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Journal of Integrative Medicine ›› 2014, Vol. 12 ›› Issue (6): 495-503.doi: 10.1016/S2095-4964(14)60052-2

• Research Article • Previous Articles     Next Articles

Effect of Saikokeishito, a Kampo medicine, on hydrogen peroxide-induced premature senescence of normal human dermal fibroblasts

Takanobu Takataa, Yoshiharu Motooa,b, Naohisa Tomosugia,c   

  1. a Medical Research Institute of Kanazawa Medical University, Ishikawa, Japan
    b Department of Medical Oncology, Kanazawa Medical University, Ishikawa, Japan
    c Department of Nephrology, Kanazawa Medical University, Ishikawa, Japan
  • Received:2014-03-27 Accepted:2014-07-18 Online:2014-11-10 Published:2014-11-15

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Objective

Saikokeishito (TJ-10) is a Kampo (traditional Japanese herbal) medicine, clinically used for hundreds of years in East Asia. Among its various mechanisms elucidated so far, TJ-10 inhibits the production of transforming growth factor-β1 (TGF-β1) and development of pancreatic fibrosis in vivo. Oxidative damage of normal human dermal fibroblasts (NHDFs) in the corium is a cause of human dermal senescence. Our aim was to determine whether TJ-10 protects NHDFs from premature senescence by hydrogen peroxide (H2O2).

Methods

Premature senescence was induced in NHDFs by 200 μmol/L H2O2 for 4 h. Cell viability and the expressions of p53, AMP-activated protein kinase α1 (AMPKα1), AMPKα2, and 14-3-3 protein sigma (14-3-3 σ) were measured in NHDFs treated with TJ-10 for 48 h before exposure to H2O2 for 4 h. 

Results

Cell viability after treatment with 200 μmol/L H2O2 for 4 h was similar (about 80%) to after pre-treatment with TJ-10. Ascorbic acid as a control did not protect NHDFs from damage by 200 μmol/L H2O2. Treatment with 200 μmol/L H2O2 tended to up-regulate p53 and to down-regulate SIRT1 and AMPKα1, but had no effect on AMPKα2 and 14-3-3 σ expression. Pretreatment with TJ-10 inhibited H2O2-induced up-regulation of p53 and enhanced AMPKα1 expression. 

Conclusion

It is suggested that Saikokeishito has a protective effect on oxidative stress-induced senescence of NHDFs.

Key words: Saikokeishito, Medicine, Kampo, Premature senescence, p53, Fibroblasts

Figure 1

Premature senescence induced by hydrogen peroxide and analysis of cell viability The MTT assay was done in 8 wells. Data are presented as mean ± standard deviation, n=8; **P<0.01, vs control."

Figure 2

Western blot analysis on the effects of H2O2 on the expressions of senescence-associated proteins"

Figure 3

Quantitative analysis on the effects of H2O2 on the expressions of senescence-associated proteins A: p53; B: SIRT1; C: AMPKα1; D: 14-3-3 σ; E: AMPKα2. Data are presented as mean ± standard deviation, n=3; *P<0.05, **P<0.01, vs control."

Figure 4

Cell viability of NHDFs treated with ascorbic acid and TJ-10 Cells were incubated for 24 h and the medium was changed to new medium containing ascorbic acid or TJ-10. Then cells were incubated for 48 h. The MTT assay was done in 8 wells. Data are presented as mean ± standard deviation, n=8; **P<0.01, vs control."

Figure 5

Cell viability of NHDFs treated with ascorbic acid and TJ-10 before H2O2 exposure Cells were incubated for 24 h and the medium was changed to new medium containing ascorbic acid or TJ-10. Then, cells were incubated for 48 h, the medium was changed to serum-free medium, and the cells were exposed to 200 μmol/L H2O2 stress. ‘A’ indicates ascorbic acid. The MTT assay was done in 8 wells. Data are presented as mean ± standard deviation, n=8; *P<0.05, vs blank control; △P<0.05, vs H2O2 control."

Figure 6

Effects of ascorbic acid and TJ-10 on H2O2 stress-induced changes in the expressions of senescence-associated proteins"

Figure 7

Effects of ascorbic acid and TJ-10 on H2O2-induced changes in the expression of p53, SIRT1, and AMPKα1 Cells were incubated for 48 h and the medium was changed. Then cells were exposed to 200 μmol/L H2O2 for 4 h. ‘A’ indicates ascorbic acid at 200 μmol/L. The data are presented as mean ± standard deviation, n=3; **P<0.01, vs blank control; △P<0.05, △△P<0.01, vs H2O2 control."

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