ALI/ARDS治疗的新途径：肾素-血管紧张素系统
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摘要
目的
由于目前尚无特异有效的治疗方法,急性肺损伤(Acute lung injury,ALI)和急性呼吸窘迫综合征(Acute respiratory distress syndrome,ARDS)的死亡率一直居高不下。最近,肾素-血管紧张素系统(Renin-angiotensin system,RAS)引起关注,有证据提示RAS与ALI/ARDS存在重要关联。为系统研究RAS与ALI/ARDS的关系,我们首先建立盲肠结扎穿孔术(Cecal ligation and puncture,CLP)诱导的ALI/ARDS动物模型,以观察ALI/ARDS中RAS系统的变化规律,并通过干预RAS系统观察动物病情和预后的改变;在此基础上,进一步研究RAS药物氯沙坦干预ALI/ARDS炎症反应通路的机制;同时,还观察ALI/ARDS模型的血管内皮细胞超微结构,并在体外实验验证氯沙坦干预人脐静脉内皮细胞(HUVEC)凋亡的作用。期望通过这一系列的实验,为ALI/ARDS治疗探索新的途径。
研究内容和方法
第一部分: ALI/ARDS动物模型中RAS系统的变化
建立CLP诱导的ALI/ARDS动物模型,在手术后18小时观察小鼠的症状、血气分析、肺湿/干重比(W/D)和肺组织病理等指标;采用免疫组织化学、蛋白质免疫印迹法(Western blotting)和放射免疫等方法研究ALI/ARDS动物模型中RAS系统几种关键酶(ACE、ACE2和AngⅡ)的变化规律。
第二部分:RAS药物对ALI/ARDS的影响
小鼠ALI/ARDS模型复制成功后,分别给小鼠腹腔注射卡托普利(ACE拮抗剂)、重组小鼠ACE2(recombinant mouse ACE2,rm ACE2)和氯沙坦(AngⅡ的AT1受体阻滞剂),观察它们对ALI/ARDS动物的影响,在此基础上,着重观察RAS下游阻滞剂氯沙坦对ALI/ARDS的治疗效果。
第三部分:氯沙坦治疗ALI/ARDS的可能机制
收集假手术组、手术组和氯沙坦组的小鼠肺组织,分别提取肺组织细胞胞浆蛋白和胞核蛋白,采用Western blotting方法检测胞浆IκB-α表达的变化,采用电泳迁移率变动分析实验(Electrophoretic Mobility Shift Assay,EMSA)检测胞核中NF-κB的表达变化;另外提取肺组织总蛋白,采用Western blotting方法检测肺组织中丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)信号通路的变化(包括ERK1/2、p38和JNK)。
第四部分:氯沙坦对人脐静脉内皮细胞凋亡的影响
先用透视电镜观察ALI/ARDS肺血管内皮细胞的变化,提示ALI/ARDS病理变化中存在血管内皮细胞的变化;在此基础上,在体外培养原代人脐静脉内皮细胞(HUVEC),培养成功后先用AngⅡ刺激细胞,采用流式细胞术观察细胞的凋亡率;然后再加入氯沙坦与AngⅡ共同作用于HUVEC,再次测定细胞的凋亡率,评价氯沙坦对血管内皮细胞的保护作用。
研究结果
第一部分: ALI/ARDS动物模型中RAS系统的变化
1、CLP能够诱导小鼠在手术后18小时出现典型的ALI/ARDS改变,手术组较假手术组小鼠肺水含量明显增多(W/D:6.08±0.64 vs 4.38±0.93,P<0.01),明显缺氧(PaO2:40.8±5.03 vs 72.8±4.32,P<0.01),氧合指数明显下降(PaO2/FiO2:194.3±23.9 vs 346.7±20.5,P<0.01),且肺组织病理出现明显的肺损伤改变。
2、Western blotting和免疫组化的结果显示,与假手术组比较,ALI/ARDS小鼠肺组织的ACE2表达下降;而ACE则没有明显差异,手术组和假手术组肺组织中ACE蛋白量均很高。
3、在ALI/ARDS小鼠肺组织和血浆中AngⅡ的量均明显增高,而且加入相关RAS药物后AngⅡ出现变化;与手术组比较,卡托普利(ACE拮抗剂)和rmACE2(重组小鼠ACE2)都能不同程度的抑制体内AngⅡ的生成(肺,1.58±0.16,1.65±0.21 vs 2.38±0.41;血,178.04±17.87,153.74±10.24 vs 213.38±25.44)。
第二部分:RAS药物对ALI/ARDS的影响
1、与手术组比较,卡托普利组小鼠肺W/D明显降低(5.35±0.25 vs 6.06±0.22,P﹤0.05),rmACE2组与手术组的W/D比较虽然无明显统计学差异,但也出现下降趋势(5.61±0.59 vs 6.06±0.22,P=0.096)。相比而言,氯沙坦能更显著的减少ALI/ARDS小鼠肺W/D (5.35±0.25 vs 6.06±0.22,P﹤0.01)。
2、氯沙坦改善肺损伤的程度是与其浓度有关的,在5mg/kg至15mg/kg的范围内,ALI/ARDS小鼠的氧合指数和W/D均得到改善,其改善程度随氯沙坦的浓度增加而增加,但当氯沙坦的浓度超过15mg/kg时,氧合指数和W/D反而逐渐变差,氯沙坦浓度为15mg/kg时治疗ALI/ARDS效果最好。
3、氯沙坦能抑制ALI/ARDS小鼠血浆中TNF-α、IL-1β、IL-6等炎症因子的表达,减轻肺损伤,且能一定程度的改善小鼠7天生存率。
第三部分:氯沙坦治疗ALI/ARDS的可能机制
1、CLP手术诱导的ALI/ARDS小鼠肺组织胞浆中的IκB-α减少,而胞核NF-κB表达明显增强;腹腔注射氯沙坦后,小鼠IκB-α降解程度减少,相应的,细胞核活化的NF-κB也减少了。
2、在CLP手术诱导的ALI/ARDS小鼠肺组织中,ERK1/2、JNK和p38的磷酸化明显增强,而增加氯沙坦后MAPK磷酸化得到不同程度的抑制。
第四部分:氯沙坦对人脐静脉内皮细胞凋亡的影响
1、CLP诱导的ALI/ARDS小鼠肺血管内皮细胞出现水肿或溶解,核周间隙分离增宽,内皮细胞间连接增宽。
2、人脐静脉内皮细胞(Human umbilical vein endothelial cell,HUVEC)加入AngⅡ刺激24小时后,细胞凋亡呈浓度依赖型,AngⅡ(10-7mol/l、10-6mol/l、10-5mol/l)的细胞凋亡率分别是7.85%、9.96%和10.4%。加入10-6mol/l氯沙坦后,HUVEC凋亡率得到改善,细胞凋亡率分别变为:7.95%、8.62%、8.67%。
结论
1、CLP诱导的ALI/ARDS存在RAS系统激活,表现为机体AngⅡ含量明显增加。
2、RAS系统中ACE和ACE2对AngⅡ生成有不同的调节作用,ACE起正调节作用而ACE2起负调节作用;卡托普利和rmACE2都能在一定程度上减少AngⅡ的分泌,改善肺损伤。
3、AngⅡ的AT1受体阻滞剂氯沙坦能够明显改善CLP诱导的ALI/ARDS症状,减轻肺损伤,并能提高7天生存率。
4、氯沙坦治疗ALI/ARDS的可能机制:干预NF-κB和MAPK信号通路,减轻ALI/ARDS的全身性炎症反应。
5、CLP诱导的ALI/ARDS小鼠出现血管内皮细胞损伤;氯沙坦可能通过影响血管内皮细胞的凋亡,减轻血管渗透性,发挥其治疗ALI/ARDS的作用。
6、肾素-血管紧张素系统可能是治疗ALI/ARDS的新途径。
OBJECTIVE:
The lack of specific and efficient therapies is a major cause for the high mortality rate observed in the acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Recently, much research interest was directed towards the role renin-angiotensin system (RAS) played in the pathogenesis of ALI/ARDS. Therefore, this study was undertaken to clarify the relation of ALI/ARDS and RAS by using a mouse cecal ligation and puncture (CLP) model. We also investigated the impacts of RAS drugs on ALI/ARDS and its’mechanisms. In addition, to further elucidate the mechanisms, of losartan prevent lung injury, we looked at the ultrastructure of vascular endothelial cell in ALI/ARDS model, and investigated the impacts of losartan on HUVEC apoptosis in vitro. We hope that these findings can aid in the development of a new therapeutic strategy for ALI/ARDS in future studies.
METHODS:
Part 1. The changes of RAS on ALI/ARDS models
ALI/ARDS animal models were induced by the CLP operaten. The acute lung injury of these models was assessed by measuring blood gas, wet/dry lung weight rate (W/D), and lung tissue histology 18 h after operaten. After the building of the ALI/ARDS models is successful, immunohistochemistry, Western blotting and radioimmunity were used to investigate the changes of several key enzymes of RAS, such as ACE, ACE2 and Ang II.
Part 2. The impacts of RAS drugs on ALI/ARDS
The mice were than divided into three group. Each group received a separate intraperitoneal injection of angiotensin-converting enzyme (ACE) inhibitor captopril, or recombinant mouse ACE2 (rmACE2), or AT1 inhibitor losartan after CLP. These treatments would help in illustrating the effects these drugs have on the ALI/ARDS models, especially losartan.
Part 3. The possible mechanisms of losartan prevented lung injury
Lung tissue samples were collected from sacrificed samples of the SHAM group, CLP group and losartan group. Then, nuclear protein and cytosol protein extraction were performed. Then the extraction is subjected to Western blotting or EMSA (Electrophoretic Mobility Shift Assay) which NF-ΚB activations, IκB-degradations, phosphorylations of p38 MAPK, ERK1/2, and JNK expressions are evaluated.
Part 4. The impacts of losartan on HUVEC apoptosis
To further elucidate the injure pervention mechanisms of losartan in the lung, we observed the ultrastructure of lung vascular endothelial cell in ALI/ARDS model. Moreover, to investigate the impacts of losartan on HUVEC (Human umbilical vein endothelial cell), we conducted the apoptosis in vitro experiments on the models. HUVEC apoptosis rate was measured by the flow cytometry at 24 hours after AngII treatment. Losartan was then added to the Ang II treated HUVEC. Then it was measured again for the apoptosis rate as described previously.
RESULTS:
Part 1. The changes of RAS on ALI/ARDS models
1. The common lung injuries induced by CLP challenge typically features the thickening of the alveolar septa, alveolar hemorrhage, and infiltration of the inflammatory cells. All these features can be observed in the lung tissue from CLP-treated animals 18 h after operation. Furthermore, the CLP-induced ALI/ARDS led to an increase in the wet/dry weight rate (W/D:6.08±0.64 vs 4.38±0.93, P<0.01. vs sham group) of the lung tissues, and a decrease in the PaO2/FiO2( 194.3±23.9 vs 346.7±20.5. P<0.01, vs sham group).
2. Immunohistochemistry and Western blotting tests of the lung tissues from CLP-treated animals showed a decrease in the ACE2 protein level. However, in both the CLP and sham mice there is a very high level of ACE protein present, and there is no significant differences between the two groups.
3. CLP markedly increased Ang II level in lungs and plasma of mice, and several RAS drugs significantly impacted the Ang II levels of mice. Compared with the CLP group, captopril or rmACE2 led to decreasing of the Ang II level in mice (Lung,1.58±0.16, 1.65±0.21 vs 2.38±0.41; Plasma,178.04±17.87, 153.74±10.24 vs 213.38±25.44).
Part 2. The impacts of RAS drugs on ALI/ARDS
1. Compared with the CLP group, W/D rate of lung tissue decreased significantly in captopril group (5.35±0.25 vs 6.06±0.22, P<0.05). But the W/D rate of rmACE2 group was no significant different from controls, whereas a decreased tendency was observed (5.61±0.59 vs 6.06±0.22, P=0.096). The losartan administration had led to a more significant decrease in W/D rate of the models when compared with the captopril group and rmACE2 group.
2. The effectiviness of losartan in the prevention of the ALI/ARDS is partialy depended on the concentrate of losartan administered. The index of lung injury measured shown a persistent improving trend in the mice that received intraperitoneal injections of 5 mg/kg -15 mg/kg losartan. However, a decreasing trend is also observed in mice that received a dose of over 15 mg/kg. The maximum improvement was observed at 15 mg/kg losartan injection.
3. Losartan treatment significantly attenuated TNF-α, IL-6, and IL-1β6 h after CLP, prevented histopathologic appearance of ALI/ARDS after CLP, and significantly improved 7-day survival in mice.
Part 3. The possible mechanisms of losartan prevented lung injury
1. Western blot analysis showed that CLP mice lung cytosolic extracts exhibited a degradation in the amount of sham IκB-αlevels in the cytosol , whereas similar degradation was not observed in the CLP+losartan lung tissues. Accordingly, CLP-induced activation of NF-κB signaling was inhibited significantly by losartan treatment 6 h after CLP in an EMSA experiment.
2. Losartan given post - CLP led to the inhibition of the phosphorylation of p38MAPK, ERK1/2, and JNK pathways, which are critical for cytokine release in ALI/ARDS models.
Part 4. The impacts of losartan on HUVEC apoptosis
1. Cell edema or dissolved, separated widened perinuclear space, widened intercellular junctions of cells was detected in the lung vascular endothelial cell of CLP-induced ALI/ARDS mice.
2. 24 hours after, the Ang II stimulation of the HUVEC cell apoptosis was dose-dependent. The cell apoptosis rate of three AngII groups (10-7mol/l, 10-6mol/l,10-5mol/l) were 7.85 %, 9.96% and 10.4 % respectively. After adding 10-6mol/l losartan, HUVEC apoptosis rate was decreased to 7.95%, 8.62%, 8.67% accordingly.
CONCLUSIONS :
1. RAS activation is one of the casusative agent of CLP-induced ALI/ARDS in mice models. And they are manifested as an increased secretion of AngⅡ.
2. ACE and ACE2 in RAS have a different role in the regulation of the synthesis AngⅡ. While ACE has a positive effect in generating AngⅡ, ACE2 shows a negative effect. At the same time, captopril (ACE inhibitor) and rmACE2 can also partly reduce the secretion of AngⅡ, and thus prevent lung injuries of CLP-induced ALI/ARDS.
3. The AT1 inhibitor, Losartan, can prevent the manifestation of blood gas and histopathologic in ALI/ARDS after CLP, and significantly improved the 7-day survival rate.
4. The possible mechanisms of the prevention lung injury by Losartan are: the intervention of NF-κB and MAPK signaling pathway that reduced the ALI/ARDS systemic inflammatory responses.
5. Since vascular endothelial cell injuries are observed in CLP-induced ALI/ARDS mice, Losartan’s ability to decrease vascular endothelial cell apoptosis and reduces vascular permeability, can potiential become an effective treatment for ALI / ARDS.
6. RAS may be a new therapeutic strategy for ALI/ARDS.
由于目前尚无特异有效的治疗方法,急性肺损伤(Acute lung injury,ALI)和急性呼吸窘迫综合征(Acute respiratory distress syndrome,ARDS)的死亡率一直居高不下。最近,肾素-血管紧张素系统(Renin-angiotensin system,RAS)引起关注,有证据提示RAS与ALI/ARDS存在重要关联。为系统研究RAS与ALI/ARDS的关系,我们首先建立盲肠结扎穿孔术(Cecal ligation and puncture,CLP)诱导的ALI/ARDS动物模型,以观察ALI/ARDS中RAS系统的变化规律,并通过干预RAS系统观察动物病情和预后的改变;在此基础上,进一步研究RAS药物氯沙坦干预ALI/ARDS炎症反应通路的机制;同时,还观察ALI/ARDS模型的血管内皮细胞超微结构,并在体外实验验证氯沙坦干预人脐静脉内皮细胞(HUVEC)凋亡的作用。期望通过这一系列的实验,为ALI/ARDS治疗探索新的途径。
研究内容和方法
第一部分: ALI/ARDS动物模型中RAS系统的变化
建立CLP诱导的ALI/ARDS动物模型,在手术后18小时观察小鼠的症状、血气分析、肺湿/干重比(W/D)和肺组织病理等指标;采用免疫组织化学、蛋白质免疫印迹法(Western blotting)和放射免疫等方法研究ALI/ARDS动物模型中RAS系统几种关键酶(ACE、ACE2和AngⅡ)的变化规律。
第二部分:RAS药物对ALI/ARDS的影响
小鼠ALI/ARDS模型复制成功后,分别给小鼠腹腔注射卡托普利(ACE拮抗剂)、重组小鼠ACE2(recombinant mouse ACE2,rm ACE2)和氯沙坦(AngⅡ的AT1受体阻滞剂),观察它们对ALI/ARDS动物的影响,在此基础上,着重观察RAS下游阻滞剂氯沙坦对ALI/ARDS的治疗效果。
第三部分:氯沙坦治疗ALI/ARDS的可能机制
收集假手术组、手术组和氯沙坦组的小鼠肺组织,分别提取肺组织细胞胞浆蛋白和胞核蛋白,采用Western blotting方法检测胞浆IκB-α表达的变化,采用电泳迁移率变动分析实验(Electrophoretic Mobility Shift Assay,EMSA)检测胞核中NF-κB的表达变化;另外提取肺组织总蛋白,采用Western blotting方法检测肺组织中丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)信号通路的变化(包括ERK1/2、p38和JNK)。
第四部分:氯沙坦对人脐静脉内皮细胞凋亡的影响
先用透视电镜观察ALI/ARDS肺血管内皮细胞的变化,提示ALI/ARDS病理变化中存在血管内皮细胞的变化;在此基础上,在体外培养原代人脐静脉内皮细胞(HUVEC),培养成功后先用AngⅡ刺激细胞,采用流式细胞术观察细胞的凋亡率;然后再加入氯沙坦与AngⅡ共同作用于HUVEC,再次测定细胞的凋亡率,评价氯沙坦对血管内皮细胞的保护作用。
研究结果
第一部分: ALI/ARDS动物模型中RAS系统的变化
1、CLP能够诱导小鼠在手术后18小时出现典型的ALI/ARDS改变,手术组较假手术组小鼠肺水含量明显增多(W/D:6.08±0.64 vs 4.38±0.93,P<0.01),明显缺氧(PaO2:40.8±5.03 vs 72.8±4.32,P<0.01),氧合指数明显下降(PaO2/FiO2:194.3±23.9 vs 346.7±20.5,P<0.01),且肺组织病理出现明显的肺损伤改变。
2、Western blotting和免疫组化的结果显示,与假手术组比较,ALI/ARDS小鼠肺组织的ACE2表达下降;而ACE则没有明显差异,手术组和假手术组肺组织中ACE蛋白量均很高。
3、在ALI/ARDS小鼠肺组织和血浆中AngⅡ的量均明显增高,而且加入相关RAS药物后AngⅡ出现变化;与手术组比较,卡托普利(ACE拮抗剂)和rmACE2(重组小鼠ACE2)都能不同程度的抑制体内AngⅡ的生成(肺,1.58±0.16,1.65±0.21 vs 2.38±0.41;血,178.04±17.87,153.74±10.24 vs 213.38±25.44)。
第二部分:RAS药物对ALI/ARDS的影响
1、与手术组比较,卡托普利组小鼠肺W/D明显降低(5.35±0.25 vs 6.06±0.22,P﹤0.05),rmACE2组与手术组的W/D比较虽然无明显统计学差异,但也出现下降趋势(5.61±0.59 vs 6.06±0.22,P=0.096)。相比而言,氯沙坦能更显著的减少ALI/ARDS小鼠肺W/D (5.35±0.25 vs 6.06±0.22,P﹤0.01)。
2、氯沙坦改善肺损伤的程度是与其浓度有关的,在5mg/kg至15mg/kg的范围内,ALI/ARDS小鼠的氧合指数和W/D均得到改善,其改善程度随氯沙坦的浓度增加而增加,但当氯沙坦的浓度超过15mg/kg时,氧合指数和W/D反而逐渐变差,氯沙坦浓度为15mg/kg时治疗ALI/ARDS效果最好。
3、氯沙坦能抑制ALI/ARDS小鼠血浆中TNF-α、IL-1β、IL-6等炎症因子的表达,减轻肺损伤,且能一定程度的改善小鼠7天生存率。
第三部分:氯沙坦治疗ALI/ARDS的可能机制
1、CLP手术诱导的ALI/ARDS小鼠肺组织胞浆中的IκB-α减少,而胞核NF-κB表达明显增强;腹腔注射氯沙坦后,小鼠IκB-α降解程度减少,相应的,细胞核活化的NF-κB也减少了。
2、在CLP手术诱导的ALI/ARDS小鼠肺组织中,ERK1/2、JNK和p38的磷酸化明显增强,而增加氯沙坦后MAPK磷酸化得到不同程度的抑制。
第四部分:氯沙坦对人脐静脉内皮细胞凋亡的影响
1、CLP诱导的ALI/ARDS小鼠肺血管内皮细胞出现水肿或溶解,核周间隙分离增宽,内皮细胞间连接增宽。
2、人脐静脉内皮细胞(Human umbilical vein endothelial cell,HUVEC)加入AngⅡ刺激24小时后,细胞凋亡呈浓度依赖型,AngⅡ(10-7mol/l、10-6mol/l、10-5mol/l)的细胞凋亡率分别是7.85%、9.96%和10.4%。加入10-6mol/l氯沙坦后,HUVEC凋亡率得到改善,细胞凋亡率分别变为:7.95%、8.62%、8.67%。
结论
1、CLP诱导的ALI/ARDS存在RAS系统激活,表现为机体AngⅡ含量明显增加。
2、RAS系统中ACE和ACE2对AngⅡ生成有不同的调节作用,ACE起正调节作用而ACE2起负调节作用;卡托普利和rmACE2都能在一定程度上减少AngⅡ的分泌,改善肺损伤。
3、AngⅡ的AT1受体阻滞剂氯沙坦能够明显改善CLP诱导的ALI/ARDS症状,减轻肺损伤,并能提高7天生存率。
4、氯沙坦治疗ALI/ARDS的可能机制:干预NF-κB和MAPK信号通路,减轻ALI/ARDS的全身性炎症反应。
5、CLP诱导的ALI/ARDS小鼠出现血管内皮细胞损伤;氯沙坦可能通过影响血管内皮细胞的凋亡,减轻血管渗透性,发挥其治疗ALI/ARDS的作用。
6、肾素-血管紧张素系统可能是治疗ALI/ARDS的新途径。
OBJECTIVE:
The lack of specific and efficient therapies is a major cause for the high mortality rate observed in the acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Recently, much research interest was directed towards the role renin-angiotensin system (RAS) played in the pathogenesis of ALI/ARDS. Therefore, this study was undertaken to clarify the relation of ALI/ARDS and RAS by using a mouse cecal ligation and puncture (CLP) model. We also investigated the impacts of RAS drugs on ALI/ARDS and its’mechanisms. In addition, to further elucidate the mechanisms, of losartan prevent lung injury, we looked at the ultrastructure of vascular endothelial cell in ALI/ARDS model, and investigated the impacts of losartan on HUVEC apoptosis in vitro. We hope that these findings can aid in the development of a new therapeutic strategy for ALI/ARDS in future studies.
METHODS:
Part 1. The changes of RAS on ALI/ARDS models
ALI/ARDS animal models were induced by the CLP operaten. The acute lung injury of these models was assessed by measuring blood gas, wet/dry lung weight rate (W/D), and lung tissue histology 18 h after operaten. After the building of the ALI/ARDS models is successful, immunohistochemistry, Western blotting and radioimmunity were used to investigate the changes of several key enzymes of RAS, such as ACE, ACE2 and Ang II.
Part 2. The impacts of RAS drugs on ALI/ARDS
The mice were than divided into three group. Each group received a separate intraperitoneal injection of angiotensin-converting enzyme (ACE) inhibitor captopril, or recombinant mouse ACE2 (rmACE2), or AT1 inhibitor losartan after CLP. These treatments would help in illustrating the effects these drugs have on the ALI/ARDS models, especially losartan.
Part 3. The possible mechanisms of losartan prevented lung injury
Lung tissue samples were collected from sacrificed samples of the SHAM group, CLP group and losartan group. Then, nuclear protein and cytosol protein extraction were performed. Then the extraction is subjected to Western blotting or EMSA (Electrophoretic Mobility Shift Assay) which NF-ΚB activations, IκB-degradations, phosphorylations of p38 MAPK, ERK1/2, and JNK expressions are evaluated.
Part 4. The impacts of losartan on HUVEC apoptosis
To further elucidate the injure pervention mechanisms of losartan in the lung, we observed the ultrastructure of lung vascular endothelial cell in ALI/ARDS model. Moreover, to investigate the impacts of losartan on HUVEC (Human umbilical vein endothelial cell), we conducted the apoptosis in vitro experiments on the models. HUVEC apoptosis rate was measured by the flow cytometry at 24 hours after AngII treatment. Losartan was then added to the Ang II treated HUVEC. Then it was measured again for the apoptosis rate as described previously.
RESULTS:
Part 1. The changes of RAS on ALI/ARDS models
1. The common lung injuries induced by CLP challenge typically features the thickening of the alveolar septa, alveolar hemorrhage, and infiltration of the inflammatory cells. All these features can be observed in the lung tissue from CLP-treated animals 18 h after operation. Furthermore, the CLP-induced ALI/ARDS led to an increase in the wet/dry weight rate (W/D:6.08±0.64 vs 4.38±0.93, P<0.01. vs sham group) of the lung tissues, and a decrease in the PaO2/FiO2( 194.3±23.9 vs 346.7±20.5. P<0.01, vs sham group).
2. Immunohistochemistry and Western blotting tests of the lung tissues from CLP-treated animals showed a decrease in the ACE2 protein level. However, in both the CLP and sham mice there is a very high level of ACE protein present, and there is no significant differences between the two groups.
3. CLP markedly increased Ang II level in lungs and plasma of mice, and several RAS drugs significantly impacted the Ang II levels of mice. Compared with the CLP group, captopril or rmACE2 led to decreasing of the Ang II level in mice (Lung,1.58±0.16, 1.65±0.21 vs 2.38±0.41; Plasma,178.04±17.87, 153.74±10.24 vs 213.38±25.44).
Part 2. The impacts of RAS drugs on ALI/ARDS
1. Compared with the CLP group, W/D rate of lung tissue decreased significantly in captopril group (5.35±0.25 vs 6.06±0.22, P<0.05). But the W/D rate of rmACE2 group was no significant different from controls, whereas a decreased tendency was observed (5.61±0.59 vs 6.06±0.22, P=0.096). The losartan administration had led to a more significant decrease in W/D rate of the models when compared with the captopril group and rmACE2 group.
2. The effectiviness of losartan in the prevention of the ALI/ARDS is partialy depended on the concentrate of losartan administered. The index of lung injury measured shown a persistent improving trend in the mice that received intraperitoneal injections of 5 mg/kg -15 mg/kg losartan. However, a decreasing trend is also observed in mice that received a dose of over 15 mg/kg. The maximum improvement was observed at 15 mg/kg losartan injection.
3. Losartan treatment significantly attenuated TNF-α, IL-6, and IL-1β6 h after CLP, prevented histopathologic appearance of ALI/ARDS after CLP, and significantly improved 7-day survival in mice.
Part 3. The possible mechanisms of losartan prevented lung injury
1. Western blot analysis showed that CLP mice lung cytosolic extracts exhibited a degradation in the amount of sham IκB-αlevels in the cytosol , whereas similar degradation was not observed in the CLP+losartan lung tissues. Accordingly, CLP-induced activation of NF-κB signaling was inhibited significantly by losartan treatment 6 h after CLP in an EMSA experiment.
2. Losartan given post - CLP led to the inhibition of the phosphorylation of p38MAPK, ERK1/2, and JNK pathways, which are critical for cytokine release in ALI/ARDS models.
Part 4. The impacts of losartan on HUVEC apoptosis
1. Cell edema or dissolved, separated widened perinuclear space, widened intercellular junctions of cells was detected in the lung vascular endothelial cell of CLP-induced ALI/ARDS mice.
2. 24 hours after, the Ang II stimulation of the HUVEC cell apoptosis was dose-dependent. The cell apoptosis rate of three AngII groups (10-7mol/l, 10-6mol/l,10-5mol/l) were 7.85 %, 9.96% and 10.4 % respectively. After adding 10-6mol/l losartan, HUVEC apoptosis rate was decreased to 7.95%, 8.62%, 8.67% accordingly.
CONCLUSIONS :
1. RAS activation is one of the casusative agent of CLP-induced ALI/ARDS in mice models. And they are manifested as an increased secretion of AngⅡ.
2. ACE and ACE2 in RAS have a different role in the regulation of the synthesis AngⅡ. While ACE has a positive effect in generating AngⅡ, ACE2 shows a negative effect. At the same time, captopril (ACE inhibitor) and rmACE2 can also partly reduce the secretion of AngⅡ, and thus prevent lung injuries of CLP-induced ALI/ARDS.
3. The AT1 inhibitor, Losartan, can prevent the manifestation of blood gas and histopathologic in ALI/ARDS after CLP, and significantly improved the 7-day survival rate.
4. The possible mechanisms of the prevention lung injury by Losartan are: the intervention of NF-κB and MAPK signaling pathway that reduced the ALI/ARDS systemic inflammatory responses.
5. Since vascular endothelial cell injuries are observed in CLP-induced ALI/ARDS mice, Losartan’s ability to decrease vascular endothelial cell apoptosis and reduces vascular permeability, can potiential become an effective treatment for ALI / ARDS.
6. RAS may be a new therapeutic strategy for ALI/ARDS.
引文
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2. Vincent, J. L., Sakr, Y. & Ranieri, V. M. Epidemiology and outcome of acute respiratory failure in intensive care unit patients. Crit. Care Med. 31 (suppl.), S296–-S299 (2003).
3. Douglas GC, O′Bryan MK, Hedger MP, Lee DK,Yarsk MA, Smith A I , and Lew RA. The novel angiotensin-converting enzyme (ACE) homolog, ACE2, is selectively expressed by adult Leydig cells of the testis. Endocrinology,2004,145, 4703– 4711.
4. Tipnis SR, Hooper NM, Hyde R, Karran E,Christie G, and Turner AJ. A human homolog of angiotensin converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase. J Biol Chem,2000,275, 33238– 33243.
5. Imai Y , Kuba K , Rao S , Huan Y , Guo F, Guan B, Yang P, Sarao R, Wada T, Leong-Poi H, Crackower MA, Fukamizu A, Hui CC, Hein L, Uhlig S, Slutsky AS, Jiang C, and Penninger JM. Angiotensin-converting enzyme 2 protects from severe acute lung failure. Nature 2005. 436, 112– 116.
6. Lavoie JL, Sigmund CD. Minireview: overview of the renin-angiotensin system-an endocrine and paracrine system. Endocrinology, 2003;144:2179-2183.
7. Filippatos G, Tilak M, Pinillos H, Uhal BD. Regulation of apoptosis by angiotensin II in the heart and lungs. Int J Mol Med, 2001,7 :273-280.
8. Orte C, Polak JM, Haworth SG, Yacoub MH, Morrell NW. Expression of pulmonary vascular angiotensin-converting enzyme in primary and secondary plexiform pulmonary hypertension. J Pathol,2000,192,379-384.
9. Otsuka M, Takahashi H, Shiratori M, Chiba H, Abe S: Reduction of bleomycin induced lung fibrosis by candesartan cilexetil, an angiotensin II type 1 receptor antagonist. Thorax 2004, 59:31-38.
10.陆艳辉,惠汝太,赵彦芬,刘国仗,柳志红,陈白屏,叶珏,张春玲.ACE基因插入/缺失多态与国人肺血栓栓塞症的关联研究.中华结核和呼吸杂志2001 , 24,265-268。
11.Marshall RP, Webb S, Bellingan GJ,Montgomery HE, Chaudhari B, McAnulty RJ, Humphries SE, Hill MR, and Laurent GJ. Angiotensin converting enzyme insertion/deletion polymorphism is associated with susceptibility and outcome in acute respiratory distress syndrome. Am J Respir Crit Care Med. 2002 166, 646– 650.
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