血必净对内毒素血症大鼠炎症反应的保护作用及心肌AngⅡ和Toll受体-4表达的影响

目的探讨血必净(XBJ)对内毒素血症(ETM)大鼠炎症反应的保护作用及心肌Toll受体-4(TLR-4)表达的影响。方法选取8周龄的雄性SD大鼠30只并采用阴茎背静脉注射脂多糖(LPS)诱导ETM大鼠模型。将ETM大鼠随机分为LPS组:LPS+XBJ组和LPS+地塞米松(DEX)组(n=10),LPS+XBJ组和LPS+DEX组在注射LPS的同时分别给予XBJ 5 ml/kg和DEX 5 mg/kg。选取同期的10只正常大鼠作对照,仅给予等体积的生理盐水。3 h后断头取血并取心肌于液氮保存,采用酶联免疫吸附法检测各组血浆炎症因子(TNF-α、CRP和IL-6)水平,放射免疫法检测心肌组织AngⅡ水平;采用RT-PCR法和免疫印迹法检测心肌组织中TLR-4 mRNA和蛋白水平。结果与对照组相比,LPS组的血浆炎症因子及心肌组织AngⅡ、TLR-4 mRNA和蛋白水平均升高(P<0.05);除血浆CRP外,XBJ处理可降低LPS诱导的其余指标升高(P<0.05),但与对照组的差异仍有统计学意义(P<0.05);除血浆TNF-α和IL-6外,LPS+XBJ组的其余指标均高于LPS+DEX组。结论血必净可抑制ETM大鼠的炎症反应及AngⅡ,同时可降低TLR-4表达,对ETM有较好的保护作用。     

脓毒症大鼠心肌组织Toll样受体4和肿瘤坏死因子-α表达及细胞凋亡研究

目的:检测脂多糖(LPS)诱导的脓毒症大鼠心肌组织中Toll样受体4(TLR4)、肿瘤坏死因子-α(TNF-α)的表达和心肌细胞凋亡情况。方法:取72只大鼠随机分为3组,每组24只。实验1组、实验2组分别按10、12 mg/kg腹腔注射LPS建立脓毒症大鼠模型,对照组按10 mL/kg腹腔注射生理盐水。3组分别在注射后1、6、24 h,各取8只大鼠处死,处死前眼眶静脉丛采血,检测血清TNF-α水平,处死后取心脏组织,检测心肌组织中TNF-α阳性细胞率,TLR4、TNF-αmRNA表达水平及心肌细胞凋亡指数(AI)。结果:2个实验组血清TNF-α水平、心肌组织TNF-α阳性细胞率及TLR4、TNF-αmRNA相对表达量随时间均呈显著上升趋势(P 0. 05),2个实验组上述指标均高于对照组,且实验2组显著高于实验1组(P 0. 05); 2个实验组心肌细胞AI随时间呈显著上升趋势(P 0. 05),且心肌细胞各时间段AI均显著高于对照组(P 0. 05),实验2组AI水平显著高于实验1组(P 0. 05)。结论:LPS诱导的脓毒症大鼠心肌细胞凋亡水平随病情发展显著增加,心肌损害随病情程度逐渐加重,推测与TLR4、TNF-α在血清及心肌组织内的异常高表达有关。     

TLR4/NF-κB信号通路在黄芪甲苷抑制异丙肾上腺素诱导大鼠心肌肥厚中的作用

目的 探讨TLR4/NF-κB信号通路在黄芪甲苷抑制异丙肾上腺素诱导大鼠心肌肥厚中的作用.方法 以异丙肾上腺素5 mg/(kg·d)腹腔注射制备大鼠心肌肥厚模型.60只SD大鼠随机分为6组,每组10只:正常对照组、异丙肾上腺素组、异丙肾上腺素+黄芪甲苷20 mg/(kg·d)、异丙肾上腺素+黄芪甲苷40 mg/(kg·d)、异丙肾上腺素+黄芪甲苷80 mg/(kg·d)及异丙肾上腺素+普萘洛尔40 mg/(kg·d).给药组连续灌胃3周,并于灌胃1天后腹腔注射异丙肾上腺素2周.给药3周后,分别检测各组大鼠全心质量指数(HMI)、左心质量指数(LVMI);取左心室组织进行HE染色,测量左心室心肌细胞横径;RT-PCR检测心肌组织ANP和TLR4的mRNA表达;Western blot检测心肌组织TLR4、p65和IκBα的蛋白表达;ELISA检测血清中TNF-α、IL-6含量.结果 同正常对照组相比,异丙肾上腺素组大鼠表现为HMI和LVMI显著增加,左心室心肌细胞横径增加,ANP和TLR4 mRNA表达增加,TLR4和p65蛋白含量增加以及IκBα蛋白含量减少,血清中TNF-α、IL-6含量明显增加.与异丙肾上腺素组相比,黄芪甲苷不同剂量组表现为HMI和LVMI降低,左心室心肌细胞横径缩小,ANP和TLR4 mRNA表达减少,TLR4和p65蛋白含量减少以及IκBα蛋白含量增加,血清中TNF-α、IL-6含量减少,且呈一定的剂量依赖性.结论 黄芪甲苷对异丙肾上腺素诱导的心肌肥厚有保护作用,其机制可能与抑制TLR4/NF-κB信号通路有关.     

高血压大鼠左室重构时Toll样受体4的表达及作用

目的探讨Toll样受体4(toll like receptor 4,TLR4)在高血压大鼠左室重构中的表达及作用。方法健康雄性sprague-dawley(SD)大鼠6~8周龄,体重160~190g。采用两肾一夹法建立Goldblatt鼠模型,45只SD大鼠随机分为高血压组(25只)和假手术组(20只)。每周测1次尾动脉压,每两周测1次超声心动图。术后第8周,应用RT-PCR及Western-blot从mRNA及蛋白水平检测左室心肌TLR4的表达情况;放免法检测血管紧张素Ⅱ(AngⅡ)的含量。结果与假手术组相比,术后8周高血压组尾动脉压、收缩末期经线室壁应力(Meridional end-systolic wall stress,MESS)及左室重量指数(left ventricle mass index,LVMI)、相对室壁厚度(relative wall thickness,RWT)和心肌AngⅡ含量均明显增高(P<0.01);高血压组大鼠左室心肌组织中TLR4 mRNA及蛋白表达水平也明显升高(P<0.01)。相关性分析TLR4蛋白表达与LVMI、RWT呈正相关;同时与动脉压、MESS及局部AngⅡ含量相关。结论高血压左室重构时TLR4表达明显增高,表明TLR4及其介导的免疫、炎症反应可能参与高血压左室重构的发生发展,持续的压力负荷及心肌局部AngⅡ含量可能对左室心肌TLR4表达起到重要调节作用。     

血管活性肠肽对内毒素性休克大鼠肠道TLR4、MD2和BD3 mRNA表达的影响

目的探讨血管活性肠肽(vasoactive intestinal peptide,VIP)对细菌脂多糖(lipopolysaccharide,LPS)致休克大鼠肠道组织TLR4、MD2和BD3 mRNA表达的影响。方法 40只SD大鼠,随机分为LPS组(15只)、LPS+VIP组(15只)和对照组(10只)。LPS组尾静脉注射LPS(E.coli O55B5)10 mg/kg;LPS+VIP组尾静脉注射LPS 10 mg/kg后注射VIP 5 nmol/kg;对照组尾静脉注射等容量生理盐水。分别于注射后6 h和24 h处死,留取结肠组织标本,RT-PCR检测结肠组织TLR4、MD2和BD3 mRNA表达,光镜下观察24 h时肠组织病理变化。结果①肠组织病理改变:注射LPS后大鼠肠黏膜坏死脱落,微绒毛结构消失,结缔组织明显充血,大量的炎性细胞浸润。采用VIP治疗后病变明显减轻。②TLR4、MD2和BD3 mRNA表达:注射VIP后6 h、24 h,肠组织TLR4、MD2和BD3 mRNA表达升高(P0.05);24 h时LPS+VIP组TLR4、BD3和MD2 mRNA表达明显低于LPS组(P0.05)。结论 LPS致内毒素性休克大鼠肠道损伤时,肠组织TLR4、MD2和BD3 mRNA表达增强。VIP可减轻LPS所致肠道黏膜损伤,其机制可能与下调重要的炎症基因TLR4、MD2和BD3 mRNA表达有关。     

参附注射液对老年脓毒症大鼠心肌保护作用的研究

目的研究参附注射液对老年脓毒症大鼠心肌的保护作用及其可能机制。方法将20月龄SD老年大鼠随机分为空白对照组、单纯脓毒症组和参附治疗组,其中,空白对照组采用腹腔注射生理盐水5mg／kg,脓毒症组采用腹腔注射LPS5mg／kg,参附治疗组在腹腔注射LPS5mg／kg后立即尾静脉注射参附注射液7．5mL／kg。用ELISA方法榆测0h、2h和4h大鼠血清中炎症细胞因子TNF-α和IL-6的变化,及用RT—PCR检测各个时间点大鼠心脏TLR4-m RNA表达,并用透射电镜观察大鼠心肌细胞超微结构变化。结果脓毒症时TNF-α和IL-6的表达明显增高,各个时间点均显著高丁对照组（P〈0．05）,而参附治疗组与单纯脓毒症组相比,TNF-α和IL-6水平则明湿降低;参附治疗组心肌组织损伤较单纯脓毒症组明显减轻;脓毒症时心肌TLR4-m RNA表达明显上调,而参附则能抑制TLR4-m RNA的表达。结论参附可能通过降低心肌组织TLR—mRNA的表达,对脓毒症诱导的老年大鼠心肌损伤具有保护作用。     

白藜芦醇抑制血管紧张素Ⅱ/TLR4/NF-KB调控鼠血管平滑肌细胞炎症和氧化应激损伤作用

目的探讨白藜芦醇(RES)抑制血管紧张素Ⅱ(AngⅡ)/TLR4/NF-KB调控大鼠血管平滑肌细胞炎症和氧化应激损伤的作用。方法采用大鼠胸主动脉平滑肌A7r5细胞株为主要受试物,分为对照组、RES组(30μmol/L)、AngⅡ组(10-4μmol/L)和实验组(30μmol/L RES预处理2 h+10-4μmol/L AngⅡ)。CCK-8法检测A7r5细胞的存活率;ELISA法检测细胞上清液中TNF-α和IL-6表达;采用试剂盒检测ROS和iNOS活性;Western blot法检测A7r5细胞AngⅡ、TLR4、P-P65,P-IKB蛋白表达水平。结果与对照组相比,AngⅡ组A7r5细胞株存活率显著升高,RES组和实验组A7r5细胞株存活率显著降低(P0.05);AngⅡ组A7r5细胞株TNF-α和IL-6表达显著升高,ROS和iNOS活性显著升高,AngⅡ、TLR4、P-P65,P-IKB蛋白含量显著升高,RES组和实验组TNF-α和IL-6表达显著降低,ROS和iNOS活性显著降低,AngⅡ、TLR4、P-P65,P-IKB蛋白含量显著升高(P0.05)。与AngⅡ组相比,RES组和实验组A7r5细胞株存活率显著降低,实验组A7r5细胞株存活率下降更为明显(P0.05);AngⅡ组A7r5细胞株TNF-α和IL-6表达显著降低,ROS和iNOS活性显著降低,AngⅡ、TLR4、P-P65,P-IKB蛋白含量显著降低,RES组和实验组TNF-α和IL-6表达显著降低,ROS和iNOS活性显著降低,AngⅡ、TLR4、P-P65,P-IKB蛋白含量显著降低(P0.05)。结论白藜芦醇可有效缓解大鼠血管平滑肌细胞的炎症和氧化应激损伤,主要通过抑制血管紧张素Ⅱ/TLR4/NF-KB信号通路。     

锌指蛋白A20抑制Toll样受体4介导的人单核细胞炎症反应的实验研究

目的:观察锌指蛋白A20过度表达对人单核细胞膜Toll样受体(TLR4),下游促炎因子肿瘤坏死因子-α(TNF-α)与白细胞介素(IL)-12,以及抗炎因子IL-10表达的影响,探讨锌指蛋白A20对单核细胞炎症反应的抑制作用及可能的调节机制。方法:Ficoll细胞分离液分离人外周血单核细胞,随机分为对照组、脂多糖(LPS)组、A20转染组与LPS+A20转染组。荧光显微镜检测GFP报告基因,免疫组织化学检测A20蛋白的表达,RT-PCR检测A20及TLR4的mRNA表达,流式细胞检测技术检测TLR4的蛋白表达,ELISA方法检测上清液TNF-α、IL-12及IL-10表达水平。结果:LPS刺激后,单核细胞TLR4和内源性A20的mRNA和蛋白、TNF-α、IL-12和IL-10表达较对照组均明显升高(均P<0.01);TNF-α/IL-10和IL-12/IL-10均明显高于对照组(均P<0.01);转染A20基因的单核细胞,在无LPS刺激的条件下,上述指标与对照组相比均差异无统计学意义;转染A20基因的单核细胞在LPS刺激后,TLR4mRNA和蛋白、TNF-α、IL-12的表达以及TNF-α/IL-10和IL-12/IL-10均显著低于LPS组,而IL-10的表达明显高于对照组和LPS组(均P<0.01)。结论:TLR4激活介导单核细胞的炎症反应,且正反馈调节其自身受体TLR4和内源性A20的表达;A20参与单核细胞TLR4激活所介导的炎症反应,其表达增加与TLR4表达的增加有关;单纯提高A20表达对未被激活的单核细胞TLR4及其信号通路影响不大;A20过表达可抑制TLR4激活所介导的单核细胞的炎症反应。     

TLR4在慢性支气管炎大鼠模型中对炎症损伤与黏液分泌的调控作用

目的观察TLR4在细菌内毒素(LPS)致慢性支气管炎大鼠模型中对气道炎症损伤与黏液分泌的调控作用。方法 SPF级雄性Wistar大鼠40只,随机分为正常对照组(NS组)、慢性支气管炎组(LPS组)、多粘菌素B干预组(LPS+PMB组)、多粘菌素B自身对照组(PMB组),每组10只。用气管内注入LPS 200μg/200μl及每日LPS溶液雾化吸入1 h的方法建立慢性支气管炎动物模型,3 w后收集支气管肺泡灌洗液(BALF)进行细胞学计数和白细胞分类检测,酶联免疫吸附实验(ELISA)法测定BALF中肿瘤坏死因子(TNF)-α含量,对大鼠肺脏进行病理学检查,肺组织阿先蓝过碘酸雪夫(AB-PAS)阳染面积,用免疫组化法观察黏蛋白Muc5AC、TLR4在支气管肺内的表达及荧光定量RT-PCR Muc5AC mRNA、TLR4 mRNA在肺内的表达。结果 LPS组大鼠BALF细胞总计数、中性粒细胞个数、TNF-α水平。AB-PAS阳染面积、Muc5AC、TLR4蛋白表达量及Muc5AC mRNA、TLR4 mRNA水平与LPS+PMB组比较有显著性差异(均P0.05),NS组和PMB无统计学差异(P0.05)。结论 TLR4介导LPS诱发的慢性呼吸道炎症反应可能导致Muc5AC蛋白与mRNA高表达。多粘菌素B可能通过抑制肺组织TLR4 mRNA及其蛋白的表达而抑制气道黏液高分泌。     

TRALI大鼠肺泡活化巨噬细胞及共刺激分子CD40、ICAM-1的表达变化分析

目的探讨输血相关急性肺损伤(TRALI)大鼠肺泡活化巨噬细胞及共刺激分子CD40、血浆及肺组织中细胞间粘附分子-1(ICAM-1)的表达变化及其意义。方法选取60只SPF级、6-7周龄的雄性SD大鼠,采用随机数字表法分为假手术组(腹腔注射等量生理盐水)、对照组(腹腔注射2mg/kg脂多糖)、模型组(静脉输注脂多糖5mg/kg)和TRALI组(腹腔注射2mg/kg脂多糖+静脉输注血浆1m L/只)各15只,采用RTPCR技术检测活化巨噬细胞中Toll样受体4(TLR4)mRNA的表达,采用Northern bolt检测CD40 mRNA的表达,采用免疫组化染色检测ICAM-1蛋白的表达,采用酶联免疫吸附法(ELISA)检测血清中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-β)、ICAM-1的水平。结果模型组和TRALI组的活化巨噬细胞中TLR4 mRNA、肺组织中CD40 mRNA、ICAM-1蛋白表达水平显著高于假手术组和对照组(P0.05),TRALI组的活化巨噬细胞中TLR4 mRNA、肺组织中CD40 mRNA、ICAM-1蛋白表达水平显著的低于模型组(P0.05);模型组和TRALI组的血清中TNF-ɑ、IL-β、ICAM-1的水平显著的高于假手术组和对照组(P0.05),TRALI组的血清中TNF-ɑ、IL-β、ICAM-1的水平显著的低于模型组(P0.05)。结论 TRALI大鼠肺TLR4 mRNA、CD40 mRNA、ICAM-1蛋白高表达,这可能与促进炎症因子释放,引起肺损伤有关。     

人参二醇组皂苷对内毒素休克大鼠体内血管活性物质的调节作用

目的 探讨人参二醇组皂苷(PDS)对内毒素休克大鼠体内血管活性物质的调节作用.方法 大鼠舌下静脉注射PDS进行预治疗,10 min后注射细菌内毒素(LPS,5 mg/kg)复制感染性休克模型.实验动物随机分为对照组(C组),内毒素休克组(L组),地塞米松预治疗组(LD组),PDS预治疗组(LP组).各组大鼠于休克2、4 h腹主动脉取血,硝酸还原酶法测定血清中一氧化氮合酶(NOS) 活性和NO-2/NO-3的变化,间接反映NO的水平.于休克后4 h处死动物,称取肝组织20 mg匀浆后测血栓素B2(TXB2)及6-酮-前列腺素F1α(6-Keto-PGF1α)的含量.结果 肝组织中TXB2、6-keto-PGF1α及6-Keto-PGF1α/TXB2在LP组均显著低于L组.注射内毒素2 h后L组的血清NOS和NO-2/NO-3明显升高,LD组和LP组NOS活性、NO-2/NO-3显著低于L组.结论 PDS能够降低内毒素休克大鼠TXA2、PGI2和NO的水平,改善微循环状态,起到抗休克的作用.     

人参二醇组皂苷对感染性休克大鼠血液流变性及微循环的影响

目的 观察人参二醇组皂苷(PDS)对感染性休克大鼠血液流变性及微循环的影响,探讨其抗感染性休克的作用机制.方法 大鼠舌下静脉注射PDS进行预治疗,10 min后注射细菌内毒素(LPS 5 mg/kg)复制感染性休克模型.实验动物随机分为对照组(S组);内毒素休克模型组(L组);地塞米松预治疗组(LD组);PDS预治疗(22.5 mg/kg,LP_小)(45 mg/kg,LP_中)(90 mg/kg,LP_大)组.各组于休克后观察肠系膜微循环的变化,2 h和4 h腹主动脉取血,测量全血黏度.结果 模型组在休克2 h、4 h全血黏度明显高于PDS各剂量组(P＜0.01).肠系膜微循环的变化:PDS各剂量组在休克2～4 h微动脉管径、微动脉血流速度及微动脉管袢数均高于模型组(P＜0.05).结论 PDS通过降低内毒素休克大鼠全血黏度,改善微循环状态,起到抗休克的作用.     

Effects of glutamine on intestinal permeability and bacterial translocation in TPN－rats with endotoxemia

AIM: To evaluate the protective effect and mechanism of glutamine on the intestinal barrier function in total parenteral nutrition (TPN) rats with trauma or endotoxernia.METHODS: To perform prospective, randomized and controlled animal experimentation of rats with surgical trauma, TPN and endotoxemia, thirty-four male, adult Sprague Dawley rats were divided into four groups: control group (n=8), TPN group (n=9), trauma and endotoxemia group (LPS, n=8) and trauma plus endotoxemia supplemented with glutamine in TPN solution group (Gin.group, n=9). All groups except the control group were given TPN solutions in 7-day experimental period. For Gin group, 1 000 mg/kg/d of glutamine was added to TPN solution during day 1-6. On the 7^th day all the animals were garaged with lactulose (66 mg) and mannitol (50 mg)in 2 ml of normal saline. Then 24 h urine with preservative was collected and kept at -20℃. On day 8, under intraperitoneal anesthesia using 100 mg/kg ketamin, the intestine, liver, mesenteric lymph nodes and blood were taken for examination.RESULTS: The body weight of LPS group decreased most among the four groups. The structure of small intestinal mucosa in TPN group, LPS group and Gin group showed impairments of different degrees, and the damage of small intestinal mucosa in Gin group was remarkably alleviated.The concentrations of interleukins in small intestine mucosa were lower (for IL-4 and IL-6) or the lowest (IL-10) in Gln group. The IgA level in the blood plasma and the mucosaof Gin group was the highest among all of the groups. The urine lactulose/mannitol test showed that the intestinal permeability in LPS group was lower than that in TPN group(P&lt;0.001), but there was no difference between LPS group and Gln group. The rate of bacterial translocation in Gln group was lower than that in LPS group (P&lt;0.02).CONCLUSION: Prophylactic treatment with glutamine could minimize the increments of intestinal permeability and bacterial translocation caused by trauma and endotoxemia in rats treated with TPN.     

内毒素血症大鼠肠黏膜免疫细胞的变化及通腑颗粒的干预作用

目的探讨内毒素血症大鼠肠黏膜免疫细胞的变化及中药通腑颗粒对其影响,为临床治疗提供理论依据。方法 120只雄性Wistar大鼠随机分为正常对照组（A组）、内毒素血症组（B组）及中药治疗组（C组）三组。B组及C组采取尾静脉一次性注射给药方法,注射1%LPS溶液（给药量为10 mg/kg）制作内毒素血症模型,A组注射等量生理盐水（1 mL/kg）;C组尾静脉注射给药后即刻给予50%通腑颗粒溶液（给药量为5 g/kg）灌胃,A组及B组给予等量蒸馏水灌胃。造模后在第2、6、12、24 h时间点分批（每次10只）麻醉后处死动物,取小肠组织进行检测。采用HE染色观察肠黏膜病理变化,采用免疫组织化学及TUNEL染色方法,分别标记并计数各组大鼠肠黏膜免疫细胞及凋亡淋巴细胞。结果与A组比较,B组肠黏膜M细胞、DC细胞、CD8＋T细胞及IgA＋B细胞计数明显减少,而CD4＋T细胞、Tr细胞及淋巴细胞凋亡计数明显增多;C组肠黏膜M细胞、CD8＋T及Tr细胞计数无明显改变,DC细胞、IgA＋B细胞及淋巴细胞凋亡计数明显减少,而CD4＋T细胞计数明显增多;与B组比较,C组肠黏膜M细胞无明显改变,Tr细胞及淋巴细胞凋亡计数明显减少,而DC细胞、CD4＋T细胞、CD8＋T细胞及IgA＋B细胞计数明显增多。结论内毒素血症时肠黏膜损伤,肠免疫不同阶段的免疫细胞均发生损害,Th1/Th2比例失调,淋巴细胞凋亡增多,导致肠黏膜局部免疫发生抑制。应用通腑颗粒可以减轻肠黏膜损伤,减轻免疫细胞损伤,改善Th1/Th2比例,减少淋巴细胞凋亡,保护局部免疫功能。     

异丙酚联合地塞米松对早期脓毒症大鼠急性肺损伤的保护作用及机制

目的探讨异丙酚联合地塞米松对早期脓毒症大鼠急性肺损伤的保护作用及可能机制。方法将25只雄性SD大鼠随机分为5组各5只,N组尾静脉注射0.9%氯化钠注射液10 mL;另四组注射脂多糖(LPS)10 mg/kg,制作脓毒症急性肺损伤模型。制模后L组不用药,D组注射地塞米松0.1 mg/kg;P组注射异丙酚5 mg/kg,继以异丙酚5 mg/(kg.h)的速度持续缓慢静脉泵注4 h;PD组注射地塞米松0.1 mg/kg和异丙酚5 mg/kg,继以异丙酚5 mg/(kg.h)的速度静脉泵注至4 h。各组均于造模后4 h麻醉处死,行动脉血气分析,测定肺组织湿/干重量比值(W/D),RT-PCR法检测肺组织水通道蛋白1(AQP1)mRNA表达水平。结果与L组比较,D、P、PD组肺组织W/D值明显降低,AQP1 mRNA表达水平明显升高,动脉血气指标明显改善;PD组各指标较D、P组改善更明显。结论异丙酚及地塞米松对早期脓毒症大鼠急性肺损伤有协同保护作用,其机制可能为上调肺毛细血管内皮AQP1 mRNA表达水平,提高肺间质内水肿液的跨内皮转运速率。     

Novel Carboxamide Derivative (IS-741) Attenuates Lung Injury in Rats with Cerulein-Induced Pancreatitis Complicated by Endotoxemia

The therapeutic effects of an intravenouslyinjected carboxamide derivative (IS-741) on lung injurywere studied in rats with cerulein-induced pancreatitiscomplicated by endotoxemia. Pancreatitis was induced by four intramuscular injections of cerulein(50 g/kg at 1-hr intervals). Pancreatitis rats wereinjected intraperitoneally with 10 mg/kg oflipopolysaccharide (LPS) 6 hr following the firstcerulein injection as a challenge of endotoxemia. Ratswere divided into four groups: group I, pancreatitiswith LPS; group II, pancreatitis with LPS treated witha continuous intravenous injection of IS-741 at 0.03 mg/kg/hr); group III, pancreatitis withLPS treated with a continuous intravenous injection ofIS-741 at 0.3 mg/kg/hr); and group IV, pancreatitis withLPS treated with a continuous intravenous injection of IS-741 at 3 mg/kg/hr). IS-741 wasadministered 30 min before the endotoxemia challenge.Intense mononuclear cell infiltration and lunghemorrhage occurred in untreated pancreatitis rats withLPS (group I), but hemorrhage was not seen in group IVrats receiving a continuous injection of IS-741 shortlybefore the induction of endotoxemia. The IS-741- treatedrats (groups II, III, and IV) had lower serum concentrations of cytokine-induced neutrophilchemoattractant (CINC), as well as fewer pulmonaryinfiltrates immunoreactive for CINC or Mac-1(CD11b/CD18). The number of neutrophils infiltrating thelung in groups II, III, and IV was significantlylower than that of group I. Conversely, CINC productionby bronchoalveolar macrophages in vitro were stimulatedby LPS but were reduced by the presence of IS-741. The carboxamide derivative IS-741 effectivelyprevented pancreatitisassociated lung injury followingthe challenge of endotoxemia.     

Cross-Tolerance Between Acute Alcohol Intoxication and Endotoxemia

This study tests two hypotheses: (1) prior exposure to LPS induces cross-tolerance for the hepatic effects of subsequent short-term alcohol intoxication; and (2) short-term alcohol intoxication renders the liver resistant to the effects of acute endotoxemia, resulting in reduced production of superoxide and tumor necrosis factor. In the first group of experiments, male Sprague-Dawley rats were treated intravenously with E. coli lipopolysaccharide (LPS) (0.5 mg/kg) 48 hr before they were given an intravenous bolus of ethanol (1.75 g/kg), followed by 250–300 mg/kg/hr) for 3–5 hr. Superoxide release in the perfused liver was measured after the 3-hr ethanol infusion. Pre-treatment with LPS attenuated ethanol-mediated superoxide anion release by the perfused liver. The stimulatory effect of phorbol myristate acetate on hepatic release of superoxide was also decreased. In the second group of experiments, rats previously treated with ethanol for 5 hr, received an intravenous injection of LPS (1 mg/kg). At 90 min after LPS, sera were collected for tumor necrosis factor a assay. Hepatic release of superoxide anion was determined 3 hr after LPS. Acute ethanol intoxication for 5 hr significantly reduced LPS-induced serum tumor necrosis factor activity and free radical release by the perfused liver. LPS-induced mortality was also decreased. In both groups of experiments serum corticosteroid levels were reduced during cross-tolerance. These results demonstrate that cross-tolerance develops between acute alcohol intoxication and endotoxemia manifesting in reduced hepatic production of cytotoxic cytokines and superoxide anions.     

Induction of a 72-kDa heat shock protein and protection against lipopolysaccharide-induced liver injury in cirrhotic rats

BACKGROUND AND AIM: A 70-kDa heat shock protein (stress-inducible HSP70, HSP72) has been reported to be a cytoprotectant in a variety of organs. It has been reported that HSP72 protected non-cirrhotic rats against endotoxemia. However, its cytoprotective effect against endotoxemia in cirrhotic rats has not yet been studied. In this study, we investigated the cytoprotective effect of HSP72 on lipopolysaccharide (LPS)-induced liver injury in carbon tetrachloride (CCl(4))-induced cirrhotic rats. METHODS: Liver cirrhosis was produced by an 8-week intraperitoneal injection of CCl(4) in male Sprague-Dawley rats. Expression of HSP72 was investigated using western blot analysis. Cirrhotic rats were given an intraperitoneal injection of LPS (10 mg/kg) with or without hyperthermia (42.5 degrees C, 15 min) preconditioning. Liver injury was assessed biochemically (aspartate transaminase, alanine transaminase, bilirubin, lactate dehydrogenase, creatinine) and histologically. The plasma tumor necrosis factor (TNF)-alpha level was determined. RESULTS: Hyperthermia preconditioning induced a 4-fold increase in HSP72 in the cirrhotic rat liver. Pre-induction of HSP72 prevented LPS-induced liver injury, as evaluated using serum biochemical parameters and histology with reduced TNF-alpha response. CONCLUSION: These findings suggest that pre-induction of HSP72 may provide therapeutic strategies for Gram-negative sepsis-induced liver injury in liver cirrhosis.     

Role of glucocorticoids in the response of the hypothalamo-corticotrope, immune and adipose systems to repeated endotoxin administration.

The present study was designed to determine whether the glucocorticoid inhibitory feedback mechanism plays a role in the well-known tolerance of the neuroendocrine-immune axis response to repeated endotoxemia. Adult male rats underwent adrenalectomy (ADX) and were implanted with a subcutaneous corticosterone (compound B, CB, 75 mg) pellet, or sham operated and implanted with a placebo pellet. On the morning of day 8 after surgery (experimental day, D1), all rats received an intravenous injection of lipopolysaccharide (LPS) (25 microg/kg body weight) which was repeated daily until D5. Blood was drawn via intravenous indwelling catheters before (sample time zero) as well as 1, 2, 3 and 4 h after LPS treatment on D1, 3 and 5 for measurements of corticotropin (ACTH), CB, tumor necrosis factor-alpha (TNF-alpha) and leptin. In sham animals, tolerance to repeated LPS administration was complete by D5 for the corticotrope axis and the immune response. In addition, LPS was found to stimulate leptin secretion on day 1 in intact rats, an effect that also disappeared thereafter. ADX + CB rats showed only a partial tolerance of the corticotrope axis on D5, whereas tolerance of the immune response was similar to that found in sham animals. Interestingly, the acute stimulation of leptin secretion by LPS in ADX + CB rats was qualitatively similar to that of intact controls on D1, but plasma leptin levels were significantly reduced on D3 and 5 compared to controls. Our results demonstrate that the adrenal response tolerance of the hypothalamo-pituitary-adrenal axis to repeated endotoxemia. In addition, our finding that TNF-alpha secretion follows the same pattern in sham-operated and in adrenalectomized animals suggests that unlike the corticotrope axis, tolerance of the immune response does not depend upon stimulated CB levels. The decrease in circulating levels of leptin following ADX is consistent with the stimulatory effects of glucocorticoids on leptin secretion. However, our finding of an acute stimulation of leptin secretion by LPS in ADX + CB animals demonstrates that this effect of endotoxemia is at least partially glucocorticoid independent.