严重烧伤大鼠肝脏p38丝裂原活化蛋白激酶对肿瘤坏死因子α表达的调控及其在肝损伤中的作用

目的观察大鼠严重烧伤后肝脏p38丝裂原活化蛋白激酶(MAPK)对肿瘤坏死因子(TNF)α表达的调控及其在肝损伤中的作用。方法将健康成年雄性SD大鼠随机分为假伤组;烧伤 SB203580组:30%TBSAⅢ度烫伤(以下称烧伤)后15min和12h静脉注射p38MAPK的特异性抑制剂SB203580(10mg/kg);烧伤对照组:同前致伤后给予等量等渗盐水,每组8只。测定3组大鼠伤后24h血清天冬氨酸转氨酶(AST)和丙氨酸转氨酶(ALT)活性的变化,并分别采用实时逆转录聚合酶链反应(RT-PCR)法和蛋白印迹(Western blot)法检测肝脏TNF-αmRNA及p38MAPK、磷酸化p38MAPK的表达水平。结果烧伤对照组大鼠血清AST和ALT活性及肝脏TNF-αmRNA的表达水平均显著高于假伤组(P<0.05或0.01);烧伤 SB203580组此3项指标均显著低于烧伤对照组(P<0.05或0.01),但与假伤组比较差异无统计学意义(P>0.05).3组大鼠肝脏p38MAPK表达水平比较,差异无统计学意义(P>0.05);其磷酸化p38MAPK表达水平之比———假伤组∶烧伤对照组∶烧伤 SB203580组为1.00∶3.90∶1.10,烧伤 SB203580组与假伤组比较,差异无统计学意义(P>0.05),与烧伤对照组比较明显偏低(P<0.01).结论大鼠严重烧伤后,肝脏中活化的p38MAPK促进了TNF-αmRNA的表达,并参与了肝损伤的发生。     

神经病理性疼痛大鼠鞘内注射SB203580的镇痛作用

目的 观察神经病理性疼痛大鼠鞘内注射p38丝裂原活化蛋白激酶（p38MAPK）抑制剂SB203580的镇痛作用，探讨p38MAPK信号转导通路在慢性神经病理性疼痛中的作用。方法雄性SD大鼠，体重220～250g，建立坐骨神经结扎性损伤（CCI）疼痛模型，第一部分40只CCI大鼠，随机分为5组，对照组、SB0．1μg组、SB0．5μg组、SB2．5μg组和SB5μg组，（n=8），CCI后7d，鞘内注射2％二甲基亚砜或不同剂量的SB203580（0．1、0．5、2．5、5μg）10μl，在给药前和给药后0．5、3、6、12、24h用von Frey丝测定大鼠术侧后爪机械缩足反射阈值（MWT）；另外36只大鼠，随机分为6组（n=6）：Sham组、CCI组、DMSO组、SB0．1、0．5、5μg组，CCI后7d鞘内注射相应剂量SB203580 10μl，给药后6h取L4.5脊髓，用免疫组织化学方法检测脊髓背角磷酸化环磷酸腺苷反应元件结合蛋白（pCREB）的表达。结果CCI后大鼠产生了机械痛敏。鞘内注射SB203580剂量依赖性地提高了MWT。CCI后脊髓背角pCREB阳性神经元增加，鞘内注射SB0．5、5μg抑制了CCI引起的脊髓背角pCREB表达增加（P〈0．01）。结论鞘内注射SB203580能减轻CCI引起的痛敏，抑制脊髓背角CREB的磷酸化，p38MAPK信号通路参与慢性神经病理性疼痛。     

斯伐他汀对糖尿病肾病大鼠肾小球系膜细胞p38信号通路的影响

目的研究糖尿病肾病大鼠肾小球系膜细胞p38丝裂原活化蛋白激酶（MAPK）的表达及斯伐他汀对其的影响。方法分别以高糖、糖基化终产物（AGE）及过氧化氢孵育糖尿病大鼠肾小球系膜细胞（RMC），Western印迹法检测RMC的p38MAPK和TGF—β蛋白表达，p38MAPK特异性抑制剂SB203580及斯伐他汀预处理对其影响。结果高糖、AGE及过氧化氢均可单独激活p38MAPK，增加RMC的磷酸化（P）p38MAPK和TGF—β的蛋白表达；SB203580显著抑制TGF—β的蛋白表达（P〈0．05）；斯伐他汀抑制p38MAPK的活化并减少TGF—β的蛋白表达（P〈0．05）。结论p38MAPK可能是糖尿病肾病发生的始动信号之一。斯伐他汀可能通过抑制p38MAPK磷酸化而减少TGF—β的蛋白表达。     

p38 MAPK在吗啡预处理对缺血-再灌注损伤心脏保护中的作用

目的 丝裂原活化蛋白激酶（MAPK）p38（p38 MAPK）是MAPK家族中重要成员之一，在缺血预处理（IPC）的保护作用信号传导机制中起着一定的作用，本研究旨在探讨p38 MAPK在吗啡预处理（MPC）对心脏缺血-再灌注损伤保护中的角色.方法 在麻醉开胸大鼠在体心脏模型，随机将54只雄性大鼠分为八组：所有心脏都经历30 min缺血和120 min再灌注，对照组（CON组，n=9）；缺血预处理组（IPC组，n=9）：在缺血-再灌注前经历3次5 min缺血，3次5 min再灌注；吗啡预处理组（MPC组，n=6）：在缺血和再灌注前5 min内分别静注吗啡100 μg/kg，再停止5 min共三个循环；SB＋MPC组（n=6）和SB＋IPC组（n=6）分别于MPC或IPC前15 min给予p38 MAPK选择性拮抗药（SB 203580）0.2 mg/kg静注.MPC＋SB组（n=6）或IPC＋SB组（n=6）分别于缺血前5min给予SB 203580 0.2 mg/kg静注.以及SB组（n=6）：缺血-再灌注前静脉给予SB 203580 0.2mg/kg.观察血流动力学指标、心肌缺血梗死区（IS/AAR）、p38 MAPK和磷酸化的p38 MAPK（pp38 MAPK）的表达.结果 MPC或IPC前应用SB 203580可以明显消除IPC的作用，但并没有消除MPC的作用.而30 min缺血前给予SB 203580可以消除MPC和IPC降低心脏IS/AAR的作用IPC组pp38 MAPK分别在缺血5 min和再灌注30 min蛋白表达密度明显高于CON组（P＜0.01）.然而在MPC组pp38 MAPK仅在再灌注30 min明显高于CON组（P＜0.01）.结论 p38 MAPK的激活是MPC对大鼠缺血后心肌保护信号机制中的介导因子而不是触发因子.     

七氟烷诱导大鼠海马HO－1基因表达信号转导通路的研究

目的：探讨七氟烷诱导大鼠海马血红素氧合酶－1（NO－1）表达的信号转导通路。 方法：40只Wistar大鼠随机分为5组（n=8）：正常培养组（C组）、0．6MAC七氟烷组（s组）．0．6MAC七氟烷＋SB203580组（SB组）、0．6MAC七氟烷＋U-0126组（u组）、0．6MAC七氟烷＋SP600125（SP组）。C组大鼠正常喂养。S组大鼠吸入0．6MAC七氟烷60min后继续培养24h。SB组、U组和SP组在大鼠吸入0．6MAC七氟烷时分别腹腔注100mg／kg SB203580、100mg／kg U-0126或1mg／kg SP600125后同S组处理。检测大鼠海马组织中HO－1－mRNA和磷酸化ERK1／2（p^ERK1／2）、磷酸化P^38（PP^38）．磷酸化JNK（P^JNK）、磷酸化HO-1（P^HO-1）蛋白的表达。 结果：与C组比较，S组大鼠海马HO-1～mRNA的表达增加（P〈0．05），p^ERK1／2（P〈0．05），PP38（P〈0．05）和HO^-1（p〈0.05）蛋白表达增加。与S组比较，SB组大鼠海马HO^-1 -mRNA的表达减少（P〈0．05），PP^38（p〈0．05）和PHO^-1（P〈0．05）蛋白表达减少，P^ERK1／2（p〈0．05）和P^JNK（p〈0．05）蛋白表达变化不明显（P〉0．05）。与S组比较，U组大鼠海马HO^-1-mRNA的表达减少（p〈0．05），P^ERK1／2（P〈0．05）和PHO^-1（P〈-．05）蛋白表达减少．PP^38（p〉0．05）和P^JNK（p〉0．05）蛋白表达变化不明显。与S组比较，SP组大鼠海马HO^-1-mRNA的表达变化不明显（p〉0．05），p^JNK蛋白表达减少（P〈0．01）．P^ERK1／2（p〉0．05），PP^38（p〉0.05）和PHO^-1（P〉0．05）蛋白表达变化不明显。 结论：七氟烷通过^ERK1／2和P^38信号转导通路诱导大鼠海马HO－1－mRNA的表达。     

p38丝裂原活化蛋白激酶信号转导通路在严重烧伤大鼠枯否细胞肿瘤坏死因子α和白细胞介素1β产生中的作用

目的 探讨p38丝裂原活化蛋白激酶(MAPK)信号转导通路在严重烧伤大鼠枯否细胞(KCs)促炎性细胞因子肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)产生中的作用。方法 健康成年的雄性SD大鼠32只,随机分为：假烫组;假烫 SB203580组;烧伤对照组;烧伤 SB203580组,每组8只。假烫或烧伤24h后分离出肝脏KCs,培养18h后加入50ng／ml的LPS进行刺激,18h后取上清液,用酶联免疫吸附法(ELISA)测定TNF-α和IL-1β的含量,并收集KCs,实时逆转录聚合酶链反应检测KCs内TNF-α和IL-1β mRNA表达的改变,蛋白印迹(Western blot)法检测KCs中p38MAPK和JNK活性的变化。结果 烧伤大鼠分离出的KCs培养上清液中TNF-α和IL-1β含量、KCs中TNF-α和IL-1β mRNA的表达均较假烫组的明显增强,同时KCs中p38 MAPK活性和JNK活性升高,SB203580能显著抑制大鼠KCs上清液中TNF-α和IL-1β含量、KCs中TNF-α和IL-1β mRNA的表达和p38MAPK活性的升高,对JNK活性无影响。结论p38MAPK信号转导通路介导了严重烧伤大鼠KCs促炎性细胞因子TNF-α和IL-1β的产生。     

P38 MAPK抑制剂在高糖诱导系膜细胞CTGF表达和基质蛋白分泌中的作用

目的:探讨P38 MAPK抑制剂SB203580在高糖诱导大鼠肾小球系膜细胞对丝裂原活化蛋白激酶磷酸酶-1(mitogen-activated protein kinase phosphatase-1,MKP-1)与结缔组织生长因子(connective tissue growth factor,CTGF)表达和细胞外基质蛋白分泌的作用。方法:体外培养大鼠肾小球系膜细胞分为4组:正常对照组(NG组,5. 5 mmol/L葡萄糖);渗透压对照组(NG+M组,5. 5 mmol/L葡萄糖+24. 5 mmol/L甘露醇);高糖组(HG组,30 mmol/L葡萄糖);高糖+SB203580组(HG+SB203580组,30 mmol/L葡萄糖+10μmol/L SB203580),分别给予不同刺激。48 h收集细胞,分别提取系膜细胞蛋白、RNA及细胞上清液。采用Western blot检测MKP-1、p38 MAPK及磷酸化p38MaPK的表达; RT-PCR检测p38 MAPK、MKP-1、CTGF和FN mRNA的表达; ELISA法测定细胞上清CTGF和纤维黏连蛋白(fibronectin,FN)含量,放免法测定细胞上清液Ⅳ型胶原的含量。结果:与NG组相比,HG组系膜细胞MKP-1表达下调,p38 MAPK蛋白表达无明显差别,但磷酸化的p38 MAPK表达明显升高,MKP-1 mRNA表达下降,p38 MAPK、CTGF和FN mRNA的表达增加,细胞上清液中CTGF、FN和Ⅳ型胶原含量增加;与HG组相比,HG+SB203580组MKP-1表达升高,p38 MAPK蛋白表达无明显差别,但磷酸化的p38MAPK表达明显下降,p38 MAPK、CTGF和FN mRNA的表达下降,系膜细胞上清液中CTGF、FN和Ⅳ型胶原含量下降。结论:p38 MAPK抑制剂SB203580通过增强MKP-1的表达,增强p38 MAPK去磷酸化,使p38 MAPK活性下降,从而阻断CTGF的合成和细胞外基质蛋白表达,在糖尿病肾病细胞外基质重构中发挥保护作用。     

颈交感神经阻滞对复合伤大鼠小肠黏膜血流量及屏障功能的影响

目的 了解颈交感神经阻滞（SB）对放烧复合伤大鼠小肠黏膜血流量及屏障功能的影响。方法 将SD大鼠分为正常（18只）、复合伤组（100只）和复合伤＋SB组（100只）。后2组制成5Gy^60co γ射线全身性照射＋15％TBSAⅢ度烧伤的复合伤模型，并设伤后即刻和伤后1、3、5、7d为观察时相点（每时相点20只大鼠）。复合伤＋SB组在特定时间颈后双侧注射盐酸罗哌卡因致SB，复合伤组同法给予等量等渗盐水，不造成SB。检测各组大鼠不同时相点小肠黏膜组织血流量、肠绒毛高度及隐窝深度，并测定肠黏膜Na^＋、K^＋-腺苷三磷酸（ATP）酶活性和小肠通透性的变化。结果 与正常组大鼠小肠黏膜血流量[（1．26±0．23）ml·min·g^-1]比较，复合伤组明显下降[伤后1d为（0．29±0．07）ml·min^-1·g^-1，P〈0．01]，且绒毛高度显著降低、隐窝细胞大量破坏，肠黏膜Na^＋、K^＋ -ATP酶活性下降和小肠通透性显著升高。与复合伤组各项指标比较，复合伤＋SB组损伤程度有所减轻[小肠黏膜血流量伤后即刻为（0．82±0．11）ml·min^-1·g^-1，P〈0．01]。结论 SB 可增加放烧复合伤大鼠小肠黏膜血流量、促进肠上皮修复、改善肠道屏障功能。     

阻断p38MAPK信号通路降低脑死亡大鼠肾脏免疫原性

目的 探讨降低脑死亡大鼠肾脏免疫原性的有效途径.方法 雄性Wistar大鼠30只,体质量180～200 g,随机均分3组,脑死亡+SB203580组:诱导大鼠脑死亡时,静脉注射SB203580(1 mg/100 g·Wt);脑死亡组:诱导大鼠脑死亡.脑死亡后机械呼吸6 h,如大鼠血压>80mm Hg(1 mm Hg=0.133 kPa),取肾脏;对照组:正常大鼠麻醉后取肾脏.逆转录-聚合酶链反应(RT-PCR)检测肾脏肿瘤坏死因子(TNF)-α仅和白细胞介素(IL)-1βmRNA表达,Western blot检测肾脏磷酸化p38丝裂原活化蛋白激酶(p38MAPK)以及TNF-α和IL-1β蛋白表达.结果 肾脏TNF-α和IL-1βmRNA和蛋白表达,以及磷酸化p38MAPK蛋白表达,脑死亡组比对照组显著增加(P<0.01);脑死亡+SB203580组比脑死亡组显著下降(P<0.05),但比对照组明显增加(P<0.01).结论 SB203580能阻断p38MAPK信号通路,减少脑死亡大鼠肾脏磷酸化p38MAPK和促炎细胞因子表达,可望成为降低其免疫原性的一条有效途径.     

热休克预处理对严重烧伤大鼠胃黏膜的保护作用及机制

目的观察热休克预处理（HS）对严重烧伤大鼠胃黏膜热休克蛋白（HSP）60、HSP70表达及线粒体超氧化物歧化酶（SOD）、细胞色素氧化酶（CCO）活性的影响，探讨HS对严重烧伤大鼠胃黏膜的保护作用及机制。方法将Wistar大鼠随机分为烧伤组（40只）：烧伤后即制作急性胃黏膜损伤模型；另取8只大鼠不致伤作为空白对照。HS组（40只）：于烧伤前20h行HS，另取8只大鼠仅行HS不烧伤，作为实验对照。放线菌素D组（40只）：在HS前30min静脉注射放线菌素D0．1mg／kg，随后的处理同HS组；另取8只大鼠只注射放线菌素D不烧伤，作为实验对照。于伤后3、6、12、24、48h处死各组大鼠（每组每时相点8只）。取胃黏膜组织检测胃黏膜损伤指数（UI）、HSP70 mRNA、HSP60、HSP70的表达及SOD、CCO的活性。结果大鼠烧伤后uI呈时间依赖性增加，烧伤组伤后24h胃黏膜损伤程度最严重，UI为12．8±1．9。除伤后3h外，HS组大鼠各时相点uI均低于烧伤组（P＜0．05或0．01）。放线菌素D组大鼠胃黏膜损伤程度明显重于烧伤组及HS组（P＜0．05）。烧伤组、HS组除伤后24、48h外其余各时相点HSP70 mRNA均增加，而放线菌素D组伤后24、48h有所增加。与烧伤组比较，HS组大鼠胃黏膜HSP60及HSP70表达显著增加，除伤后48h外其余各时相点比较，差异均有统计学意义（P＜0．05或0．01）。放线菌素D组大鼠HSP60和HSP70表达明显受抑（P＜0．05）。大鼠烧伤后胃黏膜CCO、SOD活性不断降低，经HS后CCO及SOD下降不明显，在伤后6、12、24h均高于同时相点烧伤组（P＜0．05或0．01）。结论HS对大鼠严重烫伤后急性胃黏膜损害具有保护作用，其机制可能与HSP60、HSPT0诱导表达增强，线粒体SOD及CCO活性增加等有关。     

大鼠烧伤后早期心肌组织核因子κB活化对细胞因子表达及心肌功能的影响

目的观察核因子(NF)κB活化在大鼠烧伤后早期心肌组织表达肿瘤坏死因子(TNF)α及心肌功能损害中的作用,进一步阐明烧伤后早期心肌功能损害的发生机制。方法将170只Wistar大鼠随机分为对照组(20只)、烧伤组(90只)、吡咯烷二硫代氨基甲酸盐(PDTC)组(60只)。后两组大鼠均于背部造成35％TBSAⅢ度烧伤后,立即腹腔注射等渗盐水,且PDTC组同时皮下注射PDTC 250 mg／kg。对照组除不烧伤外,其余处理同烧伤组。伤后3、6、12、24 h采用八导生理记录仪记录左心室收缩压(LVSP),左心室舒张末期压(LVEDP),室内压最大上升、下降速率( dp／dtmax、-dp／dtmax);逆转录聚合酶链反应和原位杂交法观察心肌组织TNF-αmRNA的表达。伤后1、3、6、12、24 h采用凝胶电泳迁移率变动分析法检测心肌组织NF-κB活性,以积分吸光度(A)值表示。对照组作相同检测。结果伤后3～24 h烧伤组大鼠LVSP、±dp／dtmax低于对照组(P<0．01),而LVEDP高于对照组(P<0．01)。伤后3 h烧伤组大鼠心肌组织TNF-αmRNA表达明显高于对照组, 6 h达峰值(P<0．01),它在心肌细胞中表达尤为明显。伤后1 h烧伤组大鼠心肌组织NF-κB活性迅速升高[积分A值为(20．3±3．4)×104],明显高于对照组积分A值(2．2±0．4)×104,3 h时达峰值,24 h时仍高于对照组(P<0．01)。与烧伤组比较,PDTC组上述指标有较明显的改善(P< 0．01)。结论大鼠严重烧伤后心肌组织NF-κB被活化,使其表达和释放促炎细胞因子,在心肌功能损害发生过程中起重要作用。     

Inhibition of p38 MAP kinase improves survival of cardiac myocytes with hypoxia and burn serum challenge

This study was aimed to investigate the effects of SB203580, the specific p38 mitogen-activated protein (MAP) kinase inhibitor, on cardiac myocyte survival and secretion of cytokines in an in vitro model of hypoxia and burn serum challenge. Results demonstrated that hypoxia and burn serum induced a persistent activation of p38 MAP kinase in primary cultured neonatal rat cardiomyocytes during the 12h period of stimulation, concomitant with a time-dependent increased expression of tumor necrosis factor (TNF)-alpha and inducible nitric oxide (iNOS), a progressively developed oxidative stress reflected by malondialdehyde (MDA) production, and myocytes injury evidenced by the increased levels of released lactate dehydrogenase (LDH) and the decreased myocyte viability. Furthermore, hypoxia and burn serum resulted in a significant increase in myocyte apoptosis, which may account for the impairment of myocyte viability as observed. SB203580 abolished p38 MAP kinase activation, blunted the upregulation of TNF-alpha, iNOS and the subsequent nitric oxide (NO) production, reduced oxidative stress, and alleviated hypoxia and burn serum-induced myocytes injury or apoptosis. These results demonstrated for the first time that inhibition of p38 MAP kinase improves survival of cardiac myocytes with hypoxia and burn serum challenge possibly via reducing the production of cytokines, such as TNF-alpha and NO, and the subsequent oxidative stress, providing strong evidence that the excessive inflammatory cytokines produced by cardiomyocytes themselves may be sufficient to cause myocardial injury after burn.     

p38丝裂原活化蛋白激酶在烧伤大鼠急性肺损伤中的作用机制

目的　研究 p38丝裂原活化蛋白激酶 (MAPK)信号转导通路在严重烧伤大鼠肺部促炎细胞因子肿瘤坏死因子α(TNF α)、白细胞介素 1β(IL 1β)产生及肺血管内皮细胞损伤中的作用和相关机制。　方法　健康成年雄性SD大鼠 4 8只 ,随机分为假烫 (A)组、烫伤对照 (B)组和烫伤 p38MAPK抑制剂SB2 0 35 80(C)组 ,每组各 16只。观察烫伤 (以下称烧伤 ) 2 4h后大鼠血清和支气管肺泡灌洗液 (BALF)中TNF α和IL 1β含量、血浆和肺脏微血管vonWillebrand因子 (vWF)含量、肺脏激活蛋白 1(AP 1)活性等指标的改变。　 结果　B组大鼠烧伤后 2 4h血清和BALF中TNF α和IL 1β含量明显增高 ,血浆vWF含量为 (194 .2± 2 8.3) % ,显著高于A组的 (93.2± 14 .3) % (P <0.0 1);肺脏微血管vWF含量的积分值为 1.1± 0 .3,显著低于A组的 3.3± 0 .4 (P <0.0 1);其肺脏AP 1活性上升。C组血清和BALF中TNF α和IL 1β含量、血浆及肺脏微血管vWF含量、肺脏AP 1的活性较B组变化幅度明显偏小。　结论　p38MAPK活化后 ,通过活化转录因子AP 1,介导了严重烧伤后肺脏促炎性细胞因子TNF α和IL 1β的产生和肺血管内皮细胞的损伤     

p38 mitogen-activated protein kinase inhibition attenuates burn-induced liver injury in rats

This study was made to evaluate the effect of SB203580, a specific p38 MAP kinase inhibitor, on burn-induced hepatic injury as well as the activation of nuclear factor (NF)-kappaB in severely burned rats. Sprague-Dawley rats were divided into three groups: (1) sham group, rats underwent sham burn; (2) burn group, rats given third-degree burns over 30% total body surface area (TBSA) and treated with vehicle plus lactated Ringer solution for resuscitation 4 ml/(kg% TBSA); and (3) burn plus SB203580 group, rats given burn injury and fluid resuscitation plus SB203580 (10 mg/kg i.v., 15 min and 12 h after burn). Hepatocellular injury (measured by serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) and hepatocellular function (determined by the indocyanine green dye retention rate (ICG R15)) were assessed at 24 h post-burn. Liver histologic changes were also analyzed. Burn trauma resulted in increased serum aminotransferases concentrations, decreased ICG R15, elevated serum tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta levels and hepatic TNF-alpha and IL-1beta mRNA expressions, and worsen histologic condition. The level of Nuclear Factor (kappa) inhibitor (IkappaBalpha) in liver was decreased and DNA-binding activity of Nuclear Factor-kappaB (NF-kappaB) was increased after thermal injury. p38 MAP kinase was more significantly activated in liver harvested from burn rats than from shams. SB203580 inhibited the activation of p38 MAP kinase, reduced the levels of TNF-alpha and IL-1beta, and prevented burn-mediated liver injury. Both the IkappaBalpha level and NF-kappaB activity in the liver following burns was not affected by administration with SB203580. These findings suggest that (1) p38 MAP kinase activation is one important aspect of the signaling event that may mediate the release of TNF-alpha and IL-1beta and contributes to burn-induced liver injury and (2) p38 MAP kinase does not influence the activation of NF-kappaB directly in the liver of severely burned rats.     

Role of p38 mitogen-activated protein kinase in Kupffer cell secretion of the proinflammatory cytokines after burn trauma

This study was designed to investigate the role of p38 mitogen-activated protein (MAP) kinase on Kupffer cells (KCs) secretion of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta and hepatic injury following burn trauma. Sprague-Dawley rats were randomized into four groups: (1) sham burn rats given vehicle, (2) sham burn rats given the p38 MAP kinase inhibitor SB203580 (10mg/kg i.v., 15min and 12h after sham burn), (3) rats given a 30% total body surface area (TBSA) full-thickness burn and fluid resuscitation plus vehicle, and (4) burn rats given injury and fluid resuscitation plus SB203580. Rats from each group were killed at 24h post-burn to examine plasma aspartate transaminase (AST) and alanine transaminase (ALT) and KCs were isolated. The KCs secretion of TNF-alpha and IL-1beta and p38 MAP kinase activity (by Western blot analysis) were also examined. These studies showed by more significant activation of p38 MAP kinase in KCs harvested from burn rats than from shams. Burn trauma resulted in hepatic dysfunction and promoted KCs secretion of TNF-alpha and IL-1beta. SB203580 inhibited p38 MAP kinase activity, reduced KCs secretion of proinflammatory cytokines, and alleviated burn-mediated hepatic dysfunction. These data suggest p38 MAP kinase activation is one important aspect of the signaling event that may mediate the KCs secretion of proinflammatory cytokines TNF-alpha and IL-1beta following burn trauma.     

烧伤血清激活p38丝裂原活化蛋白激酶信号转导通路和核因子kB诱导血管内皮细胞血管细胞黏附分子1的表达

目的观察烧伤血清对血管内皮细胞血管细胞黏附分子(VCAM)1表达的影响,探讨其信号转导机制。方法采用人脐静脉内皮细胞(HUVEC)培养模型,根据刺激物的不同分为正常血清组(正常血清刺激)、烧伤血清组(烧伤血清刺激)、SB203580组(烧伤血清 SB203580刺激)和吡咯烷二硫代氨基甲酸盐(PDTC)组(烧伤血清 PDTC刺激)。SB203580组和PDTC组在烧伤血清刺激前1h分别加入10μmol/L SB203580及10mmol/L PDTC.于烧伤血清刺激后即刻(0)、6、12、24和36h检测HUVEC内VCAM-1mRNA的转录水平。检测血清刺激24h后HUVEC膜表面VCAM-1表达水平、上清液中VCAM-1含量及HUVEC与外周血单核细胞(PBMC)之间的黏附情况。结果烧伤血清组刺激后VCAM-1mRNA的表达水平明显升高,在刺激24h时达高峰,随后下降。SB203580组及PDTC组刺激24h时HUVEC中VCAM-1mRNA表达明显下降,与正常血清组比较,差异无统计学意义(P>0.05).HUVEC膜表面VCAM-1表达水平:刺激24h时;烧伤血清组(66.5±6.2)显著高于正常血清组(19.1±1.9,P<0.05);而SB203580组及PDTC组,分别为21.7±2.3、23.1±2.4,与烧伤血清组比较明显降低(P<0.05),与正常血清组比较,差异无统计学意义(P>0·05).培养上清液中VCAM-1含量:刺激24h时,烧伤血清组(125±10)ng/L显著高于正常血清组(23±3)ng/L(P<0.05);而SB203580组(27±5)ng/L及PDTC组(29±5)ng/L均低于烧伤血清组(P<0.05).刺激24h时,烧伤血清组PBMC与HUVEC之间的黏附数为(197±11)%,较正常血清组(100±4)%显著增加(P<0·05),而SB203580组[(113±7)%]及PDTC组[(97±112)%]却明显低于烧伤血清组(P<0·05),与正常血清组比较,差异无统计学意义(P>0.05).结论烧伤血清可通过p38丝裂原活化蛋白激酶(MAPK)信号转导通路增强血管内皮细胞VCAM-1的表达,核因子(NF)κB系统参与了这一调控过程。     

Smad7 inhibits TGF-β1-induced MCP-1 upregulation through a MAPK/p38 pathway in rat peritoneal mesothelial cells

Purpose

The TGF-β1/Smad signaling pathway is subject to inhibition by Smad7. High expression of Smad7 in the peritoneum of rats can delay and attenuate not only peritoneal fibrosis, but also monocyte infiltration into the peritoneum. The aim of this study was to investigate the anti-inflammatory mechanism of Smad7 in peritoneal fibrosis.

Methods

Rat peritoneal mesothelial cells were stimulated with TGF-β1, and the expression of MCP-1 protein and mRNA was measured. Furthermore, the expression of MCP-1 was determined following inhibition of TGF-β/Smad or p38 signaling using Smad7 transfection or SB203580 (10 μmol/L), respectively. The effect of exogenous Smad7 and SB203580 on activation of the TGF-β/Smad and p38 signaling pathways was also studied.

Results

TGF-β1 significantly upregulated the expression of MCP-1 at both the protein and mRNA level in a time-dependent manner. Exogenous Smad7 and SB203580 markedly inhibited TGF-β1-induced MCP-1 expression. Moreover, high expression of exogenous Smad7 not only inhibited phosphorylation of Smad2 and Smad3, but also diminished the level of phosphorylated p38. However, SB203580 had no effect on the phosphorylation of Smad2 and Smad3.

Conclusions

TGF-β1 exhibits pro-inflammatory effects through the upregulation of MCP-1 in peritoneal fibrosis. Smad7 inhibits TGF-β1 induced MCP-1 upregulation through a MAPK/p38-dependent pathway.