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鳖甲煎丸通过p62/Keap1/NRF2信号通路调控肝癌细胞铁死亡的作用机制研究

Biejiajian Pill Regulates Ferroptosis in Hepatocellular Carcinoma Cells via p62/Keap1/NRF2 Signaling Pathway: A Mechanism Study

    摘要
  • 摘要:
    目的  探讨鳖甲煎丸通过p62/Keap1/NRF2通路调控肝癌细胞铁死亡的作用机制，为该方防治肝癌提供实验依据。
    方法  将肝癌Huh7细胞分为对照组、鳖甲煎丸含药血清组、Erastin（铁死亡诱导剂）组、鳖甲煎丸含药血清+Erastin组、鳖甲煎丸含药血清+Ferrostatin-1（Fer-1，铁死亡抑制剂）组。通过动物实验制备鳖甲煎丸含药血清，并通过CCK-8实验筛选最佳药物干预浓度及时间。检测细胞内铁（Fe）、还原型谷胱甘肽（GSH）、脂质过氧化物（MDA）、活性氧（ROS）的含量；Western blot检测FTH1、GPX4、xCT、SLC40A1、p62、Keapl、NRF2的表达水平，JC-1染色检测线粒体膜电位，细胞免疫荧光检测p62、Keap1表达情况。
    结果  CCK-8实验表明，鳖甲煎丸高剂量（2.2 g/kg）细胞抑制率最高（P<0.001），用高剂量鳖甲煎丸药物血清处理48 h，Huh7细胞抑制率最高，因此采用高剂量鳖甲煎丸药物血清浓度、48 h为后续实验剂量和处理时间。与对照组比较，鳖甲煎丸含药血清组、Erastin组、鳖甲煎丸含药血清+Erastin组铁含量升高，GSH含量减低，MDA、ROS水平升高，同时FTH1、GPX4、xCT、SLC40A1、p62、NRF2表达降低，Keap1表达升高，细胞中线粒体膜电位降低（P<0.05）。
    结论 鳖甲煎丸可通过抑制p62/Keap1/NRF2通路调控肝癌Huh7细胞的铁死亡，从而达到防治肝癌的目的。

     

    Abstract:
    Objective  To investigate the mechanism by which Biejiajian Pill (BJJP) regulates ferroptosis in hepatocellular carcinoma (HCC) cells through the p62/Keap1/NRF2 pathway and to provide an experimental basis for its application in the prevention and treatment of HCC.
    Methods  Huh7 HCC cells were divided into a normal control group, a BJJP drug serum group, an erastin (a ferroptosis inducer) group, a BJJP drug serum + erastin group, and BJJP drug serum + ferrostatin-1 (Fer-1) (a ferroptosis inhibitor) group. BJJP drug serum was prepared with animals treated with BJJP and CCK-8 assay was performed to determine the optimal concentration and duration of BJJP intervention. The levels of intracellular iron (Fe), reduced glutathione (GSH), lipid peroxides (MDA), and reactive oxygen species (ROS) were measured. Western blot was performed to determine the expression levels of FTH1, GPX4, xCT, SLC40A1, Keapl, p62, and NRF2. JC-1 staining was performed to measure mitochondrial membrane potential, and cell immunofluorescence was performed to determine the expression of p62 and Keap1.
    Results  According to the CCK-8 assay results, the cell inhibition rate was highest when BJJP was administered at a high dose of 2.2 g/kg (P < 0.001). Furthermore, the inhibition rate of Huh7 cells was highest when Huh7 cells were treated with high-dose BJJP drug serum for 48 h. Therefore, the serum concentration of high-dose BJJP and 48 h were selected as the treatment dose and duration for the subsequent experiment. Compared with the control group, the BJJP drug serum group, the erastin group, and the BJJP drug serum + erastin group showed increased iron content, decreased GSH content, increased MDA levels, increased ROS aggregation, decreased FTH1, GPX4, xCT, SLC40A1, p62, and NRF2 contents, increased Keap1 content, and decreased mitochondrial membrane potential (P < 0.05).
    Conclusion  BJJP regulates ferroptosis in Huh7 HCC cells by inhibiting the p62/Keap1/NRF2 pathway, demonstrating potentials as a therapeutic agent for HCC.

     

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