野艾蒿总黄酮对HepG2细胞脂质代谢中AMPK蛋白相关信号转导通路的作用研究

目的：通过研究野艾蒿总黄酮对腺苷酸蛋白活化激酶（AMPK）相关信号转导通路的影响，从而探明其调节HepG2细胞脂质代谢过程中存在的作用机制。方法：以HepG2细胞作为研究对象建立脂质堆积模型，将实验细胞分为对照组和药物组，Western blot检测经药物作用后该模型中乙酰辅酶A羧化酶（acetyl-CoA carboxylase，ACC）磷酸化表达情况以及AMPK上游钙调蛋白依赖性激酶（Ca²⁺ dependentprotein kinase kinase，CaMKK）活性改变对药物激活AMPK的影响；超高效液相色谱法检测野艾蒿总黄酮激活AMPK对HepG2细胞内AMP/ATP水平的影响。结果：（1）油红染色结果显示，野艾蒿总黄酮能够通过AMPK来促进HepG2细胞脂质代谢。（2）Western blot结果显示，药物组与对照组相比，ACC的磷酸化表达均具有显著性差异（P<0.05）且表达水平升高，但该过程可以被AMPK抑制剂所阻断。（3）野艾蒿总黄酮在CaMKK特异性抑制剂作用下仍能够上调HepG2细胞中AMPK的磷酸化表达。（4）超高效液相色谱结果显示药物组与对照组相比，一磷酸腺苷（adenosine monophosphate，AMP）、二磷酸腺苷（adenosine diphosphate，ADP）、三磷酸腺苷（adenosine triphosphate，ATP）含量及AMP/ATP含量比值均未发生显著变化，差异无统计学意义（P>0.05）。结论：野艾蒿总黄酮能够依靠药物-AMPK-ACC这一调脂通路来影响HepG2细胞脂质代谢，但其激活AMPK途径与CaMKK活性和AMP/ATP含量比值变化无关。

Objective: To investigate the effects of total flavonoids from Artemisia lavandulaefolia on adenosine monophosphate-activated protein kinase (AMPK)protein-related signal transduction pathway in order to explore its mechanism of action in regulating lipid metabolism pathways in HepG2 cells. Methods: The HepG2 cells were used as research objects and lipid accumulation model was established. The experimental cells were divided into the control group and the drug group. Western blot was used to detect the phosphorylation of ACC protein expression in this model and the effect of changes in AMPK upstream kinase Ca²⁺ dependent protein kinase kinase (CaMKK)activity on drug-activated AMPK after drug action. Ultra-high performance liquid chromatography was used to detect the effects of activation of AMPK of the total flavonoids from Artemisia lavandulaefolia on the AMP/ATP ratio in HepG2 cells. Results: (1)Oil red staining results showed that the total flavonoids from Artemisia lavandulaefolia could promote lipid metabolism through AMPK. (2)The results of Western blot showed that there was significant difference in the phosphorylation of ACC protein between the drug group and the control group (P<0.05). The expression level increased and the process could be blocked by AMPK inhibitors. (3) The total flavonoids of Artemisia lavandulaefolia could significantly up-regulate the phosphorylation of AMPK protein in cells under the action of CaMKK-specific inhibitors. (4)There was no significant difference (P>0.05) in contents of adenosine triphosphate (ATP), adenosine diphosphate (ADP)and adenosine monophosphate (AMP)and AMP/ATP ratio between the drug group and the control group. Conclusion: The total flavonoids of Artemisia lavandulaefolia can affect the lipid metabolism of HepG2 cells through the drug-AMPK-ACC lipidregulating pathway, but its mechanism of activating AMPK protein is not related to changes in CaMKK activity and AMP/ATP ratio.

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