Effect of melatonin on altered expression of vasoregulatory genes during hepatic ischemia/reperfusion

The production of reactive oxygen species during hepatic ischemia/reperfusion (I/R) can help create disturbances in microcirculation. This study examined the effect of melatonin, a pineal secretory product and a potent antioxidant, on the expression of vascular stress genes during hepatic I/R. Rats were subjected to 60 min of hepatic warm ischemia followed by 5 h reperfusion. Melatonin (10 mg/kg) was administered intraperitoneally 15 min before ischemia and immediately before reperfusion. The serum alanine aminotransferase and hepatic malondialdehyde levels increased markedly after I/R. These increases were significantly inhibited by melatonin. The levels of endothelin-1 (ET-1) and its receptor, ET(B) mRNA, were elevated by I/R but attenuated by melatonin. The mRNA levels of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and heme oxygenase-1 were significantly higher after I/ R. Melatonin augmented the increase in the eNOS mRNA level, whereas it reduced the increase in the iNOS mRNA level. The expression of tumor necrosis factor-alpha was increased markedly by I/R. This increase was also attenuated by melatonin. These results suggest that melatonin ameliorates the imbalanced expression of the vascular stress genes during hepatic I/R through its antioxidant property.     

吡咯烷二硫代氨基甲酸盐对糖尿病大鼠血管内皮功能不全的影响及其机制

目的探讨吡咯烷二硫代氨基甲酸盐(PDTC)对糖尿病大鼠血管内皮依赖性舒张功能损害的保护作用及其机制。方法雄性SD大鼠一次性ip给予链脲佐菌素60 mg·kg-1制备糖尿病模型,通过饮水中给予PDTC 10 mg·kg-1,连续治疗8周。检测血糖、血脂和血清内源性一氧化氮合酶(NOS)抑制物非对称性二甲基精氨酸(ADMA)浓度。用含有人二甲基精氨酸二甲胺水解酶2(h DDAH2)基因的重组腺病毒(Ad5CMV-h DDAH2)体外感染糖尿病大鼠血管环,分别检测感染前后血管环对乙酰胆碱诱导的最大舒张反应(Emax)、半数有效量(EC50)及血管组织DDAH活性。结果与正常组比较,糖尿病大鼠血糖明显升高,血清ADMA浓度从正常组的(1.14±0.26)μmol·L-1升至(2.18±0.52)μmol·L-1(P<0.01);血管组织DDAH活性也从正常组的(0.10±0.02)U·g-1蛋白降至(0.05±0.01)U·g-1蛋白(P<0.01);血管内皮依赖性舒张功能损伤,表现为Emax由正常组的(93.6±4.4)%降至(50.8±4.9)%(P<0.01),EC50由正常组的(88±22)nmol·L-1升至(240±45)nmol·L-1(P<0.01)。PDTC治疗降低血糖和血清ADMA浓度分别至(13.2±3.5)mmol·L-1和(1.40±0.25)μmol·L-1(P<0.01),增加血管DDAH活性至(0.08±0.02)U·g-1蛋白(P<0.01),改善内皮依赖性血管舒张功能,使Emax增至(84.6±4.5)%,EC50降至(134±27)nmol·L-1(P<0.01)。糖尿病大鼠血管转染DDAH2基因后,血管DDAH活性及Emax和EC50的变化与PDTC治疗组相似。结论 PDTC对糖尿病大鼠血管内皮依赖性舒张功能具有明显的保护作用,其机制可能与上调血管DDAH活性,降低内源性NOS抑制物ADMA蓄积有关。     

百里醌通过抑制氧化应激信号通路减轻肝缺血再灌注损伤

目的探讨百里醌对肝缺血再灌注损伤的影响及其机制。方法建立小鼠肝缺血再灌注损伤模型。30只C57小鼠随机分为3组,每组10只,分别为假手术组、缺血/再灌注损伤组(IRI组)和百里醌预处理组(百里醌组)。IRI组、百里醌组小鼠肝缺血90 min再灌注4 h时采血和收集肝脏标本,分别生化检测血清中丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)的含量,以及肝组织中活性氧自由基(ROS)、丙二醛(MDA)含量,过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GPx)、超氧化物歧化酶(SOD)活性,观察肝组织的病理形态。结果与假手术组比较,IRI组ALT、AST、ROS、MDA含量提高,而CAT、GPx、SOD活性降低,病理改变加重。与IRI组比较,百里醌组ALT、AST、ROS、MDA含量降低,而CAT、GPx、SOD活性提高,病理改变减轻。结论百里醌能通过抑制氧化应激减轻肝缺血再灌注损伤。     

Effects of Trolox on the activity and gene expression of cytochrome P450 in hepatic ischemia/reperfusion

1. The aim of this study was to investigate the effect of Trolox on hepatic microsomal cytochrome P450 (CYP) activity and gene expression during ischemia and reperfusion (I/R). 2. Rats were subjected to 60 min of hepatic ischemia, and 5 h (acute phase) and 24 h (subacute phase) of reperfusion. Rats were treated intravenously with Trolox (2.5 mg kg(-1)) or vehicle, 5 min before reperfusion. 3. The serum alanine aminotransferase level and lipid peroxidation were increased as a result of I/R. These increases were attenuated by Trolox. Reduced glutathione concentration decreased in I/R group, and this decrease was inhibited by Trolox. 4. Both total hepatic CYP content and NADPH-cytochrome P450 reductase activity decreased after I/R, which were restored by Trolox. 5. CYP1A1 activity and its protein level decreased 24 h after reperfusion; decreases which were prevented by Trolox. Both the activity and mRNA expression of CYP1A2 decreased 24 h after reperfusion. The decrease in CYP1A2 mRNA was prevented by Trolox. CYP2B1 activity and mRNA expression decreased 5 h after reperfusion. The decrease in CYP2B1 activity was prevented by Trolox. In contrast, the CYP2E1 activity and its protein level increased 5 h after reperfusion and this increase was prevented by Trolox. 6. The expression of TNF-alpha and iNOS mRNAs increased after I/R. Trolox inhibited increase in iNOS mRNA expression. 7. Trolox ameliorates hepatic drug-metabolizing dysfunction, as indicated by abnormalities in CYP isoforms during I/R, and this protection is likely due to the scavenging of reactive oxygen species.     

吡咯烷二硫代氨基甲酸盐下调基质金属蛋白酶9表达机制的研究

目的探讨TNF-α诱导的肺泡巨噬细胞(AM)性基质金属蛋白酶9(MMP-9)表达的信号通路以及吡咯烷二硫代氨基甲酸盐(PDTC)对MMP-9表达的影响机制。方法从慢性阻塞性肺疾病患者支气管肺泡灌洗液中分离与培养AM,以PDTC预处理AM,以TNF-α或IL-1刺激AM。半定量逆转录-聚合酶链反应法检测MMP-9 mRNA的表达;Western blot检测MMP-9蛋白的表达及TNF-α或IL-1诱导的IκBα磷酸化水平。凝胶阻滞分析实验检测NF-κB活性。结果TNF-α上调AM源性MMP-9 mRNA和蛋白的表达(P<0.05);PDTC抑制TNF-α诱导的MMP-9的表达(P<0.05)。PDTC对TNF-α或IL-1诱导的IκBα的磷酸化均无抑制作用(P>0.05)。PDTC对TNF-α或IL-1诱导的NF-κB的活化均有抑制作用(P<0.05);且PDTC不能在体外直接抑制NF-κB的DNA结合活性(P>0.05)。结论NF-κB在TNF-α诱导的AM源性MMP-9的表达中起着重要作用;PDTC可能通过抑制泛素化-蛋白酶小体途径来下调TNF-α诱导的AM源性MMP-9的表达。     

An insight into the possible protective effect of pyrrolidine dithiocarbamate against lipopolysaccharide-induced oxidative stress and acute hepatic injury in rats

Lipopolysaccharide (LPS) is a major cell wall molecule of Gram-negative bacteria known to stimulate the synthesis and secretion of several toxic metabolites, such as reactive oxygen species. In this study, the effect of pyrrolidine dithiocarbamate (PDTC), an antioxidant with nuclear factor-κB inhibitor activity, was evaluated in LPS-induced oxidative stress and acute hepatic injury in rats. Animals were pretreated for 3 consecutive days with PDTC (200 mg/kg/day, i.p.) or saline and animals were then challenged with LPS (6 mg/kg, i.p.) or saline. Six hours after LPS injection, animals were decapitated and blood and liver samples were collected to assess the chosen biochemical parameters. Saline-pretreated animals challenged with LPS revealed extensive liver damage, as evidenced by increases in serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma glutamyl transferase (γ-GT). Also, LPS treatment resulted in significant increases in serum lactate dehydrogenase (LDH), tumor necrosis factor-alpha (TNF-α) and nitrite levels. Furthermore, LPS challenge caused oxidative stress as indicated by an increase in hepatic lipid peroxidation measured as thiobarbituric acid reactive substances (TBARS) and a decrease in hepatic reduced glutathione concentration (GSH) as well as decreased activities of superoxide dismutase (SOD) and catalase in hepatic tissues. The administration of PDTC prior to LPS challenge resulted in improved liver functions as evidenced by the decline in serum AST, ALT, γ-GT levels and reduction in serum LDH, TNF-α and nitrite levels. Moreover, PDTC reduced the chosen lipid peroxidation marker, TBARS and increased GSH concentration, and SOD and catalase activities in hepatic tissues. These results indicate that PDTC may be a useful pharmacological agent in alleviating LPS-induced oxidative stress and acute hepatic injury.     

肝缺血再灌注损伤大鼠P-选择素的表达

目的 通过测定肝缺血-再灌注损伤（HIRI）模型大鼠中P-选择素在肝脏组织的表达,探讨HIRI的发病机制.方法 将32只健康雌性SD大鼠随机分为四组（每组8只）：假手术组（SO组）、缺血30 min组（Ⅰ组）、缺血30 min再灌注30 min组（IR 30 min组）、缺血30 min再灌注1 h组（IR 1 h组）.采用Pringle 法制作HIRI模型.采用SP试剂盒测定肝组织中P-选择素含量.结果 缺血30 min后,肝组织中P-选择素的表达较SO组无明显差异;再灌注1 h后,P-选择素在肝组织血管内皮、肝细胞内广泛表达,较SO组差异显著.结论 在大鼠肝缺血-再灌注过程中,肝组织中P-选择素的表达逐渐升高.     

Inhibiton of NF-kappaB activation during ischemia reduces hepatic ischemia/reperfusion injury in rats

The aim of this study was to determine whether nuclear factor-kappaB (NF-kappaB) inhibitors are efficient against hepatic ischemia/reperfusion (I/R) injury. We previously demonstrated that xanthine oxidase-derived reactive oxygen species activate NF-kappaB during ischemia. However, the role of NF-kappaB activation during ischemia in post-reperfusion injury remains unclear. Therefore, while we examined the effects of NF-kappaB inhibitors, sulfasalazine and pyrrolidinedithiocarbamate on hepatic I/R injury using a rat lobar hepatic I/R model, we estimated the relationship between NF-kappaB activation during ischemia and following hepatic damage caused by reperfusion. The portal vein and the hepatic artery were clamped for 1 hr followed by reperfusion for up to 24 hr. NF-kappaB activation was determined by Western blot analysis. NF-kappaB activation was observed in the ischemic lobe of the liver, and the activation was prevented by pre-administration with NF-kappaB inhibitors. Although the serum ALT level, hepatic MPO activity and BSP clearance, as an index of hepatic injury, were increased after reperfusion, the increase was attenuated by pre-administration with NF-kappaB inhibitors. These findings suggest that NF-kappaB activation during ischemia is relevant to hepatic I/R injury. Moreover, we first showed that pre-administration with NF-kappaB inhibitors is effective against hepatic I/R injury.     

七氟醚对大鼠缺血再灌注心肌凋亡相关基因表达的影响

目的探讨七氟醚预处理对大鼠心脏缺血再灌注过程中心肌细胞Bcl-2及Bax基因表达的影响。方法24只SD大鼠随机分成3组（n=8）：假手术对照组（C组）、缺血再灌注对照组（IR组）和七氟醚预处理组（s组）。IR组与S组接受左冠脉3h阻断和3h再灌注。七氟醚预处理组在缺血前吸入七氟醚,30min后洗脱15min。取心肌缺血区组织,RT-PCR测Bcl-2及Bax基因的mRNA表达,免疫印迹法（Western-blot）测蛋白表达。结果与C组相比较,IR组和S组Bcl-2的mRNA和蛋白表达均下调,而S组高于IR组;Bax的mRNA和蛋白表达IR及S组均高于C组,IR组则高于S组。结论七氟醚预处理抑制心肌细胞凋亡可能与上调Bd-2基因的表达、下调Bax基因的表达有关。     

Effect of alpha-tocopherol on the expression of hepatic vascular stress genes in response to sepsis

Sepsis is the leading cause of death in critically ill surgical patients. Septic hepatic dysfunction, an important determinant of outcome, although poorly understood, includes inappropriate expression of vasoregulatory genes. In this study the effect of alpha-tocopherol was determined on the expression of hepatic vascular stress genes in response to sepsis. Rats were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP). Rats received either vehicle or alpha-tocopherol (AT, 15 mg/kg), intraperitoneally injected for 3 d prior to CLP procedure. Serum aminotransferase activities and hepatic lipid peroxides levels markedly increased 24 h after CLP, and this rise was attenuated by AT treatment. The hepatic concentrations of reduced glutathione decreased in CLP animals, which was inhibited by AT. CLP significantly increased mRNA levels of endothelin (ET)-1 and ET(B) receptor in livers, which was not prevented by AT treatment. There were no significant changes in ET(A) mRNA expression among any of the experimental groups. There were significant increases in the mRNA expression of inducible nitric oxide synthase and heme oxygenase-1 in livers of CLP animals, and this was prevented by AT treatment. The expression of tumor necrosis factor-alpha and cyclooxygenase-2 mRNA increased 4.9-fold and 4.4-fold, respectively, in livers of CLP animals. This increase was attenuated by AT treatment. Our data suggest that sepsis induces an imbalance in hepatic vasoregulatory gene expression and that AT ameliorates altered expression of vasodilators through its free radical scavenging activity.     

苯妥英钠对大鼠脑缺血再灌注后脑内Fas表达的抑制作用

目的观察脑缺血再灌注后Fas基因在脑内的表达及苯妥英钠对其的影响。方法采用夹闭大鼠双侧颈总动脉的急性前脑缺血再灌注模型和免疫组织化学染色法。结果脑缺血再灌注后神经细胞过度地表达Fas,尤以缺血半暗带明显,且Fas表达分布广泛;苯妥英钠给药组(30、60mg/kg)Fas阳性细胞的表达数显著低于不给药的生理盐水对照组(P<0.01),且两剂量组之间差异无统计学意义。结论①苯妥英钠对缺血再灌注脑损伤有保护作用;②苯妥英钠拮抗缺血再灌注脑损伤的机制与其抑制Fas表达从而减少缺血诱导的神经细胞凋亡有关。     

Effect of antioxidants on apoptosis induced by influenza virus infection: inhibition of viral gene replication and transcription with pyrrolidine dithiocarbamate

Influenza virus (IV) infection induced apoptotic DNA fragmentation and the moderate overproduction of reactive oxygen species (ROS) in primary cultured chorion cells prepared from human fetal membranes, and IV particles were released from the infected cells. The antioxidant pyrrolidine dithiocarbamate (PDTC) inhibited the induced DNA fragmentation, ROS overproduction and IV particle release. Although Trolox inhibited ROS overproduction, it did not inhibit DNA fragmentation or IV production. The inhibitory effect of PDTC on DNA fragmentation was manifested when added up to 3 h after infection or by exposing the infected cells to it for only 1 h after infection. PDTC inhibited IV hemagglutinin (HA) viral (vRNA) and complementary (cRNA and mRNA) RNAs synthesis until 6 h after infection and delayed and decreased HA protein synthesis. However, HA RNA synthesis resumed after 12 h even in the presence of PDTC. These results suggested that PDTC inhibited apoptosis by inhibiting viral macromolecule synthesis rather than through its antioxidant effect, because Trolox did not inhibit apoptosis or IV production, although ROS overproduction was inhibited. The synthesis of specific viral macromolecules at the early stage of infection may play a critical role in the mechanism of apoptosis induction and moderate ROS overproduction may not be involved in the mechanism.     

雌激素与雌激素受体在大鼠肝脏缺血再灌注损伤中的作用

目的 观察雌激素与雌激素受体在大鼠肝脏缺血再灌注损伤中的作用.方法 用Pringle's大鼠全肝缺血再灌注损伤模型,选取健康清洁级雄性SD大鼠120只,随机分成假手术组、肝缺血再灌注组和雌激素干预组(预处理+肝缺血再灌注组)3组,每组各40只;检测血清谷丙转氨酶(ALT)水平,HE染色观察肝脏组织形态学变化,ELISA法测定血清肿瘤坏死因子-a(TNF-a)水平,免疫组化方法检测肝脏组织核转录因子(NF-KB)和雌激素受体(ER)的表达情况.结果 假手术组,血清ALT,TNF-a水平升高不明显,ER,NF-KB表达无明显变化,肝脏组织形态学改变不明显;缺血再灌组、雌激素干预组的ALT,TNF-a水平与ER,NF-KB表达显著升高;但雌激素干预组ER表达明显高于缺血再灌组,缺血再灌组的NF-KB表达明显高于雌激素干预组,缺血再灌组的肝脏组织损伤程度显著高于雌激素干预组.结论 雌激素能刺激雌激素受体表达,进而降低TNF-a水平、抑制NF-KB表达,来减轻肝脏缺血再灌注损伤.     

Erk activation is required for Nrf2 nuclear localization during pyrrolidine dithiocarbamate induction of glutamate cysteine ligase modulatory gene expression in HepG2 cells.

Pyrrolidine dithiocarbamate (PDTC) induction of the human glutamate cysteine ligase modulatory (GCLM) gene is dependent on activation of the mitogen-activated protein kinases (MAPKs) extracellular regulated kinase (Erk) and p38, and is not affected by protein kinase C (PKC) or PI3K inhibitors. Nrf2 binding to the electrophile response element (EpRE) located within the GCLM promoter is decreased after MAPK inhibition, suggesting that Nrf2 could be a downstream target of activated MAPK. To evaluate this hypothesis, a series of Nrf2 proteins harboring mutations in conserved consensus MAPK phosphorylation sites were developed and used in multiple functional assays. All mutated Nrf2 proteins tested interacted with the cytoplasmic repressor Keap1 in a manner indistinguishable from wild-type Nrf2. Furthermore, the mutant and wild-type Nrf2 proteins were similarly capable of transactivating an EpRE-containing GCLM/luciferase reporter transgene. Collectively these functional assays suggest that Nrf2 is not likely to be a direct downstream target of activated MAPK in vivo. However, treatment of HepG2 cells with MAPK inhibitors PD98059 and/or SB202190 prior to exposure to PDTC, reduced Nrf2 translocation to the nucleus, suggesting that MAPK-directed phosphorylation is a requirement for nuclear localization during PDTC induction of GCLM gene expression.     

二硫代氨基甲酸吡咯烷对小鼠免疫性肝损伤的抑制作用

目的探讨二硫代氨基甲酸吡咯烷(PDTC)对免疫性肝损伤的抑制作用及其机制。方法设正常对照、脂多糖(LPS)、卡介苗(BCG)、BCG+LPS、PDTC和BCG+PDTC+LPS组。除正常对照、LPS和PDTC组外,其余各组小鼠经尾静脉注射BCG(每只2.5mg)。10d后,LPS和BCG+LPS组分别给予LPS(0.2mg·kg-1,ip),PDTC和BCG+PDTC+LPS组在给予LPS前24和2h分别给予PDTC(100mg·kg-1,ip),对照组给予等体积生理盐水。每组15只小鼠用于观察LPS处理后72h的死亡率;每组6只小鼠于LPS处理后1.5h处死,取肝脏,用RT-PCR检测肝脏组织肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)mRNA表达水平,用凝胶电泳迁移率分析法测定肝脏核因子κB(NF-κB)结合活性;每组12只小鼠于LPS处理后6h取血,处死,留取肝脏,测定血清谷丙转氨酶(GPT)活性、一氧化氮(NO)水平和肝组织还原型谷胱甘肽(GSH)含量,制备肝组织切片进行HE染色,观察组织病理变化。结果与正常对照组比较,BCG和LPS组小鼠肝脏炎症细胞明显增加,血清GPT活性升高,肝脏GSH含量显著下降,肝脏TNF-α与IL-1β mRNA表达增强,各组均未见小鼠死亡;PDTC组除血清GPT活性升高外,上述其他指标均未发生明显改变。与BCG和LPS组比较,BCG+LPS组血清GPT活性进一步升高,并伴有大面积肝脏坏死和大量炎症细胞浸润,肝脏NF-κB结合活性显著升高,TNF-α和IL-1β表达进一步增强,GSH水平下降,血清NO水平增加,小鼠死亡率40%。与BCG+LPS组比较,PDTC预处理明显抑制BCG+LPS引起的肝脏NF-κB活性、TNF-α及IL-1β mRNA表达增强,升高肝脏GSH含量,降低血清GPT活性和NO水平,减轻BCG+LPS引起的肝脏炎症和坏死,未见小鼠死亡。结论PDTC可抑制BCG+LPS引起的小鼠免疫性肝损伤,其机制可能与其抗炎和抗氧化作用有关。