晚期氧化蛋白产物对THP-1巨噬细胞源性泡沫细胞ABCA1表达的影响

目的探讨晚期氧化蛋白产物对THP-1巨噬细胞源性泡沫细胞三磷酸腺苷结合盒转运体A1表达及胆固醇流出的影响。方法用160nmol/L的佛波酯诱导THP-1细胞24h,使其贴壁并转化为巨噬细胞,并以50mg/L的氧化型低密度脂蛋白孵育细胞使其转化为泡沫细胞,再用不同浓度的晚期氧化蛋白产物(0、50、100、200μmol/L)处理细胞24h,或以100μmol/L的晚期氧化蛋白产物处理细胞不同的时间(0、12、24、48h)。采用液体闪烁计数法观察细胞内胆固醇的流出....     

β受体介导肾上腺素对THP-1源性巨噬细胞清道夫受体AI与ABCA1基因表达的影响

目的 探讨肾上腺素对THP-1源巨噬细胞ATP结合盒转运体A1与清道夫受体AI mRNA表达影响的β受体介导作用.方法 分别用β1受体拮抗剂美托洛尔(10 nmol/L～100 μmol/L)和β2受体拮抗剂丁氧胺(2.5mol/L～25 μmol/L)预处理细胞1 h,再加入终浓度为1 μmol/L肾上腺素继续孵育24 h,检测受试基因的表达.结果 1 μmol/L的肾上腺素上调清道夫受体AI mRNA水平和下调ATP结合盒转运体A1 mRNA水平,与各自对照组比较差异有统计学意义(P<0.05).而终浓度为1 μmol/L肾上腺素分别与不同浓度的β肾上腺素能受体拮抗剂美托洛尔和丁氧胺联合作用时,随着美托洛尔和丁氧胺浓度加大,各组ATP结合盒转运体A1 mRNA的表达升高而清道夫受体AI mRNA的表达随浓度升高呈下降趋势,与1 μmol/L肾上腺素组比较差异有统计学意义(P<0.05),且丁氧胺对肾上腺素的拮抗作用在2.5 μmol/L达到平台期.结论 肾上腺素可能通过β1和β2肾上腺素能受体介导从而上调THP-1源性巨噬细胞清道夫受体AI mRNA水平和下调ATP结合盒转运体AI mBNA水平.     

PHF-2在氧化型低密度脂蛋白诱导THP-1源性

目的　观察氧化型低密度脂蛋白对THP-1巨噬细胞植物同源结构域指蛋白2（PHF-2）表达的影响,探讨表观遗传调节在氧化型低密度脂蛋白诱导巨噬细胞炎症反应中的作用。方法　用不同浓度（0、50、100、150　mg/L）氧化型低密度脂蛋白处理THP-1巨噬细胞4　h,采用酶联免疫吸附法检测肿瘤坏死因子α和巨噬细胞炎性蛋白1β的表达;采用实时荧光定量聚合酶链反应和Western　blot分别检测PHF-2　mRNA和蛋白质的表达;采用免疫共沉淀法检测PHF-2和p65核因子κB的结合。结果　100～150　mg/L氧化型低密度脂蛋白处理THP-1巨噬细胞4　h后,肿瘤坏死因子α和巨噬细胞炎性蛋白1β的表达明显上调;氧化型低密度脂蛋白呈浓度依赖性上调THP-1巨噬细胞PHF-2　mRNA和蛋白质的表达,并促进PHF-2和p65核因子κB的结合。结论　　PHF-2参与氧化型低密度脂蛋白诱导THP-1巨噬细胞核因子κB激活和炎症因子表达。     

丹红注射液对氧化型低密度脂蛋白诱导的THP-1巨噬细胞脂质蓄积的影响

目的　观察丹红注射液对氧化型低密度脂蛋白（ox-LDL）诱导的THP-1巨噬细胞脂质蓄积的影响,并探讨其机制。方法　160　nmol/L　PMA孵育THP-1巨噬细胞24　h后,细胞分为三组：对照组、ox-LDL组（100　mg/L）和丹红组（100　mg/L　ox-LDL+30　mL/L丹红注射液）。高效液相色谱分析法检测细胞内胆固醇水平,采用液体闪烁计数法观察细胞内胆固醇的流出,Western　blot检测细胞ATP结合盒转运体A1（ABCA1）和肝X受体α（LXRα）的蛋白表达,定量　PCR　检测细胞ABCA1和LXRα的mRNA　表达。结果　丹红注射液抑制ox-LDL诱导的THP-1巨噬细胞脂质蓄积,显著降低细胞总胆固醇（TC）、游离胆固醇（FC）和胆固醇酯（CE）的含量,增加载脂蛋白AⅠ（ApoAⅠ）介导的胆固醇流出,促进THP-1巨噬细胞ABCA1和LXRα的表达。结论　丹红注射液抑制ox-LDL诱导的THP-1巨噬细胞脂质蓄积,可能与其促进LXRα-ABCA1途径介导的胆固醇流出有关。     

过氧化体增殖物激活型受体γ对巨噬细胞脂质蓄积及CD36表达的影响

目的观察过氧化体增殖物激活型受体γ激动剂和拮抗剂对THP-1巨噬细胞胆固醇蓄积及CD36表达的影响。方法实验分对照组、氧化型低密度脂蛋白组、Ciglitazone处理组和GW9662处理组,后两组用50 mg/L氧化型低密度脂蛋白分别与过氧化体增殖物激活型受体γ激动剂Ciglitazone(10μmol/L)及拮抗剂GW9662(10μmol/L)共同孵育24 h,高效液相色谱分析法检测细胞总胆固醇蓄积情况,RT-PCR和Western blot分别检测THP-1巨噬细胞CD36 mRNA和蛋白的表达。结果与对照组(76.28±10.36 mg/g)相比,氧化型低密度脂蛋白(121.63±13.32 mg/g)能使细胞总胆固醇含量显著增加,而Ciglitazone能使氧化型低密度脂蛋白处理的细胞总胆固醇含量进一步增加(136.23±14.78 mg/g),GW9662能使氧化型低密度脂蛋白处理的细胞总胆固醇含量减少(98.52±11.45 mg/g)。过氧化体增殖物激活型受体γ拮抗剂GW9662使巨噬细胞CD36 mRNA和蛋白的表达下调及胆固醇蓄积减少,过氧化体增殖物激活型受体γ激动剂Ciglitazone使巨噬细胞CD36 mRNA和蛋白的表达上调及胆固醇蓄积增多。结论过氧化体增殖物激活型受体γ拮抗剂使THP-1巨噬细胞胆固醇蓄积减少及氧化型低密度脂蛋白诱导的CD36表达下调。     

阿托伐他汀对THP-1巨噬细胞脂质蓄积及CD36表达的影响

目的观察阿托伐他汀对氧化型低密度脂蛋白诱导的THP-1巨噬细胞脂质蓄积及CD36表达的影响。方法用50mg/L氧化型低密度脂蛋白与不同浓度的阿托伐他汀(0、0.312、1.25和5μmol/L)共同孵育THP-1巨噬细胞24h,以空白组作对照,用液体闪烁计数法检测细胞[3H]胆固醇流入情况,油红O染色观察细胞内脂质蓄积情况,高效液相色谱分析法检测细胞内总胆固醇水平,逆转录聚合酶链反应与免疫印迹分析法分别检测THP-1巨噬细胞CD36mRNA和蛋白的表达。结果阿托伐他汀使THP-1巨噬细胞胆固醇流入减少,对照组胆固醇流入为35.90%±2.36%,0、0.312、1.25和5μmol/L阿托伐他汀组胆固醇流入分别为47.10%±3.18%、41.20%±2.88%、35.10%±2.35%和28.30%±1.98%;阿托伐他汀能抑制THP-1巨噬细胞对氧化型低密度脂蛋白的摄取,油红O染色可见氧化型低密度脂蛋白 阿托伐他汀组细胞内脂滴较氧化型低密度脂蛋白组明显减少,且脂滴颗粒体积变小;高效液相色谱分析发现,对照组细胞总胆固醇含量为78.24±11.35mg/g,0、0.312、1.25和5μmol/L阿托伐他汀组细胞总胆固醇含量分别为123.13±15.92mg/g、115.36±13.18mg/g、107.52±12.05mg/g和98.03±10.24mg/g。阿托伐他汀使氧化型低密度脂蛋白诱导的THP-1巨噬细胞CD36mRNA和蛋白的表达下调。结论阿托伐他汀引起氧化型低密度脂蛋白诱导的THP-1巨噬细胞CD36表达下调,并使脂质蓄积减少。     

NO/PKG对THP-1巨噬细胞ABCA1基因mRNA表达和胆固醇含量的影响

目的:探讨THP-1巨噬细胞泡沫化过程中NO/PKG的变化,以及NO/PKG对THP-1巨噬细胞ATP结合盒转运体A1(ABCA1)基因mRNA表达和胆固醇含量的影响。方法:THP-1细胞诱导分化为巨噬细胞,用氧化低密度脂蛋白(ox-LDL)处理,油红O染色观察巨噬细胞脂滴,酶标仪测定一氧化氮(NO)的释放;用一氧化氮供体L-精氨酸和硝普钠(SNP)、乙二醇二乙醚二胺四乙酸(EGTA)、PKG激动剂处理巨噬细胞,RT-PCR法测定细胞ABCA1基因mRNA的表达,高效液相色谱法(HPLC)测定细胞内胆固醇的含量变化。结果:ox-LDL可导致THP-1巨噬细胞脂质的蓄积和NO释放的增加,实验组NO释放量较对照组显著升高(P<0.05),实验组中50mg/L组、100mg/L组、200mg/L组与25mg/L组相比显著升高(P<0.05)。用PKG激动剂处理巨噬细胞后促进ABCA1基因mRNA表达上调(P<0.05);用PKG激动剂、L-精氨酸、SNP处理后的巨噬细胞内胆固醇较对照组显著减少(P<0.05)。EGTA能显著增加细胞内胆固醇含量(P<0.05)。结论:NO/PKG能降低细胞内胆固醇,其机制可能是PK...     

单核—巨噬细胞和ECV304内皮细胞表达死亡相关蛋白

为研究单核——巨噬细胞和ECV304细胞是否表达死亡相关蛋白以及影响死亡相关蛋白表达的因素。分别用氧化型低密度脂蛋白(100mg／L)孵育THP-1细胞，H2O2(1mmol／L)孵育ECV304细胞，用逆转录聚合酶链反应检测死亡相关蛋白mRNA的表达。结果发现，THP-1细胞及ECV304细胞均表达死亡相关蛋白，氧化型低密度脂蛋白和H2O2能分别诱导THP-1细胞和ECV304细胞死亡相关蛋白mRNA表达增高。实验结果提示死亡相关蛋白可能参与调节氧化应激诱导的单核—巨噬细胞和内皮细胞的凋亡。     

高糖对THP-1巨噬细胞CD36表达及脂质蓄积的影响

目的 观察高糖对THP-1巨噬细胞清道夫受体CD36表达和脂质蓄积的影响。方法 用不同浓度的D-葡萄糖（分别为5.6、11、20、30及35 mmol/L）、50 mg/L氧化型低密度脂蛋白（ox-LDL）、50 mg/L ox-LDL＋20 mmol/L D-葡萄糖孵育THP-1巨噬细胞24 h,油红O染色观察细胞内脂质蓄积情况,高效液相色谱分析法检测细胞内总胆固醇水平,定量PCR与免疫印迹分析法分别检测THP-1巨噬细胞CD36 mRNA和蛋白的表达。结果 随着D-葡萄糖处理THP-1巨噬细胞浓度的增加,CD36 mRNA和蛋白的表达逐渐增加（P<0.05）;高糖可协同ox-LDL诱导THP-1巨噬细胞CD36 mRNA和蛋白的表达上调（P<0.05）,并增加细胞内总胆固醇水平（P<0.05）。结论 高糖可诱导THP-1巨噬细胞CD36的表达上调,并促进细胞内脂质蓄积。     

JAK抑制剂阻断干扰素γ减少THP1巨噬细胞源性泡沫细胞胆固醇流出和ATP结合盒转运体A1的表达

目的本文观察JAK抑制剂AC490对干扰素γ处理后的THP1巨噬细胞源性泡沫细胞内胆固醇流出和ATP结合盒转运体A1表达的影响。方法用160nmol/L的佛波酯诱导THP1细胞24h，使其贴壁并转化为巨噬细胞，在含有50mg/L氧化型低密度脂蛋白培养液中继续培养细胞48h使其转化为泡沫细胞，以不加任何处理因素的细胞作为空白对照，用干扰素γ单独或联合JAK抑制剂AG490处理细胞24h。     

Cytochrome 1A1 and 1B1 gene diversity in the Zanzibar islands

Amodiaquine (AQ) is a 4‐aminoquinoline widely used in the treatment of malaria as part of the artemisinin combination therapy (ACT). AQ is metabolised towards its main metabolite desethylamodiaquine mainly by cytochrome P450 2C8 (CYP2C8). CYP1A1 and CYP1B1 play a minor role in the metabolism but they seem to be significantly involved in the formation of the short‐lived quinine‐imine. To complete the genetic variation picture of the main genes involved in AQ metabolism in the Zanzibar population, previously characterised for CYP2C8, we analysed in this study CYP1A1 and CYP1B1 main genetic polymorphisms. The results obtained show a low frequency of the CYP1A1*2B/C allele (2.4%) and a high frequency of CYP1B1*6 (approximately 42%) followed by CYP1B1*2 (approximately 27%) in Zanzibar islands. Genotype data for CYP1A1 and CYP1B1 show a low incidence of fast metabolisers, revealing a relatively safe genetic background in Zanzibar’s population regarding the appearance of adverse effects.     

CYP1A1 , GSTM1 , GSTT1 and NQO1 polymorphisms and colorectal adenomas in Japanese men

AIM: To investigate the role of functional genetic poly-morphisms of metabolic enzymes of tobacco carcinogens in the development of colorectal adenomas. METHODS: The study subjects were 455 patients with colorectal adenomas and 1052 controls with no polyps who underwent total colonoscopy in a preretirement health examination at two Self Defense Forces hospitals. The genetic polymorphisms studied wereCYP1A1*2A (rs 4646903), CYP1A1*2C (rs 1048943), GSTM1 (null or non-null genotype), GSTT1 (null or non-null genotype) and NQO1 C609T (rs 1800566). Genotypes were determined by the polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR method using genomic DNA extracted from the buffy coat. Cigarette smoking and other life-style factors were ascertained by a self-administered questionnaire. The associations of the polymorphisms with colorectal adenomas were examined by means of OR and 95%CI, which were derived from logistic regression analysis. Statistical adjustment was made for smoking, alcohol use, body mass index and other factors. The gene-gene interaction and effect modification of smoking were evaluated by the likelihood ratio test. RESULTS: None of the five polymorphisms showed a significant association with colorectal adenomas, nor was the combination of GSTM1 and GSTT1 . A borderline significant interaction was observed for the combination of CYP1A1*2C and NQO1 (P = 0.051). The OR associated with CYP1A1*2C was significantly lower than unity among individuals with the NQO1 609CC genotype. The adjusted OR for the combination of the CYP1A1*2C allele and NQO1 609CC genotype was 0.61 (95%CI: 0.42-0.91). Although the interaction was not statistically significant (P = 0.24), the OR for individuals carrying the CYP1A1*2C allele and GSTT1 null genotype decreased significantly compared with those who had neither CYP1A1*2C allele nor GSTT1 null genotype (adjusted OR: 0.69, 95%CI: 0.49-0.97). Smoking did not modify the associations of the individual polymorphisms with colorectal adenomas. There w     

Inhibition of procarcinogen-bioactivating human CYP1A1, CYP1A2 and CYP1B1 enzymes by melatonin

Abstract:  Administration of melatonin to rodents decreases the incidence of tumorigenesis initiated by benzo[ a ]pyrene or 7,12-dimethylbenz[ a ]anthracene, which requires bioactivation by cytochrome P450 enzymes, such as CYP1A1, CYP1A2 and CYP1B1, to produce carcinogenic metabolites. The present study tested the hypothesis that melatonin is a modulator of human CYP1 catalytic activity and gene expression. As a comparison, we also investigated the effect of melatonin on the catalytic activity of CYP2A6, which is also a procarcinogen-bioactivating enzyme. Melatonin (3–300 μ m ) decreased 7-ethoxyresorufin O -dealkylation catalyzed by human hepatic microsomes and recombinant CYP1A1, CYP1A2 and CYP1B1, whereas it did not affect coumarin 7-hydroxylation catalyzed by hepatic microsomes or recombinant CYP2A6. Melatonin inhibited CYP1 enzymes by mixed inhibition, with apparent K _i values (mean ± S.E.M.) of 59 ± 1 (CYP1A1), 12 ± 1 (CYP1A2), 14 ± 2 (CYP1B1) and 46 ± 8 μ m (hepatic microsomes). Additional experiments indicated that melatonin decreased benzo[ a ]pyrene hydroxylation catalyzed by hepatic microsomes and CYP1A2 but not by CYP1A1 or CYP1B1. Treatment of MCF-10A human mammary epithelial cells with melatonin (up to 300 μ m ) did not affect basal or benzo[ a ]pyrene-inducible CYP1A1 or CYP1B1 gene expression. Consistent with this finding, melatonin did not influence reporter activity in aryl hydrocarbon receptor-dependent pGudluc6.1-transfected MCF-10A cells treated with or without benzo[ a ]pyrene, as assessed in an in vitro cell-based luciferase reporter gene assay. Overall, melatonin is an in vitro inhibitor of human CYP1 catalytic activity, and it may be useful to develop potent analogues of melatonin as potential cancer chemopreventive agents that block CYP1-mediated chemical carcinogenesis.     

Pandemic H1N1 influenza

The 2009 H1N1 influenza A virus that has targeted not only those with chronic medical illness, the very young and old, but also a large segment of the patient population that has previously been afforded relative protection - those who are young, generally healthy, and immune naive. The illness is mild in most, but results in hospitalization and severe ARDS in an important minority. Among those who become critically ill, 20-40% will die, predominantly of severe hypoxic respiratory failure. However, and potentially in part due to the young age of those affected, intensive care with aggressive oxygenation support will allow most people to recover. The volume of patients infected and with critical illness placed substantial strain on the capacity of the health care system and critical care most specifically. Despite this, the 2009 pandemic has engaged our specialty and highlighted its importance like no other. Thus far, the national and global critical care response has been brisk, collaborative and helpful - not only for this pandemic, but for subsequent challenges in years ahead.     

1-Naphthyl isocyanate and 1-naphthylamine as metabolites of 1-naphthylisothiocyanate

Abstract: The importance of the bioactivation of 1-naphthylisothiocyanate was studied. Forty minutes after 1-naphthylisothiocyanate administration to rats, bile was collected over a 2.5-h period; the liver was then excised and homogenized. 1-naphthylisothiocyanate and its metabolites in bile and liver of rats were identified and quantified using coupled gas chromatography-mass spectrometry. Three main compounds were found in all 1-naphthylisothiocyanate-treated animals. They were identified as 1-naphthyl isocyanate, 1-naphthylamine and the parent compound, 1-naphthylisothiocyanate. When rats were given cycloheximide, which attenuates 1-naphthylisothiocyanate toxicity, 30 min before 1-naphthylisothiocyanate (300 mg/kg), 1-naphthyl isocyanate concentration was significantly lower than in rats receiving only 1-naphthylisothiocyanate. The appearance of 1-naphthylamine was also inhibited by cycloheximide, although not to the same extent as 1-naphthyl isocyanate. On the other hand, phenobarbital, which potentiates 1-naphthylisothiocyanate hepatotoxicity, enhanced 1-naphthyl isocyanate and 1-naphthylamine formation. It is suggested that 1-naphthyl isocyanate, 1-naphthylamine and the highly reactive sulfur released from 1-naphthylisothiocyanate might be involved in the hepatotoxic effect of 1-naphthylisothiocyanate.     

Sulfotransferase 1A1 (SULT1A1) polymorphism and susceptibility to primary brain tumors

Purpose Sulfotransferase 1A1 is a member of sulfotransferase family that plays an important role in the biotransformation of numerous carcinogenic and mutagenic compounds through sulfation. The present study has investigated the association between SULT1A1 polymorphism and primary brain tumor incidence. Methods SULT1A1 genotypes were successfully detected using the PCR-RFLP assay in 60 primary brain tumor patients and 156 hospital-based healthy control individuals with no history of cancer or precancerous disorder. Results There was a significant difference in genotypes distribution (GG vs. GA + AA) between brain tumor patients (GG genotype frequency = 48.3%) and control population (GG genotype frequency = 65.4%; OR = 2.019, 95% CI = 1.103–3.695; P = 0.022). In order to determine the association between SULT1A1 polymorphism and specific types of brain tumors, the patients were classified according to the type of brain tumors they suffer from: glial and non-glial. Results of the statistical analyses of each group of patients in comparison with the control individuals showed a significant difference only between SULT1A1 polymorphism and non-glial brain tumors (OR = 2.615; 95% CI = 1.192–5.739; P = 0.014) but glial tumors (OR = 1.535; 95% CI = 0.688–3.425; P = 0.293). When non-glial tumors were classified as meningiomal and others (pituitary adenoma, craniopharyngioma, acoustic neuroma and hemangioblastoma), statistical analysis showed that this significance is only due to the meningiomal tumors (OR = 3.238; CI = 1.205–8.704; P = 0.015). We also estimated a reduced risk of brain tumor in non-smokers (OR = 1.700; CI = 0.800–3.615) in comparison to smokers (OR = 2.773; CI = 0.993–7.749), but this was not statistically significant. Conclusion Our findings have suggested that there was a significant association between brain tumor and SULT1A1*2 allele (A allele that is also known as His allele) and this allele is an important risk factor in the development of meningiomal brain tumors.     

The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1)

Ezetimibe is a potent inhibitor of cholesterol absorption that has been approved for the treatment of hypercholesterolemia, but its molecular target has been elusive. Using a genetic approach, we recently identified Niemann-Pick C1-Like 1 (NPC1L1) as a critical mediator of cholesterol absorption and an essential component of the ezetimibe-sensitive pathway. To determine whether NPC1L1 is the direct molecular target of ezetimibe, we have developed a binding assay and shown that labeled ezetimibe glucuronide binds specifically to a single site in brush border membranes and to human embryonic kidney 293 cells expressing NPC1L1. Moreover, the binding affinities of ezetimibe and several key analogs to recombinant NPC1L1 are virtually identical to those observed for native enterocyte membranes. KD values of ezetimibe glucuronide for mouse, rat, rhesus monkey, and human NPC1L1 are 12,000, 540, 40, and 220 nM, respectively. Last, ezetimibe no longer binds to membranes from NPC1L1 knockout mice. These results unequivocally establish NPC1L1 as the direct target of ezetimibe and should facilitate efforts to identify the molecular mechanism of cholesterol transport.