熊果酸对HepG2细胞AMPK和LPIN1表达的影响
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摘要
目的:研究野艾蒿(Artemisia lavandulaefolia DC.)中萜类化合物熊果酸(ursolic acid)对HepG2细胞AMPK和LPIN1表达的影响,从分子水平探明其降血脂的有效成分。方法:以HepG2细胞为研究对象,用细胞毒性实验(MTT法)观察熊果酸在不同用药浓度下对HepG2细胞增殖的影响,并筛选最佳实验条件。将细胞分为对照组和药物组,分别抽提两组细胞总RNA与总蛋白,RT-PCR检测LPIN1,AMPKα1和AMPKα2 mRNA表达量,Western Blot检测LPIN1,AMPKα1,AMPKα2和p-AMPK蛋白表达量。结果:熊果酸可不同程度抑制HepG2细胞的增殖。RT-PCR结果显示,熊果酸AMPKα1,AMPKα2 mRNA表达量与对照组比较均无显著差异(P> 0. 05); LPIN1 mRNA表达量与对照组比较有显著性差异(P <0. 01)。Western Blot结果显示,熊果酸AMPKα1蛋白表达量与对照组比较无显著性差异(P> 0. 05),AMPKα2表达量有显著性差异(P <0. 05); LPIN1表达量有显著性差异(P <0. 01); p-AMPK表达量有显著性差异(P <0. 01),且表达水平升高。结论:熊果酸可不同程度抑制HepG2细胞的增殖。熊果酸对HepG2细胞AMPKα1基因和蛋白表达均无显著性差异,但可提高AMPKα2蛋白表达量。同时可显著降低LPIN1基因和蛋白表达水平;可使p-AMPK蛋白表达量升高,提示熊果酸可能是通过激活AMPK磷酸化,使其活化。
Objective: To investigate the effects of ursolic acid in Artemisia lavandulaefolia on the expression of AMPK and LPIN1 in Hep G2 cells,and identify its active lipid-lowering ingredients at molecular level. Methods:Using MTT method,the effects of ursolic acid in Artemisia lavandulaefolia on the proliferation of HepG2 cells were measured at different drug concentrations and the best experimental conditions were screened. The HepG2 cells were divided into control group and ursolic acid group,and total RNA and protein were extracted from the two groups,respectively. The mRNA levels of LPIN1,AMPKα1,and AMPKα2 were measured by RT-PCR,while the protein expression levels of LPIN1,AMPKα1,AMPKα2 and p-AMPK were examined by Western blot. Results:Ursolic acid could inhibit the proliferation of HepG2 cells. The results of RT-PCR indicated that no significant differences in the expression of AMPKα1 and AMPKα2 mRNA existed between the ursolic acid group and control group( P > 0. 05). The expression of LPIN1 mRNA was significantly decreased in the five ursolic acid groups( P < 0. 01). The Western blot results showed no significant differences in the expression of AMPKα1 between the ursolic acid groups and control group( P > 0. 05). The expression of AMPKα2 and p-AMPK in the ursolic acid group was significantly increased than the control group( P < 0. 05). The expression of LPIN1 in the ursolic acidgroup was significantly decreased than the control group( P < 0. 01). Conclusion: Ursolic acid can inhibit the proliferation of HepG2 cells. In HepG2 cells,ursolic acid has no obvious effects on the expression of AMPKα1 mRNA and protein. Ursolic acid can increase the expression levels of AMPKα2 protein and p-AMPK protein and decrease the expression levels of LP1 N1 mRNA and protein,suggesting that ursolic acid may activate AMPK by activating its phosphorylation.
Objective: To investigate the effects of ursolic acid in Artemisia lavandulaefolia on the expression of AMPK and LPIN1 in Hep G2 cells,and identify its active lipid-lowering ingredients at molecular level. Methods:Using MTT method,the effects of ursolic acid in Artemisia lavandulaefolia on the proliferation of HepG2 cells were measured at different drug concentrations and the best experimental conditions were screened. The HepG2 cells were divided into control group and ursolic acid group,and total RNA and protein were extracted from the two groups,respectively. The mRNA levels of LPIN1,AMPKα1,and AMPKα2 were measured by RT-PCR,while the protein expression levels of LPIN1,AMPKα1,AMPKα2 and p-AMPK were examined by Western blot. Results:Ursolic acid could inhibit the proliferation of HepG2 cells. The results of RT-PCR indicated that no significant differences in the expression of AMPKα1 and AMPKα2 mRNA existed between the ursolic acid group and control group( P > 0. 05). The expression of LPIN1 mRNA was significantly decreased in the five ursolic acid groups( P < 0. 01). The Western blot results showed no significant differences in the expression of AMPKα1 between the ursolic acid groups and control group( P > 0. 05). The expression of AMPKα2 and p-AMPK in the ursolic acid group was significantly increased than the control group( P < 0. 05). The expression of LPIN1 in the ursolic acidgroup was significantly decreased than the control group( P < 0. 01). Conclusion: Ursolic acid can inhibit the proliferation of HepG2 cells. In HepG2 cells,ursolic acid has no obvious effects on the expression of AMPKα1 mRNA and protein. Ursolic acid can increase the expression levels of AMPKα2 protein and p-AMPK protein and decrease the expression levels of LP1 N1 mRNA and protein,suggesting that ursolic acid may activate AMPK by activating its phosphorylation.
引文
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[2] DAS PP,MAIHOTRA S,CHAKRABARTI S,et al. Elevated total cholesterol in severely depressed patients,Role in cardiovascular risk[J]. Biol Psychi,2010,35(11):321-328.
[3]吴刚,庞东卫,苗利,等.蒙药童格勒格-1对大鼠高脂血症的影响[J].中国现代应用药学,2006,23(4):272-274.
[4]周成江,和彦苓,周立社,等.蒙药童格勒格-1四种粗提物对大鼠肝细胞BRL株低密度脂蛋白受体基因表达的影响[J].中国现代应用药学,2008,25(3):186-189.
[5]王晓琴,周成江,张娜,等.野艾蒿化学成分研究[J].中药材,2011,34(2):234-236.
[6] THOMSON DM,WINDER WW. AMP-activated protein kinase control of fat metabolism in skeletal muscle[J]. Acta Physiol(Oxf),2009,196(1):147-154.
[7] HUYPEN P,MOENS K,HEIMBERG H,et al. Adiponectin-mediated stimulation of AMP-activated protein kinase(AMPK)in Pancreatic beta cells[J]. Life Sci,2015,77(11):1273-1282.
[8] LIZCANO JM,GORANSSON O,TOTH R,et al. LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily,including MARK/PAR-1[J]. EMBO,2004,23(4):833-843.
[9] JANSENANSEN M,TENEN KLOOSTERLOOSTER JP. LKB1and AMPK family signaling:the intimate link between cell polarity and energy metabolism[J]. Physiol Rev,2009,89(3):777-798.
[10] HARDIE DG. New roles for the LKB1-AMPK path way[J]. Curr Opin Cell Biol,2005,17(2):167-173.
[11]牛茂源. LPIN1基因多态性与奶牛脂肪肝的相关性分析[D].泰山:山东农业大学,2014:1-70.