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目的:研究野艾蒿(Artemisia lavandulaefolia DC.)中木犀草素、柚皮素、槲皮素、芹菜素4种黄酮类单体化合物对HepG2细胞的腺苷酸活化蛋白激酶(AMPK)和磷脂酸磷酸水解酶1(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)观察野艾蒿中4种单体黄酮类化合物在不同用药时间和浓度时对HepG2细胞增殖的影响,并从中筛选出合适的用药浓度和作用时间。将实验分为对照组和药物组,根据筛选出的用药时间和浓度对培养细胞进行药物干预,分别抽提对照组和实验组细胞总RNA与总蛋白,实时荧光定量PCR检测LPIN1、AMPKα1和AMPKα2 mRNA表达情况,蛋白质印迹法(Western blot法)检测LPIN1、AMPKα1、AMPKα2和p-AMPK蛋白表达情况。结果:(1)4种黄酮类单体化合物均可不同程度抑制HepG2细胞的增殖,并在用药质量浓度0.01mg·mL~(-1),作用时间24h(药物对细胞的抑制率在15%~25%)为最适合作用条件。(2)实时荧光定量PCR检测结果显示,药物组AMPKα1mRNA表达与对照组比较无显著差异(P>0.05);药物组AMPKα2 mRNA表达与对照组比较,柚皮素、槲皮素有统计学意义(P<0.05),且表达水平升高;LPIN1 mRNA表达中,药物组与对照组比较均有显著性差异(P<0.01),且表达量均显著降低。(3)Western blot结果显示,药物组AMPKα1蛋白表达与对照组比较,均无显著性差异(P>0.05);AMPKα2蛋白表达中,药物组均有显著性差异(P<0.05),且表达量升高;LPIN1蛋白表达中,药物组与对照组比较均有显著性差异(P<0.01),且表达量均降低;p-AMPK蛋白表达均有显著性差异(P<0.01),且表达水平升高,提示野艾蒿乙酸乙酯提取的黄酮类化合物可通过磷酸化使AMPK活化。结论:(1)野艾蒿乙酸乙酯提取的4种黄酮类单体化合物可不同程度抑制HepG2细胞的增殖。(2)4种黄酮类单体化合物对HepG2细胞AMPKα1基因和蛋白表达均无明显作用;柚皮素和槲皮素可提高HepG2细胞内AMPKα2基因表达水平;4种黄酮类单体化合物均使AMPKα2蛋白表达量升高。同时均可显著降低HepG2细胞内LPIN1基因和蛋白表达水平;4种黄酮类单体化合物均可增加p-AMPK蛋白表达,提示均可激活AMPK磷酸化,使其活化。
OBJECTIVE: To study the effect of four flavonoid compounds, luteolin, naringenin, quercetin and apigenin, on the expression of AMPK and phosphatidic acid in HepG2 cells in Artemisia lavandulaefolia DC. Phosphorylase 1 (LPIN1) expression in the molecular level of its effective components of anti-hyperlipidemia. Methods: HepG2 cells were used to study the effects of four mononuclear flavonoids on the proliferation of HepG2 cells under different administration time and concentration by cytotoxicity assay (MTT method), and the appropriate drug concentration And the role of time. The experiment was divided into the control group and the drug group. According to the selected time and concentration of the drug, the cultured cells were intervened. The total RNA and protein of the control group and the experimental group were extracted. The expression of LPIN1, AMPKα1 and AMPKα2 The mRNA expression of LPIN1, AMPKα1, AMPKα2 and p-AMPK were detected by Western blot. Results: (1) All four flavonoid compounds could inhibit the proliferation of HepG2 cells to varying degrees. When the drug concentration was 0.01 mg · mL -1 and the action time was 24h (the inhibition rate of the drug to the cells was 15% ~ 25%) for the most suitable conditions. (2) The result of real-time fluorescence quantitative PCR showed that the expression of AMPKα1 mRNA in the drug group had no significant difference compared with the control group (P> 0.05). Compared with the control group, the expression of AMPKα2 mRNA in the drug group had statistical significance (naringin and quercetin, P <0.05), and the expression of LPIN1 mRNA was significantly increased (P <0.01), and the expression level of LPIN1 mRNA was significantly lower than that of the control group. (3) Western blot results showed that the expression of AMPKα1 in the drug group was not significantly different from that in the control group (P> 0.05). The expression of AMPKα2 protein in the drug group was significantly different (P <0.05) (P <0.01), and the expression of LPIN1 protein was significantly lower than that of the control group (P <0.01); the expression of p-AMPK protein was significantly different (P <0.01), and the expression level Elevated, suggesting wild Yehao ethyl acetate extraction of flavonoids can phosphorylation of AMPK activation. Conclusions: (1) Four flavonoid compounds extracted from Artemisia annua with ethyl acetate can inhibit the proliferation of HepG2 cells to varying degrees. (2) The four flavonoid compounds had no significant effect on the expression of AMPKα1 gene and protein in HepG2 cells. Naringenin and quercetin could increase the expression of AMPKα2 gene in HepG2 cells. The four flavonoid compounds all induced AMPKα2 Protein expression increased. At the same time, the expression of LPIN1 gene and protein in HepG2 cells was significantly reduced. Four kinds of flavonoid compounds could increase the expression of p-AMPK protein, which could both activate AMPK phosphorylation.