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

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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