番石榴苷通过激活AdipoR-1/AMPK信号通路减轻大鼠肝细胞瘤H4ⅡE细胞脂质沉积的作用机制研究
|
摘要
目的:观察番石榴苷(guaijaverin, GAN)对油酸诱导的大鼠H4ⅡE细胞脂质沉积的作用,并从的油酸诱导大鼠H4ⅡE细胞脂质沉积模型,采用0.3 mM的番石榴苷干预24小时,采用GPO?DAOS评估细胞甘油三酯的含量,以DCTM法检测总蛋白含量,以甘油三酯/蛋白含量评估脂质沉积的情况;同时采用western blotting法检测肝细胞内乙酰辅酶A羧化酶(Acetyl-CoA arboxylase,ACC)以及其各自磷酸化蛋白表达水平;以实时荧光定量PCR法检测细胞AdipoR-1mRNA含量。结果:番石榴苷具有减少肝细胞内脂质沉积、提高AMPK、ACC磷酸化水平、提高AdipoR-1 mRNA相对表达量的作用,且其减少肝细胞内脂质沉积作用可被AMPK阻滞剂Compound C所逆转。结论:番石榴苷可改善油酸诱导的大鼠H4ⅡE细胞内脂质沉积,其作用机制与通过激活AdipoR-1/AMPK信号通路有关。
Objective: To observe the effect of GAN(guaijaverin) on oleic acid induced lipid deposition in rat H4 IIE cells,and to explore its action mechanism from AdipoR-1/AMPK signaling pathway. Methods: Used 0.2 mmol/L oleic acid to induce rat H4 IIE cells lipid deposition model. Used 0.3 mM GAN to intervene for 24 hours. Used GPO?DAOS to evaluate the level of triglyceride in cells. Measured total protein level using DCTM assay. Adopted triglyceride/protein level to assess the degree of lipid deposition. Adopted Western blotting method to detect AMPK ACC, and their respective phosphorylated protein expression level in hepatocytes. Adopted Real-time fluorescent quantitative PCR method to detect cellular AdipoR-1 mRNA level. Results: GAN had the function of reducing the lipid deposition in hepatocytes, increasing the phosphorylation level of AMPK and ACC, and increasing the relative expression of AdipoR-1 mRNA. Its effect on reducing lipid deposition in hepatocytes could be reversed by AMPK blocker Compound C.Conclusion: GAN could improve the lipid deposition in rat H4 IIE cells induced by oleic acid, and its mechanism was related to the activation of AdipoR-1/AMPK signaling pathway.
Objective: To observe the effect of GAN(guaijaverin) on oleic acid induced lipid deposition in rat H4 IIE cells,and to explore its action mechanism from AdipoR-1/AMPK signaling pathway. Methods: Used 0.2 mmol/L oleic acid to induce rat H4 IIE cells lipid deposition model. Used 0.3 mM GAN to intervene for 24 hours. Used GPO?DAOS to evaluate the level of triglyceride in cells. Measured total protein level using DCTM assay. Adopted triglyceride/protein level to assess the degree of lipid deposition. Adopted Western blotting method to detect AMPK ACC, and their respective phosphorylated protein expression level in hepatocytes. Adopted Real-time fluorescent quantitative PCR method to detect cellular AdipoR-1 mRNA level. Results: GAN had the function of reducing the lipid deposition in hepatocytes, increasing the phosphorylation level of AMPK and ACC, and increasing the relative expression of AdipoR-1 mRNA. Its effect on reducing lipid deposition in hepatocytes could be reversed by AMPK blocker Compound C.Conclusion: GAN could improve the lipid deposition in rat H4 IIE cells induced by oleic acid, and its mechanism was related to the activation of AdipoR-1/AMPK signaling pathway.
引文
1中华医学会肝病学分会脂肪肝和酒精性肝病学组.非酒精性脂肪性肝病诊治指南.中华肝病杂志, 2010, 18(3):163-166.
2 郭翔宇,刘铜华,朱寅荻等.番石榴叶化学成分及其治疗2型糖尿病研究进展.世界科学技术-中医药现代化, 2014, 16(5):1029-1034.
3 Guo, Xiangyu, Yoshitomi, Hisae, Gao, et al. Guava leaf extracts promote glucose metabolism in SHRSP.Z-Leprfa/Izm rats by improving insulin resistance in skeletal muscle, BMC Complement Altern Med, 2013, 13:52-52.
4 Yoshitomi H, Guo X, Liu T, et al. Guava leaf extracts alleviate fatty liver via expression of adiponectin receptors in SHRSP.Z-Leprfa/Izm rats,Nutr Metab(Lond). 2012 Feb 20; 9:13
5 陈标,满玉蓉,高柳玲,等. AMPK调控能量代谢研究进展.生物学杂志, 2017, 34(5):78-82.
6 Chen L, Xin FJ, Wang J, et al. Conserved regulatory elements in AMPK[J/OL]. Nature, 2013, 498(7453):E8-10. doi:10.1038/nature12189.
7 侯祥红.共轭亚油酸对HepG2细胞脂质代谢的影响及其机制研究.西安:第四军医大学, 2009.
8 王瑾.外周大麻素Ⅰ型受体抑制剂的筛选及减肥作用机制的研究.镇江:江苏大学, 2016.
9 Oakhill JS, Chen ZP, Scott JW,et al.β-Subunit myristoylation is the gatekeeper for initiating metabolic stress sensing by AMP-activated protein kinase(AMPK). Proc Natl Acad SciUSA, 2010, 107(45):19237-19241.
10 安平,王安平,母义明.脂联素与胰岛素抵抗研究进展.生物技术通讯, 2017, 28(3):360-365.
11 Wojtaszewski JF, Hansen BF, Kien B, et al. Insulin signaling in human skeletal muscle time course and effect of exercise. Diabetes, 1997, 46(11):1775-1781.
12 Jackson S, Bataff S, Yeasman S J, et al. Decreased insulin responsiveness of glucose uptake in cultured human skelet al muscle cells from insulin resistant nondiabetic relatives of Type2 diabetic families.Diabetes,2000, 49(7):1169-1177.
13 Awazawa M, Ueki K, Inabe K, et al. Kadowaki T:Adiponectin suppresses hepatic SREBP1c expression in an AdipoR1/LKB1/AMPK dependent pathway. Biochem Biophys Res Commun 2009, 382:51-56.
14 李璇,杨蕾,王娟飞,等.番石榴叶提取物对高脂性肥胖小鼠的减肥降脂作用的研究.现代生物医学进展. 2012, 12(31):6001-6005.
2 郭翔宇,刘铜华,朱寅荻等.番石榴叶化学成分及其治疗2型糖尿病研究进展.世界科学技术-中医药现代化, 2014, 16(5):1029-1034.
3 Guo, Xiangyu, Yoshitomi, Hisae, Gao, et al. Guava leaf extracts promote glucose metabolism in SHRSP.Z-Leprfa/Izm rats by improving insulin resistance in skeletal muscle, BMC Complement Altern Med, 2013, 13:52-52.
4 Yoshitomi H, Guo X, Liu T, et al. Guava leaf extracts alleviate fatty liver via expression of adiponectin receptors in SHRSP.Z-Leprfa/Izm rats,Nutr Metab(Lond). 2012 Feb 20; 9:13
5 陈标,满玉蓉,高柳玲,等. AMPK调控能量代谢研究进展.生物学杂志, 2017, 34(5):78-82.
6 Chen L, Xin FJ, Wang J, et al. Conserved regulatory elements in AMPK[J/OL]. Nature, 2013, 498(7453):E8-10. doi:10.1038/nature12189.
7 侯祥红.共轭亚油酸对HepG2细胞脂质代谢的影响及其机制研究.西安:第四军医大学, 2009.
8 王瑾.外周大麻素Ⅰ型受体抑制剂的筛选及减肥作用机制的研究.镇江:江苏大学, 2016.
9 Oakhill JS, Chen ZP, Scott JW,et al.β-Subunit myristoylation is the gatekeeper for initiating metabolic stress sensing by AMP-activated protein kinase(AMPK). Proc Natl Acad SciUSA, 2010, 107(45):19237-19241.
10 安平,王安平,母义明.脂联素与胰岛素抵抗研究进展.生物技术通讯, 2017, 28(3):360-365.
11 Wojtaszewski JF, Hansen BF, Kien B, et al. Insulin signaling in human skeletal muscle time course and effect of exercise. Diabetes, 1997, 46(11):1775-1781.
12 Jackson S, Bataff S, Yeasman S J, et al. Decreased insulin responsiveness of glucose uptake in cultured human skelet al muscle cells from insulin resistant nondiabetic relatives of Type2 diabetic families.Diabetes,2000, 49(7):1169-1177.
13 Awazawa M, Ueki K, Inabe K, et al. Kadowaki T:Adiponectin suppresses hepatic SREBP1c expression in an AdipoR1/LKB1/AMPK dependent pathway. Biochem Biophys Res Commun 2009, 382:51-56.
14 李璇,杨蕾,王娟飞,等.番石榴叶提取物对高脂性肥胖小鼠的减肥降脂作用的研究.现代生物医学进展. 2012, 12(31):6001-6005.