p38MAPK信号通路在内毒素性休克诱发急性肺损伤大鼠肺组织HO-1表达上调中的作用

目的 评价p38丝裂原活化蛋白激酶(p38MAPK)信号通路在内毒素性休克诱发急性肺损伤大鼠肺组织血红素加氧酶-1(HO-1)表达上调中的作用.方法 雄性SD大鼠48只,8周龄,体重180～ 200g,采用随机数字表法,将其随机分为4组(n=12):对照组(C组)、内毒素性休克组(LS组)、内毒素性休克+ p38MAPK特异性抑制剂SB203580组(LSS组)和SB203580组(SB组).C组和SB组股静脉注射生理盐水0.5 ml;LS和LSS组股静脉注射LPS 10 mg/kg(溶于0.5ml生理盐水);2h内MAP下降至基础值的75％时,C组和IS组股静脉输注10％二甲基亚砜0.1ml;LSS组和SB组股静脉输注SB203580 5 μmol/kg(溶于0.1 ml 10％二甲基亚砜),输注速率0.01 ml/min.给予LPS或生理盐水后6h时采集动脉血样,进行血气分析,计算氧合指数(PaO2/FiO2);然后处死大鼠取肺组织,光镜下观察病理学结果,并进行病理学损伤评分,计算肺含水率,测定SOD活性、MDA含量、HO-1 mRNA及其蛋白、p38MAPK蛋白和磷酸化p38MAPK(p-p38MAPK)蛋白的表达.结果 与C组比较,IS组和LSS组氧合指数和SOD活性降低,病理学损伤评分、肺含水率和MDA含量升高,肺组织HO-1 mRNA及其蛋白和p-p38MAPK蛋白的表达上调(P＜0.05),p38MAPK蛋白表达差异无统计学意义,SB组各指标差异无统计学意义(P＞0.05);与LS组比较,LSS组氧合指数和SOD活性升高,病理学损伤评分、肺含水率和MDA含量降低,肺组织HO-1 mRNA及其蛋白表达上调,p-p38MAPK蛋白表达下调(P＜0.05),p38MAPK蛋白表达差异无统计学意义(P＞0.05).结论 抑制p38MAPK信号通路可导致内毒素性休克诱发急性肺损伤大鼠肺组织HO-1表达上调.     

p38MAPK信号转导通路在大鼠内毒素性急性肺损伤中的作用

目的 探讨p38丝裂原活化蛋白激酶(p38MAPK)信号转导通路在大鼠内毒素性急性肺损伤中的作用.方法 成年雄性SD大鼠60只,体重180～230 g,采用随机数字表法,将大鼠随机分为4组:对照组(C组,n=6)、急性肺损伤组(ALI组,n=24)、p38MAPK特异性抑制剂SB203580+ALI组(SB+ALI组,n=24)和SB203580组(SB组,n=6).ALI组尾静脉注射内毒素5 mg/kg制备大鼠急性肺损伤模型,C组给予等容量生理盐水,SB+ALI组于注射内毒素前30 min经尾静脉注射SB20358010 mg/kg.ALI组和SB+ALI组于注射内毒素后1、3、6 h(T1-3)时随机取8只大鼠,C组和SB组分别于给予生理盐水、SB203580后1 h处死取肺组织,检测磷酸化p38MAPK(p-p38MAPK)蛋白表达.T3时回收支气管肺泡灌洗液(BALF),测定蛋白浓度.计算细胞凋亡指数,观察肺组织病理学结果.另取32只大鼠,采用随机数字表法,将大鼠随机分为2组(n=16):ALI组和SB+ALI组,观察48 h内大鼠生存情况.结果 与C组相比,ALI组和SB+ALI组BALF中蛋白浓度、细胞凋亡指数、肺组织p-p38MAPK蛋白表达水平升高(P＜0.05);与ALI组相比,SB+ALI组上述指标降低(P＜0.05).SB+ALI组病理学损伤程度较ALI组明显减轻.ALI组大鼠生存率较SB+ALI组降低(P＜0.01).结论 p38MAPK信号转导通路参与了大鼠内毒素性急性肺损伤的发生和发展,可能与肺组织细胞凋亡有关.

Abstract：

Objective To investigate the role of p38 mitogen-activated protein kinase (MAPK) signal transduction pathway in lipopolysaccharide (LPS)-induced acute lung injury (ALI).Methods Sixty male SD rats weighing 180-230 g were randomly divided into 4 groups: control group (group C, n = 6), ALI group ( n = 24),p38MAPK specific inhibitor SB203580 + ALI group (group SB + ALI, n = 24), SB203580 group (group SB,n =6). LPS 5 mg/kg was injected intravenously via tail vein in group ALI and SB + ALI, while the equal volume of normal saline was given instead in group C. Group SB + ALI received iv injection of SB203580 10 mg/kg via tail vein 30 min before LPS administration. Group SB received injection of SB203580. The rats were sacrificed at 1, 3 and6 h agter LPS administration (T1-3) in group ALI and SB + ALI (8 rats at each time point) andat 1 h after administration in C and SB groups. The lungs were immediately removed for microscopic examination and determination of phosphorylated p38MAPK (p-p38MAPK) expression, the concentration of protein in bronchoalveolar lavage fluid (BALF) and apoptotic index (AI). Another 32 rats were selected and randomly divided into 2 groups for survival study: ALI group and SB + ALI group ( n = 16 each), and then they were treated as mentioned above and observed for 48 h. Results The concentration of protein in BALF, AI and p-p38MAPK expression were significantly increased in group ALI and SB + ALI compared with group C, while decreased in group SB + ALI compared with group ALI ( P ＜ 0. 05 ). LPS-induced pulmonary histological changes were significantly attenuated in group SB + ALI compared with group ALI. The survival rate was significantly decreased in group ALI compgred with group SB + ALI ( P ＜ 0.01 ). Conclusion p38 MAPK signal transduction pathway is involved in LPS-induced ALI, which may be related to the apoptosis in the cells in the lung.     

p38MAPK/iNOS/HO-1信号通路在赤芍减轻大鼠内毒素性急性肺损伤中的作用

目的 评价p38MAPK/iNOS/HO-1信号通路在赤芍减轻大鼠内毒素性急性肺损伤(AL1)中的作用.方法 健康清洁级雄性Wistar大鼠40只,随机分为5组(n=8):生理盐水对照组(C组)、内毒素组(L组)、赤芍组(R组)、赤芍预处理组(PR组)和SB203580组(S组).气管内滴注脂多糖(LPS)制备大鼠ALI模型.L组气管内滴注1 ml LPS溶液(2.5 mg/kg);C组滴注等容量生理盐水;R组、PR组分别于气管内滴注LPS后、滴注前2 h,经股静脉输注赤芍注射液15 mg·kg-1·h-1 2 h;S组于气管内滴注LPS前3 h,经股静脉输注SB203580溶液2.5 μmol·kg-1·h-1 3 h.于气管内滴注LPS后6 h时,经颈动脉采血样2 ml,行血气分析及测定血清NO浓度;颈动脉放血处死大鼠,测定支气管肺泡灌洗液蛋白浓度,计数中性粒细胞及细胞总数,检测肺组织MDA含量,p38MAPK、HO-1及iNOS的表达.观察肺组织病理学结果 .结果 与C组比较,其余各组肺组织p38MAPK、iNOS及HO-1表达上调,支气管肺泡灌洗液中性粒细胞计数比、蛋白浓度、肺组织MDA含量及血清NO浓度升高.PaO2和HCO1浓度降低(P<0.01);与L组比较,R组、PR组和S组p38MAPK及iNOS表达下调,HO-1表达上调.支气管肺泡灌洗液中性粒细胞计数比、蛋白浓度、肺组织MDA含量及血清NO浓度降低,PaO2和HCO3-升高(P<0.05);R组、PR组和S组肺组织损伤程度较L组减轻.结论 赤芍可减轻大鼠内毒素性急性肺损伤,可能与抑制p38MAPK/iNOS/HO-1信号通路有关.     

缝隙连接在兔失血性休克复苏诱发肺损伤中的作用

目的 评价缝隙连接在兔失血性休克复苏诱发肺损伤中的作用.方法 健康家兔24只,体重1.5～2.2 kg,经股动脉放血制备失血性休克模型.随机分为2组(n=12),传统治疗组(A组)静脉输注乳酸钠林格氏液1.5 ml·kg-1·min-1 30 min(T3);辛醇组(B组)静脉输注乳酸钠林格氏液1.5 ml·kg-1·min-1 30 min(T3),同时腹腔注射99.5%辛醇5 mmol/kg.然后两组回输全部放血及等放血量的乳酸钠林格氏液后静脉输注乳酸钠林格氏液2.5 ml·kg-·h-1 150 min(T4).于放血前(T1)、模型制备成功即刻(T2)、T3、T4时记录左心室收缩压(LVSP)和HR,记录复苏期间兔的病死情况.于T时处死,测定肺组织Na+-K+-ATP酶和Ca2+-ATP酶的活性,计算肺通透性指数和肺湿重/干重(W/D)比,光镜下观察肺组织病理学结果.结果 与A组比较,B组T3时HR和LVSP降低,T4时LVSP升高,HR降低,Na+-K+-ATP酶和Ca2+-ATP酶活性升高,肺通透性指数降低,肺组织W/D比和病死率降低(P＜0.05或0.01).B组肺组织病理学损伤程度较A组明显减轻.结论 缝隙连接参与了兔失血性休克复苏诱发肺损伤.     

环腺苷酸-蛋白激酶A在大鼠内毒素性急性肺损伤时血红素加氧酶-1表达上调中的作用

目的 探讨环腺苷酸-蛋白激酶A(cAMP-PKA)在大鼠内毒素性急性肺损伤时血红素加氧酶-1(HO-1)表达上调中的作用.方法 健康清洁级雄性SD大鼠48只,体重180 ～ 220 g,2.5 ～ 3.0月龄,采用随机数字表法,将大鼠随机分为4组(n=12)∶正常对照组(C组)股静脉注射生理盐水(LPS溶媒)0.5 ml,2h后皮下注射生理盐水(H89溶媒)0.5 ml;内毒素性急性肺损伤组(ALI组)股静脉注射10 mg/kg LPS 0.5 ml,2h后皮下注射生理盐水0.5 ml; H89+内毒素性急性肺损伤组(H+ALI组),股静脉注射10 mg/kg LPS 0.5 ml,2h后皮下注射5 mg/kg H89 0.5 ml; H89组(H组)股静脉注射生理盐水0.5 ml,2h后皮下注射5 mg/kg H89 0.5 ml.静脉注射LPS后6h时处死大鼠,取肺组织,行病理学评分,测定肺组织含水量;采用Western blot法测定HO-1和PKA表达;采用荧光定量PCR法测定HO-1mRNA表达.结果 与C组比较,ALI组和H+ALI组肺组织含水量和病理学评分升高,肺组织PKA、HO-1和HO-1 mRNA表达上调(P＜0.05),H组上述各指标差异无统计学意义(P＞0.05);与ALI组比较,H+ALI组肺组织含水量和病理学评分升高,肺组织PKA、HO-1和HO-1 mRNA表达下调(P＜0.05).结论 大鼠内毒素性急性肺损伤时HO-1表达上调的机制与cAMP-PKA信号通路活化有一定关系.     

Toll样受体4在失血性休克复苏致小鼠急性肺损伤中的作用

目的 探讨Toll样受体4(TLR4)在失血性休克复苏致小鼠急性肺损伤中的作用.方法 TLR4基因突变型C3H/HeJ小鼠和野生型C3H/HeN小鼠各24只,两种品系小鼠各随机分为2组:假手术组(S组,n=6)、失血性休克复苏组(HSR组,n=18)制备失血性休克复苏模型,并于复苏后6、24、48 h时各取6只小鼠颈动脉放血处死后开胸取肺组织,免疫组织化学法检测p38 MAPK表达水平,酶联免疫吸附法测定白细胞介素(IL)-10和IL-6含量,透射电镜下观察肺组织超微结构.结果 与C3H/HeN小鼠比较,C3H/HeJ小鼠复苏后肺组织p38 MAPK表达下调,IL-6和IL-10含量降低(P<0.05或0.01),病理损伤程度减轻.两种品系小鼠中,与S组比较,HSR组复苏后24 h时肺组织IL-6和IL-10含量增加,复苏后48 h时肺组织IL-6含量增加(P<0.05或0.01);C3H/HeN小鼠中,与S组比较,HSR组复苏后6 h和24 h时肺组织p38 MAPK蛋白表达上调,复苏后48 h时肺组织IL-10含量增加(P<0.01).结论 TLR4参与小鼠失血性休克复苏致急性肺损伤的发生,其机制与激活p38 MAPK信号转导通路有关.     

戊乙奎醚预处理对脓毒症小鼠肺损伤时MAPK信号转导通路的影响

目的 探讨戊乙奎醚(PHC)预处理对脓毒症小鼠肺损伤时丝裂原活化蛋白激酶(MAPK)信号转导通路的影响.方法 健康雌性昆明小鼠105只,体重20～25 g,随机分为3组(n=35):假手术组(S组)、脓毒症(CLP)组和戊乙奎醚(PHC)组.采用盲肠结扎并穿孔法制备脓毒症模型.PHC组于造模前1 h腹腔注射戊乙奎醚0.45 mg/kg,s组和CLP组于造模前1 h注射等容量生理盐水.于造模后即刻测定肺微血管通透性;造模后12 h时进行动脉血气分析,观察肺组织病理结果,测定肺组织丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性和磷酸化的p38丝裂原活化蛋白激酶(p38MAPK)、细胞外信号调节激酶(ERK1/ERK2)和c-jun氨基末端蛋白激酶(JNK)表达.结果 与S组比较,CLP组PaO2、PaO2/FiO2和pH值降低,肺微血管通透性和肺组织MDA含量升高,SOD活性降低,磷酸化的p38MAPK、ERK1/ERK2和JNK表达上调(P<0.05或0.01);与CLP组比较,PHC组PaO2、PaO2/FiO2和pH值升高,肺微血管通透性和肺组织MDA含量降低,SOD活性升高,磷酸化的p38MAPK和ERK1/ERK2表达下调(P<0.05或0.01).结论 戊乙奎醚预处理可通过抑制MAPK信号转导通路(p38MAPK和ERK1/ERK2)的激活,从而减轻脓毒症小鼠肺损伤.     

MLK3-MKK3/6-p38MAPK信号转导通路在大鼠内毒素性急性肺损伤中的作用

目的 探讨MLK3-MKK3/6-p38MAPK信号转导通路在大鼠内毒素性急性肺损伤中的作用.方法 健康成年雄性SD大鼠78只,体重200～250 g,随机分为4组:对照组(C组,n=6)、急性肺损伤组(ALI组,n=24)、MLK3抑制剂K252a组(MK组,n=24)和p38MAPK特异性抑制剂SB203580组(MS组,n=24).ALI组尾静脉注射内毒素5 mg/kg制备大鼠急性肺损伤模型,C组给予等容量生理盐水,MK组和MS组于注射内毒素前30 min经尾静脉分别注射K252a 75μg/kg、SB203580 10 mg/kg.ALI组、MK组和MS组于注射内毒素后1、3、6、12 h(1-4)时各组随机取6只大鼠,C组于给予生理盐水后即刻处死取肺,采用ELISA法测定支气管肺泡灌洗液中TNF-α浓度,称重后计算肺湿干重比,采用Western b1ot法测定p-MLK3、p-MKK3/6及p-p38MAPK的表达,观察肺组织病理学结果.结果 与C组比较,ALI组、MK组和MS组各时点支气管肺泡灌洗液中TNF-α浓度、肺湿干重比、p-MLK3、p-MKK3/6及p-p38MAPK的表达水平升高(P＜0.01);与ALI组比较,MK组上述指标降低,MS组支气管肺泡灌洗液中TNF-α浓度、肺湿干重比、p-p38MAPK表达水平降低(P＜0.05).病理学结果显示:MK组和MS组肺组织损伤较ALI组减轻.结论 MLK3-MKK3/6-p38MAPK信号转导通路在大鼠内毒素性急性肺损伤中起重要作用.     

c-Jun氨基末端激酶在大鼠内毒素性急性肺损伤中的作用

目的 评价c-Jun氨基末端激酶(JNK)在大鼠内毒素性急性肺损伤中的作用.方法 雄性成年SD大鼠80只,体重250～300 g,采用随机数字表法,将其随机分为4组(n=20):对照组(C组)、急性肺损伤组(ALI组)、SP600125组(S组)和二甲基亚砜组(D组).ALI组、S组和D组尾静脉注射LPS 5 mg/kg,C组尾静脉注射等容量生理盐水;S组和D组给予LPS后,分别尾静脉注射JNK抑制剂SP600125 30 mg/kg或二甲基亚砜0.2 ml.于给予LPS后4 h时,各组处死10只大鼠,回收支气管肺泡灌洗液(BALF)并取肺组织,采用ELISA法检测BALF中TNF-α和IL-1β的浓度,计算肺组织湿重/干重比(W/D比),观察肺组织病理学结果,并进行肺损伤评分.各组其余10只大鼠观察至给予LPS后48 h,记录大鼠生存情况.结果 与C组比较,其余各组BALF中TNF-α和IL-1β的浓度、肺组织W/D比和肺损伤评分升高,生存率降低(P<0.05或0.01);与ALI组比较,S组BALF中TNF-α和IL-1度、肺组织W/D比和肺损伤评分降低,生存率升高(P<0.01),D组差异无统计学意义(P>0.05).结论 JNK的活化参与了大鼠内毒素性急性肺损伤的发生发展.

Abstract：

Objective To evaluate the role of c-Jun N-terminal kinase (JNK) in lipopolysaccharide (LPS)-induced acute lung injury ( ALI) in rats.Methods Eighty male SD rats weighing 250-300 g were randomly divided into 4 groups ( n = 20 each) : control group (group C) ; ALI group; LPS + SP600125 (JNK inhibitor)group (group S) and LPS+ DMSO (the solvent) group (group DMSO) . ALI was induced by intravenous LPS 5mg/kg. In S and DMSO groups, SP600125 30 mg/kg and DMSO 0.2 ml were injected intravenously after LPS administration respectively. Ten animals were sacrificed by exsanguinafions at 4 h after LPS administration in each group. The broncho-alveolar lavage fluid (BALF) was colleted. The TNF-α and IL-1β concentrations in BALF were measured. The lungs were removed for microscopic examination and determination of W/D lung weight ratio. The other 10 animals in each group were observed for 48 h survival rate. Results Intravenous LPS significantly increased TNF-α and IL-1β concentrations in BALF and W/D lung weight ratio, decreased 48 h survival rate and induced histologic damage. Intravenous SP600125 30 mg/kg significantly attenuated the above-mentioned LPS-induced changes. Conclusion Activation of JNK is involved in the development of endotoxin-induced ALI in rats.     

p38MAPK信号传导通路与重症急性胰腺炎

丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号传导通路是细胞外信号引起细胞反应的共同通路,其中p38MAPK在介导炎症、应激等细胞反应中最为引人注目.本文综述了p38MAPK这一信号传导通路的结构特征、激活与生物学效应,及其在重症急性胰腺炎发病机制中的作用,旨在为重症急性胰腺炎的诊治研究提供新的思路.     

活化蛋白-1在大鼠内毒素性肺损伤时血红素加氧酶-1上调中的作用

目的 评价活化蛋白-1(AP-1)在大鼠内毒素性肺损伤时血红素加氧酶-1(HO-1)上调中的作用.方法 健康清洁级雄性SD大鼠48只,体重200 ～ 220 g,2.5 ～ 3.0月龄,采用随机数字表法,将其随机分为4组(n=12):正常对照组(C组)腹腔注射0.1％二甲基亚砜(姜黄素溶媒)0.5 ml,30 min时股静脉注射生理盐水(LPS溶媒)0.5 ml;内毒素性肺损伤组(ALI组)腹腔注射0.1％二甲基亚砜0.5ml,30 min时股静脉注射10 mg/kg LPS0.5 ml;姜黄素+内毒素性肺损伤组(Cur+ ALI组)腹腔注射20mg/kg姜黄素0.5 ml,30 min时股静脉注射10 mg/kg LPS 0.5 ml;姜黄素组(Cur组)腹腔注射姜黄素20mg/kg,30 min时股静脉注射生理盐水0.5 ml.静脉注射LPS 6 h时处死大鼠取肺组织,行病理学评分,测定MDA含量和SOD活性;采用Western blot法测定HO-1和AP-1表达;采用荧光定量PCR法测定HO-1 mRNA表达.结果 与C组比较,ALl组和Cur +ALI组肺组织病理学评分和MDA含量升高,SOD活性降低,HO-1 mRNA、HO-1和AP-1表达上调(P＜0.05),Cur组上述各指标差异无统计学意义(P＞0.05);与ALl组比较,Cur+ ALl组肺组织病理学评分和MDA含量升高,SOD活性降低,HO-1mRNA、HO-1和AP-1表达下调(P＜0.05).结论 内毒素性肺损伤时HO-1上调的机制与转录因子AP-1活化有一定关系.     

瑞芬太尼对失血性休克兔急性肺损伤时脂质过氧化反应的影响

目的 探讨瑞芬太尼对失血性休克兔急性肺损伤时脂质过氧化反应的影响.方法 健康成年兔32只,随机分为4组(n=8):假手术组(S组)、急性肺损伤组(ALI组)、低剂量瑞芬太尼组(LR组)和高剂量瑞芬太尼组(HR组).ALI组、LR组和HR组经10 min股动脉放血至35～45 mm Hg制备急性肺损伤模型,LR组、HR组分别于放血前15 min静脉输注瑞芬太尼0.66、1.32μg·kg-1·min-1至处死动物.ALI组给予等容量生理盐水.于放血即刻(T1)、放血开始后20(T2)、70(T3)和100 min(T4)时经左侧股动脉采集血样,行血气分析,随后处死,取肺组织,光镜下观察肺组织病理学结果,计算肺湿干重比(W/D),测定MDA含量和SOD活性.结果 与S组比较,ALI组W/D、MDA含量升高,SOD活性降低(P＜0.05);与ALI组比较,LR组T2时pH值、T2～4时PaO2升高,HR组T2-4时pH值和PaCO2升高,LR组和HR组W/D、MDA含量降低,SOD活性升高(P＜0.05);与LR组比较,HR组W/D、MDA含量降低,SOD活性升高(P＜0.05).LR组和HR组肺组织病理学损伤程度较ALI组减轻,HR组较LR组减轻.结论 瑞芬太尼可通过抑制脂质过氧化反应减轻失血性休克兔急性肺损伤.     

Effect of TLR-4 and HO-1 on acute lung injury induced by hemorrhagic shock in mice

Objective： To examine whether TLR-4 has an ettect on hemorrhage induced changes in lung, and to investigate the change of heme oxygenase-1 （HO-1） on acute lung injury （ALl） induced by hemorrhagic shock in mice.
Methods： Forty-eight male mice, including C3H/HeN mice and C3H/HeJ mice, were randomly divided into sham group （n=12）, hemorrhagic shock group with twelve mice in each phase. Blood pressure （BP） was monitored continuously by attaching carotid artery catheter to a strain gauge pressure transducer/ polygraph. Arterial blood samples were taken for blood gas analysis. A mouse model of non-lethal hemorrhagic shock and resuscitation was used to observe pulmonary myeloperoxidase （MPO） activity and wet/dry weight ratio （W/D）. The expression of HO- 1 was observed by means of RT-PCR and immunohistochemistry. IL-6 and IL-10 in lung tissue homogenate were assayed by enzyme-linked immunosorbent assay （ELISA）. The pulmo- nary pathologic changes were observed under electron microscope and light microscope.
Results： Compared with sham group, the expression of HO- 1 in lung tissue was significantly higher in Hem 24 h and Hem 48 h of C3H/HeN mice （P〈0.01）. The expression of HO-1 mRNA and the levels of IL-6, IL-10 and MPO in lung tissue were markedly increased in Hem 24 h （P〈0.01 or P〈0.05）; Compared with C3H/HeN mice, the expression of HO- 1 rnRNA and the levels of IL-6 and IL-10 in C3H/HeJ mice significantly decreased in Hem 24 h and Hem 48 h （P〈0.01 or P〈O.05）, and the W/D, MPO in C3H/HeJ mice were obvi- ously lower in Hem 24 h （P〈0.05）. The injuries of lung tissues after hemorrhagic shock have been demonstrated by histological examination with electron microscope and light microscope.
Conclusions： TLR-4 and HO-1 might modulate the bal- ance of pro- and anti-inflammatory processes in inflamma- tory reaction of hemorrhagic shock-induced ALl, and the activation of Toll-like receptor might induce the transcrip- tion activity of HO- 1, which may play a k