Change in cytokine levels after administration of saikokaryuukotsuboreito or testosterone in patients with symptoms of late-onset hypogonadism

Abstract

The purpose of this study was to evaluate plasma cytokine levels after treatment with saikokaryukotsuboreito (SKRBT), which is a herbal medicine, or androgen replacement treatment (ART), for patients with late-onset hypogonadism (LOH)-related symptoms. Thirty-one patients over 40 years of age with LOH-related symptoms were included in this study. SKRBT was given orally three times daily to a total of 7.5 g/day for 15 eugonadal patients and ART was give to 16 hypogonadal patients by intramuscular injection of testosterone enanthate at 125 mg each time every 2 weeks. Plasma levels of testosterone and 18 cytokines, as well as LOH-related symptoms scored according to the Aging Males' Symptoms (AMS) scale, were compared before and more than 2 months after treatment. In the ART group, the total AMS score was decreased and testosterone was increased significantly after treatment. No cytokine variables were altered significantly after the treatment. In the SKRBT group, although the total AMS score was significantly decreased, testosterone did not change. From the evaluation of cytokines, a significant increase was found in interleukin (IL)- 8, IL-13, interferon-γ and tumour necrosis factor-α. We conclude that SKRBT might improve LOH-related symptoms in eugonadal patients through the beneficial effect of cytokines, a mechanism that is quite different from ART.

Keywords:

• Cytokine
• saikokaryukotsuboreito
• testosterone replacement
• LOH

Introduction

It is well known that the serum androgen level declines with age. Thus, late-onset hypogonadism (LOH), a biochemical syndrome associated with advancing age and characterised by a deficiency in serum androgen with or without decreased genomic sensitivity to androgen, has received widespread attention in the popular and medical media [1]. The first-line treatment for LOH is androgen replacement therapy (ART), which aims to substitute the deficient hormone with a perfect copy of the natural hormone at a dosing schedule that generates physiological hormone levels 24 h a day [2]. There have been reports that ART can improve LOH-related symptoms by enhancing sexual function, libido, and the sense of well-being and that bone and muscle mass are maintained [3–7]. However, it is also well known that many eugonadal patients complain of LOH-related symptoms, as we previously reported [8]. Currently, alternatives to ART for eugonadal patients with LOH-related symptoms are problematic. We recently reported the possibility of the herbal medicine, saikokaryukotsuboreito (SKRBT), as another treatment option [9]. SKRBT is an important Japanese herbal preparation (a kampo medicine; KAMPO) and generally indicated for the relief of hypertension, arteriosclerosis, chronic renal disease, neurasthenia, neurotic palpitations, epilepsy, hysteria and erectile dysfunction (ED). In our previous study, the total score in the Aging Males' Symptoms (AMS) scale [10] and each of the AMS subscores (psychological, somatovegetative, sexual) were significantly decreased by treatment with SKRBT, despite no significant changes in laboratory values and endocrinological variables in response to SKRBT treatment. Recently, another study has shown effective responses of LOH-related symptoms to several preparations of KAMPO, including SKRBT [11]. However, the mechanism by which SKRBT can improve LOH-related symptoms remains unclear.

Recently, it has been reported that some types of KAMPO such as kamishoyosan and chaihuguizhiganjiangtang improved depressed moods in climacteric women by regulating the concentration of both tumour necrosis factor-α (TNF-α) and interleukin (IL)- 6 [12,13]. These interesting studies of menopause gave us clues for exploring the mechanism of SKRBT action in LOH. In the present study, we evaluate symptom scores by questionnaire, and serum concentrations of cytokines before and after treatment with SKRBT and ART, to clarify the relationship between cytokines and treatment efficacy.

Materials and methods

Patients

Thirty-one patients over 40 years of age were included in the study with at least one of seven characteristic LOH symptoms: (1) diminished sexual desire and erectile quality and frequency; (2) changes in mood including fatigue, a depressed mood and irritability as well as concomitant decreases in intellectual activity, cognitive functions and spatial orientation ability; (3) sleep disturbances; (4) decrease in lean body mass with associated diminution in muscle volume and strength; (5) increase in visceral fat; (6) decrease in body hair and skin alterations and (7) decreased bone mineral density and an AMS total score of >26 [10]. No patients with a serious disease such as malignancy, suicidal depression, high prostate-specific antigen (PSA) levels (more than 4.0 ng/ml), or who refused treatment were included in the study. Patients ranged in age from 40 to 66 years (52.9 ± 8.2 years). All patients provided written informed consent for participation in this study. Sixteen patients in whom the serum concentration of total testosterone (TT) was lower than 3.0 ng/ml underwent ART according to the clinical practice manual of the Japanese Urological Association and the Japanese Society for the Study of the Aging Male [14]. Regarding ART, testosterone enanthate (ASKA Pharmaceutical Co., Ltd., Tokyo, Japan) is generally administered intramuscularly at 125 mg every 2 weeks for more than 2 months. For the other 15 patients, in whom the serum concentration of TT was more than 3.0 ng/ml, SKRBT (Table I), which was supplied in the form of a water-extracted dried powder (Tsumura & Co., Tokyo, Japan), was given orally three times a day (before meals) to a total dose of 7.5 g/day for more than 2 months. General symptoms of LOH were judged according to the AMS score. Serum TT and analogue ligand-free testosterone (aFT) levels were examined. Calculated bioavailable testosterone (cBT) was determined on the basis of TT and sex hormone binding globulin levels according to the formula provided by the International Society for the Study of the Aging Male (available at http://www.issam.ch/freetesto.htm) [15]. All blood samples were collected between 9:00 AM and 11:00 AM for monitoring of endocrinological variables. Patient age and the AMS score did not differ between two treatment groups, shown in Table II. Endocrinological values and LOH-related symptoms determined before and more than 2 months after treatment were compared.

Table I. Components of saikokaryukotsuboreito.

Pinellia Tuber	4.0 g
Cinnamon Bark	3.0 g
Poria Sclerotium	3.0 g
Scutellaria Root	2.5 g
Jujube	2.5 g
Ginseng	2.5 g
Oyster Shell	2.5 g
Longgu	2.5 g
Ginger	1.0 g
Bupleurum Root	5.0 g

7.5 g of this product contains 4.5 g of dried extract obtained from mixed raw herbs in the preceding.

Table II. Patient characteristics.

Characteristic	ART, n = 16	SKRBT, n = 15	p
Age	52.4 ± 7.5	53.5 ± 9.2	NS
AMS	48.8 ± 10.3	52.2 ± 11.8	NS
Psychological	13.9 ± 4.50	15.9 ± 6.30	NS
Somatovegetative	19.9 ± 5.20	21.5 ± 5.20	NS
Sexual	14.9 ± 4.10	15.1 ± 4.30	NS
Androgen
TT (ng/ml)	2.09 ± 0.48	4.61 ± 1.03	<0.01
aFT (pg/ml)	7.0 ± 2.0	11.2 ± 2.50	<0.01
cBT (ng/ml)	1.10 ± 0.24	1.90 ± 0.51	<0.01

ART, androgen replacement treatment; SKRBT, saikokaryukotsuboreito; NS, not significant; AMS, Aging Male Symptoms; TT, total testosterone; aFT, analogue-ligand free testosterone; cBT, calculated bioavailable testosterone.

Measurement of cytokine concentration

Concentration of 18 cytokines, including IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-γ, monocyte chemoattractant protein (MCP)- 1, macrophage inflammatory protein (MIP)- 1β, TNF-α and vascular endothelial growth factor (VEGF), were determined using the Bio-Plex Protein Array System (Bio-Rad, Hercules, CA). Cytokine-specific antibody-coated beads (Bio-Rad) were used for these experiments. The assay quantitates cytokines/growth factors over a broad range (0.2–32,000 pg/ml) and eliminates multiple dilutions of high-concentration samples. The samples were prepared and incubated with the antibody-coupled beads for 1 h with continuous shaking. The beads were washed three times with wash buffer to remove unbound protein and then incubated with biotinylated detection of cytokine-specific antibodies for 1 h with continuous shaking. The beads were washed once more and were then incubated with streptavidin–phycoerythrin for 10 min. After incubation, the beads were washed and resuspended in assay buffer, and the constituents of each well were drawn up into the flow-based Bio-Plex Suspension Array System, which identifies each different colour bead as a protein population and quantifies each protein target based on secondary antibody fluorescence. Cytokine concentrations were automatically calculated by Bio-Plex Manager software using a standard curve derived from a recombinant cytokine standard. Concentrations of cytokine before and after treatment were also compared.

Statistical analysis

Data of age, testosterone, AMS score and cytokine concentration are presented as mean ± SD. The comparison of patient background was analysed by the Mann–Whitney U test and differences in values determined before and more than 2 months after treatment were analysed by Wilcoxon's rank sum test. A p value of less than 0.05 was considered statistically significant.

Results

In the ART group, the total AMS score was significantly decreased after treatment, as shown in Table III. Furthermore, the psychological subscore of the AMS was also significantly decreased, whereas differences in the somatovegetative and sexual subscores did not reach significance. With respect to testosterone, TT, aFT and cBT were significantly increased. In the SKRBT group, the total AMS score and psychological and somatovegetative subscores of the AMS were significantly decreased, whereas the sexual subscore was not significantly different after treatment (Table IV). With respect to testosterone, no variables were altered after treatment with SKRBT, as we reported previously [9].

Table III. AMS scores and androgen values before and after androgen replacement treatment.

	Before	After	p
AMS	48.8 ± 10.3	44.1 ± 11.9	<0.05
Psychological	13.9 ± 4.50	11.3 ± 4.60	<0.01
Somatovegetative	19.9 ± 5.20	19.3 ± 5.60	NS
Sexual	14.9 ± 4.10	13.5 ± 4.70	NS
Androgen
TT (ng/ml)	2.09 ± 0.48	4.49 ± 1.96	<0.01
aFT (pg/ml)	7.0 ± 2.0	15.1 ± 10.3	<0.01
cBT (ng/ml)	1.10 ± 0.24	2.48 ± 1.52	<0.01

AMS, Aging Male Symptoms; TT, total testosterone; aFT, analogue-ligand free testosterone; cBT, calculated bioavailable testosterone.

Table IV. AMS scores and androgen values before and after treatment with saikokaryukotsuboreito.

	Before	After	p
AMS	52.2 ± 11.8	44.8 ± 12.6	<0.01
Psychological	15.9 ± 6.30	12.5 ± 5.70	<0.05
Somatovegetative	21.5 ± 5.20	18.0 ± 5.80	<0.05
Sexual	15.1 ± 4.30	14.3 ± 4.60	NS
Androgen
TT (ng/ml)	4.61 ± 1.03	4.69 ± 1.03	NS
aFT (pg/ml)	11.2 ± 2.50	10.1 ± 1.70	NS
cBT (ng/ml)	1.90 ± 0.51	1.78 ± 0.33	NS

AMS, Aging Male Symptoms; TT, total testosterone; aFT, analogue-ligand free testosterone; cBT, calculated bioavailable testosterone.

For the evaluation of cytokines, the concentrations of IL-1β, IL-5 and IL-10 were below the detection limit of the assay. Among the remaining 15 cytokines, no variables were altered significantly after the treatment in the ART group. However, in the SKRBT group, a significant increase was found in IL-8 (from 8.80 pg/ml to 17.99 pg/ml), IL-13 (from 1.20 pg/ml to 1.73 pg/ml), IFN-γ (from 144.63 pg/ml to 179.78 pg/ml) and TNF-α (from 12.07 pg/ml to 18.33 pg/ml; Figure 1). There was no cytokines that significantly decreased after treatment with SKRBT.

Figure 1.  Cytokines with a significant increase after the treatment with saikokaryukotsuboreito. Error bar, standard error.

Discussion

The beneficial effects of short-term ART for patients with LOH has been reported [3–7], although the efficacy, according to long-term ART data on the older population, is still controversial except for effects on body composition and bone mass [16–18]. In general, androgens play many physiological roles in various organs and tissues in which androgen receptors exist. We previously reported a significant improvement in LOH-related symptoms resulting from increased testosterone concentrations after treatment with human chorionic gonadotropin [19]. In the present study, we also found that the AMS score was significantly improved and that TT, aFT and cBT were significantly increased by ART. However, we found that no cytokine variables were altered after ART. Thus, it seemed that the improvement of LOH-related symptoms may be caused by the direct effect of increased serum testosterone, regardless of alterations in plasma cytokines. Conversely, no significant change was found in testosterone after treatment with SKRBT, despite the finding that the total AMS score was significantly decreased, an AMS score response that was similar to ART. The effect of SKRBT on LOH-related symptoms does not depend on the patient's serum testosterone concentration. In addition, we found significant increases in several kinds of cytokine in the SKRBT group. Thus, we speculate that such increases may lead to the mechanism by which SKRBT improves LOH-related symptoms.

Cytokines are involved in various functions of lean body mass, the central nervous system and several organs including bone. In the present study, we found that the four cytokines, IL-8, IL-13, IFN-γ and TNF-α, increased after treatment with SKRBT. IL-8 is produced mainly by macrophages and monocytes and plays a role in modulating an inflammatory response [20]. Recently, it was reported that adipocytes also synthesise and secrete IL-8 [21,22], which may be related to insulin sensitivity and altherosclerosis [23,24]. Although the plasma level of IL-8 is generally higher in obese people than lean subjects [24], it is the plasma levels of IL-8 which have been reported to increase after weight loss by exercise and a weight-maintenance diet [25] and that weight reduction results in an increase in the messenger RNA expression of IL-8 in peripheral blood mononuclear cells [26]. In the present study, we showed an improvement in the somatovegetative subscore of the AMS. Thus, we speculate that an increased IL-8 after treatment with SKRBT may be related to beneficial effects on body composition, such as weight loss, although alteration in weight was not recorded in the present study.

IL-13 also plays a major role in various inflammatory diseases including cancer, asthma and allergy [27]. It mediates a variety of different effects on various cell types including B cells, monocytes, natural killer cells, endothelial cells and fibroblasts. IL-13 is well known as a critical mediator of asthma pathology [28–30]. Recently, it was reported that the plasma concentration of IL13 in women with major depressive disorders was significantly lower than in controls [31], as it is well known that several circulating cytokines are dysregulated in major depression [32]. In the present study, we showed an improvement in psychological subscores of the AMS. Thus, we speculate that increased IL-13 after the treatment with SKRBT may be related with improvement of psychological disorders such as depression. A study showing a low plasma concentration of IL13 in women with major depressive disorders also revealed that their bone mineral density was also significantly lower than that in control group [31]. With respect to bone mineral density, the involvement of another cytokine, IFN-γ, was reported [33]. IFN-γ is produced from many immune cell types such as CD4+, CD8+ cytotoxic T lymphocytes, natural killer cells and natural killer T cells, and from antigen-presenting cells like macrophages, dendritic cells and B lymphocytes [34]. It generally plays a critical role in innate and adaptive immunity against viral and bacterial infections [34]. It was reported that peripheral CD4+ cytotoxic T lymphocytes of women with osteoporosis secreted significantly less IFN-γ than the control group [33]. Thus, the increased IL-13 and IFN-γ after the treatment with SKRBT may provide beneficial effects in bone.

TNF-α is a proinflammatory cytokine produced by macrophages and circulating monocytes that has been implicated in many inflammatory infections and immune disorders. TNF-α affects central processes through stimulation of vagal afferents and acts as a central nervous system modulator [35,36]. Systemic administration of TNF-α was reported to result in increased serotonin levels in the prefrontal cortex and decreased serotonin levels in the paraventriclular nucleus [37]. Thus, these serotonin reactions associated with serum TNF-α levels have been considered to be involved in the depressive status. Indeed, several studies have shown that the serum TNF-α level is higher in adults with major depressive disorders than in the control groups. However, there is a contrary study showing that plasma TNF-α is decreased in depressed patients [38]. In addition, several studies have reported that antidepressants increase the production of several cytokines [38,39]. Furthermore, Gabbay et al. recently reported that suicidal adolescents with major depressive disorders had significantly decreased serum TNF-α concentrations compared to non-suicidal adolescents with major depressive disorders [40]. Thus, the relationship between TNF-α and psychological status is still controversial. Recently, an interesting study has been reported that administration of Kamishoyosan, a herbal medicine, increased plasma TNF-α levels in depressed menopausal patients and improved the score of the Hamilton Rating Scale for depression [12]. Our finding in the present study that administration of SKRBT increased plasma TNF-α and improved psychological subscores of the AMS is very similar to the Kamishoyosan study. Thus, we speculate that the increase in TNF-α as well as IL-13 after herbal medicine treatments may be related with improvement of psychological disorders such as depression.

As already reported, LOH-related symptoms are not only related to serum testosterone concentrations [8], and so we are faced in clinical settings with many eugonadal patients that have LOH-related symptoms. For a long time it has been problematic how to treat such patients by means other than a testosterone preparation. As one potential solution for this problem, we have shown the efficacy of SKRBT for eugonadal patients with LOH-related symptoms in the previous [9] and present studies.

Conclusion

We found that the treatment with SKRBT increased plasma levels of IL-8, IL-13, IFN-γ and TNF-α without alteration of plasma testosterone, whereas ART does not affect any cytokine production. We conclude that herbal medicines including SKRBT have the potential to improve LOH-related symptoms in eugonadal patients by a quite different mechanism to ART, through exploitation of the beneficial effects of cytokines.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.