Suppressive effects of 1,4-dihydroxy-2-naphthoic acid administration on bone resorption
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- Matsubara, M., Yamachika, E., Tsujigiwa, H. et al. Osteoporos Int (2010) 21: 1437. doi:10.1007/s00198-009-1075-y
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The main component of the metabolic by-products of fermentation by Propionibacterium freudenreichii ET-3 is 1,4-dihydroxy-2-naphthoic acid (DHNA), which has a naphthoquinone skeleton, as in vitamin K2. This study showed that DHNA improved bone mass reduction with osteoporosis model mice caused by FK506.
Growth of the intestinal bacterium Lactobacillus bifidus is specifically facilitated by DHNA. The present study used osteoporosis model mice to investigate the effects of DHNA on bone remodeling.
FK506, an immunosuppressant, was used to prepare osteoporosis model mice. Thirty mice were divided into three groups: FK group, FK+DHNA group, and control group. In the FK group, FK506 was administered to induce bone mass reduction. In the FK-DHNA group, FK506 and DHNA were administered concurrently to observe improvements in bone mass reduction. To ascertain systemic and local effects of DHNA, we investigated systemic pathological changes in colon, kidney function and cytokine dynamics, and morphological and organic changes in bone and osteoclast dynamics as assessed by culture experiments.
Compared to the FK group without DHNA, colon damage and kidney dysfunction were milder for FK+DHNA group, and production of inflammatory cytokines (interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α) was more suppressed. Furthermore, compared to the group without DHNA, histological analyses and radiography showed that bone resorption was suppressed for the DHNA group. Culture experiments using osteoclasts from murine bone marrow showed osteoclast suppression for the DHNA group compared to the group without DHNA.
These results show that DHNA has some effects for improving bone mass reduction caused by FK506.
KeywordsBone metabolismBone resorptionDHNAFK506OsteoporosisOsteoporosis treatment
Bone is the key motor organ, but is also a complex organ involved with calcium metabolism, hematopoietic cell maintenance, and hematological organ function. Bone maintains biological function and blood calcium concentration through constant remodeling involving osteoblast-related bone formation and osteoclast-related bone resorption. Homeostasis of bone remodeling is regulated by the endocrine, nervous, and immune systems, and bone diseases occur when this balance is disrupted. One such disease is osteoporosis [1, 2].
In the field of clinical transplantation, recent studies have documented bone diseases caused by immunosuppressive therapy following organ transplantation [3, 4]. Studies have documented that long-term administration of immunosuppressants following organ transplantation causes osteoporosis [5, 6]. We have administered FK506 daily to mice to prepare osteoporosis model mice  and reported that these mice develop high-turnover osteoporosis due to abnormal expression of receptor activator of nuclear factor-κB ligand (RANKL) .
Like cyclosporin A (CsA), FK506 is classified as a suppressor of T cell activity [9, 10]. Both agents hinder calcineurin activities. FK506 binds with a 12-kDa binding protein (FKBP12) in antigen-stimulated T cells. CsA binds with an immunophilin called cyclophilin A to form the CsA–cyclophilin A complex . FK506 forms the FK506–FKBP12 complex to hinder the nuclear transfer of nuclear factor of activated T cell, a transcription factor. Production of interleukin (IL)-2 and other cytokines is subsequently suppressed. Ultimately, T cell activation is hindered to suppress the immune system [12, 13].
As mentioned above, osteoporosis may occur after immunosuppressive therapy [3–7]. As a general rule, pharmacotherapy is administered for this form of osteoporosis [14–18]. However, side effects represent a significant issue with some drugs, and systemic conditions of patients sometimes make drug administration difficult [19–25].
Since vitamin K facilitates bone formation and is produced by microbes, we focused on 1,4-dihydroxy-2-naphthoic acid (DHNA), a bacterial substance with a naphthoquinone skeleton. DHNA is the main component of the metabolic by-products of fermentation by Propionibacterium freudenreichii ET-3 (PC) [26–28]. In addition, DHNA has been reported as a bifidogenic growth stimulator that specifically facilitates the growth of Lactobacillus bifidus, part of the normal intestinal flora [29, 30]. DHNA regulates intestinal function and has been used to treat colitis in clinical settings [29, 30]. DHNA has already been added to foods in Japan. With regard to safety, toxicity studies (Kitasato Laboratory, Toxicity Study Report 1994, Bozo Research Center, Toxicity Study Report 2002) have been conducted and biological non-toxicity has been confirmed . Furthermore, as with vitamin K2, DHNA has a naphthoquinone skeleton. Vitamin K2 is produced by microbes and improves bone metabolism by facilitating calcium fixation . Since DHNA has a naphthoquinone skeleton, we assumed that like vitamin K2, DHNA would be therapeutically effective for osteoporosis.
In the present study, DHNA was administered concurrently with FK506 to osteoporosis model mice to ascertain effects on bone mass reduction. The effects of DHNA on local osseous changes, systemic changes in bone metabolism-related organs, and the dynamics of bone metabolism regulators were ascertained. Pathological changes in systemic conditions such as colon and renal function and cytokine dynamics were examined, morphological and organic changes in osseous tissue were analyzed, and osteoclast dynamics were assessed in culture experiments.
Materials and methods
This study used 30 male ICR mice (6 weeks old; CLEA Japan, Tokyo, Japan) divided into three groups (n = 10 each): FK group, FK+DHNA group, and control group. In the FK group, FK506 (Astellas, Tokyo, Japan) was administered intraperitoneally to induce low bone mass, and dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) was administered orally. In the FK-DHNA group, to observe improvements in low bone mass, FK506 and DHNA (Meiji Milk, Odawara, Japan) were administered concurrently, with FK506 administered intraperitoneally and DHNA administered orally. In the control group, physiological saline was administered intraperitoneally and DMSO administered orally.
During the study, solid food (MF; Oriental Yeast, Tokyo, Japan) and water were provided. Each cage housed two mice in the same group. Mice were weighed every week, and food intake was measured for each group.
The dose of FK506 was set at 1.0 mg kg−1 day−1. DHNA was diluted using 0.002% DMSO, and dose was also set at 1.0 mg kg−1 day−1 to match FK506. This was the minimal dose with which effects were confirmed in the preliminary study. In each group, drugs were administered daily for 5 weeks. Based on the preliminary study, the administration period was also set at 5 weeks.
At the end of the administration period, ether anesthesia was induced and blood was collected from the orbital venous plexus. The femur and colon were then excised. Additionally, in seven of the ten mice in each group, femur was collected for bone analysis and radiographic imaging, and bone marrow cells were collected from three of the ten mice in each group for culture experiments.
The present study was approved by the Animal Study Committee at Okayama University and performed according to the guidelines of the committee.
Shifts in body weight of mice
In all groups, the weight of each mouse and food intake was measured every week. Taking into account errors caused by body movements, body weight was measured three times and an average was calculated. Each cage housed two mice. A set amount of the food was given per cage, and the amount of leftover food was measured every week to calculate average food intake per group.
Osseous tissue observation
In each group, the right femur was collected from seven mice and immediately fixed in 4% paraformaldehyde for 24 h. Fixed tissues were demineralized using 10% ethylenediaminetetraacetic acid for 30 days, dehydrated in the conventional manner, and embedded in paraffin. Paraffinized tissue was sliced into 5-μm sections and stained using hematoxylin and eosin (HE) for histological analysis and tartrate-resistant acid phosphatase (TRAP) stain for osteoclast observation. In addition, in each group, the left femur was collected and analyzed by soft X-ray imaging (Type SRO-M50; Sofron, Tokyo, Japan) under the following conditions: tube current, 3 mA; tube voltage, 40 kV; and irradiation time, 3 s. All samples were placed on a single film. Furthermore, using an animal bone mineral density (BMD) analyzer (DCS-600EX-IIIA; ALOKA, Tokyo, Japan), bone mineral content of the femur were measured. In addition, a micro-CT machine (Scan X mate-A80; Scancom, Tokyo, Japan) was used to obtain 10-μm sections of the distal epiphyseal femur. The resulting images were subjected to analysis using software (TRI/3D-BON; RATOC, Tokyo, Japan) to reconstruct 3D images for bone morphology analysis.
Histological observation of the colon
The excised colon was cut open to eliminate the contents and soaked and fixed in 4% paraformaldehyde for 24 h. Fixed tissues were embedded in paraffin, sliced into 5-μm sections, and stained using HE for histological analysis.
Biochemical analysis of murine serum samples
Blood samples were centrifuged in the conventional manner to separate serum. Using the serum, blood urea nitrogen (BUN), blood creatinine (Cr), serum calcium (Ca), and 23 kinds of cytokines were measured. Cr was determined using the creatinine test ® (Wako, Tokyo, Japan) by the Jaffe method, and BUN was determined using the BUN test ® (Wako) by the urease indophenol method. These two parameters were measured using a DU640 absorption spectrometer (Beckman, Fullerton, CA, USA) according to the manual.
Ca was measured using the Calcium Assay Kit® (Quantichrom, Hayward, CA, USA) based on absorbency using a microplate reader (model 680; Bio-Rad Laboratories, Hercules, CA, USA).
A total of 23 cytokines (IL-1α,IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-17, eotaxin, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-g, keratinocyte chemoattractant, monocyte chemoattractant protein, macrophage inflammatory protein (MIP)-1β, MIP-1β, regulated on activation normal T cell expressed and secreted (RANTES), TNF-α)were measured using the Bio-Plex cytokine assay (23-Plex Panel; Bio-Rad Laboratories) and the model 680 microplate reader (Bio-Rad Laboratories) according to the manual.
In each group, the femur was collected from three mice, and the proximal and distal ends were cut to collect bone marrow cells. For incubation, minimum essential medium alpha (Invitrogen, Carlsbad, CA, USA) enriched with 10% fetal bovine serum (JRH Biosciences, Lenexa, KS, USA) and 1% penicillin–streptomycin (Invitrogen) was used. In each well of a 24-well cell incubation plate (BD Labware, Franklin Lakes, NJ, USA), 500 µl of culture solution and 5 × 105 bone marrow cells were placed, and RANKL (Pepro Tech EC, London, UK) and macrophage colony-stimulating factor (M-CSF; Techne Corporation, Minneapolis, MN, USA) were added. Every 48 h, half of the culture solution containing RANKL and M-CSF was exchanged. Final concentrations were 40 ng/ml for RANKL and 300 ng/ml for M-CSF. From each mouse, culture tests were conducted in six wells each.
Assessment of osteoclast differentiation and formation
After 10 days of incubation, cultured cells were subjected to TRAP staining using acid phosphatase 387A (Sigma-Diagnostics, St. Louis, MO, USA) in accordance with the instructions from the manufacturer. After mounting using glycerin, cells were observed under light microscopy, and numbers of osteoclasts (multinucleated cells with ≥3 TRAP-positive nuclei) and osteoclast precursor cells (TRAP-positive mononuclear cells) were counted.
All data are expressed as mean ± SEM and were analyzed by one-way factorial measures ANOVA for multiple group comparison. Fisher’s least significant difference test was used to specify differences between groups. A p value of less than 0.05 was considered significant. Scheffe’s test for non-parametric paired samples was used for comparison of the semiquantitative evaluation of the repair.
Measurement of body weight and food intake
Compared to the control group, the degree of body weight increase was significantly lower for both the FK and FK-DHNA groups. No significant difference existed between FK and FK-DHNA groups. In addition, no clear differences existed in food intake among the three groups (data not shown).
Colon tissue observation
Biochemical analysis of murine serum samples
After the 5-week administration period, serum Cr, BUN, Ca, and 23 kinds of cytokines were measured. Cr was significantly higher for the FK group than for the FK-DHNA and control groups (p < 0.05), but no significant difference existed between FK-DHNA and control groups. BUN was significantly higher for the FK group than for the FK-DHNA and control groups (p < 0.05), but no significant difference existed between FK-DHNA and control groups. Ca was significantly higher for the FK group than for the FK-DHNA and control groups (p < 0.05), but again, no significant difference existed between FK-DHNA and control groups.
The present study focused on DHNA as a bifidogenic growth stimulator with a naphthoquinone skeleton similar to vitamin K. We investigated the effects of DHNA on bone and measured factors related to bone metabolism. In other words, as mentioned earlier, the present study investigated systemic pathological changes in the colon, kidney function and cytokine dynamics, and morphological and organic changes in bone and osteoclast dynamics as assessed by culture experiments.
Morphological and organic changes in bone
The effects of daily FK506 administration on bone were assessed by examining murine femora by soft X-ray imaging and histological analysis. Compared to the control group, radiolucency was more marked near the medial and distal epiphyses of the femur for the FK group, and bone mass reduction was confirmed. For the FK-DHNA group, although slight radiolucency was confirmed, the findings resembled those for the control group. In addition, using an animal BMD analyzer and micro-CT machine, BMD was measured and bone morphology analysis was performed. As seen by soft X-ray imaging, BMD for the FK group was low. BV/TV was also lower for the FK group. However, these values were higher for the FK-DHNA group, suggesting the suppressive effects of DHNA on bone resorption. Femoral specimens were also analyzed by HE and TRAP staining. Compared to the control group, bone mass reduction was seen for the FK group, and many TRAP-positive multinucleated cells were identified. For the FK-DHNA group, histological findings confirmed improved bone mass reduction and decreases in TRAP-positive multinucleated cells, suggesting that DHNA alleviated FK506-induced bone destruction.
Systemic pathological changes in the colon, kidney function, and cytokines
Compared to the control group, body weight was lower for the FK and FK-DHNA groups. Food intake was also measured, but no significant changes were seen among groups, suggesting the suppressive effects of FK506 on growth.
Regarding the systemic effects of DHNA on bone metabolism, the present study focused on the colon and kidney. In the gastrointestinal tract, 1,25(OH)2D produced by renal proximal uriniferous tubules accelerates the absorption of calcium and phosphoric acid . The kidney is also closely involved with maintaining calcium balance in the body by activating vitamin D. The intestine and kidney are thus organs that are involved with bone metabolism . However, the intestine and kidney are affected by the side effects of FK506, and a study reported that the incidence of gastric ulcer, duodenal ulcer, and colitis was <5%, and the incidence of renal dysfunction (increased creatinine and BUN) was >15% .
For the FK-DHNA group, colon tissue damage was milder, and histological findings for the FK-DHNA group resembled those for the control group. This suggested that DHNA improved colitis and ulcer caused by FK506. DHNA specifically facilitates the growth of L. bifidus to aid intestinal function. Studies have also shown that DHNA increases immune function in the intestine and improves enteritis [29, 30]. Improvements in colon tissue are believed to be beneficial for maintaining the normal balance of bone metabolism.
The effects of DHNA on renal function were assessed by measuring blood Cr and BUN. In the present study, Cr and BUN levels for the control group that did not receive FK506 were considered normal. Cr and BUN for the FK group were higher than those for the FK-DHNA and control groups, confirming that FK506 caused renal dysfunction. Cr and BUN were significantly lower for the FK-DHNA group than for the FK group, suggesting that DHNA improved renal function.
Moreover, calcium metabolism was assessed in relation to colon and kidney dysfunction by measuring serum calcium. Hypercalcemia accompanying bone resorption is present with high-turnover osteoporosis. Serum calcium was significantly higher for the FK group than for the FK-DHNA group. For the FK group, bone resorption increased blood calcium concentration, and for the FK-DHNA group, the suppressive effects of DHNA on bone resorption lowered blood calcium concentration.
To determine the effects of systemic bone metabolism on osteoclasts, we measured cytokines in murine serum and IL-1β, IL-6, TNF-α, IL-5, and GM-CSF increased in the FK group. For the FK-DHNA group, these cytokines were decreased, thus suggesting that FK506 increased these cytokines while DHNA lowered levels. IL-1β facilitates bone resorption by increasing the production of prostaglandin E2, collagenase, IL-6, and IL-11[36, 37]. In addition, TNF-α acts directly on marrow-derived macrophages (which are osteoclast precursor cells) to induce osteoclast differentiation [38–40]. In the present study, DHNA lowered levels of bone resorption-related cytokines, i.e., IL-1β, IL-6, and TNF-α, thus suggesting involvement with the suppression of bone resorption.
Furthermore, DHNA may improve colon inflammation by suppressing these cytokines, although the relationship of IL-5 and GM-CSF to bone metabolism remains unclear.
Dynamics of osteoclasts by culture tests
In vitro experiments were performed by adding M-CSF and RANKL, which are essential for osteoclast differentiation and induction. For the FK group, appearance of osteoclasts was marked, but far fewer osteoclasts were seen for the FK-DHNA group. This suggests that DHNA administration suppressed osteoclast differentiation and formation.
The above findings clarified that DHNA suppresses bone resorption. DHNA has been recognized to facilitate the growth of the intestinal bacteria L. bifidus and regulate intestinal function. DHNA has also been shown to improve the overall immune system and bring about beneficial effects. However, no previous studies have investigated the effects of DHNA on bone. Long-term administration of immunosuppressants causes high-turnover osteoporosis and side effects such as nephropathy and colitis. DHNA alleviates these conditions. Improvements in the kidney and colon appeared to have suppressed metabolic bone resorption. Interestingly, inflammatory cytokines that were suppressed by DHNA were elevated by FK506. While details of the mechanisms underlying these changes could not be clarified, DHNA indeed suppressed these inflammatory cytokines. In the present study, DHNA was found to suppress osteoclasts. Furthermore, DHNA has the same skeleton as vitamin K2. DHNA may thus be used in the treatment of osteoporosis. At present, pharmacotherapy is mostly performed in the treatment of osteoporosis. However, the factors for osteoporosis are complicated, and with long-term drug therapy, secondary damage is another factor that may hinder therapy. With regard to the safety of DHNA, toxicity studies have already confirmed non-toxicity. During the present study, no toxic symptoms or side effects of DHNA were identified in study animals, suggesting safety of the drug.
Conflicts of interest