烟酸通过下调PCSK9的表达促进HepG2细胞摄取LDL-C

目的探讨烟酸对HepG2细胞摄取及代谢LDL-C的影响,寻找烟酸改善血脂异常,减缓动脉粥样硬化进程的相关分子机制,为其临床用药提供指导。方法采用油红O染色观察细胞内脂质蓄积;酶法检测细胞内胆固醇含量;荧光流式检测细胞表面LDLR丰度分布;qPCR及Western blot检测LDLR、SREBP2以及PCSK9的mRNA及蛋白含量变化。结果经烟酸处理的HepG2细胞,油红O阳性细胞数及细胞内红染脂滴增多,细胞内TC、FC水平明显增高(P<0.05);LDLR mRNA含量无差异(P>0.05),而细胞膜表面LDLR分布丰度及细胞内LDLR的含量明显增加(P<0.05);烟酸对SREBP2的表达无影响,却能明显下调PCSK9的表达。结论烟酸可能通过下调PCSK9的表达,减少PCSK9成熟体蛋白含量,使得LDLR降解减少,进而增加LDLR含量,促进HepG2细胞摄取LDL-C。     

黄连素通过对高脂小鼠肝脏、胰腺中PCSK9和LDLR的不同调控改善高脂血症和高胰岛素血症

目的 观察黄连素对高脂小鼠肝脏、胰腺中PCSK9和LDLR的不同调控，探讨其减缓高脂血症和高胰岛素血症的新作用机制。方法 SPF级C57BL/6J小鼠随机分为3组：正常对照组(普通饮食喂养)、高脂饲料组(高脂饮食喂养)、黄连素组(高脂饮食喂养同时灌胃黄连素)。自动血糖仪检测空腹血糖值；酶联免疫法(ELISA)测空腹血清胰岛素水平，计算血糖与胰岛素比值。ELISA测空腹血清PCSK9水平；酶标仪和相应的试剂盒检测血清中的甘油三酯、总胆固醇、高密度胆固醇、低密度胆固醇的含量。HE染色法观察肝脏和胰腺的组织形态变化。Western blotting检测肝脏和胰腺中PCSK9和LDLR蛋白的表达。结果 黄连素能降低血清中胆固醇的含量，同时明显降低血浆中PCSK9的含量，减少肝脏PCSK9蛋白表达，增加肝脏表面LDLR蛋白表达；改善高脂诱导的胰岛素抵抗，减少胰岛细胞肥大及空泡数量，增加胰腺表面PCSK9蛋白表达，减少胰腺表面LDLR蛋白表达。结论 黄连素调节高脂饮食小鼠PCSK9的表达和分泌，具有组织特异性，在肝脏中增强LDLR介导的胆固醇细胞摄取，同时在胰腺中抑制LDLR介导的胆固醇摄取，从而改善高脂饮食诱导的肝脏脂肪性病变和胰岛素抵抗。     

孔石莼多糖及其硫酸酯化衍生物降血脂机制的体外研究

目的 通过研究不同浓度孔石莼多糖(U)及硫酸酯化孔石莼多糖(HU)对人肝癌细胞HepG2中内源性低密度脂蛋白受体(LDLR)表达和LDLR mRNA转录的影响,探讨U和HU在受体及基因水平上的调脂作用机制。方法 利用Western-Blotting技术测定U和HU对HepG2细胞LDLR表达量的影响,采用荧光定量PCR技术分析不同浓度的U和HU对人肝癌细胞HepG2 LDLR mRNA表达的影响。结果HepG2细胞经不同浓度的U和HU作用后,U和HU用药组的LDLR表达量显著高于空白对照组(P<0.05),证明从海藻孔石莼中提取的U和HU可显著提高人肝癌细胞HepG2中LDLR的表达水平。同时,HU药物组的LDLR表达量明显高于U药物组(P<0.05)。U和HU用药组其LDLR mRNA的含量与空白对照组相比均明显升高(P＜0.05)。其中U药物组(0.1 mg/mL)对HepG2细胞中LDLR mRNA的升高作用最明显,与空白对照组相比提高了约7.72倍。HU药物组(0.1 mg/mL)对HepG2细胞中LDLR mRNA的升高作用弱于U药物组(0.1 mg/mL),与空白对照组相比提高了约3.89倍。结论 U和HU可能通过促进HepG2细胞中LDLR mRNA,增加肝细胞LDLR蛋白表达量,从而降低LDL浓度。     

FGF-21通过上调肝脏LDL受体的表达降低血浆中LDL水平

目的成纤维细胞生长因子-21(FGF-21)可降低实验动物血浆中的低密度脂蛋白(LDL)的浓度,但其作用机制尚不清楚,本实验试图通过研究FGF-21对LDLR的调节作用揭示FGF-21调节血浆LDL浓度的机制。方法向谷氨酸钠(MSG)肥胖大鼠连续注射FGF-21蛋白40d后,检测其血浆中LDL的变化,并用荧光定量PCR的方法结合免疫荧光检测FGF-21对肝脏组织中LDLR mRNA及蛋白的影响。结果 MSG肥胖鼠经FGF-21处理,血浆内的LDL降低18%,其肝脏组织中LDLR mRNA含量提高9倍;HepG2细胞内的LDLR mRNA表达量随FGF-21处理时间增加而提高,且呈剂量依赖性;流式细胞仪检测显示,经FGF-21处理后HepG2细胞表面LDLR蛋白数量增加。结论 FGF-21通过增加肝细胞的LDLR表达量从而降低血浆中LDL浓度。     

PCSK9抑制剂的研究进展

前蛋白转化酶枯草溶菌素9(PCSK9)通过促进低密度脂蛋白受体LDLR降解来调节血浆低密度脂蛋白胆固醇(LDL-C)代谢。抑制PCSK9可以显著降低血浆LDL-C水平,因此PCSK9作为新的降脂靶点受到越来越多的关注。本文对新降脂靶点PCSK9及其抑制剂最新研发现状及临床研究进展进行综述。     

白藜芦醇对高血脂模型小鼠的预防作用和对模型金黄地鼠的改善作用研究

目的:研究白藜芦醇对高血脂模型小鼠的预防作用和对模型金黄地鼠(以下简称地鼠)的改善作用。方法:以高脂高胆固醇饲料饲养诱导高血脂动物模型。将小鼠和地鼠均分为模型组和白藜芦醇低、中、高剂量组(小鼠给予40、80、160 mg/kg,地鼠给予25、50、100 mg/kg),每组10只,同时设立10只相应动物为正常对照组。除正常对照组动物喂食正常饲料和ig生理盐水外,其余小鼠在喂食高脂高胆固醇饲料的同时ig相应药物;其余地鼠先建模2周,成模后再ig相应药物,给药期间继续喂食高脂高胆固醇饲料。持续给药4周,每周检测各组小鼠和地鼠血清中总胆固醇(TC)和低密度脂蛋白胆固醇(LDL-C)水平,给药4周后检测各组小鼠和地鼠血清中前蛋白转化酶枯草溶菌素9(PCSK9)mRNA及蛋白、微小RNA-27a(miRNA-27a)表达,以及小鼠肝组织中低密度脂蛋白受体(LDLR)蛋白表达。结果:与正常对照组比较,模型组小鼠从给药2周后开始血清中TC、LDL-C、PCSK9 mRNA及蛋白、miRNA-27a水平均明显升高(P<0.05),肝组织中LDLR蛋白水平明显降低(P<0.05);模型组地鼠给药期间血清中TC、LDLC、PCSK9 mRNA及蛋白、miRNA-27a水平均明显升高(P<0.05)。与模型组比较,各给药组小鼠和地鼠上述指标均明显改善(P<0.05),且与剂量呈正相关。结论:白藜芦醇对高血脂模型小鼠有预防作用,对模型地鼠有改善作用。其机制可能是通过下调血清中miRNA-27a表达与PCSK9蛋白表达,进而升高肝组织中LDLR蛋白水平,最终降低血清中LDL-C水平。     

芒果苷单钠盐对高胆固醇血症大鼠肝脂质代谢及PCSK9/LDLR途径的影响

目的 观察芒果苷单钠盐对高胆固醇血症大鼠肝脂质代谢及前蛋白转化酶枯草溶菌素9(PCSK9)/低密度脂蛋白受体(LDLR)的作用。方法 选取SD大鼠40只,将其按体质量随机分为对照组、模型组、阿托伐他汀钙片组、芒果苷单钠盐组,每组10只。除对照组外,其余各组采用高脂饲料喂养法建立高胆固醇血症模型。造模成功后,阿托伐他汀钙片组ig阿托伐他汀钙片5 mg/kg,芒果苷单钠盐组ig 80 mg/kg芒果苷单钠盐,给药8周后,观察各组体质量及肝脏指数变化,生化法检测各组血脂指标[三酰甘油(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)],肾功能、血清酶指标[肌酐(CRE)、血清尿素(UREA)、肌酸激酶(CK)、乳酸脱氢酶(LDH)]及肝组织游离胆固醇(FC)、总胆汁酸(TBA)的含量,苏木精-伊红染色(HE)观察大鼠肝组织形态学变化,RT-PCR检测肝组织中白细胞介素-1β(IL-1β)和CD68 mRNA表达,Western blotting及RT-PCR检测肝组织中PCSK9、LDLR的蛋白及mRNA表达。结果 与模型组相比,各给药组大鼠体质量和肝脏指数明显降低(P<0.05);血清中TC、LDL-C及CRE、UREA、CK、LDH水平显著降低,HDL-C水平显著升高(P<0.05);肝组织中FC含量降低,TBA含量升高(P<0.05);肝脏脂肪变性改善明显;肝组织I L-1β、CD68 mRNA表达明显减少(P<0.05),同时显著降低了PCSK9的蛋白和mRNA水平,提升了LDLR的蛋白和mRNA水平(P<0.05)。结论 芒果苷单钠盐可有效调节高胆固醇血症大鼠肝胆固醇代谢,减轻高脂造成的心肾功能损害,其作用可能与调节肝脏PCSK9/LDLR信号通路的表达,改善脂质代谢紊乱相关。     

硫化氢对动脉粥样硬化胆固醇代谢的调节作用

目的观察H2S对动脉粥样硬化(AS)胆固醇代谢的影响,并探究其作用机制。方法用高脂饲料结合维生素D3的方法建立AS大鼠模型,并给与腹腔注射NaHS 56μmol·kg-1.d-1,持续12周。检测血脂水平及肝胆固醇含量。HepG2细胞给与NaHS 100μmol·L-16 h后,用RT-PCR方法检测羟甲基戊二酰辅酶A还原酶(HMGCoA reductase)和低密度脂蛋白受体(LDLR)mRNA表达。结果 NaHS组血清总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-Ch)和低密度脂蛋白胆固醇(LDL-Ch)均较模型组降低,肝胆固醇含量下降。NaHS使HepG2细胞HMGCoA Re-ductase mRNA表达下降,LDLR mRNA表达增加。结论H2S对AS胆固醇代谢具有调节作用,能下调肝HMGCoAReductase,使胆固醇合成下降;上调肝脏LDLR表达,降低血清总胆固醇水平。     

姜黄素对293细胞固醇调控报告系统的作用

目的在人胚肾细胞系HEK-293中建立低密度脂蛋白受体基因表达的固醇调控报告系统,通过姜黄素的干预培养,从基因调控水平上阐明姜黄素降脂作用的机制。方法将绿色荧光蛋白报告基因构建在调控低密度脂蛋白受体基因表达的固醇调控元件的下游,经磷酸钙沉降法导入HEK-293细胞,在用含姜黄素的培养液中培养2 d后,用荧光显微镜及流式细胞仪对绿色荧光蛋白的表达情况进行定性定量分析。结果姜黄素诱导了绿色荧光蛋白的表达。结论姜黄素可以通过影响固醇调控元件的作用上调低密度脂蛋白受体基因的表达。     

银杏叶提取物GBE50对培养细胞内胆固醇代谢的影响

目的研究银杏叶提取物GBE50对不同培养细胞内胆固醇含量的影响,及对HepG2细胞内胆固醇代谢相关基因HMGCR和SREBF2表达的调节。方法采用MTT法观察了GBE50对HepG2细胞生长的影响,用胆固醇氧化酶终点法检测GBE50对培养细胞HepG2、HUVEC、SH-SY5Y总胆固醇含量的影响;用荧光实时定量PCR技术检测了药物处理后HMGCR和SREBF2基因表达水平在HepG2细胞中的改变。结果GBE50在不影响HepG2细胞的增殖的剂量下可不同程度降低培养细胞HepG2,HUVEC内总胆固醇含量。对HepG2细胞内总胆固醇的影响呈一定的剂量及时间-效应关系,同时可以调节其中HMGCR、SREBF2基因的表达。结论GBE50可有效降低部分培养细胞内总胆固醇的含量,部分胆固醇代谢相关基因表达量的改变可能是GBE50调节HepG2细胞内胆固醇水平的机制之一。     

Quantitative in vivo and in vitro sex differences in artemisinin metabolism in rat

1. The pharmacokinetics of the antimalarial compound artemisinin were compared in the male and female Sprague-Dawley rat after single dose i.v. (20 mg.kg) or i.p. (50 mg.kg) administration of an emulsion formulation. 2. Plasma clearance of artemisinin was 12.0 (95% confidence interval: 10.4, 13.0) l.h. kg in the male rat and 10.6 (95% CI: 7.5, 15.0) l.h. kg in the female rat suggesting high hepatic extraction in combination with erythrocyte uptake or clearance. Artemisinin half-life was 0.5 h after both routes of administration in both sexes. Values for plasma clearance and half-lives did not statistically differ between the sexes. 3. After i.p. administration artemisinin AUCs were 2-fold higher in the female compared with male rat (p 0.001). Artemisinin disappearance was 3.9-fold greater in microsomes from male compared with female livers and it was inhibited in male microsomes by goat or rabbit serum containing antibodies against CYP2C11 and CYP3A2 but not CYP2B1 or CYP2E1. 4. The unbound fraction of artemisinin in plasma was lower (p 0.001) in plasma obtained from the male (8.8 2.0%) compared with the female rat (11.7 2.2%). 5. The possibility of a marked sex difference, dependent on the route of administration, has to be taken into account in the design and interpretation of toxicological studies of artemisinin in this species.     

Quantitative in vivo and in vitro sex differences in artemisinin metabolism in rat

1. The pharmacokinetics of the antimalarial compound artemisinin were compared in the male and female Sprague-Dawley rat after single dose i.v. (20 mg x kg(-1)) or i.p. (50 mg x kg(-1)) administration of an emulsion formulation. 2. Plasma clearance of artemisinin was 12.0 (95% confidence interval: 10.4, 13.0) 1 x h(-1) x kg(-1) in the male rat and 10.6 (95% CI: 7.5, 15.0) 1 x h(-1) x kg(-1) in the female rat suggesting high hepatic extraction in combination with erythrocyte uptake or clearance. Artemisinin half-life was approximately 0.5 h after both routes of administration in both sexes. Values for plasma clearance and half-lives did not statistically differ between the sexes. 3. After i.p. administration artemisinin AUCs were 2-fold higher in the female compared with male rat (p < 0.001). Artemisinin disappearance was 3.9-fold greater in microsomes from male compared with female livers and it was inhibited in male microsomes by goat or rabbit serum containing antibodies against CYP2C11 and CYP3A2 but not CYP2B1 or CYP2E1. 4. The unbound fraction of artemisinin in plasma was lower (p < 0.001) in plasma obtained from the male (8.8 +/- 2.0%) compared with the female rat (11.7 +/- 2.2%). 5. The possibility of a marked sex difference, dependent on the route of administration, has to be taken into account in the design and interpretation of toxicological studies of artemisinin in this species.     

Theophylline kinetics in peripheral tissues in vivo in humans

Several biochemical and cellular effects have been described for methylxanthines under in vitro conditions. However, it is unknown, whether threshold concentrations required to exert these effects are attained in target tissues in vivo. We therefore employed the microdialysis technique for measuring theophylline concentrations in peripheral tissues under in vivo conditions.Following in vitro and in vivo calibration, microdialysis probes were inserted into the medial vastus muscle and into the periumbilical subcutaneous adipose layer of healthy volunteers. Following single oral dose administration of 300 mg or i.v. infusion of 240 mg theophylline, in vivo time courses of theophylline concentrations were monitored in tissues and plasma. Major pharmacokinetic parameters (c_max, t_max, AUC) were calculated for plasma and tissue time courses. The mean AUC_tissue /AUC_plasma-ratio was 0.56 (p.o.) and 0.55 (i.v.) for muscle and 0.55 (p.o.) and 0.72 (i.v.) for subcutaneous adipose tissue.We conclude that microdialysis provides important information on the distribution and the tissue pharmacokinetics of theophylline.Abbreviations FPIA Fluorescence polarisation immuno assay - AUC Area under the curve - t_max Time to peak concentration - c_max Peak concentration     

休克时血中氧自由基的变化

本实验测定10名休克患者血浆和红细胞的丙二醛(MDA)、血浆总抗的氧化活性(AOA)的含量。结果表明:休克病人红细胞膜和血浆 MDA 含量(4.298±0.722;5.348±0.834)与对照组(3.235±0.682;4.356±1.081)比较明显增高(P<0.05);血浆 AOA(39.65±7.858)与对照组(48.21±10.81)比较明显降低(P<0.01)。提示:休克时,患者机体内自由基反应增强是引起组织细胞损伤的原因之一。     

Interindividual variability in metabolic activation in humans in vivo

In assessing interindividual variability in metabolic activation, the toxic metabolite is often too unstable for conventional analysis. Possible alternatives include a stable product of the reactive metabolite e.g. cysteinyl derivatives of N-acetyl-4-benzoquinoneimine, the toxic metabolite of paracetamol, adducts with DNA or protein, and indirect measurement of the activity of the enzyme(s) producing the active metabolite. An example of the last approach is the use of furafylline, a highly specific inhibitor of human CYP1A2, to determine the extent of the metabolic activation of the cooked food mutagens PhIP and MeIQx. The extent of inhibition, determined from levels of unchanged amine in urine, is an indirect measure of the activity of the activation pathway. Further refinement of this approach, allied to improved measures of the biological process of interest should prove of value in evaluating interindividual variability and its role in the risk assessment process.     

Changes in angiopoietin expression in glomeruli involved in glomerulosclerosis in rats with daunorubicin-induced nephrosis

AIM: To study the potential pathological role of endogenous angiopoietins in daunorubicin-induced progressive glomerulosclerosis in rats. METHODS: Seventy male Wistar rats were allocated randomly into a daunorubicin group (DRB; n=40) or a control group (n=30). The rats in the DRB group were injected with DRB (15 mg/kg), in their tails. Subsequently, at intervals of 1, 2, 4, 6, 8, and 12 weeks, 5 male Wistar rats in each group were chosen randomly for 24 h urinary protein quantitative measurements (24 h UPQM), and determination of plasma tumor necrosis factor alpha (TNF-alpha), angiopoietin-1 (Ang1), and angiopoietin-2 (Ang2) levels. Kidney sections were examined by electron microscopy, Periodic Acid Schiff (PAS) staining, immunohistochemical staining and in situ hybridization histochemistry. RESULTS: As glomerulosclerosis progressed in the DRB group, expression of Ang1 mRNA and protein in glomeruli decreased and expression of TNF-alpha protein, Ang2 mRNA and protein in glomeruli increased. Expression of Ang1 mRNA and protein in glomeruli were negatively correlated with 24 h UPQM, Fn protein expression, and mean area of extracellular matrix (MAECM). In comparison, expression of Ang2 mRNA and protein in glomeruli were positively correlated with 24 h UPQM, Fn protein expression and MAECM; furthermore, there was a positive correlation between plasma Ang2 and 24 h UPQM. Plasma TNF-alpha and expression of TNF-alpha in glomeruli were positively correlated with expression of Ang2 mRNA and protein in glomeruli. There was a negative correlation between Ang1 protein expression and Ang2 protein expression in glomeruli. CONCLUSION: During DRB-induced glomerulosclerosis, podocyte injury led to a shift in the balance of Ang1 and Ang2 in glomeruli. Increased TNF-alpha in plasma and glomeruli may upregulate Ang2 expression in glomeruli. Elevated Ang2 in both plasma and glomeruli may mediate protein permeability through the glomerular filtration barrier. Moreover, local expression of Ang2 may facilitate the progress of glomerulosclerosis by upregulating a component expression of extracellular matrix.     

Hyperkalaemia in patients in hospital

A survey of all laboratory blood specimens with a plasma potassium concentration greater than or equal to 5.5 mmol/L was conducted over a three month period. Of 331 specimens with hyperkalaemia, 71 were excluded because the specimens was haemolysed, old or contaminated. The laboratory served a population of 348,561 and during this time measured the plasma potassium on 25,016 occasions. Sixty-six outpatients and 20 neonates were not evaluated. The survey was undertaken on 86 of 102 inpatients (46 males), 48 of whom were over 66 years of age. Fifty-seven patients were admitted under a medical service and 29 under a surgical service. Fifty-nine had a single episode of hyperkalaemia. Thirty-two underwent a surgical procedure. The commonest contributing factor was impaired renal function which was present in 71 (83%) patients. Although a definitive causative role for drugs could be identified in only five patients, in 52 (60%) patients drugs were a contributing factor (potassium supplements 24, ACE inhibitors 16, nonsteroidal antiinflammatory drugs 12). Thirty-five of the 86 (41%) patients died during their hospital admission. Nineteen of the 35 deaths occurred within three days of the hyperkalaemia being recorded. A normal plasma potassium was eventually documented in 50 of the 86 patients. Of the remaining 36 patients, 25 (69%) subsequently died. In general the treatment of patients with hyperkalaemia focused on identifying and treating the underlying cause. Hyperkalaemia must always be considered seriously and regard given to the overall clinical status of the patient, with particular attention to drug therapy, renal and cardiac function, acid base status and the possibility of sepsis.