【作者】 李慧；

【导师】 欧阳冬生；

【作者基本信息】 中南大学 ， 药学， 2013， 硕士

【摘要】 背景：高甘油三酯血症是心血管疾病剩留风险的主要危险因素之一,目前,临床上针对性控制血浆甘油三酯的药物种类较少,疗效和安全性存在缺陷,开发新的降甘油三酯药物具有重要意义。杜仲含有多种药理活性成分,具有降血压、降血脂、抗氧化、抗动脉粥样硬化、保护心肌等作用。杜仲木脂素是从杜仲中提取的有效部位。在治疗高血压疾病的临床应用中,杜仲木脂素可降低伴随有高血脂的患者的血浆甘油三酯。因此,研究杜仲木脂素对甘油三酯代谢的影响,阐明其机制,将为杜仲木脂素降甘油三酯功效的开发和临床应用提供理论依据。目的：研究杜仲木脂素对油酸孵育的HepG2细胞甘油三酯聚集的影响及其机制。方法：本课题采用HepG2细胞作为实验材料。在研究杜仲木脂素对HepG2生长的影响实验中,实验分为7组(空白对照组,15mg/L,37.5mg/L,75mg/L,150mg/L,300mg/L,600mg/L木脂素干预组)。采用MTT一步法检测A490值来评估细胞数目。在研究杜仲木脂素对OA孵育的HepG2细胞甘油三酯聚集的影响实验中,实验共分为6组：空白对照组；油酸(OA)孵育模型组(0.5mM OA);低浓度(0.5mM OA+15mg/L杜仲木脂素)、中浓度(0.5mM OA+30mg/L杜仲木脂素)和高浓度杜仲木脂素组(0.5mM OA+45mg/L杜仲木脂素)；非诺贝特组(0.5mM OA+100μmo/L非诺贝特)。采用酶法测定细胞的甘油三酯含量；采用实时荧光定量聚合酶链式反应法检测细胞PPARα、FAS、CPTlA的mRNA,蛋白质印迹法检测细胞FAS、CPT1A、 PPARα蛋白表达水平。结果：与正常对照组相比,15mg/L～300mg/L杜仲木脂素组细胞数目差异不具有统计学意义,无细胞毒性。而600mg/L杜仲木脂素组的细胞数减少,差异具有统计学意义(0.83±0.17vs1.00±0.08,P=0.011)。与空白对照组相比,用0.5mM的OA孵育的HepG2细胞内甘油三酯的含量增加(3.13±0.27vs1.00±0.00,P<0.001)；中高浓度杜仲木脂素可以抑制HepG2细胞内甘油三酯的聚集(2.25±0.12νs3.13±O.27,P<0.000；2.42±0.13νs3.13±0.27,P<0.001).与正常对照组相比,模型组的CPTlA mRNA表达增加,差异具有统计学意义(1.62±0.07νs1.00±0.02.P<0.001)；PPARα mRNA表达减少(0.85±0.10vs1.00±0.03,P=0.019)；与模型组相比,低中高不同浓度的杜仲木脂素组可增加CPTlAmRNA的表达,并具有剂量依赖性,差异具有统计学意义(1.89±0.07,2.06±0.11,2.26±0.11νs1.62±0.07,P=0.013,P<0.001,P<0.001)；PPARαmRNA表达增加,差异具有统计学意义(1.06±0.05,1.19±0.03,1.24±0.06νs0.85±0.10,P=0.002,P<0.001,P<0.001)；与模型组相比,非诺贝特组可显著增加CPTlA.FAS和PPARα mRNA的表达,差异具有统计学意义(2.78±0.21νs1.62±0.07, P=0.001；1.53±0.15νs0.88±0.19,P<0.001；0.97±0.09νs0.85±0.10,P=0.046).与正常对照组相比,模型组的CPTlA蛋白表达增加(1.14±0.03νs1.00±0.01,P<0.001),PPARα表达增加(1.22±0.01vs1.00±0.03,P<0.001),差异具有统计学意义；与模型组相比,低中高不同浓度的杜仲木脂素组可促进CPTlA(1.42±0.04,1.90±0.01,2.04±0.04νs1.14±0.03, P<0.001,P<0.001,P<0.001).FAS(1.11±0.01,1.39±0.01,1.50±0.03νs1.00±0.01,P=0.011,P<0.001,P=0.011)和PPARα(1.32±0.03,1.52±0.01,1.83±0.04νs1.22±0.01,P<0.001)的蛋白表达,差异具有统计学意义；与模型组相比,非诺贝特组可增加CPTlA.FAS和PPARα的蛋白表达(2.51±0.10νs1.14±0.03,P=0.010,1.21±0.01νs1.00±0.01,P=0.002,1.33±0.02νs1.22±0.01,P<0.001).结论：,杜仲木脂素可以抑制OA孵育的HepG2细胞的甘油三酯聚集,其机制可能与激活PPARα,上调CPTlA,促进脂肪酸氧化有关。更多还原

【Abstract】 Background:Hypertriglyceridemia is a major risk factor of cardiovascular diseases. There are few drugs in controlling plasma triglyceride clinically. Owing to the defects of drugs in effect and safety treating hypertriglyceridemia, it is significant to develop triglyceride-lowering drugs. Eucommia ulmoides contains many pharmacological activities:anti-hypertension, lipid-lowering, anti-oxidation, anti-atherosclerosis, and myocardial protection. Eucommia Lignans is active fraction extracted from Eucommia Ulmoides, reducing plasma triglyceride in hypertension patients with hyperlipidemia in clinical application. Investigating the effects and the mechanisms of lignans from Eucommia ulmoides on the metabolism of triglyceride will provide evidence for the developing lipid-lowering of Eucommia Lignans and clinic application.Objective:To investigate the effects and the mechanism of lignans from Eucommia ulmoides on the metabolism of triglyceride in HepG2cells incubated in oleic acid.Method:HepG2cells was used in this research.To observed the effect of Eucommia Lignans on the HepG2cells growth, HepG2cells were cultured in vitro in7groups:control,15mg/L,37.5mg/L,75mg/L,150mg/L,300mg/L,600mg/L treated by lignans; then determinate the A490value by MTT one-step detection to assess the number of cells.HepG2cells were cultured in vitro to evaluate the effects and mechanisms of lignans on the accumulation of triglyceride in HepG2cells in6groups:control, OA model (0.5mM OA), low concentration lignans (0.5mM OA+15mg/L lignans), middle concentration lignans (0.5mM OA+30mg/L lignans), high concentration lignans (0.5mM OA+45mg/L lignans), and fenofibrate group (0.5mM OA+100μmol/L fenofibrate). The determination of triglyceride content in the cells was using kits. The mRNA and protein level of PPARou FAS、CPT1A in HepG2were determined by using real-time quantitative polymerase chain reaction method and Western blot.Result:Lignans from Eucommia ulmoides was safe to the proliferation of HepG2incubated with different concentrations lignans(15mg/L～300mg/L). While600mg/L Eucommia lignans inhibited the proliferation of cells signifcantly(0.83±0.17νs1.00±0.08P=0.011).The content of triglyceride increased in HepG2incubated with0.5mM OA compared with the control,(3.13±0.27νs1.00±0.00,P<0.001)；The middle and high concentration of Eucommia Lignans can suppresse intracellular triglyceride accumulation significantly compared with the model group,(2.25±0.12and2.42±0.13νs3.13±0.27,P<0.0001and P<0.001).0.5mM OA induced the expression of CPTlA mRNA in HepG2significantly compared with control(1.62±0.071νs1.00±0.02,P<0.001), while suppressing the PPARα mRNA(0.85±0.10νs1.00±0.03,P=0.019)；The Eucommia Lignans can increase the CPT1A mRNA in different concentration in a dose-dependent manner significantly compared with the model group,(1.89士0.07,2.06±0.11,2.26±0.11νs1.62±0.07,P=0.013,P<0.001,P<0.001)； and PPARα[mRNA in lignans group was increased(1.06±0.05,1.19±0.03,1.24±0.06νs0.85±0.10,P=0.002,P<0.001,P<0.001)；Compared with the model group,fenofibrate increased the level of CPTlA and FAS mRNA(2.78±0.21vs1.62±0.07,P=0.001；1.53±0.15νs0.88±0.19,P<0.001；0.97±0.09vs0.85±0.10,P=0.046),significantly.The protein level of CPTlA and PPARα induced by OA was increased significantly(1.14±0.03νs1.00±0.01,P<0.001and1.22±0.01νs1.00±0.03,P<0.001)；Different concentration of Eucommia Lignans increased the protein level of CPTlA,FAS,PPARα significantly,compared with the model group,(1.42±0.04,1.90±0.01,2.04±0.04νs1.14±0.03,P<0.001,P<0.001, P<0.001in CPTlA；1.11±0.01,1.39±0.01,1.50±0.03νs1.00±0.01,P=0.011,P<0.001,P=0.011in FAS；1.32±0.03,1.52±0.01,1.83±0.04νs1.22±0.01,P<0.001in PPARα)；fenofibrate increased the expression of CPTlA, FAS and PPARα in protein significantly compared with the OA group,(2.51± 0.10νs1.14±0.03, P=0.010,1.21±0.01νs1.00±0.01, P=0.002,1.33±0.02vs1.22±0.01, P<0.001).Conclusions:Lignans from Eucommia ulmoides can suppress accumulation of intracellular triglyceride in HepG2cells, which may be related to the activation of PPARa and the increase of CPTlA, induced the P-oxidation of fatty acid.更多还原