Search JIM Advanced Search

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


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).


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. 


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. 


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."

[1] Motoo Y, Xie MJ, Su SB, Sawabu N . Molecular mechanisms of therapeutic effects of Saikokeishito on spontaneous chronic pancreatitis in the WBN/Kob rat[J]. J Tradit Med, 2003,20(4):143-149
[2] Motoo Y, Su SB, Xie MJ, Taga H, Sawabu N . Effect of herbal medicine Saiko-keishi-to(TJ-10) on rat spontaneous chronic pancreatitis: comparison with other herbal medicines[J]. Int J Pancreatol, 2000,27(2):123-129
doi: 10.1385/IJGC:27:2
[3] Pillai S, Oresajo C, Hayward J . Ultraviolet radiation and skin aging: roles of reactive oxygen species, inflammation and protease activation, and strategies for prevention of inflammation-induced matrix degradation — a review[J]. Int J Cosmet Sci, 2005,27(1):17-34
doi: 10.1111/ics.2005.27.issue-1
[4] Cross CE, Halliwell B, Borish ET, Pryor WA, Ames BN, Saul RL , McCord JM, Harman D.Oxygen radicals and human disease[J]. Ann Int Med, 1987,107(4):526-545
doi: 10.7326/0003-4819-107-4-526
[5] Rai P, Onder TT, Young JJ , McFaline JL, Pang B, Dedon PC, Weinberg RA.Continuous elimination of oxidized nucleotides is necessary to prevent rapid onset of cellular senescence[J]. Proc Natl Acad Sci U S A, 2009,106(1):169-174
doi: 10.1073/pnas.0809834106
[6] Hayflick L, Moorhead PS . The serial cultivation of human diploid cell strains[J]. Exp Cell Res, 1961,25:585-621
doi: 10.1016/0014-4827(61)90192-6
[7] Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O . A biomarker that identifies senescent human cells in culture and in aging skin in vivo[J]. Proc Natl Acad Sci U S A, 1995,92(20):9363-9367
doi: 10.1073/pnas.92.20.9363
[8] Taniguchi M, Arai N, Kohno K, Ushio S, Fukuda S . Anti-oxidative and anti-aging activities of 2-O-α-glucopyranosyl-L-ascorbic acid on human dermal fibroblasts[J]. Eur J Pharmacol, 2012,674(2-3):126-131
doi: 10.1016/j.ejphar.2011.11.013
[9] Ido Y, Duranton A, Lan F, Cacicedo JM, Chen TC, Breton L, Ruderman NB . Acute activation of AMP-activated protein kinase prevents H2O2-induced premature senescence in primary human keratinocytes[J]. PLoS One, 2012,7(4):e35092
doi: 10.1371/journal.pone.0035092
[10] Price NL, Gomes AP, Ling AJ, Duarte FV, Martin-Montalvo A, North BJ, Agarwal B, Ye L, Ramadori G, Teodoro JS, Hubbard BP, Varela AT, Davis JG, Varamini B, Hafner A, Moaddel R, Rolo AP, Coppari R , Palmeira CM, de Cabo R, Baur JA, Sinclair DA. SIRT1 is required for AMPK activation and the beneficial effects of resveratrol on mitochondrial function[J]. Cell Metab, 2012,15(5):675-690
doi: 10.1016/j.cmet.2012.04.003
[11] Li J, Qu X, Ricardo SD, Bertram JF, Nikolic-Paterson DJ . Resveratrol inhibits renal fibrosis in the obstructed kidney: potential role in deacetylation of Smad3[J]. Am J Pathol, 2010,177(3):1065-1071
doi: 10.2353/ajpath.2010.090923
[12] Kume S, Haneda M, Kanasaki K, Sugimoto T, Araki S, Isshiki K, Isono M, Uzu T, Guarente L, Kashiwagi A, Koya D . SIRT1 inhibits transforming growth factor β-induced apoptosis in glomerular mesangial cells via Smad7 deacetylation[J]. J Biol Chem, 2007,282(1):151-158
doi: 10.1074/jbc.M605904200
[13] Brunet A, Sweeney LB, Sturgill JF, Chua KF, Greer PL, Lin Y, Tran H, Ross SE, Mostoslavsky R, Cohen HY, Hu LS, Cheng HL, Jedrychowski MP, Gygi SP, Sinclair DA, Alt FW, Greenberg ME . Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase[J]. Science, 2004,303(5666):2011-2015
doi: 10.1126/science.1094637
[14] Langley E, Pearson M, Faretta M, Bauer UM, Frye RA, Minucci S, Pelicci PG, Kouzarides T . Human SIR2 deacetylates p53 and antagonizes PML/p53-induced cellular senescence[J]. EMBO J, 2002,21(10):2383-2396
doi: 10.1093/emboj/21.10.2383
[15] Mhawech P, Greloz V, Assaly M, Herrmann F . Immunohistochemical expression of 14-3-3σ protein in human urological and gynecological tumors using a multi-tumor microarray analysis[J]. Pathol Int, 2005,55(2):77-82
doi: 10.1111/pin.2005.55.issue-2
[16] Rajagopalan S, Sade RS, Townsley FM, Fersht AR . Mechanistic differences in the transcriptional activation of p53 by 14-3-3 isoforms[J]. Nucleic Acids Res, 2010,38(3):893-906
doi: 10.1093/nar/gkp1041
[17] Yu AL, Fuchshofer R, Kook D, Kampik A, Bloemendal H, Welge-Lüssen U . Subtoxic oxidative stress induces senescence in retinal pigment epithelial cells via TGF-β release[J]. Invest Ophthalmol Vis Sci, 2009,50(2):926-935
doi: 10.1167/iovs.07-1003
[18] Yasuda K, Aoshiba K, Nagai A . Transforming growth factor-β promotes fibroblast apoptosis induced by H2O2[J]. Exp Lung Res, 2003,29(3):123-134
doi: 10.1080/01902140303773
[19] Elston R, Inman GJ . Crosstalk between p53 and TGF-β signalling[J]. J Signal Transduct, 2012,2012:294097
[20] Kubota A, Okamura S, Shimoda K, Harada N, Omori F, Niho Y . A traditional Chinese herbal medicine, Juzen-Taiho-To, augments the production of granulocyte/macrophage colony-stimulating factor from human peripheral blood mononuclear cells in vitro[J]. Int J Immunother, 1992,8(4):191-195
[21] Ito K, Satoh T, Watanabe Y, Ikarashi N, Asano T, Morita T, Sugiyama K . Effects of Kampo medicines on CYP and P-gp activity in vitro[J]. Biol Pharm Bull, 2008,31(5):893-896
doi: 10.1248/bpb.31.893
[22] Yasui T, Matsuzaki T, Ogata R, Kiyokawa M, Ushigoe K, Uemura H, Kuwahara A, Ikawa H, Maegawa M, Furumoto H, Aono T, Irahara M . The herbal medicine Unkei-to stimulates the secretion of a cytokine-induced neutrophil chemoattractant, CINC/gro, in the rat ovarian cell culture[J]. Am J Reprod Immunol, 2003,49(1):14-20
doi: 10.1034/j.1600-0897.2003.01145.x
[23] Yano H, Mizoguchi A, Fukuda K, Haramaki M, Ogasawara S, Momosaki S, Kojiro M . The herbal medicine sho-saiko-to inhibits proliferation of cancer cell lines by inducing apoptosis and arrest at the G0/G1 phase[J]. Cancer Res, 1994,54(2):448-454
[24] Ando H, Asanuma Y, Sato Y, Koyama K . The effect of TJ-9, TJ-41, and TJ-114 against the proliferation of HepG2[J]. Kanzo, 1995,36(3):171-172
doi: 10.2957/kanzo.36.171
[25] Kirschner M, Montazem A, Hilaire HS, Radu A . Long-term culture of human gingival keratinocyte progenitor cells by down-regulation of 14-3-3 σ[J]. Stem Cells Dev, 2006,15(4):556-565
doi: 10.1089/scd.2006.15.556
[26] Lim JY, Oh MA, Kim WH, Sohn HY, Park SI . AMP-activated protein kinase inhibits TGF-β-induced fibrogenic responses of hepatic stellate cells by targeting transcriptional coactivator p300[J]. J Cell Physiol, 2012,227(3):1081-1089
doi: 10.1002/jcp.22824
[27] Kim KH, Park GT, Lim YB, Rue SW, Jung JC, Sonn JK, Bae YS, Park JW, Lee YS . Expression of connective tissue growth factor, a biomarker in senescence of human diploid fibroblasts, is up-regulated by a transforming growth factor-β-mediated signaling pathway[J]. Biochem Biophys Res Commun, 2004,318(4):819-825
doi: 10.1016/j.bbrc.2004.04.108
[28] Yu AL, Fuchshofer R, Kook D, Kampik A, Bloemendal H, Welge-Lüssen U . Subtoxic oxidative stress induces senescence in retinal pigment epithelial cells via TGF-β release[J]. Invest Ophthalmol Vis Sci, 2009,50(2):926-935
doi: 10.1167/iovs.07-1003
[29] Frippiat C, Chen QM, Zdanov S, Magalhaes JP, Remacle J, Toussaint O . Subcytotoxic H2O2 stress triggers a release of transforming growth factor-β1, which induces biomarkers of cellular senescence of human diploid fibroblasts[J]. J Biol Chem, 2001,276(4):2531-2537
doi: 10.1074/jbc.M006809200
[30] Jiang S, Park DW, Stigler WS, Creighton J, Ravi S, Darley-Usmar V, Zmijewski JW . Mitochondria and AMP-activated protein kinase-dependent mechanism of efferocytosis[J]. J Biol Chem, 2013,288(36):26013-26026
doi: 10.1074/jbc.M113.489468
[31] Hu R, Yan H, Hao X, Liu H, Wu J . Shizukaol D isolated from Chloranthus japonicas inhibits AMPK-dependent lipid content in hepatic cells by inducing mitochondrial dysfunction[J]. PLoS One, 2013,8(8):e73527
doi: 10.1371/journal.pone.0073527
[32] Dugan LL, You YH, Ali SS, Diamond-Stanic M, Miyamoto S , DeCleves AE, Andreyev A, Quach T, Ly S, Shekhtman G, Nguyen W, Chepetan A, Le TP, Wang L, Xu M, Paik KP, Fogo A, Viollet B, Murphy A, Brosius F, Naviaux RK, Sharma K.AMPK dysregulation promotes diabetes-related reduction of superoxide and mitochondrial function[J]. J Clin Invest, 2013,123(11):4888-4899
doi: 10.1172/JCI66218
[33] Filomeni G, Piccirillo S, Graziani I, Cardaci S , Da Costa Ferreira AM, Rotilio G, Ciriolo MR.The isatin-Schiff base copper(II) complex Cu(isaepy)2 acts as delocalized lipophilic cation, yields widespread mitochondrial oxidative damage and induces AMP-activated protein kinase-dependent apoptosis[J]. Carcinogenesis, 2009,30(7):1115-1124
doi: 10.1093/carcin/bgp105
[1] Brody Slostad, Tejinder Khalsa, Kathleen Young, Hildalicia Guerra, Anjali Bhagra. A case-based approach to integrative medicine for cardiovascular disease prevention. Journal of Integrative Medicine, 2020, 18(2): 159-162.
[2] Ameena Yasmeen, Ghulamuddin Sofi, Kaleemullah Khan. Aamar-e-Advia (shelf-lives) of drugs in Unani system of medicine: A conceptual review. Journal of Integrative Medicine, 2020, 18(2): 114-124.
[3] Louisa Sylvia, Emerson West, Allyson M. Blackburn, Carina Gupta, Eric Bui, Tara Mahoney, Geraldine Duncan, Edward C. Wright, Simon Lejeune, Thomas J. Spencer. Acceptability of an adjunct equine-assisted activities and therapies program for veterans with posttraumatic stress disorder and/or traumatic brain injury. Journal of Integrative Medicine, 2020, 18(2): 169-173.
[4] Sherman Gu, Arthur Yin Fan. Controversial conclusions from two randomized controlled trials for acupuncture's effects on polycystic ovary syndrome or in vitro fertilization support. Journal of Integrative Medicine, 2020, 18(2): 89-91.
[5] Xue-qing Yu, Shu-guang Yang, Yang Xie, Jian-sheng Li. Traditional Chinese medicine in the treatment of idiopathic pulmonary fibrosis based on syndrome differentiation: Study protocol of exploratory trial. Journal of Integrative Medicine, 2020, 18(2): 163-168.
[6] Chang-quan Ling. Traditional Chinese medicine is a resource for drug discovery against 2019 novel coronavirus (SARS-CoV-2). Journal of Integrative Medicine, 2020, 18(2): 87-88.
[7] Sajjad Sadeghi, Farzaneh Ghaffari, Ghazaleh Heydarirad, Mehdi Alizadeh. Galen’s place in Avicenna’s The Canon of Medicine : Respect, confirmation and criticism. Journal of Integrative Medicine, 2020, 18(1): 21-25.
[8] Maryam Naeimia, Narjes Gorjib, Zahra Memarianib, Reihaneh Moeinib, Mohammad Kamalinejadc, Fatemeh Kolangid. Gastroprotective herbs for headache management in Persian medicine: A comprehensive review. Journal of Integrative Medicine, 2020, 18(1): 1-13.
[9] Mohadeseh Ostovar, Abolfazl Akbari, Mohammad Hossein Anbardar, Aida Iraji, Mohsen Salmanpour, Salar Hafez Ghoran, Mojtaba Heydari, Mesbah Shams. Effects of Citrullus colocynthis L. in a rat model of diabetic neuropathy. Journal of Integrative Medicine, 2020, 18(1): 59-67.
[10] Preecha Nootim, Nattiya Kapol, Waranee Bunchuailua, Panoopat Poompruek, Parankul Tungsukruthai . Current state of cancer patient care incorporating Thai traditional medicine in Thailand: A qualitative study. Journal of Integrative Medicine, 2020, 18(1): 41-45.
[11] Md. Anzar Alam, Mohd Aleemuddin Quamri, Ghulamuddin Sofi, Barkati Md.Tarique. Understanding hypothyroidism in Unani medicine. Journal of Integrative Medicine, 2019, 17(6): 387-391.
[12] Naghmeh Yazdi, Alireza Salehi, Mina Vojoud, Mohammad Hossein Sharifi, Ayda Hoseinkhani. Use of complementary and alternative medicine in pregnant women: a cross-sectional survey in the south of Iran. Journal of Integrative Medicine, 2019, 17(6): 392-395.
[13] Wen Qiang Lee, Jeremy Teoh, Pei Zheng Kenneth Lee, Zhi Xiong Gerard Low, Xueling Sim, Foong Fong Mary Chong, Norbert Ludwig Wagner. Factors influencing communication of traditional Chinese medicine use between patients and doctors: a multisite cross-sectional study. Journal of Integrative Medicine, 2019, 17(6): 396-403.
[14] Zienab Mahmoudpour, Javad Shokri, Mohammad Kamalinejad, Neda Meftah, Soraya Khafri, Seyyed Ali Mozaffarpur, Hoda Shirafkan. The efficacy of a Persian herbal formulation on functional bloating: A double-blind randomized controlled trial. Journal of Integrative Medicine, 2019, 17(5): 344-350.
[15] Claire L. Hoban, Roger W. Byard, Ian F. Musgrave. Analysis of spontaneous adverse drug reactions to echinacea, valerian, black cohosh and ginkgo in Australia from 2000 to 2015. Journal of Integrative Medicine, 2019, 17(5): 338-343.
Full text



[1] Zhao-guo Li. Discussion on English translation of commonly used sentences in traditional Chinese medicine: part one. Journal of Chinese Integrative Medicine, 2008, 6(1): 107-110
[2] Jun Hu, Jian-ping Liu. Non-invasive physical treatments for chronic/recurrent headache. Journal of Chinese Integrative Medicine, 2008, 6(1): 31
[3] Dong Yang, Yong-ping Du, Qing Shen, Wei Chen, Yan Yu, Guang-lei Chen. Expression of alpha-smooth muscle actin in renal tubulointerstitium in patients with kidney collateral stasis. Journal of Chinese Integrative Medicine, 2008, 6(1): 41-44
[4] Xue-mei Liu, Qi-fu Huang, Yun-ling Zhang, Jin-li Lou, Hong-sheng Liu, Hong Zheng. Effects of Tribulus terrestris L. saponion on apoptosis of cortical neurons induced by hypoxia-reoxygenation in rats. Journal of Chinese Integrative Medicine, 2008, 6(1): 45-50
[5] Zhi-chun Jin. Problems in establishing clinical guideline for integrated traditional Chinese and Western medicine. Journal of Chinese Integrative Medicine, 2008, 6(1): 5-8
[6] SUN Ming-yu, ZUO Jian, DUAN Ji-feng, HAN Jun, FAN Shi-ming, ZHANG Wei, ZHU Li-fang, YAO Ming-hui. Antitumor activities of kushen flavonoids in vivo and in vitro. Journal of Chinese Integrative Medicine, 2008, 6(1): 51-59
[7] Wei Zhang, Xiang-feng Lu, Xiao-mei Zhang, Jian-jun Wu, Liang-duo Jiang. A rat model of pulmonary fibrosis induced by infusing bleomycin quickly through tracheal intubation. Journal of Chinese Integrative Medicine, 2008, 6(1): 60-67
[8] Jin-zhou Tian, Jing Shi, Xin-qing Zhang, Qi Bi, Xin Ma, Zhi-liang Wang, Xiao-bin Li, Shu-li Shen, Lin Li, Zhen-yun Wu, Li-yan Fang, Xiao-dong Zhao, Ying-chun Miao, Peng-wen Wang, Ying Ren, Jun-xiang Yin, Yong-yan Wang, Beijing United Study Group on MCI of the Capital Foundation of Medical Developments. Guiding principles of clinical research on mild cognitive impairment (protocol). Journal of Chinese Integrative Medicine, 2008, 6(1): 9-14
[9] Bo Wang , Wei Yan , Li-hui Hou, Xiao-ke Wu. Disorder of Tiangui (kidney essence) and reproductive dysfunction in patients with polycystic ovary syndrome. Journal of Chinese Integrative Medicine, 2010, 8(11): 1018-1022
[10] Yan-bo Zhu , Qi Wang, Cheng-yu Wu, Guo-ming Pang, Jian-xiong Zhao, Shi-lin Shen, Zhong-yuan Xia , Xue Yan . Logistic regression analysis on relationships between traditional Chinese medicine constitutional types and overweight or obesity. Journal of Chinese Integrative Medicine, 2010, 8(11): 1023-1035