Journal of Bone and Mineral Metabolism Glycirrhizic acid (GCA) as 11β-hydroxysteroid dehydrogenase inhibitor exerts protective effect against glucocorticoid-induced osteoporosis. --Manuscript Draft-Manuscript Number:
JBMM-D-12-00177R1
Full Title:
Glycirrhizic acid (GCA) as 11β-hydroxysteroid dehydrogenase inhibitor exerts protective effect against glucocorticoid-induced osteoporosis.
Article Type:
Original Article
Corresponding Author:
Ima N Soelaiman UKM Kuala Lumpur, MALAYSIA
Corresponding Author Secondary Information: Corresponding Author's Institution:
UKM
Corresponding Author's Secondary Institution: First Author:
Elvy Suhana Mohd Ramli, M.D
First Author Secondary Information: Order of Authors:
Elvy Suhana Mohd Ramli, M.D Farihah Suhaimi, PhD Siti Fadziyah Asri, M.D Fairus Ahmad, Masters Ima N Soelaiman
Order of Authors Secondary Information: Abstract:
Rapid onset of bone loss is a frequent complication of systemic glucocorticoid therapy which may lead to fragility fractures. Glucocorticoid action in bone depends upon the activity of 11β-hydroxysteroid dehydrogenase type 1 enzyme (11β-HSD1). Regulations of 11β-HSD1 activity may protect the bone against bone loss due to excess glucocorticoids. Glycirrhizic acid (GCA) is a potent inhibitor of 11β-HSD. Treatment with GCA led to significant reduction in bone resorption markers. In this study we determined the effect of GCA on 11β-HSD1 activity in bones of glucocorticoid-induced osteoporotic rats. Thirty six male Sprague-Dawley rats (aged 3 month and weighing 250-300 g) were divided randomly into groups of ten. (i) G1, sham operated group; (ii) G2, adrenalectomized rats administered with intramuscular dexamethasone 120 µg/kg/day and oral vehicle normal saline vehicle; and (iii) G3, adrenalectomized rats administered with intramuscular dexamethasone 120 µg/kg/day and oral GCA 120mg/kg/day The results showed that GCA reduced plasma corticosterone concentration. GCA also reduced serum concentration of the bone resorption marker, pyridinoline and induced 11β-HSD1 dehydrogenase activity in the bone. GCA improved bone structure, which contributed to stronger bone. Therefore GCA has the potential to be used as an agent to protect the bone against glucocorticoid induced osteoporosis. Key Words: Glycirrhizic acid, 11β-hydroxysteroid dehydrogenase type 1, dexamethasone, glucocorticoids, osteoporosis.
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Original article Glycirrhizic acid (GCA) as 11β-hydroxysteroid dehydrogenase inhibitor exerts protective effect against glucocorticoid-induced osteoporosis. Elvy Suhana MR, Farihah HS, Siti Fadziyah A, Faizah O, Fairus A, Ima Nirwana S.
Running title: 11-beta-HSD1 and glucocorticoid induced osteoporosis Conflict of interest: None
Department of Anatomy ,Faculty of Medicine, Universiti Kebangsaan Malaysia Jalan Raja Muda Abdul Aziz, 50300, Kuala Lumpur, Malaysia. -
Elvy Suhana MR, MBBCh, BAO, MMedSc, Lecturer
-
Farihah HS, MBBS, MMedSc, PhD, Associate Professor
-
Faizah O, MD, PhD, Associate Professor
-
Fairus A, MBBCh, BAO, MMedSc, Lecturer
-
Siti Fadziyah A, MBBCh, Student
Department of Pharmacology, Faculty of Medicine, Universiti Kebangsaan Malaysia Jalan Raja Muda Abdul Aziz, 50300, Kuala Lumpur, Malaysia. -
Ima-Nirwana S, MBBS, PhD, Professor
Corresponding author: Prof. Dr Ima Nirwana Soelaiman . Tel: (60)3-92897514 Fax: (60)3-26938205 Email:
[email protected]
Abstract
Rapid onset of bone loss is a frequent complication of systemic glucocorticoid therapy which may lead to fragility fractures. Glucocorticoid action in bone depends upon the activity of 11β-hydroxysteroid dehydrogenase type 1 enzyme (11β-HSD1). Regulations of 11β-HSD1 activity may protect the bone against bone loss due to excess glucocorticoids. Glycirrhizic acid (GCA) is a potent inhibitor of 11β-HSD. Treatment with GCA led to significant reduction in bone resorption markers. In this study we determined the effect of GCA on 11β-HSD1 activity in bones of glucocorticoid-induced osteoporotic rats. Thirty six male Sprague-Dawley rats (aged 3 month and weighing 250-300 g) were divided randomly into groups of ten. (i) G1, sham operated group; (ii) G2, adrenalectomized rats administered with intramuscular dexamethasone 120 µg/kg/day and oral vehicle normal saline vehicle; and (iii) G3, adrenalectomized rats administered with intramuscular dexamethasone 120 µg/kg/day and oral GCA
120mg/kg/day
The
results
showed
that
GCA
reduced
plasma
corticosterone concentration. GCA also reduced serum concentration of the bone resorption marker, pyridinoline and induced 11β-HSD1 dehydrogenase activity in the bone. GCA improved
bone structure, which contributed to
stronger bone. Therefore GCA has the potential to be used as an agent to protect the bone against glucocorticoid induced osteoporosis.
Key Words: Glycirrhizic acid, 11β-hydroxysteroid dehydrogenase type 1, dexamethasone, glucocorticoids, osteoporosis.
Introduction
Osteoporosis is characterized by decreased bone mineral density (BMD) which leads to bone fragility and increased fracture risk due to reduced bone strength [1,2]. This is due to the imbalance of bone remodeling process [1]. Glucocorticoids are crucial for normal bone development but excessive level of glucocorticoids exerts diverse effects on bone microstructure, integrity and mineral metabolism [3,4]. Rapid onset of bone loss and fragility fractures are common complications of long term systemic glucocorticoids treatment and glucocorticoid induced osteoporosis affects up to 50% of patients taking long term glucocorticoid therapy [1,5].
Bone strength is determined by bone size, shape and material properties [6]. Relevant modification of bone quality will lead to change in bone biomechanical properties which ultimately lead to fracture [7]. Non-traumatic or fragility fractures result from weakened bone. Bone fragility is the susceptibility of the bone to fracture which depends on several biomechanical properties. As the load exceeds bone strength, and the energy from the fall exceeds the mechanical energy that the bone can absorb fracture will occur [8]. Bones are considered fragile if they are unable to absorb the energy due to excessive brittleness. Bone fragility can result from: (a) failure of production of optimal mass and strength during growth; (b) excessive bone resorption and (c) inadequate bone formation [1]. Bone fragility is determined by three components: strength, brittleness and work to failure. Stiffness does not directly measure fragility but is used to assess mechanical integrity. Bone quality can be
determined by measuring its intrinsic biomechanical properties [8]. Changes are more pronounced at the appendicular skeleton compared to the spine and bone loss in glucocorticoid induced osteoporosis is most pronounced in trabecular bone and in the cortical shell of the vertebral body [1]. These rapid-onset bone loss will lead to fragility fractures
Histomorphometric studies of glucocorticoid treated patients showed that reduction in bone mass and decreased bone formation is the most significant event leading to chronic bone loss [9-11]. Glucocorticoids decrease the osteoblastic lineage cell number and impair the differentiation of stromal cells to cells of the osteoblastic lineage [12,13]. It also reduces the number of mature osteoblasts by preventing terminal differentiation of quasi-mature osteoblastic cells [14] Studies also have shown that glucocorticoids shift stromal cells away from osteoblasts towards adipocytes, leading to adipogenesis
[14,15].
Decrease in osteoblast number and function leads to reduced synthesis of bone matrix and consequently reduction in trabecular thickness (Tb.Th) [16]. Chavassieux et al. [17] also reported that bone volume and mineralizing surface were significantly lower in the glucocorticoid treated patients.
Glucocorticoids
inhibit
osteoblastogenesis
and
promote
apoptosis
of
osteoblasts and osteocytes which correlates with decrease in bone formation [18]. Studies have shown that glucocorticoids suppress apoptosis and prolong the survival and life span of osteoclasts [19-21]. Apart from modulating proliferation, apoptosis and maturation of the osteoblasts, glucocorticoids also
inhibit the production of a major autocrine stimulator of osteogenes, insulin like growth factor 1 by osteoblasts [22]. Glucocorticoids promotes bone resorption by increasing the expression of RANKL and CSF-1 which together with the decrease in osteoprotegerin expression in osteoblastic and stromal cells, resulted in increased osteoclastogenesis [23,24].
11β-hydroxysteroid dehydrogenase (11β HSD) has been established as a prereceptor signaling pathway in corticosteroid hormone action and has been shown to be an important mediator of glucocorticoid hormone action in several tissues [25,26]. There are two isonzymes of 11β-HSD, 11β-HSD1 and 11βHSD2 which regulate glucocorticoids hormone action [26].11β-HSD1 is a low affinity bidirectional enzyme that catalyzes the interconversion of active cortisol to inactive cortisone. However, in intact tissues it acts predominantly as a reductase, converting cortisone to active cortisol. In contrast 11β-HSD2 is a high affinity dehydrogenase enzyme inactivating cortisol to cortisone [27]. Physiological reductase activity has not been seen in 11β-HSD2. Studies using primary human bone samples have shown that normal adult osteoblasts and osteoclasts express predominantly 11β-HSD1 and the activities of this enzyme vary between individuals. 11β-HSD1 activity plays a positive role in the modulation of osteoclastic activity as inhibition of this enzyme in vivo decreased bone resorption markers [28]. Both reductase and dehydrogenase were evident in human bone but studies focused more on dehydrogenase activity in tissue homogenates which resulted in loss of reductase activity. Reductase activity generates active glucocorticoids in the tissue but dehydrogenase activity
attenuates local active glucocorticoid level. Reductase activity is prominent in maintenance of the physiological level of glucocorticoids but dehydrogenase activity will increase when the active glucocorticoid level exceed the local needs [28]. Defective dehydrogenase activity of 11β-HSD1 leads to increased conversion of cortisosone to cortisol [29]. An in vitro study had shown that glucocorticoids increased the 11β-HSD1 expression in osteoblast [30]. Both isoenzymes are inhibited by liquorice and its derivatives, carbenoxolone and glycyrrhetinic acid. Treatment with carbenoxolone results in a significant fall in bone resorption markers by blocking the osteoclastic 11β-HSD1 activity. However, treatment with carbenoxolone does not affect bone formation markers [28].
Liquorice (glycirrhiza) is a tall erect perennial plant with pinnate leaves foliage bluish-purple flowers. Glycyrrhizic acid isolated from the licorice root is the main active component of licorice. It is widely applied as sweetener in the food and chewing tobacco. It is metabolized to glycyrrhetinic acid, which inhibits 11 betahydroxysteroid dehydrogenase and other enzymes involved in the metabolism of corticosteroids. Glycyrrhetinic acid is 200-1000 times more potent as an inhibitor of 11-beta-hydroxysteroid dehydrogenase compared to glycyrrhizic acid [31]. Carbenoxolone is a synthetic derivatives of glycyrrhetinic acid and inhibits both the 11β-HSD isoenzyme [31]. Studies by Cooper et al. [28] have shown that carbenoxolone treatment did not affect to bone formation markers but significantly reduced bone resorption marker, pyridinoline. However, short term exposure to high levels of glucococorticoids did not change the pyridinoline level
[33,34]. The resorption marker was found to be reduced by blocking
osteoclastic 11β-HSD1 activity. Eijken et al. [35] reported that 11β-HSD1 reductase activity was blocked by 18β-glycirrhetinic acid (GA) in vitro.
The aim of this study was to determine the effect of glycirrhizic acid on the bone structure and strength of male rats given long term glucocorticoid treatment. Since 11β-HSD1 activity determines the sensitivity of bone to therapeutic glucocorticoids,
inhibition
of
this
enzyme
may
be
protective
against
glucocorticoid induced osteoporosis. Glycirrhizic acid which is a known 11βHSD inhibitor may be able to prevent glucocorticoid induced osteoporosis through inhibition of this enzyme. Materials and methods Animals and treatment
All procedures were carried out in accordance with the institutional guidelines for animal research surgical procedures of the Universiti Kebangsaan Malaysia UKM
Research
and
Animal
Ethics
Committee
(UKMAEC)
(No:
ANT/2007/FARIHAH/14-NOV/201-NOV-2007-SEPT-2010)
Thirty six, 3 month-old male Sprague-Dawley rats weighing 250-300 grams were obtained from the Universiti Kebangsaan Malaysia (UKM) Animal Breeding Centre. The GCA was diluted in normal saline.
24 rats were
adrenalectomized and the other 12 rats underwent sham procedure. Adrenalectomy was performed two days after receiving the animals. The rats were anaesthetized with Ketapex and Xylazil (Troy Laboratories, Australia) before dorsal midline and bilateral flank muscle incisions were made.
We
identified the adrenal glands and ligated the vessels to secure the bleeding before removing the glands. The incisions were sutured and the wounds were cleaned. Poviderm Cream (Hoe Pharmaceuticals, Malaysia) was applied to the wound daily for five days to prevent infection and also to aid wound healing. The rats were also given intramuscular injection of Baytril 5% (Bayer Health Care, Thailand) for 5 days. The sham-operated rats underwent a similar procedure except that the adrenal glands were left in-situ.
The adrenalectomized rats were divided randomly into two groups of 12 and another group of 12 were the sham operated rats. The treatment was started 2 weeks after adrenalectomy. The rats were given the following treatments: G1: sham operated control and given intramuscular (IM) vehicle olive oil at 0.05 ml/100 g, G2: adrenalectomised (adrx) and given IM dexamethasone 120 µg/kg/day; G3: adrx and given IM dexamethasone 120 µg/kg/day and glycirrhizic acid 120 mg/kg/day (GCA) by oral gavage. Dexamethasone (Sigma, USA) was dissolved in olive oil (Bertolli, Italy) and administered intramuscularly (120 µg/kg/day) for 6 days a week. The dose and duration of treatment was determined by a pilot study. GCA (Sigma, USA) were dissolved in normal saline and administered by oral gavage at the dose of 120 mg/kg/day, respectively for two months. The sham-operated rats were administered equivalent volumes of vehicle olive oil intramuscularly and vehicle normal saline by oral gavage. The dexamethasone treated adrenalectomized rats (G3) were also administered with vehicle normal saline by oral gavage at 0.1 ml/100 g.
The animals were placed in clean cages under natural sunlight and darkness at night. They were fed with rat pellets (Gold Coin, Malaysia) ad libitium. The adrenalectomized animals were given normal saline to drink ad libitium to replace the salt loss due to mineralocorticoid deficiency post-adrenalectomy while the sham operated animals were given tap water. 2 months after the beginning of treatment the animals were sacrificed under anaesthesia.
The following parameters were measured at the end of two months of treatment- plasma corticosterone, osteocalcin and pyridinoline, 11β-HSD1 enzyme activity and expression in femoral bone, bone biomechanical strength and histomorphometry.
Sample collection
Both femoral bones of each rat were cleared from the soft tissues. The right femoral bones were wrapped in gauze soaked with phosphate buffer saline (PBS) and frozen at -70°C until used for biomechanical testing. The left femoral bones were cut at the mid shaft with a rotary blade (Black & Decker) to separate the distal and proximal parts. The distal part was cut longitudinally to separate the bones into medial and lateral parts. The lateral part of the bones then underwent decalcification process in a mixture of EDTA and 10% formalin.
Plasma corticosterone level measurements
Plasma corticosterone levels were analyzed using the High Performance Liquid Chromatography (HPLC) (Waters, USA) machine utilizing the mobile phase which consists of 60:40 of methanol and deionized water. 100 µl dichloromethyl containing 0.025mg/L cortexolone was added to 100 µl of plasma. The mixture was shaken vigorously for 10 minutes and centrifuged for 5 minutes at 1000 g. 0.05M NaOH was added after discarding the upper aqueous phase and centrifuged for 5 minutes at 1000g. The aqueous phase was again discarded, and this process was repeated. Following the washing steps, the dichloroethane layer was transferred into another tube and evaporated to dryness in a vacuum concentrator. The extract was then resuspended in 1 ml HPLC mobile phase and placed in the vials. Steroids were detected at 254 nm using Empower software (Waters, USA).
Measurement of plasma bone biochemical markers.
Bone biochemical markers were analyzed using the ELISA techniques. The kits used for plasma osteocalcin and Pyd measurements were ELISA Rat-MIDTM rat osteocalcin (Nordic Bioscience Diagnostics A/S, Denmark) and EIA Metra serum PYD kit (Quidel Corp., San Diego, USA) respectively.
Assay for 11β-HSD1 dehydrogenase activity
11β-HSD1 dehydrogenase activity was measured by the method used by previous studies with some modification [36,37]. The right femoral bones were dissected, cleared from soft tissues and washed extensively in phosphatebuffered saline to reduce the fat content. The right femoral bones were grounded into small pieces before being suspended in Krebs-Ringer bicarbonate buffer and homogenized overnight at 4ºC. The bone homogenate was centrifuged at 12,100 g for 20 min at 4 °C and the supernatant was decanted. The total protein content was estimated calorimetrically (Bio-Rad, Hercules, CA, USA). Two hundred micromolar NADP and 12nM [1,2,6,7-3H] corticosterone (specific activity:84 Ci/mmol; Amersham, Buckinghamshire, England) were added to the tissue homogenates containing 0.5 mg protein. Krebs-Ringer bicarbonate buffer containing 0.2% glucose and 0.2% BSA was then added to make up the total assay volume of 250 µl. The required protein concentration and incubation period were determined by the standard curve using various concentrations. After incubation in a water bath at 37ºC for 2 hours, the reaction was terminated by the addition of ethyl acetate and the steroids were then extracted. The organic layer was separated by centrifugation at 3000 rpm and 4ºC for 10 min. The top layer was then transferred into new test tubes and evaporated to dryness at 55 ºC in a vacuum concentrator. Steroid residues were dissolved in an ethanol containing nonradioactive carrier of 11-dehydrocorticosterone and corticosterone. They were then separated by
thin layer chromatography, TLC (Whatman, UK) in 92:8 ratio of chloroform and 95% ethanol. The fractions corresponding to the steroids were located by UV lamp absorption at 240 nm, scraped, transferred into scintillation vials and counted in scintillation fluid (Cocktail T) in a Kontron Betamatic fluid scintillation counter (Merck, Germany). Enzyme activity was calculated as the percentage conversion
of
the
active
[3H]
corticosterone
to
inactive
[3H]
11-
dehydrocorticosterone from the radioactivity of each fraction.
Measurement of 11β-HSD1 expression
11β-HSD1 expression was measured using immunohistochemistry technique. The formalin-fixed embedded bone sections were
deparaffinized and
rehydrated. The sections were incubated in 0.01M citrate buffer at 90ºC for 5 minutes. For antigen retrieval, the sections then were incubated in 0.3% hydrogen peroxide for 10 minutes to block the endogenous peroxide activity, and subsequently incubated in 1:50 normal goat serum (Vector Laboratories; Burlingame, CA) for 20 minutes to block nonspecific antibody binding. Sections were then incubated for 60 minutes with primary rabbit 11β-Hydroxysteroid Dehydrogenase (Type1) Polyclonal Antibody, and detected by goat anti-rabbit peroxidase (Vector Laboratories, Burlinghame California) using DAB (3,3’diaminobezidine) as a chromogen, and counterstained with haematoxylin. Controls were done by using positive tissues (liver and adipose tissue) and omissions of primary antibody. Photomicrographs taken were graded by two observers by double blind technique at 25 times and 50 times magnification. 5
slides from each group were graded for intensity and percentage of immunopositive stained area by two independent viewers. The scores for the percentage of cells stained were as follows: 0 represents no staining in the majority of cells; 1 represents staining in 25-50% of cells; 3 represents staining in 50-75% of cells; 4 represents staining in more than 75% of cells. The grades were added and the averages were taken as the readings. This procedure followed previous studies with some modifications [38]. The 11β-HSD1 evaluations from the two independent viewers were tested by Pearson correlation prior to the normality test.
Bone biomechanical test
The biomechanical properties of the femoral bones were assess using an Instron Universal testing Machine (model 5560, Instron, Canton, MA, USA) equipped with the Bluehill 2 software. The femoral bones were placed in the three-point bending configuration, each bone was placed on two lower supports that are 5 mm apart [39]. The force was applied at mid-diaphysis on the anterior surface such that the anterior surfaces were in compression and the posterior surface in tension until it fractures.
The load, displacement stress and strain parameters were recorded by the software. Graphs of load against displacement and stress against stress were plotted. The slope-value of the load-displacement curve represented the modulus of elasticity of the femur. The main parameters of the bone mechanical
test can be divided into extrinsic and intrinsic parameters: The extrinsic parameters (load, displacement and stiffness) measure the properties of whole bone and the intrinsic parameters (stress, strain and modulus of elasticity) measure the material of bone [8].
Bone histomorphometric analysis
For structural parameter measurements, which included trabecular bone volume (BV/TV) and trabecular thickness (Tb.Th), undecalcified bone samples were embedded in the mixture of Osteo Bed Resin Solution A (Polysciences Inc., PA, Germany) with Benzoyl Peroxide Plasticized (Catalyst) (Polysciences Inc., PA, Germany) in the ratio of 100 ml of Osteo Bed Resin Solution A: 1.4g of Benzoyl Peroxide Plasticized (Catalyst). The samples were sectioned at 9 μm thickness using a microtome (Leica RM2155, Nussloch, Germany) and stained with the Von Kossa method. Structural parameters were analyzed by an image analyzer (Leica DMRXA2, Wetzlar, Germany) using the VideoTest-Master software (VT, St. Petersburg, Russia).
Histomorphometric parameter measurements were performed randomly at the metaphyseal region, which was located 3–7 mm from the lowest point of the growth plate and 1 mm from the lateral cortex, excluding the endocortical region. The selected area is known as the secondary spongiosa area, which is rich in trabecular bone. All parameters were measured according to the guidelines set by the
American Society of
Bone Mineral Research
Histomorphometry Nomenclature Committee (1987) [40].
Statistical Analysis
The statistical software used for data analysis was the Statistical Package for Social Sciences (SPSS) version 12. The 11β-HSD1 evaluations from the two independent viewers were tested by Pearson correlation prior to the normality test. The data was tested for normality using the Kolmogrov-Smirnov test. Since the groups were found to be normally distributed, the data was analyzed using parametric statistics, i.e. the ANOVA test followed by the Tukey pos-hoc test for comparison between treatment groups. P values < 0.05 were taken as significant. Data was presented as mean + standard error of the mean (SEM).
3.
Results
Effects of GCA on plasma corticosterone level in dexamethasone-treated adrenalectomised rats.
Plasma corticosterone level had increased significantly after 2 months in the dexamethasone-treated adrenalectomized rats (G2) compared to the sham operated
group
(G1).
Supplementing
the
dexamethasone
treated
adrenalectomized rats with GCA (G3) had decreased the corticosterone level in the plasma significantly compared to G2. The G2 group also had significantly higher post-treatment corticosterone level. This was not seen in the G1 and G3 groups (Figure 1).
Plasma osteocalcin level
The plasma osteocalcin levels decreased significantly after two months of dexamethasone treatment in adrenalectomized rats compared to the prêtreatment level in all the treatment groups including the sham-operated group. However, no significant difference between the groups was observed before and after the treatment (Figure.2).
Plasma pyridinoline
The pyridinoline levels decreased after two months compared to the pretreatment levels in G1 and G3 but not G2, i.e the adrenalectomized group treated with dexamethasone where the levels did not show any obvious changes. The post-treatment pyridinoline levels of the dexamethasone treated adrenalectomized group supplemented with GCA (G3) were significantly lower compared to G2 (Figure 3).
Effects of GCA on 11β-HSD1 activity in dexamethasone-induced osteoporotic bone
Two months treatment with intramuscular injection of dexamethasone had significantly reduced the 11β-HSD1 dehydrogenase activity in the bone of adrenalectomized rat as seen in G2 compared to the sham-operated G1. Supplementing the dexamethasone treated adrenalectomised rats with GCA
(G3) has increased the 11β-HSD1 dehydrogenase activity towards the sham operated levels G1 (Figure 4).
Effects
of
GCA
on 11β-HSD1
expression
in
dexamethasone-induced
osteoporotic bone
The
adrenalectomized
rats
treated
with
intramuscular
injection
of
dexamethasone (G2) had significantly higher 11β-HSD1 expression compared to the sham-operated group (G1). The 11β-HSD1 dehydrogenase expression was significantly higher in the dexamethasone treated group supplemented with GCA (G3) compared to the sham operated (G1) group (Figure 5). The changes are illustrated in the photomicrograph in Figure 6.
Effects of GCA on bone structural histomorphometry in dexamethasoneinduced osteoporotic bone.
The Bone Volume/Tissue Volume (BV/TV) (Fig.7a), Trabecular thickness (Tb.Th) (Fig76b) and Trabecular Number (Tb.N) (Fig. 7c) were significantly reduced after 2 months treatment with dexamethasone in adrenalectomized rats (G2) compared to the sham operated rats (G1). 2 months treatment with dexamethasone also had significantly increased the Trabecular Separation (Tb.Sp)
(Fig.7d)
of
the
adrenalectomized
rats.
Supplementing
the
dexamethasone treated adrenalectomized rats with GCA 120mg/kg/day had prevented the changes in structural histomorphometric parameter due to
dexamethasone treatment. The changes are illustrated in the photomicrograph in Figure 8.
Effects of GCA on bone biomechanical strength in dexamethasone-induced osteoporotic bone.
2 months dexamethasone treatment had significantly compromised both the intrinsic (flexure modulus, stress and strain) and extrinsic properties (energy, load, and flexure extension). Rats supplemented with GCA (G3) had prevented the changes in both the intrinsic (flexure modulus and flexure strain) and extrinsic properties (energy and flexure extension) due to dexamethasone. The GCA also had improved the stress and load but they were not statistically significant (Figure 9 and 10) .
Discussion
The rats were adrenalectomized to remove the endogenous glucocorticoids that were
subjected
to
circadian
rhythm,
physical
and
emotional
stress.
Predetermined doses of dexamethasone were administered as a replacement for endogenous glucocorticoid to ensure a constant high level of glucocorticoids in the body. No replacement of mineralcorticoids was considered necessary as it was shown earlier to have no influence on bone metabolism [41] However, normal saline ad libitium was given to the animals to maintain normal sodium homeostasis. The dose and duration of dexamethasone treatment (120 µg/kg)
were determined by previous studies [41,42]. The doses of the GCA was also determined by previous studies [43].
In this study we found that the 2 month treatment of adrenalectomized rats with dexamethasone had increased the corticosterone level in the plasma and 11βHSD1 expression in the bone. Consistent with the changes in plasma corticoserone levels, dexamethasone treatment had lowered the 11β-HSD1 dehydrogenase activity and increased the 11β-HSD1 expression in bone. These results were associated with an increase in the serum bone resorption marker, pyridinoline but the bone formation marker, osteocalcin level did not differ from the sham group at the end of 2 months treatment. The dexamethasone induced bone loss was confirmed by the histomorphometric and biochemical evidence of decreased bone formation, increased bone resorption and impaired bone strength.
These findings appear consistent with a previous study, where glucocorticoid treatment was found to cause a time and dose dependent increase in 11βHSD1 reductase activity [44]. Inhibition of 11β-HSD1 dehydrogenase activity in the bone could be associated with an increased in 11β-HSD1 reductase activity leading to increased availability local active glucocorticoid availability [28]. This finding was already explained by Philipov et al. [28], who postulated that defective dehydrogenase activity of 11β-HSD1 lead to increase conversion of cortisone to cortisol. High level of active glucocorticoid concentration in bone inhibits
osteoblastogenesis
while
enhancing
osteoclastogenesis
and
adipogenesis leading to reduce bone formation and increase bone resorption
activity [45]. Glucocorticoids inhibit the synthesis of type I collagen and β1 integrin as well as modify the expression of the osteocalcin gene which contributes to decrease in bone formation [46,47]. Receptor activator of nuclear factor kappa B ligand (RANKL) is an essential cytokine for osteoclast differentiation and activation while osteoprotegerin (OPG) is its natural occurring inhibitor preventing osteoclastogenic activity [48,49]. Both cytokines are expressed by osteoblast. The RANKL to OPG ratio is the determinant of osteoclast cell biology and bone resoprtion [50]. Glucocorticoids influence the RANKL to OPG ratio by enhancing the expression of RANKL and at the same time down-regulating the expression of OPG [23,51]. 11β-HSD1 dehydrogenase may be also suppressed systemically, leading to systemic increased in 11βHSD1 reductase activity, which may explain the increase in plasma corticosterone levels [44]. These changes support the observation of increased bone resorption leading to osteoporosis in the condition of glucocorticoid excess [28].
Supplementing the dexamethasone-treated adrenalectomized rats with GCA had maintained the plasma corticosterone at the pre-treatment level which was also seen in the sham operated group. This result correlates with the higher 11β-HSD1 dehydrogenase activity in bone as well as lower plasma PYD levels which indicated reduced bone resorption. Despite that, the 11β-HSD1 expression in the bone supplemented with GCA was found to be the highest. These results suggested that GCA was able to enhance the 11β-HSD1 expression but a higher proportion of 11β-HSD1 in the bone was demonstrating
dehydrogenase activity. Higher dehydrogenase activity in the bone coupled with lower reductase activity that may have reduced the levels of local active glucocorticoids levels. The inhibition may also be occurring in the systemic circulation, where it was able to maintain normal circulating plasma corticosterone levels. However, GCA did not show any significant effect on the bone formation markers, plasma osteocalcin. The effects of GCA on bone biochemical markers in this study were consistent with a previous study by [28,33,34]. As a potent 11β-HSD1 inhibitor, GCA improved the bone biomechanical strength in both the intrinsic and extrinsic parameters. We carried out
histomorphometric analysis to evaluate structural changes in the
bone that may have contributed to the increase in bone strength. The histomorphometric analysis showed that GCA supplementation produced bone with better trabecular volume, thickness and number that were more compact compared to the adrenalectomized group replaced by dexamethasone.
GCA increased the levels of 11β-HSD1 dehydrogenase activity in the bone that may have reduced the active levels of glucocorticoids in the bone as reported by previous studies [35,44]. By lowering the active glucocorticoids levels in bones GCA may have suppressed the RANKL expression and inhibited the suppression of OPG. This will lead to lower RANKL to OPG ratio that could have reduced osteoclastogenesis that may explain the reduction in bone resorption. GCA had increased the 11β-HSD1 expression in the bone, which exhibit more prominent dehydrogenase activity. Therefore, the reduction of
active glucocorticoids in the bone due to GCA resulted in reduced bone resorption and improved the bone mass, structure and strength.
Conclusion
GCA which is a potent 11β-HSD inhibitor, can inhibit bone resorption and improve the bone structure and biomechanical strength of rats receiving longterm glucocorticoids therapy. However the role of GCA as a prophylaxis in glucocorticoid-induced osteoporosis need to be weighed against its potential complications.
Acknowledgement
This work was supported by Malaysian Ministry of Science Technology and Innovation (MOSTI) and Universiti Kebangsaan Malaysia (UKM) Research Grant. The authors gratefully acknowledge the technical assistance of staffs of Anatomy and Pharmacology Department of Universiti Kebangsaan Malaysia especially Mrs Azizah Othman, Mrs Mazlidiyana and Mr Rafizul.
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Figure legends
Figure 1 The effect of supplementation of GCA (120 mg/kg/day) on plasma corticosterone level of dexamethasone treated adrenalectomized rats (120 µg/kg/day) for two months. Data presented as mean + SEM. Same alphabets indicate significant difference between groups at p< 0.05. * indicate significant difference before and after treatment for the same group. G1= sham operated control; G2= adrenalectomized (adrx) and given intramuscular dexamethasone 120 µg/kg/day (DEX); G3= adrx and given intramuscular DEX 120 µg/kg/day and oral GCA 120 mg/ kg/day
Figure 2 The effect of supplementation of GCA (120 mg/kg/day) on bone formation marker osteocalcin of dexamethasone treated adrenalectomized rats (120 µg/kg/day) for two months. Data presented as mean + SEM. * indicate significant difference before and after treatment for the same group at p
