Abstract: Background and Objective: Marine products rich in calcium became promising supplements used in combination with medication for osteoporosis treatment. This study aimed to use natural products extracted from marine organisms in osteoporosis treatment as novel approach in drug discovery. Therefore, calcium carbonate isolated from Brachidontes variabilis (B. variabilis) shells were used against bone loss in ovariectomized rats. Materials and Methods: Ovariectomized (OVX) rat model was used to test treatment strategy for osteoporosis. Bivalve, Brachidontes variabilis, is a biomineral lamellar composite of calcite and/or aragonite (CaCO3) embedded within an organic framework. Efficaciously advanced novel class of CaCO3 particles isolated from Brachidontes variabilis shells (BVC) were used against bone loss in ovariectomized rats. All obtained data were analyzed using the general liner models (GLM) technique of statistical analysis system. Results: The present study demonstrates that both the X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) could be feasible methods for structural identifications of the CaCO3. The present study showed that treatment of OVX-rats with low and high doses of BVC revealed significant p<0.05 increase in the proximal, distal and total bone mineral density (BMD), elevation in the serum osteoprotegerin (OPG) level and reduction in serum receptor activator of nuclear factor-κB ligand (RANKL) and β2-microglobulin levels. Results revealed that treatment of OVX-rats with low and high doses of BVC prevented significantly the expression alterations of bone resorption and bone formation genes compared with those in OVX-rats. Conclusion: These results suggested that CaCO3 isolated from B. variabilis shells may indeed have a promising significant potential in clinical translation, which then are exploited as a carrier for efficient loading of different types of therapeutic strategies.
INTRODUCTION
Calcium is an important structural component of bone and teeth and is required for the normal function of all muscles and nerves. Calcium carbonate is widely used medicinally as an inexpensive dietary calcium supplement. Calcium carbonate is composed of three important elements that are present in almost all organic and inorganic materials carbon, oxygen and calcium. It exists in three types of crystalline form and this phenomenon is called polymorphism. The crystalline forms of CaCO3 are calcite, aragonite and vaterite. Calcite is the primary constituent of many marine organisms like plankton, sponges and parts of the shells of some bivalves1. The skeleton of corals is commonly believed to be composed entirely of aragonite due to the current Mg/Ca molar ratio of the seawater, which thermodynamically favors the deposition of this polymorph of calcium carbonate (CaCO3)2. CaCO3 is one of most abundant biominerals in nature, it has been considered as a potential inorganic precursor to induce the formation of bone minerals such as hydroxyapatite [Ca10(PO4)6(OH)2]3.
Pathologically, osteoporosis is defined as a skeletal disorder characterized by absolute decrease in the amount of bone, leading to fractures after minimal trauma4. During menopause, osteoporosis is a most important age related health problem for women who often have a negative calcium balance. Decline of calcium is most probably due to decreased intestinal calcium absorption, insufficient dietary calcium intake, as well as increased urinary calcium loss associated with estrogen deficiency5. As a result of bone loss, the main underlying cause of fractures in osteoporosis increased the fragility of bone. Fracture risk is determined by absolute bone mineral density (BMD), regardless of age. The most important risk factor that can cause osteoporosis is the low BMD6.
Arjmandi et al.7, used the OVX rat as a model for studying postmenopausal bone loss. There are many matches between bone loss in rats after ovariectomy and postmenopausal bone loss in women, like (1) Bone resorption exceeding bone formation, (2) Improved bone loss virtually followed by slower bone formation and (3) Higher loss of cancellous bone than cortical bone7. The most mutual animals that are used for experimental studies are rodents, mainly rats and mice. Ovariectomized (OVX) rats were used to study postmenopausal osteoporosis8.
In humans, there are 3 types osteoporosis, Type I: Postmenopausal osteoporosis, Type II: Age-related osteoporosis and Type III: Secondary osteoporosis. Type I typically affects women by estrogen deficiency within 15-20 years after menopause. Wilkosz et al.9 indicated the fact that the decline of ovarian function is more important than age. Therefore, postmenopausal administration of estrogen decreases the occurrence of fractures associated with osteoporosis by about one half.
The treatment of osteoporosis using drugs can be classified into two groups, anti-resorptive or stimulators of bone formation. Antiresorptive drugs include calcium, estrogen, calcitonin and bisphosphonates10,11. Also plant estrogen has been used for prevention of bone resorption. Stoppage of estrogen administration primes to the recurrence of bone resorption and the degree is analogous to the bone resorption after ovariectomy. However, recent findings suggest that bisphosphonates prompt osteonecrosis of the jaw, atrial fibrillation, raising safety and ethical concerns around the use of bisphosphonates12. Therefore, development of new drugs was needed for the prevention of osteoporosis, particularly in postmenopausal women13. Excessive bone degradation in osteoporosis result from an increased activity of resorptive osteoclasts, which express proteases such as metalloproteinase (MMP-9) and cathepsin K. Cathepsin K, is considered as a key player in the process of bone resorption14. Regulation of the osteoclast differentiation and function was obtained by the receptor activator of nuclear factor kappa B ligand (RANKL)15.
Sigma anti-bonding molecule calcium carbonate (SAC; activated ionic calcium) as isolated from oyster shells, this calcium has a weak bonding force with other molecules, radicals or atoms16. In addition, SAC has been used for treatment against bone loss in ovariectomized rats. The aim of the present work was designed to find the best and cheaper source for calcium carbonate. Calcium carbonate isolated from Brachidontes variabilis shells, was used against bone loss in ovariectomized rats. These calcium molecules are expected to increase the animal metabolic system by being absorbed into the cell. Moreover, the present work is conducted to compare between the effect of SAC and BVC as natural products against bone loss in rats model for osteoporosis.
MATERIALS AND METHODS
Characterization Phase analysis: This study has been conducted at Ceramics, Refractories and Building Materials Department, National Research Centre, Egypt, during August-December, 2015, where the isolation of calcium carbonate from Brachidontes variabilis was carried out. Structural identifications have been confirmed using both the X-ray diffraction (XRD) analysis and fourier transform infrared spectroscopy analyses (FT-IR)17-20. Functional nature of the samples was obtained in a Diano X-ray diffractometer. Fourier transform infrared spectroscopy analyses (FT-IR) was attained using [FT/IR-4600 type A (JASCO, USA) and detector TGS]21-24.
Drugs and chemicals: Sigma anti-bonding molecule calcium carbonate (SAC; activated ionic calcium) was purchased from Sigma-Aldrich Corporation, USA. TRIzol reagent was bought from Invitrogen (Germany). The reverse transcription and PCR kits were obtained from Fermentas (USA). SYBR Green Mix was purchased from Stratagene, USA.
Preparation of Brachidontes variabilis calcium carbonate: For administration of the bivalve calcium carbonate, Brachidontes variabilis were collected from the coast of Port Said, Egypt. Bivalve shell, the exoskeleton was finely grounded and kept under -20°C until use.
Experimental animals: Sixty adult albino female rats, weight 250±13 g and aged 14-15 weeks were purchased from the Animal House Colony, Giza, Egypt and maintained on standard laboratory diet and water ad libitum at the Animal House Laboratory, National Research Center (NRC), Dokki, Giza, Egypt. After an acclimation period of 1 week, animals were divided into 6 groups (10 rats/group) and housed individually in filter-top polycarbonate cages. All animals received humane care in compliance with the guidelines of the Animal Care and Use Committee of National Research Center, Egypt.
Experimental design: The acclimatized rats underwent either bilateral laparotomy (sham group, n = 10) or bilateral ovariectomy (OVX, n = 50). Three weeks after recovering from surgery, the OVX rats were randomly allocated into 5 groups as following: Vehicle-treated (OVX, n = 10): SAC1-treated (OVX+SAC1, n = 10): Animals were given drinking water containing 0.0012% SAC for 12 weeks following 3 weeks post-operation, SAC2-treated (OVX+SAC2, n = 10): Animals were given drinking water containing 0.0024% SAC for 12 weeks, BVC1-treated (OVX+BVC1, n = 10): Animals were given drinking water containing 0.0012% BVC for 12 weeks and BVC2-treated (OVX+BVC2, n = 10): Animals were given drinking water containing 0.0024% BVC for 12 weeks.
Sample collections: At the end of the experimental period, blood samples from fasting rats were withdrawn from retro-orbital venous plexus under diethyl ether [(C2H5)2O] anesthesia in dry clean centrifuge. Blood samples were centrifuged and clear sera were separated and immediately stored at -20°C until analyses. The animals were then rapidly sacrificed and the right femurs were harvested. Each right femur bone was carefully cleaned, length and weight were recorded and then stored in formalin buffer 10% for dual energy X-ray absorptiometry (DEXA) [a means of measuring bone mineral density (BMD)]. Bone mineral density of each right femur were measured by DEXA using Norland XR46, version 3.9.6/2.3.1 instrument (Norland X-R-46 version 3.9.6, Peachtree City, GA, USA) equipped with dedicated software for small animal measurements. Such technique provided an integrated measure of right femur proximal, distal and total areas.
Analytical determinations: Serum osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) levels were determined via enzyme linked immunosorbent assay (ELISA) technique as described by O'Brien et al.25 and Teng et al.26, respectively. Serum β2-microglobulin level was assayed by ELISA procedure using International Immuno-Diagnostics kit (Orgentec Diagnostika GmbH, Mainz, Germany) as described by Khalil et al. 27.
Isolation of total RNA: Total RNA was extracted from the bone samples obtained from the intertrochanteric region of the proximal femurof female rats by the standard TRIzolreg; Reagent extraction method (Invitrogen, Germany). Total RNA was treated with 1 U of RQ1 RNAse-free DNAse (Invitrogen, Germany) to digest DNA residues, re-suspended in DEPC-treated water. Purity of total RNA was assessed by the 260/280 nm ratio (between 1.8 and 2.1). Aliquots were used immediately for reverse transcription (RT).
Reverse transcription (RT) reaction: The complete Poly(A)+ RNA isolated from female rat bone samples was reverse transcribed into cDNA in a total volume of 20 μL using RevertAidTM first strand cDNA Synthesis Kit (MBI Fermentas, Germany). The synthesized cDNA containing tubes were flash-cooled in an ice chamber immediately after reaction termination and stored under -20°C until being used for DNA amplification through quantitative real time-polymerase chain reaction (qRT-PCR).
Quantitative real time-polymerase chain reaction (qRT-PCR): The PCR reactions were set up in 25 μL reaction mixtures containing 12.5 μL 1×SYBRreg; Premix Ex TaqTM (TaKaRa, Biotech. Co. Ltd., Germany), 0.5 μL 0.2 μM sense primers, 0.5 μL 0.2 μM antisense primer, 6.5 μL distilled water and 5 μL of cDNA template. Each experiment included a distilled water control28.
| Table 1: | Primer sequences of bone resorption and formation related genes |
The quantitative values of RT-PCR (qRT-PCR) of bone resorption (receptor activator of nuclear factor-κB, RANKL and Matrix metallopeptidase 9, MMP9) and bone formation (Bone gamma-carboxyglutamate protein 2/osteocalcin, BGLAP2 and Collagen type I alpha 1, COL1a1) genes were normalized on the bases of β-actin expression (Table 1). The relative quantification of the target to the reference was determined by using the 2–ΔΔCT method29.
HPLC measurement of 8-Hydroxy-2-deoxyguanosine (8-OHdG) and 2-deoxyguanosine (2-dG): The DNA was extracted from female rat bone marrow by homogenization in buffer containing 1% sodium dodecyl sulphate, 10 mM Tris, 1 mM EDTA (pH 7.4) and an overnight incubation in 0.5 mg mL–1 proteinase K at 55°C. Homogenates were analyzed according to Patel et al.30 and Mazlumoglu et al.31.
Statistical analysis: All data were analyzed using the general liner models (GLM) procedure of statistical analysis system32 followed by Scheffé-test to assess significant differences between groups. The values are expressed as Mean±SEM. All statements of significance were based on probability of p<0.05.
RESULTS
Phase analysis of the shells calcium carbonate: The infrared (FT-IR) signal positions for CO2–3 ions and their assignments are shown in Fig. 1. Characteristic carbonate bands of aragonite were shown at wave number of 1082 cm–1 (υ1) and 860 cm–1 (υ2). The broad band positioned at 1473 cm–1 (υ3) was assigned to asymmetric stretching mode of CO2–3 ions whereas, the peak at about 712 cm–1 (υ4) was unique indications of the calcite crystals. Moreover, the results showed extra IR frequencies corresponding to another chemical species in addition to carbonate ions.
Figure 2 shows the XRD pattern, the calcite robust peak was shown at 2θ = 29.494 ≈ 30° of intensity 100%. However, such band indicated the existence of calcite and comparatively small other peaks at 2θ = 36.094°, 45.098° and 48.46°. Aragonite peak was shown at 2θ = 33o of intensity 28% and several minor peaks at 2θ = 45.908° of intensity 32.1% and at 2θ = 26.283 of intensity 49.4%. The calcite peak is typically larger than that of aragonite peaks.
Levels of serum OPG, RANKL and β2-microglobulin in female rats: The supplementation effect of Brachidontes variabilis calcium carbonate (BVC) and Sigma anti-bonding molecule calcium carbonate (SAC) in ovariectomized rats (OVX) on serum osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL) and beta 2-microglobulin (β2- microglobulin) levels are summarized in Table 2. The results revealed that OVX rats exhibited alteration in levels of different bone related markers compared with sham rats. The OPG level in OVX rats decreased significantly (p<0.05), while levels of serum RANKL and β2-microglobulin increased significantly (p<0.05) in comparison with the sham rats. In contrary, treatment of OVX-rats with SAC exhibited increase in serum OPG level and change in the other two parameters as compared with OVX rats but this effect was significant only (p<0.05) with the high dose of SAC. Furthermore, supplementation of OVX-rats with low and high doses of BVC revealed significant increase (p<0.01) in serum OPG level and decrease (p<0.01) in serum RANKL and β2-microglobulin levels as compared with OVX-rats.
Levels of bone mineral density (BMD) in female rats: Table 3 shows the results of bone mineral density levels in OVX-rats supplemented with BVC and SAC. The results found that OVX-rats showed significant p<0.05 decrease in bone mineral density of proximal (BMD- proximal), distal (BMD- distal) and total (BMD- total) areas of femur bones in comparison with the sham rats. In contrast, OVX-rats treated with low dose of BVC increased slightly the three measurements of BMD compared with OVX-rats.
| Fig. 1: | FTIR spectra of Brachidontes variabilis explained a common phase of the mineral calcium carbonate |
| Fig. 2: | X-ray diffraction (XRD) of the of polymorphs of CaCO3 of Brachidontes variabilis |
| Table 2: | Levels of serum OPG, RANKL and β2-microglobulin in female rats treated with different doses of SAC and BVC |
a,b,c,dMean values within column with unlike superscript letters were significantly different (ap<0.01, b,c,dp<0.05, Scheffé-test), OPG: Osteoprotegerin, RANKL: Receptor activator of nuclear factor-κB ligand, OVX: Ovariectomized, SAC: Sigma anti-bonding molecule calcium carbonate, BVC: Bivalve calcium carbonate, results are expressed as Mean±SEM | |
| Table 3: | Bone mineral density (BMD) in proximal, distal and total areas of femur bones of female rats treated with different doses of SAC and BVC |
a,bMean values within column with unlike superscript letters were significantly different (ap<0.01, bp<0.05, Scheffé-test), OVX: Ovariectomized, SAC: Sigma anti-bonding molecule calcium carbonate, BVC: Bivalve calcium carbonate, results are expressed as Mean±SEM |
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| Fig. 3: | Alterations of RANKL mRNA in bone tissues isolated from female rats treated with different doses of SAC and BVC |
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a,b,cPercentage values within tissue with unlike superscript letters were significantly different (ap<0.01, b,cp<0.05), results are expressed as Mean±SEM of data from at least 10 samples
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While, OVX-rats treated with high dose of SAC induced significant p<0.05 increase in BMD of the three areas as compared with OVX-rats. On the other hand, treatment of OVX-rats with low and high doses of BVC SAC induced highly significant increase in BMD of the three areas as compared with OVX-rats.
Expression alteration in the genes encoding bone resorption and bone formation: The expression results of bone resorption (RANKL and MMP9) and bone formation (BGLAP2 and COL1a1) genes are presented in Fig. 3-6. The results showed that OVX-rats exhibited significantly p<0.05 higher expression values of bone resorption (RANKL and MMP9) genes and lower expression values of bone formation (BGLAP2 and COL1a1) genes in comparison to the sham female rats. However, OVX-rats treated with the low dose of SAC inhibited slightly the expression alterations of bone resorption and bone formation genes compared with those in OVX-rats. Moreover, treatment of OVX-rats with high dose of SAC suppressed significantly the expression alterations of bone resorption and bone formation genes compared with those in OVX-rats.
| Fig. 4: | Alterations of MMP9 mRNA in bone tissues isolated from female rats treated with different doses of SAC and BVC |
a,b,cPercentage values within tissue with unlike superscript letters were significantly different (ap<0.01, b,cp<0.05), results are expressed as Mean±SEM of data from at least 10 samples |
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Furthermore, treatment of OVX-rats with low and high doses of BVC prevented significantly the expression alterations of bone resorption and bone formation genes compared with those in OVX-rats.
Generation of 8-hydroxy-2-deoxyguanosine (8-OhdG): Determination the levels of "8-OHdG" generation in bone marrow cells of female rat genome following SAC and BVC treatment is summarized in Fig. 7.
The results indicated that generation of 8-OHdG/2-dG ratio in OVX female rats increased with highly significant in comparison to sham female rats. However, OVX-rats treated with low dose of SAC induced insignificant decrease in the -OHdG/2-dG ratio compared with OVX-rats. However, treatment of OVX-rats with high dose of SAC decreased p<0.05 significantly the ratio of 8-OHdG/2-dG generation compared with those in OVX-rats.
| Fig. 5: | Alterations of COL1a1 mRNA in bone tissues isolated from female rats treated with different doses of SAC and BVC |
a,b,cPercentage values within tissue with unlike superscript letters were significantly different (ap<0.01, b,cp<0.05), results are expressed as Mean±SEM of data from at least 10 samples |
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| Fig. 6: | Alterations of BGLAP2 mRNA in bone tissues isolated from female rats treated with different doses of SAC and BVC |
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a,b,cPercentage values within tissue with unlike superscript letters were significantly different (ap<0.01, b,cp<0.05), results are expressed as Mean±SEM of data from at least 10 samples
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Moreover, treatment of OVX-rats with low and high doses of BVC decreased with highly significant differences the ratio of 8-OhdG/2-dG generation compared with those in OVX-rats.
DISCUSSION
The physical measurements of B. variabilis calcium carbonate indicated that characteristic carbonate band of aragonite was shown at wavenumber of 1082 cm–1 (υ1) and 860 cm–1 (υ2) as they are infrared inactive for carbonate ions in calcite structure.
| Fig. 7: | Generation of 8-OHdG in rat bone marrow genome following SAC and BVC treatments. DNA damage was expressed as the ratio of oxidized DNA base (8-OHdG) to non-oxidized base (2-dG) in bone marrow DNA |
Results are expressed as Mean±SEM of data from at least 10 samples, a,b,cMean values within cell samples with unlike superscript letters were significantly different (ap<0.01, b,cp<0.05) |
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The broadband positioned at 1473 cm–1 (υ3) was assigned to asymmetric stretching mode of CO2–3 whereas, the peak at about 712 cm–1 (υ4) was unique indications of the calcite crystals, in-plane bending mode, which may be attributed to a change in the local symmetry of carbonate ions17. Besides, a wide band was observed at high wave number related to OH at 3413 cm–1 correlated to the stretching vibrations of structural hydroxyl from the skeletal ring designating the presence of water. More importantly, authors have observed extra IR frequencies corresponding to another chemical species in addition to carbonate ions. IR band at about 2922 cm–1 was attributed to C-H stretching mode18,19. It was clearly found that the fresh specimen was made up of aragonite [a common phase of the mineral calcium carbonates20].
Furthermore, the XRD pattern indicated an easy way to distinguish between the two polymorphs of CaCO3, aragonite and calcite. The calcite robust peak at 2θ = 29.494° ≈ 30° of intensity 100%, indicated the existence of calcite and comparatively small other peaks at 2θ = 36.094°, 45.098° and 48.46°21. Aragonite peak at 2θ = 33o of intensity 28% and several minor peaks at 2θ = 45.908° of intensity 32.1% and at 2θ = 26.283° which represent intensity of 49.4%. Calcite peak was typically larger than that of aragonite peaks22. It’s clear that calcite provides higher intensity in XRD than aragonite23. Overall, from the FT-IR and XRD results, it may be concluded that the components of these products were mainly calcium carbonate24.
The biological study in this work was designed to understand the action mechanism of Brachidontes variabilis calcium carbonate (BVC) on bone tissue formation in OVX-rats. To get a good understanding for the action mechanism of BVC against bone loss in OVX-rats, a biological marker which has high susceptibility and possibility of applying is required to assess the bone transformation in osteoporosis. Therefore, several biological analyses were carried out to evaluate the effect of BVC against bone loss in OVX-rats. The results revealed that treatment of OVX-rats showed decrease in the proximal, distal and total bone mineral density (BMD), decline in the serum OPG level and increase in serum RANKL and β2-microglobulin levels. These findings were in consistent with other studies which reported that ovariectomized rats showed significant p<0.05 increase in serum RANKL and osteoclast surface and with decline of volumetric and areal BMD33-35.
Several studies tried to explain the relationship between exclusion of the ovaries and bone loss in female laboratory animal model. Thus, the primary change in OVX rats after separation of the ovaries coincided with estrogen deficiency is an augment in the depletion of calcium from the skeleton5. In addition, as a result of the fast bone loss in OVX rats, a decrease in the parathyroid hormone secretion takes a place which induces a decline in the absorption of calcium in the intestine and resulting in the loss of calcium from the whole body and bone36.
On the other hand, the present study showed that treatment of OVX-rats with low and high doses of BVC revealed significant p<0.05 increase in the proximal, distal and total BMD, elevation in the serum OPG level and decrease in serum RANKL and β2-microglobulin levels. However, similar effect on BMD, OPG level, RANKL and β2-microglobulin levels was observed with the high dose of SAC. In the same line with our results, Bae and Kim37 reported that OVX rats administrated with a diet having sufficient calcium showed significantly p<0.05 higher lumbar spine BMC compared with OVX rats fed diet without calcium. They reported also that OVX rats showed higher OPG and RANKL levels compared with the control sham rats in which these findings concludes that bone transformation increases rapidly after isolation of ovaries in OVX rats.
Heaney et al.38 reported that calcium carbonate and calcium citrate have been used widely in bone loss disease because they are considered a main ordinary calcium supplements due to well absorption when taken with food. Yang et al.18 demonstrated that absorption of calcium can be secured by intake with food. Moreover, Wright et al.39 reported that when calcium carbonate is taking in meal a well absorption was observed. Furthermore, Loke et al.40 reported that administration of CaCO3 in meal for more than 2 year in bone loss patients after menopause was more efficient in decreasing bone shortage.
Several studies investigated the effect of Ca on the activation and formation of bone tissues using molecular markers such as the mRNA levels37. Therefore, to understand the biological action of BVC on the molecular biology basis authors have used the mRNA expression tool for analysis the activity of genes related to bone resorption (RANKL and MMP9) and bone formation (BGLAP2 and COL1a1)41. The results revealed that treatment of OVX-rats with low and high doses of BVC prevented significantly the expression alterations of bone resorption and bone formation genes compared with those in OVX-rats. RANKL is considered to be one of molecules which regulate the bone metabolism. It is one of the cytokines belong to the TNF (tumor necrosis factor) which play an essential role in development and bone formation42. It is also reported that the main roles of RANKL are condensate on the biology of bone, in particular in bone metabolism such as osteoclastogenesis.
It has complicated role with M-CSF in the differentiation of mature osteoclasts starting of monocyte progenitor from the reservoir of the hematopoietic myeloid. RANKL activates the osteoclast in which it is the main responsible for bone resorption.
During the resorption of bone matrix numerous proteolytic enzymes play degradation role in this process. From these proteolytic enzymes are MMPs (matrix metalloproteinases) and cysteine proteases which considered as the most important proteases in the bone resorption process. Andersen et al.43 reported that among the MMPs gene family, MMP-9 was showed at high expression level during bone resorption process. It has been found that the effect of MMP-9 during the bone resorption is considered as (a) removing the layers of collagen from the bone surface and then (b) demineralization from bone tissues44. Moreover, Logar et al.45 suggested that the cysteine proteases degrade the collagenous layers and then the MMPs act on the bone resorption pits to clean it from the residual of collagen. In contrary, degradation of the bone tissue matrix was prevented in the existence of using MMP inhibitors45.
Osteocalcin, namely bone gamma-carboxyglutamic acid protein 2 (BGLAP2) is produced only by specific bone cells called osteoblasts46. BGLAP2 is considered to play important role in building of bone and regulation of the biological processes associated with body metabolism47. In addition, BGLAP2 is coinciding with regulation of bone mineralization during the bone formation48.
The chemical structure of collagen is a protein which supports and builds up several issues in the body49. In the present study have selected the COL1A1 gene which responsible for production of collagen from the type I, namely pro-a1 (I) chain50. The pro-a1 (I) chain is associated with several kinds of pro-chains such as pro-a1 (I) chain of another type and pro-a2 (I) chain (which coded by COL1A2 gene) to build a complex component of procollagen51. This complex molecule is responsible for formation of strong type of collagen fibers.
In the current study, evaluation the relationship between bone loss and oxidative stress as determined by generation of 8-OHdG was conducted. The results showed that generation of 8-OHdG in OVX female rats increased p<0.05 significantly in comparison to sham female rats. However, treatment of OVX-rats with low and high doses of BVC decreased significantly the ratio of 8-OHdG/2-dG generation compared with those in OVX-rats. For our knowledge, no available data are published concerning he genetic toxicity in OVX rats. However, Donida et al.52 studied the correlation between 8-OHdG generation and RANKL to OPG ratio in women with bone loss. They found that high level of genetic toxicity (in the form of 8-OHdG generation) was correlated with increase of the ratio of RANKL to OPG in serum of women with osteoporosis. Thus, the mechanism of DNA damage in OVX rats may be correlated with the ratio of RANKL to OPG53,54. Further study should be conducted to investigate the relationship between the molecular osteoporosis markers such as ration of RANKL to OPG and oxidative stress inducing DNA damage.
CONCLUSION
This study exhibited positive results for osteoporosis treatment using B. variabilis CaCO3 on the basis of bone mineral density parameters and on the expression of genes related to bone resorption and bone formation. These findings suggested that CaCO3 isolated from B. variabilis shells may have a promising significant potential in clinical translation, which then are exploited as a carrier for efficient loading of different types of therapeutic strategies.
SIGNIFICANCE STATEMENTS
This study discovers the potential effective impact of Brachidontes variabilis calcium carbonate that can be beneficial for osteoporosis treatment. This study will assist the researchers in medicine to uncover natural compounds isolated from marine organisms as safer drugs which enhance the immune system in bone loss patients that many researchers were not able to explore. Thus, a new theory on the calcium carbonate molecules from Brachidontes variabilis as a novel compounds and possibly other components of marine organisms, may be arrived at new composition of drug discovery.
ACKNOWLEDGMENTS
This study was supported by Science and Technology Development Fund (STDF), Academy of Scientific Research and Technology, Egypt, through the project number 5622. Authors thank to all the colleagues in the Animal House, National Research Centre, Dokki, Giza, for taking care of the animals throughout the experiments.