Juzentaihoto Suppresses Muscle Atrophy in Streptozotocin-Induced Diabetic Mice

Abstract

In diabetic patients, skeletal muscle atrophy occurs due to increased oxidative stress and inflammation. Skeletal muscle atrophy reduces the QOL of patients and worsens life prognosis. Therefore, development of preventive therapy for muscle atrophy in hyperglycemic state is eagerly awaited. Juzentaihoto is a medicinal herb that has a function to supplement physical strength, and it is expected to prevent muscle atrophy. To determine the preventive effect of juzentaihoto on muscle atrophy in hyperglycemic state, streptozotocin (STZ) was administered to induce diabetes in mice and the preventive effect of juzentaihoto was evaluated. Mice that received juzentaihoto extract (JTT) showed that the decrease in muscle fiber cross-sectional area in the gastrocnemius muscle was reversed. Additionally, the expression level of tumor necrosis factor α (TNF-α), an inflammatory cytokine, in serum decreased, and that of ubiquitin ligase (atrogin-1, muscle RING-finger protein-1) mRNA in skeletal muscle decreased. An anti-inflammatory cytokine interleukin-10 showed increased levels in the serum and increased levels in spleen cell culture supernatant collected from mice that received JTT. JTT had no effect on the blood glucose level. These results suggest that prophylactic administration of JTT to STZ-induced diabetic mice affects immune cells such as in spleen, causing an anti-inflammatory effect and inhibiting excessive activation of the ubiquitin-proteasome system, to reverse muscle atrophy.

INTRODUCTION

In diabetic patients, it is reported that skeletal muscle atrophy occurs. In particular, muscle atrophy occurs markedly in type I diabetes. Skeletal muscle atrophy reduces the QOL of patients and worsens life prognosis.^1,2) In diabetic patients, the expression level of inflammatory cytokines increases due to persistence of hyperglycemia state.^3,4) Inflammatory cytokines increase the expression of ubiquitin ligase (muscle RING finger-1 (MuRF1), atrogin-1) and induce atrophy of skeletal muscle.^1,5–9)

Juzentaihoto¹⁰⁾ is a traditional herbal medicine that originated in Ho-chi-chü-fang’s writings at the time of the Song Dynasty in China. Juzentaihoto was reported to have antioxidant, anticancer, and immunostimulatory effects,^11–14) and it is also used against weakness, fatigue, and malaise. Juzentaihoto is expected to prevent muscle atrophy.

In mice intraperitoneally injected with streptozotocin (STZ), pancreatic islets of Langerhans are destroyed and insulin secretion failure occurs, resulting in symptoms similar to type 1 diabetes.^15,16) In STZ-induced diabetic mice, inflammation and oxidative stress are elevated, and ubiquitin ligase expression is increased. This causes skeletal muscle atrophy.^17,18) In this study, diabetic model mice were prepared by STZ injection and juzentaihoto extract (JTT) was administered prophylactically. Skeletal muscle mass and anti-inflammatory effects were then evaluated.

MATERIALS AND METHODS

Preparation of Food Containing JTT

Juzentaihoto extract¹⁰⁾ was provided as the spray-dried product by Tsumura (Tokyo, Japan). Five grams of the extract was obtained from 28.5 g of crude drug mixture shown in Table 1. JTT was mixed with normal feed (CLEA, Shizuoka, Japan) at a rate of 4% (w/w).^19,20)

Table 1. Crude Drug Contents of JTT¹⁰⁾

Crude drug name	Origin	One-day dose (g)
Angelicae Radix	The dried root of Angelica acutiloba (SIEBOLD & ZUCC.) KITAG.	3
Astragali Radix	The dried root of Astragalus propinquus SCHISCHKIN	3
Atractylodis Lanceae Rhizoma	The dried rhizome of Atractylodes lancea DE CANDOLLE	3
Cinnamomi Cortex	The dried bark of Cinnamomum cassia (LINNÉ) J. PRESL	3
Cnidii Rhizoma	The dried rhizome of Cnidium officinale MAKINO	3
Ginseng Radix	The dried root of Panax ginseng C. A. MAYER	1.5
Glycyrrhizae Radix	The dried root and stolon of Glycyrrhiza uralensis FISHER	3
Poria	The dried sclerotium of Wolfiporia cocos RYVARDEN et GILBERTSON	3
Processi Aconiti Radix	The dried tuberous root of Aconitum carmichaeli DEBEAUX prepared by autoclaving	3
Rehmanniae Radix	The dried root of Rehmannia glutinosa (GAERTNER) A. DE CANDOLLE	3

Animals

Animal experiments were conducted with permission of Kochi University Animal Experiment Ethics Committee. ICR mice (8 weeks old, male) were purchased from Japan SLC (Shizuoka, Japan).

STZ was intraperitoneally injected at a dose of 150 mg/kg 4 d before the start of JTT administration (day −4). Blood glucose and body weight were measured on the JTT administration start date (day 0), and mice were divided into two groups (STZ or STZ + JTT) to ensure that the average body weight and blood glucose levels were the same, and each group received normal feed or JTT-mixed feed. Additionally, the control group was fed normal feed.

Sample Preparation

Mice underwent laparotomy under anesthesia on day 35 after starting JTT administration (day 0), and whole blood was collected from the descending vena cava. The collected blood was centrifuged (8000 rpm, 4°C) and the supernatant was used as a serum sample. After collecting the whole blood, the spleen and gastrocnemius muscle were excised and these tissue samples were analyzed.

Blood Glucose

When blood glucose was measured, mice were fasted for 6 h before blood collection. Blood was collected from the tail vein and measured using Glu-TEST-STRIPS (Nova Biomedical, Tokyo, Japan). Blood glucose levels were measured every 7 d. Mice in which the blood glucose level did not exceed 300 mg/mL on day 0 in the STZ-induced mice were excluded from the experiment.

Myocyte Cross-Sectional Area

The excised gastrocnemius muscle was fixed with 10% formalin, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin. A cross-section of the gastrocnemius muscle was traced using computer analysis software WinROOF (Mitani Corporation, Ohtsu, Japan), and the cross-sectional area of each fiber was measured. Cross-sectional areas of 100 muscle fibers from each mouse were randomly measured, and the average value was used in the analysis.

Spleen Cells Culture

The excised spleen was pressed through a 70 µm EASY strainer (Falcon, New York, U.S.A.) to obtain a cell suspension. The obtained cell suspension was stained with a 0.4% (w/v) trypan blue solution (FUJIFILM Wako, Osaka, Japan) and the number of viable cells was counted. Thereafter, the cell suspension was seeded onto a 48-well plate, with 1.0 × 10⁵ cells per well. Cells were cultured for 48 h with RPMI 1640 mixed with 1.0 µg/mL concanavalin A, 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Then, the culture supernatant was collected, and interleukin (IL)-10 levels were measured using an enzyme-linked immunosorbent assay (ELISA).

ELISA

Tumor necrosis factor (TNF)-α levels in the serum were measured using a TNF-α ELISA kit (R&D Systems, Minneapolis, U.S.A.). Interleukin (IL)-10 serum and culture supernatant levels were measured using a Novex IL-10 ELISA kit (Invitrogen, Carlsbad, CA, U.S.A.).

Quantitative RT-PCR of MuRF1 and Atrogin-1

Gastrocnemius was cut to 0.1 cm width and then homogenized with POLYTRON RT-MR 3100 (Central Science Trade, Tokyo, Japan). After that, it was centrifuged (8000 rpm, 4°C) and the supernatant was collected. Total RNA was collected from the supernatant of homogenized gastrocnemius using an RNeasy Fibrous Tissue Mini Kit (Qiagen, Hilden, Germany). The collected RNA was reverse transcribed using a PrimeScript RT reagent Kit (TaKaRa Bio, Ohsu, Japan) to obtain cDNA. TaqMan quantitative PCR was performed using StepOnePlus Real-Time PCR Systems (Applied Biosystems, CA, U.S.A.). MuRF1 and atrogin-1 mRNA expression levels were normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression level. PCR primers with TaqMan probe for GAPDH (Mm99999915_g1), MuRF1 (Mm01185221_m1) and Atrogin1 (Mm00499523_m1) and Taq Man Universal PCR Master Mix were from Applied Biosystems (Foster City, CA, U.S.A.).

Statistical Analysis

The transition of blood glucose was analyzed by two-way ANOVA, and other experimental results were analyzed by one-way ANOVA, followed by multiple comparison test using Dunnet’ t-test. These statistical analyses were conducted using Stat Flex program (View Flex, Tokyo, Japan). A p-value of <0.05 was considered statistically significant.

RESULTS

Blood Glucose Levels

We measured blood glucose levels every 7 d from the start of JTT administration to confirm the influence of JTT on blood glucose. There was no difference in the blood glucose level in each group at the time of intraperitoneal STZ injection 4 d before JTT administration (day −4) (Fig. 1). In the period from the JTT administration start date (day 0) to the administration end date (day 35), the blood glucose level was significantly elevated in the STZ induced group (STZ group) compared to the control group at day 0, 7, 14, 21, 28, 35 (p < 0.01). Meanwhile, there was no significant difference in the JTT administration group (STZ + JTT group) compared with the STZ group at each day (Fig. 1). Two way ANOVA results indicated that these effects was significant about the time (F_6,84 = 104, p < 0.01), drug-treatment (F_2,84 = 665, p < 0.01) and interaction of the time and drug-treatment (F_12,84 = 10.7, p < 0.01).

Fig. 1. Effect of JTT on the Level of Blood Glucose

Blood glucose levels were measured at −4, 0, 7, 14, 21, 28, 35 d after JTT administration. The results were expressed as mean ± standard deviation (S.D.) (n = 5). The data was evaluated by two-way ANOVA, and the comparison among drug-treatment groups in each day evaluated by Dunnett’s multiple comparison test. ** p < 0.01 vs. control group.

Myocyte Cross-Sectional Area

The cross-sectional area of muscle fibers in the gastrocnemius extracted 35 d after JTT administration was compared among the groups to confirm the effect of JTT on preventing skeletal muscle atrophy. In the STZ group, the gastrocnemius muscle fiber cross-sectional area was significantly reduced compared with the control group (p < 0.01), and this reduction was significantly lower in the JTT group (p < 0.01) (Figs. 2A, B). One way ANOVA result indicated that the effect was significant (F_2,12 = 16.9, p < 0.01).

Fig. 2. Myocyte Cross-Sectional Area of Gastrocneminus

Gastrocnemius muscle was removed from mice 35 d after the start of JTT administration. After that, stained with Haematoxylin and Eosin. (A) Myocyte cross-sections of gastrocnemius, (B) the summary data of myocyte cross sectional area. These data were expressed as mean ± S.D. (n = 5), and evaluated by one way ANOVA, and followed by Dunnett’s multiple comparison test. ** p < 0.01 between the groups. (Color figure can be accessed in the online version.)

Inflammatory Cytokines

TNF-α levels in serum collected 35 d after the start of JTT administration were measured to confirm the effect of JTT on inflammatory cytokines. In the STZ group, serum TNF-α levels were significantly increased compared with the control group (p < 0.01), but this increase was significantly inhibited in the STZ + JTT group (p < 0.05) (Fig. 3A). One way ANOVA result indicated that the effect was significant (F_2,12 = 8.35, p < 0.01).

Fig. 3. The Levels of Cytokines in the Serum of Mice

Blood was collected from mice at 35 d after the start of JTT administration. And, (A) the levels of TNF-α and (B) the level of IL-10 in the serum of mice. These data were expressed as mean ± S.D. (n = 5), and evaluated by one way ANOVA, and followed by Dunnett’s multiple comparison test. ** p < 0.01, * p < 0.05 between the groups.

Anti-inflammatory Cytokines

IL-10 anti-inflammatory cytokine levels after JTT administration were measured. In the STZ group, serum IL-10 levels were significantly decreased compared with the control group (p < 0.01), but the decrease was prevented in the STZ + JTT group (p < 0.05) (Fig. 3B). One way ANOVA result indicated that the effect was significant (F_2,12 = 15.4, p < 0.01). Additionally, cells were harvested from the spleen of these mice and cultured, and IL-10 levels contained in the culture supernatant were measured. Thus, IL-10 levels were significantly decreased in the STZ group compared with the control group (p < 0.05), but this decrease tended to be prevented in the STZ + JTT group (p = 0.066) (Fig. 4). One way ANOVA result indicated that the effect was significant (F_2,12 = 4.30, p < 0.05).

Fig. 4. The Levels of IL-10 in the Supernatant Following the Culture of Spleen Cells Obtained from Mice

Spleen was removed from mice 35 d after the start of JTT administration. And, the cells were obtained from spleen and cultured for 48 h with 1.0 µg/mL concanavalin A. After that, the culture supernatant was collected, and IL-10 levels were measured. These data were expressed as mean ± S.D. (n = 5), and evaluated by one way ANOVA, and followed by Dunnett’s multiple comparison test. * p < 0.05 between the groups.

Ubiquitin Ligase mRNA Expression

Total RNA was extracted from the gastrocnemius muscle that was excised from the mouse lower limb, and ubiquitin ligase mRNA expression levels (atrogin-1, MuRF1) were measured. In the STZ group, MuRF1 and Atrogin1 expression levels were significantly increased compared with the control group (p < 0.01), but this increase in both MuRF1 and Atrogin1 levels was significantly reduced in the STZ + JTT group (p < 0.05) (Figs. 5A, B). One way ANOVA result indicated that these effects were significant (MuRF1: F_2,12 = 8.81, p < 0.01, atrogin-1: F_2,12 = 7.92, p < 0.01).

Fig. 5. The Levels of Ubiquitin Ligase mRNA Expression in Gastrocneminus

Gastrocnemius muscle was removed from mice 35 d after the start of JTT administration. After that, mRNA in the gastrocnemius was extracted. And, mRNA expressions of (A) atrogin-1 and (B) MuRF1 were measured. These data were expressed as mean ± S.D. (n = 5), and evaluated by one way ANOVA, and followed by Dunnett’s multiple comparison test. ** p < 0.01, * p < 0.05 between the groups.

DISCUSSION

The most important finding in this study is that skeletal muscle atrophy was prevented by prophylactic administration of JTT to STZ-induced diabetic mice (Figs. 2A, B). However, there was no improvement in the hyperglycemic condition resulting from JTT administration (Fig. 1), suggesting that JTT improved muscle atrophy regardless of the blood glucose level.

The inflammatory cytokine expression level increases under hyperglycemic conditions.^3,4) Inflammatory cytokines increase ubiquitin ligase expression (MuRF1, atrogin-1) and cause skeletal muscle atrophy.^5,6) In STZ-induced mice, skeletal muscle atrophy occurs through elevated ubiquitin ligase expression, which results from an increase in inflammatory cytokine levels.^17,18) The same phenomenon with STZ injection also occurred in this study. However, prophylactic JTT administration to STZ-induced diabetic mice reduced serum levels of the inflammatory cytokine TNF-α (Fig. 3A). Additionally, the increase in Atrogin1 and MuRF1 expression was also suppressed (Figs. 5A, B). Thus, we suggest that JTT inhibits skeletal muscle atrophy by suppressing inflammatory cytokine levels and the enhancing the ubiquitin proteasome system.

IL-10 is an anti-inflammatory cytokine that is known to suppress inflammatory cytokine levels and reduce inflammation that is mainly produced from Th2 cells, B cells, and mast cells.^21,22) IL-10 levels decrease in diabetic patients, and this is considered to cause worsening of inflammatory diseases.²³⁾ It is also known to improve diabetic symptoms and skeletal muscle atrophy by increasing the IL-10 levels in diabetic mice.^24–26) It is expected to be a target factor for the treatment of diabetes and its complications. Additionally, in this study, IL-10 levels decreased with injection of STZ, and this decrease was reversed by prophylactic JTT administration (Fig. 3B). Most IL-10 is produced in the spleen,^27,28) and diabetic complications are exacerbated by removal of the spleen.^29,30) In this study, cells harvested from spleens isolated from these mice were cultured and IL-10 levels in the culture supernatant were measured. IL-10 expression was shown to decrease in the STZ-induced group, but in mice that received JTT, decrease tended to be prevented (Fig. 4). It is thought that JTT acts on immune cells such as spleen and has an action to promote the release of IL-10. However, as the reason that changes in IL-10 did not become significant in this study, much of IL-10 is produced in the spleen, but IL-10 production also occurs from tissues other than spleen. Therefore, it was thought that serum IL-10 concentration could not be explained only by those derived from spleen cells. JTT may also act on immune tissues other than spleen, such as adipocytes, lymph nodes, and thymus.

Similar to this study, there are several prior studies examining the effect of preventing effect for skeletal muscle atrophy in diabetic mice. For example, flavonoids from Glycyrrhiza species and panaxatriol from Panax species have been reported to suppress inflammatory cytokines and muscle atrophy.^31–33) Juzentaihoto contains root of Glycyrrhiza uralensis and root of Panama ginseng,¹⁰⁾ and, these compounds may be one of the active ingredients of juzentaihoto. But, the doses in previous studies of these active ingredients are higher compared to the JTT doses in this study. Therefore, it is unlikely that juzentaihoto alleviates muscle atrophy of diabetes by effect of these active ingredient, and juzentaihoto may contain other active ingredients.

Thus, we suggest that JTT acts on immune cells, shows an anti-inflammatory effect and decreases ubiquitin ligase expression. And this suppresses skeletal muscle atrophy. In clinical practice, it is expected that juzentaihoto inhibits inflammation and prevents muscle atrophy in diabetic patients.

Conflict of Interest

Mitsuhiko Miyamura received a research grant from Tsumura Co., Ltd.

Supplementary Materials

The online version of this article contains supplementary materials.

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