Skip to main content
Journal of Clinical Laboratory Analysis logoLink to Journal of Clinical Laboratory Analysis
. 2001 Jun 27;15(4):199–209. doi: 10.1002/jcla.1028

Semi‐quantitative analysis of cytokine mRNA expression induced by the herbal medicine Sho‐saiko‐to (TJ‐9) using a Gel Doc system

Xian‐Xi Huang 1, Masayoshi Yamashiki 1,, Kaname Nakatani 1, Tsutomu Nobori 1, Akihito Mase 2
PMCID: PMC6807871  PMID: 11436203

Abstract

The RT‐PCR method was employed to determine the cytokine mRNA expression of human peripheral lymphocytes induced by the Japanese herbal medicine Sho‐saiko‐to (TJ‐9). The results showed that the mRNA expression of IL‐12, IL‐1β, IL‐10, TNF‐α, G‐CSF, and IFN‐γ increased after 6 hr in culture. This is the first reported finding that TJ‐9 is an IFN‐γ inducer. Next, cytokine mRNA expression was semi‐quantitatively measured using the Gel Doc system with a CCD camera and then statistically analyzed in order to determine which component of TJ‐9 was the true cytokine inducer. The results showed that the scutellaria root is the main component inducing the cytokines, while the glycyrrhiza root is the secondary component. When the cytokine concentrations in the supernatants of cell cultures were measured by ELISA, the levels of IL‐12, IL‐1β, IL‐10, TNF‐α, and G‐CSF reflected mRNA expression levels in the cell fraction. However, the level of IFN‐γ was below the detectable limit. The effects of various reagents on many different kinds of cytokine mRNA expression could be analyzed objectively in a short time using the Gel Doc system. Many important findings could be demonstrated by this simple, easy, sensitive, and cheap method. After the clinical significance of cytokine analysis is confirmed, this method may become a useful clinical examination tool. J. Clin. Lab. Anal. 15:199–209, 2001. © 2001 Wiley‐Liss, Inc.

Keywords: cytokine mRNA, RT‐PCR method, semi‐quantitative analysis, Gel Doc system, ELISA, Sho‐saiko‐to (TJ‐9), cytokine inducer

REFERENCES

  • 1. Yamashiki M, Nishimura A, Suzuki H, Sakaguchi S, Kosaka Y. 1997. Effects of the Japanese herbal medicine ‘Sho‐saiko‐to’ (TJ‐9) on in vitro interleukin 10 productions by peripheral blood mononuclear cells of chronic active hepatitis C patients. Hepatology 25:1390–1397. [DOI] [PubMed] [Google Scholar]
  • 2. Yamashiki M, Kosaka Y, Nishimura A, Watanabe S, Nomoto M, Ichida F. 1998. Analysis of serum cytokine levels in primary biliary cirrhosis patients and healthy adults. J Clin Lab Anal 12:77–82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Yamashiki M, Kosaka Y, Nishimura A, Tameda Y, Takase K, Watanabe S, Kaito M, Nishioka J, Suzuki H, Nomoto M. 1998. Flow cytometric analysis of IL‐6 receptors on peripheral lymphocytes in patients with primary biliary cirrhosis. J Clin Lab Anal 12:83–87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. De Caestecker MP, Bottomley M, Telfer BA, Hutchinson IV, Vose BM, Ballardie FW. 1993. Detection of abnormal peripheral blood mononuclear cell cytokine networks in human IgA nephropathy. Kidney Int 44:1298–1308. [DOI] [PubMed] [Google Scholar]
  • 5. Okayama Y, Petit‐Frére C, Kassel O, Semper A, Quint D, Tunon‐de‐lara MJ, Bradding P, Holgate ST, Church MK. 1995. IgE‐dependent expression of mRNA for IL‐4 and IL‐5 in human lung mast cells. J Immunol 155:1796–1808. [PubMed] [Google Scholar]
  • 6. De Vita S, Dolcetti R, Ferraccioli G, Pivetta B, De Re V, Gloghini A, D’Agosto A, Bartoli E, Carbone A, Boiocchi M. 1995. Local cytokine expression in the progression toward B cell malignancy in Sjögren’s syndrome. J Rheumatol 22:1674–1680. [PubMed] [Google Scholar]
  • 7. Agostini C, Trentin L, Facco M, Sancetta R, Cerutti A, Tassinari C, Cimarosto L, Adami F, Cipriani A, Zambello R, Semenzato G. 1996. Role of IL‐15, IL‐12, and their receptors in the development of T cell alveolitis in pulmonary sarcoidosis. J Immunol 157:910–918. [PubMed] [Google Scholar]
  • 8. Kotake S, Schumacher HR Jr, Wilder RL. 1996. A simple nested RT‐PCR method for quantitation of the relative amounts of multiple cytokine mRNAs in small tissue samples. J Immunol Methods 199:193–203. [DOI] [PubMed] [Google Scholar]
  • 9. Tanabe BK, Abe LM, Kimura LH, Reinker KA, Yamaga KM. 1996. Cytokine mRNA repertoire of articular chondrocytes from arthritic patients, infants, and neonatal mice. Rheumatol Int 16:67–76. [DOI] [PubMed] [Google Scholar]
  • 10. Llorente L, Richaud‐Patin Y, Alcocer‐Castillejos N Ruiz‐Soto R, Mercado MA, Orozco H, Bamboa‐Dominguez A, Alcocer‐Varela J. 1996. Cytokine gene expression in cirrhotic and non‐cirrhotic human liver. J Hepatol 24:555–563. [DOI] [PubMed] [Google Scholar]
  • 11. Weyand CM, Tetzlaff N, Björnsson J, Brack A, Younge B, Goronzy J. 1997. Disease patterns and tissue cytokine profiles in giant cell arteritis. Arthritis Rheum 40:19–26. [DOI] [PubMed] [Google Scholar]
  • 12. Harville TO, Adams DM, Howard TA, Ware RE. 1997. Oligoclonal expansion of CD45RO+ T lymphocytes in Omenn syndrome. J Clin Immunol 17:322–332. [DOI] [PubMed] [Google Scholar]
  • 13. Yano N, Endoh M, Nomoto Y, Sakai H, Fadden K, Rifai A. 1997. Phenotypic characterization of cytokine expression in patients with IgA nephropathy. J Clin Immunol 17:396–403. [DOI] [PubMed] [Google Scholar]
  • 14. Ajjan RA, Watson PF, Weetman AP. 1997. Detection of IL‐12, IL‐13, and IL‐15 messenger ribonucleic acid in the thyroid of patients with autoimmune thyroid disease. J Clin Endocrinol Metab 82:666–669. [DOI] [PubMed] [Google Scholar]
  • 15. Soslau G, Morgan DA, Jaffe JS, Brodsky I, Wang YH. 1997. Cytokine mRNA expression in human platelets and a megakaryocytic cell line and cytokine modulation of platelet function. Cytokine 9:405–411. [DOI] [PubMed] [Google Scholar]
  • 16. Bulfone‐Paus S, Dürkop H, Paus R, Krause H, Pohl T, Onu A. 1997. Differential regulation of human T lymphoblast functions by IL‐2 and IL‐15. Cytokine 9:507–513. [DOI] [PubMed] [Google Scholar]
  • 17. Yanagihara Y, Kajiwara K, Basaki Y, Ikizama K, Akiyama K, Saito H. 1997. Induction of human IgE synthesis in B cells by a basophilic cell line, KU812. Clin Exp Immunol 108:295–301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Sospedra M, Tolosa E, Armengol P, Ashhab Y, Urlinger S, Lucas‐Martin A, Foz‐Sala M, Jaraquemada D, Pujol‐Borrell R. 1997. Hyperexpression of transporter in antigen processing‐1 (TAP‐1) in thyroid glands affected by autoimmunity: a contributory factor to the breach of tolerance to thyroid antigens? Clin Exp Immunol 109:98–106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Oka H, Fujiwara K, Oda T. 1984. Xiao‐Chai‐Hu‐Tang and Gui‐Zhi‐Fu‐Ling‐Wan for treatment of chronic hepatitisIn: Recent Advances in Traditional Medicine in the East Asia. Amsterdam: Excerpta Medica; p 232–237. [Google Scholar]
  • 20. Hirayama C, Okumura M, Tanikawa K, Yano M, Mizuta M, Ogawa N. 1989. A multicenter randomized controlled clinical trial of Sho‐saiko‐to in chronic active hepatitis. Gastroenterol Jpn 24:715–719. [DOI] [PubMed] [Google Scholar]
  • 21. Yano H, Mizoguchi A, Fukuda K, Haramaki M, Ogasawara S, Momosaki S, Kojiro M. 1994. The herbal medicine Sho‐saiko‐to inhibits proliferation of cancer cell lines by inducing apoptosis and arrest at the G0/G1 phase. Cancer Res 54:448–454. [PubMed] [Google Scholar]
  • 22. Oka H, Yamamoto S, Kuroki T, Harihara S, Marumo T, Kim SR, Monna T, Kobayashi K, Tango T. 1995. Prospective study of chemotherapy of hepatocellular carcinoma with Sho‐saiko‐to (TJ‐9). Cancer 76:743–749. [DOI] [PubMed] [Google Scholar]
  • 23. Sakaida I, Matsumura Y, Akiyama K, Hayashi K, Ishige A, Okita K. 1998. Herbal medicine Sho‐saiko‐to (TJ‐9) prevents liver fibrosis and enzyme‐altered lesions in rat liver cirrhosis induced by a choline‐deficient l‐amino acid‐defined diet. J Hepatol 28:298–306. [DOI] [PubMed] [Google Scholar]
  • 24. Kayano K, Sakaida I, Uchida K, Okita K. 1998. Inhibitory effects of the herbal medicine Sho‐saiko‐to (TJ‐9) on cell proliferation and procollagen gene expressions in cultured rat hepatic stellate cells. J Hepatol 29:642–649. [DOI] [PubMed] [Google Scholar]
  • 25. Shimizu I, Ma YR, Mizobuchi Y, Liu F, Miura T, Nakai Y, Yasuda M, Shiba M, Horie T, Amagaya S, Kawada N, Hori H, Ito S. 1999. Effects of Sho‐saiko‐to, a Japanese Herbal Medicine, on hepatic fibrosis in rats. Hepatology 29:149–160. [DOI] [PubMed] [Google Scholar]
  • 26. Yamashiki M, Kosaka Y, Nishimura A, Takase K, Ichida F. 1992. Efficacy of a herbal medicine ‘Sho‐saiko‐to’ on the improvement of impaired cytokine production of peripheral blood mononuclear cells in patients with chronic viral hepatitis. J Clin Lab Immunol 37:111–121. [PubMed] [Google Scholar]
  • 27. Yamashiki M, Kosaka Y, Nishimura A, Okuda Y, Hamaguchi K, Kondo I, Ichida F. 1993. The Japanese herbal medicine ‘Sho‐saiko‐to’ improves cytokine production of peripheral blood mononuclear cells in patients with liver cirrhosis. Curr Ther Res Clin E 54:86–97. [Google Scholar]
  • 28. Yamashiki M, Nishimura A, Sakaguchi S, Suzuki H, Kosaka Y. 1996. Effects of the Japanese herbal medicine ‘Sho‐saiko‐to’ as a cytokine inducer. Environ Toxicol Pharmacol 2:301–306. [DOI] [PubMed] [Google Scholar]
  • 29. Yamashiki M, Nishimura A, Nomoto M, Suzuki H, Kosaka Y. 1996. Herbal Medicine ‘Sho‐saiko‐to’ induces tumor necrosis factor‐alfa and granulocyte colony‐stimulating factor in vitro in peripheral blood mononuclear cells of patients with hepatocellular carcinoma. J Gastroenterol Hepatol 11:137–142. [DOI] [PubMed] [Google Scholar]
  • 30. Yamashiki M, Nishimura A, Nobori T, Nakabayashi S, Takagi T, Inoue K, Ito M, Matsushita K, Ohtaki H, Kosaka Y. 1997. In vitro effects of Sho‐saiko‐to on production of granulocyte colony‐stimulating factor by mononuclear cells from patients with chronic hepatitis C. Int J Immunopharmacol 19:381–385. [DOI] [PubMed] [Google Scholar]
  • 31. Yamashiki M, Nishimura A, Huang XX, Nobori T, Sakaguchi S, Suzuki H. 1999. Effects of the Japanese herbal medicine Sho‐saiko‐to (TJ‐9) on interleukin 12 production in patients with HCV‐positive liver cirrhosis. Dev Immunol 7:17–22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Yamashiki M, Mase A, Matsushita K, Inoue K. 2000. The Japanese herbal medicine Sho‐saiko‐to (TJ‐9) induces IL‐10 production in vivo. J Hepatol 32:125. [Google Scholar]
  • 33. Chomczynski P, Sacchi N. 1987. Single‐step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal Biochem 162:156–159. [DOI] [PubMed] [Google Scholar]
  • 34. Nakayama H, Yokoi H, Fujita L. 1992. Quantification of mRNA by non‐radioactive RT‐PCR and CCD imaging system. Nucleic Acids Res 20:4939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Utsugisawa K, Tohgi H, Yoshimura M, Nagane Y, Ukitsu M. 1999. Quantification of nicotinic acetylcholine receptor subunits α4 and β2 messenger RNA in postmortem human brain using a non‐radioactive RT‐PCR and CCD imaging system. Brain Res Protocols 4:92–96. [DOI] [PubMed] [Google Scholar]
  • 36. Monpoeho S, Dehee A, Mignotte B, Schwartzbrod L, Marechal V, Nicolas JC, Billaudel S, Ferre V. 2000. Quantification of enterovirus RNA in sludge samples using single tube real‐time RT‐PCR. BioTechniques 29:89–93. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Laboratory Analysis are provided here courtesy of Wiley

RESOURCES