Mechanisms of Ageing and Development 122 (2001) 341– 352 www.elsevier.com/locate/mechagedev Immunological restoration and anti-tumor effect by Japanese herbal medicine in aged mice Masanori Utsuyama a,b, Heinrich Seidlar a, Masanobu Kitagawa a, Katsuiku Hirokawa a,* a Department of Pathology and Immunology, Aging and De6elopmental Sciences, Tokyo Medical and Dental Uni6ersity Graduate School, 1 -5 -45, Yushima, Bunkyo-ku, Tokyo 113 -8519, Japan b Department of Membrane Biochemistry, Tokyo Metropolitan Institute of Gerontology, 35 -2, Sakaecho, Itabashi-ku, Tokyo 173 -0015, Japan Received 3 July 2000; received in revised form 14 November 2000; accepted 15 November 2000 Abstract We examined the effect of two Japanese herbal medicines (Kampo-Hozai) on immunological functions and anti-tumor activity in old mice. Hochu-ekki-to (TJ-41) was remarkably effective in the restoration of impaired immune functions of old mice, in terms of number of T cells and NK cells, and anti-SRBC antibody response, while it was not effective in enhancing immune functions of young mice. Juzen-taiho-to was also effective in increasing the number of T cells, remarkably, and NK cells, slightly, in the aged mice. While a significant increase was not observed in young mice. NK activity increased both in young and old mice with the treatment of TJ-48. A significant decrease was observed in metastatic pulmonary colonies of B16 melanoma cells both in young and old mice treated with Juzen-taiho-to for 16 weeks. These results suggested that some of Japanese herbal medicines were useful in restoration of impaired immune functions of old mice and could be recommended for human elderly. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Japanese herbal medicines; Kampo-Hozai; Immune functions; Aging; NK activity; Anti-tumor effect * Corresponding author. Tel.: 81-3-58035173; fax: +81-3-38131790. E-mail address: hirokawa.pth2@med.tmd.ac.jp (K. Hirokawa). 0047-6374/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 2 4 9 - 9 342 M. Utsuyama et al. / Mechanisms of Ageing and De6elopment 122 (2001) 341–352 1. Introduction A group of Japanese herbal medicine, so-called ‘Kampo-Hozai’ have been used to improve the physical condition of various patients. Among various KampoHozais, we focused on Hochu-ekki-to (TJ-41) and Juzen-taiho-to (TJ-48). TJ-41 is known as a useful drug to recover immune function and was reported to be useful in infection (Mori et al., 1999; Yamaoka et al., 2000), oncostatics-induced leukopenia (Kaneko et al., 1999), and allergy (Suzuki et al., 1999). TJ-48 is useful not only for the recovery of immune function (Abe et al., 1998), but also for the enhancement of anti-tumor effect (Onishi et al., 1998; Saiki et al., 1999). We have been interested in methods reversing and restoring immune functions. There are about four methods for this purpose: (1) transplantation of cells or tissues, (2) dietary manipulations, (3) chemicals or drugs such as anti-oxidants, (4) endocrine manipulations (Hirokawa, 1997). In a previous communication, we reported that vitamin E was effective in enhancing immune vigor of young, but not old mice (Wakikawa et al., 1999). On the contrary, cyclophosphamide, a well known anti-tumor drug was effective in restoring immune functions of old mice, but not young ones (Ishiyama et al., 1999). In the present study, we examined efficacy of Kampo-Hozai in terms of immunological restoration and inhibition of tumor metastasis and compared the effects between young and old mice. 2. Material and methods 2.1. Mice C57BL/6 mice were purchased from SLC (Shizuoka, Japan) and maintained in a SPF colony of Tokyo Metropolitan Institute of Gerontology. In this study, young mice were 3 months of age and old ones were 18–24 months of age. 2.2. Japanese herbal medicine (Kampo-Hozai) Hochu-ekki-to (TJ-41) was prepared as a spray-dried powder of hot-water extract obtained from ten medical plants in the ratio of Astragali Radix (4.0), Atractylodis Lanceae Rhizoma (4.0), Ginseng Radix (4.0), Angelicae Radix (3.0), Bupleuri Radix (2.0), Zizyphi Fructus (2.0), Auranti Nobilis Pericarpium (2.0), Glycyrrhizae Radix (1.5), Cimicifugae Rhizoma (1.0) and Gingiberis Rhizoma (0.5). Juzen-taihoto (TJ-48) was prepared in the same way from ten medical plants in the ratio of Astragali Radix (3.0), Cinamomi Cortex (3.0), Rehmanniae Radix (3.0), Paeoniae Radix (3.0), Cnidii Rhizoma (3.0), Angelicae Radix (3.0), Ginseng Radix (3.0), Hoelen (3.0), Glycyrrhizae Radix (1.5) and Atractylodis Lanceae Rhizoma (3.0). An-chu-san (TJ-5) was prepared from six medical plants in the ratio of Cinamomi Cortex (4.0), Corydalis Tuber (3.0), Ostreae Testa (4.0), Foeniculi Fructus (1.5), Glycyrrhizae Radix (1.5), Amomi Semen (1.0) and Alpinae Offic Rhizoma (0.5). M. Utsuyama et al. / Mechanisms of Ageing and De6elopment 122 (2001) 341–352 343 2.3. Experimental protocol 2.3.1. Experiment 1 TJ-41 was suspended in water and administered orally using gavage. Young and old mice were separated into three groups, respectively; high dose (2000 mg/kg per day), low dose (500 mg/kg per day) and saline as control. Each group was composed of six mice. TJ-41 was given once a day for 14 days and mice were sacrificed 1 day after the last administration. Sheep red blood cells (SRBC) were i.p. given 4.5 days before sacrifice for the assessment of anti-SRBC antibody (Fig. 1a). The total amount of TJ-41 per mouse was 940 mg in young high dose, 226 mg in young low dose, 860 mg in old high dose and 222 mg in old low dose. 2.3.2. Experiment 2 Young or old mice were separated into three groups, respectively, and fed the original diet without Kampo-Hozai or the diet containing TJ-48 or TJ-5 as mentioned below. Each group was composed of ten mice. The diets were given for 16 weeks and mice were sacrificed for various assessments. B 16 melanoma cells (F10, 5×104) were intravenously injected 3 weeks before the sacrifice (Fig. 1b). 2.4. Diet Regular diet, CE-2 (Clea, Japan) was mixed with 1.6% of Anchu-san (TJ-5 diet) or Juzen-taiho-to (TJ-48 diet). Young and old mice were separated into three groups, respectively and given either regular diet (control), TJ-5 diet or TJ-48 diet for 16 weeks. The amount of diet taken per mouse was approximately 3.6 g/day in both young and old mice. Accordingly, the amount of KampoHozai taken per mouse for 16 weeks was estimated at approximately 6.5 g in total. 2.5. Flow cytometry Cell suspension was prepared from spleen. The percentage of lymphocyte subsets was assessed by FACScan (Becton Dickinson, Mountain View, CA). Monoclonal antibodies employed were anti-CD3, anti-NK1.1, anti-CD4, anti-CD8 and anti-B220 (PhaMingen, San Diego, CA). 2.6. Anti-SRBC antibody response Mice were injected intraperitoneally (i.p.) with 0.5 ml of 2% sheep red blood cells (SRBC) in saline and sacrificed 4.5 days later. The spleen cells suspensions were prepared and used for the assessment of direct plaque-forming cells (PFC) as described previously (Wakabayashi et al., 1999). 344 M. Utsuyama et al. / Mechanisms of Ageing and De6elopment 122 (2001) 341–352 Fig. 1. Experimental protocol. (a) Oral administration of TJ-41 by gavage for 14 days. Sheep red blood cells (SRBC) was intraperitoneally injected 4.5 days before the sacrifice. Control mice were given saline in the same way. (b) Feeding of diet containing TJ-48 or TJ-5 for 16 weeks, ad libitum. Mouse melanoma cells, B-16 (F10), was intravenously injected 3 weeks before the sacrifice. Control mice were fed the regular diet without drug. M. Utsuyama et al. / Mechanisms of Ageing and De6elopment 122 (2001) 341–352 345 2.7. NK acti6ity A fixed number of 51Cr-labeled YAC-1 target cell (4× 104) was mixed with either 0.5, 1, 2 or 4×106 spleen cells in a total volume of 0.2 ml in microplate with round bottomed wells. The plate was incubated for 5 h, centrifuged and the radioactivity of the supernatant was counted by a gamma-counter (ARC-380, Aloka, Japan). 2.8. Assessment of lung metastasis of B16 melanoma B16 melanoma F10 cells (established by ten repetitions of an intravenous injection and recovery of cells from a lung metastasis in young C57BL/6 mice) were frozen and kept at −80°C. The number of 5× 104 of F10 cells was intravenously injected into the tail vein of the young and old mice after feeding of the diet with or without Kampo-Hozai mentioned above. The mice were sacrificed 3 weeks later and lungs were perfused with buffered formalin, then transferred to Bouin’s solution. Metastatic nodules on the surface of the lungs were traced, and the numbers were measured. 3. Results 3.1. Effect of Hochu-ekki-to (TJ-41) on the immunological functions of young and old mice, by oral administration using ga6age TJ-41, at high and low doses, was orally given to young and old mice for 14 days and the effect on the immune system was examined. Number of T cells (CD3+ cells) in the spleen did not change in young mice, but significantly increased in old mice by both high and low dose treatments (Fig. 2a). The ratio of CD4/CD8 in T cells did not change greatly in young mice, but significantly increased in old mice at dose dependent manner, approaching to the level of young mice (Fig. 2b). Number of NK1.1+ cells slightly increased in young mice at dose dependent manner. In old mice, number of NK1.1+ cells was very low in control mice, but it significantly increased by treatment with TJ-41, although the level was still lower as compared with the young level (Fig. 2c). Anti-SRBC antibody response did not change greatly by administration of TJ-41 in young mice. In old mice however, the low level of anti-SRBC antibody response significantly increased at dose dependent manner (Fig. 3a). As reflected with a slight increase of NK1.1+ cells, NK activity also slightly increased in young mice. In old mice however, NK activity was very low in control mice and did not change after the treatment (Fig. 3b). We thought that the low NK activity in old mice was caused by stress of firm grasp during oral administration of herbal medicine through gavage. Therefore, in the next experiment, we employed the feeding of diet containing herbal medicine to avoid the stress. 346 M. Utsuyama et al. / Mechanisms of Ageing and De6elopment 122 (2001) 341–352 3.2. Effect of Juzen-taiho-to (TJ-48) on the immunological functions of young and old mice, by dietary feeding In the 2nd experiment, mice were fed ad limitum the diet containing Juzen-taihoto (TJ-48) for 16 weeks. TJ-48, rather than TJ-41, was employed for its stronger capacity to restore anti-tumor activity. Number of T cells (CD3+ cells) in the spleen did not change in young mice fed TJ-48 diet. In old mice, the level of T cell number was low in old mice fed control diet or TJ-5 diet, but significantly increased in the group fed TJ-48 diet (Fig. 4a). Number of NK1.1+ cells in the spleen did not change greatly in young mice fed TJ-48 diet, as compared with other groups. It was of interest to note that number of NK1.1+ in old control mice was slightly higher than in young mice (Fig. 4b) and significantly higher than in old control mice of the first experiment (Fig. 2c). In the Fig. 2. Effect of oral administration of TJ-41 by gavage on immune system of young and aged mice. (a) Number of CD3+ cells in spleen. (b) The ratio of CD4+/CD8+ T cells in spleen. (c) Number of NK1.1+ cells in spleen. Open columns, control. Gray columns, low dose of TJ-41. Solid columns, high dose of TJ-41. Each group, six mice. Vertical bars, S.E.M. M. Utsuyama et al. / Mechanisms of Ageing and De6elopment 122 (2001) 341–352 347 Fig. 3. Effect of oral administration of TJ-41 by gavage on ant-SRBC antibody response (a) and NK activity (b) of young and aged mice. Open columns, control. Gray coloumns, low dose of TJ-41. Solid columns, high dose of TJ-41. Each group, six mice. Vertical bars, S.E.M. 348 M. Utsuyama et al. / Mechanisms of Ageing and De6elopment 122 (2001) 341–352 Fig. 4. Effect of feeding of TJ-5 diet and TJ-48 diet on the immune system of young and aged mice. (a) Number of CD3+ cells in spleen. (b) Number of NK1.1+ cells in spleen. Open columns, control. Hatched columns, TJ-5. Gray columns, TJ-48. Each group, ten mice. Vertical bars, S.E.M. 2nd experiment, a trend of increase was observed in the old group fed TJ-48 diet (Fig. 4b). NK activity significantly increased in young mice fed TJ-48 diet as compared with control (Fig. 5). The level in control group was slightly higher in young than in old mice. It significantly increased in old mice fed TJ-48 diet and approached to the young level (Fig. 5). It was important to note that the level of NK activity in old control mice of the 2nd experiment (Fig. 5) was significantly higher than in old control mice of the first experiment (Fig. 3b). Number of metastatic colony in lungs significantly low in young mice fed either TJ-5 diet or TJ-48 diet as compared with control (Fig. 6). A significant decrease of colony number was also observed in old mice fed TJ-48 diet (Fig. 6). Fig. 7 shows examples of lung metastatic colonies indicating that a significant decrease was observed in both young and old mice fed TJ-48 diet. 4. Discussion One major problem in the elderly people is susceptibility to infection mainly due to immunological insufficiencies (Hirokawa, 1998). Elucidation of mechanism of immunological insufficiencies in the elderly is a very important field. But at the same time, it would be urgent to develop methods to mending immunological insufficiencies of the elderly. Thus, we have been testing various methods to reverse and restore impaired immune functions of old mice. M. Utsuyama et al. / Mechanisms of Ageing and De6elopment 122 (2001) 341–352 349 In animal experiments, the level of immune functions of old mice can be restored to a level approaching that of young adult mice by grafting both newborn thymus and bone marrow cells of young donor mice (Hirokawa et al., 1976). However this method is not easy for human application, because it is almost impossible to find donors of newborn thymus of the same MHC type. Effect of dietary caloric or energy restriction in rodents are pronounced in terms of elongation of life span and immunological restoration (Effros et al., 1990). This method appears to be useful for middle-aged adult in preventing immunological decline in the rest of life, but not applicable to the elderly with immune deficiencies. Various thymic peptides such as thymosin, THF, thymopentin and thymulin (Dardenne and Bach, 1988) are possible candidates for substances to restore immune functions, but reports are still conflicting (Hirokawa, 1997). Solid effect of anti-oxidants or free radical scavengers may be expected in the restoration of immune deficiencies of the elderly. Vitamin E is believed to be useful for immunological restoration (Bendich, 1996). In our experiment using mouse model, however, vitamin E is effective in young, but not in old mice (Wakikawa et al., 1999). There are still many anti-oxidants and research in this area would be promising. Fig. 5. Effect of feeding of TJ-5 diet and TJ-48 diet on NK activity of young and aged mice. Open columns, control. Hatched columns, TJ-5. Gray columns, TJ-48. Each group, ten mice. Vertical bars, S.E.M. 350 M. Utsuyama et al. / Mechanisms of Ageing and De6elopment 122 (2001) 341–352 Fig. 6. Effect of feeding of TJ-5 diet and TJ-48 diet on the number of metastatic colonies of B16 melanomas in lungs. Open columns, control. Hatched columns, TJ-5. Gray columns, TJ-48. Each group, ten mice. Vertical bars, S.E.M. Japanese herbal medicine (Kampo-Hozai) is a mixture of many plant materials, and thus the mechanism of effect appears to be complicated. We selected two Kampo-Hozais, TJ-41 and TJ-48, to test the capacity to restore the immune function and anti-tumor activity, respectively. There have been numerous reports describing the beneficial effect of TJ-41 on immune functions (e.g. Kaneko et al., 1999; Mori et al., 1999; Yamaoka et al., 2000). TJ-48 is known to enhance anti-tumor activity (Onishi et al., 1998; Saiki et al., 1999), in addition to the activity to restore immune functions (Abe et al., 1998). In these experiments, the effect of Kampo-Hozais were tested in young animals in which immune deficiencies were experimentally induced by either stress, anti-tumor drugs or infection. The present study is one of a few studies employing old mice having definitely depressed immune function. Iijima et al. also employed old mice to see the effect of Juzen-taiho-to on the modulation of type 1 and type 2 immune response in old mice (Iijima et al., 1999). We tested the effect of Kampo-Hozai in these old mice in terms of immunological restoration and anti-tumor effect. In the first experiment, TJ-41 was administered orally using gavage by firmly grasping mouse, every day, for 14 days. Although the restoration of T cell number and anti-SRBC response were observed, the number and activity of NK cells in old mice was very low even in control. We thought that this was caused by stress of firm grasp during oral administration of herbal medicine through gavage. In this respect, we obtained the M. Utsuyama et al. / Mechanisms of Ageing and De6elopment 122 (2001) 341–352 351 Fig. 7. Effect of feeding of diet containing TJ-41 and TJ-48 on the gross appearance of pulmonary metastases in young and old mice. Randomly selected five lobes of the lungs are shown for control diet, TJ-5 diet and TJ-48 diet. similar results by the experiment to see the effect of restraint stress on immune system of old mice (Kanno et al., 1997). Therefore in the next experiment to see the effect of TJ-48, mice were fed ad libitum the diet containing TJ-48 for 16 weeks. In this experiment, a significant increase in the number of T cells was again observed in old experimental group and besides the number of NK cells was well preserved in old control mice and slightly enhanced in old experimental mice. NK activity was enhanced definitely in old mice and slightly in young mice. Reflecting the increase of NK activity, number of metastatic colonies in lungs was significantly decreased in both young and old mice. The results indicated that NK cells of old mice were susceptible to stress. Oral administration of foods or drug through gavage must be avoided in experiments to see data, which are susceptible to stress. Both TJ-41 and TJ-48 are useful to restore some of immunological indices of old mice. The magnitude of enhancement is more pronounced in old mice than in young mice. TJ-41 and TJ-48 are already used in patients suffering from cancers or infections to activate immune functions. 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