牛磺酸脱氧胆酸诱导HepG2细胞凋亡及坏死的研究

目的:探讨牛磺酸脱氧胆酸(TDCAO)对HepG2细胞凋亡和坏死的影响。方法:应用台盼蓝拒染试验判断细胞坏死,应用HE染色、Hoechst 33 258染色、电镜和DNA电泳对细胞凋亡定性;应用流式细胞仪对细胞凋亡定量。结果:TDCA 200 μmol/L孵育15 h可抑制细胞生长及增殖;400 μmol/L孵育 12 h可诱导显著 HepG2细胞凋亡,凋亡率为50.35±2.20％;600μmol/L孵育6 h即显著抑制细胞生长和增殖,随孵育时间延长脱落坏死细胞明显增多;800μmol/L细胞不能增殖,孵育6 h即可引起细胞坏死。结论:TDCA与HepC2细胞孵育时,即可引起细胞坏死,又可诱导细胞凋亡,取决于浓度及孵育时间的长短;在低浓度,TDCA对细胞的毒性主要表现在为抑制细胞生长增殖和诱导细胞凋亡,在高浓度则以引起细胞坏死为主。     

胆汁酸对胃癌细胞株MKN-28生长的影响

胆汁反流是常见现象，其对胃上皮细胞生长的影响尚不明确。目的：探讨胆汁酸体外对胃癌细胞株MKN-28生长的影响。方法：采用Mr丌法测定不同浓度牛磺石胆酸钠（TLC）和甘氨鹅脱氧胆酸钠（GCDC）对胃癌细胞株MKN．28生长的影响。结果：第1-6d，60-300μmol／LTLC可明显抑制MKN-28细胞生长，且呈剂量依赖性。300μmol／LGCDC干预24h可显著抑制MKN-28细胞生长。结论：脂溶性胆汁酸TLC和水溶性胆汁酸GCDC体外均可直接抑制胃癌细胞生长。     

牛磺酸熊脱氧胆酸对细胞色素C介导HepG2细胞凋亡的作用

目的 探讨牛磺酸熊脱氧胆酸(TUDCA)对牛磺酸脱氧胆酸(TDCA)诱导HepG2细胞凋亡阻抑作用及分子机制。 方法 应用Hoechst 3325 8染色、电镜和DNA电泳对细胞凋亡进行定性;应用流式细胞仪对凋亡细胞进行定量;检测TDCA单独孵育及与TUDCA共孵育时,胞浆中细胞色素C含量及Caspase-3、8、9蛋白酶活性的变化。 结果 TDCA 400μmol/L孵育12 h可诱导显著HepG2细胞凋亡,凋亡率为(50.35±2.20)％,细胞经TUDCA与TDCA共孵育后,细胞凋亡率明显下降,为(13.78±0.84)％;TUDCA能显著抑制TDCA引起的细胞色素C释放及Caspase-9、3蛋白酶活性升高,孵育12 h时,Caspase-3活性分别下降54.9％(t=16.88,P<0.01)和52.5％(t≥13.00,P<0.01),轻度降低Caspase-8活性升高,活性下降24.5％(t=1.94,P>0.05)。结论 稳定线粒体膜、阻止细胞色素C释放及随后Caspase-9、Caspase-3活化,是TUDCA抗凋亡的主要机制。抗凋亡作用可能是TUDCA治疗胆汁淤积性肝病取得显著疗效的重要机制之一。     

PARP-1在脱氧胆酸钠促结肠癌细胞增殖中的作用

结肠癌的发生与肠腔内脱氧胆酸引起的DNA损伤有关,聚腺苷二磷酸核糖聚合酶-1（PARP-1）对DNA损伤具有修复作用.目的：探讨PARP-1在脱氧胆酸钠促结肠癌细胞增殖中的作用及其可能机制.方法：选用不同浓度的脱氧胆酸钠、PARP-1抑制剂5-氨基异喹啉酮（5-AIQ）或两者联合分别作用于人结肠腺癌细胞株HT-29.MTT实验检测细胞增殖情况,流式细胞仪检测细胞周期和细胞凋亡,免疫细胞化学染色检测环氧合酶-2（COX-2）、caspase-3、PARP-1蛋白表达.结果：10～50 μmol/L脱氧胆酸钠能促进HT-29细胞增殖,增加COX-2、PARP-1蛋白表达,减低caspaae-3蛋自表达.100μmol/L 5-AIQ单独作用对HT-29细胞增殖无明显影响,但能减低COX-2、PARP-1蛋白表达.与单独作用相比,10μmol/L脱氧胆酸钠与100 μmol/L 5-AIQ联合能显著抑制HT-29细胞生长,阻滞细胞周期,诱导细胞凋亡,COX-2、PARP-1蛋白表达减低,caspase-3蛋白表达无明显变化.结论：COX-2与PARP-1在脱氧胆酸钠促结肠癌细胞增殖的过程中可能存在相互作用,PARP-1抑制剂5-AIQ可能用于预防脱氧胆酸诱发的结肠癌.     

熊去氧胆酸诱导肝癌细胞株HepG2凋亡及对Survivin、Caspase-3表达的影响

目的探讨熊去氧胆酸（UDCA）对肝癌细胞株HepG2增殖、凋亡及Survivin、Caspase-3表达的影响。方法UDCA孵育细胞后，分别采用MTT法、流式细胞仪、激光共聚焦显微镜、半定量RT-PCR法等观测相关指标。结果UDCA能抑制HepG2增殖，诱导其凋亡，并具有浓度、时间依赖性。随着Survivin表达下调，Caspase-3表达上调（r=-0．9959，P〈0．01）、细胞增殖抑制（r=0．9883，P〈0．05）、凋亡指数增加（r=-0．9521，P〈0．05）。结论UDCA可能通过干预Survivin的表达，减弱后者对Caspase-3的负调控作用，而影响肝癌细胞的增殖、凋亡。     

脱氧胆酸调节环氧合酶-2对结肠癌细胞增殖的影响

目的 通过观察不同浓度脱氧胆酸对结肠腺癌细胞SW1116生长的效应，以及在相应状态下细胞内环氧合酶(COX)-2蛋白表达量的改变，探讨脱氧胆酸钠对结肠癌细胞的作用机制。方法 用MTT法测定细胞增殖活性；免疫组化及Western blot方法检测细胞内COX-2蛋白的表达。结果 10～100μmol／L的脱氧胆酸钠具有明显的促进结肠腺癌细胞生长的作用；大于100μmol／L时则表现出抑制作用。脱氧胆酸钠在10、50和100μmol／L的浓度下均可促进COX-2蛋白的表达，10μmol／L的效应可以持续72h，但后两者在48h后COX-2蛋白表达开始下降。结论 脱氧胆酸对结肠癌细胞SW1116增殖的影响呈双向调节作用，脱氧胆酸促进COX-2蛋白表达可能是其促进结肠癌细胞增殖的作用途径。     

葡萄冻干粉在线粒体应激介导肝细胞凋亡中的作用

目的 了解葡萄冻干粉（FDG）对线粒体应激介导的肝细胞凋亡的阻抑作用,探讨其治疗肝细胞损伤的作用机制。方法用牛磺酸脱氧胆酸（TDCA）诱导Huh7细胞,建立线粒体应激凋亡模型,用不同剂量FDG进行干预。通过MTT、DNA ladder、Western blot、细胞凋亡率检测等方法,了解FDG对Huh7细胞生长、对TDCA诱导的Huh7细胞的凋亡率、凋亡信号蛋白表达的影响。结果FDG（2μg／ml、20μg／ml、200μg／ml、4（10μg／ml和800μg／ml）与Huh7细胞共孵育,FDG可提高Huh7细胞的活力,以FDG400μg／ml时细胞活力最高。用400pMTDCA诱导Huh7细胞,随着诱导时间延长细胞活力下降、凋亡率增加,出现凋亡条带;Caspase-9和7蛋白酶原被活化,PCNA表达下调。用FDG干预TDCA诱导Huh7细胞48h后发现,FDG明显提高Huh7细胞活力、使细胞凋亡减少、凋亡信号蛋白的表达减少。结论TDCA能触发Huh7细胞线粒体氧化应激凋亡,而FDG能促进细胞生长,减轻肝细胞损伤,阻断线粒体氧化应激介导的肝细胞凋亡。     

人参皂甙Rg1和Rb1对培养大鼠室管膜前下区神经干细胞谷氨酸兴奋毒性的保护作用及其与STAT3表达的关系

目的：探讨人参皂甙Rg1和Rb1对大鼠室管膜前下区神经干细胞（SVZa NSCs）谷氨酸兴奋毒性的保护作用及其与STAT3表达的关系。方法：培养的胚胎大鼠SVZaNSCs用含Rg1或Rb1的培养液孵育24h后暴露于谷氨酸（500μmol／L）1h。用台盼蓝和MTT染色检测细胞存活率,TUNEL法检测细胞凋亡率,免疫细胞化学法检测STAT3表达。结果：人参皂甙Rg1（40μmol／L）和Rb1（40μmol／L）能显著提高SVZaNSCs存活率,增加STAT3表达。结论：人参皂甙Rg1和Rb1对SVZa NSCs谷氨酸兴奋毒性有保护作用,其机制可能与STAT3表达增加有关。     

熊脱氧胆酸、腺苷蛋氨酸、地塞米松治疗妊娠合并肝内胆汁淤积症疗效比较

目的评估熊脱氧胆酸（UDCA）、腺苷蛋氨酸（SAMe）、地塞米松（DEX）治疗妊娠期肝内胆汁淤积症（ICP）的效果。方法选择90例ICP患者，随机分成3组：A组采用UDCA 250mg/次，4次／d；B组采用SAMe 1．0g加5％葡萄糖溶液500ml静滴，1次／d；C组口服地塞米松10mg／次，1次／d，10d后减量，每2d减去原药量的1／3。3组患者均观察1个疗程（14d），比较3组患者的疗效。结果治疗后3组患者瘙痒症状评分间差异有显著性意义（P〈0．01）；血清甘胆酸、ALT、TBiL、DBiL含量间的差别有显著性意义（P〈0．05）。妊娠结局：A组新生儿早产率、羊水粪染率与B组、C组间差别无显著性意义（P〉0．05）。结论UDCA是治疗ICP的安全有效的方法，可改善妊娠预后。     

尾加压素Ⅱ对ox-LDL诱导的EVC-304细胞凋亡的影响

目的探讨尾加压素Ⅱ(UⅡ)对ox-LDL诱导的EVC-304细胞凋亡的影响,并探讨其可能机制。方法体外培养人脐静脉内皮细胞系ECV304,将不同浓度UⅡ及ox-LDL与内皮细胞共同孵育24 h后,应用倒置显微镜观察细胞形态变化,四甲基偶氮唑蓝(MTT)法检测内皮细胞活性,TUNEL、流式细胞仪检测内皮细胞凋亡,免疫组化法检测内皮细胞凋亡基因bcl-2,bax,caspase-3蛋白表达水平。结果 ox-LDL(20μg/ml)处理24 h后,可诱导内皮细胞凋亡;同时给予不同浓度的UⅡ(20μg/ml ox-LDL+10-10mol/L UⅡ,20μg/ml ox-LDL+10-9mol/L UⅡ,20μg/ml ox-LDL+10-8mol/L UⅡ,20μg/ml ox-LDL+10-7mol/L UⅡ)处理24 h后,可显著增强ox-LDL诱导内皮细胞凋亡作用,并呈浓度依赖性。ox-LDL处理内皮细胞24 h后,bcl-2蛋白阳性表达率降低,而bax、caspase-3阳性表达率升高。同时给予UⅡ可增强促凋亡作用,与ox-LDL组比较,ox-LDL+UⅡ组的bcl-2蛋白阳性表达率显著降低(P0.05),而bax、caspase-3阳性表达率显著升高(P0.05)。结论 UⅡ可增强ox-LDL诱导的内皮细胞凋亡,并呈浓度依赖性,其作用可能与bcl-2、bax、caspase-3调节有关。     

核糖核酸治疗慢性肝炎肝硬化的临床和病理研究

目的:应用核糖核酸(RNA)对慢性肝炎、肝硬化进行抗肝纤维化治疗,以改善肝脏组织学指标,恢复肝功能。方法:用RNA治疗慢性肝炎、肝硬化83例,与辅酶Q_(10)治疗的43例对照,疗程3个月。结果:RNA提高血清白蛋白、降低血清谷丙转氨酶(ALT)及血清透明质酸(HA)含量的疗效明显优于对照组。治疗前后2次肝活检光镜和电镜检查对比观察,RNA治疗后肝脏炎症、坏死程度和胶原纤维定量所反映的肝纤维化程度均明显减轻,对照组则无变化,其纤维化程度反而加重。统计学上两组有明显差异。临床与病理结果一致。结论:RNA确有保护肝细胞和抗肝纤维化作用。     

Serum sialic acid levels are increased during relapse to alcohol drinking: a pilot study

BACKGROUND: Sialic acid has been suggested to be a potential marker for alcohol abuse. A previous study showed that sialic acid levels were significantly higher in serum among alcoholics as compared with social drinkers. In addition, serum sialic acid concentrations decreased after a treatment program aiming at abstinence. In this study, sialic acid was investigated as a possible marker for relapse to alcohol drinking. METHODS: Serum from alcohol-dependent patients in outpatient treatment ( n = 8) was analyzed for sialic acid by a colorimetric assay. A baseline sample was taken when the subject had been abstinent for longer than 4 weeks. A second sample was taken after relapse within 3 days after cessation of drinking. A relapse was defined as two or more days with daily drinking of more than 60 g of pure alcohol. RESULTS: The sialic acid levels were significantly increased by 21% (median; range, 6-33%; < 0.01; n = 8) after a relapse as compared with the level after 4 weeks or longer of abstinence. CONCLUSIONS: This study suggests that serum sialic acid levels are significantly increased even after a short period of heavy drinking and may be a potential marker for relapse.     

Development of alcoholic fatty liver and fibrosis in rhesus monkeys fed a low n-3 fatty acid diet

BACKGROUND: The amount and type of dietary fat seem to be important factors that modulate the development of alcohol-induced liver steatosis and fibrosis. Various alcohol-feeding studies in animals have been used to model some of the symptoms that occur in liver disease in humans. METHODS: Rhesus monkeys (Macaca mulatta) were maintained on a diet that had a very low concentration of alpha-linolenic acid and were given free access to an artificially sweetened 7% ethanol solution. Control and ethanol-consuming animals were maintained on a diet in which the linoleate content was adequate (1.4% of energy); however, alpha-linoleate represented only 0.08% of energy. Liver specimens were obtained, and the fatty acid composition of the liver phospholipids, cholesterol esters, and triglycerides of the two groups were compared at 5 years and histopathology of tissue samples were compared at 3 and 5 years. RESULTS: The mean consumption of ethanol for this group over a 5-year period was 2.4 g.kg.day. As a consequence of the ethanol-dietary treatment, there were significantly lower concentrations of several polyunsaturated fatty acids in the liver phospholipids of the alcohol-treated group, including arachidonic acid and most of the n-3 fatty acids and particularly docosahexaenoic acid, when compared with dietary controls. Liver specimens from animals in the ethanol group at 5 years showed a marked degree of steatosis, both focal and diffuse cellular necrosis, and an increase in the development of fibrosis compared with specimens obtained at 3 years and with those from dietary controls, in which there was no evidence of fibrotic lesions. CONCLUSION: These findings suggest that the advancement of ethanol-induced liver disease in rhesus monkeys may be modulated by the amount and type of dietary essential fatty acids and that a marginal intake of n-3 fatty acids may be a permissive factor in the development of liver disease in primates.     

Formic acid, a novel metabolite of chronic ethanol abuse, causes neurotoxicity, which is prevented by folic acid

Background: Methanol is endogenously formed in the brain and is present as a congener in most alcoholic beverages. Because ethanol is preferentially metabolized over methanol (MeOH) by alcohol dehydrogenase, it is not surprising that MeOH accumulates in the alcohol‐abusing population. This suggests that the alcohol‐drinking population will have higher levels of MeOH’s neurotoxic metabolite, formic acid (FA). FA elimination is mediated by folic acid. Neurotoxicity is a common result of chronic alcoholism. This study shows for the first time that FA, found in chronic alcoholics, is neurotoxic and this toxicity can be mitigated by folic acid administration. Objective: To determine if FA levels are higher in the alcohol‐drinking population and to assess its neurotoxicity in organotypic hippocampal rat brain slice cultures. Methods: Serum and CSF FA was measured in samples from both ethanol abusing and control patients, who presented to a hospital emergency department. FA’s neurotoxicity and its reversibility by folic acid were assessed using organotypic rat brain hippocampal slice cultures using clinically relevant concentrations. Results: Serum FA levels in the alcoholics (mean ± SE: 0.416 ± 0.093 mmol/l, n = 23) were significantly higher than in controls (mean ± SE: 0.154 ± 0.009 mmol/l, n = 82) (p < 0.0002). FA was not detected in the controls’ CSF (n = 20), whereas it was >0.15 mmol/l in CSF of 3 of the 4 alcoholic cases. Low doses of FA from 1 to 5 mmol/l added for 24, 48 or 72 hours to the rat brain slice cultures caused neuronal death as measured by propidium iodide staining. When folic acid (1 μmol/l) was added with the FA, neuronal death was prevented. Conclusions: Formic acid may be a significant factor in the neurotoxicity of ethanol abuse. This neurotoxicity can be mitigated by folic acid administration at a clinically relevant dose.     

Normal lysosomal enzyme levels in hypertrophied and failing dog hearts

The activity levels of three lysosomal acid hydrolases, acid phosphatase, acid ribonuclease, and cathepsin, were determined in whole homogenates of right and left ventricular myocardium from normal and chronically stressed dog hearts. Stressed hearts were from four dogs with right ventricular hypertrophy and congestive failure induced by progressive constriction of the main pulmonary artery and from four dogs with acquired right ventricular hypertrophy secondary to pulmonary hypertension resulting from infestation with heartworms (Dirofilaria immitis). Control hearts were from four unoperated and four sham operated normal dogs. No significant differences were found that could be related to myocardial stress.     

Acid Secretory Potency and Elimination of the 15-Leucine Gastrin-17 Analogue in Man

The effect on gastric acid secretion and the elimination of a commercially available synthetic human gastrin analogue (15-Leucine Synthetic Human Gastrin I, 15-LSG) were studied in six healthy human subjects. On a molar basis acid secretory efficacy was approximately four times greater than pentagastrin and secretory potency ten times greater. Elimination of 15-LSG was bi-exponential, with half-lives of 5.2 ± 0.8 and 25.9 ± 7.0 min. Metabolic clearance rate was 6.9 ± 0.4 ml/kg-min and volume of distribution 52 ml/kg.     

Quantitation of the Mass of Fatty Acid Ethyl Esters Synthesized by Hep G2 Cells Incubated with Ethanol

Fatty acid ethyl esters (FAEE), esterification products of fatty acids and ethanol, have been increasingly implicated as mediators of ethanol-induced organ damage. The first goal of this study was to determine the mass of FAEE synthesized by Hep G2 cells exposed to a given dose of ethanol. The second goal was to determine whether all fatty acids in cells are equally available for FAEE synthesis. Hep G2 cells and essential fatty acid deficient Hep G2 cells (Hep G2-EFD) were used to study the synthesis of FAEE upon exposure to ethanol. A two-pool fatty acid model was created: (1) a "previously incorporated pool" formed by incubating the cells with ¹⁴C-labeled fatty acids for 24 hr; and (2) a "newly incorporated pool" formed by incubating cells with ³H-labeled fatty acids for 0.5 hr. The FAEE production from each pool was then determined. The total production of FAEE within 3 hr by Hep G2 cells in culture was 150 to 250 pmol/mg cell protein. The fatty acids most recently incorporated into the cells were preferred as substrates for FAEE synthesis because a higher percentage of fatty acids from the newly incorporated pool was used for FAEE synthesis than from the previously incorporated pool. Furthermore, a dose-response relationship was observed between the amount of fatty acid in the newly incorporated pool and FAEE production, but not between the amount of fatty acid in the previously incorporated pool and FAEE synthesis. Taken together, the results indicate that a relatively small amount of endogenously synthesized FAEE is generated from specific intracellular pools of fatty acid since not all fatty acids are equally available for FAEE synthesis. This indicates that if endogenous FAEE are toxic, they exert their toxic effect at very low intracellular FAEE concentrations.     

烟酸对实验性动脉粥样硬化兔主动脉骨桥蛋白表达的影响

目的 探讨烟酸对动脉粥样硬化兔主动脉壁骨桥蛋白表达的影响.方法 16只雄性新西兰大白兔给予高脂饮食8周后,随机分为高脂血症组与烟酸组:高脂血症组(n=8)继续饲以高脂饲料6周;烟酸组(n=8)在高脂饮食基础上给予烟酸[200 mg/(kg·d)]6周.另选8只兔给予普通饮食14周作为正常对照组.14周末处死动物进行主动脉病理学检测,采用免疫组织化学染色检测兔主动脉壁骨桥蛋白的表达水平,采用实时定量PCR检测各组兔主动脉壁骨桥蛋白mRNA的表达.结果 与正常对照组相比,高脂血症组主动脉内膜厚度和斑块面积显著增加,骨桥蛋白和mRNA表达显著增加(P＜0.01).烟酸组主动脉内膜和斑块面积显著缩小,骨桥蛋白和mRNA表达显著减少(均P＜0.01).相关性分析显示:骨桥蛋白表达量与动脉粥样硬化斑块面积(r=0.821,P＜0.01)及内膜厚度(r =0.818,P＜0.01)均呈正相关;骨桥蛋白mRNA表达量与动脉粥样硬化斑块面积(r=0.888,P＜0.01)及内膜厚度(r =0.874,P＜0.01)也均呈正相关.结论 烟酸抗动脉粥样硬化作用除与其降脂作用有关外,还可能与其降低骨桥蛋白和mRNA水平有关.     

The importance of vitamin C for hydroxylation of vitamin D3 to 1,25(OH)2D3 in man

Summary The effects of vitamin C on 1,25(OH)2D3 synthesis in humans were evaluated; the study included 20 females. They were divided into 2 groups. The first of the 10 subjects (age range 55–71) received ascorbic acid at a dose of 150 mg/die i.v. for 10 days; the second 10 subjects (age range 55–69) received a placebo i.v. for 10 days. In a later study (after a 30-day washout) the same two groups were tested for the second time with ascorbic acid at a dose of 1,000 mg/die i.v. for 10 days and placebo i.v. for 10 days. Serum calcium and phosphorus, serum Ca++, serum proteins, blood and urinary pH, serum 25(OH)D3 and 1,25(OH)2D3, serum PTH, urinary hydroxyprolin were tested before and after the treatments. In the first study a significant increase in serum 1,25(OH)2D3 was observed after ascorbic acid while no significant variation was observed for the other parameters. In the second study, a significant increase in serum Ca++ and a significant decrease in serum 1,25(OH)2D3 were observed after ascorbic acid while no significant variation was observed for the other parameters. The authors conclude that ascorbic acid promotes 1,25(OH)2D3 synthesis at a paraphysiologic dose (150 mg/die) in humans but this synthesis is inhibited at higher doses (1,000 mg/die). The latter effect by Ca++ or by an effect of ascorbate on 1 alpha-hydroxylase enzyme could be mediated.