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。     

PCSK9抑制剂在降低心血管事件风险中的研究进展

心血管疾病已成为影响人们生活和生命健康的主要危险因素,多种心血管事件的发生严重威胁患者生命,其主要诱因是循环中低密度脂蛋白胆固醇(LDL-C)的升高.前蛋白转化酶枯草杆菌蛋白酶/kexin 9型抑制剂(PCSK9i)作为一种新型降脂药物,在降低患者血清及血浆LDL-C及降低患者的心血管事件(CV)风险中有重要作用,可能...     

PCSK9抑制剂依洛尤单抗在降低心血管事件风险中的研究进展

2015年,前蛋白转化酶枯草杆菌蛋白酶/kexin 9（proprotein convertase subtilisin kexin 9,PCSK9）抑制剂依洛尤单抗被美国食品药品监督管理局（the Food and Drug Administration,FDA）批准上市,为高脂血症及心血管高危患者的治疗带来了新的希望。本文综述了依洛尤单抗的药学性质及相关临床试验结果,并介绍其在真实世界中的应用效果,以期为临床药物治疗提供参考。     

田蓟苷对HepG2细胞胆固醇代谢的作用机制研究

目的 探讨田蓟苷对人肝癌细胞株(HepG2)胆固醇代谢的作用机制.方法 用氧化型低密度脂蛋白(ox-LDL)诱导HepG2细胞脂质堆积模型.用四甲基偶氮唑蓝法检测细胞活力,根据细胞活力选取田蓟苷100μmol·L-1浓度(田蓟苷-2组)进行后续实验.将HepG2细胞分为4组:对照组(溶剂)、模型组(ox-LDL 50 ...     

香樟3-羟基-3-甲基戊二酰辅酶A还原酶(CcHMGRs)基因家族的克隆及表达分析

3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGR)是萜类甲羟戊酸途径(mevalonic acid pathway, MVA)上的第一个限速酶,是细胞质中萜类代谢途径中的重要调控位点。本研究以5种化学型的香樟(Cinnamomum camphora)作为实验材料,基于转录组数据,从香樟cDNA中克隆出2个HMGR基因,分别命名为CcHMGR1 (GenBank登录号:MN163055)和CcHMGR2 (GenBank登录号:MN163056)。基因包含开放阅读框(ORF)分别为1 689 bp和1 683 bp,编码562和560个氨基酸残基,生物信息学分析推测其分子质量为59.819 kDa和59.397 kDa,等电点(theoretical pI)为8.20和8.61,均不含信号肽,存在2个跨膜结构。结合蛋白质保守区以及进化树分析,证实该基因确为HMGR家族基因。利用实时荧光定量PCR检测, CcHMGR1和CcHMGR2在香樟5种化学型中的表达模式类似,均在油樟中的表达量要高于另外...     

藏药珊瑚七十味丸对高脂血症模型大鼠的降脂作用及机制研究

目的研究藏药珊瑚七十味丸对高脂血症(HLP)模型大鼠的降脂作用,并初步探讨其作用机制。方法将60只SD大鼠按体质量随机分为正常组、模型组、辛伐他汀组(阳性对照,20 mg/kg)和珊瑚七十味丸低、中、高剂量组(50、100、200 mg/kg),每组10只。正常组大鼠饲喂常规饲料,其余各组大鼠饲喂高脂饲料建立HLP模型,连续喂养4周。各给药组大鼠在造模同时即开始灌胃相应药物,正常组和模型组大鼠灌胃等体积生理盐水,每天1次,连续4周。末次灌胃后,检测各组大鼠血清中总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)水平,观察各组大鼠肝组织病理变化,测定各组大鼠肝组织中AMP活化蛋白激酶1(AMPK)、磷酸化AMPK(p-AMPK)、肝激酶B1(LKB1)、3-羟基-3-甲基戊二酸单酰辅酶A还原酶(HMGCR)蛋白的表达情况。结果低、中、高剂量珊瑚七十味丸均可显著降低HLP模型大鼠血清中TC、TG、LDL-C水平以及肝组织中HMGCR蛋白表达水平(P<0.05),显著升高HLP模型大鼠血清中HDL-C水平及肝组织中AMPK磷酸化水平、LKB1蛋白表达水平(P<0.05),不同程度地改善HLP模型大鼠肝组织的病理变化。结论珊瑚七十味丸能降低HLP模型大鼠的血脂水平;其机制可能与抑制LKB1/AMPK信号通路的传导,从而调节脂代谢相关。     

地菍总黄酮抗2型糖尿病作用机制的网络药理学研究及实验验证

目的 利用网络药理学方法研究地菍总黄酮抗2型糖尿病的作用机制，通过动物实验对靶点进行验证。方法 通过文献检索收集地菍总黄酮的化学成分，并在Swiss Target Prediction平台进行筛选；依据Swiss Target Prediction、GeneCards、TTD、DrugBank数据库预测和筛选地菍总黄酮成分治疗2型糖尿病的作用靶点；由Cytoscape 3.7.1构建“成分–疾病–靶点”网络；String数据库构建靶点蛋白相互作用（PPI）网络；R语言对靶点基因进行基因本体（GO）富集分析及京都基因和基因组百科全书（KEGG）通路富集分析。以高脂饮食诱导和ip链脲佐菌素（STZ）方法构建2型糖尿病模型大鼠，将2型糖尿病模型造模成功的40只大鼠随机分成模型组，二甲双胍组（0.2 g/kg）组，地菍总黄酮（0.34、0.45、0.60 g/kg）组，每组8只，继续提供高脂饲料喂养，另设对照组。各组每天ig相应药物，1次/d，对照组和模型组ig 0.5% CMC-Na溶液，连续给药5周。记录大鼠体质量、饮水量和摄食量，检测大鼠血糖及血脂的相关指标，运用qPCR法检测地菍总黄酮对2型糖尿病大鼠肝组织中磷脂酰肌醇-3-激酶（PI3K）、蛋白激酶B（Akt）、腺苷酸活化蛋白激酶（AMPK）作用靶点的调控作用。Western blotting法检测各组大鼠肝脏中Akt、AMPK、固醇调节元件结合蛋白-1C（SREBP-1C）的蛋白相对表达量。结果 从地菍总黄酮成分中筛选得到槲皮素、芹菜素、柚皮素等8种活性成分，作用于98个2型糖尿病靶点，GO功能与KEGG富集分析表明，地菍总黄酮抗糖尿病主要通过调节PI3K/Akt信号通路、AMPK相关信号通路等来发挥抗2型糖尿病的作用。动物实验证实，地菍总黄酮可降低2型糖尿病大鼠的空腹血糖（FBG），抑制血脂升高，上调2型糖尿病大鼠肝脏PI3K、Akt、AMPK mRNA表达，显著增加Akt、AMPK蛋白表达含量，降低SREBP-1C蛋白表达含量（P＜0.01、0.05），对糖、脂代谢紊乱均具有改善作用，具有良好的抗2型糖尿病作用。结论 验证了地菍总黄酮多成分、多靶点、多途径治疗2型糖尿病的特点，其抗糖尿病的作用机制可能与调节PI3K、Akt、AMPK、SREBP-1C靶点的表达水平，从而改善糖脂代谢紊乱有关，为后续更进一步研究作用机制提供参考。     

HBx蛋白激活肝细胞表达的FasL的生理效应

目的：观察HepG2细胞表达的FasL蛋白的生理效应，期望能进一步阐明乙型病毒性肝炎的分子发病机制。方法：(1)建立转染HBx基因的HepG2细胞系。(2)HtepC2x细胞表达的FasL生理效应的分析：采用细胞混合培养后，测定LDH释放、DNA Ladder及流式细胞仪方法检测细胞凋亡情况。结果：HepC2x细胞可表达FasL，HepC2x细胞与HepC2o、Jurkat细胞混合培养48小时后，HepG2o及Jurkat细胞特异性LDH释放率均等于零oDNAladder及流式细胞仪检测APO2.7抗体标记细胞结果表明，与HepC2x混合培养后，HepG2o、Jurkat细胞发生凋亡；增强HepG2o细胞表达Fas，细胞凋亡程度也增加。结论：HepC2x细胞表达的FasL具有致表达Fas的肝细胞或Jurkat细胞凋亡的作用。     

匹伐他汀和阿托伐他汀诱发HepG2细胞发生胰岛素抵抗

目的研究匹伐他汀和阿托伐他汀诱发Hep G2细胞发生胰岛素抵抗的作用。方法 Hep G2细胞分别给予胰岛素0.5μmol·L-1,匹伐他汀1,10和100μmol·L-1,阿托伐他汀1,10和100μmol·L-1,以及阿托伐他汀100μmol·L-1+MK886 100μmol·L-1。48,72和96 h后检测细胞残余[125I]胰岛素结合率,[3H]D-葡萄糖摄取率,糖原和三酰甘油(TG)含量;作用48 h时细胞载脂蛋白A5(Apo A5)mRNA、Apo A5蛋白和Akt信号通路。结果与阴性对照组相比,匹伐他汀和阿托伐他汀对残余[125I]胰岛素结合率和糖原含量无明显作用。阿托伐他汀100μmol·L-1增加TG含量(mg·g蛋白:0.71±0.04 vs 1.51±0.05,P<0.05),降低[3H]D-葡萄糖摄取率〔37.2±3.2 vs(26.7±1.9)μmol·g-1·h-1,P<0.05),减少磷酸化Akt表达水平(0.92±0.09 vs 0.32±0.02,P<0.05),升高Apo A5 mRNA(0.30±0.02 vs 0.69±0.06,P<0.05)和蛋白表达水平(0.30±0.04 vs0.91±0.03,P<0.05),与胰岛素0.5μmol·L-1组类似。阿托伐他汀100μmol·L-1上述作用与作用时间呈正相关(r=0.729,P<0.05),而MK886可拮抗这些作用。结论阿托伐他汀100μmol·L-1时间依赖性地增强Apo A5表达,导致细胞糖脂代谢异常,诱发Hep G2细胞胰岛素抵抗。     

NADPH-cytochrome P-450 reductase is involved in flunitrazepam reductive metabolism in Hep G2 and Hep 3B cells

Flunitrazepam (FNTZ), like other benzodiazepines, has a high affinity for the benzodiazepine receptor within the gama-aminobutyric acid (GABA) complex. These affinities correlate with the pharmacological and therapeutic potencies of the drug. FNTZ is a drug commonly abused by young adults. In humans, FNTZ is oxidized to the major metabolites N-demethylflunitrazepam (DM FNTZ) and 3-hydroxyflunitrazepam (3-OH FNTZ) and reduced to 7-aminoflunitrazepam (7A FNTZ). Human CYP2C19 and CYP3A4 are the principal P-450 cytochromes involved in DM FNTZ and 3-OH FNTZ formation. However, it is not clear which enzyme is responsible for the reduction of FNTZ to 7-aminoflunitrazepam (7A FNTZ). In this study, the involvement of NADPH-cytochrome P-450 reductase in the conversion of FNTZ to 7A FNTZ was investigated in two human hepatoma cell lines, human lymphoblast microsomes specifically expressing human NADPH-cytochrome P-450 reductase and purified recombinant human HADPH-cytochrome P-450 reductase. Significantly more FNTZ was converted to 7A FNTZ in Hep G2 than in Hep 3B cells, and this difference was associated with the catalytic activity and protein levels of NADPH-cytochrome P-450 reductase in these cells. In Hep G2 cells, conversion of FNTZ to 7A FNTZ was effectively inhibited by alpha-lipoic acid, an NADPH-cytochrome P-450 reductase inhibitor. In addition, formation of 7A FNTZ by the microsomal fraction of Hep G2 cells was specifically inhibited by antibody against NADPH-cytochrome P-450 reductase. Under hypoxia (N2 85%; CO2 5%; H2 10%), human lymphoblast microsomes specifically expressing human NADPH-cytochrome P-450 reductase and purified recombinant human NADPH-P-450 reductase catabolized FNTZ to 7A FNTZ in a concentration-dependent manner. These results suggest that NADPH-cytochrome P-450 reductase is involved in the reductive metabolism of FNTZ to 7A FNTZ under hypoxic conditions.     

拉米夫定对HepG2.2.15细胞MMP-9及p53表达的影响

目的通过检测拉米夫定作用于人肝癌细胞系(Hep G2.2.15)后对其基质金属蛋白酶-9(MMP-9)及p53表达的变化,探讨拉米夫定对原发性肝癌发生发展的抑制作用。方法不同浓度(100μg/ml、200μg/ml、300μg/ml)拉米夫定作用于Hep G2.2.15细胞不同时间(3 d及6 d)后,四甲基偶氮唑盐(MTT)比色法测定细胞增殖活性;双抗体夹心酶联免疫吸附实验(ELISA)测定细胞培养上清及胞质中MMP-9及p53蛋白含量。结果经拉米夫定作用后的Hep G2.2.15细胞生长未受明显抑制;细胞中MMP-9及p53的表达均出现不同程度降低。结论拉米夫定通过降低Hep G2.2.15细胞中MMP-9及p53的表达来抑制肝癌的侵袭及转移。     

The toxicity of menadione and mitozantrone in human liver-derived Hep G2 hepatoma cells

The cytotoxic properties of quinone drugs such as menadione and adriamycin are thought to be mediated through one-electron reduction to semiquinone free radicals. Redox cycling of the semiquinones results in the generation of reactive oxygen species and in oxidative damage. In this study the toxicity of mitozantrone, a novel quinone anticancer drug, was compared with that of menadione in human Hep G2 hepatoma cells. Mitozantrone toxicity in these cells was not mediated by the one-electron reduction pathway. In support of this, inhibition of the enzymes glutathione reductase and catalase, responsible for protecting the cells from oxidative damage, did not affect the response of the Hep G2 cells to mitozantrone, whereas it exacerbated menadione toxicity. In addition, the toxicity of menadione was preceded by depletion of reduced glutathione which was probably due to oxidation of the glutathione. Mitozantrone did not cause glutathione depletion prior to cell death. DT-diaphorase activity and intracellular glutathione were found to protect the cells from the toxicity of both quinones. Inhibition of epoxide hydrolase potentiated mitozantrone toxicity but did not affect that of menadione. Our experiments indicate that mitozantrone toxicity may involve activation to an epoxide intermediate. Both quinone drugs inhibited cytochrome P-450-dependent mixed-function oxidase activity, although menadione was more potent in this respect.     

Modulation of lipoprotein production in Hep G2 cells by fenofibrate and clofibrate.

Fenofibrate and other fibrate derivatives are commonly used to treat hyperlipidemia. It is not yet clear how they exert their modulatory effects on plasma lipoproteins. To investigate whether these drugs act on the liver to primarily inhibit very low density lipoprotein production, we utilized the highly differentiated human hepatoma cell line, Hep G2. At concentrations greater than 15 micrograms/mL, fenofibrate caused a 30% decrease in secreted apolipoprotein B (apo B) after 4 days of treatment. Pulse-chase studies demonstrated that this was not due to inhibition of apo B synthesis. Triglyceride synthesis by fenofibrate-treated Hep G2 cells was decreased by 30%, and the amount secreted into the medium was reduced by 50%. At a low concentration of drug (5 micrograms/mL), triglyceride secretion was reduced markedly while apo B secretion remained unchanged. Thus, apo B secretion is less sensitive to fenofibrate than the synthesis and secretion of triglyceride, and may be secondary to changes in the latter. Fenofibrate has also been shown to raise plasma high density lipoprotein concentrations. We found that low concentrations of fenofibrate caused a 20-101% increase in secreted apolipoprotein AI (apo AI), and pulse-chase immunoprecipitation studies showed that this was due to an increase in apo AI synthesis. Fenofibrate was compared to clofibrate to investigate whether their relative effects on lipoprotein production in Hep G2 cells were comparable to their relative effects on plasma lipoproteins. Both fibrates decreased the secretion of apo B to the same extent, but only fenofibrate increased apo AI secretion. Fenofibrate was more effective than clofibrate in inhibiting the secretion of lipids by these cells. Thus, the known effects of fenofibrate on plasma lipoproteins can be attributed to its direct modulation of lipoprotein synthesis in the liver cell. Hep G2 cells may thus be useful in testing the relative efficacy of fibric acid derivatives in vitro.     

Rhein inhibits the growth and induces the apoptosis of Hep G2 cells

The effects of rhein on the human hepatoblastoma G2 (Hep G2) cell line were investigated in this study. The results showed that rhein not only inhibited Hep G2 cell growth but also induced apoptosis and blocked cell cycle progression in the G1 phase. An ELISA assay demonstrated that rhein significantly increased the expression of p53 and p21/WAF1 protein, which caused cell cycle arrest. An enhancement in CD95 and its two forms of ligands, membrane-bound CD95 ligand (mCD95L) and soluble CD95 ligand (sCD95L), might be responsible for the apoptotic effect induced by rhein. Taken together, p53 and the CD95/CD95L apoptotic system possibly participated in the antiproliferative activity of rhein in Hep G2 cells.     

Effects of perfluorooctanoate and perfluorooctane sulfonate exposure on hepatoma Hep G2 cells

Perfluorinated compounds (PFCs) are emerging compounds of concern. They are widely distributed in the environment, wildlife and human. Concern has been raised over their possible adverse effects on human health. This study was designed to determine cytotoxic effects of two important PFCs, perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS), in a single and a mixture of them exposure to Hep G2 cells. The results showed that PFOA and PFOS (50–200 μmol/l) induced production of reactive oxygen species (ROS), dissipation of mitochondria membrane potential and apoptosis of Hep G2 cells. Moreover, activities of superoxide dismutase, catalase and glutathione reductase were increased, whereas activities of glutathione-S-transferase and glutathione peroxidase were decreased. Glutathione content was reduced. Differential expression of genes, such as p53, Bcl-2, caspase-9, was evident in PFOA or PFOS exposure groups. The possible mechanism was that they could overwhelm homeostasis of antioxidative systems, boost ROS generation, impact mitochondria, and affect genes expression of apoptotic regulators, which resulted in start-ups of apoptosis program. Cells exposed to mixture of PFOA and PFOS and each of them showed non-apoptotic rate significant difference, which indicated that the combined effect of two compounds was summation effect, but neither synergistic nor antagonistic effect.