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KMID : 0352319990220010119
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Journal of KyungHee Oriental Medicine College
1999 Volume.22 No. 1 p.119 ~ p.140
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The Effects of Injinsaryungsan on Cell Viability, Cell Cycle Progression and Fas-mediated Apoptosis of Hepatocytes
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Pyo Im-Jeng
Lee Jang-Hoon
Woo Hong-Jung
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Abstract
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To evaluate the effects of Injinsaryungsan on cell viability, cell cycle progression, and Fas-mediated apotosis, MTT assay, cell cycle analysis, PCNA expression analysis and tryphan blue exclusion assay, flow cytometry, Cpp32 protease assay, DNA fragmemtation assay were performed. And for quantitative evaluation of gene expression, we performed RT-PCR. The results were as followed.
1. In MTT assay, Injinsaryungsan treatment increases cellular viability of the cells and this increase showed strong correlation with the treatment doses of Injinsaryungsan.
2. Cell cycle analysis showed a significant reduction of G₁phase, and PCNA analysis showed that cell cycle progression of the HepG2 cells is positively regulated by Injinsaryungsan. And a dosage-and time-dependent induction of PCNA expression was recognized.
3. Using tryphan blue exclusion, flow cytometric analysis of sub G₁phase, a significant and dosage-dependent reduction of apoptosis were observed in Injinsaryungsan-treated cells compared to untreated control cells.
4. DNA fragmentation assay showed significantly reduction of DNA fragmentation in Injinsaryungsan-treated cells, decreases of CPP32 protease activities were observed in CPP32 protease assay. Which indicates that Injinsaryungsan carry inhibitory effects on apoptosis.
5. A quantitative RT-PCR analysis demonstrated that Fas and CPP32 mRNA expression were dramatically reduced and Bcl-2, Bcl-X?? mRNA expression were induced by the treatment of Injinsaryungsan while expression of Fadd, Fap-1, and ICE were not affected by Injinsaryungsan.
These results implies that Injinsaryungsan increase hepatocyte viability, cell cycle progression and protect hepatocyte from damage by regulating the expression of genes associated with Fas-mediated apoptosis, which explains the mechanism of the clinical effect of Injinsaryungsan on liver diseases.
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