中国中药杂志

2020, v.45(21) 5265-5272

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土鳖虫活性肽DP17对高脂血症大鼠的降脂作用机制研究
Study on lipid-lowering mechanism of active peptide DP17 from Eupolyphaga steleophaga in hyperlipidemia rats

姜珊;董萍萍;李浩然;徐静;李华健;于盈盈;代龙;高鹏;王少平;张加余;
JIANG Shan;DONG Ping-ping;LI Hao-ran;XU Jing;LI Hua-jian;YU Ying-ying;DAI Long;GAO Peng;WANG Shao-ping;ZHANG Jia-yu;School of Pharmacy, Binzhou Medical University;School of Pharmacy,Shandong University of Traditional Chinese Medicine;Yantai Affiliated Hospital of Binzhou Medical University;

摘要(Abstract):

研究土鳖虫活性肽DP17治疗高脂血症大鼠的作用机制。运用HPLC和MADIL-TOF/TOF-MS对经仿生酶解、分离纯化得到的土鳖虫活性肽组分进行氨基酸序列分析并对其进行固相合成。采用高脂饲料诱导建立高脂血症SD大鼠模型。造模20 d后,灌胃给药,空白组与模型组给予生理盐水,其余组给予相应药物。给药4周后,检测大鼠血清甘油三酯(TG)、总胆固醇(TC)和低密度脂蛋白(LDL)的含量,肝脏组织匀浆液TG、TC、一磷酸腺苷(AMP)和三磷酸腺苷(ATP)含量以及粪便TG含量;苏木素-伊红(HE)染色观察肝脏组织病理变化;采用实时荧光定量PCR法检测肝脏乙酰辅酶A羧化酶(ACC)和羧甲戊二酸辅酶A还原酶(HMGCR) mRNA表达情况;Western blot法检测肝脏腺苷酸活化蛋白激酶(AMPK)、雷帕霉素靶蛋白复合物1(mTORC1)蛋白的表达。经分析,土鳖虫活性肽单体氨基酸序列为DAVPGAGPAGCHPGAGP(DP17)。药理实验结果表明,DP17给药后,大鼠血清TG,TC和LDL含量以及肝脏组织匀浆液TG和TC含量均显著下降(P<0.05);肝脏组织匀浆液AMP,ATP含量和粪便TG含量显著升高(P<0.05);大鼠肝脏脂肪变性明显减轻;肝脏ACC和HMGCR mRNA表达以及mTORC1蛋白表达显著降低,AMPK磷酸化蛋白表达显著上升(P<0.05)。土鳖虫活性肽DP17可明显抑制大鼠肝脏脂肪堆积现象,主要通过调控机体内能量代谢平衡并激活AMPK/mTOR信号通路来发挥降血脂的作用。
The aim of this paper was to investigate the mechanism of the active peptide DP17 of Eupolyphaga steleophaga in the treatment of hyperlipidemia rats. HPLC and MADIL-TOF/TOF-MS were used for the amino acid sequence analysis and solid-phase synthesis on the active peptide of E. steleophaga which were obtained by biomimetic enzymatic hydrolysis, separation and purification. The hyperlipidemia model was established by feeding with high-fat diet.Twenty days later, the rats in the blank group and the model group were given the saline and the rats in remaining groups were given the corresponding drugs by oral administration. After administration for 4 weeks, the levels of triglyceride(TG), total cholesterol(TC) and low density lipoprotein(LDL) in serum, the levels of TG, TC, adenosine monophosphate(AMP), adenosine triphosphate(ATP) in liver tissues and TG in feces were detected, respectively. Hematoxylin-eosin(HE) staining was used to observe the pathological changes of liver tissues. The Real-time fluorescence quantitative PCR method was used to detect the expression of acetyl coa carboxylase(ACC) and hydroxymethylglutaryl-coa reductase(HMGCR) mRNA in liver tissues. The expression of mammalian target of rapamycin(mTORC1) protein and adenosine 5′-monophosphate-activated protein kinase(AMPK) in liver tissues were detected by Western blot. The analysis showed that the amino acid sequence of active peptide from E. steleophaga was DAVPGAGPAGCHPGAGP(DP17). The results of pharmacological experiments showed that after oral administration of DP17 in rats, the levels of TG, TC and LDL in serum as well as TG and TC levels in liver tissues were significantly decreased(P<0.05), while the levels of AMP, ATP in liver tissues and TG content in feces were significantly increased(P<0.05); the liver steatosis of rats was significantly relieved; the expression of ACC, HMGCR mRNA and mTORC1 protein in liver tissues were significantly reduced, while the expression of AMPK phosphorylated protein was significantly increased(P<0.05). DP17, the active peptide of E. steleophag can significantly reduce lipid accumulation in liver tissues, and it may play a role in reducing blood lipids by regulating the energy metabolism balance in the body and activating AMPK/mTOR signaling pathway.

关键词(KeyWords): 土鳖虫;活性肽;高脂血症;AMPK;mTORC1
Eupolyphaga steleophaga;active peptide;hyperlipidemia;AMPK;mTORC1

Abstract:

Keywords:

基金项目(Foundation): 滨州医学院科研启动项目(BY2018KYQD11,2019KYQD05,2019KYQD06);; 中央本级重大增减支项目(2060302-1907-08);; 山东省中药资源普查项目(2019-1020);; 烟台市校地融合发展项目——中药大健康产业化平台项目(2019XDRHXMPT18);; 山东省青创人才引育计划项目(10073004)

作者(Author): 姜珊;董萍萍;李浩然;徐静;李华健;于盈盈;代龙;高鹏;王少平;张加余;
JIANG Shan;DONG Ping-ping;LI Hao-ran;XU Jing;LI Hua-jian;YU Ying-ying;DAI Long;GAO Peng;WANG Shao-ping;ZHANG Jia-yu;School of Pharmacy, Binzhou Medical University;School of Pharmacy,Shandong University of Traditional Chinese Medicine;Yantai Affiliated Hospital of Binzhou Medical University;

Email:

DOI: 10.19540/j.cnki.cjcmm.20200709.403

参考文献(References):

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