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

CHEN Weiguang, HE Chunyu, WEN Bin, SUN Haitao, YANG Xuemei, CHEN Weicong, LIU Yang, ZHONG Binglian, H

Abstract

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.

 

Keywords: Biejiajian Pill, Hepatocellular carcinoma, Ferroptosis, p62/Keap1/NRF2

 

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