硫化氢对大鼠内毒素性急性肺损伤的作用及其机制研究
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摘要
急性肺损伤(acute lung injury,ALI)是指由心源性以外的各种肺内外致病因素所导致的急性、进行性、缺氧性呼吸衰竭,主要表现为顽固性呼吸困难、低氧血症、肺顺应性降低等。细菌感染所致的脓毒血症是引起ALI的主要原因,革兰氏阴性菌(gramnegative bacterium,G-)细胞壁的主要成分脂多糖(1ipopolysaccharide,LPS)是主要的致病因素,利用LPS复制ALI的动物模型进行研究是科研工作中的常用手段之一。
内源性硫化氢(hydrogen sulfide,H2S)主要由半胱氨酸等含硫氨基酸在胱硫醚-β-合成酶(cystathionine-β-synthase,CBS)和胱硫醚-γ-裂解酶(cystathionine-γ-lyase,CSE)作用下生成,它对神经系统特别是海马的功能具有调节作用,并可以调节消化道和血管平滑肌的张力。随着对H2S研究的逐渐深入,越来越多的证据证实它参与多种呼吸系统疾病的发生发展过程,是继一氧化氮(nitric oxide,NO)和一氧化碳(carbon monoxide,CO)之后的一种新型气体信使分子。本实验首先观察了在内毒素性ALI病程中H2S和CSE的时程性变化,在此基础上分别给予H2S供体氢硫化钠(sodium hydrosulfide,NaHS)和CSE抑制剂炔丙基甘氨酸(DL-propargylglycine,PPG),观察它们对肺组织氧化应激、炎症细胞、炎症介质、细胞凋亡和肺表面活性物质(pulmoalverolar surfactant,PS)等方面的影响,从细胞、基因、蛋白水平多层次、多方面探讨H2S在肺损伤中的作用及机制,为临床治疗肺损伤提供理论依据。
第一部分LPS致大鼠急性肺损伤硫化氢/胱硫醚-γ-裂解酶体系的变化
目的:观察大鼠内毒素性ALI过程中白介素-1β(IL-1β)、白介素-10(IL-10)和H2S/CSE体系的时程性变化及关系,探讨LPS诱导ALI的发病机制。
方法:健康雄性SD大鼠(270±20g)共80只,随机分为6组,Ⅰ空白对照组;ⅡLPS 1 h组;ⅢLPS 3 h组;ⅣLPS 6 h组;ⅤLPS 9 h组;ⅥLPS 12 h组,空白对照组共40只(不同时间点各8只)。用20%的乌拉坦(5 ml/kg)麻醉大鼠,LPS 1 h、3 h、6 h、9 h和12 h组舌静脉注射LPS(5 mg/kg)。分别于给药1 h、3 h、6 h、9 h及12 h后采集样品;检测肺系数和肺湿/干重比的变化;去蛋白法检测血浆中H2S含量、亚甲基蓝法检测肺组织CSE活性、酶联免疫吸附法(ELISA)检测血清中IL-1β和IL-10的变化;光镜、电镜观察肺组织病理变化。
结果:
1.空白对照组大鼠肺系数和肺湿/干重比在1 h ~ 12 h之间无明显变化。给予LPS 1 h后大鼠肺系数和肺湿/干重比与空白对照组比较无显著差异(0.47±0.02 VS 0.46±0.03,4.65±0.34 VS 4.41±0.33,P > 0.05),给予LPS 3 h后肺系数和肺湿/干重比开始明显升高(0.52±0.03 VS 0.47±0.03,5.24±0.45 VS 4.43±0.21,P < 0.05),给予LPS 6 h、9 h和12 h后肺系数和肺湿/干重比均比空白对照组升高且维持在较高水平(P < 0.05或P < 0.01)。
2.空白对照组大鼠血清中IL-1β和IL-10浓度在1 h ~ 12 h之间无明显变化。给予LPS 1 h后大鼠血清中IL-1β浓度较空白对照组显著升高(11.18±0.59 VS 9.26±1.37,P < 0.05)并达高峰,给予LPS 3 h、6 h、9 h及12 h后血清中IL-1β浓度较LPS 1 h随时程延长逐渐下降,但与空白对照组比较均明显升高(P < 0.05或P < 0.01)。给予LPS 1 h后大鼠血清中IL-10浓度较空白对照组显著升高(213.46±12.84 VS 11.84±1.25,P < 0.01),给予LPS 3 h时达高峰(230.46±9.49 VS 10.73±9.97,P < 0.01),6 h、9 h及12 h后血清中IL-10浓度较LPS 1 h和3 h时降低,并随时程延长逐渐下降,但与空白对照组比较均明显升高(P < 0.01)。
3.空白对照组大鼠血浆中H2S含量和肺组织CSE活性在1 h ~ 12 h之间无明显变化。给予LPS 1 h后大鼠血浆中H2S含量和肺组织CSE活性较空白对照组无显著差异(35.10±3.81 VS 37.22±4.48,4.61±0.75 VS 4.68±0.63,P > 0.05),给予LPS 3 h后血浆中H2S含量和肺组织CSE活性明显降低(24.90±2.21 VS 36.67±5.35,3.35±0.98 VS 4.61±0.68,P < 0.01),给予LPS 6 h、9 h和12 h后血浆中H2S含量和肺组织CSE活性与空白对照组比较均明显降低(P < 0.01)。
4.高倍光镜下显示,空白对照组肺泡结构完整,给予LPS 1 h后无明显变化,给予LPS 3 h、6 h、9 h及12 h后肺组织明显受损,可见肺泡间隔增宽,肺泡完整性被破坏,腔内有大量渗出,炎性细胞浸润。电镜下显示,空白对照组肺泡Ⅱ型上皮细胞超微结构基本正常,给予LPS 1 h后无明显变化,给予LPS 3 h、6 h、9 h及12 h后肺组织超微结构明显受损,粗面内质网脱颗粒,线粒体水肿、嵴减少嗜锇性板层小体的板层结构显著融合或消失。
结论:大鼠静脉注射LPS后,1 h时肺组织未出现明显损伤,但炎性细胞因子显著升高。给予LPS 3 h至12 h时均存在肺损伤,IL-1β和IL-10均明显升高,内源性H2S和CSE均明显降低。IL-1β、IL-10和H2S/ CSE体系可能参与内毒素性ALI的病理生理过程。
第二部分硫化氢对大鼠内毒素性急性肺损伤组织氧化应激的影响
目的:观察H2S对大鼠内毒素性ALI组织过氧化物和抗氧化物酶的影响,并探讨其作用机制。
方法:健康成年雄性SD大鼠(270±20g)共48只,随机分为6组,每组8只,分别为Ⅰ空白对照组;ⅡLPS组;ⅢLPS+NaHS低剂量组;ⅣLPS+NaHS中剂量组;ⅤLPS+NaHS高剂量组;ⅥLPS+PPG组。用20%的乌拉坦(5 ml/kg)麻醉大鼠,LPS组舌静脉注射LPS,LPS+NaHS低、中、高剂量组分别于舌静脉注射LPS 3 h时腹腔注射0.78 mg/kg、1.56 mg/kg和3.12 mg/kg的NaHS,LPS+PPG组于舌静脉注射LPS 3 h时腹腔注射30 mg/kg的PPG,空白对照组舌静脉注射等容量的生理盐水。各组大鼠均于给予LPS或生理盐水6 h时处死。分别测定各组大鼠肺系数和肺湿/干重比的变化;去蛋白法测定血浆中H2S含量、亚甲基蓝法测定肺组织CSE活性、硫代巴比妥酸法测定肺组织丙二醛(MDA)含量、黄嘌呤氧化酶法测定肺组织超氧化物歧化酶(SOD)活性和酶促反应谷胱甘肽消耗法测定肺组织谷胱甘肽过氧化酶(GSH-PX)活性;光镜、电镜观察肺组织病理变化。
结果:
1.与空白对照组比较,LPS组大鼠肺系数和肺湿/干重比明显升高(P < 0.01)。与LPS组比较,LPS+NaHS低、中、高剂量组大鼠肺系数和肺湿/干重比均明显降低(P < 0.05或P < 0.01),而LPS+PPG组上述两指标均明显升高(P < 0.05或P < 0.01)。
2.与空白对照组比较,LPS组大鼠血浆中H2S含量和肺组织中CSE活性明显降低(P < 0.01)。与LPS组比较,LPS+NaHS低、中、高剂量组血浆中H2S含量和肺组织中CSE活性均显著升高(P < 0.05或P < 0.01),而LPS+PPG组上述两项指标均明显降低(P < 0.05)。
3.与空白对照组比较,LPS组肺组织MDA含量明显升高(4.04±1.21 VS 8.86±1.36,P < 0.01)、SOD和GSH-PX活性均明显降低(28.35±1.94 VS 16.38±4.42,21.45±2.99 VS 11.45±2.39,P < 0.01)。与LPS组比较,LPS+NaHS低、中、高剂量组肺组织MDA含量降低、SOD和GSH-PX活性均显著升高(P < 0.05或P < 0.01);LPS+PPG组肺组织MDA含量升高、SOD活性降低(P < 0.05或P < 0.01),GSH-PX活性虽有所降低但无统计学意义。
4.光镜下观察显示,空白对照组大鼠肺泡结构完整,LPS组肺组织明显受损,可见肺泡间隔增宽,肺泡完整性被破坏,腔内有大量渗出,炎性细胞浸润。LPS+NaHS低、中、高剂量组上述肺损伤程度较LPS组有所减轻;LPS+PPG组肺损伤程度加重。电镜下观察显示,空白对照组大鼠肺泡Ⅱ型上皮细胞超微结构基本正常。LPS组肺组织超微结构明显受损,粗面内质网脱颗粒,线粒体水肿、嵴减少嗜锇性板层小体的板层结构显著融合或消失。LPS+NaHS低、中、高剂量组大鼠肺组织超微结构与LPS组比较肺损伤程度有所减轻,LPS+PPG组仍存在严重肺损伤。结论:ALI早期应用NaHS后,CSE活性增强,H2S生成增加,肺系数和肺湿/干重比降低,脂质过氧化物MDA生成减少,抗过氧化物酶SOD和GSH-PX活性增强,肺损伤减轻。而应用PPG后,CSE活性降低,H2S生成减少,肺系数和肺湿/干重比升高,MDA生成增加,SOD活性降低,肺损伤加重。
第三部分硫化氢对大鼠内毒素性急性肺损伤组织炎症细胞因子的影响
目的:观察H2S对大鼠内毒素性ALI组织炎症细胞因子、核内转录因子和粘附分子的影响,并探讨其作用机制。
方法:健康成年雄性SD大鼠(270±20g)共48只,随机分为6组,每组8只,分别为Ⅰ空白对照组;ⅡLPS组;ⅢLPS+NaHS低剂量组;ⅣLPS+NaHS中剂量组;ⅤLPS+NaHS高剂量组;ⅥLPS+PPG组。用20%的乌拉坦(5 ml/kg)麻醉大鼠,LPS组舌静脉注射LPS,LPS+NaHS低、中、高剂量组分别于舌静脉注射LPS 3 h时腹腔注射0.78 mg/kg、1.56 mg/kg和3.12 mg/kg的NaHS,LPS+PPG组于舌静脉注射LPS 3 h时腹腔注射30 mg/kg的PPG,空白对照组舌静脉注射等容量的生理盐水。各组大鼠均于给予LPS或生理盐水6 h时处死。采用酶联免疫吸附法(ELISA)测定血清中IL-1β和IL-10含量;逆转录聚合酶链反应法(RT-PCR)测定肺组织中IL-1β、IL-10和细胞间黏附分子-1(ICAM-1)mRNA基因表达;凝胶迁移电泳(EMSA)法测定核内转录因子-κBp65(NF-κBp65)的活性。
结果:
1.与空白对照组比较,LPS组大鼠血清中IL-1β和IL-10浓度明显升高(P < 0.01)。与LPS组比较,LPS+NaHS低、中、高剂量组血清中IL-1β浓度明显降低(13.96±0.71 VS 15.01±0.81,12.45±1.35 VS 15.01±0.81,12.24±0.90 VS 15.01±0.81,P < 0.05或P < 0.01),LPS+NaHS中、高剂量组血清中IL-10浓度明显升高(137.68±6.27 VS 127.04±5.30,138.56±6.29 VS 127.04±5.30,P < 0.05)。与LPS组比较,LPS+PPG组血清中IL-1β浓度明显升高(21.58±3.29 VS 15.01±0.81,P < 0.05),血清中IL-10浓度明显降低(113.28±9.68 VS 127.04±5.30,P < 0.05)。
2.空白对照组大鼠肺组织可见到IL-1β、IL-10和ICAM-1 mRNA基因表达,及NF-κBp65蛋白表达。与空白对照组比较,LPS组肺组织中IL-1β、IL-10和ICAM-1 mRNA基因表达和NF-κBp65活性均明显升高(P < 0.05或P < 0.01)。与LPS组比较,LPS+NaHS低、中、高剂量组肺组织中IL-1βmRNA基因表达和NF-κBp65活性均明显降低,LPS+NaHS中、高剂量组ICAM-1 mRNA基因表达明显降低、IL-10 mRNA基因表达明显升高(P < 0.05或P < 0.01)。与LPS组比较,LPS+PPG组肺组织中IL-1β和ICAM-1 mRNA基因表达及NF-κBp65活性均明显升高,IL-10 mRNA基因表达明显降低(P < 0.05)。
结论:ALI早期给予NaHS后,IL-1β和ICAM-1 mRNA基因表达及NF-κBp65活性减弱,IL-10 mRNA基因表达增强,肺损伤减轻;而给予PPG后,IL-1β和ICAM-1 mRNA基因表达及NF-κBp65活性增强,IL-10 mRNA基因表达减弱,肺损伤加重。
第四部分硫化氢对大鼠内毒素性急性肺损伤组织细胞凋亡的影响
目的:观察H2S对大鼠内毒素性ALI组织细胞凋亡的影响,并探讨其作用机制。
方法:健康成年雄性SD大鼠(270±20g)共48只,随机分为6组,每组8只,分别为Ⅰ空白对照组;ⅡLPS组;ⅢLPS+NaHS低剂量组;ⅣLPS+NaHS中剂量组;ⅤLPS+NaHS高剂量组;ⅥLPS+PPG组。用20%的乌拉坦(5 ml/kg)麻醉大鼠,LPS组舌静脉注射LPS,LPS + NaHS低、中、高剂量组分别于舌静脉注射LPS 3 h时腹腔注射0.78 mg/kg、1.56 mg/kg和3.12 mg/kg的NaHS,LPS+PPG组于舌静脉注射LPS3 h时腹腔注射30 mg/kg的PPG,空白对照组舌静脉注射等容量的生理盐水。各组大鼠均于给予LPS或生理盐水6 h时处死。采用流式细胞术(FCM)检测肺细胞凋亡;免疫组化法检测肺组织Bcl-2和Bax的表达;Western blotting法检测肺组织Caspase-3和Caspase-9的表达。
结果:
1. LPS组肺组织细胞凋亡率较空白对照组明显升高(P < 0.01)。与LPS组比较,LPS+NaHS中、高剂量组肺组织细胞凋亡率明显降低(P < 0.01),LPS+PPG组肺组织细胞凋亡率明显升高(P < 0.05)。
2.免疫组化结果显示,Bcl-2和Bax蛋白阳性染色呈棕黄色,主要在肺泡和支气管上皮细胞,血管内皮细胞和炎性细胞也有表达,空白对照组可见Bcl-2和Bax蛋白阳性细胞。与空白对照组比较,LPS组Bcl-2蛋白表达明显减弱,Bax蛋白表达明显增强,Bcl-2/Bax比值明显降低(P < 0.01)。与LPS组比较,LPS+NaHS高剂量组Bcl-2阳性细胞表达明显增强(P < 0.05),LPS+NaHS中、高剂量组Bax阳性细胞表达明显减弱(P < 0.05),LPS+NaHS低、中、高剂量组Bcl-2/Bax比值明显增加(P < 0.05或P < 0.01)。与LPS组比较,LPS+PPG组Bcl-2阳性细胞表达明显减弱(P < 0.05),Bax阳性细胞表达明显增强(P < 0.05),Bcl-2/Bax比值明显降低(P < 0.01)。
3. Western blotting法检测结果显示,空白对照组仅有少量的Caspase-3和Caspase-9的表达,LPS组Caspase-3和Caspase-9蛋白表达较空白对照组明显增强(P < 0.05或P < 0.01)。与LPS组比较,LPS+NaHS高剂量组Caspase-3和Caspase-9蛋白表达明显减弱(P < 0.05),LPS+PPG组Caspase-3和Caspase-9蛋白表达明显增强(P < 0.05)。
结论:ALI早期给予NaHS后,肺细胞凋亡率降低,Bax、Caspase-3和Caspase-9蛋白表达减弱,Bcl-2蛋白表达增强,肺损伤减轻;给予PPG后,肺细胞凋亡率升高,Bax、Caspase-3和Caspase-9蛋白表达增强,Bcl-2蛋白表达减弱,肺损伤加重。
第五部分硫化氢对大鼠内毒素性急性肺损伤肺泡表面活性物质的影响
目的:观察H2S对大鼠内毒素性ALI肺泡表面活性物质(PS)的影响,探讨其作用机制。
方法:健康成年雄性SD大鼠(270±20 g)共48只,随机分为6组,每组8只,分别为Ⅰ空白对照组;ⅡLPS组;ⅢLPS+NaHS低剂量组;ⅣLPS+NaHS中剂量组;ⅤLPS+NaHS高剂量组;ⅥLPS+PPG组。用20%的乌拉坦(5 ml/kg)麻醉大鼠,LPS组舌静脉注射LPS,LPS+NaHS低、中、高剂量组分别于舌静脉注射LPS 3 h时腹腔注射0.78 mg/kg、1.56 mg/kg和3.12 mg/kg的NaHS,LPS+PPG组于舌静脉注射LPS 3 h时腹腔注射30 mg/kg的PPG,空白对照组舌静脉注射等容量的生理盐水。各组大鼠均于给予LPS或生理盐水6 h时处死。应用RT-PCR法测定肺组织中肺泡表面活性蛋白-A、B、C(SP-A、B、C)mRNA基因表达的变化;测定各组大鼠肺泡灌洗液(BALF)中总蛋白(TP)和总磷脂(TPL)含量的变化。
1.空白对照组大鼠肺组织可见到SP-A、SP-B和SP-C mRNA基因表达。与空白对照组比较,LPS组肺组织中SP-A、SP-B和SP-C mRNA基因表达均明显降低(0.93±0.05 VS 1.35±0.11,1.18±0.10 VS 1.12±0.10,0.72±0.05 VS 0.81±0.08, P < 0.05或P < 0.01)。与LPS组比较,LPS+NaHS中、高剂量组肺组织中SP-A和SP-B mRNA基因表达升高(P < 0.05或P < 0.01),LPS+NaHS各剂量组SP-C mRNA基因表达无明显变化。与LPS组比较,LPS+PPG组SP-A、SP-B和SP-C mRNA基因表达均明显降低( 0.85±0.08 VS 0.93±0.05 , 0.74±0.06 VS 0.82±0.08 , 0.66±0.05 VS 0.72±0.05,P < 0.05)。
2.空白对照组大鼠BALF中均可检测到TP和TPL。与空白对照组比较,LPS组BALF中TP含量明显升高(0.39±0.33 VS 0.24±0.05,P < 0.01),TPL含量明显降低(0.29±0.04 VS 0.38±0.06,P < 0.05)。与LPS组比较,LPS+NaHS高剂量组BALF中TP含量明显降低,LPS+NaHS中、高剂量组BALF中TPL含量明显升高(P < 0.05)。与LPS组比较,LPS+PPG组BALF中TP含量明显升高,TPL含量明显降低(0.43±0.04 VS 0.24±0.05,0.24±0.03 VS 0.29±0.04,P < 0.05)。
结论:ALI早期给予NaHS后,SP-A和SP-B mRNA基因表达上调,TP生成降低,TPL生成增加,肺损伤减轻。给予PPG后,SP-A、SP-B和SP-C mRNA基因表达下调,TP生成增加,TPL生成降低,肺损伤加重。
Acute lung injury (ALI) is acute, progressive and hypoxia respiratory failure by endopathic and exopathic cause of lung rather than by cardiogenic one. The major clinical manifestations of ALI are obstinate dyspnea, hypoxemia, lung compliance decreased and so on. Lipopolysaccharide (LPS) is the main composition of gramnegative bacterium (G-) and is the most common cause of this syndrome. So it is one of the common ways that cause ALI by LPS in scientific research.
Endogenous hydrogen sulfide (H2S) is produced from cysteine by cystathionine-β-synthase (CBS) and/or cystathionine-γ-lyase (CSE). It was reported as a neuromodulator in the brain because of its regulation to hippocampus, and it also can regulate the tesion of smooth muscle in gastrointestinal tract and blood vessel. According to many studies, H2S was suggested that it may have effects on many respiratory diseases and be a new endogenous signaling gasotransmitter after nitric oxide (NO) and carbon monoxide (CO). In the present study, we observed the chronological changes of H2S/CSE system in LPS-induced ALI in rats, investigated the effects of H2S on hyperoxidation, inflammatory factor, apoptosis and pulmoalverolar surfactant, and explored the possible mechanisms.
Part 1 Change of endogenous hydrogen sulfide/cystathionine-γ-lyase system while acute lung injury induced by lipopolysaccharide in rats
Objective: To observe the chronological changes of interleukin-1β(IL-1β), interleukin-10 (IL-10) and H2S/CSE system in LPS-induced acute lung injury in rats, and to investigate the mechanisms of ALI.
Methods: Eighty male rats were randomly divided into six groups.ⅠControl group;ⅡLPS 1 h group;ⅢLPS 3 h group;ⅣLPS 6 h group;ⅤLPS 9 h group;ⅥLPS 12 h group. There were fourty rats in control group (There were eight rats in per time point). The ALI model were induced by LPS in rats. The rats were respectively killed at 1, 3, 6, 9 or 12 hour after administration of LPS. The lung coefficient and wet-to-dry weight ratio (W/D) were respectively measured. The level of H2S in plasma, the activity of CSE in lung tissue and the contents of IL-1βand IL-10 in serum were respectively detected. The morphological changes of lung tissue were observed by light and electron mircroscope.
Results:
1. In control group, the lung coefficient and W/D were not altered from 1 hour to 12 hour. The lung coefficient and W/D were not altered at 1 hour after administration of LPS compared with those of control group (0.47±0.02 VS 0.46±0.03, 4.65±0.34 VS 4.41±0.33, P > 0.05). The lung coefficient and W/D were significantly increased at 3 hour after administration of LPS compared with those of control group (0.52±0.03 VS 0.47±0.02, 5.24±0.45 VS 4.43±0.21, P < 0.05). During 6 hour to 12 hour after administration of LPS, the lung coefficient and W/D were significantly increased compared with those of control group (P < 0.05 or P < 0.01).
2. In control group, the contents of IL-1βand IL-10 were not altered from 1 hour to 12 hour. The content of IL-1βwas significantly increased at 1 hour after administration of LPS compared with that of control group (11.18±0.59 VS 9.26±1.37, P < 0.01). During 3 hour to 12 hour after administration of LPS, the content of IL-1βwas decreased compared with that of LPS 1 h group while was increased compared with that of control group (P < 0.05 or P < 0.01). The content of IL-10 was significantly increased at 1 hour after administration of LPS compared with that of control group (213.46±12.84 VS 11.84±1.25, P < 0.01). The content of IL-10 was significantly increased at 3 hour after administration of LPS compared with that of LPS 1 h group and control group. During 6 hour to 12 hour after administration of LPS, the content of IL-10 was decreased compared with that of LPS 3 h group while was significantly increased compared with control group (P < 0.01).
3. In control group, the level of H2S in plasma and the activity of CSE in lung tissue were not altered from 1 hour to 12 hour. The level of H2S in plasma and the activity of CSE in lung tissue were not altered at 1 hour after administration of LPS compared with those of control group (35.10±3.81 VS 37.22±4.48, 4.61±0.75 VS 4.68±0.63, P > 0.05). The level of H2S in plasma and the activity of CSE in lung tissue were significantly decreased at 3 hour after administration of LPS compared with those of control group (24.90±2.21 VS 36.67±5.35, 3.35±0.98 VS 4.61±0.68, P < 0.01). During 6 hour to 12 hour after administration of LPS, the level of H2S in plasma and the activity of CSE in lung tissue were significantly decreased compared with those of control group (P < 0.01).
4. The structure of pulmonary alveoli were intact in control group. The structure of pulmonary alveoli were not altered at 1 hour after administration of LPS compared with control group. In LPS 3 h, 6 h, 9 h and 12 h groups, the lung tissues were injuried, the pulmonary alveoli were damaged, much exudate in the alveolar sacs and mang inflammatory cells infiltrated. The microvillus of typeⅡalveolar cells were orderly arranged, and the structure of mitochondria, the rough surfaced endoplasmic reticulums and the osmiophilic lamellar bodies were intact in control group. The structure of typeⅡalveolar cells were not altered at 1 hour after administration of LPS compared with those of control group. In LPS 3 h, 6 h, 9 h and 12 h groups, the microvillus of typeⅡalveolar cells were fusion, the mitochondrial swelling, the rough surfaced endoplasmic reticulums were degranulated and the osmiophilic lamellar bodies were fusion or disappear.
Conlunsion:The IL-1βand IL-10 were increased while the lung tissue was not significantly injuried. During 3 hour to 12 hour after administration of LPS, the contents of IL-1βand IL-10 were increased, while the level of H2S in plasma and the activity of CSE in lung tissue were markedly decreased. It could be concluded that IL-1β, IL-10 and H2S/CSE system may be play a role in the physiopathologic process of ALI induced by LPS.
Part 2 Effects of hydrogen sulfide on hyperoxidation in acute lung injury induced by lipopolysaccharide in rats
Objective: To study the effect of H2S on hyperoxidation in LPS-induced acute lung injury rats and explore the possible mechanism.
Methods: Fourty-eight male rats were randomly divided into six groups (n=8).ⅠControl group;ⅡLPS group;ⅢLPS+NaHS Low dose group;ⅣLPS+NaHS Middle dose group;ⅤLPS+NaHS High dose group;ⅥLPS+PPG group. LPS was administrated in LPS group. Saline was administrated in control group. In LPS+NaHS Low, Middle and High dose groups or LPS+PPG group, sodium hydrosulfide (NaHS) or DL- propargylglycine (PPG) were respectively administrated at 3 hour after administration of LPS. The rats were respectively killed at 6 hour after administration of LPS or saline. The lung coefficient, W/D, the content of H2S in plasma and the activity of CSE in lung tissue were respectively detected. The content of malondialdehyde (MDA), and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) in lung tissue were respectively measured. The morphological changes of lung tissue were respectively observed by light and electron microscopes.
Results:
1. The lung coefficient and W/D were significantly increased in LPS group compared with those of control group (P < 0.01). Compared with LPS group, the lung coefficient and W/D were respectively decreased in LPS+NaHS Low, Middle and High dose groups (P < 0.05 or P < 0.01), while were significantly increased in LPS+PPG group (P < 0.05 or P < 0.01).
2. The level of H2S in plasma and the activity of CSE in lung tissue were significantly decreased in LPS group compared with those of control group (P < 0.01). Compared with LPS group, the level of H2S in plasma and the activity of CSE in lung tissue were significantly increased in the LPS+NaHS Low, Middle and High dose groups (P < 0.05 or P < 0.01), while were decreased in the LPS+PPG group (P < 0.05).
3. Compared with control group, the content of MDA was significantly increased (4.04±1.21 VS 8.86±1.36, P < 0.01), and the activities of SOD and GSH-PX were significantly decreased (28.35±1.94 VS 16.38±4.42,21.45±2.99 VS 11.45±2.39, P < 0.01) in lung tissue in LPS group. Compared with LPS group, the content of MDA was significantly decreased, and the activities of SOD and GSH-PX were significantly increased in LPS+NaHS Low, Middle and High dose groups(P < 0.05 or P < 0.01). Compared with LPS group, the content of MDA was increased, the activity of SOD was decreased and the activity of GSH-PX was not altered in LPS+PPG group (P < 0.05 or P < 0.01).
4. The structure of pulmonary alveoli were intact in control group. In LPS group, the lung tissue was injuried, the pulmonary alveoli were damaged, much exudate in the alveolar sacs and mang inflammatory cells infiltrated. In LPS+NaHS Low, Middile and High dose groups, the injury of the lung tissue was markedly ameliorated compared with that of LPS group. In LPS + PPG group, the injury of the lung tissue was markedly aggragated compared with that of LPS group. The microvillus of typeⅡalveolar cells were orderly arranged, and structure of the mitochondria, the rough surfaced endoplasmic reticulums and the osmiophilic lamellar bodies were intact in control group. In LPS group, the microvillus of typeⅡalveolar cells were fusion, the mitochondrial swelling, the rough surfaced endoplasmic reticulums were degranulated and the osmiophilic lamellar bodies were fusion or disappear. In LPS + NaHS Low, Middle and High dose groups, the injury of the lung tissue was ameliorated compared with that of LPS group. In LPS + PPG group, the injury of the lung tissue was markedly aggragated compared with LPS group. Conclusion: Relatively early administration of NaHS could ameliorate LPS-induced ALI in rats. The possible mechanism is that the H2S synthesis, and the activities of CSE, SOD and GSH-PX were increased, while the lung coefficient, W/D and the MDA synthesis were decreased after administration of NaHS. But relatively early administration of PPG could aggragate ALI. The possible mechanism is that the activities of CSE and SOD, and the H2S synthesis were decreased, while the lung coefficient, W/D and the MDA synthesis were increased after administration of PPG.
Part 3 Effects of hydrogen sulfide on inflammatory factor in acute lung injury induced by lipopolysaccharide in rats
Objective: To study the effect of H2S on inflammatory factor in LPS-induced acute lung injury rats and explore the possible mechanism. Methods: Fourty-eight male rats were randomly divided into six groups (n=8).ⅠControl group;ⅡLPS group;ⅢLPS+NaHS Low dose group;
ⅣLPS+NaHS Middle dose group;ⅤLPS+NaHS High dose group;ⅥLPS+PPG group. LPS was administrated in LPS group. Saline was administrated in control group. In LPS+NaHS Low, Middle and High dose groups or LPS+PPG group, NaHS or PPG were respectively administrated at
3 hour after administration of LPS. The rats were respectively killed at 6 hour after administration of LPS or saline. The contents of interleukin-1β(IL-1β) and interleukin-10 (IL-10) in serum, and their mRNA expression in lung tissue were respectively measured. The expression of intercellular adhesion molecule-1 (ICAM-1) mRNA and the nuclear factor-κB (NF-кBp65) activity in lung tissue were respectively detected.
Results:
1. Compared with control group, the contents of IL-1βand IL-10 in serum were significantly increased in LPS group. Compared with LPS group, the content of IL-1βin serum was significantly deceased in LPS+NaHS Low, Middle and High dose groups (13.96±0.71 VS 15.01±0.81,12.45±1.35 VS 15.01±0.81,12.24±0.90 VS 15.01±0.81,P < 0.05 or P < 0.01), and the content of IL-10 in serum was significantly increased in LPS+NaHS Middle and High dose groups (137.68±6.27 VS 127.04±5.30, 138.56±6.29 VS 127.04±5.30, P < 0.05). Compared with LPS group, the content of IL-1βin serum was significantly increased (21.58±3.29 VS 15.01±0.81, P < 0.05) and the content of IL-10 in serum was significantly decreased (113.28±9.68 VS 127.04±5.30, P < 0.05) in LPS+PPG group.
2. The expression of IL-1β, IL-10 and ICAM-1mRNA, and the activity of NF-кBp65 in lung tissue were detectable in control group. Compared with control group, the expression of IL-1β, IL-10 and ICAM-1 mRNA, and the activity of NF-кBp65 in lung tissue were significantly increased in LPS group. Compared with LPS group, the expression of IL-1βmRNA and the activity of NF-кBp65 in lung tissue were significantly decreased in LPS+NaHS Low, Middle and High dose groups (P < 0.05 or P < 0.01), the expression of ICAM-1 mRNA was significantly decreased and the expression of IL-10 mRNA was significantly increased in LPS+NaHS Middle and High dose groups (P < 0.05 or P < 0.01). Compared with LPS group, the expression of IL-1βand ICAM-1 mRNA, and the activity of NF-кBp65 in lung tissue were significantly increased, while the expression of IL-10 mRNA was significantly decreased in the LPS+PPG group (P < 0.05 or P < 0.01).
Conclusion: Relatively early administration of NaHS could ameliorate LPS-induced ALI in rats. The possible mechanism is that the content of IL-1β, the expression of IL-1βand ICAM-1 mRNA, and the activity of NF-κBp65 were decreased, while the content of IL-10 and the expression of IL-10 mRNA were increased after administration of NaHS. But relatively early administration of PPG could aggragate ALI. The possible mechanism is that the content of IL-1β, the expression of IL-1βand ICAM-1 mRNA, and the activity of NF-κBp65 were increased, while the content of IL-10 and the expression of IL-10 mRNA were decreased after administration of PPG. Part 4 Effects of hydrogen sulfide on apoptosis in acute lung injury induced by lipopolysaccharide in rats
Objective: To study the effect of H2S on apoptosis in LPS-induced acute lung injury rats and explore the possible mechanism.
Methods: Fourty-eight male rats were randomly divided into six groups (n=8).ⅠControl group;ⅡLPS group;ⅢLPS+NaHS Low dose group;ⅣLPS+NaHS Middle dose group;ⅤLPS+NaHS High dose group;ⅥLPS+PPG group. In LPS group, LPS was administrated. Saline was administrated in control group. In LPS+NaHS Low, Middle and High dose groups or LPS + PPG group, NaHS or PPG were respectively administrated at 3 hour after administration of LPS. The rats were respectively killed at 6 hour after administration of LPS or saline. The apoptosis of lung cells was detected by FCM. The expressions of Bcl-2 and Bax were respectively detected by immunohistochemisty (IHC). The expressions of Caspase-3 and Caspase-9 were respectively analysised by Western blotting.
Results:
1. The rate of apoptosis was significantly increased in LPS group compared with that of control group (P < 0.01). Compared with LPS group, the rate of apoptosis was significantly decreased in LPS+NaHS Middle and High dose groups (P < 0.05), and was significantly increased in the LPS+PPG group (P < 0.01).
2. The expressions of Bcl-2 and Bax showed buffy positive staining, and the Bcl-2 and Bax were expressed in endochylema of alveolar and airway epithelial cells in control group. In LPS group, a few positive expression of Bcl-2 and considerable positive expression of Bax were observed. In LPS group the expression of Bcl-2 and the ratio of Bcl-2 and Bax were significantly decreased, while Bax was significantly increased (P < 0.01). Compared with LPS group, the expression of Bcl-2 was significantly increased in LPS+NaHS High dose group (P < 0.05), the expression of Bax was significantly decreased in LPS+NaHS Middle and High dose groups (P < 0.05), the ratio of Bcl-2 and Bax was significantly increased in LPS+NaHS Low, Middle and High dose groups (P < 0.05 or P < 0.01). Compared with LPS group, the expression of Bax was significantly increased (P < 0.05), while the expression of Bcl-2 and the ratio of Bcl-2 and Bax were significantly decreased in LPS+PPG group (P < 0.05 or P < 0.01).
3. The results by Western blotting showed that the expression of Caspase-3 and Caspase-9 were markedly increased in LPS group compared with those of control group (P < 0.05). Compared with LPS group, the expression of Caspase-3 and Caspase-9 were significantly decreased in LPS+NaHS High dose group and were significantly increased in LPS+PPG group (P < 0.05).
Conlusion: Relatively early administration of NaHS could ameliorate LPS-induced ALI in rats. The possible mechanism is that the rate of apoptosis was decreased, the expression of Bax, Caspase-3 and Caspase-9 were decreased, while the expression of Bcl-2 was increased after administration of NaHS. But relatively early administration of PPG could aggragate ALI. The possible mechanism is that the rate of apoptosis was increased, the expression of Bax, Caspase-3 and Caspase-9 were increased, while the expression of Bcl-2 was decreased after administration of PPG.
Part 5 Effects of hydrogen sulfide on pulmoalverolar surfactant in acute lung injury induced by lipopolysaccharide in rats
Objective: To study the effect of H2S on pulmoalverolar surfactant (PS) in LPS-induced acute lung injury rats and explore the possible mechanism. Methods: Fourty-eight male rats were randomly divided into six groups (n=8).ⅠControl group;ⅡLPS group;ⅢLPS+NaHS Low dose group;ⅣLPS+NaHS Middle dose group;ⅤLPS+NaHS High dose group;ⅥLPS+PPG group. In LPS group, LPS was administrated. Saline was administrated in control group. In LPS+NaHS Low, Middle and High dose groups or LPS+PPG group, NaHS or PPG were respectively administrated at 3 hour after administration of LPS. The rats were respectively killed at 6 hour after administration of LPS or saline. The expression of SP-A, SP-B and SP-C mRNA in lung tissue were respectively analysised. The contents of total protein (TP) and total phospholipids (TPL) in bronchoalveolar lavage fluid (BLAF) were respectively measured.
Results:
1. The expression of SP-A, SP-B and SP-C mRNA in the lung tissue were significantly decreased in LPS group compared with those of control group (0.93±0.05 VS 1.35±0.11, 0.82±0.08 VS 1.12±0.10, 0.72±0.05 VS 0.81±0.08, P < 0.05 or P < 0.01). Compared with LPS group, the expression of SP-A and SP-B mRNA in lung tissue were significantly increased in LPS+NaHS Middle and High dose groups (P < 0.05 or P < 0.01), and the expression of SP-C mRNA was not altered in LPS+NaHS Low, Middle and High dose groups. Compared with LPS group, the expression of SP-A, SP-B and SP-C mRNA were significantly decreased in LPS+PPG group (0.85±0.08 VS 0.93±0.05, 0.74±0.06 VS 0.82±0.08, 0.66±0.05 VS 0.72±0.05, P < 0.05).
2. The content of TP was increased and TPL was decreased in BLAF in LPS group compared with those of control group (0.39±0.33 VS 0.24±0.05,0.29±0.04 VS 0.38±0.06, P < 0.05 or P < 0.01). Compared with LPS group, the content of TP in BLAF was significantly decreased in LPS+NaHS High dose group (P < 0.05), and the content of TPL in BLAF was significantly increased in LPS+NaHS Middle and High dose groups (P < 0.05). Compared with LPS group, the contents of TP was significantly increased and TPL was significantly decreased in BLAF in LPS+PPG group (0.43±0.04 VS 0.24±0.05,0.24±0.03 VS 0.29±0.04, P < 0.05).
Conclusion: Relatively early administration of NaHS could ameliorate LPS-induced ALI in rats. The possible mechanism is that the expression of SP-A、SP-B and SP-C mRNA were increased, the content of TP was decreased and the content of TPL in BALF was increased after administration of NaHS. But relatively early administration of PPG could aggragate ALI. The possible mechanism is that the expression of SP-A、SP-B and SP-C mRNA were decreased, the content of TP was increased and the content of TPL was decreased in BALF after administration of PPG.
内源性硫化氢(hydrogen sulfide,H2S)主要由半胱氨酸等含硫氨基酸在胱硫醚-β-合成酶(cystathionine-β-synthase,CBS)和胱硫醚-γ-裂解酶(cystathionine-γ-lyase,CSE)作用下生成,它对神经系统特别是海马的功能具有调节作用,并可以调节消化道和血管平滑肌的张力。随着对H2S研究的逐渐深入,越来越多的证据证实它参与多种呼吸系统疾病的发生发展过程,是继一氧化氮(nitric oxide,NO)和一氧化碳(carbon monoxide,CO)之后的一种新型气体信使分子。本实验首先观察了在内毒素性ALI病程中H2S和CSE的时程性变化,在此基础上分别给予H2S供体氢硫化钠(sodium hydrosulfide,NaHS)和CSE抑制剂炔丙基甘氨酸(DL-propargylglycine,PPG),观察它们对肺组织氧化应激、炎症细胞、炎症介质、细胞凋亡和肺表面活性物质(pulmoalverolar surfactant,PS)等方面的影响,从细胞、基因、蛋白水平多层次、多方面探讨H2S在肺损伤中的作用及机制,为临床治疗肺损伤提供理论依据。
第一部分LPS致大鼠急性肺损伤硫化氢/胱硫醚-γ-裂解酶体系的变化
目的:观察大鼠内毒素性ALI过程中白介素-1β(IL-1β)、白介素-10(IL-10)和H2S/CSE体系的时程性变化及关系,探讨LPS诱导ALI的发病机制。
方法:健康雄性SD大鼠(270±20g)共80只,随机分为6组,Ⅰ空白对照组;ⅡLPS 1 h组;ⅢLPS 3 h组;ⅣLPS 6 h组;ⅤLPS 9 h组;ⅥLPS 12 h组,空白对照组共40只(不同时间点各8只)。用20%的乌拉坦(5 ml/kg)麻醉大鼠,LPS 1 h、3 h、6 h、9 h和12 h组舌静脉注射LPS(5 mg/kg)。分别于给药1 h、3 h、6 h、9 h及12 h后采集样品;检测肺系数和肺湿/干重比的变化;去蛋白法检测血浆中H2S含量、亚甲基蓝法检测肺组织CSE活性、酶联免疫吸附法(ELISA)检测血清中IL-1β和IL-10的变化;光镜、电镜观察肺组织病理变化。
结果:
1.空白对照组大鼠肺系数和肺湿/干重比在1 h ~ 12 h之间无明显变化。给予LPS 1 h后大鼠肺系数和肺湿/干重比与空白对照组比较无显著差异(0.47±0.02 VS 0.46±0.03,4.65±0.34 VS 4.41±0.33,P > 0.05),给予LPS 3 h后肺系数和肺湿/干重比开始明显升高(0.52±0.03 VS 0.47±0.03,5.24±0.45 VS 4.43±0.21,P < 0.05),给予LPS 6 h、9 h和12 h后肺系数和肺湿/干重比均比空白对照组升高且维持在较高水平(P < 0.05或P < 0.01)。
2.空白对照组大鼠血清中IL-1β和IL-10浓度在1 h ~ 12 h之间无明显变化。给予LPS 1 h后大鼠血清中IL-1β浓度较空白对照组显著升高(11.18±0.59 VS 9.26±1.37,P < 0.05)并达高峰,给予LPS 3 h、6 h、9 h及12 h后血清中IL-1β浓度较LPS 1 h随时程延长逐渐下降,但与空白对照组比较均明显升高(P < 0.05或P < 0.01)。给予LPS 1 h后大鼠血清中IL-10浓度较空白对照组显著升高(213.46±12.84 VS 11.84±1.25,P < 0.01),给予LPS 3 h时达高峰(230.46±9.49 VS 10.73±9.97,P < 0.01),6 h、9 h及12 h后血清中IL-10浓度较LPS 1 h和3 h时降低,并随时程延长逐渐下降,但与空白对照组比较均明显升高(P < 0.01)。
3.空白对照组大鼠血浆中H2S含量和肺组织CSE活性在1 h ~ 12 h之间无明显变化。给予LPS 1 h后大鼠血浆中H2S含量和肺组织CSE活性较空白对照组无显著差异(35.10±3.81 VS 37.22±4.48,4.61±0.75 VS 4.68±0.63,P > 0.05),给予LPS 3 h后血浆中H2S含量和肺组织CSE活性明显降低(24.90±2.21 VS 36.67±5.35,3.35±0.98 VS 4.61±0.68,P < 0.01),给予LPS 6 h、9 h和12 h后血浆中H2S含量和肺组织CSE活性与空白对照组比较均明显降低(P < 0.01)。
4.高倍光镜下显示,空白对照组肺泡结构完整,给予LPS 1 h后无明显变化,给予LPS 3 h、6 h、9 h及12 h后肺组织明显受损,可见肺泡间隔增宽,肺泡完整性被破坏,腔内有大量渗出,炎性细胞浸润。电镜下显示,空白对照组肺泡Ⅱ型上皮细胞超微结构基本正常,给予LPS 1 h后无明显变化,给予LPS 3 h、6 h、9 h及12 h后肺组织超微结构明显受损,粗面内质网脱颗粒,线粒体水肿、嵴减少嗜锇性板层小体的板层结构显著融合或消失。
结论:大鼠静脉注射LPS后,1 h时肺组织未出现明显损伤,但炎性细胞因子显著升高。给予LPS 3 h至12 h时均存在肺损伤,IL-1β和IL-10均明显升高,内源性H2S和CSE均明显降低。IL-1β、IL-10和H2S/ CSE体系可能参与内毒素性ALI的病理生理过程。
第二部分硫化氢对大鼠内毒素性急性肺损伤组织氧化应激的影响
目的:观察H2S对大鼠内毒素性ALI组织过氧化物和抗氧化物酶的影响,并探讨其作用机制。
方法:健康成年雄性SD大鼠(270±20g)共48只,随机分为6组,每组8只,分别为Ⅰ空白对照组;ⅡLPS组;ⅢLPS+NaHS低剂量组;ⅣLPS+NaHS中剂量组;ⅤLPS+NaHS高剂量组;ⅥLPS+PPG组。用20%的乌拉坦(5 ml/kg)麻醉大鼠,LPS组舌静脉注射LPS,LPS+NaHS低、中、高剂量组分别于舌静脉注射LPS 3 h时腹腔注射0.78 mg/kg、1.56 mg/kg和3.12 mg/kg的NaHS,LPS+PPG组于舌静脉注射LPS 3 h时腹腔注射30 mg/kg的PPG,空白对照组舌静脉注射等容量的生理盐水。各组大鼠均于给予LPS或生理盐水6 h时处死。分别测定各组大鼠肺系数和肺湿/干重比的变化;去蛋白法测定血浆中H2S含量、亚甲基蓝法测定肺组织CSE活性、硫代巴比妥酸法测定肺组织丙二醛(MDA)含量、黄嘌呤氧化酶法测定肺组织超氧化物歧化酶(SOD)活性和酶促反应谷胱甘肽消耗法测定肺组织谷胱甘肽过氧化酶(GSH-PX)活性;光镜、电镜观察肺组织病理变化。
结果:
1.与空白对照组比较,LPS组大鼠肺系数和肺湿/干重比明显升高(P < 0.01)。与LPS组比较,LPS+NaHS低、中、高剂量组大鼠肺系数和肺湿/干重比均明显降低(P < 0.05或P < 0.01),而LPS+PPG组上述两指标均明显升高(P < 0.05或P < 0.01)。
2.与空白对照组比较,LPS组大鼠血浆中H2S含量和肺组织中CSE活性明显降低(P < 0.01)。与LPS组比较,LPS+NaHS低、中、高剂量组血浆中H2S含量和肺组织中CSE活性均显著升高(P < 0.05或P < 0.01),而LPS+PPG组上述两项指标均明显降低(P < 0.05)。
3.与空白对照组比较,LPS组肺组织MDA含量明显升高(4.04±1.21 VS 8.86±1.36,P < 0.01)、SOD和GSH-PX活性均明显降低(28.35±1.94 VS 16.38±4.42,21.45±2.99 VS 11.45±2.39,P < 0.01)。与LPS组比较,LPS+NaHS低、中、高剂量组肺组织MDA含量降低、SOD和GSH-PX活性均显著升高(P < 0.05或P < 0.01);LPS+PPG组肺组织MDA含量升高、SOD活性降低(P < 0.05或P < 0.01),GSH-PX活性虽有所降低但无统计学意义。
4.光镜下观察显示,空白对照组大鼠肺泡结构完整,LPS组肺组织明显受损,可见肺泡间隔增宽,肺泡完整性被破坏,腔内有大量渗出,炎性细胞浸润。LPS+NaHS低、中、高剂量组上述肺损伤程度较LPS组有所减轻;LPS+PPG组肺损伤程度加重。电镜下观察显示,空白对照组大鼠肺泡Ⅱ型上皮细胞超微结构基本正常。LPS组肺组织超微结构明显受损,粗面内质网脱颗粒,线粒体水肿、嵴减少嗜锇性板层小体的板层结构显著融合或消失。LPS+NaHS低、中、高剂量组大鼠肺组织超微结构与LPS组比较肺损伤程度有所减轻,LPS+PPG组仍存在严重肺损伤。结论:ALI早期应用NaHS后,CSE活性增强,H2S生成增加,肺系数和肺湿/干重比降低,脂质过氧化物MDA生成减少,抗过氧化物酶SOD和GSH-PX活性增强,肺损伤减轻。而应用PPG后,CSE活性降低,H2S生成减少,肺系数和肺湿/干重比升高,MDA生成增加,SOD活性降低,肺损伤加重。
第三部分硫化氢对大鼠内毒素性急性肺损伤组织炎症细胞因子的影响
目的:观察H2S对大鼠内毒素性ALI组织炎症细胞因子、核内转录因子和粘附分子的影响,并探讨其作用机制。
方法:健康成年雄性SD大鼠(270±20g)共48只,随机分为6组,每组8只,分别为Ⅰ空白对照组;ⅡLPS组;ⅢLPS+NaHS低剂量组;ⅣLPS+NaHS中剂量组;ⅤLPS+NaHS高剂量组;ⅥLPS+PPG组。用20%的乌拉坦(5 ml/kg)麻醉大鼠,LPS组舌静脉注射LPS,LPS+NaHS低、中、高剂量组分别于舌静脉注射LPS 3 h时腹腔注射0.78 mg/kg、1.56 mg/kg和3.12 mg/kg的NaHS,LPS+PPG组于舌静脉注射LPS 3 h时腹腔注射30 mg/kg的PPG,空白对照组舌静脉注射等容量的生理盐水。各组大鼠均于给予LPS或生理盐水6 h时处死。采用酶联免疫吸附法(ELISA)测定血清中IL-1β和IL-10含量;逆转录聚合酶链反应法(RT-PCR)测定肺组织中IL-1β、IL-10和细胞间黏附分子-1(ICAM-1)mRNA基因表达;凝胶迁移电泳(EMSA)法测定核内转录因子-κBp65(NF-κBp65)的活性。
结果:
1.与空白对照组比较,LPS组大鼠血清中IL-1β和IL-10浓度明显升高(P < 0.01)。与LPS组比较,LPS+NaHS低、中、高剂量组血清中IL-1β浓度明显降低(13.96±0.71 VS 15.01±0.81,12.45±1.35 VS 15.01±0.81,12.24±0.90 VS 15.01±0.81,P < 0.05或P < 0.01),LPS+NaHS中、高剂量组血清中IL-10浓度明显升高(137.68±6.27 VS 127.04±5.30,138.56±6.29 VS 127.04±5.30,P < 0.05)。与LPS组比较,LPS+PPG组血清中IL-1β浓度明显升高(21.58±3.29 VS 15.01±0.81,P < 0.05),血清中IL-10浓度明显降低(113.28±9.68 VS 127.04±5.30,P < 0.05)。
2.空白对照组大鼠肺组织可见到IL-1β、IL-10和ICAM-1 mRNA基因表达,及NF-κBp65蛋白表达。与空白对照组比较,LPS组肺组织中IL-1β、IL-10和ICAM-1 mRNA基因表达和NF-κBp65活性均明显升高(P < 0.05或P < 0.01)。与LPS组比较,LPS+NaHS低、中、高剂量组肺组织中IL-1βmRNA基因表达和NF-κBp65活性均明显降低,LPS+NaHS中、高剂量组ICAM-1 mRNA基因表达明显降低、IL-10 mRNA基因表达明显升高(P < 0.05或P < 0.01)。与LPS组比较,LPS+PPG组肺组织中IL-1β和ICAM-1 mRNA基因表达及NF-κBp65活性均明显升高,IL-10 mRNA基因表达明显降低(P < 0.05)。
结论:ALI早期给予NaHS后,IL-1β和ICAM-1 mRNA基因表达及NF-κBp65活性减弱,IL-10 mRNA基因表达增强,肺损伤减轻;而给予PPG后,IL-1β和ICAM-1 mRNA基因表达及NF-κBp65活性增强,IL-10 mRNA基因表达减弱,肺损伤加重。
第四部分硫化氢对大鼠内毒素性急性肺损伤组织细胞凋亡的影响
目的:观察H2S对大鼠内毒素性ALI组织细胞凋亡的影响,并探讨其作用机制。
方法:健康成年雄性SD大鼠(270±20g)共48只,随机分为6组,每组8只,分别为Ⅰ空白对照组;ⅡLPS组;ⅢLPS+NaHS低剂量组;ⅣLPS+NaHS中剂量组;ⅤLPS+NaHS高剂量组;ⅥLPS+PPG组。用20%的乌拉坦(5 ml/kg)麻醉大鼠,LPS组舌静脉注射LPS,LPS + NaHS低、中、高剂量组分别于舌静脉注射LPS 3 h时腹腔注射0.78 mg/kg、1.56 mg/kg和3.12 mg/kg的NaHS,LPS+PPG组于舌静脉注射LPS3 h时腹腔注射30 mg/kg的PPG,空白对照组舌静脉注射等容量的生理盐水。各组大鼠均于给予LPS或生理盐水6 h时处死。采用流式细胞术(FCM)检测肺细胞凋亡;免疫组化法检测肺组织Bcl-2和Bax的表达;Western blotting法检测肺组织Caspase-3和Caspase-9的表达。
结果:
1. LPS组肺组织细胞凋亡率较空白对照组明显升高(P < 0.01)。与LPS组比较,LPS+NaHS中、高剂量组肺组织细胞凋亡率明显降低(P < 0.01),LPS+PPG组肺组织细胞凋亡率明显升高(P < 0.05)。
2.免疫组化结果显示,Bcl-2和Bax蛋白阳性染色呈棕黄色,主要在肺泡和支气管上皮细胞,血管内皮细胞和炎性细胞也有表达,空白对照组可见Bcl-2和Bax蛋白阳性细胞。与空白对照组比较,LPS组Bcl-2蛋白表达明显减弱,Bax蛋白表达明显增强,Bcl-2/Bax比值明显降低(P < 0.01)。与LPS组比较,LPS+NaHS高剂量组Bcl-2阳性细胞表达明显增强(P < 0.05),LPS+NaHS中、高剂量组Bax阳性细胞表达明显减弱(P < 0.05),LPS+NaHS低、中、高剂量组Bcl-2/Bax比值明显增加(P < 0.05或P < 0.01)。与LPS组比较,LPS+PPG组Bcl-2阳性细胞表达明显减弱(P < 0.05),Bax阳性细胞表达明显增强(P < 0.05),Bcl-2/Bax比值明显降低(P < 0.01)。
3. Western blotting法检测结果显示,空白对照组仅有少量的Caspase-3和Caspase-9的表达,LPS组Caspase-3和Caspase-9蛋白表达较空白对照组明显增强(P < 0.05或P < 0.01)。与LPS组比较,LPS+NaHS高剂量组Caspase-3和Caspase-9蛋白表达明显减弱(P < 0.05),LPS+PPG组Caspase-3和Caspase-9蛋白表达明显增强(P < 0.05)。
结论:ALI早期给予NaHS后,肺细胞凋亡率降低,Bax、Caspase-3和Caspase-9蛋白表达减弱,Bcl-2蛋白表达增强,肺损伤减轻;给予PPG后,肺细胞凋亡率升高,Bax、Caspase-3和Caspase-9蛋白表达增强,Bcl-2蛋白表达减弱,肺损伤加重。
第五部分硫化氢对大鼠内毒素性急性肺损伤肺泡表面活性物质的影响
目的:观察H2S对大鼠内毒素性ALI肺泡表面活性物质(PS)的影响,探讨其作用机制。
方法:健康成年雄性SD大鼠(270±20 g)共48只,随机分为6组,每组8只,分别为Ⅰ空白对照组;ⅡLPS组;ⅢLPS+NaHS低剂量组;ⅣLPS+NaHS中剂量组;ⅤLPS+NaHS高剂量组;ⅥLPS+PPG组。用20%的乌拉坦(5 ml/kg)麻醉大鼠,LPS组舌静脉注射LPS,LPS+NaHS低、中、高剂量组分别于舌静脉注射LPS 3 h时腹腔注射0.78 mg/kg、1.56 mg/kg和3.12 mg/kg的NaHS,LPS+PPG组于舌静脉注射LPS 3 h时腹腔注射30 mg/kg的PPG,空白对照组舌静脉注射等容量的生理盐水。各组大鼠均于给予LPS或生理盐水6 h时处死。应用RT-PCR法测定肺组织中肺泡表面活性蛋白-A、B、C(SP-A、B、C)mRNA基因表达的变化;测定各组大鼠肺泡灌洗液(BALF)中总蛋白(TP)和总磷脂(TPL)含量的变化。
1.空白对照组大鼠肺组织可见到SP-A、SP-B和SP-C mRNA基因表达。与空白对照组比较,LPS组肺组织中SP-A、SP-B和SP-C mRNA基因表达均明显降低(0.93±0.05 VS 1.35±0.11,1.18±0.10 VS 1.12±0.10,0.72±0.05 VS 0.81±0.08, P < 0.05或P < 0.01)。与LPS组比较,LPS+NaHS中、高剂量组肺组织中SP-A和SP-B mRNA基因表达升高(P < 0.05或P < 0.01),LPS+NaHS各剂量组SP-C mRNA基因表达无明显变化。与LPS组比较,LPS+PPG组SP-A、SP-B和SP-C mRNA基因表达均明显降低( 0.85±0.08 VS 0.93±0.05 , 0.74±0.06 VS 0.82±0.08 , 0.66±0.05 VS 0.72±0.05,P < 0.05)。
2.空白对照组大鼠BALF中均可检测到TP和TPL。与空白对照组比较,LPS组BALF中TP含量明显升高(0.39±0.33 VS 0.24±0.05,P < 0.01),TPL含量明显降低(0.29±0.04 VS 0.38±0.06,P < 0.05)。与LPS组比较,LPS+NaHS高剂量组BALF中TP含量明显降低,LPS+NaHS中、高剂量组BALF中TPL含量明显升高(P < 0.05)。与LPS组比较,LPS+PPG组BALF中TP含量明显升高,TPL含量明显降低(0.43±0.04 VS 0.24±0.05,0.24±0.03 VS 0.29±0.04,P < 0.05)。
结论:ALI早期给予NaHS后,SP-A和SP-B mRNA基因表达上调,TP生成降低,TPL生成增加,肺损伤减轻。给予PPG后,SP-A、SP-B和SP-C mRNA基因表达下调,TP生成增加,TPL生成降低,肺损伤加重。
Acute lung injury (ALI) is acute, progressive and hypoxia respiratory failure by endopathic and exopathic cause of lung rather than by cardiogenic one. The major clinical manifestations of ALI are obstinate dyspnea, hypoxemia, lung compliance decreased and so on. Lipopolysaccharide (LPS) is the main composition of gramnegative bacterium (G-) and is the most common cause of this syndrome. So it is one of the common ways that cause ALI by LPS in scientific research.
Endogenous hydrogen sulfide (H2S) is produced from cysteine by cystathionine-β-synthase (CBS) and/or cystathionine-γ-lyase (CSE). It was reported as a neuromodulator in the brain because of its regulation to hippocampus, and it also can regulate the tesion of smooth muscle in gastrointestinal tract and blood vessel. According to many studies, H2S was suggested that it may have effects on many respiratory diseases and be a new endogenous signaling gasotransmitter after nitric oxide (NO) and carbon monoxide (CO). In the present study, we observed the chronological changes of H2S/CSE system in LPS-induced ALI in rats, investigated the effects of H2S on hyperoxidation, inflammatory factor, apoptosis and pulmoalverolar surfactant, and explored the possible mechanisms.
Part 1 Change of endogenous hydrogen sulfide/cystathionine-γ-lyase system while acute lung injury induced by lipopolysaccharide in rats
Objective: To observe the chronological changes of interleukin-1β(IL-1β), interleukin-10 (IL-10) and H2S/CSE system in LPS-induced acute lung injury in rats, and to investigate the mechanisms of ALI.
Methods: Eighty male rats were randomly divided into six groups.ⅠControl group;ⅡLPS 1 h group;ⅢLPS 3 h group;ⅣLPS 6 h group;ⅤLPS 9 h group;ⅥLPS 12 h group. There were fourty rats in control group (There were eight rats in per time point). The ALI model were induced by LPS in rats. The rats were respectively killed at 1, 3, 6, 9 or 12 hour after administration of LPS. The lung coefficient and wet-to-dry weight ratio (W/D) were respectively measured. The level of H2S in plasma, the activity of CSE in lung tissue and the contents of IL-1βand IL-10 in serum were respectively detected. The morphological changes of lung tissue were observed by light and electron mircroscope.
Results:
1. In control group, the lung coefficient and W/D were not altered from 1 hour to 12 hour. The lung coefficient and W/D were not altered at 1 hour after administration of LPS compared with those of control group (0.47±0.02 VS 0.46±0.03, 4.65±0.34 VS 4.41±0.33, P > 0.05). The lung coefficient and W/D were significantly increased at 3 hour after administration of LPS compared with those of control group (0.52±0.03 VS 0.47±0.02, 5.24±0.45 VS 4.43±0.21, P < 0.05). During 6 hour to 12 hour after administration of LPS, the lung coefficient and W/D were significantly increased compared with those of control group (P < 0.05 or P < 0.01).
2. In control group, the contents of IL-1βand IL-10 were not altered from 1 hour to 12 hour. The content of IL-1βwas significantly increased at 1 hour after administration of LPS compared with that of control group (11.18±0.59 VS 9.26±1.37, P < 0.01). During 3 hour to 12 hour after administration of LPS, the content of IL-1βwas decreased compared with that of LPS 1 h group while was increased compared with that of control group (P < 0.05 or P < 0.01). The content of IL-10 was significantly increased at 1 hour after administration of LPS compared with that of control group (213.46±12.84 VS 11.84±1.25, P < 0.01). The content of IL-10 was significantly increased at 3 hour after administration of LPS compared with that of LPS 1 h group and control group. During 6 hour to 12 hour after administration of LPS, the content of IL-10 was decreased compared with that of LPS 3 h group while was significantly increased compared with control group (P < 0.01).
3. In control group, the level of H2S in plasma and the activity of CSE in lung tissue were not altered from 1 hour to 12 hour. The level of H2S in plasma and the activity of CSE in lung tissue were not altered at 1 hour after administration of LPS compared with those of control group (35.10±3.81 VS 37.22±4.48, 4.61±0.75 VS 4.68±0.63, P > 0.05). The level of H2S in plasma and the activity of CSE in lung tissue were significantly decreased at 3 hour after administration of LPS compared with those of control group (24.90±2.21 VS 36.67±5.35, 3.35±0.98 VS 4.61±0.68, P < 0.01). During 6 hour to 12 hour after administration of LPS, the level of H2S in plasma and the activity of CSE in lung tissue were significantly decreased compared with those of control group (P < 0.01).
4. The structure of pulmonary alveoli were intact in control group. The structure of pulmonary alveoli were not altered at 1 hour after administration of LPS compared with control group. In LPS 3 h, 6 h, 9 h and 12 h groups, the lung tissues were injuried, the pulmonary alveoli were damaged, much exudate in the alveolar sacs and mang inflammatory cells infiltrated. The microvillus of typeⅡalveolar cells were orderly arranged, and the structure of mitochondria, the rough surfaced endoplasmic reticulums and the osmiophilic lamellar bodies were intact in control group. The structure of typeⅡalveolar cells were not altered at 1 hour after administration of LPS compared with those of control group. In LPS 3 h, 6 h, 9 h and 12 h groups, the microvillus of typeⅡalveolar cells were fusion, the mitochondrial swelling, the rough surfaced endoplasmic reticulums were degranulated and the osmiophilic lamellar bodies were fusion or disappear.
Conlunsion:The IL-1βand IL-10 were increased while the lung tissue was not significantly injuried. During 3 hour to 12 hour after administration of LPS, the contents of IL-1βand IL-10 were increased, while the level of H2S in plasma and the activity of CSE in lung tissue were markedly decreased. It could be concluded that IL-1β, IL-10 and H2S/CSE system may be play a role in the physiopathologic process of ALI induced by LPS.
Part 2 Effects of hydrogen sulfide on hyperoxidation in acute lung injury induced by lipopolysaccharide in rats
Objective: To study the effect of H2S on hyperoxidation in LPS-induced acute lung injury rats and explore the possible mechanism.
Methods: Fourty-eight male rats were randomly divided into six groups (n=8).ⅠControl group;ⅡLPS group;ⅢLPS+NaHS Low dose group;ⅣLPS+NaHS Middle dose group;ⅤLPS+NaHS High dose group;ⅥLPS+PPG group. LPS was administrated in LPS group. Saline was administrated in control group. In LPS+NaHS Low, Middle and High dose groups or LPS+PPG group, sodium hydrosulfide (NaHS) or DL- propargylglycine (PPG) were respectively administrated at 3 hour after administration of LPS. The rats were respectively killed at 6 hour after administration of LPS or saline. The lung coefficient, W/D, the content of H2S in plasma and the activity of CSE in lung tissue were respectively detected. The content of malondialdehyde (MDA), and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) in lung tissue were respectively measured. The morphological changes of lung tissue were respectively observed by light and electron microscopes.
Results:
1. The lung coefficient and W/D were significantly increased in LPS group compared with those of control group (P < 0.01). Compared with LPS group, the lung coefficient and W/D were respectively decreased in LPS+NaHS Low, Middle and High dose groups (P < 0.05 or P < 0.01), while were significantly increased in LPS+PPG group (P < 0.05 or P < 0.01).
2. The level of H2S in plasma and the activity of CSE in lung tissue were significantly decreased in LPS group compared with those of control group (P < 0.01). Compared with LPS group, the level of H2S in plasma and the activity of CSE in lung tissue were significantly increased in the LPS+NaHS Low, Middle and High dose groups (P < 0.05 or P < 0.01), while were decreased in the LPS+PPG group (P < 0.05).
3. Compared with control group, the content of MDA was significantly increased (4.04±1.21 VS 8.86±1.36, P < 0.01), and the activities of SOD and GSH-PX were significantly decreased (28.35±1.94 VS 16.38±4.42,21.45±2.99 VS 11.45±2.39, P < 0.01) in lung tissue in LPS group. Compared with LPS group, the content of MDA was significantly decreased, and the activities of SOD and GSH-PX were significantly increased in LPS+NaHS Low, Middle and High dose groups(P < 0.05 or P < 0.01). Compared with LPS group, the content of MDA was increased, the activity of SOD was decreased and the activity of GSH-PX was not altered in LPS+PPG group (P < 0.05 or P < 0.01).
4. The structure of pulmonary alveoli were intact in control group. In LPS group, the lung tissue was injuried, the pulmonary alveoli were damaged, much exudate in the alveolar sacs and mang inflammatory cells infiltrated. In LPS+NaHS Low, Middile and High dose groups, the injury of the lung tissue was markedly ameliorated compared with that of LPS group. In LPS + PPG group, the injury of the lung tissue was markedly aggragated compared with that of LPS group. The microvillus of typeⅡalveolar cells were orderly arranged, and structure of the mitochondria, the rough surfaced endoplasmic reticulums and the osmiophilic lamellar bodies were intact in control group. In LPS group, the microvillus of typeⅡalveolar cells were fusion, the mitochondrial swelling, the rough surfaced endoplasmic reticulums were degranulated and the osmiophilic lamellar bodies were fusion or disappear. In LPS + NaHS Low, Middle and High dose groups, the injury of the lung tissue was ameliorated compared with that of LPS group. In LPS + PPG group, the injury of the lung tissue was markedly aggragated compared with LPS group. Conclusion: Relatively early administration of NaHS could ameliorate LPS-induced ALI in rats. The possible mechanism is that the H2S synthesis, and the activities of CSE, SOD and GSH-PX were increased, while the lung coefficient, W/D and the MDA synthesis were decreased after administration of NaHS. But relatively early administration of PPG could aggragate ALI. The possible mechanism is that the activities of CSE and SOD, and the H2S synthesis were decreased, while the lung coefficient, W/D and the MDA synthesis were increased after administration of PPG.
Part 3 Effects of hydrogen sulfide on inflammatory factor in acute lung injury induced by lipopolysaccharide in rats
Objective: To study the effect of H2S on inflammatory factor in LPS-induced acute lung injury rats and explore the possible mechanism. Methods: Fourty-eight male rats were randomly divided into six groups (n=8).ⅠControl group;ⅡLPS group;ⅢLPS+NaHS Low dose group;
ⅣLPS+NaHS Middle dose group;ⅤLPS+NaHS High dose group;ⅥLPS+PPG group. LPS was administrated in LPS group. Saline was administrated in control group. In LPS+NaHS Low, Middle and High dose groups or LPS+PPG group, NaHS or PPG were respectively administrated at
3 hour after administration of LPS. The rats were respectively killed at 6 hour after administration of LPS or saline. The contents of interleukin-1β(IL-1β) and interleukin-10 (IL-10) in serum, and their mRNA expression in lung tissue were respectively measured. The expression of intercellular adhesion molecule-1 (ICAM-1) mRNA and the nuclear factor-κB (NF-кBp65) activity in lung tissue were respectively detected.
Results:
1. Compared with control group, the contents of IL-1βand IL-10 in serum were significantly increased in LPS group. Compared with LPS group, the content of IL-1βin serum was significantly deceased in LPS+NaHS Low, Middle and High dose groups (13.96±0.71 VS 15.01±0.81,12.45±1.35 VS 15.01±0.81,12.24±0.90 VS 15.01±0.81,P < 0.05 or P < 0.01), and the content of IL-10 in serum was significantly increased in LPS+NaHS Middle and High dose groups (137.68±6.27 VS 127.04±5.30, 138.56±6.29 VS 127.04±5.30, P < 0.05). Compared with LPS group, the content of IL-1βin serum was significantly increased (21.58±3.29 VS 15.01±0.81, P < 0.05) and the content of IL-10 in serum was significantly decreased (113.28±9.68 VS 127.04±5.30, P < 0.05) in LPS+PPG group.
2. The expression of IL-1β, IL-10 and ICAM-1mRNA, and the activity of NF-кBp65 in lung tissue were detectable in control group. Compared with control group, the expression of IL-1β, IL-10 and ICAM-1 mRNA, and the activity of NF-кBp65 in lung tissue were significantly increased in LPS group. Compared with LPS group, the expression of IL-1βmRNA and the activity of NF-кBp65 in lung tissue were significantly decreased in LPS+NaHS Low, Middle and High dose groups (P < 0.05 or P < 0.01), the expression of ICAM-1 mRNA was significantly decreased and the expression of IL-10 mRNA was significantly increased in LPS+NaHS Middle and High dose groups (P < 0.05 or P < 0.01). Compared with LPS group, the expression of IL-1βand ICAM-1 mRNA, and the activity of NF-кBp65 in lung tissue were significantly increased, while the expression of IL-10 mRNA was significantly decreased in the LPS+PPG group (P < 0.05 or P < 0.01).
Conclusion: Relatively early administration of NaHS could ameliorate LPS-induced ALI in rats. The possible mechanism is that the content of IL-1β, the expression of IL-1βand ICAM-1 mRNA, and the activity of NF-κBp65 were decreased, while the content of IL-10 and the expression of IL-10 mRNA were increased after administration of NaHS. But relatively early administration of PPG could aggragate ALI. The possible mechanism is that the content of IL-1β, the expression of IL-1βand ICAM-1 mRNA, and the activity of NF-κBp65 were increased, while the content of IL-10 and the expression of IL-10 mRNA were decreased after administration of PPG. Part 4 Effects of hydrogen sulfide on apoptosis in acute lung injury induced by lipopolysaccharide in rats
Objective: To study the effect of H2S on apoptosis in LPS-induced acute lung injury rats and explore the possible mechanism.
Methods: Fourty-eight male rats were randomly divided into six groups (n=8).ⅠControl group;ⅡLPS group;ⅢLPS+NaHS Low dose group;ⅣLPS+NaHS Middle dose group;ⅤLPS+NaHS High dose group;ⅥLPS+PPG group. In LPS group, LPS was administrated. Saline was administrated in control group. In LPS+NaHS Low, Middle and High dose groups or LPS + PPG group, NaHS or PPG were respectively administrated at 3 hour after administration of LPS. The rats were respectively killed at 6 hour after administration of LPS or saline. The apoptosis of lung cells was detected by FCM. The expressions of Bcl-2 and Bax were respectively detected by immunohistochemisty (IHC). The expressions of Caspase-3 and Caspase-9 were respectively analysised by Western blotting.
Results:
1. The rate of apoptosis was significantly increased in LPS group compared with that of control group (P < 0.01). Compared with LPS group, the rate of apoptosis was significantly decreased in LPS+NaHS Middle and High dose groups (P < 0.05), and was significantly increased in the LPS+PPG group (P < 0.01).
2. The expressions of Bcl-2 and Bax showed buffy positive staining, and the Bcl-2 and Bax were expressed in endochylema of alveolar and airway epithelial cells in control group. In LPS group, a few positive expression of Bcl-2 and considerable positive expression of Bax were observed. In LPS group the expression of Bcl-2 and the ratio of Bcl-2 and Bax were significantly decreased, while Bax was significantly increased (P < 0.01). Compared with LPS group, the expression of Bcl-2 was significantly increased in LPS+NaHS High dose group (P < 0.05), the expression of Bax was significantly decreased in LPS+NaHS Middle and High dose groups (P < 0.05), the ratio of Bcl-2 and Bax was significantly increased in LPS+NaHS Low, Middle and High dose groups (P < 0.05 or P < 0.01). Compared with LPS group, the expression of Bax was significantly increased (P < 0.05), while the expression of Bcl-2 and the ratio of Bcl-2 and Bax were significantly decreased in LPS+PPG group (P < 0.05 or P < 0.01).
3. The results by Western blotting showed that the expression of Caspase-3 and Caspase-9 were markedly increased in LPS group compared with those of control group (P < 0.05). Compared with LPS group, the expression of Caspase-3 and Caspase-9 were significantly decreased in LPS+NaHS High dose group and were significantly increased in LPS+PPG group (P < 0.05).
Conlusion: Relatively early administration of NaHS could ameliorate LPS-induced ALI in rats. The possible mechanism is that the rate of apoptosis was decreased, the expression of Bax, Caspase-3 and Caspase-9 were decreased, while the expression of Bcl-2 was increased after administration of NaHS. But relatively early administration of PPG could aggragate ALI. The possible mechanism is that the rate of apoptosis was increased, the expression of Bax, Caspase-3 and Caspase-9 were increased, while the expression of Bcl-2 was decreased after administration of PPG.
Part 5 Effects of hydrogen sulfide on pulmoalverolar surfactant in acute lung injury induced by lipopolysaccharide in rats
Objective: To study the effect of H2S on pulmoalverolar surfactant (PS) in LPS-induced acute lung injury rats and explore the possible mechanism. Methods: Fourty-eight male rats were randomly divided into six groups (n=8).ⅠControl group;ⅡLPS group;ⅢLPS+NaHS Low dose group;ⅣLPS+NaHS Middle dose group;ⅤLPS+NaHS High dose group;ⅥLPS+PPG group. In LPS group, LPS was administrated. Saline was administrated in control group. In LPS+NaHS Low, Middle and High dose groups or LPS+PPG group, NaHS or PPG were respectively administrated at 3 hour after administration of LPS. The rats were respectively killed at 6 hour after administration of LPS or saline. The expression of SP-A, SP-B and SP-C mRNA in lung tissue were respectively analysised. The contents of total protein (TP) and total phospholipids (TPL) in bronchoalveolar lavage fluid (BLAF) were respectively measured.
Results:
1. The expression of SP-A, SP-B and SP-C mRNA in the lung tissue were significantly decreased in LPS group compared with those of control group (0.93±0.05 VS 1.35±0.11, 0.82±0.08 VS 1.12±0.10, 0.72±0.05 VS 0.81±0.08, P < 0.05 or P < 0.01). Compared with LPS group, the expression of SP-A and SP-B mRNA in lung tissue were significantly increased in LPS+NaHS Middle and High dose groups (P < 0.05 or P < 0.01), and the expression of SP-C mRNA was not altered in LPS+NaHS Low, Middle and High dose groups. Compared with LPS group, the expression of SP-A, SP-B and SP-C mRNA were significantly decreased in LPS+PPG group (0.85±0.08 VS 0.93±0.05, 0.74±0.06 VS 0.82±0.08, 0.66±0.05 VS 0.72±0.05, P < 0.05).
2. The content of TP was increased and TPL was decreased in BLAF in LPS group compared with those of control group (0.39±0.33 VS 0.24±0.05,0.29±0.04 VS 0.38±0.06, P < 0.05 or P < 0.01). Compared with LPS group, the content of TP in BLAF was significantly decreased in LPS+NaHS High dose group (P < 0.05), and the content of TPL in BLAF was significantly increased in LPS+NaHS Middle and High dose groups (P < 0.05). Compared with LPS group, the contents of TP was significantly increased and TPL was significantly decreased in BLAF in LPS+PPG group (0.43±0.04 VS 0.24±0.05,0.24±0.03 VS 0.29±0.04, P < 0.05).
Conclusion: Relatively early administration of NaHS could ameliorate LPS-induced ALI in rats. The possible mechanism is that the expression of SP-A、SP-B and SP-C mRNA were increased, the content of TP was decreased and the content of TPL in BALF was increased after administration of NaHS. But relatively early administration of PPG could aggragate ALI. The possible mechanism is that the expression of SP-A、SP-B and SP-C mRNA were decreased, the content of TP was increased and the content of TPL was decreased in BALF after administration of PPG.
引文
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12 陈晓波, 杜军保, 张春雨, 等. 新型气体信号分子硫化氢对低氧大鼠肺动脉平滑肌细胞凋亡的影响[J]. 北京大学学报(医学版),2004, 36 (4): 341-344
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2 王凤英, 王玉梅, 曹红卫, 等. TRH,EGF 及地塞米松促进未成熟胎肺组织分化及表面活性物质合成的研究[J]. 重庆医学, 2002, 31 (1):4-7
3 Putman E, van Golde LM, Haagsman HP. Toxic oxidant species and their impact on the pulmonary surfactant system[J]. Lung, 1997, 175 (2): 75-103
4 Hamm H, Kroegel C, Hohlfeld J. Surfatant: a review of its functions and relevance in respiratory disorders[J]. Respir Med. 1996, 90 (5): 251-270
5 Whitsett JA, Baatz JE. Hydrophobic surfactant proteins SP-B and SP-C: molecular biology, structure and function. In: Robertson B, Van Golde L,Batenburg J (eds) Pulmonary surfactant. Elsevier, Amsterdam, 1992, 55- 77
6 王祥瑞, 杭燕南. 急性肺损伤: 基础医学与临床. 中国协和医科大学出版社. 8- 9
7 Rubio F, Cooley J, Accurso FJ, et al. Linkage of neutrophil serine proteases and decreased surfactant protein-A (SP-A) levels in inflammatory lung disease[J]. Thorax, 2004, 59 (4) : 318-323
8 McCormack FX, Whitsett JA. The pulmonary collectins, SP - A and SP- D, orchestrate innate immunity in the lung[J]. J Clin Invest, 2002, 109 (6): 707-712
9 Wu Y, Adam S, Hamann L, et al. Accumulation of inhibitory kappaB -alpha as a mechanism contributing to the anti - inflammatory effects of surfactant protein-A[J]. Am J Respir Cell Mol Biol, 2004, 31 (6): 587-594
1 Hosoki R, Matsuki N, Kimura H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide[J]. Biochem Biophys Res Commun, 1997, 237 (3): 527-531
2 Stipanuk MH, Beck PW. Characterization of the enzymatic capacity for cysteine desulphhydration in liver and kindney of the rat[J]. Biochem J, 1982, 206 (2): 267-277
3 Kamoun P. Endogenous Production of hydrogen sulfide in mammals[J]. Amino Aeiels, 2004, 26(3): 243-254
4 Eto K, Kimura H. The production of hydrogen sulfide is regulated by testosterone and S-adenosyl-L-methionine in mouse brain[J]. J Neurochem, 2002, 83 (1): 80-86
5 Bao L, Vleek C, Paces V, et al. Idetification and tissue distribution of human cystathionine beta-synthase mRNA isoforms[J]. Arch Biochem Biophys, 1998, 350 (1): 95-103
6 Zhao W, Zhang J, Lu Y, et al. The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener[J]. EMBO J, 2001, 20 (21): 6008-6016
7 Abe K, Kimura H. The possible role of hydrogen sulfide as an endogenous neuromodulator[J]. J Neurosci, 1996, 16 (3): 1066-1071
8 Mitchell TW, Savaga JC, Gould DH. High-performance liquid chromatography detection of sulfide in tissues from sulfide-treated mice[J]. J Appl Toxicol, 1993, 13 (6): 389-394
9 Warenycia MW, Goodwin LR, Benishin CG, et al. Acute hydrogen sulfide poison: demonstation of selective up take of sulfide by the brainsrem by measurement of brain sulfide levels[J]. Biochem Pharmacol, 1989, 38 (6): 973-981
10 Wang R. The gasotransmitter role of hydrogen sulfide[J]. Antioxid Redox Sianal, 2003, 5 (4): 493-501
11 Richardson CJ, Magee EA, Cwmmings JH. A new method for the determinatermination of sulphide in gastrointestinal contents and whole blood by microdistillation and ion chromatography[J]. Clin Chim Acta, 2000, 293 (1-2 ): 115-125
12 Beauchamp RJ, Bus JS, Popp JA, et al. A critical review of the literature on hydrogen sulfide toxicity[J]. Crit Rev Toxicol, 1984, 13 (1): 25-97
13 Furne J, Springfield J, Koening T, et al. Oxidation of hydrogen sulfide and methanethiol to thiosulfate by rat tissues: a specialized function of the colonic mucosa[J]. Biochem Pharmacol, 2001, 62 (2): 255-259
14 Dorman DC, Moulin FJ, Mcmanus TB, et al. Cytochrome oxidase inhibition induced by acute hydrogen sulfide inhalation: correlation with tissue sulfide concentrations in the rat brain, liver, lung, and nasal epithelium [J]. Toxicol Sci, 2002, 65 (1): 18-25
15 Simonneau G, Galie N, Robin LJ, et al. Clinical classification of pulmonary hypertension[J]. J Am Coll Candiol, 2004, 43(12 Suppl S ) : 5S- 12S
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18 张春雨, 丛柏林, 宫丽敏, 等. 硫化氢体系在大鼠低氧肺动脉平滑肌细胞增殖中的意义[J]. 实用儿科临床, 2005, 20 (2): 140-143
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22 魏红玲, 杜军保, 唐朝枢, 等. 硫化氢对低氧性肺动脉高压中氧化应激的调节作用[J]. 北京大学学报(医学版), 2007, 39(6): 565- 569
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12 陈晓波, 杜军保, 张春雨, 等. 新型气体信号分子硫化氢对低氧大鼠肺动脉平滑肌细胞凋亡的影响[J]. 北京大学学报(医学版),2004, 36 (4): 341-344
1 王凤君, 赵云, 谢尔凡, 等. 高效液相色谱法测定生物样品中磷脂含量[J]. 第三军医大学学报, 1996, 18: 67
2 王凤英, 王玉梅, 曹红卫, 等. TRH,EGF 及地塞米松促进未成熟胎肺组织分化及表面活性物质合成的研究[J]. 重庆医学, 2002, 31 (1):4-7
3 Putman E, van Golde LM, Haagsman HP. Toxic oxidant species and their impact on the pulmonary surfactant system[J]. Lung, 1997, 175 (2): 75-103
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7 Rubio F, Cooley J, Accurso FJ, et al. Linkage of neutrophil serine proteases and decreased surfactant protein-A (SP-A) levels in inflammatory lung disease[J]. Thorax, 2004, 59 (4) : 318-323
8 McCormack FX, Whitsett JA. The pulmonary collectins, SP - A and SP- D, orchestrate innate immunity in the lung[J]. J Clin Invest, 2002, 109 (6): 707-712
9 Wu Y, Adam S, Hamann L, et al. Accumulation of inhibitory kappaB -alpha as a mechanism contributing to the anti - inflammatory effects of surfactant protein-A[J]. Am J Respir Cell Mol Biol, 2004, 31 (6): 587-594
1 Hosoki R, Matsuki N, Kimura H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide[J]. Biochem Biophys Res Commun, 1997, 237 (3): 527-531
2 Stipanuk MH, Beck PW. Characterization of the enzymatic capacity for cysteine desulphhydration in liver and kindney of the rat[J]. Biochem J, 1982, 206 (2): 267-277
3 Kamoun P. Endogenous Production of hydrogen sulfide in mammals[J]. Amino Aeiels, 2004, 26(3): 243-254
4 Eto K, Kimura H. The production of hydrogen sulfide is regulated by testosterone and S-adenosyl-L-methionine in mouse brain[J]. J Neurochem, 2002, 83 (1): 80-86
5 Bao L, Vleek C, Paces V, et al. Idetification and tissue distribution of human cystathionine beta-synthase mRNA isoforms[J]. Arch Biochem Biophys, 1998, 350 (1): 95-103
6 Zhao W, Zhang J, Lu Y, et al. The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener[J]. EMBO J, 2001, 20 (21): 6008-6016
7 Abe K, Kimura H. The possible role of hydrogen sulfide as an endogenous neuromodulator[J]. J Neurosci, 1996, 16 (3): 1066-1071
8 Mitchell TW, Savaga JC, Gould DH. High-performance liquid chromatography detection of sulfide in tissues from sulfide-treated mice[J]. J Appl Toxicol, 1993, 13 (6): 389-394
9 Warenycia MW, Goodwin LR, Benishin CG, et al. Acute hydrogen sulfide poison: demonstation of selective up take of sulfide by the brainsrem by measurement of brain sulfide levels[J]. Biochem Pharmacol, 1989, 38 (6): 973-981
10 Wang R. The gasotransmitter role of hydrogen sulfide[J]. Antioxid Redox Sianal, 2003, 5 (4): 493-501
11 Richardson CJ, Magee EA, Cwmmings JH. A new method for the determinatermination of sulphide in gastrointestinal contents and whole blood by microdistillation and ion chromatography[J]. Clin Chim Acta, 2000, 293 (1-2 ): 115-125
12 Beauchamp RJ, Bus JS, Popp JA, et al. A critical review of the literature on hydrogen sulfide toxicity[J]. Crit Rev Toxicol, 1984, 13 (1): 25-97
13 Furne J, Springfield J, Koening T, et al. Oxidation of hydrogen sulfide and methanethiol to thiosulfate by rat tissues: a specialized function of the colonic mucosa[J]. Biochem Pharmacol, 2001, 62 (2): 255-259
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