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Characterization of in vitro tumor necrosis factor-⍺-induced hepatic cell death |
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| รหัสดีโอไอ | |
| Title | Characterization of in vitro tumor necrosis factor-⍺-induced hepatic cell death |
| Creator | Thanawan Soimanee |
| Contributor | Chareeporn Akekawatchai, Advisor |
| Publisher | Thammasat University |
| Publication Year | 2568 |
| Keyword | Hepatocyte death, TNF-α, Cycloheximide, Apoptosis, Cell cycle, S-phase arrest, Proteomics |
| Abstract | Hepatocyte death triggers liver inflammation and fibrosis, ultimately driving the progression to hepatocellular carcinoma. Various mediators in the inflammatory liver microenvironment have been identified as inducers of hepatocyte death, including the proinflammatory cytokine tumor necrosis factor-ɑ (TNF-ɑ). While TNF-ɑ-induced hepatocyte death is established as a potential therapeutic target for chronic liver diseases, its molecular mechanisms remain unclear. This study aimed to characterize an in vitro model of TNF-ɑ-mediated hepatocyte death using the HepG2 cell line. The cells were treated with various concentrations of TNF-ɑ in combination with cycloheximide (CHX), a protein synthesis inhibitor, at different time points. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay). Additionally, cell cycle arrest and apoptotic cell death were evaluated using DNA-binding dye staining and AnnexinV-7AAD labeling, respectively, followed by flow cytometric analysis. Further molecular characterization was performed using Western blot analysis, coupled with comprehensive proteomic and bioinformatic evaluations. Treatment with TNF-α alone reduced cell viability, but did not have any effect on cell growth, cell cycle and apoptotic cell death. Western blot analysis indicated no cleavage of the apoptotic markers, caspase-3 and PARP-1, and no expected shift in cell cycle regulation. Notably, TNF-ɑ treated cells may exhibit SA-β-gal activity, a hallmark of cellular senescence, without cell cycle or growth arrest. Cells treated with CHX alone exhibited decreased viability, growth and S-phase cell cycle arrest, and a limited degree of apoptosis. Consistently, molecular analysis revealed no cleavage of caspase-3 and PARP-1 and downregulated p21 expression. Importantly, cells treated with a combination of TNF-ɑ and CHX showed an additive reduction of viability, growth and S-phase cell cycle arrest, but notably enhanced induction of apoptosis (up to 75.8% from 50 M CHX and 100 ng/mL TNF- co-treatment). The co-treatment also caused a clear cleavage of caspase-3 and PARP-1, with p21 downregulation. The results indicate that TNF- alone does not induce cell cycle arrest or apoptosis, while the translational inhibitor, CHX removes the survival pathways, effectively allowing TNF- to drive the cells toward caspase-3/PARP-1-dependent apoptosis. Additionally, proteomic analysis using MetaboAnalyst 6.0 revealed six candidate proteins from a total of 19,482 proteins expressed differentially in all treatments: phospholipase A2 homolog, ZNF5930S, zinc finger protein 557, oligodendrocyte transcription factor 2, haloacid dehalogenase-like hydrolase domain-containing 5 and adenine phosphoribosyltransferase. The protein-protein interaction network generated by STITCH 5.0 predicted interactions among the latter three proteins, CHX and TNF-α, suggesting their potential roles in TNF-ɑ/CHX-induced cell cycle and apoptosis. Collectively, this molecular characterization of TNF-α/CHX-induced hepatocyte apoptosis provided a valuable in vitro model for investigating hepatocyte stress and death in inflammatory environments. |
