A novel fibrinogenase from Agkistrodon acutus venom protects against DIC via direct degradation of thrombosis and activation of protein C

The incidence of disseminated intravascular coagulation (DIC), which leads to multiple organ dysfunction and high mortality, has remained constant in recent years. At present, treatments of DIC have focused on preventing cytokine induction, inhibiting coagulation processes and promoting fibrinolysis. Recent clinical trials have supported the use of antithrombin and activated protein C supplementation in DIC. To better understand the mechanism of treatment on DIC, we here report a novel fibrinogenase from Agkistrodon acutus (FIIa) that effectively protected against LPS-induced DIC in a rabbit model, and detected the tissue factors expression in HUVE cells after using FIIa. In vivo, administration of FIIa reduced hepatic and renal damage, increased the concentration of fibrinogen, the activities of protein C, the platelet count, APTT, PT, FDP, the level of AT-III and t-PA, decreased the level of PAI-1, and increased survival rate in LPS-induced DIC rabbits. In vitro experiments, we further confirmed that FIIa up-regulated the expression of t-PA and u-PA, down-regulated the expression of PAI-1, and directly activated protein C. Our findings suggest that FIIa could effectively protect against DIC via direct degradation of microthrombi and activation of protein C as well as provide a novel strategy to develop a single proteinase molecule for targeting the main pathological processes of this disease.     

板蓝根对脂多糖刺激鼠释放炎性细胞因子的影响

目的探讨板蓝根抗内毒素活性部位F022对脂多糖(LPS)刺激鼠单核细胞释放炎性细胞因子的影响。方法取BALB/C小鼠腹腔内单核细胞,实验设计为6组。其中,实验组根据F022浓度分为1%、0.5%、0.25%、0.125%4组,分别加入板蓝根F022部位液后再加入LPS液;LPS阳性组仅加入LPS液;阴性组加入1%F022液。之后检测细胞培养上清液中3种炎性细胞因子肿瘤坏死因子-α(TNF-α)、白细胞介素6(IL-6)和一氧化氮(NO)水平。结果LPS可刺激鼠单核细胞过度释放炎性细胞因子TNF-α、IL-6和NO;与阳性组比较,实验组炎性细胞因子水平降低,且呈剂量依赖性。结论板蓝根抗内毒素活性部位F022对LPS刺激鼠单核细胞过度释放炎性细胞因子具有抑制作用。     

The antipyretic effects of baicalin in lipopolysaccharide-evoked fever in rabbits

Evidence has accumulated to indicate that systemic administration of lipopolysaccharide (LPS), in addition to elevating tumor necrosis factor-alpha (TNF-alpha) as well as fever, induces overproduction of both glutamate and hydroxyl radicals in the rabbit's hypothalamus. Current investigation was attempted to determine whether baicalin exerts its antipyresis by suppressing overproduction of circulating TNF-alpha and hypothalamic glutamate and hydroxyl radicals in rabbits. The microdialysis probes were stereotaxically and chronically implanted into the preoptic anterior hypothalamus of rabbit brain for determination of both glutamate and hydroxyl radicals in situ. It was found that systemic administration of LPS (0.5-10 microg/kg) induced dose-related increased levels of both core temperature and hypothalamic levels of both glutamate and hydroxyl radicals accompanied by increased plasma levels of TNF-alpha. The rise in both the core temperature and hypothalamic glutamate and hydroxyl radicals could also be induced by direct injection of TNF-alpha (1-20 ng) into the lateral ventricle of rabbit brain. Pretreatment with baicalin (2-20 mg/kg, i.v.) one hour before an i.v. dose of LPS significantly reduced the LPS-induced overproduction of circulating TNF-alpha and brain glutamate and hydroxyl radicals. Both the febrile response and overproduction of both glutamate and hydroxyl radicals in the hypothalamus caused by central administration of TNF-alpha could be suppressed by baicalin. These findings suggest that systemic administration of baicalin may exert its antipyresis by inhibiting the N-methyl-D-aspartate receptor-dependent hydroxyl radicals pathways in the hypothalamus and circulating TNF-alpha accumulation during LPS-fever.     

Hemorrhagic activity and mechanism of FⅡa′a fibrinolytic enzyme from Agkistrodon acutus venom

AIM: To study the local hemorrhagic activity of a fibrinolytic enzyme (FⅡa) from Agkistrodon acutus venom and its mechanism. METHODS: The local hemorrhagic activity was determined by subcutaneous injection on the back of mouse. The effects of FⅡa on factor X, prothrombin, gelatin, and collagen were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Platelet aggregation assays were performed in rat platelet-rich plasma (PRP). Human umbilical vein endothelial cells (HUVEC) were cultured and passaged in complete M 199 medium. Cell viability and nuclear morphology change were determined by fluorescein diacetate (FDA) staining and Hoechst 33258 staining, respectively. RESULTS: The minimum hemorrhagic dose (MHD) of FⅡa was 89 μg.In vitro, FⅡa (0.25 g/L) degraded factor X, prothrombin, collagen, and gelatin, and dose-dependently (0.25, 0.50,0.75, and 1.00 g/L) inhibited the platelet aggregation induced by ADP in rat PRP. When HUVEC in culture treated with FⅡa, HUVEC showed detachment in a dose-dependent manner, but no apoptosis sign was observed.CONCLUSION: FⅡa had local hemorrhagic activity, and the mechanism was related to the degradation of factor X,prothrombin, gelatin, and collagen, the inhibition of ADP-induced platelet aggregation, and inducement of HUVEC detachment.     

脂多糖加速饮食所致非酒精性脂肪肝小鼠的炎症和氧化应激

目的本研究旨在探讨脂多糖在非酒精性肝炎(NASH)模型中的作用。方法采用胆碱蛋氨酸缺乏(Methionine choline deficient,MCD)饮食诱导的小鼠NASH模型。18只雄性C57BL/6小鼠分为3组,MCS+Saline组给予正常饮食+腹腔生理盐水,MCD+Saline组给予胆碱蛋氨酸缺乏饮食+腹腔注射生理盐水,MCD+LPS组给予胆碱蛋氨酸缺乏饮食+腹腔注射1 mg/kg脂多糖,共2周。在最后一次注射的6 h之后处死小鼠,取血清和肝组织。进行肝脏HE染色和Sirius Red染色,观察肝组织病理学变化。并测定血清中血清丙氨酸氨基转移酶(ALT)和肿瘤坏死因子-α(TNF-α)的含量。结果 MCD造成小鼠肝脏中大量脂肪滴的沉积和炎症细胞浸润,血清ALT升高,脂多糖注射进一步加重肝细胞凋亡,肝脏中TBARS进一步升高,血清TNF-α含量显著增加。结论在MCD所致非酒精性脂肪肝炎模型小鼠中,腹腔注射脂多糖引起血清中TNF-α升高,进一步加重细胞凋亡,因此,脂多糖在非酒精性脂肪肝炎的进程中具有重要作用,提示临床预防或治疗非酒精性脂肪肝炎需要关注患者肠道菌群失调症状。     

~(131)I标记尖吻蝮蛇毒凝血酶样酶在动物体内的分布

用氯胺T法对尖吻蝮(Agkistrodon acutus)蛇毒的抗血栓组分凝血酶样酶进行~(131)Ⅰ标记,观察其在大、小鼠体内的分布。以实验组和给予同剂量凝血酶样酶混合Na~(131)Ⅰ的对照组的放射性参入量(脏器与血液放射比)的比值作为凝血酶样酶在组织中分布的依据。结果表明,一次快速ⅳ凝血酶样酶后2h分布最多的为肾、其次为肺、脾、肾上腺和肝。给药后4h,肾、脾、肺和肝比2h时高,心脏含量较少,其它组织未见有分布。尿中含量高,表明其主要经肾脏随尿排泄;经胆汁排出则很少。     

急性心肌缺血大鼠肺组织肿瘤坏死因子-α及mRNA表达的研究

目的观察急性心肌缺血大鼠肺组织中肿瘤坏死因子-α(TNF-α)及TNF-αmRNA的表达,探讨TNF-α及TNF-αmRNA在急性心肌缺血诱发肺损伤中的作用。方法健康成年雄性SD大鼠36只,体质量(250±20)g,随机分为两组:手术对照组(S组)和结扎冠状动脉缺血组(CAO组)。S组动物仅开胸但不结扎冠状动脉左前降支;CAO组动物开胸后结扎冠状动脉左前降支。S组、CAO组又各分为1、3、6 h三个不同的时点组。各组在开胸或扎闭冠状动脉左前降支后分别计时1 h,3 h,6 h取出右肺下叶。采用免疫组织化学、原位杂交两种方法观察TNF-α及TNF-αmRNA在各组动物肺组织中的表达情况。结果CAO组各时间点大鼠的支气管上皮细胞、肺泡Ⅱ型细胞和血管内皮细胞均可见到TNF-α及TNF-αmRNA的均呈强阳性表达(P<0.01),在3 h达高峰。结论TNF-α及TNF-αmRNA参与了急性心肌缺血诱发肺损伤的发生和发展。     

Infliximab联合免疫球蛋白治疗噬血细胞综合征疗效观察

目的:观察抗肿瘤坏死因子单克隆抗体(infliximab)联合免疫球蛋向治疗噬血细胞综合征疗效.方法:大剂量免疫球蛋白(10 g/dx4 d)联合2次Infliximab(100mg/d).结果:1例有多个脏器功能损害的患者诊断为柯萨奇病毒感染合并噬血细胞综合征,治疗后病情完全缓解.结论:噬血细胞综合征本身只是一种过度免疫反应,infliximab可能成为阻断细胞因子的重要治疗手段.     

Expression, purification and molecular modeling of recombinant fibrinogenase [IV], a metalloproteinase from Deinakistrodon acutus venom.

A novel metalloproteinase, recombinant fibrinogenase IV (rFIV(a)), was expressed and purified from Deinakistrodon acutus venom. It was a single chain protein with an apparent molecular weight 27 kDa and an isoeletric point of pH 7.1. RFIV(a) cleaved preferentially the Aalpha-chain and also cleaved Bbeta, gamma-chains of fibrinogen when the incubation time was prolonged. The proteolytic activity was inhibited by EDTA, l-cysteine, and DTT, indicating rFIV(a) was a metalloproteinase requiring disulfide bonds for its activity. It kept above 85% of the initial activity from pH 4.5-11, showed an equal maximum activity at the temperature range from 30 to 50 degrees C, and was inactivated by Zn2+, Cu2+ and Cd2+. Homology modeling of rFIV(a) showed that two highly conserved disulfide bonds (Cys159-Cys164 and Cys117-Cys197) was maintained from its structure, and it exhibited the characteristic conserved motif H142E143XXH146XXGXXH152, whose three histidine residues were involved in binding of the catalytically essential zinc ion. This work demonstrates the expression, purification and characterization of recombinant fibrinogenase IV, which belongs to class P-I metalloproteinase from D. acutus venom.     

再生障碍性贫血患者血清IL-8、TNF-α水平的检测及其意义

目的 探讨再生障碍性贫血(AA)患者血清IL-8、TNF-α的变化及其意义.方法 应用酶联免疫吸附试验检测45例AA患者及30名健康人血清中IL-8、TNF-α水平.结果 血清IL-8、TNF-α水平AA组及急性AA(AAA)亚组均明显高于对照组(P＜0.05),慢性AA(CAA)亚组与对照组差异无统计学意义(P＞0.05);AAA亚组明显高于CAA亚组(P＜0.05).AA组血清IL-8与TNF-α水平呈正相关(r=0.682,P＜0.05).结论 AA患者存在细胞免疫功能紊乱,IL-8和TNF-α过量分泌可能在AA特别是在AAA的发病中起一定的作用.     

Isolation and structural characterization of a new fibrin(ogen)olytic metalloproteinase from Bothrops moojeni snake venom.

A proteinase, named BmooMPalpha-I, from the venom of Bothrops moojeni, was purified by DEAE-Sephacel, Sephadex G-75 and heparin-agarose column chromatography. The enzyme was purified to homogeneity as judged by its migration profile in SDS-PAGE stained with coomassie blue, and showed a molecular mass of about 24.5 kDa. Its complete cDNA was obtained by RT-PCR and the 615 bp codified for a mature protein of 205 amino acid residues. The multiple alignment of its deduced amino acid sequence and those of other snake venom metalloproteinases showed a high structural similarly, mainly among class P-IB proteases. The enzyme cleaves the Aalpha-chain of fibrinogen first, followed by the Bbeta-chain, and shows no effects on the gamma-chain. On fibrin, the enzyme hydrolyzed only the beta-chain, leaving the gamma-dimer apparently untouched. It was devoid of phospholipase A(2), hemorrhagic and thrombin-like activities. Like many venom enzymes, it is stable at pH values between 4 and 10 and stable at 70 degrees C for 15 min. The inhibitory effects of EDTA on the fibrinogenolytic activity suggest that BmooMPalpha-I is a metalloproteinase and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Aprotinin and benzamidine, specific serine proteinase inhibitors, had no effect on BmooMPalpha-I activity. Since the BmooMPalpha-I enzyme was found to cause defibrinogenation when administered i.p. on mice, it is expected that it may be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis.     

危重症患者血清肿瘤坏死因子-α、白细胞介素-6及白细胞介素-10变化的研究

目的探讨危重症患者的血清肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）、白细胞介素-10（IL-10）的变化，为判断危重症患者病情的严重程度提供新的客观指标。方法41例危重症患者中好转或治愈30例为存活组，死亡11例为死亡组，健康体检者20例为对照组。患者入住ICU后第1、7天清晨空腹抽取静脉血3ml，对照组抽空腹静脉血3ml，应用双抗体夹心酶联免疫吸附法检测血清中TNF-α、IL-6和IL-10的浓度。同时按急性生理和慢性健康评分系统（APACHEⅢ评分）方法进行危重症评分。结果危重症患者早期血清中促炎因子TNF-α、IL-6及抗炎因子IL-10的浓度均较对照组明显升高，存活组和死亡组与对照组比较均有统计学意义；TNF-α、IL-6、IL-10及APACHEⅢ评分四项指标两两之间呈显著性相关（P〈0．05）。结论促炎因子TNF-α、IL-6和抗炎因子IL-10在危重症患者中起重要作用，决定患者的预后。TNF-α、IL-6和IL-10浓度同APACHEⅢ评分一样可作为危重症预警的价值参数。     

细胞因子TNF-α、IL-6与老年高血压患者合并颈动脉粥样硬化的关系

目的:探讨细胞因子TNF-α、IL-6与老年高血压患者颈动脉粥样硬化发生之间的关系。方法:选择142例老年高血压患者,男104例、女38例,平均年龄(75.63±3.93)岁,应用颈动脉超声测量颈动脉内中膜厚度(IMT),观察有无斑块形成,用免疫学方法测定血清中TNF-α、IL-6的浓度。结果:合并颈动脉粥样硬化的老年高血压患者血清TNF-α、IL-6的含量明显高于无颈动脉硬化患者(P<0.05=;有颈动脉斑块的患者血清TNF-α、IL-6的含量明显高于颈动脉内中膜增厚及内中膜正常患者(P<0.05,P<0.01)。结论:细胞因子TNF-α、IL-6参与高血压患者颈动脉粥样硬化的发生发展过程。     

过敏性紫癜患儿血清IL-8、IL-12及TNF-α表达的意义

目的探讨血清白细胞介素-8（IL-8）、白细胞介素-12（IL-12）及肿瘤坏死因子-α（TNF-α）在过敏性紫癜（HSP）患儿中表达及其临床意义。方法对本研究采用ELISA方法检测了48例过敏性紫癜患儿（其中22例合并肾脏损害）以及30例正常健康儿童的血清IL-8、IL-12及TNF-α水平，分别比较急性期和缓解期以及有无合并肾损害的过敏性紫癜患儿的细胞因子水平；分析IL-8、IL-12与TNF-α是否存在相关关系。结果HSP患儿血清IL-8、IL-12与TNF-α水平高于健康对照组（P〈0．01）；无肾损害HSP与紫癜性肾炎（HSPN）组血清IL培、IL-12水平均无统计学差异；HSPN组肿瘤坏死因子-α水平高于无肾损害HSP组；HSP血清TNF-α水平与IL-8正相关（r=0．524P〈0．01）。HSP患儿血清TNF-α水平与IL-12正相关（r=0．670，P〈0．01）。结论细胞因子IL-8、IL-12及TNF-α可能参与HSP／HSPN发病过程。     

Protective effects of curcumin against hepatic fibrosis induced by carbon tetrachloride: Modulation of high-mobility group box 1, Toll-like receptor 4 and 2 expression

The aim of the study was to investigate the effect of curcumin on the liver fibrosis induced by carbon tetrachloride (CCl₄) in rats, and to elucidate its underlying molecular mechanisms. Rats were administered with CCl₄ together with or without curcumin for 6 weeks. Hepatic damage was evaluated by analysis of liver function tests in serum. Hepatic histopathology and collagen content were employed to quantify liver fibrosis; and activated hepatic stellate cells were assessed. Moreover, the mRNA and protein expression levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, high-mobility group box 1 (HMGB1), Toll like receptor (TLR) 2 and TLR4 were determined by quantitative real time PCR, Western blot or immunohistochemistry. Treatment with curcumin significantly attenuated CCl₄-induce liver injury, hepatic inflammation and reduced the levels of proinflammatory mediators (TNF-α, IL-6 and MCP-1). Moreover, curcumin significantly inhibited extracellular matrix deposition, reduced the number of activated stellate cells, and decreased the levels of HMGB1, TLR4 and TLR2 expression in the rat model of fibrogenesis. These results suggest that curcumin could be an effective agent for preventing liver fibrosis and its mechanism may in part be a consequence of the reduction TLR2, TLR4 and HMGB1 expression.     

多索茶碱对支气管哮喘患者血清细胞因子和肺功能的影响

目的观察多索茶碱对支气管哮喘患者血清肿瘤坏死因子-α（TNF-α）、白细胞介素-10（IL-10）水平和肺功能的影响。方法将72例急性发作期支气管哮喘患者随机均分为两组,治疗组采用多索茶碱治疗,对照组采用氨茶碱治疗,治疗前后观察血清细胞因子（TNF-α、IL-10）、肺功能的变化。结果与治疗前比较,两组治疗后的TNF-α水平明显下降,治疗组下降更明显（P〈0.01）;两组治疗后的血清IL-10水平均明显上升,治疗组上升更明显（P〈0.01）;两组治疗后的肺功能指标明显改善,且治疗组改善更明显（P〈0.01）。结论多索茶碱可降低TNF-α水平、上调IL-10水平,从而抑制气道炎症、缓解气道痉挛、降低气道高反应性,改善肺功能。     

KF24345, an adenosine uptake inhibitor,ameliorates the severity and mortality of lethal acute pancreatitis via endogenous adenosine in mice

Adenosine protects against cellular damage and dysfunction under several adverse conditions including inflammation and ischemia. In this study, we examined the effects of 3-[1-(6,7-diethoxy-2-morpholinoquinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1H,3H)-quinazolinedione hydrochloride (KF24345), an adenosine uptake inhibitor, on experimental acute pancreatitis induced by choline-deficient and ethionine-supplemented diet in mice. KF24345, administered with the diet onset and every 24 h thereafter, prevented hyperamylasemia, acinar cell injury and serum tumor necrosis factor-alpha elevation and ultimately decreased mortality. Therapeutic treatment with KF24345, which started 32 h after the diet onset, also decreased mortality. The beneficial effect of KF24345 on mortality was abolished by the pretreatment with 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385), a selective adenosine A(2A) receptor antagonist. An intravenous injection of KF24345 at 48 h after the diet onset increased plasma adenosine concentrations in mice with acute pancreatitis. These results suggest that KF24345 shows anti-pancreatitis effects via endogenous adenosine and adenosine A(2A) receptors. The adenosine uptake inhibition could be a new therapeutic approach for acute pancreatitis.     

丹参对大鼠重症急性胰腺炎肠道微循环及炎性因子的实验研究

目的 确良观察重症急性胰腺炎（SAP）时肠道微循环血流量和内毒素、肿瘤坏死因子α（TNF-α）、白细胞介素（IL）-1β及IL-6的变化以及丹参的作用.方法 将雄性SD大鼠192只,完全随机分为对照组、SAP组和治疗组各64只,各组又按模型制备后2、6、12、24 h分为4个亚组,每组16只,其中8只用于肠道血流量测定,其余8只用于抽取血样及组织学观察.以4%牛磺胆酸钠逆行胰胆管注射复制SAP模型,治疗组经颈静脉注射丹参.采用放射生物微球技术在造模后2、6、12、24 h分别测定肠道组织微循环血流量,同时检测血浆内毒素及血清TNF-α、IL-1β、IL-6,并观察肠黏膜病理改变.结果 SAP组在造模后2、6、12、24 h肠道组织血流量[分别为（0.52±0.14）、（0.43±0.04）、（0.38±0.53）、（0.33±0.47）ml/（min·g）]较对照组[分别为（1.24±0.13）、（1.27±0.27）、（1.36±0.42）、（1.14±0.63）ml/（min·g）]明显减少（P＜0.01）,血浆内毒素及血清TNF-α、IL-1β、IL-6水平较对照组明显升高（P＜0.01）,肠黏膜损伤程度较对照组明显加重（P＜0.01） 治疗组2、6、12 h肠道组织血流量[分别为（0.72±0.21）、（0.68±0.35）、（0.64±0.28）ml/（min·g）]与SAP组比较明显升高（P＜0.01）,24 h变化不明显.血清TNF-α、IL-1β、IL-6活性2、6、12 h时段与SAP组比较明显减少（P＜0.01）,24 h变化不明显.肠黏膜损伤程度较SAP组明显改善（P＜0.01）.结论 SAP时肠组织血流量减少同时伴有内毒素血症及炎性因子TNF-α、IL-1β、IL-6的升高,丹参能通过提高肠道血流量改善微循环,减轻肠道损伤.     

R-130823, a novel inhibitor of p38 MAPK, ameliorates hyperalgesia and swelling in arthritis models

We found that a novel compound, R-130823 {2-(4-fluorophenyl)-4-(1-phenethyl-1,2,3,6-tetrahydropyridin-4-yl)-3-(pyridin-4-yl)-1H-pyrrole}, had highly selective inhibition against mitogen-activated protein kinase p38alpha (IC50=22 nM). The release of tumor necrosis factor-alpha, interleukin-1beta, -6 and -8 was inhibited in lipopolysaccharide-stimulated human blood pretreated by R-130823, with IC50 values of 0.089, 0.066, 0.95 and 0.16 microM, respectively. R-130823 reduced the established hind paw swelling in rat adjuvant-induced arthritis, while methotrexate showed no suppression. In the same model, R-130823 ameliorated adjuvant-induced hyperalgesia with rapid onset and long duration comparable to a cyclooxygenase-2 inhibitor, celecoxib. In murine collagen-induced arthritis, R-130823 blocked the progress of arthritis when administered just after the onset of the arthritis. Histological analysis of the knee joints showed that proliferation of fibroblasts and synoviocytes and infiltration of neutrophils were ameliorated. In conclusion, R-130823 is expected to be an efficacious treatment for rheumatoid arthritis by blocking the p38 pathway.