[鳖甲煎丸通过p62/Keap1/NRF2信号通路调控肝癌细胞铁死亡的机制研究]
[Biejiajian Pill Regulates Ferroptosis in Hepatocellular Carcinoma Cells via p62/Keap1/NRF2 Signaling Pathway: A Mechanism Study].
作者信息
Chen Weiguang, He Chunyu, Wen Bin, Sun Haitao, Yang Xuemei, Chen Weicong, Liu Yang, Zhong Binglian, He Songqi
机构信息
（ 510515） School of Chinese Medicine, Southern Medical University, Guangzhou 510515, China.
出版信息
Sichuan Da Xue Xue Bao Yi Xue Ban. 2025 Jan 20;56(1):51-58. doi: 10.12182/20250160502.
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 ( < 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 ( < 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.
目的
探讨鳖甲煎丸（BJJP）通过p62/Keap1/NRF2通路调节肝癌（HCC）细胞铁死亡的机制，为其在HCC防治中的应用提供实验依据。
方法
将Huh7肝癌细胞分为正常对照组、BJJP药物血清组、艾拉司群（一种铁死亡诱导剂）组、BJJP药物血清+艾拉司群组和BJJP药物血清+铁抑素-1（Fer-1，一种铁死亡抑制剂）组。用BJJP处理动物制备BJJP药物血清，并进行CCK-8测定以确定BJJP干预的最佳浓度和持续时间。测量细胞内铁（Fe）、还原型谷胱甘肽（GSH）、脂质过氧化物（MDA）和活性氧（ROS）水平。进行蛋白质免疫印迹法以测定FTH1、GPX4、xCT、SLC40A1、Keapl、p62和NRF2的表达水平。进行JC-1染色以测量线粒体膜电位，并进行细胞免疫荧光测定以确定p62和Keap1的表达。
结果
根据CCK-8测定结果，当BJJP以2.2 g/kg的高剂量给药时，细胞抑制率最高（<0.001）。此外，当用高剂量BJJP药物血清处理Huh7细胞48小时时，Huh7细胞的抑制率最高。因此，选择高剂量BJJP的血清浓度和48小时作为后续实验的治疗剂量和持续时间。与对照组相比，BJJP药物血清组、艾拉司群组和BJJP药物血清+艾拉司群组的铁含量增加，GSH含量降低，MDA水平升高，ROS聚集增加，FTH1、GPX4、xCT、SLC40A1、p62和NRF2含量降低，Keap1含量增加，线粒体膜电位降低（<0.05）。
结论
BJJP通过抑制p62/Keap1/NRF2通路调节Huh7肝癌细胞的铁死亡，显示出作为HCC治疗药物的潜力。
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