GPs 抑 制 mirna122 表 达 及 调 节 脂 代 谢 酶 活 性 降 血 脂 作 用 研 究 1 2 2 1 (1. 遵 义 医 学 院 遵 义 563004;2. 遵 义 医 学 院 附 属 医 院 遵 义 563004) 摘 要 Gypenosides GPs mirna122 48 SD GPs G C 3 GPs 0.3% CMC-Na 0.3% CMC-Na 1 ml/100 g G GPs 160 mg kg -1 5 mg kg -1 8 12 h 7% TC TG HDL-C LDL-C RNA Real time-pcr mirna-122 HL LPL HMG-CoA C M TC TG LDL-C P<0.01 HDL-C P<0.05 M S G TC TG LDL-C P<0.05 HDL-C P<0.05 M S G P<0.05 M S G mirna-122 P<0.05 M S G HMG-CoA P<0.05 HL LPL P<0.05 GPs GPs mirna-122 关 键 词 mirna-122 doi 10.11842/wst.2015.08.016 R543.5 A Hyperlipidemia [4,5] [1-3] Gynostemma pentaphyllum GP Gypenosides GPs GP GPs 2015-03-27 2015-04-01 Triglyceride TG Total Cholesterol TC Low Density Lipoprotein Cholesterol LDL-C J 2009 2-178 World Science and Technology/Modernization of Traditional Chinese Medicine and Materia Medica 1679
2015 Vol.17 No.8 High Density Lipoprotein HDL [6-10] GPs 160 mg kg -1 8 1.5 检 测 指 标 及 检 测 方 法 1.5.1 动 物 处 置 及 标 本 采 集 GPs 12 h 7% RNA microrna mirna 5 ml 4 GPs mirna-122 GPs 1.5.2 大 鼠 血 脂 的 测 定 1 材 料 与 方 法 1.1 实 验 动 物 SD SPF 60 200 1.5.3 大 鼠 肝 指 数 的 测 定 20 g = / 100% SCXK 2012-0005 12 h 1.5.4 Real time PCR 检 测 大 鼠 肝 脏 mirna-122 1.2 主 要 实 验 材 料 GPs Trizol 98.06% Trizol 3136568 Trizol Invitrogen USA RT- PCR TakaRa 2014AK5301 HL LPL HMG-CoA GAACGATACAGAGAAGATT-3 U6 snrna R 20130410-37-902 20130410-37-903 20130410-37-904 1.3 主 要 仪 器 设 备 Thermo USA PCR Eppendorf CFX96 PCR 95 3 min 40 cycles 95 12 s 62 40 s U6 BIO-RAD mirna-122 Tokyo Eppendorf Real-time PCR Ct 2 ΔΔCT 1.4 实 验 分 组 及 模 型 建 立 1 1.5.5 大 鼠 肝 脏 肝 脂 肪 酶 (Hepatic Lipase,HL) C M 脂 蛋 白 本 酶 (Lipoprteinlipase,LPL) 3- 羟 基 -3- S G 4 C 甲 基 戊 二 酰 辅 酶 A(3-hydroxy-3-methy glutaryl 10% coenzyme A,HMG-CoA) 还 原 酶 活 性 的 测 定 2% 5% 0.5% 0.2% 82.3% 4 3 000 rpm 10 min 10 min 3 000 rpm 10 min [11] GPs 0.3% CMC-Na HL LPL HMG-CoA Na 30 mg kg -1 S 5 mg kg -1 G 4 3 000 rpm 15 min TC TG HDL-C LDL-C 的 表 达 RNA RT-PCR cdna 20 μl mirna-122 C M 0.3% CMC- PCR mirna-122 U6 snrna F Primer 5 -ATTG- Primer 5 -GGAACGCTTCACGAATTTG-3 hsamir-122 F Primer 5 -CAAGCGTTGGAGTGTGACA- 3 hsa-mir-122 R Primer: 5 -CGTCCTACCATTCT- CCAGC-3 25 30 min 42 30 min 85 5 min 4 hold Real time PCR PBS 1680 World Science and Technology/Modernization of Traditional Chinese Medicine and Materia Medica
1.5.6 GPs 对 离 体 胆 固 醇 微 胶 粒 形 成 的 抑 制 作 用 1 1 C M TC TC 5 TG LDL-C P<0.01 HDL-C mg kg -1 GPs 40 mg kg -1 80 mg kg -1 160 mg kg -1 +GPs G TC TG LDL-C P<0.05 6 15 ml 0.1 ml HDL-C P<0.05 2.5 mg 2.2 GPs 对 高 血 脂 模 型 大 鼠 肝 指 数 变 化 的 影 响 2 ml TC 2 2 TC P<0.01 GPs G 5.0 ml ph=6.2 37 P<0.05 P<0.05 M 1 h 4 8 000 rpm 30 min 2.3 GPs 对 高 血 脂 模 型 大 鼠 肝 脏 mirna-122 表 达 的 影 响 U6 Real time-pcr mirna-122 3 3 1.6 统 计 学 处 理 mirna-122 P<0.01 S G SPSS17.0 mirna-122 P<0.05 G mirna-122 LSD P<0.05 2.4 GPs 对 高 血 脂 模 型 大 鼠 肝 脏 HL LPL HMG- P<0.01 CoA 还 原 酶 活 性 的 影 响 ELISA HL LPL HMG- 2 结 果 CoA 4 4 S 2.1 GPs 对 高 血 脂 模 型 大 鼠 血 清 血 脂 水 平 的 影 响 HMG-CoA P<0.01 HL LPL P<0.01 表 1 各 组 大 鼠 血 脂 水 平 (mmol L -1, ) n TG TC LDL-C HDL-C C 10 0.43 0.12 2.26 0.20 0.53 0.10 1.69 0.10 M 10 1.39 0.07 ** 12.62 1.95 ** 10.37 1.33 ** 1.35 0.32 * S 10 0.59 0.18 6.57 2.44 4.84 1.93 1.53 0.28 G 11 0.77 0.26 6.89 1.91 5.15 1.67 1.57 0.20 C * P<0.05 P<0.01 M P<0.05 表 2 各 组 大 鼠 肝 指 数 的 变 化 ( ) n /% C 10 2.74 0.14 M 11 4.11 0.17 ** S 11 3.28 0.24 G 11 3.37 0.31 ** C P<0.01 M P<0.05 图 1 各 组 大 鼠 血 清 的 血 脂 水 平 图 2 各 组 大 鼠 肝 指 数 的 变 化 World Science and Technology/Modernization of Traditional Chinese Medicine and Materia Medica 1681
2015 Vol.17 No.8 表 3 各 组 大 鼠 肝 脏 mirna-122 表 达 的 变 化 ( ) n mirna-122/u6 C 10 1.029 0.434 M 11 3.516 0.778 ** S 11 2.043 0.550 G 11 2.691 0.732 ** C P<0.01 M P<0.05 图 3 各 组 大 鼠 肝 脏 肝 脏 mirna-122 表 达 的 变 化 表 4 各 组 大 鼠 肝 脏 HL LPL HMG-CoA 还 原 酶 活 性 (U ml -1, ) n HL LPL HMG-CoA C 10 1.813 0.360 3.280 0.099 1.929 0.188 M 11 0.701 0.110 ** 1.737 0.534 ** 7.359 1.807 ** S 11 1.151 0.104 2.576 0.062 3.932 0.234 G 11 0.967 0.077 2.464 0.053 4.146 0.259 ** C P<0.01 M P<0.05 G HMG-CoA P<0.05 HL LPL P<0.05 G HL LPL HMG-CoA 图 4 各 组 大 鼠 肝 脏 HL LPL HMG-CoA 还 原 酶 活 性 2.5 GPs 对 离 体 胆 固 醇 微 胶 粒 形 成 的 抑 制 作 用 的 影 响 GPs A mg mg 100% GPs TC 0.399 0.989 1.511 S 0.628 1.557 0.943 5 37.566 G-L 0.490 1.215 1.285 14.928 G-M 0.570 1.413 1.087 28.051 1.511 mg G-H 0.620 1.537 0.963 36.254 0.05% GHS 0.713 1.767 0.733 51.510 GPs GPs TC P<0.05 P<0.01. 表 5 GPs 对 离 体 胆 固 醇 微 胶 粒 形 成 的 抑 制 作 用 的 影 响 1682 World Science and Technology/Modernization of Traditional Chinese Medicine and Materia Medica
P<0.05 mirna-122 GPs GPs1.60% mirna-122 36.254% GPs mirna-122 GPs 51.510 P<0.01 3 讨 论 TG C M TG TC LDL-C P<0.01 M CoA P<0.01 HMG-CoA M G TC TG LDL-C P<0.05 HL P<0.05 HL GPs TG Phospholipid PL LPL mirna VLDL β-vldl VLDL RNA TG mrna mrna [12,13] GPs HMG-CoA mirna P<0.05 HL LPL P<0.05 [14] mirna-122 GPs mirna 70% mirna-122 [15] mirna-122 GPs [16] M G mirna-122 LPL TG CM VLDL HMG- GPs 参 考 文 献 1,,., 2011, 33(3): 260-262. LDL. -, 2014, 4 Dopazo C, Bilbao I, Lázaro J L et al. Severe rhabdomyolysis and 16(2): 393-397. 2,,,.. rapamycin plus tacrolimus in liver transplant patient. Transplant Proc, -, 2008, 10(1): 103-106. acute renal failure secondary to concomitant use of simvastatin with 2009, 41(3): 1021-1024. 3,,.. 5,.., World Science and Technology/Modernization of Traditional Chinese Medicine and Materia Medica 1683
2015 Vol.17 No.8 2009, 18(4): 612-614. 6..., 2010, 30(4): 403-406., 2007, 29(15): 1497-1499. 7,,,., 2013, 25(7): 1446-1452.., 2008, 24(2): 13,,,.MicroRNA. 205-208. 8,,,. 14 Elmen J, Lindow M, Schutz S, et al. LNA-mediated microrna., 2014, 28(2): 60-64. 9,,,. 15 Krützfeldt J, Rajewsky N, Braich R, et al. Silencing of micrornas in. vivo with antagomirs. Nature, 2005, 438(7068): 685-689., 2013, 11(6): 655-657. 10,. metabolism revealed by in vivo antisense targeting. Cell Metabolism,., 2011, 35(1): 37-39. 11,. 12,,,.MiRNA. ( ), 2013, 38(3): 318-322. silencing in non-human primates. Nature, 2008, 452(7189): 896-899. 16 Esau C, Davis S, Murray S F, et al. MiR-122 regulation of lipid 2006, 3(2): 87-98. Research on GPs Inhibition of mirna-122 Expression and Lipid-lowering Effect Via Regulation of Lipid Metabolism Enzyme Activity Wu Liusong 1,2, Feng Yonghuai 2, Qian Minzhang 1 (1. Zunyi Medical College, Zunyi 563004, China; 2. Affiliated Hospital of Zunyi Medical College, Zunyi 563004, China) Abstract: This study was aimed to explore whether gypenosides (GPs) can inhibit the expression of mirna-122 and regulate the lipid metabolism enzyme activity to play a role in lipid-lowering effect. A total of 48 healthy male SD rats were randomly divided into 4 groups, which were the normal control group (C), hyperlipidemic model group (M), simvastatin group (S) and the GPs group (G). All groups were fed with high-fat diets except the normal control group which was fed with normal diets. The GPs, which were dissolved in 0.3% sodium carboxymethyl cellulose (CMC-Na) solution, were given by the intragastric administration. The C group and M group were given 0.3% CMC-Na solution (1 ml/100 g) daily. The G group was given 160 mg kg -1 of GPs daily. The S group was given 5 mg kg -1 of simvastatin daily. The experiment was continued for 8 weeks. After the last medication, rats were fasted for 12 hours. Rats were anesthetized with chloral hydrate (7%). Abdominal arterial blood samples were collected to detect the total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C). The wet weight of liver was weighed and the liver index was measured. The liver total RNA was extracted to determine the expression of mirna-122 by the real-time PCR. The homogenates of liver tissues were prepared for the determination of hepatic lipase (HL), lipoprotein lipase (LPL) and HMG-CoA reductase activity. Cholesterol micelle formation experiments were implemented in vitro. The results showed that compared with the normal control group, TC, TG and LDL-C levels of the model group were significantly increased (P < 0.01), while the HDL-C levels in each group were obviously decreased (P < 0.05). Compared with the model group, TC, TG and LDL-C levels of the S group and G group were obviously decreased (P < 0.05), and the HDL-C level was obviously increased (P < 0.05). Compared with the model group, the liver indexes of the S group and G group were obviously decreased (P < 0.05). Compared with the hyperlipidemia model group, the expressions of mirna-122 of the S group and G group were significantly reduced (P < 0.05). Compared with the hyperlipidemia model group, the activity of HMG-CoA reductase was obviously reduced in the S group and G group (P < 0.05), but the HL and LPL activities were obviously increased (P < 0.05). GPs can 1684 World Science and Technology/Modernization of Traditional Chinese Medicine and Materia Medica
inhibit the formation of cholesterol micelles to some extent. It was concluded that GPs can effectively reduce the blood lipid level in hyperlipidemic rats, in order to relieve the hepatic fatty lesions. Its lipid-lowering mechanism was related to its inhibition of mirna-122 expression and regulation of lipid metabolism enzyme activity, as well as the inhibition on the formation of cholesterol micelles. Keywords: Gypenosides, hyperlipidemia, mirna-122, lipid metabolism enzyme World Science and Technology/Modernization of Traditional Chinese Medicine and Materia Medica 1685