊乱是肺泡和肺微血管纤维蛋白沉积的主要原因。纤溶和凝血紊乱影响ALI/ARDS的发生发展和预后,成为治疗ALI/ARDS的新目标。PAI-1增高与ALI/ARDS肺局部纤维蛋白沉积密切相关,因此抑制PAI-1基因的表达可作为急性肺损伤的新的干预点。吸入一氧化氮(iNO)在临床上作为严重ARDS的急救手段,往往在ARDS的晚期机械通气等方法难以控制时使用。有研究表明早期iNO可以预防内毒素引起的血管内皮细胞功能失调。也有资料显示一氧化氮(NO)可抑制PAI-1的表达。我们将采用早期iNO,探讨治疗剂量NO干预对急性肺损伤PAI-1表达的影响。严重ARDS的病人往往高浓度氧疗。研究表明长时间高氧暴露的小鼠过度表达PAI-1。有动物实验提示iNO可降低长期高氧暴露下新生大鼠的病死率。iNO对高氧治疗下急性肺损伤的PAI-1表达的影响尚不明了。体内内皮细胞、上皮细胞、血小板、中性粒细胞、巨噬细胞、神经组织在一定刺激下由NOS合成酶(NOS)合成NO。外源性的NO势必对内源性NO系统产生影响,这种影响对急性肺损伤PAI-1的表达的意义值得进一步探讨。
目的:
探讨吸入一氧化氮和高氧对内毒素(LPS)二次打击诱导大鼠急性肺损伤PAI-1表达的影响及其意义;初步探讨吸入一氧化氮和高氧对急性肺损伤大鼠PAI-1相关因子表达的影响;探讨吸入一氧化氮和高氧后急性肺损伤大鼠内源性NO系统的变化及其与PAI-1表达的关系。
方法:
4～5周清洁级SD雄性大鼠(180～200g)随机分为生理盐水对照组简称C,内毒素造模组简称LPS。采用腹腔注射LPS 0.1mg/kg 16h后气管滴入LPS 1mg/kg的二次打击方法诱导大鼠急性肺损伤模型,生理盐水组相应予等量生理盐水处理。造模后C组和LPS组分别随机给予吸入空气(A)、20ppmNO(NO)、95%氧气(O)、20ppmNO加95%氧气(ONO)4种气体,不同气体干预4h、24h及干预24h观察至造模48h。C组每个时点6～8只大鼠,LPS组每个时点8～10只。实时荧光定量PCR测定肺组织PAI-1及uPA、MMP-9、HGF mRNA表达水平,免疫组化法测定肺组织PAI-1及uPA、MMP-9、HGF蛋白表达水平;改良MSB染色法进行肺纤维素染色,HE染色肺病理评分;测定肺组织总一氧化氮合酶(tNOS)、构成型一氧化氮合酶(cNOS)和诱导型一氧化氮合酶(iNOS)活性以及NO含量。
结果:
1.一氧化氮吸入与高氧对急性肺损伤PAI-1及相关因子表达的影响:
①肺组织PAI-1 mRNA表达在造模4h、24h时,LPS-A组和LPS-O组较相应C组增高,iNO干预的LPS-NO组和LPS-ONO组较LPS-A和LPS-O组降低;造模48h时,LPS-A组表达降至正常对照水平,而高氧干预的LPS-O组表达仍持续增高,高于C-O组,iNO干预的LPS-ONO组较LPS-O组降低(P＜0.05)。
肺组织PAI-1蛋白在造模4h时,iNO干预的LPS-NO组表达低,与对照组无显著差异,并低于其他气体干预的LPS组(P＜0.05);造模24h时,所有LPS组较相应C组表达增高(P＜0.05),iNO干预的LPS-NO组和LPS-ONO组较LPS-A组和LPS-O组有下降趋势(P＞0.05);造模48h时,iNO干预的LPS-NO组和LPS-ONO组降至正常对照水平(P＞0.05),LPS-NO组较其他气体干预的LPS组降低,LPS-ONO组较LPS-O组降低(P＜0.05)。
②肺组织uPA mRNA表达在造模4h、24h、48h各时点,所有LPS组与相应C组比较差异无统计学意义(P＞0.05)。
肺组织uPA蛋白表达在造模24h时LPS-A组和LPS-O组分别低于相应C组(P＜0.05);LPS-NO组和LPS-ONO组较LPS-A组和LPS-O组升高(P＜0.05)。
③肺组织PAI-1 mRNA/uPA mRNA在造模4h时,所有LPS组大鼠较相应C组增高;造模24h、48h时,仅LPS-A组和LPS-O组高于相应C组(P＜0.05),iNO干预的LPS-NO组和LPS-ONO组已降至正常对照水平,其中造模48h时LPS-NO组和LPS-ONO组较LPS-A组和LPS-O组降低(P＜0.05)。
肺组织PAI-1/uPA蛋白比值在造模4h时,iNO干预的LPS-NO组比值低,与对照组比较无显著差异(P＞0.05),其他气体干预的LPS组则高于相应C组(P＜0.05),LPS-NO组较LPS-A组,LPS-ONO组较LPS-O组有降低趋势,但差异无统计学意义(P＞0.05);造模24h时,所有LPS组均高于相应C组(P＜0.05),造模48h时,只有LPS-NO组降至正常对照水平;在造模24h和48h时iNO干预的LPS-NO组和LPS-ONO组较LPS-A组和LPS-O组降低(P＜0.05)。
④肺组织MMP-9mRNA在造模24h时,所有LPS组高于相应C组(P＜0.05);不同气体干预的LPS组肺组织PAI-1mRNA表达差异无统计学意义(P＞0.05)。
肺组织MMP-9蛋白在造模4h和24h时,所有LPS组较相应C组有增高趋势;造模48h时,LPS-O组高于C-O组(P＜0.05),其他LPS组均在正常对照水平。
⑤肺组织HGFmRNA表达在造模4h和24h时,所有LPS组较相应C组有增高趋势(P＞0.05),其中LPS-NO组高于C-NO组(P＜0.05);造模48h时,所有LPS组HGF mRNA表达下降至正常对照水平。
肺组织HGF蛋白表达在造模4h和24h时,所有LPS组较相应C组增高(P＜0.05),造模48h时,LPS组大鼠HGF蛋白表达均降至正常对照水平。
2.iNO与高氧对急性肺损伤内源性NO系统的影响及与PAI-1表达的关系:
①肺组织iNOS活性在造模4h、24h和48h,所有LPS组较相应C组均有增高趋势,其中在4h、24h时LPS-A组和LPS-O组以及48h时LPS-A组较相应C组增高,差异有统计学意义(P＜0.05);一氧化氮干预后iNOS活性有下降趋势,在造模24h时LPS-NO和LPS-ONO组低于LPS-A组(P＜0.05)。
②肺组织cNOS活性在造模4h和24h时,LPS-A组和LPS-O组大鼠较相应C组有下降趋势;其中造模24h时,LPS-A组较C-A组下降,差异有统计学意义(P＜0.05),LPS-A组较其他气体干预下的LPS组肺组织cNOS活性降低(P＜0.05);造模48h时,LPS-NO组肺组织cNOS活性高于LPS-A组(P＜0.05)。
③肺组织tNOS活性在造模4h时LPS组与相应C组大鼠比较差异无统计学意义;在造模24h和48h时LPS组肺组织tNOS活性较相应C组有增高趋势(P＞0.05)。
④肺组织NO含量以NO_2~-和NO_3~-之和表示。NO含量在造模4h、48h时所有LPS组高于相应C组(P＜0.05);在造模24h时LPS-NO组NO含量下降至正常对照水平,并较其LPS-A组下降(P＜0.05)。其他气体干预的LPS组仍高于相应C组(P＜0.05)。
⑤在造模4h、24h、48h时,iNOS活性与PAI-1 mRNA和蛋白表达均呈正相关(P＜0.05),而cNOS活性与PAI-1 mRNA和蛋白表达均呈负相关(P＜0.05)。在各时点,肺组织NO含量与PAI-1 mRNA和蛋白表达均呈正相关(P＜0.05)。
3.一氧化氮吸入与高氧对肺组织病理学的影响:
肺病理评分在造模4h、24h、48h各时点,所有LPS组均大于相应C组(P＜0.05)。LPS-NO组、LPS-ONO较LPS-A和LPS-O组肺病理评分有下降趋势(P＞0.05),其中24h时,LPS-NO组较LPS-A组下降差异有统计学意义(P＜0.05)。
肺组织纤维素染色C组肺组织未见明显纤维蛋白沉着,LPS组可见肺泡腔,小血管腔及间质有纤维蛋白沉积,LPS-NO组和LPS-ONO组较LPS-A组和LPS-O组有所减轻。
结论:
1.早期20ppmNO吸入可抑制内毒素所致大鼠急性肺损伤PAI-1的高表达,纠正高氧暴露后PAI-1高表达延长,缓解纤溶失衡,减轻肺损伤,同时也为临床急性肺损伤早期iNO干预治疗提供实验依据。
2.iNO降低急性肺损伤肺组织iNOS活性和NO产量,这种作用与PAI-1mRNA和蛋白表达下调密切相关。
PartⅠExpression and Role of PAI-1 and Correlation Factors in a Rat Model of Acute Lung injury
Backgroud:
Acute lung injury(ALI) and acute respiratory distress syndrome(ARDS) are common,life-threatening causes of acute hypoxemic respiratory failure that is associated with both intrapulmonary and extrapulmonary disorders and with no left atrial hypertension.It is well established that acute lung injury and acute respiratory distress syndrome are characterized by local and intense inflammatory responses. Fibroproliferation,with deposition of collagen is an early response to lung injury and an important therapeutic target as well as anti-inflammation.Plasminogen activator inhibitor(PAI)-1 is the key inhibitor of the plasminogen activation system(PAS). The level of PAI-1 gene expression correlates closely with the amount of collagen deposition in lung tissues.PAI-1 blocking MMPs may prevent the extracellular matrix degradation.Enhanced plasminogen activation leads to increased levels of active hepatocyte growth factor(HGF) within the alveolar spaces and contributes to reducing the extent of subsequent fibrosis.Inhibition of the plasminogen system by increased expression of PAI-1 in lung injury leads to abnormal accumulation of fibrin and play an essential role in lung remodeling.
Objectives:
The objectives of the experimental study are 1) to observe changes of PAI-1 expression in a rat model of acute lung injury so as to search suitable therapeutic opportunity;2) to explore expression and role of the factors related to PAI-1 in acute lung injury.
Methods:
Male SD rats aged 4～5weeks(clean conventional rats,180～200g) were primed by lipopolysaccharide(LPS)(0.1mg/kg) via intraperitoneal challege and then followed by a second dose of LPS(1mg/kg) that were given intratracheally 16h later,wheras control groups of rats were given normal saline.LPS-treated rats were sacrificed at 2h、4h、8h、16h、24h、48h and saline-treated rats at 2～4h(marked as baseline)(n=8～10).Measuring of blood gas analysis,histopathological lung injury score,total proteins(TP) and white blood cell(WBC) count in bronchoalveolar lavage fluid(BALF),pulmonary myeloperoxidase(MPO), pulmonary malondialdehyde(MDA) and wet-to-dry lung weight(another six rats sacrificed at baseline,at 4h and 24h respectively) were measured.PAI-1 antigen levels in both plasma and BALF were also measured.Meanwhile,expression of PAI-1, uPA,MMP-9 and HGF mRNA of the lung tissue were evaluated by real-time polymerise chain reaction;PAI-1,uPA,MMP-9 and HGF proteins were determined by immunohistochemistry.Modified MSB stains were performed to evaluate fibrin of the lung tissue.
Results:
1.Establishing acute lung injury in a rat model:At 2～48h time points,arterial partial pressure of oxygen(PaO_2)were decreased when compared to baseline(P＜0.05),and were lower than 60mmHg during 4～8h.In LPS-treated rats,the histopathological lung injury score values at 2h were significantly higherthan that of baseline and the score values in time points after 2h were higher than that of LPS group at 2h(P＜0.05).They were manifested by widerspread neutrophil infiltration, edema,hemorrhage and alveolar atelectasis in the lungs.During 2h～48h after second hit of LPS,TP concentration and WBC counts in BALF,pulmonary MPO were all higher than baseline(P＜0.05) with a trend of progressing.At 4h～48h time points, pulmonary MDA were increased compared with baseline(P＜0.05) with a trend of progressing.At 4h,24h time points,wet-to-dry lung weight showed a trend of increasing.
2.Expression of PAI-1 in the lung tissue:After 2h with two LPS attacks,PAI-1 antigen levels in plasma showed a trend of increasing and PAI-1 levels were increased significantly at 8～16h time points(P＜0.05).PAI-1 antigen levels in BALF and PAI-1 proteins were increased compared with baseline during 2～48h(P＜0.05)with a trend of progressing.Expression of PAI-1 mRNA in lung were increased gradually in LPS groups during 2h～24h(P＜0.05) and decreased at 48h.PAI-1 antigen levels in plasma and BALF,expression of PAI-1 mRNA and proteins had a significant correlation with each other;PAI-1 antigen levels in plasma and BALF,PAI-1 proteins had significant correlations with histopathological lung injury scores(P＜0.05).
3.Expression of the factors related to PAI-1 in the lung tissue:
①Expression of uPA mRNA was increased rapidly after second LPS hit,and at 2h was higher than baseline(P＜0.05).But then uPA mRNA had no significant change during 4～48 time points compared with baseline(P＞0.05).uPA proteins showed a increasing trend at 2h compared with baseline and were decreased at 4h. After 8h with two LPS attacks,uPA proteins were lower than that at 2h and at 24h lower than baseline(P＜0.05).
②PAI-1/uPAmRNA was increased at 2～48h time points in contrast to baseline and PAI-1/uPAmRNA during 8～16h was higher than that at 2h(P＜0.05). PAI-1/uPA ratio was increased at 4h compared with baseline and was significant higher after 8h than that at 4h(P＜0.05).Fibrin deposition in the lung tissure showed the semblable trend with PAI-1 protein and PAI-1/uPA.PAI-1 mRNA/uPA mRNA and PAI-1/uPA had significant correlations with histopathological lung injury scores(P＜0.01 ).
③Expression of MMP-9 mRNA tended to be increased compared with baseline at 2～24h time points and tended to be decresed at 48h in contrast to at 24h(P＞0.05).At 4～24h time points,MMP-9 proteins were higher than baseline(P＜0.05).
④Expression of HGF mRNA tended to be increased at 2～4h time points compared with baseline(P＞0.05) and was higher than baseline at 8～24h(P＜0.05).At 4～24h time points,HGF proteins were higher than baseline(P＜0.05).
Conclusions:
1.Expression of PAI-1 is early elevated in rats with two hits of LPS induced acute lung injury.The prolonged time course of the increase in PAI-1 levels causes imbalance between PAI-1 and uPA and results in depression of the plasminogen activation system in the lung tissue.PAI-1 plays a pivotal role in lung injury and repair and it is important in the progression of ALI.The elevated levels of PAI-1 antigen in BALF corresponding to PAI-1 proteins in the lung tissue predicts severity of ALI.
2.Expression of MMP-9 and HGF are increased at first and then decreased.They are decreased earlier than PAI-1 and it may contribute to abnormal deposition of extracellular matrix and lung remodeling.
PartⅡEffects of Inhaled Nitric Oxide and Hyperoxia on Expression of PAI-1 in a Rat Model of Acute Lung Injury
Backgroud:
Fibrin deposition in the alveolar and lung microvasculature likely results from disordered coagulation and fibrinolysis,triggered by inflammation in ALI.The degree of alterations in coagulation and fibrinolysis may be an important pathogenetic and prognostic determinant of mortality in ALI/ARDS.Modulation of fibrin deposition in the lung through targeting modulation of fibrinolysis may be an important therapeutic target in ALI/ARDS.Locally increased amplification of plasminogrn activator inhiitor-1(PAI-1) is largely responsible for the fibrinolytic defect in the acutely injuried lung.A variety of strategies are being explored to develop inhibitors of PAI-1. Inhibition of PAI-1 has been reported with nitric oxide.Inhaled nitric oxide(iNO) has been demonstrated to improve oxygen by selective pulmonary vasolation to alter ventilation-perfusion mismatching in refractory ARDS.It was reported that iNO prevents pulmonary vascular endothelial cell dysfunction in early-stage of acute lung injury.It's unknown that effects of early intervention with inhaled nitric oxide on expression of PAI-1 in ALI.iNO is usually used in combination with high oxygen supply in critical conditions.An animal study showed that lungs of mice exposed to hyperoxia overproduced plasminogen activator inhibitor-1(PAI-1).Whether iNO together with high oxygen supply would have effects on expression of PAI-1 in ALI remains unknown.NO is synthesized from the amino acid L-arginine by NO synthase (NOS) and released by various cells after chemical and mechanical activation. Exogenous NO may influence endogenous NO system.The relation between exogenous NO and endogenous NO system and the effect on expression of PAI-1 in ALI are still unclear.
Objectives:
The objectives of the experimental study are 1) to explore the effects of inhaled nitric oxide and/or oxygen of high concentration on expression of PAI-1 in early-stage of experimental acute lung injury in a rat model;2) to explore expression and role of correlation factors of PAI-1 with above-mentioned intervention;3) to explore the effects of inhaled nitric oxide and/or oxygen of high concentration on endogenous NO system in experimental acute lung injury.
Methods:
Male SD rats aged 4～5weeks(clean conventional rats,180～200g) received lipopolysaccharide(LPS)(0.1mg/kg ) with intraperitoneal challege followed by a second LPS(1mg/kg) attack via intratracheal injection 16h later,wheras control rats received normal saline.LPS-treated(LPS) rats(n=8～10 per subgroup) and control(C) rats(n=6～8 per subgroup) were randomly allocated to subgroups exposed to:air(A),95%oxygen(O),20ppm iNO(NO),95%oxygen and 20ppm iNO(ONO).They were sacrificed at 4h、24h、48h(NO and/or 95%oxygen exposure for 24h).Expression of PAI-1,uPA,MMP-9 and HGF mRNA of the lung tissue were evaluated by real-time polymerise chain reaction;PAI-1,uPA,MMP-9 and HGF proteins were determined by immunohistochemistry.NO production in the lung tissues and pulmonary NOS activity including total NOS(tNOS),inducible NOS(iNOS) and constitutive NOS(cNOS) were measured.Meanwhile, histopathological lung injury scores were evaluated and modified MSB stains were performed to evaluate fibrin of the lung tissues.
Results:
1.Altered expression of PAI-1 and related factors:
①PAI-1 in lung tissure:At 4 and 24h time points,expression of PAI-1 mRNA in LPS-A subgroup and LPS-O subgroup were elevated in contrast to control;with intervention of iNO,PAI-1 mRNA levels in LPS-NO subgroup and LPS-ONO subgroup were decreased compared with other LPS subgroups.At 48h,only in LPS-O subgroup PAI-1 mRNA levels were higher than control and with iNO that in LPS-ONO subgroup was lower than LPS-O subgroup(P＜0.05).
At 4h,expression of PAI-1 proteins in LPS-NO subgroup were lower than other LPS subgroups and there were no statistical difference between LPS-NO subgroup and control.At 24h,All LPS subgroups had higher PAI-1 protein levels than corresponding control subgroups(P＜0.05).It tended to be decreased in LPS-NO and LPS-ONO subgroups in contrast to other LPS subgroups(P＞0.05).At 48h,with iNO,PAI-1 protein levels in LPS-NO and LPS-ONO were decreased compared with corresponding LPS subgroups(P＜0.05).
②uPA in lung tissure:At 4h,24h and 48 time points,there were no significant difference in uPA mRNA levels between LPS subgroups and corresponding control.
At 24h,uPA protein levels in LPS-A subgroup and LPS-O subgroup were lower than correspongding control and that in LPS subgroups with iNO were increased in contrast to LPS subgroups without iNO(P＜0.05).
③PAI-1/uPA in lung tissue:At 4h,PAI-1 mRNA/uPA mRNA in all LPS subgroups was higher than correponding controls.At 24h and 48h,only in LPS-A and LPS-O subgroups the ratios were higher than correponding controls.At 48h,the ratios in LPS subgroups with iNO were decreased in contrast to LPS subgroups without iNO (P＜0.05).
At 4h,PAI-1/uPA in LPS-NO and LPS-ONO subgroups tended to be decreased compared with LPS-A and LPS-O subgroups respectively.At 24h,the ratios in all LPS subgroups were higher than corresponding controls.At 48h the radios in LPS-NO subgroup were decreased and there were no difference between LPS-NO subgroup and control.At 24h and 48h,the ratios in LPS subgroups with iNO were decreased in contrast to LPS subgroups without iNO(P＜0.05).
④MMP-9 in lung tissue:At 24h,MMP-9 mRNA levels in all LPS subgroups were elevated in contrast to corresponding controls(P＜0.05).There were no difference among all LPS subgroups(P＞0.05).
At 4h and 24h,MMP-9 protein levels in all LPS subgroups tended to be elevated in contrast to corresponding controls(P＞0.05).At 48h,only LPS-O subgroup was higher than control(P＜0.05).
⑤HGF in lung tissue:At 4h and 24h,HGF mRNA levels in all LPS subgroups appeared increasing trend compared with corresponding controls(P＞0.05),and LPS-NO subgroup were marked higher than control(P＜0.05).At 48h,there were no difference in HGF mRNA levels between all LPS subgroups and corresponding controls(P＞0.05).
At 4h and 24h,HGF protein levels in all LPS subgroups were higher than corresponding controls(P＞0.05),and at 48h,there were no difference between all LPS subgroups and corresponding controls(P＞0.05).
2.Altered endogenous NO system consistent with expression of PAI-1:
①iNOS activity in lung tissue:At 4h,24h and 48h,iNOS activity in all LPS subgroups showed a trend of increasing in contrast to corresponding controls,while LPS-A subgroups at all time points and LPS-O subgroup at 4h,24h iNOS activity was higher than corresponding controls,iNOS activity in LPS subgroups with iNO tended to decrease in contrast to LPS subgroups without iNO,and at 24h,LPS-NO and LPS-ONO subgroups were lower than LPS-A subgroup(P＜0.05).
②cNOS activity in lung tissue:At 24h,cNOS activity in LPS-A subgroup was significantly lower than control and other LPS subgroups.And at 48h,cNOS activity in LPS-NO subgroup was increased in contrast to LPS-A subgroup(P＜0.05).
③tNOS activity in lung tissue:At 24h and 48h,tNOS activity in all LPS subgroups showed a trend of increasing compared with corresponding controls(P＞0.05).
④NO productions in lung tissue:At 4h and 48h,NO productions in all LPS subgroups were increased compared with corresponding controls.At 24h,NO productions in LPS-NO subgroup were decreased in contrast to LPS-A subgroup(P＜0.05);There was no difference between LPS-NO subgroup and control(P＞0.05).
⑤Correlations between NO system and PAI-1:At 4h,24h and 48h,iNOS activity had significant correlation with expression of PAI-1 mRNA and protein in lung tissue;cNOS activity had inverse correlation with expression of PAI-1 mRNA and protein in lung tissue;NO production had significant correlation with expression of PAI-1 mRNA and protein in lung tissue(P＜0.05).
3.Histopathologic changes in lung tissue:At 4h,24h and 48h,the histopathologic lung injury scores in all LPS subgroups were increased in contrast to corresponding controls.LPS subgroups with iNO appeared a decreasing trend in contrast to LPS subgroups without iNO and at 24h,the scores in LPS-NO subgroup were lower than LPS-A subgroup(P＜0.05).
Fibrin deposition evaluated by modified MSB stains in LPS subgroups was found in alveolar space,lumen of blood vessel and mesenchymal;LPS subgroups with iNO appeared a decreasing trend in contrast to LPS subgroups without iNO.
Conclusions:
1.Inhaled nitric oxide of 20ppm can suppress elevated expression of PAI-1 in rats with two hits of LPS induced acute lung injury.Exposure to high concentration oxygen prolongs the time course of the increase in PAI-1 mRNA and protein in ALI, while iNO can reduce it.iNO can improve imbalance of plasminogen activation system and alleviate lung injury.
2.Inhaled nitric oxide down-regulates intrapulmonary iNOS activity as well as endogenous nitric oxide productions in rats with two hits of LPS induced acute lung injury.These changes also have a closed correlation with down-regulation of PAI-1 mRNA and protein.
引文
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