|Year : 2019 | Volume
| Issue : 1 | Page : 33-41
Green tea extract suppresses osteoclastogenesis and opposes glucocorticoid-induced osteoporosis in rats
Dalia A Saad1, Abd El-Hamid A Mohamed1, Shimaa A El-Gohary2
1 Department of Physiology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
|Date of Submission||22-Apr-2019|
|Date of Acceptance||26-May-2019|
|Date of Web Publication||27-Jun-2019|
Abd El-Hamid A Mohamed
Department of Physiology, Faculty of Medicine, Ain Shams University, Cairo, 11727
Source of Support: None, Conflict of Interest: None
Background/aim Glucocorticoids-induced osteoporosis (GIO) through suppression of osteoblast activity, induction of receptor activator of nuclear factor-κB ligand (RANKL), and downregulation of osteoprotegerin (OPG). Green tea has antioxidant and anti-inflammatory properties. This study aims to evaluate the effects of green tea extract (GTE) supplementation on bone proteins, mineralization, and markers in GIO.
Materials and methods The study was performed on 30 adult male Wistar rats, which were divided into three groups: control (C, n=10); GIO (n=10), which received dexamethasone 7 mg/kg body weight intramuscular once/week for 4 weeks; and GTE-supplemented osteoporotic (GT-GIO, n=10), which received dexamethasone, followed by 300 mg/kg per day of GTE by gavage for 4 weeks, with continuation of the dexamethasone. Serum calcium, phosphate, alkaline phosphatase activity, parathyroid hormone, deoxypyridinoline, osteocalcin, OPG, and RANKL were determined. Samples of left tibia were used for histopathological examination of bone.
Results A significant decrease in serum Ca++, PO4, osteocalcin, and OPG, accompanied by a significant increase in serum bone-specific alkaline phosphatase, deoxypyridinoline, and RANKL was detected in the GIO group compared with the control group. GT-GIO group showed a significant increase in serum Ca++, PO4, osteocalcin, and OPG, with a significant decrease in serum bone-specific alkaline phosphatase, deoxypyridinoline, and RANKL compared with the GIO group. Bone examination of GIO rats revealed thinning, loss of architecture, erosions, and increased osteoclasts. Bone sections of the GT-GIO group revealed significant improvement, regain of normal architecture, and decreased osteoclast number.
Conclusion GTE had an antiosteoporotic effect in GIO, and its effects are much more than its antioxidant characteristics.
Keywords: bone markers, glucocorticoids, green tea, osteoporosis
|How to cite this article:|
Saad DA, Mohamed AHA, El-Gohary SA. Green tea extract suppresses osteoclastogenesis and opposes glucocorticoid-induced osteoporosis in rats. J Arab Soc Med Res 2019;14:33-41
|How to cite this URL:|
Saad DA, Mohamed AHA, El-Gohary SA. Green tea extract suppresses osteoclastogenesis and opposes glucocorticoid-induced osteoporosis in rats. J Arab Soc Med Res [serial online] 2019 [cited 2020 Feb 25];14:33-41. Available from: http://www.new.asmr.eg.net/text.asp?2019/14/1/33/261612
| Introduction|| |
Glucocorticoids (GCs) are commonly used immunosuppressive and anti-inflammatory drugs. GCs are used frequently in the treatment of many inflammatory, allergic, and autoimmune disorders. GCs can actively decrease severity of these types of disorders, but their massive and long-term use is always associated with multiple complications, including osteoporosis .
The mechanisms involved in glucocorticoids-induced osteoporosis (GIO) are multiple. GCs suppress the formation of the bone-forming cells, osteoblasts, and induce their apoptosis . In addition, GCs inhibit the activity and differentiation of osteoblast-derived cells . Moreover, GCs induce the expression of receptor activator of nuclear factor-κB ligand (RANKL), which is essential for osteoclast development. On the contrary, GCs downregulate the decoy receptor of RANKL, osteoprotegerin (OPG) . The resulting imbalance between RANKL and OPG ultimately results in a state of increased bone resorption rate. In addition, GCs induce catabolism of the bone proteins, osteocalcin  and osteopontin , resulting in a decreased bone anabolic state. In addition, it was reported that GCs could prolong the mature osteoclasts life span .
Green tea is used for its antioxidant and anti-inflammatory beneficial effects. Green tea extract (GTE) is famous for its high contents of polyphenolic compounds especially catechins . Some literature studies based on animals demonstrated a promising role for GTE in the prevention of bone loss . However, the clear effects of green tea or its bioactive ingredients on bone cells and bone proteins, especially in conditions of GIO, are not clear.
Therefore, the present study was designed to investigate the potential benefits of the dietary antioxidants, GTE, in the prevention of bone impairment conditions, as in GIO, in addition to elucidate the possible effects of GTE supplementation on bone cells, mineralization, and bone markers.
This study was designed to evaluate the possible protective role of GTE supplementation on rats with corticosteroid-induced osteoporosis, as well as to evaluate the effects of GTE supplementation on bone proteins, bone mineralization, and bone markers.
| Materials and methods|| |
A total of 30 adult male Wistar rats, initially weighing 130–150 g, were used. Rats were purchased from the Research Institute of Ophthalmology, Giza, Egypt, and housed in animal cages (three rats/cage) with suitable ventilation, temperature of 22–25°C, 12-h light dark cycle, and free access to food and water ad libitum. The animals were allowed to climatize to the new environment for seven days before experimental procedures to decrease the possible discomfort of animals.
Animals were not exposed to unnecessary pain or stress, and animal manipulation was performed with maximal care and hygiene. At the end of the experiment, animals were killed by overdose of anesthesia. Disposal of animal remains was done by incineration.
All animal experiments were performed according to the Ethics Committee of Faculty of Medicine, Ain Shams University, and according to the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978) .
Animals were divided randomly into the following groups: control group (C, n=10): rats of this group received a regular diet with free access to water; GIO group (n=10): rats of this group received the synthetic GC decadron (dexamethasone sodium phosphate) 7 mg/kg body weight by intramuscular injection once per week for 4 weeks . GTE-supplemented osteoporotic group (GT-GIO, n=10): rats of this group received the synthetic GC decadron (dexamethasone sodium phosphate) 7 mg/kg body weight by intramuscular injection once per week for 4 weeks, followed by 300 mg/kg per day of GTE orally by gavage , for 4 weeks, with concomitant continuation of the weekly dexamethasone administration.
GTE was obtained as 300-mg tablets, in a box of 20 tablets, from Mepaco (Cairo, Egypt). Tablets were crushed, and the required dose weighed using a digital scale and dissolved in distilled water.
On the day of killing, overnight fasting rats were weighed and anesthetized with intraperitoneal injection of pentobarbital (40 mg/kg body weight). Then, abdominal midline incision performed, and blood samples from the abdominal aorta were collected. The blood was left to clot for ∼30 min and then centrifuged to obtain serum. Serum was kept at −20°C till analysis.
Serum calcium and phosphate levels were measured by colorimetric method, as described by Gindler and King  and El-Merzabani et al. , respectively, using kits supplied by Bio-diagnostics (Cairo, Egypt).
Serum alkaline phosphatase activity was measured by colorimetric method, according to the method described by Roy , using kits supplied by Sigma Co. (St Louis, Missouri, USA). Serum osteocalcin concentration was measured by an enzyme-linked immunosorbent assay, using Rat Mid, Osteocalcin ELISA kit (IDS Inc., Fountain Hills, Arizona, USA), according to the manufacturer’s instructions.
Serum deoxypyridinoline was determined to assess bone resorption activity, using ELISA kits of IDS Inc. according to the manufacturer’s instructions. Serum parathyroid hormone (PTH) was performed using an enzyme-linked immunosorbent assay (Rat ELISA kits IDS Inc.) according to the manufacturer’s instructions. Serum OPG level was determined by enzyme-linked immunosorbent assay technique (Rat Elisa kits, R&D Systems, Minnesota, USA), according to the method of O’Brien et al.  Serum RANKL level was determined by enzyme-linked immunosorbent assay technique using Elisa kits supplied by R&D Systems, according to the method of Teng et al. .
Histological examination of bone
The metaphysis of tibias was dissected out, fixed in 10% buffered formalin, and decalcified in EDTA solution for 2 weeks. When decalcified, the specimens were embedded in paraffin. Overall, 5-µm-thick sections were then deparaffinized and stained with hematoxylin and eosin , and by toluidine blue , for light microscopic examination.
Results of the present study are expressed as mean±SEM. Comparisons between means of different groups were made using one-way analysis of variance test of the Statistical Package for the Social Sciences (SPSS Inc., Chicago, Illinois, USA) program, version 20.0. Differences were considered significant when P value less than or equal to 0.05.
| Results|| |
[Table 1] showed a significant decrease in both of serum Ca++ and PO4 in the GIO group compared with the control group. On the contrary, a significant increase in serum bone-specific alkaline phosphatase (b-ALP) was detected in the GIO group as compared with the control group. GTE supplementation to the GIO (GT-GIO) group resulted in significant increase in serum Ca++ and PO4 compared with the GIO group. Moreover, GT-GIO group showed a significant decrease in serum b-ALP compared with the GIO group. Serum PTH showed nonsignificant differences among the studied groups.
|Table 1 Serum calcium (Ca++), serum phosphorus (PO4), bone-specific alkaline phosphatase, and serum parathyroid hormone in the control, glucocorticoid-induced osteoporosis, and green tea extract-supplemented glucocorticoid-induced osteoporosis groups|
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As shown in [Table 2], serum osteocalcin and OPG significantly decreased in the GIO group compared with the control group. However, all significantly increased in the GT-GIO group when compared with the GIO group. On the contrary, serum deoxypyridinoline and serum RANKL showed a significant increase in the GIO group compared with the controls, whereas it showed a significant decrease in the GT-GIO group compared with the GIO group.
|Table 2 Serum deoxypyridinoline, serum osteocalcin, serum osteoprotegerin, and serum receptor activator of nuclear factor-κB ligand in the control, glucocorticoid-induced osteoporosis, and green tea extract-supplemented glucocorticoid-induced osteoporosis groups|
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Histopathologic picture of normal bone of control group
Histopathological examination of sections of the proximal metaphysis of tibia in control rats revealed that the proximal metaphysis of tibia formed of an outer lamellar bone (cortical bone) and inner cancellous bone, showing a network of anastomosing bone trabeculae in-between the bone marrow. The cortical bone shows haversian systems, and covered by periosteum with endosteal lining. Osteocytes were detected in their lacunae in between the lamellae. The endosteal surface of trabeculae was lined by osteoblasts, as shown in [Figure 1].
|Figure 1 Proximal metaphysis of tibia of the control group using H&E (a, b, and c) and toluidine blue (d, e, f, and g). (a) A network of trabeculae of cancellous bone with bone marrow spaces (BM) are seen between the trabeculae (×100). (b) Osteoblasts (Ob) appear rimming the endosteal surface ‘black arrows’ (×400). (c) Osteocytes (OC) within their lacunae within bone trabeculae ‘black circles’ and osteoprogenitor (OP) cells lining endosteal surface ‘red arrows’. Toluidine blue-stained sections show (d) bone trabeculae with bone marrow spaces (BM) ‘black star’ in between (×200), (e and f) osteoprogenitor cells (OP) and osteoblasts (Ob) rimming bone trabeculae (black arrow) (×400), and (g) osteocytes (OC) in their lacunae within the bone trabeculae ‘black circles’ (×400).|
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Histopathological examination of the bone sections in the GIO rats revealed marked thinning of the cortical bone compared with the control group. There is loss of normal architecture of inner cancellous bone trabeculae, which appeared as thin discontinuous widely spaced bony trabeculae separated by bone marrow spaces. In the endosteal surface of some bone trabeculae, few erosion cavities were detected, together with proliferation of osteoblasts was also detected in some areas. There is increased number of osteoclasts and erosion cavities detected ([Figure 2]).
|Figure 2 Proximal metaphysic of tibia of the GIO rats using H&E (a, b, c, and d) and toluidine blue (e and f). (a) Marked thinning of cortical bone (×100), (b) loss of the normal architecture of trabeculae of cancellous bone (×200), (c) discontinuous thinned bony ossicles separated by widened bone marrow (BM) (×200), and (d) poorly calcified bone trabeculae. Toluidine blue-stained sections show (e) cancellous bone trabeculae appeared as discontinuous bony ossicles ‘black arrows’ with widened bone marrow (BM) spaces (×200) and (f) eroded surface of a trabecula with increased number of osteoclasts (OCL) and the erosion cavities detected ‘red arrows’ (×400). GIO, glucocorticoids-induced osteoporosis.|
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Histopathological examination of sections of the metaphysis of tibia in the GT-GIO rats revealed significant improvement compared with the GIO rats. The cortical bone thickness was slightly similar to the control group. The cancellous bone trabeculae partially recovered near the normal architecture and appeared more continuous with less widening of the bone marrow spaces. Insignificant osteoclast number in comparison with the GIO group was recorded ([Figure 3]).
|Figure 3 Proximal metaphysis of tibia of the green tea-GIO rats using H&E (a, b, and c) and toluidine blue (d, e, and f). (a) Showing slightly preserved architecture of trabeculae in comparison with the GIO rats. Trabeculae appear more continuous with bone marrow spaces (BM) (black star) in between (×100). (b) The bone trabeculae regaining its normal thickness with osteocytes (OC) in their lacunae within the bone trabeculae ‘black circles’ (×200). (c) Osteoprogenitor cells (OP) ‘black arrows’ and osteoblasts (Ob) ‘red arrows’ line the endosteal surface (×400). Toluidine blue-stained sections show (d) slightly preserved architecture of trabeculae in comparison with the GIO rats with marrow in between bone marrow (BM) (×200), (e) the bone trabeculae regaining its normal thickness with osteocytes (OC) in their lacunae within the bone trabeculae (×400), and (f) osteoblasts (Ob) appear rimming the endosteal surface ‘black arrows’ (×400). GIO, glucocorticoids-induced osteoporosis.|
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| Discussion|| |
This study was designed to evaluate the possible protective role of GTE supplementation on rats with corticosteroid-induced osteoporosis, as well as to evaluate the effects of GTE supplementation on bone proteins, mineralization, and markers.
The well-documented secondary osteoporosis in response to excess or prolonged GC s is multifactorial. Osteoporotic changes were evident in this study from the decreased serum levels of Ca++ and PO4 in the GIO group, demonstrating the ability of large doses of the corticosteroid dexamethasone to decrease significantly the serum mineral levels. GCs inhibit calcium resorption in the gastrointestinal tract and inhibit renal tubular calcium reabsorption with the development of hypocalcemia . In addition, GCs elevated the serum levels of b-ALP and deoxypyridinoline in the present study, reflecting the GCs-induced increase in bone resorption and turnover state .
The decreased serum osteocalcin in the GIO group in the present study reflects the inhibitory effects of GCs on osteoblast metabolism needed for bone formation. GCs exert major suppressive effects on osteoblasts. Excess GC induces osteoblast apoptosis and suppresses osteoblast differentiation and function . Osteocalcin is an osteoblastic marker of bone formation and of the metabolic activity of osteoblasts .
Moreover, in the present study, the decrease in serum OPG and the increase in serum RANKL in the GIO group demonstrates that imbalance in the OPG/RANKL ratio is present in the GIO group in favor of RANKL and increased osteoclastic bone resorptive capacity. GC s increase bone resorption by activation and prolongation of life span of osteoclasts. This occurs through upregulating RANKL and at the same time, by downregulation of the RANKL decoy receptor OPG .
Results of the present study did not detect significant changes in serum level of PTH in the different studied groups. These findings demonstrate that PTH is not involved in GIO changes, and the osteoporotic changes induced by GCs are the results of the direct effects of GCs on mineral balance and bone cells . These findings are consistent with similar previous findings that PTH is not significantly increased and is not primarily involved in the pathogenesis of GIO ,.
Adding to the results of serum markers that indicate osteoporosis in GIO rats, histopathological examination of tibia sections of GIO group of rats showed marked thinning of the cortical bone and loss of normal architecture of inner cancellous bone trabeculae that became thin, discontinuous and widely spaced, with the presence of few erosion cavities and increased number of osteoclasts. These histopathological findings add to the confirmation of osteoporotic bone dystrophic changes induced by GC.
GIO encountered in the present study is a result of imbalance between bone resorption and formation, in favor of increased resorption. This was confirmed by the increased serum markers of bone resorption RANKL, and by the deceased serum markers of bone formation OPG and osteocalcin. The histopathological findings of tibia of GIO group showed loss of normal architecture, bone cavities, erosions, and increased number of osteoclasts, which further support the bone metabolic imbalance induced by GC.
GTE supplementation in the present study resulted in normalization of serum minerals and lowering of serum deoxypyridinoline and serum b-ALP in the supplemented group, which received dexamethasone concomitantly, indicating the protective effects of GTE on the bone in rats exposed to GCs. Moreover, GTE restores the balance between OPG and RANKL in the supplemented group, by lowering the serum RANKL and increasing the serum OPG. This was associated with elevation of serum osteocalcin. In addition, there were marked structural improvements detected by histopathological examination of tibial sections of the GT-GIO group of rats. These results reflect the ability of GTE to enhance bone metabolic functions and to counteract the impaired osteoblastic function and survival in states of GIO. The decreased serum level of deoxypyridinoline and b-ALP in the GT-GIO group reflects the antiresorptive effect of GTE on GIO.
The anti-inflammatory and antioxidant effects of GTE, demonstrated in the decreased serum RANKL in the GT-GIO group of the current study, are of major importance in the counter-acting effect of GTE on GIO. RANKL is the primary regulator of osteoclast activation . Similar to the findings of a previous study by Suliburska et al. , GTE increased the serum Ca++ in the GTE-supplemented group. This finding reflects the ability of GTE to enhance intestinal Ca++ absorption. An effect opposing the effect of GCs that increase Ca++ urinary excretion is by decreasing its intestinal absorption.
Results of the present study regarding the effects of GTE supplementation are in accordance with many previous animal studies. Animal studies strongly suggest that GTE plays a role in bone preservation, increasing bone mass, increasing bone density, enhancing bone formation, and inhibiting bone resorption . Studies on different models of bone dystrophies, other than GIO, clearly demonstrated the value of GTE in mitigation of bone loss ,. In a lipopolysaccharide-induced model of chronic inflammation in female rats, GTE administration reduced oxidative stress-induced damage and inflammation and improved bone metabolic and mineral properties . In addition, in studies of osteopenic postmenopausal women, GTE supplementation resulted in a marked improvement in bone turnover and an increase in the serum biomarkers indicating bone formation . Moreover, in-vitro human primary osteoblasts incubation with GTE was found to improve cell viability and decrease oxidative and inflammatory events that induce bone-associated diseases. Interestingly, GTE also increased the expression of pro-osteogenic genes and expression of osteocalcin gene .
Although initially the biological effects of GTE were supposed to be owing to their antioxidant and anti-inflammatory activity, the results of the present study are useful in extending the concept on the mechanism of action of GTE. Since their discovery, GCs are known to act by regulation of gene expression mediated by their cytoplasmic receptors, a mechanism known as genomic mechanism .
From the results of the current study, we strongly suggest that GTE-mediated actions may be through affecting gene expression and signaling pathways involved in GC action, and so can attend and counteract the GIO bone changes. This can be clearly postulated from the lowering effect of GTE on RANKL, and by the augmenting effects on OPG and osteocalcin, resulting in restoration of bone metabolism in favor of preservation of bone and prevention of bone loss and dystrophies, as detected by the decrease in serum markers of bone resorption, and also by histopathological examination of bone of GT-GIO group.
In accordance, some previous studies pointed to the possibility that the contents of GTE may act through signaling pathways. Flavonoid biological effects in cell culture studies suggest the possibility of flavonoid interaction along the signaling pathways of cell growth and apoptosis, and this might explain their anticancerous, vascular, and cardioprotective activities . Moreover, epigallocatechin gallate content of GTE was reported to increase apoptosis  and inhibit the survival  and the differentiation of osteoclasts . Moreover, it decreases proinflammatory cytokines and nuclear factor-κB activation , and so, it is considered as an important anti-inflammatory therapeutic agent .
| Conclusion|| |
Collectively, previous studies in addition to the results of the present study could be of great value in broadening the concepts on the mechanism of action of GTE. Based on the results of serum markers and the histopathological examination, it could be strongly suggested that the biological activities of GTE are much more than the antioxidant and anti-inflammatory characteristics of GTE. GTE could play a major role in regulation of cell growth, apoptosis, gene expression, and signaling pathways. These observations could be of great value in considering the supplementation of GTE in pathological conditions of disturbed cell/organ function owing to defects in cell signaling pathways.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]