Quercetin protects liver injury induced by bile duct ligation via attenuation of Rac1 and NADPH oxidase1 expression in rats

BACKGROUND:Bile duct ligation (BDL) and subsequent cholestasis are correlated with oxidative stress,hepatocellular injury and fibrosis.Quercetin is a flavonoid with antifibrotic,and hepatoprotective properties.However,the molecular mechanism underlying quercetin-mediated hepatoprotection is not fully understood.The current study was to evaluate mechanisms of hepatoprotective effect of quercetin in BDL rat model.METHODS:We divided male Wistar rats into 4 groups (n=8 for each):sham,sham+quercetin (30 mg/kg per day),BDL,and BDL+quercetin (30 mg/kg per day).Four weeks later,the rats were sacrificed,the blood was collected for liver enzyme measurements and liver for the measurement of Racl,Racl-GTP and NOX1 mRNA and protein levels by quantitative PCR and Western blotting,respectively.RESULTS:Quercetin significantly alleviated liver injury in BDL rats as evidenced by histology and reduced liver enzymes.Furthermore,the mRNA and protein expression of Racl,Racl-GTP and NOX1 were significantly increased in BDL rats compared with those in the sham group (P＜0.05);quercetin treatment reversed these variables back toward normal (P＜0.05).Another interesting finding was that the antioxidant markers e.g.superoxide dismutase and catalase were elevated in quercetin-treated BDL rats compared to BDL rats (P＜0.05).CONCLUSION:Quercetin demonstrated hepatoprotective activity against BDL-induced liver injury through increasing antioxidant capacity of the liver tissue,while preventing the production of Racl,Racl-GTP and NOX1 proteins.     

Tiam1、Rac1在评估大肠癌远隔脏器转移中的意义

目的:观察人大肠癌和癌旁正常大肠黏膜组织中T淋巴瘤侵袭转移诱导因子1(Tiam1)和RacGTP酶激活蛋白1(Rac1)的表达,并探讨其与大肠癌根治术后发生远隔脏器转移的相关性.方法:应用SP免疫组织化学法检测87例大肠癌及40例癌旁正常组织标本中Tiam1/Rac1的表达情况;应用竞争性RT-PCR检测上述标本中Tiam1 mRNA的表达情况;应用配体沉淀法检测上述标本中Rac1蛋白活性.结果:Tiam1/Rac1在大肠癌细胞胞质内呈阳性染色.Tiam1 mRNA在大肠癌中表达明显增强(0.6±0.02vs0.24±0.02,P<0.0005),并且在术后发生远隔转移患者中的表达要高于未发生远隔转移者(0.91±0.02vs0.52±0.02,P<0.0005).Rac1蛋白活性的表达情况与Tiam1一致,在大肠癌中表达高于正常黏膜(0.17±0.01vs0.07±0.05,P<0.0005),并且在有远隔转移的大肠癌组织中活性要高于无远隔转移者(0.25±0.02vs0.15±0.01,P<0.0005).对于不同分化程度的大肠癌组织,Tiam1/Racl的含量均没有统计学差异(0.63±0.04,0.60±0.04,0.57±0.04,P=0.613;0.18±0.06,0.17±0.05,0.15±0.05,P=0.558).结论:Tiam1/Racl是大肠癌根治术后患者发生远隔转移的正性因子,可以作为预测大肠癌根治术后患者发生远隔脏器转移的有效指标.     

阴道毛滴虫Rac1蛋白的cDNA克隆和序列分析

目的　获得阴道毛滴虫Rac1蛋白的cDNA克隆 ,研究其在细胞周期中的调解作用。　方法　提取阴道毛滴虫总RNA ,构建cDNA表达文库 ,随机分离cDNA克隆并测序。用在线生物分析软件NCBIBLAST、ClustalW以及Treeview等程序进行序列分析。　结果　获得一株有 714bp的cDNA克隆。序列分析表明 ,该克隆开放阅读框具 60 0bp ,推测肽链具 2 0 0个氨基酸。该肽链与Rho家族中Rac1鸟苷三磷酸 (GTP)酶同源性最高 (>60 % ) ,并具多种RhoGTP酶的保守基序 ,如GTP结合部位、GTP酶激活蛋白作用基序、GTP分离抑制因子作用基序、鸟嘌呤核苷酸交换因子作用基序等。进化树分析显示该克隆属于Rac亚家族GTP酶 ,与原虫Rac1蛋白最接近。　结论　该克隆属RhoGTP酶的Rac亚家族 ,很可能是阴道毛滴虫的Rac1蛋白。     

17Beta-estradiol inhibits monocyte adhesion via down-regulation of Rac1 GTPase

Estrogens confer atheroprotective effects that remain poorly understood. We hypothesised that estrogens directly target monocytes, and investigated the pathways via which estrogens might impact on monocyte adhesion. In an in vitro model of the vasculature (parallel plate laminar flow chamber, 2 dynes/cm2), 17beta-estradiol (24 h, 0.1-1 microM) potently inhibits monocyte adhesion. In parallel, 17beta-estradiol down-regulates Rac1 GTPase activity in monocytes. Transfection of monocytic cells with dominant-negative Rac1N17 significantly decreases adhesion to human endothelial cells, while constitutively-active Rac1L61 augments adhesion. As determined by pull-down assays, Rac1 is rapidly activated by the chemokine stromal-derived factor-1 (SDF-1) in human monocytes (100 nM, 30 s). Within the same time period, SDF-1 mediates both ICAM-1/beta2- and VCAM-1/beta1-integrin-dependent monocyte adhesion, which is significantly decreased in cells overexpressing dominant-negative Rac1N17. Inhibitor studies revealed that Rac1-triggered monocyte adhesion is dependent upon actin rearrangement, while production of reactive oxygen species via Rac1 is not involved. Estrogen directly inhibits monocyte adhesion via down-regulation of Rac1, which is both necessary and sufficient to enhance monocyte adhesion under physiological flow conditions. These studies extend current knowledge about the mechanisms responsible for the vascular recruitment of pro-inflammatory cells, and potentially open up new avenues for the therapy of atherosclerosis.     

Rac1对L02肝细胞株上皮细胞间质转化及增殖和凋亡的影响

目的检测Rac1在TGF-β1诱导的L02细胞上皮间质转化中的作用,及其对细胞增殖和凋亡的影响。方法应用不同活性的Rac1质粒pExRed-NLS Flag（空载体组）、pExRed-NLS Flag Rac1（野生型Rac1组）、pExRed-NLS Flag Rac1T17N（显性负调控Rac1组）、pExRed-NLS Flag Rac1G12V（持续活化型Rac1组）瞬时转染L02细胞,经5 ng/ml TGF-β1处理细胞。采用免疫印迹法检测融合蛋白Flag-Rac1表达,采用细胞免疫荧光及免疫印迹法检测Ck8和Vimentin表达,采用细胞划痕实验及Transwell法检测细胞迁移能力。使用不同浓度的Rac1特异性抑制剂NSC23766处理L02细胞,采用CCK-8法检测细胞增殖,采用Annexin V-FITC/PI双染法检测细胞凋亡。结果四组质粒均成功瞬时转染到L02细胞中;与空载体组和野生型Rac1组比,持续活化型Rac1转染细胞Vimentin蛋白表达水平显著增高,CK8蛋白表达水平降低,细胞迁移能力增加;与空载体组和野生型Rac1组比,显性负调控Rac1转染细胞Vimentin蛋白表达水平降低,CK8蛋白表达水平增高,细胞迁移能力降低（P〈0.05）;在NSC23766处理L02细胞后,细胞增殖被抑制,但各处理组细胞凋亡无明显差异。结论 Rac1可促进TGF-β1诱导的L02肝细胞株上皮间质转化和细胞增殖,但对细胞凋亡无明显影响。     

阴道毛滴虫Rac1蛋白的cDNA克隆和序列分析

目的　获得阴道毛滴虫Rac1蛋白的cDNA克隆,研究其在细胞周期中的调解作用。　方法　提取阴道毛滴虫总RNA,构建cDNA表达文库,随机分离cDNA克隆并测序。用在线生物分析软件NCBIBLAST、ClustalW以及Treeview等程序进行序列分析。　结果　获得一株有714bp的cDNA克隆。序列分析表明,该克隆开放阅读框具600bp,推测肽链具200个氨基酸。该肽链与Rho家族中Rac1鸟苷三磷酸(GTP)酶同源性最高(>60 %),并具多种RhoGTP酶的保守基序,如GTP结合部位、GTP酶激活蛋白作用基序、GTP分离抑制因子作用基序、鸟嘌呤核苷酸交换因子作用基序等。进化树分析显示该克隆属于Rac亚家族GTP酶,与原虫Rac1蛋白最接近。　结论　该克隆属RhoGTP酶的Rac亚家族,很可能是阴道毛滴虫的Rac1蛋白。     

Antiplatelet activity of deferiprone through cyclooxygenase-1 inhibition

Abstract

Thalassemia patients are susceptible to both iron overload and thromboembolism. Deferiprone is an iron chelator that shows an antiplatelet activity and thus may alleviate platelet hyperactivation in thalassemia. Therefore, this study aimed to characterize the inhibitory effects and mechanisms of deferiprone on normal human platelets. The results illustrated that deferiprone inhibited platelet aggregation at the iron chelating concentrations (0.08–0.25 mmol/l). Deferiprone inhibited human platelet aggregation stimulated by arachidonic acid and ADP more potently than epinephrine and collagen, with the IC₅₀ of 0.24 mmol/l and 0.25 mmol/l vs. 3.36 mmol/l and 3.73 mmol/l, respectively. Interestingly, deferiprone significantly inhibited COX-1 activity, with the IC₅₀ of 0.33 mmol/l, and slightly increased cAMP level at the high concentration of 4 mmol/l. Moreover, the results from molecular docking showed that deferiprone interacted closely with key residues in the peroxidase active site of COX-1. These results suggested that deferiprone possessed antiplatelet activity mainly through the inhibition of COX-1 activity.     

Differential roles of the dopamine 1-class receptors,D1R and D5R,in hippocampal dependent memory

Activation of the hippocampal dopamine 1-class receptors (D1R and D5R) are implicated in contextual fear conditioning (CFC). However, the specific role of the D1R versus D5R in hippocampal dependent CFC has not been investigated. Generation of D1R- and D5R-specific in situ hybridization probes showed that D1R and D5R mRNA expression was greatest in the dentate gyrus (DG) of the hippocampus. To identify the role of each receptor in CFC we generated spatially restricted KO mice that lack either the D1R or D5R in DG granule cells. DG D1R KOs displayed significant fear memory deficits, whereas DG D5R KOs did not. Furthermore, D1R KOs but not D5R KOs, exhibited generalized fear between two similar but different contexts. In the familiar home cage context, c-Fos expression was relatively low in the DG of control mice, and it increased upon exposure to a novel context. This level of c-Fos expression in the DG did not further increase when a footshock was delivered in the novel context. In DG D1R KOs, DG c-Fos levels in the home cage was higher than that of the control mice, but it did not further increase upon exposure to a novel context and remained at the same level upon a shock delivery. In contrast, the levels of DG c-Fos expression was unaffected by the deletion of DG D5R neither in the home cage nor upon a shock delivery. These results suggest that DG D1Rs, but not D5Rs, contribute to the formation of distinct contextual representations of novel environments.The hippocampus is crucial for aversive Pavlovian conditioning, such as contextual fear conditioning (CFC) (1, 2). In CFC, the conditioned stimulus (context) is paired with the unconditioned stimulus (footshock), and after pairing, the context serves as a cue to predict a potential footshock (3, 4). Although the role of dopamine has been studied in the context of reward learning (5), evidence suggests that midbrain dopaminergic neurons are also important for aversive Pavlovian conditioning (6–9). In line with this evidence, hippocampal encoding of novel and contextual information is linked to dopamine release via excitation of dopamine neurons of the midbrain (5, 10, 11). Additionally, delivery of aversive stimuli, such as a footshock, results in increased dopaminergic neuron activity (12). Moreover, inactivation of hippocampal D1Rs and D5Rs attenuates contextual fear memory (13). Thus, it follows that delivery of an aversive stimulus activates midbrain dopamine neurons that project to the hippocampus, which is crucial for encoding novel contextual cues (12, 14, 15). Activation of hippocampal D1Rs and D5Rs may then strengthen the encoding of novel contextual information during CFC.The precise role of subregion-specific D1R or D5R activation in hippocampal-dependent learning and memory is unknown. This is in part due to the inability to discriminate between and spatially restrict D1R from D5R function (16–18), which is an important caveat because each receptor is involved in modulating distinct neuronal processes (19–22). Indeed, there is a lack of consensus of D1R and D5R expression patterns in the rodent hippocampus (23–27). Moreover, pharmacological findings are at odds with D1R and D5R global KO studies, which show that neither D1Rs nor D5Rs are required for fear conditioning (16, 17). Therefore, to reconcile these disparate findings and to test the necessity of D1R and D5R activation for CFC, it is necessary to functionally isolate and spatially restrict hippocampal D1R and D5R activity.In this study, we found that D1Rs and D5Rs exhibit overlapping expression in dentate gyrus (DG) granule cells. DG D1R activation is necessary to increase c-Fos expression in the DG and CA3 to enhance novel contextual encoding. Moreover, DG D1R activation decreases generalization of the conditioned fear response to novel contexts. However, we found no role for DG D5Rs in modulating DG c-Fos expression or contextual fear learning and memory. In using our subregion-specific KO mice, we show that the hippocampal dopamine signal plays a definitive role in CFC.     

Bves directly interacts with GEFT, and controls cell shape and movement through regulation of Rac1/Cdc42 activity

Bves is an integral membrane protein with no determined function and no homology to proteins outside of the Popdc family. It is widely expressed throughout development in myriad organisms. Here, we demonstrate an interaction between Bves and guanine nucleotide exchange factor T (GEFT), a GEF for Rho-family GTPases. This interaction represents the first identification of any protein that has a direct physical interaction with any member of the Popdc family. Bves and GEFT are shown to colocalize in adult skeletal muscle. We also demonstrate that exogenous expression of Bves reduces Rac1 and Cdc42 activity levels while not affecting levels of active RhoA. Consistent with a repression of Rac1 and Cdc42 activity, we show changes in speed of cell locomotion and cell roundness also result from exogenous expression of Bves. Modulation of Rho-family GTPase signaling by Bves would be highly consistent with previously described phenotypes occurring upon disruption of Bves function in a wide variety of model systems. Therefore, we propose Bves as a novel regulator of the Rac1 and Cdc42 signaling cascades.     

Chronic doxorubicin cardiotoxicity is mediated by oxidative DNA damage-ATM-p53-apoptosis pathway and attenuated by pitavastatin through the inhibition of Rac1 activity

Doxorubicin is known to have cumulative dose-dependent cardiotoxicity, and a tumor suppressor protein p53 has been implicated in the pathogenesis of doxorubicin cardiotoxicity. However, how p53 is induced by doxorubicin and mediates the cardiotoxic effects of doxorubicin remains elusive. In cultured cardiac myocytes, doxorubicin induced oxidative stress, DNA damage, ATM activation, and p53 induction. A free radical scavenger NAC attenuated all of these events, whereas an ATM kinase inhibitor wortmannin attenuated doxorubicin-induced ATM activation and p53 induction but not oxidative stress. Doxorubicin treatment in vivo also induced oxidative stress, DNA damage, ATM activation, and p53 accumulation. These observations suggest that p53 induction by doxorubicin is mediated by oxidative DNA damage-ATM pathway. Doxorubicin-induced contractile dysfunction and myocyte apoptosis in vivo were attenuated in heterozygous p53 deficient mice and cardiac-restricted Bcl-2 transgenic mice, suggesting that myocyte apoptosis plays a central role downstream of p53 in doxorubicin cardiotoxicity. We also tested whether pitavastatin exerts protective effects on doxorubicin cardiotoxicity. Pitavastatin attenuated doxorubicin-induced oxidative stress, DNA damage, ATM activation, p53 accumulation, and apoptosis in vitro. Pitavastatin also attenuated myocyte apoptosis and contractile dysfunction in vivo. The beneficial effects of pitavastatin were reversed by intermediate products of the mevalonate pathway that are required for the activation of Rac1, and Rac1 inhibitor exhibited cardioprotective effects comparable to those of pitavastatin. These data collectively suggest that doxorubicin-induced cardiotoxicity is mediated by oxidative DNA damage-ATM-p53-apoptosis pathway, and is attenuated by pitavastatin through its antioxidant effect involving Rac1 inhibition.     

Measurement of serum paraoxonase-1 activity in the evaluation of liver function

Paraoxonase-1 （PON1） is an esterase and lactonase synthesized by the liver and found in the circulation associated with high-density lipoproteins. The physiological function of PON1 seems to be to degrade specific oxidized cholesteryl esters and oxidized phospholipids in lipoproteins and cell membranes. PON1 is, therefore, an antioxidant enzyme. Alterations in circulating PON1 levels have been reported in a variety of diseases involving oxidative stress including chronic liver diseases. Measurement of serum PON1 activity has been proposed as a potential test for the evaluation of liver function. However, this measurement is still restricted to research and has not been extensively applied in routine clinical chemistry laboratories. The reason for this restriction is due to the problem that the substrate commonly used for PON1 measurement, paraoxon, is toxic and unstable. The recent development of new assays with non-toxic substrates makes this proposal closer to a practical development. The present editorial summarizes PON1 biochemistry and function, its involvement with chronic liver impairment, and some aspects related to the measurement of PON1 activity in circulation.     

XIAP通过调节CDK4／CDK6／cyclinD1复合物表达促进肝癌细胞增殖

目的研究肝癌细胞中XIAP与cDK4／cDK6／cyclinD1复合物之间的调控关系及在肝癌细胞增殖中的作用。方法前期研究用PathwayArray进行肝癌组织异常表达蛋白筛选,后期XIAP特异性抑制剂embelin处理Huh7细胞进行增殖、周期分析。Westernblot验证XIAP与cDK4／cDK6／cyclinD1复合物之间的调控关系。结果PathwayArray提示XIAP与CDK4、CDK6、cyclinD1之间存在联合表达。embelin引起Huh7细胞G1期阻滞、S期分布减少,抑制细胞增殖。XIAP可以调节Huh7细胞G1期蛋白CDK4、CDK6、cyclinD1表达。结论XIAP通过调节肝癌细胞G1期蛋白CDK4／CDK6／eyelinD1复合物的表达,促使细胞进入G1周期,从而促进其增殖。     

lncRNA NEAT1调控miR-206对缺氧复氧大鼠心肌细胞氧化应激损伤和凋亡的影响

目的 探讨长链非编码RNA(lncRNA)NEAT1调控miR-206对缺氧复氧诱导的心肌细胞氧化应激损伤和凋亡的影响和机制。方法 体外培养H9c2心肌细胞,构建心肌细胞缺氧复氧模型。实时荧光定量PCR(RT-qPCR)检测缺氧复氧诱导后lncRNA NEAT1和miR-206的表达水平。将lncRNA NEAT1小干扰RNA(si-lncRNA NEAT1)、miR-206模拟物(miR-206 mimics)分别转染H9c2细胞,缺氧复氧诱导后,检测细胞中丙二醛(MDA)和活性氧(ROS)含量以及细胞上清液中乳酸脱氢酶(LDH)活性,MTT法检测细胞存活率,流式细胞术检测细胞凋亡,Western blot检测含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved Caspase-3)和cleaved Caspase-9蛋白表达。利用荧光素酶报告基因实验以及RT-qPCR验证lncRNA NEAT1和miR-206的靶向结合关系。结果 缺氧复氧诱导后H9c2细胞中lncRNA NEAT1的表达显著升高,miR-206的表达显著降低(P＜0.05)。转染si-lncRNA NEAT1或miR-206 mimics处理缺氧复氧H9c2细胞,细胞存活率显著升高,MDA、ROS含量以及LDH活性显著降低,cleaved Caspase-3和cleaved Caspase-9蛋白的表达显著降低,细胞凋亡率显著降低(P＜0.05)。lncRNA NEAT1靶向miR-206并负调控miR-206表达。抑制miR-206部分逆转沉默lncRNA NEAT1对缺氧复氧诱导的心肌细胞氧化损伤和凋亡的影响(P＜0.05)。结论 沉默lncRNA NEAT1通过上调miR-206可减轻缺氧复氧诱导的心肌细胞氧化应激损伤和细胞凋亡,进而发挥心肌保护作用。