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Volume 40 Issue 11
Nov.  2024
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Article Navigation >  Journal of Clinical Hepatology  > 2024  >  40(11): 2238-2245

Role and mechanism of hepatic stellate cells in regulating the apoptosis of hepatocellular carcinoma cells through cystathionine γ-lyase/hydrogen sulfide

DOI: 10.12449/JCH241117
  • 1.

    School of Basic Medical Sciences，Capital Medical University，Beijing 100069，China

  • 2.

    Department of Hepatology and Gastroenterology，Beijing YouAn Hospital，Capital Medical University，Beijing 100069，China

  • 3.

    Oncology Center，Shanxi Bethune Hospital，Taiyuan 030000，China

Research funding:

Sino-German Cooperation Group (GZ1517)

More Information
  • Corresponding author: DING Huiguo， dinghuiugo@ccmu.edu.cn （ORCID： 0000-0002-8716-4926）
  • Received Date: 2024-08-07
  • Accepted Date: 2024-08-26
  • Published Date: 2024-11-25
    Abstract
  •   Objective  As important components in the microenvironment of hepatocellular carcinoma （HCC）， hepatic stellate cells （HSCs） and hydrogen sulfide （H2S） participate in various biological processes that regulate the development and progression of HCC. Through the co-culture of HSCs and HCC cells， this article aims to investigate the role and mechanism of HSCs in regulating the apoptosis of HCC cells by secreting H2S.  Methods  The HSC cell line （LX-2） and HCC cell lines （HepG2 and PLC/PRF/5） were used for experiment. RT-qPCR and Western Blot （WB） were used to measure the mRNA and protein expression levels of cystathionine γ-lyase （CSE）， a key synthase for H2S； ELISA was used to measure the concentration of H2S in supernatant； next-generation sequencing， cell immunofluorescence assay， chromatin immunoprecipitation （ChIP）， and WB were used to measure the JNK/JunB-TNFSF14 signaling pathway genes， binding sites， and related proteins after HepG2 cells were treated by H2S. LX-2 cells were co-cultured with HepG2 or PLC/PRF/5 cells in a Transwell chamber； CCK-8 assay and flow cytometry were used to measure the viability and apoptosis of HCC cells， and WB was used to measure the H2S-TNFSF14 signaling pathway-related proteins. All cell experiments were repeated three times. The independent-samples t test was used for comparison of continuous data between two groups； a one-way analysis of variance or the analysis of variance with repeated measures was used for comparison between multiple groups， and the Dunnett-t test was used for further comparison between two groups.  Results  LX-2 cells synthesized H2S mainly through CSE， and the concentration of H2S in supernatant of LX-2 cells gradually increased over time （22.89±0.08 pg/mL vs 28.29±0.15 pg/mL vs 36.19±1.90 pg/mL， F=79.63， P<0.05）. In LX-2 cells， the mRNA expression level of CSE was significantly higher than that of CBS and MPST （1.008±0.13 vs 0.320±0.014 vs 0.05±0.02， F=80.84， P<0.05）. When CSE was inhibited by PPG， the concentration of H2S decreased with the increase in the concentration of PPG （P<0.05）. LX-2 cells were co-cultured with HepG2 or PLC/PRF/5 cells， and over the time of culture， there were significant reductions in the viability of HepG2 cells （87.48%±0.82% vs 70.48%±0.641% vs 52.89%±0.57% vs 45.20%±0.69%， F=1 517.13， P<0.001） and PLC/PRF/5 cells （92.41%±0.48% vs 74.10%±0.73% vs 53.70%±0.60% vs 44.00%±0.27%， F=2626.21， P<0.001） and significant increases in the apoptosis of HepG2 cells （12.88%±0.64% vs 15.5%±0.16% vs 18.43%±0.37% vs 13.01%±0.58%， F=142.15， P<0.001） and PLC/PRF/5 cells （8.51±0.05 vs 12.80±0.33 vs 15.59±0.21 vs 10.72±0.30， F=676.40， P<0.001）， with the most significant changes on day 3. Next-generation sequencing showed that endogenous H2S and NaHS （endogenous H2S donor） were involved in regulating the expression of various genes in HepG2 cells. By releasing H2S， NaHS and LX2 activated the JNK/JunB signaling pathway and upregulated the expression of the apoptosis gene TNFSF14 in HCC cells， with increased binding between p-JunB and the transcriptional regulatory regions of the TNFSF14 gene.  Conclusion  In the microenvironment of HCC， HSCs activate the JNK/JunB signaling pathway in HCC cells through the signal molecules CSE/H2S， and there is an increase in the expression of TNFSF14， thereby promoting the apoptosis of HCC cells.

     

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