调血脂药物体外筛选模型的建立及应用
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摘要
目的：利用肝癌细胞HepG2建立细胞内脂质堆积模型,筛选中药中的活性成分,得到具有调脂作用的活性化合物,从吸收及代谢通路上的基因表达变化探讨其作用机制,进行体内药效研究。
方法：加入外源性的油酸(OA)与棕榈酸(PA)与HepG2细胞共同培养,造成细胞内脂质堆积；MTT法检测不同浓度脂质对细胞活性的影响,选择合适的脂质堆积模型作为筛选工具。选择造模浓度后,在造模同时加入不同的中药活性成分共同培养,利用尼罗红对细胞内脂质进行染色,借助用流式细胞仪技术检测细胞内荧光强度,来判断细胞内脂质含量的变化。利用模型筛选出具有调节脂质代谢的活性化合物；在此基础上,利用荧光实时定量RT-PCR技术考察吸收通路上的脂肪酸结合蛋白L-FABP的基因表达变化,以及在代谢通路上PPAR-α基因的表达变化,借此初步探讨药物的作用机制；利用高血脂大鼠模型对筛选出的药物进行体内药效评价。
结果：细胞培养同时加入总浓度分别为1mM、2mM、3mM的油酸(OA)与棕榈酸(PA),经MTT法检测后发现随着脂质浓度的增加,造成的细胞毒性也随之而增加,1mM组细胞活性基本无显著影响,故选择为体外脂质堆积造模浓度。经尼罗红染色后流式检测,与空白组相比较各个模型组细胞内脂质含量都得到了显著的提高,1mM组中细胞内脂质含量是空白组的2.69倍,而2mM组脂质含量能够达到空白组的3.22倍,3 mM组的达到了空白的3.69倍(P<0.01),说明外源性FFAs的浓度与细胞内脂质含量呈正相关；随着造模时间从6h、12h、18h至24h的延长,细胞内脂质含量成正比增加,从空白组的1.5倍、1.9倍、2.1倍直至2.5倍。利用1mM脂质堆积模型进行天然活性化合物的筛选,得到部分在体外对细胞内脂质堆积具有调节作用的化合物,如甘草次酸、绞股蓝皂苷、连翘苷、槲皮苷、水飞蓟素等,能分别降低1mM模型组细胞内的脂质含量达27.85%、20.13%、19.32%、13.58%、1.18%。
已知过氧化物酶体增生物激活受体PPAR-α作为与脂质氧化相关的转录因子,能够上调过氧化物酶体及与线粒体内β氧化相关的基因表达；L-FABP主要表达于肝脏组织和小肠,介导脂肪酸及多种疏水基团转运,涉及多种疾病的发病机制,故以这两个基因为考察指标,对化合物的作用机制进行讨论。选用苯扎贝特作为阳性药,对脂质堆积细胞作用24h后,苯扎贝特组的PPAR-α的表达与模型组相比提高了约3.5倍,甘草次酸组表现出了约2.4倍的提高,水飞蓟素组约为2倍,三个给药组均发挥上调作用；对L-FABP的考察结果表明,苯扎贝特组与模型组相比基因表达量提高了1.5倍左右,在各个给药组中最高,其次是甘草次酸组,其相对表达量接近1倍,而水飞蓟素组的相对表达量接近0.5倍,表现出一定的抑制作用。经过结构修饰的化合物NDHO1,低剂量组的PPAR-α的基因mRNA表达增至2.5倍,而高剂量组约为1倍；HMG-CoA还原酶的mRNA表达都得到了抑制,相对表达情况均小于1倍,高剂量组的抑制作用强于低剂量组；FABP蛋白的表达在2个剂量组中均未表现显著变化。
通过建立高脂动物模型对NDH01化合物进行体内药效考察,发现NDH01化合物高中低三个剂量组中,低剂量组(5mg/kg)与中剂量组(15mg/kg)组降低了血清中的TC、LDL-C,与模型组相比具有极显著差异(P<0.01)；高剂量组(45mg/kg)在TC水平上与其余2个剂量组同样发挥了良好的降低作用(P<0.01),对LDL-C水平均无显著影响。
结论：利用外源性游离脂肪酸与HepG2细胞共同培养,造成细胞内脂质堆积模型,利用尼罗红染色及流式细胞技术检测含量,经过时效关系与量效关系的考察,能够稳定重复出此模型,证明细胞内脂质堆积模型成功建立。利用此模型进行化合物的筛选,得到一系列对细胞内脂质堆积发挥调节作用化合物,包括天然化合物及经过结构修饰的化合物。从荧光实时定量RT-PCR的结果发现,这些药物在脂质氧化通路上发挥了一定的作用,降低了细胞内的脂质含量,减少了甘油三酯合成的前体化合物,从减少TG的合成发挥调血脂作用。利用高脂动物体内试验验证得出NDH01化合物在体内对于血脂发挥一定的调节作用,具有极高的开发利用价值。
Objective:This topic ues HepG2 cell line to establish a lipid accumulation cell model in vitro for drugs screening, in aim to get some lipid-regulating ingredients from natural compounds, discuss the drug mechanism from absorption and metabolism pathway, then use the model in vivo to research the pharmacodynamic action.
Method:we incubate HepG2 cells with exogenous oil acid and palmitic acid to induce lipid accumulation in it, detect the cytoactive by MTT to choose a suitable concentration to model. Incubate cells with FFAs and different natural active compounds, after staining cells with Nile Red, using flow cytometry to detect the fluorescence intensity in cells to judge the content of lipid in cells. This method can be used to screen the activity of lipid-regulated conpounds. Base on the result of screening, detect the gene expression changes to discuss the mechanism of drug's action by Fluorescent real-time quantitative PCR, including Fatty acid binding protein(L-FABP) in absorption pathway, as well as the peroxisome Proliferator-activated receptor a(PPAR-a) in metabolic pathway. After that, taking advantage of hyperlipemia rat model to discuss the drug action in vivo.
Result:Cells are incubated with 1mM、2 mM、3 mM of OA and PA for 24h, the cytotoxicity is exacerbated with the concentration of FFAs rising, choose the group with lmM FFAs as the screening model for the minmum cytotoxicity in the cell by MTT test. After model establishing, staining cells with Nile red, compare with the control, each models group with different FFAs concerntration has a significant increase of lipid content in cells, 1mM group's lipid content is 2.69 fold of control, the 2mM group is 3.22 fold of control, the 3mM group is 3.69 fold of control(P< 0.01), indicate the concentration of exogenous FFAs and intracellular lipid content is positively correlated. As FFAs incubating with cells for 6h、12h、18h to 24h, the content of lipid in cell is rising from 1.5 fold、1.9 fold、2.1 fold to 2.5 fold of control. Choose the lmM model as the screening model, after preliminary screening, we get some compounds which can regulate the lipid accumulaition in the cell, such as GA、 Gypenosides、phillyrin、quercitrin. Silymarin and so on, respectively reduce the lipid content of cell for 27.85%、20.13%、19.32%、13.58%、1.18% compare with the group without drug treatment.
As we know, Peroxisome proliferator-activated receptorα, a transcriptional factor, up-regulates the expression of a suite of genes that includes peroxisomal and mitochondrialβ-oxidation enzymes, L-FABP mainly experess in the liver and small intestine, mediated the transport of fatty acids and many hydrophobic groups, involving in the pathogenesis of many diseases, we choose these genes as indexes to discuss the mechanism of drug in lipid-regulation. Bezafibrate is choosen as a positive control, the expression of PPAR-αrise to 3.5 fold after 24h treatment with Bezafibrate, the group treated with GA rise to 2.5 fold, and the Silymarin group is about 2 fold increasing, all of these groups promote the expression of PPAR-α. The relative expression of L-FABP in Bezafibrate group is 1.5 fold, which is the maximum in three drug treatment group, the GA group's is closed to 1 fold, the Silymarin group is closed to 0.5 fold, showing a inbihition in gene expression. NDH01 is a structural modificate chemical compound, after treating the model with low dose, the expression of PPAR-αis rise to 2.5 fold, while the high dose group is 1 fold, the expression of HMG-CoA reductase both inhibited in two dose group, the ratio is below 1 fold. The inhibiton is obvious in high dose group. The expression of FABP has no significant change.
We establish an animal model of hyperlipemia to investigate the drug effect of NDH01 in vivo, low-dose group (5mg/kg) and middle-dose group (15mg/kg) have an significant suppression in the level of TC and LDL-C vs. control.The high-dose group compare with the other groups has the same inhibiting action, but has no impact on LDL-C.
Conclusion:Adding exogenous FFAs during HepG2 cell culture inducing lipid accumulation in cells, detect the fluorescence intensity of cells using flow cytometry after staining with Nile Red. Investigate the time-effect and dose-effect relationship, this results is reliable and stable, certificate the establishment of intracellular lipd-accumulaition model is succeed. Take advantage of this model for active drug screening, we get a series of compounds including the natral compounds and the structural modificate compounds. From the result of Fluorescent real-time quantitative PCR, these compounds may have impact on the lipid-oxidation pathway, reflect on lowing the content of lipid in cells. The reducing in the precurosor of TG synthesis directly influence the lipid-regulation. Using the animal model of hyperlipemia, we verificate NDH01 can regulate the lipid in vivo, which is worth of further research.
方法：加入外源性的油酸(OA)与棕榈酸(PA)与HepG2细胞共同培养,造成细胞内脂质堆积；MTT法检测不同浓度脂质对细胞活性的影响,选择合适的脂质堆积模型作为筛选工具。选择造模浓度后,在造模同时加入不同的中药活性成分共同培养,利用尼罗红对细胞内脂质进行染色,借助用流式细胞仪技术检测细胞内荧光强度,来判断细胞内脂质含量的变化。利用模型筛选出具有调节脂质代谢的活性化合物；在此基础上,利用荧光实时定量RT-PCR技术考察吸收通路上的脂肪酸结合蛋白L-FABP的基因表达变化,以及在代谢通路上PPAR-α基因的表达变化,借此初步探讨药物的作用机制；利用高血脂大鼠模型对筛选出的药物进行体内药效评价。
结果：细胞培养同时加入总浓度分别为1mM、2mM、3mM的油酸(OA)与棕榈酸(PA),经MTT法检测后发现随着脂质浓度的增加,造成的细胞毒性也随之而增加,1mM组细胞活性基本无显著影响,故选择为体外脂质堆积造模浓度。经尼罗红染色后流式检测,与空白组相比较各个模型组细胞内脂质含量都得到了显著的提高,1mM组中细胞内脂质含量是空白组的2.69倍,而2mM组脂质含量能够达到空白组的3.22倍,3 mM组的达到了空白的3.69倍(P<0.01),说明外源性FFAs的浓度与细胞内脂质含量呈正相关；随着造模时间从6h、12h、18h至24h的延长,细胞内脂质含量成正比增加,从空白组的1.5倍、1.9倍、2.1倍直至2.5倍。利用1mM脂质堆积模型进行天然活性化合物的筛选,得到部分在体外对细胞内脂质堆积具有调节作用的化合物,如甘草次酸、绞股蓝皂苷、连翘苷、槲皮苷、水飞蓟素等,能分别降低1mM模型组细胞内的脂质含量达27.85%、20.13%、19.32%、13.58%、1.18%。
已知过氧化物酶体增生物激活受体PPAR-α作为与脂质氧化相关的转录因子,能够上调过氧化物酶体及与线粒体内β氧化相关的基因表达；L-FABP主要表达于肝脏组织和小肠,介导脂肪酸及多种疏水基团转运,涉及多种疾病的发病机制,故以这两个基因为考察指标,对化合物的作用机制进行讨论。选用苯扎贝特作为阳性药,对脂质堆积细胞作用24h后,苯扎贝特组的PPAR-α的表达与模型组相比提高了约3.5倍,甘草次酸组表现出了约2.4倍的提高,水飞蓟素组约为2倍,三个给药组均发挥上调作用；对L-FABP的考察结果表明,苯扎贝特组与模型组相比基因表达量提高了1.5倍左右,在各个给药组中最高,其次是甘草次酸组,其相对表达量接近1倍,而水飞蓟素组的相对表达量接近0.5倍,表现出一定的抑制作用。经过结构修饰的化合物NDHO1,低剂量组的PPAR-α的基因mRNA表达增至2.5倍,而高剂量组约为1倍；HMG-CoA还原酶的mRNA表达都得到了抑制,相对表达情况均小于1倍,高剂量组的抑制作用强于低剂量组；FABP蛋白的表达在2个剂量组中均未表现显著变化。
通过建立高脂动物模型对NDH01化合物进行体内药效考察,发现NDH01化合物高中低三个剂量组中,低剂量组(5mg/kg)与中剂量组(15mg/kg)组降低了血清中的TC、LDL-C,与模型组相比具有极显著差异(P<0.01)；高剂量组(45mg/kg)在TC水平上与其余2个剂量组同样发挥了良好的降低作用(P<0.01),对LDL-C水平均无显著影响。
结论：利用外源性游离脂肪酸与HepG2细胞共同培养,造成细胞内脂质堆积模型,利用尼罗红染色及流式细胞技术检测含量,经过时效关系与量效关系的考察,能够稳定重复出此模型,证明细胞内脂质堆积模型成功建立。利用此模型进行化合物的筛选,得到一系列对细胞内脂质堆积发挥调节作用化合物,包括天然化合物及经过结构修饰的化合物。从荧光实时定量RT-PCR的结果发现,这些药物在脂质氧化通路上发挥了一定的作用,降低了细胞内的脂质含量,减少了甘油三酯合成的前体化合物,从减少TG的合成发挥调血脂作用。利用高脂动物体内试验验证得出NDH01化合物在体内对于血脂发挥一定的调节作用,具有极高的开发利用价值。
Objective:This topic ues HepG2 cell line to establish a lipid accumulation cell model in vitro for drugs screening, in aim to get some lipid-regulating ingredients from natural compounds, discuss the drug mechanism from absorption and metabolism pathway, then use the model in vivo to research the pharmacodynamic action.
Method:we incubate HepG2 cells with exogenous oil acid and palmitic acid to induce lipid accumulation in it, detect the cytoactive by MTT to choose a suitable concentration to model. Incubate cells with FFAs and different natural active compounds, after staining cells with Nile Red, using flow cytometry to detect the fluorescence intensity in cells to judge the content of lipid in cells. This method can be used to screen the activity of lipid-regulated conpounds. Base on the result of screening, detect the gene expression changes to discuss the mechanism of drug's action by Fluorescent real-time quantitative PCR, including Fatty acid binding protein(L-FABP) in absorption pathway, as well as the peroxisome Proliferator-activated receptor a(PPAR-a) in metabolic pathway. After that, taking advantage of hyperlipemia rat model to discuss the drug action in vivo.
Result:Cells are incubated with 1mM、2 mM、3 mM of OA and PA for 24h, the cytotoxicity is exacerbated with the concentration of FFAs rising, choose the group with lmM FFAs as the screening model for the minmum cytotoxicity in the cell by MTT test. After model establishing, staining cells with Nile red, compare with the control, each models group with different FFAs concerntration has a significant increase of lipid content in cells, 1mM group's lipid content is 2.69 fold of control, the 2mM group is 3.22 fold of control, the 3mM group is 3.69 fold of control(P< 0.01), indicate the concentration of exogenous FFAs and intracellular lipid content is positively correlated. As FFAs incubating with cells for 6h、12h、18h to 24h, the content of lipid in cell is rising from 1.5 fold、1.9 fold、2.1 fold to 2.5 fold of control. Choose the lmM model as the screening model, after preliminary screening, we get some compounds which can regulate the lipid accumulaition in the cell, such as GA、 Gypenosides、phillyrin、quercitrin. Silymarin and so on, respectively reduce the lipid content of cell for 27.85%、20.13%、19.32%、13.58%、1.18% compare with the group without drug treatment.
As we know, Peroxisome proliferator-activated receptorα, a transcriptional factor, up-regulates the expression of a suite of genes that includes peroxisomal and mitochondrialβ-oxidation enzymes, L-FABP mainly experess in the liver and small intestine, mediated the transport of fatty acids and many hydrophobic groups, involving in the pathogenesis of many diseases, we choose these genes as indexes to discuss the mechanism of drug in lipid-regulation. Bezafibrate is choosen as a positive control, the expression of PPAR-αrise to 3.5 fold after 24h treatment with Bezafibrate, the group treated with GA rise to 2.5 fold, and the Silymarin group is about 2 fold increasing, all of these groups promote the expression of PPAR-α. The relative expression of L-FABP in Bezafibrate group is 1.5 fold, which is the maximum in three drug treatment group, the GA group's is closed to 1 fold, the Silymarin group is closed to 0.5 fold, showing a inbihition in gene expression. NDH01 is a structural modificate chemical compound, after treating the model with low dose, the expression of PPAR-αis rise to 2.5 fold, while the high dose group is 1 fold, the expression of HMG-CoA reductase both inhibited in two dose group, the ratio is below 1 fold. The inhibiton is obvious in high dose group. The expression of FABP has no significant change.
We establish an animal model of hyperlipemia to investigate the drug effect of NDH01 in vivo, low-dose group (5mg/kg) and middle-dose group (15mg/kg) have an significant suppression in the level of TC and LDL-C vs. control.The high-dose group compare with the other groups has the same inhibiting action, but has no impact on LDL-C.
Conclusion:Adding exogenous FFAs during HepG2 cell culture inducing lipid accumulation in cells, detect the fluorescence intensity of cells using flow cytometry after staining with Nile Red. Investigate the time-effect and dose-effect relationship, this results is reliable and stable, certificate the establishment of intracellular lipd-accumulaition model is succeed. Take advantage of this model for active drug screening, we get a series of compounds including the natral compounds and the structural modificate compounds. From the result of Fluorescent real-time quantitative PCR, these compounds may have impact on the lipid-oxidation pathway, reflect on lowing the content of lipid in cells. The reducing in the precurosor of TG synthesis directly influence the lipid-regulation. Using the animal model of hyperlipemia, we verificate NDH01 can regulate the lipid in vivo, which is worth of further research.
引文
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[22]刘志锋,姜勇.报告基因技术的理论基础及其应用[J].生理科学进展,2002,33(4)：361-363.
[23]Jey Cheng, Keith Wood, Frank Fan生物发光技术在生命科学中的应用[J].生物物理学报,2009,25(5)：356-360.
[24]张华,张成刚,姜成林.Ascofuranone增加HepG2细胞LDL-R报告基因表达的生物活性的研究[J].中国抗生素杂志,2002,27(8)：449-451.
[25]胡蔚蓉,连霁虹,纪伟,等.建立一个基于报告基因的靶标筛药系统筛选上调ABCA1基因转录的中药提取物[J].华东理工大学学报,2007,33(6)：784-882.
[26]Gomez-Lechon MJ, Donato MT, Martinez-Romero A. A Human Hepatocellular In Vitro Model To Investigate Steatosis[J]. Chemico-Biological Interactions, 2007,165(2):106-116.
[27]Harmeet M, Steven Fb, Nathan Ww, Etal. Free Fatty Acids Induce Jnk-Dependent Hepatocyte Lipoapoptosis[J]. J Bio Chem.2006,281(17):12093-12101.
[28]Oksana Gavrilova, Martin Haluzik, Kimihiko Matsusue, Etal. Liver Peroxisome Proliferator-Activated Receptor Contributes To Hepatic Steatosis, Triglyceride Clearance, And Regulation Of Body Fat Mass[J]. The Journal Of Biological Chemistry,2003,278(38):34268-34276.
[29]Arend Bonen. Mechanisms and regulation of protein-mediated cellular fatty acid uptake:molecular, biochemical, and physiological evidence[J].Physiology,2007,22:15-29.
[30]唐孟萱,周万军,胡元佳,等.Hepg2细胞培养方法与条件的探讨[J].实用预防医学,2005,12(1)：71-73.
[31]Xudong Wu, Luyong Zhang, Emily Gurley, Etal. Prevention Of Free Fatty Acid-Induced Hepatic Lipotoxicity By 18β-Glycyrrhetinic Acid Through Lysosomal And Mitochondrial Pathways[J]. Heptology,2008,47(6):1905-1915.
[32]Wei Chen, Chengwu Zhang, Lirong Song, etal. A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae[J]. Journal of Microbiological Methods,2009,77(1):41-47.
[33]谢世荣,赵洁,刘琳,等.甘草次酸的研究与展望[J].大连大学学报,2005,26,(4)：85-88.
[34]陈英剑,胡成进,赵苗青.SYBR Green实时荧光定量PCR技术平台的建立[J].实用医 药杂志,2004,21(11)：997-999.
[35]Newberry, Elizabeth P, Davidson, Nicholas 0. Liver Fatty Acid Binding Protein (L-FABP) as a Target for the Prevention of High Fat Diet Induced Obesity and Hepatic Steatosis[J]. Immunology, Endocrine & Metabolic Agents,2009, 9(1):30-38.
[36]Ariel EF, Nathan WW, Ali C, etal. Free fatty acids promote hepatic lipotoxicity by stimulating TNF-α expression via a lysosomal pathway. Hepatology.2004,40(1):185-194.
[37]杨慧莹,林克荣.水飞蓟素治疗脂肪肝的作用机制[J].中华现代内科学杂志,2006,3(8)：876-878.
[38]倪鸿昌,李俊,金涌,等.大鼠实验性高脂血症和高脂血症性脂肪肝模型研究[J].中国药理学通报,2004,20：703-706.
[39]孔悦, 张冰, 刘小青,等.高甘油三酯高血糖血症大鼠脂糖代谢相关酶活性变化的实验研究[J].中国现代医学杂志,2006.16(23)：3542-3545.
[1]Camelia Stancu, Anca Sima. Statins:mechanism of action and effects[J]. J. Cell. Mol. Med,2001,5 (4):378-387.
[2]Chao-Yung Wang, Ping-Yen Liu, James K. Liao. Pleiotropic effects of statin therapy:molecular mechanisms and clinical results[J]. Trends In Molecular Medicine,2007,14(1):37-44.
[3]Thomas Helbing; Rene Rothweiler; Jennifer Heinke, et al. BMPER is upregulated by statins and modulates endothelial inflammation by intercellular adhesion molecule-1[J]. Arterioscl Throm Vas,2010,30(3):554-560.
[4]Marcoff L, Thompson Pd. The role of coenzyme Q10 in statin-associated myopathy: a systematic review[J]. J Am Coll Cardiol,2007,49(23):2231-2237.
[5]H. R. Davis, L. Zhu, L. M. Hoos, et al. Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a keymodulator of whole body cholesterol homeostasis[J]. Biol. Chem,2004,279(32):33486-33592.
[6]Stephen D. Turley. Role of Niemann-Pick C1-Like 1 (NPC1L1) in intestinal sterol absorption[J]. Journal of Clinical Lipidology,2008,2(2):S20-S28.
[7]Michalis Kalogirou, Vasilis Tsimihodimos, Moses Elisaf. Pleiotropic effects of ezetimibe:Do they really exist [J]. European Journal of Pharmacology,2010, 633(1-3):62-70.
[8]Peter P. Toth. High-density lipoprotein as a therapeutic target:clinical evidence and treatment strategies [J]. The American journal of cardiology,2005, 96 (9):50-58.
[9]L.A.Carlson. Nicotinic acid:the broad-spectrum lipid drug. A 50th anniversary review[J]. Journal of Internal Medicine,2005,258(2):94-114.
[10]Vaijinath S. Kamanna, Anthony Vo, Moti L. Kashyap. Nicotinic acid:recent developments[J]. Current Opinion in Cardiology,2008,23(4):393-398.
[11]John Y. L. Chiang. Regulation of bile acid synthesis:pathways, nuclear receptors, and mechanisms [J]. Journal of Hepatology,2004,40(3):539-551.
[12]Minako Yamaoka-Tojo, Taiki Tojo, Tohru Izumi. Beyond Cholesterol lowering: pleiotropic effects of bile acid binding resins against cardiovascular disease risk factors in patients with metabolic syndrome[J]. Current Vascular Pharmacology,2008, (4):271-281.
[13]Ashish Shah, Daniel J. Rader, John S. Millar. The effect of PPAR-α agonism on apolipoprotein metabolism in humans[J]. Atherosclerosis,2010 210(1):35-40.
[14]Takero Nakajima, Naoki Tanaka, Hiroki Kanbe, et al. Bezafibrate at clinically relevant doses decreases serum/liver triglycerides via down-regulation of sterol regulatory element-binding protein-1c in mice:a novel peroxisome proliferator-activated receptor α-independent mechanism[J]. Mol Pharmacol, 2009,75(4):782-792.
[15]Steven E. Nissen, E. Murat Tuzcu, H. Bryan Brewer, et al. Effect of ACAT inhibition on the progression of coronary atherosclerosis [J]. N Engl J Med, 2006,354(12):2616-2617.
[16]Honggang Hu, Hongli Liao, Jun Zhang, et al. First identification of xanthone sulfonamides as potent acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors[J]. Bioorganic & Medicinal Chemistry Letters (in press) 2010.
[17]Mm Hussain, P Rava, X Pan, et al. Microsomal triglyceride transfer protein in plasma and cellular lipid metabolism[J]. Current Opinion in Lipidology, 2008,19 (3):277-284.
[18]Michael H. Davidson. Novel Nonstatin Strategies to Lower Low-Density Lipoprotein Cholesterol[J]. Current Atherosclerosis Reports,2009, 11(1):67-70.
[19]Matthias Hermann, Frank T. Ruschitzka. The hypertension peril:lessons from cetp inhibitors[J]. Current Hypertension Reports,2009,11(1):76-80.
[20]Weijia Kong, Jing Wei, Parveen Abidi, et al. Berberine is a novel cholesterol-lowering drug working through a unique mechanism distinct from statins[J]. Nature Medicine,2004,10(12):1344-1351.
[21]Hu Jun, Shen Zhong-chen, Shen Yun, et al. Effects of PNS on Blood Lipids and mRNA Expression of Vascular Cell Adhesion Molecule-1 in Atherosclerotic Rats[J]. Journal of Shanghai University (Natural Science),2009,15(3):320-325.
[22]Ilker Durak, Mustafa Kavutcu, Bilal Aytac, et al. Effects of garlic extract consumption on blood lipid and oxidant/antioxidant parameters in humans with high blood cholesterol[J]. Journal of Nutritional Biochemistry,2004, 15(6):373-377.
[2]叶红燕,尹苗,商允菊,等.脂代谢相关基因在apoE基因缺失幼龄小鼠肝脏中的表达[J].生理学报,2008,60(1)：51-58.
[3]曾武威,陈保生,等。一个新的高甘油三酯血症候选基因一载脂蛋白A5[J].中国动脉硬化杂志,2007,15(11)：870-872.
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[7]谢亚利,李维琪,曹旭.高密度脂蛋白与动脉粥样硬化关系的研究进展[J].中国临床保健杂志,2004,7：228-230.
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[10]倪鸿昌,李俊,金涌,等.大鼠实验性高脂血症和高脂血症性脂肪肝模型研究[J].中国药理学通报,2004,20：703-706.
[11]李大伟, 张玲, 夏作理.建立高脂血症模型的动物选择与常用造模方法分析及改[J].中国临床康复,2006,10(48)：145-147.
[12]Cavasotto Cn, Orry Aj. Ligand Docking And Structure-Based Virtual Screening In Drug Discovery[J].Curr Top Med Chem,2007,7(10):1006-14.
[13]刘艾林,杜冠华.虚拟筛选辅助新药发现的研究进展[J].药学学报,2009,44(6)：566-570.
[14]Nancy Arguelles, Eugenia Sanchez-Sandoval, Aar6n Mendieta, Etal. Design, Synthesis, And Docking Of Highly Hypolipidemic Agents:Schizosaccharomyces Pombe As A New Model For Evaluating Alpha-Asarone-Based Hmg-Coa Reductase Inhibitors[J].Bioorg Med Chem,2010,18(12):4238-4248.
[15]Anna Tavridou, Loukas Kaklamanis, George Megaritis. Pharmacological Characterization In Vitro Of Ep2306 And Ep2302, Potent Inhibitors Of Squalene Synthase And Lipid Biosynthesis[J]. European Journal Of Pharmacology,2006, 535(1-3):34-42.
[16]Hoerer S, Schmid A, Heckel A, Et Al. Crystal Structure Of The Human Liver X Receptor Beta Ligand-Binding Domain In Complex With A Synthetic Agonist [J]. J Mol Biol,2003,334(5):853-861.
[17]Ajees Aa, Anantharamaiah Gm, Mishra Vk, Et Al. Crystal Structure Of Human Apolipoprotein A-I:Insights Into Its Protective Effect Against Cardiovascular Diseases [J]. Proc Natl Acad Sci U S A,2006,103(7):2126-2131.
[18]崔琴,鞠现霞,周红文.PCSK9在高胆固醇血症中的作用研究进展[J].医学综述,2010,16(16)：2454-2456.
[19]Hamilton Ja, Deckelbaum Rj. Crystal Structure Of CETP:New Hopes For Raising HDL To Decrease Risk Of Cardiovascular Disease [J]. Nat Struct Mol Biol,2007, 14(2):95-97.
[20]刘艾林,杜冠华.虚拟筛选辅助新药发现的研究进展[J].药学学报,2009,44(6)：566-570.
[21]Cho MC, Lee S, Choi HS, et al. Optimization of an enzyme-linked immunosorbent assay to screen ligand of Peroxisome proliferator-activated receptor alpha[J]. Immunopharmacol Immunotoxicol,2009,3(4):1-9.
[22]刘志锋,姜勇.报告基因技术的理论基础及其应用[J].生理科学进展,2002,33(4)：361-363.
[23]Jey Cheng, Keith Wood, Frank Fan生物发光技术在生命科学中的应用[J].生物物理学报,2009,25(5)：356-360.
[24]张华,张成刚,姜成林.Ascofuranone增加HepG2细胞LDL-R报告基因表达的生物活性的研究[J].中国抗生素杂志,2002,27(8)：449-451.
[25]胡蔚蓉,连霁虹,纪伟,等.建立一个基于报告基因的靶标筛药系统筛选上调ABCA1基因转录的中药提取物[J].华东理工大学学报,2007,33(6)：784-882.
[26]Gomez-Lechon MJ, Donato MT, Martinez-Romero A. A Human Hepatocellular In Vitro Model To Investigate Steatosis[J]. Chemico-Biological Interactions, 2007,165(2):106-116.
[27]Harmeet M, Steven Fb, Nathan Ww, Etal. Free Fatty Acids Induce Jnk-Dependent Hepatocyte Lipoapoptosis[J]. J Bio Chem.2006,281(17):12093-12101.
[28]Oksana Gavrilova, Martin Haluzik, Kimihiko Matsusue, Etal. Liver Peroxisome Proliferator-Activated Receptor Contributes To Hepatic Steatosis, Triglyceride Clearance, And Regulation Of Body Fat Mass[J]. The Journal Of Biological Chemistry,2003,278(38):34268-34276.
[29]Arend Bonen. Mechanisms and regulation of protein-mediated cellular fatty acid uptake:molecular, biochemical, and physiological evidence[J].Physiology,2007,22:15-29.
[30]唐孟萱,周万军,胡元佳,等.Hepg2细胞培养方法与条件的探讨[J].实用预防医学,2005,12(1)：71-73.
[31]Xudong Wu, Luyong Zhang, Emily Gurley, Etal. Prevention Of Free Fatty Acid-Induced Hepatic Lipotoxicity By 18β-Glycyrrhetinic Acid Through Lysosomal And Mitochondrial Pathways[J]. Heptology,2008,47(6):1905-1915.
[32]Wei Chen, Chengwu Zhang, Lirong Song, etal. A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae[J]. Journal of Microbiological Methods,2009,77(1):41-47.
[33]谢世荣,赵洁,刘琳,等.甘草次酸的研究与展望[J].大连大学学报,2005,26,(4)：85-88.
[34]陈英剑,胡成进,赵苗青.SYBR Green实时荧光定量PCR技术平台的建立[J].实用医 药杂志,2004,21(11)：997-999.
[35]Newberry, Elizabeth P, Davidson, Nicholas 0. Liver Fatty Acid Binding Protein (L-FABP) as a Target for the Prevention of High Fat Diet Induced Obesity and Hepatic Steatosis[J]. Immunology, Endocrine & Metabolic Agents,2009, 9(1):30-38.
[36]Ariel EF, Nathan WW, Ali C, etal. Free fatty acids promote hepatic lipotoxicity by stimulating TNF-α expression via a lysosomal pathway. Hepatology.2004,40(1):185-194.
[37]杨慧莹,林克荣.水飞蓟素治疗脂肪肝的作用机制[J].中华现代内科学杂志,2006,3(8)：876-878.
[38]倪鸿昌,李俊,金涌,等.大鼠实验性高脂血症和高脂血症性脂肪肝模型研究[J].中国药理学通报,2004,20：703-706.
[39]孔悦, 张冰, 刘小青,等.高甘油三酯高血糖血症大鼠脂糖代谢相关酶活性变化的实验研究[J].中国现代医学杂志,2006.16(23)：3542-3545.
[1]Camelia Stancu, Anca Sima. Statins:mechanism of action and effects[J]. J. Cell. Mol. Med,2001,5 (4):378-387.
[2]Chao-Yung Wang, Ping-Yen Liu, James K. Liao. Pleiotropic effects of statin therapy:molecular mechanisms and clinical results[J]. Trends In Molecular Medicine,2007,14(1):37-44.
[3]Thomas Helbing; Rene Rothweiler; Jennifer Heinke, et al. BMPER is upregulated by statins and modulates endothelial inflammation by intercellular adhesion molecule-1[J]. Arterioscl Throm Vas,2010,30(3):554-560.
[4]Marcoff L, Thompson Pd. The role of coenzyme Q10 in statin-associated myopathy: a systematic review[J]. J Am Coll Cardiol,2007,49(23):2231-2237.
[5]H. R. Davis, L. Zhu, L. M. Hoos, et al. Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a keymodulator of whole body cholesterol homeostasis[J]. Biol. Chem,2004,279(32):33486-33592.
[6]Stephen D. Turley. Role of Niemann-Pick C1-Like 1 (NPC1L1) in intestinal sterol absorption[J]. Journal of Clinical Lipidology,2008,2(2):S20-S28.
[7]Michalis Kalogirou, Vasilis Tsimihodimos, Moses Elisaf. Pleiotropic effects of ezetimibe:Do they really exist [J]. European Journal of Pharmacology,2010, 633(1-3):62-70.
[8]Peter P. Toth. High-density lipoprotein as a therapeutic target:clinical evidence and treatment strategies [J]. The American journal of cardiology,2005, 96 (9):50-58.
[9]L.A.Carlson. Nicotinic acid:the broad-spectrum lipid drug. A 50th anniversary review[J]. Journal of Internal Medicine,2005,258(2):94-114.
[10]Vaijinath S. Kamanna, Anthony Vo, Moti L. Kashyap. Nicotinic acid:recent developments[J]. Current Opinion in Cardiology,2008,23(4):393-398.
[11]John Y. L. Chiang. Regulation of bile acid synthesis:pathways, nuclear receptors, and mechanisms [J]. Journal of Hepatology,2004,40(3):539-551.
[12]Minako Yamaoka-Tojo, Taiki Tojo, Tohru Izumi. Beyond Cholesterol lowering: pleiotropic effects of bile acid binding resins against cardiovascular disease risk factors in patients with metabolic syndrome[J]. Current Vascular Pharmacology,2008, (4):271-281.
[13]Ashish Shah, Daniel J. Rader, John S. Millar. The effect of PPAR-α agonism on apolipoprotein metabolism in humans[J]. Atherosclerosis,2010 210(1):35-40.
[14]Takero Nakajima, Naoki Tanaka, Hiroki Kanbe, et al. Bezafibrate at clinically relevant doses decreases serum/liver triglycerides via down-regulation of sterol regulatory element-binding protein-1c in mice:a novel peroxisome proliferator-activated receptor α-independent mechanism[J]. Mol Pharmacol, 2009,75(4):782-792.
[15]Steven E. Nissen, E. Murat Tuzcu, H. Bryan Brewer, et al. Effect of ACAT inhibition on the progression of coronary atherosclerosis [J]. N Engl J Med, 2006,354(12):2616-2617.
[16]Honggang Hu, Hongli Liao, Jun Zhang, et al. First identification of xanthone sulfonamides as potent acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors[J]. Bioorganic & Medicinal Chemistry Letters (in press) 2010.
[17]Mm Hussain, P Rava, X Pan, et al. Microsomal triglyceride transfer protein in plasma and cellular lipid metabolism[J]. Current Opinion in Lipidology, 2008,19 (3):277-284.
[18]Michael H. Davidson. Novel Nonstatin Strategies to Lower Low-Density Lipoprotein Cholesterol[J]. Current Atherosclerosis Reports,2009, 11(1):67-70.
[19]Matthias Hermann, Frank T. Ruschitzka. The hypertension peril:lessons from cetp inhibitors[J]. Current Hypertension Reports,2009,11(1):76-80.
[20]Weijia Kong, Jing Wei, Parveen Abidi, et al. Berberine is a novel cholesterol-lowering drug working through a unique mechanism distinct from statins[J]. Nature Medicine,2004,10(12):1344-1351.
[21]Hu Jun, Shen Zhong-chen, Shen Yun, et al. Effects of PNS on Blood Lipids and mRNA Expression of Vascular Cell Adhesion Molecule-1 in Atherosclerotic Rats[J]. Journal of Shanghai University (Natural Science),2009,15(3):320-325.
[22]Ilker Durak, Mustafa Kavutcu, Bilal Aytac, et al. Effects of garlic extract consumption on blood lipid and oxidant/antioxidant parameters in humans with high blood cholesterol[J]. Journal of Nutritional Biochemistry,2004, 15(6):373-377.