松花粉提取物对胰岛素抵抗HepG2细胞糖脂代谢的影响
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摘要
研究松花粉提取物对胰岛素抵抗Hep G2细胞糖脂代谢的影响,探究其潜在分子机制。采用游离脂肪酸构建Hep G2细胞胰岛素抵抗模型,经油红O染色确认模型成功后,采用MTT法研究不同极性的松花粉提取物对细胞增殖的影响。测定各组上清葡萄糖,胞内糖原,上清甘油三酯(TG),胞内TG以及胞内总胆固醇(TC)的含量,实时荧光PCR定量检测细胞裂解液中的调控糖脂代谢关键基因In SR、PI3K以及GLUT-4的mRNA表达水平。结果表明,不同极性的松花粉提取物在质量浓度为100～500μg/m L时,对细胞无明显毒性;不同极性的松花粉提取物能够促进细胞吸收葡萄糖,抑制糖原降解,抑制TG和TC的形成,促进TG向细胞外转运,从而改善Hep G2胰岛素抵抗状况。各松花粉处理组细胞PI3K以及GLUT-4 mRNA表达量均上调,说明松花粉能够激活PI3K/Akt/GLUT-4通路从而促进糖代谢;乙酸乙酯相(AE),正丁醇相(BU)和水相(WT)组细胞In SR mRNA表达量显著上调,说明这3种提取物能够通过提高胰岛素的敏感性改善胰岛素抵抗。
This study aimed to evaluate the effects of pine pollen extracts on glycolipid metabolism in insulin-resistant HepG2 cells,and explore its underlying molecular mechanisms. The insulin-resistant HepG2 cell model was established by free fatty acids and validated using Oil red O staining. MTT assay was used to study the effects of pine pollen extracts with different polarities on cell proliferation. Levels of glucose,glycogen,and triglycerides( TG) in cell supernatants were measured. TG and total cholesterol( TC) in cytoplasm were also determined. Quantitative realtime PCR( qRT-PCR) was used to detect the mRNA expression levels of glucose and lipid metabolism-related genes in cell lysis buffer,such as InSR,PI3 K and GLUT-4. The results showed that there were no obvious toxic effects of pine pollen extracts on HepG2 cells at 100-500 μg/m L. The absorption of glucose and transhipment of TG were promoted,and the degradation of glycogen and formations of TG and TC were suppressed after pine pollen extracts treatment,indicating that the insulin-resistance of HepG2 was improved. Meanwhile,the mRNA expressions of PI3 K and GLUT-4 significantly increased in all pine pollen extracts treatment groups,which means that pine pollen could promote glucose metabolism by activating the PI3 K/Akt/GLUT-4 pathway. It was also found that pine pollen after ethyl acetate,butanol and water treatments could attenuate insulin resistance through enhancing insulin sensitivity,because the mRNA expression of InSR up-regulated significantly among these three groups.
This study aimed to evaluate the effects of pine pollen extracts on glycolipid metabolism in insulin-resistant HepG2 cells,and explore its underlying molecular mechanisms. The insulin-resistant HepG2 cell model was established by free fatty acids and validated using Oil red O staining. MTT assay was used to study the effects of pine pollen extracts with different polarities on cell proliferation. Levels of glucose,glycogen,and triglycerides( TG) in cell supernatants were measured. TG and total cholesterol( TC) in cytoplasm were also determined. Quantitative realtime PCR( qRT-PCR) was used to detect the mRNA expression levels of glucose and lipid metabolism-related genes in cell lysis buffer,such as InSR,PI3 K and GLUT-4. The results showed that there were no obvious toxic effects of pine pollen extracts on HepG2 cells at 100-500 μg/m L. The absorption of glucose and transhipment of TG were promoted,and the degradation of glycogen and formations of TG and TC were suppressed after pine pollen extracts treatment,indicating that the insulin-resistance of HepG2 was improved. Meanwhile,the mRNA expressions of PI3 K and GLUT-4 significantly increased in all pine pollen extracts treatment groups,which means that pine pollen could promote glucose metabolism by activating the PI3 K/Akt/GLUT-4 pathway. It was also found that pine pollen after ethyl acetate,butanol and water treatments could attenuate insulin resistance through enhancing insulin sensitivity,because the mRNA expression of InSR up-regulated significantly among these three groups.
引文
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[29]龙琼先,吴才良,唐治蓉,等.卵巢上皮性癌中血管内皮生长因子-C和酪氨酸激酶受体B表达与微淋巴管密度的关系[J].四川医学,2015(7):927-930.
[30]高婷,王子旭,陈祝茗,等. ROS介导的氧化应激与自噬[J].中国畜牧兽医,2018,45(3):656-662.
[2] YALOW R S,et al. Immunoassay of endogenous plasma insulin inman[J]. J Clin Invest,1960,39(7):1 157-1 175.
[3]祝红梅. Fox O1在氧化应激诱导糖异生/胰岛素抵抗机制中的作用[D].泸州:西南医科大学泸州医学院,2011.
[4] MAHADEV K,MOTOSHIMA H,WU Xiangdong,et al.The NAD(P)H oxidase homolog Nox4 modulates insulinstimulated generation of H202 and plays an integral role in insulin signal transduction[J]. Mol Cell Biol,2004,24(5):1 844-1 854.
[5] AVRAMOGLU R K,BASEIANO H,ADELI K. Lipid and lipoprotein dysregulation in insulin resistant states[J].Clinica Chimica Acta,2006,368(1-2):l-19
[6] LI Y,WANG J,GU T,et al. Oleanolic acid supplement attenuates liquid fructose-induced adipose tissue insulin resistance through the insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt signaling pathway in rats[J].Toxicol Appl Pharmacol,2014,277(2):155-163.
[7]赵金香,胡章云,李耀华,等.菊粉联合游泳训练对代谢综合征大鼠骨骼肌胰岛素受体底物1及葡萄糖转运子4表达的影响[J].中国糖尿病杂志,2017,25(9):831-835.
[8]蔡双朋,李俊,黄成,等.丹参酮Ⅰ对Hep G2细胞胰岛素抵抗作用及机制的研究[J].安徽医科大学学报,2016,51(7):974-977.
[9] MATSUDA S,NAKANISHI A,WADA Y,et al. Roles of PI3K/AKT/PTEN pathway as a target for pharmaceutical therapy[J]. Open Med Chem J,2013,7:23-29.
[10]石明隽,肖瑛,桂华珍,等.糖尿病大鼠肾组织中AKT的表达及意义[J].中国老年学杂志,2011,31(22):4 375-4 378.
[11] ALBURY-WARREN T M,PANDEY V,SPINEL L P,et al. Prediabetes linked to excess glucagon in transgenic mice with pancreatic active AKT1[J]. J Endocrinol,2016,228(1):49-59.
[12]樊柏林,刘烈刚.破壁松花粉对大鼠降血脂效应及作用机制研究[J].职业与健康,2005,21(6):809-811.
[13]潘小玲,竹剑平.破壁松花粉的降血糖作用[J].中国医院药学杂志,2006,26(6):777-778.
[14]毛永强.松花粉的营养成分及保健功能综述[J].中国食物与营养,2008(3):50-52.
[15]刘杰尔.松花粉成分分析及其轻身减肥功能初探[D].杭州:浙江大学,2008.
[16] CAI Y Z,LUO Q,SUN M,et al. Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer[J]. Life Science,2004,74:2 157-2 184.
[17]俞卓裕.竹叶药用有效部位提取、分离、精制工艺技术的研究[D].杭州:浙江大学,2003.
[18]郑婧.肉桂多酚对Hep G2细胞脂质代谢的影响及其机制[J].山东医药,2017(1):38-40.
[19]欧露,麻燕妮,张彩平,等.烟酸通过下调PCSK9的表达促进Hep G2细胞摄取LDL-C[J].中国药理学通报,2017(2):243-248.
[20]李欣,王帅,周岳,等.羟基肉桂酸对Hep G2和C2C12细胞糖脂代谢的调节作用[J].国际药学研究杂志,2017(7):693-698.
[21]陈亚蓝,王雪青,王怡雯,等.普洱茶茶色素对Hep G2细胞脂质代谢的影响及作用机理[J].食品科学,2017,38(17):203-209.
[22] SHULMAN G I. Cellular mechanisms of insulin resistance[J]. Journal of Clinical Investigation,2000,106(2):171-176.
[23] KNOWLES B B,HOWE C C,ADEN D P. Human hepatocellular careinoma cell lines secrete the major plasma protein and hepatitis B surface antigen[J]. Science,1980,209(4 455):497-499.
[24]柳嘉,郭孝萱,吴薇,等.高糖高脂诱导胰岛素抵抗Hep G2细胞模型的建立及活性成分的功能评价[J].食品科技,2012,37(3):73-78.
[25] PICKUP J C. Inflammation and activated innate immunity in the pathogenesis of type2diabetes[J]. Diabetes Care,2004,27(3):813-823.
[26] NOLAN C J,RUDERMAN N B,KAHN S E,et al. Insulin resistance as a physiological defense against metabolic stress:implications for the management of subsets of type 2 diabetes[J]. Diabetes,2015,64(3):673-686.
[27]刘颐轩,臧莎莎,宋光耀.二甲双胍分子机制的研究进展[J].中国糖尿病杂志,2015,23(4):375-377.
[28] XIAO B,SANDERS M J,UNDERWOOD E,et al.Structure of mammalian AMPK and its regulation by ADP[J]. Nature,2011,472(7342):230-233.
[29]龙琼先,吴才良,唐治蓉,等.卵巢上皮性癌中血管内皮生长因子-C和酪氨酸激酶受体B表达与微淋巴管密度的关系[J].四川医学,2015(7):927-930.
[30]高婷,王子旭,陈祝茗,等. ROS介导的氧化应激与自噬[J].中国畜牧兽医,2018,45(3):656-662.