- Email: [email protected]
The protective effects of silibinin in the treatment of streptozotocin-induced diabetic osteoporosis in rats
Biomedicine & Pharmacotherapy 89 (2017) 681–688
Available online at
ScienceDirect www.sciencedirect.com
Original article
The protective effects of silibinin in the treatment of streptozotocin-induced diabetic osteoporosis in rats Te Wanga,1, Leyi Caia,1, Yangyang Wangb , Qingqing Wanga , Di Lua , Hua Chena , Xiaozhou Yinga,* a b
Department of Orthopaedic Surgery, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325000, China Department of Endocrinology, Wenzhou Hospital of Integrated Traditional Chinese and Western Medicine, Wenzhou 325000, China
A R T I C L E I N F O
Article history: Received 18 December 2016 Received in revised form 2 February 2017 Accepted 7 February 2017 Keywords: Silibinin Diabetic osteoporosis Microarchitecture Oxidative stress
A B S T R A C T
Diabetic osteoporosis (DO) is a complication of diabetes mellitus. Our previous study showed that silibinin can attenuate high glucose mediated human bone marrow stem cells dysfunction through antioxidant effect. However, no study has yet investigated the effect of silibinin in diabetic rats. Therefore, we assessed the effects of silibinin on bone characteristics in streptozotocin-induced diabetic rats. The aim of our study was to determine whether providing silibinin in the different supplementation could prevent bone loss in diabetic rats or not. Rats were randomly divided into four groups: (1) control group (CG) (n = 10); (2) diabetic group (DG) (n = 10); (3) diabetic group with 50 mg kg 1day 1 of silibinin orally (DG-50) (n = 10); and (4) diabetic group with 100 mg kg 1day 1 of silibinin orally (DG-100) (n = 10). 12 weeks after streptozotocin (STZ) injection, the femora from all rats were assessed and oxidative stress was evaluated. Bone mineral density was significantly decreased in diabetic rats; these effects were prevented by treatment with silibinin (100 mg kg 1 day 1 orally). Similarly, in the DG and DG-50 groups, changes in microarchitecture of femoral metaphysis assessed by microcomputed tomography demonstrated simultaneous existence of diabetic osteoporosis; these impairments were prevented by silibinin (100 mg kg 1 day 1 orally). In conclusion, silibinin supplementation may have potential use as a possible therapy for maintaining skeletal health and these results can enhance the understanding of diabetic osteoporosis induced by diabetes. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Diabetes mellitus is a devastating and life-altering disease, affecting over 20 million people in the USA [1]. Diabetes mellitus causes many complications such as nephropathy, neuropathy, and retinopathy. Diabetic osteoporosis (DO) is also a complication of diabetes mellitus [2–4], which is caused by reduced bone mineral content due to the abnormal levels of sugar, protein, fat, and microelements. Many human and experimental studies on the complications of diabetes mellitus have demonstrated extensive alterations in bone
* Corresponding author at: NO.109, XueYuan West Road, LuCheng District, Wenzhou, Zhejian Province, China. E-mail addresses: [email protected] (T. Wang), [email protected] (L. Cai), [email protected] (Y. Wang), [email protected] (Q. Wang), [email protected] (D. Lu), [email protected] (H. Chen), [email protected] (X. Ying). 1 These authors contributed equally to this work and should be considered cofirst authors. http://dx.doi.org/10.1016/j.biopha.2017.02.018 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
and mineral metabolism [5,6]. The mechanisms responsible for diabetic osteoporosis have been reported partly. Increasing evidence suggested that oxidation played a role in the pathogenesis of diabetic bone disease. Studies showed the increase in oxidative stress may partly contribute to the development of diabetic osteoporosis [7,8]. And in our previous study, we found that high glucose suppressed osteogenic differentiation of human bone marrow stem cells, manifested by an increase of oxidative damage markers [9]. The optimal therapies for DO include hormone therapy, bisphosphonates, calcium and vitamin D, while none of these therapies have been found to decrease the oxidation induced by high glucose. Given the prevalence of diabetic osteoporosis and the lack of effective therapies, there is a need to develop harmless and affordable alternative therapies for preventing osteoporosis. Natural products and dietary components have positive effects on bone remodeling, particularly by inhibiting bone resorption [10,11]. Silibinin, the active component of silymarin, has been shown to have a broad spectrum of pharmacological activities such as
682
T. Wang et al. / Biomedicine & Pharmacotherapy 89 (2017) 681–688
hepatoprotective activity [12], anti-cancer [13], antioxidant [12,14], neuroprotective [15] and cardioprotective effects [16]. Milic et al. have summarized the effects of silibinin and suggested that the milk thistle may be a possible agent in the prevention or treatment of cancer, atherosclerosis, hepatitis, and cirrhosis [17]. Further, It has been reported that silibinin had the potential to prevent bone loss in vitro by suppressing osteoclastogenesis and enhancing osteoblastogenesis in murine MC3T3-E1 pre-osteoblastic cells [18–20]. In addition, we found that silibinin could increase osteogenic effect by stimulating osteogenic biomarkers of alkaline phosphatase, type I collagen and osteocalcin expression in bone marrow stem cells (hBMSCs) [21]. Further we found that silibinin can attenuate high glucose-mediated hBMSCs dysfunction through antioxidant effect and modulation of PI3K/Akt pathway [9]. However, the action mechanisms of silibinin for promoting bone formation process in the state of high glucose in vivo still remain unclear. Hence, we investigate whether silibinin supplementation can be protective against bone loss induced by diabetes mellitus or not. To investigate the effects of silibinin in DO, we focused on the streptozotocin (STZ)-induced diabetic rat, which is one of the most common animal models of type 2 diabetes mellitus. In the current study, we attempted to determine whether silibinin could prevent diabetic osteoporosis, assessed the bone microarchitecture and evaluated oxidative stress in STZ-induced diabetic rats. 2. Materials and methods 2.1. Animal feeding study All animal procedures were approved by the Animal Care and Use Committee at Wenzhou Medical University and were conducted in accordance with the policies of the Ethics Committee for Animal Research. This study employed STZ injection technique for inducing diabetes mellitus and diabetic osteoporosis. Female Wistar rats, purchased from the Shanghai Animal Experimental Center (Shanghai, China), 180 g–190 g, 7–8 weeks of age, were used for the experiment. Upon arrival in the animal care facilities, rats were individually caged and kept in rooms maintained at 22 2 C with a 12 h light – 12 h dark cycle (lights on at 06:00, lights off at 18:00). The animals were allowed to acclimatize for a week before beginning experiments. Following acclimation, rats were randomly assigned as follows: (1) control group (CG) (n = 10); (2) diabetic group (DG) (n = 10); (3) diabetic group with 50 mg kg 1day 1 of silibinin (sigma) orally (DG-50) (n = 10); and (4) diabetic group with 100 mg kg 1day 1 of silibinin orally (DG-100) (n = 10). Rats in diabetic and silibinin-treated diabetic groups were fed with high fat diet (Diet #MD45%fat; Mediscience Ltd, China) for a period of 4 weeks to induce insulin resistance. After the 4 weeks of dietary manipulation, the animals were intraperitoneally injected with STZ (Sigma-Aldrich, St Louis, MO, USA) (35 mg/kg body weight in 100 mL of sterile citrate buffer, pH 4.5) on two consecutive days to induce the type II diabetes mellitus [22,23]. Rats in the CG group were fed with standard chow and water ad libitum and injected with citrate vehicle alone. After 72 h of STZ injection, animals with venous blood glucose levels of over 16.7 mmol/L were considered diabetic and selected for further studies. Venous blood glucose was measured every week to ensure the blood glucose level more than 16.7 mmol/L. All rats had free access to standard chow and tap water throughout the experiment. All rats were provided a vehicle control or silibinin for 12 weeks after STZ injection. Due to its low solubility in water, silibinin was suspended in 0.5% sodium carboxymethyl cellulose. Silibinin was administered at a daily dose of 50 mg kg 1day 1 in the DG-50 group and 100 mg kg 1day 1 in the DG-100 group by oral gavage while the controls were administered 1 mL of deionized distill water (ddH2O) in 0.5%
CMC-Na by oral gavage for 12weeks. The dose of silibinin was according to previous published study [24]. At 12 weeks after oral gavage administration of silibinin, rats were anesthetized by administration of 2% (w/v) pentobarbital sodium (35 mg/kg, Solarbio Science & Technology, Beijing, China) via intraperitoneal injection. The bones were collected from each animal and dissected with care being taken to protect the periosteum. Each bone was individually wrapped in ddH2Osoaked gauze and stored at 20 C until analyzed. 2.2. Bone morphometry and microarchitecture Bone morphometry measurements of length, width and height were determined using a vernier caliper. Bones were dried at 110 C for 48 h and then weighed. The femoral length was measured from the medial condyle to greater trochanter. The femoral width was measured from the lateral condyle to medial condyle and the femoral height was measured from the top to the bottom of the lateral condyle in this study. Morphometry measurements were averaged after no bilateral differences were determined using a paired t-test with significance level set at P < 0.05. Bone radiographs of excised femur were taken with Dualenergy X-ray absorptiometry (DXA) scans (Kubetic) on the right femora. For microarchitecture, trabecular bone architecture was determined on the left femora using microcomputed tomography (mCT) (MicroCTm100, SCANCO Medical, Switzerland). The trabecular bone within the distal femur metaphysis was scanned and 100 images (14.8 mm/slice or 1.48 mm) were analyzed with semiautomatically drawn contours beginning 50 slices (740 mm) away from the growth plate within the volume of interest (VOI). The femora were scanned at 70KV and 200 mA and 300 ms exposure time to obtain image resolution of 14.8 mm. Next, the reconstructed images were analyzed using the software (Ray V4.0-4, Switzerland). The VOI was assessed for structural parameters including trabecular bone volume fraction (BV/TV), trabecular number (TbN), trabecular thickness (TbTh), trabecular separation (TbSp) and structure model index. 2.3. Histological examination For Harris hematoxylin and Shandon Instant eosin (H&E) staining, the right femur were excised, fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.2) for 3 days at room temperature, decalcified with decalcifying solution (SigmaAldrich, St Louis, MO, USA) for 4 weeks. The tissue samples were then dehydrated in a graded series of ethanol solutions for 18 h, embedded in paraffin and cut into 5 mm sections in thickness. The specimens were subjected to histomorphometric analysis under a light microscope with a micrometer, using an image analyzer (Olympus, Japan). 2.4. Serum and urine biochemical analyses After 12 weeks of silibinin treatment, rats were anesthetized by administration of 2% (w/v) pentobarbital sodium (35 mg/kg) via intraperitoneal injection, blood was obtained using cardiac puncture, and serum samples obtained by centrifugation (3000 rpm, 10 min) were stored at 80 C until analysis. Plasma osteocalcin, a marker of bone formation, was measured by a commercially available rat-specific enzyme immunoassay (EIA) (Shanghai Haling biological technology, PR China). Serum concentrations of parathyroid hormone (PTH) were determined using a rat PTH ELISA kit (Immutopics Inc., San Clemente, CA, USA). Serum samples were analyzed for their calcium and phosphorus contents by the arsenazo-3 dye and ammonium molybdate colorimetric methods respectively. Plasma C-reactive protein
T. Wang et al. / Biomedicine & Pharmacotherapy 89 (2017) 681–688
Fig. 1. Body weight and serum glucose of rats in each group. CG control group, DG diabetic group, DG-50 diabetic group with 50 mg kg group with 100 mg kg 1day 1 of silibinin. Means standard deviations were indicated at each week and each group.
683
1
day
1
of silibinin, DG-100 diabetic
Shanghai, China). In this study, serum GOT and GPT levels were not influenced by the administration of silibinin in the DG-50 and DG-100 group, indicating that they did not induce liver toxicity.
(CRP) was determined using a commercially available rat CRP enzyme-linked immunosorbent assay kit purchased from the Shanghai Langka Company (Shanghai, China). All samples were performed in duplicate for the assays. To evaluate the extent of oxidative stress, urinary 8-OHdG, a sensitive indicator of oxidative DNA damage, was measured. After 12 weeks of silibinin treatment, a 24 h-urine collection from each rat using metabolic cages was conducted. The urine was kept at 20 C until the concentrations of 8-OHdG were measured. Urinary 8-OHdG was determined using a sandwich enzyme-linked immunosorbent assay kit (Uscnlife Life Sciences, Inc). Serum levels of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) were measured by using enzymatic kits (Shanghai Kehua Bio-engineering Co., Ltd.,
2.5. Statistical analysis Results are expressed as means standard error of the mean. The statistical significant of difference between groups was obtained by the student’s t-test using SPSS software (ver. 19.0; SPSS Inc., Chicago, IL). Differences were considered significant at P < 0.05. The relationship between plasma osteocalcin and urinary excretion of 8-OHdG was determined by linear regression of individual animals in the DG-50 and DG-100 group. All experiments were repeated at least three times.
Table 1 Femoral morphometry, femoral trabecular bone microstructure and biochemistry in diabetic rats administered silibinin or vehicle control. parameters
Control group (n = 10)
1
Diabetic group + 50 mg kg
0.61 0.08b 4.89 0.07b 2.93 0.05b 2.71 0.05b
0.63 0.06b 4.91 0.07b 2.95 0.07b 2.73 0.06b
0.72 0.05a 5.06 0.05a,b 3.08 0.05a,b 2.81 0.05a,b
Femoral trabecular bone microstructure BV/TV (%) 0.41 0.02 0.30 0.01b TbN (mm) 3.53 0.11 3.07 0.13b TbTH (mm) 0.098 0.005 0.096 0.004 TbSp (mm) 0.25 0.02 0.32 0.02b SMI 1.39 0.05 1.85 0.09b
0.29 0.01b 3.03 0.17b 0.097 0.002 0.32 0.01b 1.78 0.05b
0.36 0.02a,b 3.46 0.07a 0.105 0.007 0.28 0.01a 1.46 0.07a
biochemistry Osteocalcin (nmol/L) ALP (IU/L) Ca (mmol/L) P (mmol/L) PTH(pg/ml) CRP (mg/mL) 8-OHdG (ng/day) GOT (IU/L) GPT (IU/L)
21.4 4.7b 153 15b 8.8 1.1 5.8 0.9 43.8 6.8 44.8 4.8b 41.60 18.65b 45.44 5.56 25.00 4.53
27.3 4.5a 231 14a,b 8.7 0.85 6.1 1.1 40.4 5.3 36.4 4.6a,b 20.10 7.88a,b 46.36 5.25 24.42 4.36
Femoral morphometry Dry mass (g) 0.79 0.07 Width (mm) 5.24 0.06 Depth (mm) 3.21 0.05 Length (cm) 2.95 0.04
31.3 4.7 182 14 8.7 0.5 5.7 0.6 40.1 4.8 10.8 2.0 2.65 0.33 49.36 5.40 25.82 4.98
18.3 2.5b 135 13b 9.1 0.5 6.0 0.8 42.5 8.5 47.2 3.9b 72.98 23.10b 48.34 5.75 25.10 4.59
day
1
Diabetic group (n = 10)
of silibinin (n = 10)
Diabetic group + 100 mg kg
1
day
1
of silibinin (n = 10)
Values are expressed as the mean SEM of n = 10 groups. The statistical significant of difference between groups was obtained by the student’s t-test comparison using SPSS software. BV/TV trabecular bone volume per unit of total volume, TbN trabecular number, TbTh trabecular thickness, TbSp trabecular separation, SMI structure model index. ALP alkaline phosphatase, Ca calcium, P phosphorus, PTH parathyroid, CRP C-reactive protein, 8-OHdG 8-hydroxydeoxyguanosine, GOT glutamic oxaloacetic transaminase, GPT glutamic pyruvic transaminase. a P < 0.05 vs. the DG group. b P < 0.05 vs. the CG group.
684
T. Wang et al. / Biomedicine & Pharmacotherapy 89 (2017) 681–688
Fig. 2. Femoral X-ray and bone mineral density (BMD). CG control group, DG diabetic group, DG-50 diabetic group with 50 mg kg with 100 mg kg 1day 1 of silibinin. a P < 0.05 vs. the DG group. b P < 0.05 vs. the CG group.
3. Results 3.1. General features Body weights and serum glucose of experimental rats were showed in Fig. 1. The data of body weight and serum glucose from week 1 to week 16 were present as mean SEM. Body weights in all groups increased until administration of STZ. After injection of STZ, body weights of diabetic and silibinin-treated diabetic groups decreased, while body weights in the CG group kept on growing. There were no significant differences in body weights among the three diabetic groups after STZ injection (P > 0.05). Serum glucose suddenly exceeded the normal level in diabetic and silibinin-treated diabetic groups after STZ injection. Comparing with the DG and DG-50 groups, the serum glucose decreased 5 weeks later after the administration of silibinin in the DG-100 group (Fig. 1(B)), which meant hyperglycemia in diabetic rats could be partly reversed by silibinin. 3.2. Bone morphometry and bone mineral density The dry mass, length, width and height of femur were showed in Table 1. The length, width and height of femur in the DG group and DG-50 group decreased significantly compared with that in the CG group. Comparing with the DG and DG-50 groups, the length, width and height of femur increased significantly in the DG-100 group (P < 0.05). 12 weeks after silibinin administration, rats showed severe osteopenia in the femur by X-ray (Fig. 2(A)) and femoral BMD in the DG and DG-50 group was 35% and 32% lower respectively than that in the CG group (P < 0.05). In the DG-100 group, the femoral BMD was 25% lower compared with the CG group (P < 0.05) and was 19% higher compared with the DG group (P < 0.05) (Fig. 2(B)). 3.3. Bone microarchitecture The effects of diabetes mellitus with or without silibinin were evaluated using microarchitectural parameters in the distal femoral metaphysic (Table 1). The reconstructed images of the femur from rats in the four groups were constructed using micro-CT (Fig. 3(A), (B)). There were no significant effects between the DG and DG-50 rats
1
day
1
of silibinin, DG-100 diabetic group
on femoral trabecular bone microarchitectures. Similarly, there were no significant effects between the CG and DG-100 rats. The femoral trabecular bone microarchitectures in the DG and DG-50 rats had significant differences compared with that in the CG and DG-100 group. BV/TV and TbN, which decreased with diabetes mellitus, was significantly increased in the DG-100 group (P < 0.05). On the other hand, increased TbSp and SMI in femur were significantly reduced by administration of sibilinin at 100 mg kg 1day 1 (P < 0.05 or less). TbTh of the CG group was slightly greater than that of the DG and DG50 group and was slightly smaller than that of the DG-100 group; however, there was no significant difference in TbTh between the four groups (P > 0.05). 3.4. Histological examination Morphologic changes of the femoral metaphysis stained with the hematoxylin and eosin’s method were observed under light microscopy. In the twelve weeks after administration of silibinin, the histological cross sections of the femur showed that the bony meshwork appeared in the metaphysis of each group (Fig. 4). There was an apparent reduction in trabecular (cancellous) bones in the DG and DG-50 group. In contrast, the cancellous bones appeared to be added in the metaphyseal area of diabetic group treated with silibinin. Oral administration of 100 mg kg 1day 1 silibinin might protect from bone loss led by diabetes mellitus. 3.5. Serum biochemistry The effects of diabetes mellitus with or without silibinin were evaluated using biochemical parameters in serum (Table 1). The concentrations of serum osteocalcin, markers of bone formation, was significantly increased in diabetes mellitus (p < 0.01), which were suppressed by the administration of silibinin (P < 0.05). Since ALP is a biomarker for the matrix maturation of osteoblasts, this study investigated the osteoblastic activity of silibinin. The ALP concentration was significantly decreased in the DG and DG-50 group (p < 0.01), which was positively influenced by the administration with 100 mg kg 1day 1 silibinin (p < 0.01). No significant differences in serum Ca, P and PTH were found among the groups. Moreover, there was no significant difference in serum GOT and GPT indicating that silibinin did not induce liver toxicity.
T. Wang et al. / Biomedicine & Pharmacotherapy 89 (2017) 681–688
685
Fig. 3. The longitudinal Micro-CT images (a) and three-dimensional Micro-CT images (b) of the distal femur in each treatment group. Compared with the CG group, changes of trabecular bones microarchitecture diabetic group were confirmed. In contrast, the changes in diabetic condition were partly restored in the DG-100 group.
3.6. Assessment of oxidative and inflammatory stress
3.7. Association between oxidative stress and bone formation
To evaluate the extent of oxidative stress in the animals, urinary 8- OHdG was determined. Urinary 8-OHdG was markedly (p < 0.01) higher in the DG and DG-50 group than in the CG group. However, silibinin-treated diabetic rats in the DG-100 group showed suppressed excretion of the marker, resulting in an absence of any significant difference with the control rats. Serum CRP, a sensitive marker of systemic inflammation, was significantly higher in the DG and DG-50 group than in the CG group (p < 0.01). Whereas it was positively influenced by the treatment with 100 mg kg 1day 1 silibinin (p < 0.01). Taken together, these results demonstrate that oxidative stress and inflammatory stress both existed in diabetes mellitus. Moreover, the results demonstrate that silibinin treatment rescues both increased oxidative stress and increased inflammatory stress.
Linear regression was performed to assess the association between the parameters of bone formation (osteocalcin) and the markers of oxidative stress (urinary excretion of 8-OHdG) in silibinin-treated group (Fig. 5). There were a negative relationship (R2 = 0.827, P < 0.01), which means that 8-OHdG were inversely associated with plasma osteocalcin. 4. Discussion Diabetes is the theme of 2016 World Health Day and WHO has published the Global Report on Diabetes to raise awareness and spark momentum for action at the necessary scale. People with all types of diabetes are at risk of developing a range of complications and the high costs of care increase the risk of catastrophic medical
686
T. Wang et al. / Biomedicine & Pharmacotherapy 89 (2017) 681–688
Fig. 4. Hematoxylin and eosin stain. Femurs were removed after 12 weeks of silibinin or vehicle treatment. Compared with control rats and 100 mg kg 1day 1 silibinintreated diabetic rats, decreases of trabecular bones in diabetic rats and 50 mg kg 1day 1 silibinin-treated diabetic rats were confirmed. Magnification is 200 and the scale bar is 200 mm.
expenditure [25]. Sustained hyperglycemia and long-term metabolic disorders can lead to the damage, dysfunction and failure in body tissues and organs. DO, characterized by low bone mass, is a complication of diabetes mellitus [2–4] and frequent in diabetic patients but is asymptomatic until the fracture. Oei et al., found that fracture risk in type II diabetes patients with poor glycemic control increased by 47%–62% compared with non-diabetic patients and those with good glycemic control [26]. Bone width increases by tissue mineralization of the periosteal bone surface by osteoblasts. Increased bone formation by
Fig. 5. Relationship between serum osteocalcin and urinary 8-OHdG. There were 20 individual rats in the DG-50 and DG-100 group. A negative relationship could be found between urinary excretion of 8-OHdG and plasma osteocalcin.
osteoblasts was indicated by higher plasma osteocalcin concentration. In our study, the width of femur was greater in the DG-100 group than in the DG and DG-50 groups and silibinin supplementation in dose of 100 mg kg 1day 1 could significantly increase plasma osteocalcin (P < 0.05), a marker of bone formation. These results suggest that silibinin have the capability to promote bone formation resulting in prevention of bone loss following diabetic osteoporosis. Currently, mCT is considered the gold standard for assessing BMD and trabecular bone microarchitecture. Bouxsein et al. asserted that at least four parameters (i.e., BV/TV, TbTh, TbSp, and TbN) must be used to describe trabecular bone microarchitecture [27]. Therefore, these indicators were used as the assessment indicators in our study. In previous studies of osteoporosis, trabecular bone microarchitecture at the distal femoral metaphysic was the VOI [28]. Therefore, this anatomical region was selected as the VOI in the study. Bone volume (BV) was calculated using tetrahedrons corresponding to the enclosed volume of the triangulated surface [29]. Total tissue volume (TV) was the volume of entire scanned sample. BV/TV was then calculated from these values. TbTh, TbN and TbSp were estimated [30,31]. SMI is an index for trabecular structure and expresses the ratio of plate-like to rod-like structures. An SMI of 0 represents a perfect plate-like structure, and an SMI of 3 represents a perfect rod-like structure. Except for TbTh, diabetes accelerated loss of bone microarchitecture indicated by significantly decreased BV/TV and TbN, and increased TbSp and SMI in the DG group. These results indicated that bone formation was suppressed in STZ-induced diabetic rats, which simulated diabetic osteoporosis. To exclude potential effects of silibinin treatment, we also examined two silibinin-treated diabetic groups. Compared with diabetic rats in the DG group, the changes in BV/TV, TbN, TbSp, and SMI of rats in the DG-100 group were 20.0%, 12.7%, 12.5%, and 21.1%, respectively.
T. Wang et al. / Biomedicine & Pharmacotherapy 89 (2017) 681–688
However, the changes in BV/TV, TbN, TbSp, and SMI of rats in the DG-50 group were 0.03%, 0.01%, 0%, and 0.04%, respectively. This indicated that silibinin supplementation of diabetic rats had significant effects on bone microarchitecture and the significant effects may rely on the dose of silibinin. Unfortunately, this study observed no significant difference in TbTh between the DG and DG100 groups (p = 0.053). The main reason may be that the number of the rats used in this study was small. Few studies related to diabetic osteoporosis have been conducted; thus, no comparison was made between previous studies and this study. As to the mechanisms of diabetic osteoporosis, in vitro studies have reported that high glucose influences osteoblast differentiation [32], impairs bone formation [33], and inhibits bone mineralization [34]. In addition, experimental and clinical studies have demonstrated that osteoblast function is impaired under diabetic conditions [35,36]. Our previous studies have reported that high glucose suppressed osteogenic differentiation of human bone marrow stem cells, manifested by an increase of oxidative damage markers including reactive oxygen species and all of the observed oxidative damage can be inhibited by silibinin [9]. Concerning the pathogenesis of diabetic osteoporosis, oxidative stress may be an important factor. In the present study, urinary excretion of 8-OHdG was elevated in STZ-induced diabetic rats, while the marker was significantly suppressed to control levels in silibinin-treated diabetic rats. Our animal study demonstrated a negative relationship (R2 = 0.827, P < 0.01) between urinary 8OHdG and bone formation (plasma osteocalcin), which means the deleterious effects of skeletal unloading on bone may be suggested to be due, in part, to increased oxidation. These findings indicate that oxidative stress in diabetic conditions was suppressed in silibinin-treated diabetic rat. In vitro studies have shown that oxidative stress inhibits osteoblastic differentiation [37,38] and induces osteoblast apoptosis [39,40], which is consistent with our previous study [9]. In vivo study has shown that the increase in oxidative stress may at least partly contribute to the development of diabetic osteoporosis [7]. It is in accordance with our study that the deleterious effects of diabetes on bone may be suggested to increased oxidation and silibinin supplementation to female rats had in part bone protective effects. This study had several limitations. Specifically, we measured only the trabecular bone microarchitecture in the femur but did not explore osteoporosis of the spine or wrist. Another limitation of our study is the relatively small number of rats. This limits the power of the study, particularly due to the fact that the group is divided into four groups for analyses. Finally, we mainly used micro-CT to analyze bone mass but did not perform a biomechanical test to measure bone strength. In the future, we will carry out more research to overcome these limitations and understand the mechanism of diabetic osteoporosis. Based on our study findings, diabetes can cause osteoporosis in diabetic rats. Large-dose silibinin supplementation can attenuate demineralization and microstructure deterioration. The bone protective effects of silibinin appeared to be mediated by decreased oxidation and increased bone formation. Therefore, silibinin supplementation may have potential use as a possible therapy for maintaining skeletal health and the study may enhance the understanding of pathophysiology for diabetic osteoporosis. Conflict of interest The authors declare no competing financial interest. Acknowledgments The authors thank all the staff in the Laboratory of Orthopedic Research Institute and Scientific Research Center of Second
687
Affiliated Hospital of Wenzhou Medical University. This work was supported by grants from the National Natural Science Foundation of China (81402980) and the Wenzhou Technology Project (Y20140154). References [1] L.R. McCabe, Understanding the pathology and mechanisms of type I diabetic bone loss, J. Cell. Biochem. 102 (6) (2007) 1343–1357. [2] M.J. Kayath, S.A. Dib, J.G. Vieira, Prevalence and magnitude of osteopenia associated with insulin-dependent diabetes mellitus, J. Diabetes Complications 8 (2) (1994) 97–104. [3] S.A. Kemink, A.R. Hermus, L.M. Swinkels, J.A. Lutterman, A.G. Smals, Osteopenia in insulin-dependent diabetes mellitus; prevalence and aspects of pathophysiology, J. Endocrinol. Invest. 23 (5) (2000) 295–303. [4] P.J. Lopez-Ibarra, M.M. Pastor, F. Escobar-Jimenez, M.D. Pardo, A.G. Gonzalez, J. D. Luna, M.E. Requena, M.A. Diosdado, Bone mineral density at time of clinical diagnosis of adult-onset type 1 diabetes mellitus, Endocr. Pract. 7 (5) (2001) 346–351. [5] M.E. Levin, V.C. Boisseau, L.V. Avioli, Effects of diabetes mellitus on bone mass in juvenile and adult-onset diabetes, N. Engl. J. Med. 294 (5) (1976) 241–245. [6] Y. Seino, H. Ishida, Diabetic osteopenia: pathophysiology and clinical aspects, Diabetes Metab. Rev. 11 (1) (1995) 21–35. [7] Y. Hamada, S. Kitazawa, R. Kitazawa, H. Fujii, M. Kasuga, M. Fukagawa, Histomorphometric analysis of diabetic osteopenia in streptozotocin-induced diabetic mice: a possible role of oxidative stress, Bone 40 (5) (2007) 1408– 1414. [8] J. Liu, J. Yang, Uncarboxylated osteocalcin inhibits high glucose-induced ROS production and stimulates osteoblastic differentiation by preventing the activation of PI3K/Akt in MC3T3-E1 cells, Int. J. Mol. Med. 37 (1) (2016) 173– 181. [9] X. Ying, X. Chen, H. Liu, P. Nie, X. Shui, Y. Shen, K. Yu, S. Cheng, Silibinin alleviates high glucose-suppressed osteogenic differentiation of human bone marrow stromal cells via antioxidant effect and PI3K/Akt signaling, Eur. J. Pharmacol. 765 (2015) 394–401. [10] A. Shirwaikar, S. Khan, S. Malini, Antiosteoporotic effect of ethanol extract of Cissus quadrangularis Linn. on ovariectomized rat, J. Ethnopharmacol. 89 (2– 3) (2003) 245–250. [11] C.L. Shen, J.K. Yeh, J.J. Cao, O.L. Tatum, R.Y. Dagda, J.S. Wang, Green tea polyphenols mitigate bone loss of female rats in a chronic inflammationinduced bone loss model, J. Nutr. Biochem. 21 (10) (2010) 968–974. [12] L. Al-Anati, E. Essid, R. Reinehr, E. Petzinger, Silibinin protects OTA-mediated TNF-alpha release from perfused rat livers and isolated rat Kupffer cells, Mol. Nutr. Food Res. 53 (4) (2009) 460–466. [13] R.P. Singh, R. Agarwal, Mechanisms and preclinical efficacy of silibinin in preventing skin cancer, Eur. J. Cancer 41 (13) (2005) 1969–1979. [14] H.L. Ha, H.J. Shin, M.A. Feitelson, D.Y. Yu, Oxidative stress and antioxidants in hepatic pathogenesis, World J. Gastroenterol. 16 (48) (2010) 6035–6043. [15] S. Kittur, S. Wilasrusmee, W.A. Pedersen, M.P. Mattson, K. Straube-West, C. Wilasrusmee, B. Lubelt, D.S. Kittur, Neurotrophic and neuroprotective effects of milk thistle (Silybum marianum) on neurons in culture, J. Mol. Neurosci. 18 (3) (2002) 265–269. [16] M. Agoston, R.G. Cabello, A. Blazovics, J. Feher, A. Vereckei, The effect of amiodarone and/or antioxidant treatment on splenocyte blast transformation, Clin. Chim. Acta 303 (1–2) (2001) 87–94. [17] N. Milic, N. Milosevic, L. Suvajdzic, M. Zarkov, L. Abenavoli, New therapeutic potentials of milk thistle (Silybum marianum), Nat. Prod. Commun. 8 (12) (2013) 1801–1810. [18] C.V. Kavitha, G. Deep, S.C. Gangar, A.K. Jain, C. Agarwal, R. Agarwal, Silibinin inhibits prostate cancer cells- and RANKL-induced osteoclastogenesis by targeting NFATc1, NF-kappaB, and AP-1 activation in RAW264.7 cells, Mol. Carcinog. 53 (3) (2014) 169–180. [19] J.H. Kim, K. Kim, H.M. Jin, I. Song, B.U. Youn, J. Lee, N. Kim, Silibinin inhibits osteoclast differentiation mediated by TNF family members, Mol. Cells 28 (3) (2009) 201–207. [20] J.L. Kim, S.H. Park, D. Jeong, J.S. Nam, Y.H. Kang, Osteogenic activity of silymarin through enhancement of alkaline phosphatase and osteocalcin in osteoblasts and tibia-fractured mice, Exp. Biol. Med. (Maywood) 237 (4) (2012) 417–428. [21] X. Ying, L. Sun, X. Chen, H. Xu, X. Guo, H. Chen, J. Hong, S. Cheng, L. Peng, Silibinin promotes osteoblast differentiation of human bone marrow stromal cells via bone morphogenetic protein signaling, Eur. J. Pharmacol. 721 (1–3) (2013) 225–230. [22] M.S. Islam, T. Loots du, Experimental rodent models of type 2 diabetes: a review, Methods Find Exp. Clin. Pharmacol. 31 (4) (2009) 249–261. [23] E.J. Henriksen, Invited review: effects of acute exercise and exercise training on insulin resistance, J. Appl. Physiol. (1985) 93 (2) (2002) 788–796. [24] J.L. Kim, Y.H. Kim, M.K. Kang, J.H. Gong, S.J. Han, Y.H. Kang, Antiosteoclastic activity of milk thistle extract after ovariectomy to suppress estrogen deficiency-induced osteoporosis, Biomed. Res. Int. 2013 (2013) 919374. [25] C.M. Smith-Spangler, J. Bhattacharya, J.D. Goldhaber-Fiebert, Diabetes, its treatment, and catastrophic medical spending in 35 developing countries, Diabetes Care 35 (2) (2012) 319–326. [26] L. Oei, M.C. Zillikens, A. Dehghan, G.H. Buitendijk, M.C. Castano-Betancourt, K. Estrada, L. Stolk, E.H. Oei, J.B. van Meurs, J.A. Janssen, A. Hofman, J.P. van
688
[27]
[28]
[29] [30]
[31]
[32]
[33]
T. Wang et al. / Biomedicine & Pharmacotherapy 89 (2017) 681–688 Leeuwen, J.C. Witteman, H.A. Pols, A.G. Uitterlinden, C.C. Klaver, O.H. Franco, F. Rivadeneira, High bone mineral density and fracture risk in type 2 diabetes as skeletal complications of inadequate glucose control: the Rotterdam Study, Diabetes Care 36 (6) (2013) 1619–1628. M.L. Bouxsein, S.K. Boyd, B.A. Christiansen, R.E. Guldberg, K.J. Jepsen, R. Muller, Guidelines for assessment of bone microstructure in rodents using microcomputed tomography, J. Bone. Miner. Res. 25 (7) (2010) 1468–1486. A. Xiang, M. Kanematsu, S. Kumar, D. Yamashita, T. Kaise, H. Kikkawa, S. Asano, M. Kinoshita, Changes in micro-CT 3D bone parameters reflect effects of a potent cathepsin K inhibitor (SB-553484) on bone resorption and cortical bone formation in ovariectomized mice, Bone 40 (5) (2007) 1231–1237. W.E. Lorensen, H.E. Cline, Marching cubes a high resolution 3D surface construction algorithm, Comput. Graph. 21 (1987) 163–169. T. Hildebrand, P. Rüegsegger, A new method for the model-independent assessment of thickness in three-dimensional images, J. Microsc. 185 (1) (1997) 67–75. A.M. Parfitt, C.H.E. Mathews, A.R. Villanueva, Relationships between surface, volume, and thickness of iliac trabecular bone in aging and in osteoporosis. Implications for the microanatomic and cellular mechanisms of bone loss, J. Clin. Invest. 72 (4) (1983) 1396–1409. M. Zayzafoon, C. Stell, R. Irwin, L.R. McCabe, Extracellular glucose influences osteoblast differentiation and c-Jun expression, J. Cell. Biochem. 79 (2) (2000) 301–310. M. Terada, M. Inaba, Y. Yano, T. Hasuma, Y. Nishizawa, H. Morii, S. Otani, Growth-inhibitory effect of a high glucose concentration on osteoblast-like cells, Bone 22 (1) (1998) 17–23.
[34] E. Balint, P. Szabo, C.F. Marshall, S.M. Sprague, Glucose-induced inhibition of in vitro bone mineralization, Bone 28 (1) (2001) 21–28. [35] K.M. Thrailkill, L. Liu, E.C. Wahl, R.C. Bunn, D.S. Perrien, G.E. Cockrell, R.A. Skinner, W.R. Hogue, A.A. Carver, J.L. Fowlkes, J. Aronson, C.K. Lumpkin Jr., Bone formation is impaired in a model of type 1 diabetes, Diabetes 54 (10) (2005) 2875–2881. [36] R. Bouillon, M. Bex, E. Van Herck, J. Laureys, L. Dooms, E. Lesaffre, E. Ravussin, Influence of age, sex, and insulin on osteoblast function: osteoblast dysfunction in diabetes mellitus, J. Clin. Endocrinol. Metab. 80 (4) (1995) 1194– 1202. [37] N. Mody, F. Parhami, T.A. Sarafian, L.L. Demer, Oxidative stress modulates osteoblastic differentiation of vascular and bone cells, Free Radic. Biol. Med. 31 (4) (2001) 509–519. [38] X.C. Bai, D. Lu, J. Bai, H. Zheng, Z.Y. Ke, X.M. Li, S.Q. Luo, Oxidative stress inhibits osteoblastic differentiation of bone cells by ERK and NF-kappaB, Biochem. Biophys. Res. Commun. 314 (1) (2004) 197–207. [39] R.M. Chen, G.J. Wu, H.C. Chang, J.T. Chen, T.F. Chen, Y.L. Lin, T.L. Chen, 2,6Diisopropylphenol protects osteoblasts from oxidative stress-induced apoptosis through suppression of caspase-3 activation, Ann. N. Y. Acad. Sci. 1042 (2005) 448–459. [40] A.A. Fatokun, T.W. Stone, R.A. Smith, Hydrogen peroxide-induced oxidative stress in MC3T3-E1 cells: the effects of glutamate and protection by purines, Bone 39 (3) (2006) 542–551.
Available online at
ScienceDirect www.sciencedirect.com
Original article
The protective effects of silibinin in the treatment of streptozotocin-induced diabetic osteoporosis in rats Te Wanga,1, Leyi Caia,1, Yangyang Wangb , Qingqing Wanga , Di Lua , Hua Chena , Xiaozhou Yinga,* a b
Department of Orthopaedic Surgery, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325000, China Department of Endocrinology, Wenzhou Hospital of Integrated Traditional Chinese and Western Medicine, Wenzhou 325000, China
A R T I C L E I N F O
Article history: Received 18 December 2016 Received in revised form 2 February 2017 Accepted 7 February 2017 Keywords: Silibinin Diabetic osteoporosis Microarchitecture Oxidative stress
A B S T R A C T
Diabetic osteoporosis (DO) is a complication of diabetes mellitus. Our previous study showed that silibinin can attenuate high glucose mediated human bone marrow stem cells dysfunction through antioxidant effect. However, no study has yet investigated the effect of silibinin in diabetic rats. Therefore, we assessed the effects of silibinin on bone characteristics in streptozotocin-induced diabetic rats. The aim of our study was to determine whether providing silibinin in the different supplementation could prevent bone loss in diabetic rats or not. Rats were randomly divided into four groups: (1) control group (CG) (n = 10); (2) diabetic group (DG) (n = 10); (3) diabetic group with 50 mg kg 1day 1 of silibinin orally (DG-50) (n = 10); and (4) diabetic group with 100 mg kg 1day 1 of silibinin orally (DG-100) (n = 10). 12 weeks after streptozotocin (STZ) injection, the femora from all rats were assessed and oxidative stress was evaluated. Bone mineral density was significantly decreased in diabetic rats; these effects were prevented by treatment with silibinin (100 mg kg 1 day 1 orally). Similarly, in the DG and DG-50 groups, changes in microarchitecture of femoral metaphysis assessed by microcomputed tomography demonstrated simultaneous existence of diabetic osteoporosis; these impairments were prevented by silibinin (100 mg kg 1 day 1 orally). In conclusion, silibinin supplementation may have potential use as a possible therapy for maintaining skeletal health and these results can enhance the understanding of diabetic osteoporosis induced by diabetes. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Diabetes mellitus is a devastating and life-altering disease, affecting over 20 million people in the USA [1]. Diabetes mellitus causes many complications such as nephropathy, neuropathy, and retinopathy. Diabetic osteoporosis (DO) is also a complication of diabetes mellitus [2–4], which is caused by reduced bone mineral content due to the abnormal levels of sugar, protein, fat, and microelements. Many human and experimental studies on the complications of diabetes mellitus have demonstrated extensive alterations in bone
* Corresponding author at: NO.109, XueYuan West Road, LuCheng District, Wenzhou, Zhejian Province, China. E-mail addresses: [email protected] (T. Wang), [email protected] (L. Cai), [email protected] (Y. Wang), [email protected] (Q. Wang), [email protected] (D. Lu), [email protected] (H. Chen), [email protected] (X. Ying). 1 These authors contributed equally to this work and should be considered cofirst authors. http://dx.doi.org/10.1016/j.biopha.2017.02.018 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
and mineral metabolism [5,6]. The mechanisms responsible for diabetic osteoporosis have been reported partly. Increasing evidence suggested that oxidation played a role in the pathogenesis of diabetic bone disease. Studies showed the increase in oxidative stress may partly contribute to the development of diabetic osteoporosis [7,8]. And in our previous study, we found that high glucose suppressed osteogenic differentiation of human bone marrow stem cells, manifested by an increase of oxidative damage markers [9]. The optimal therapies for DO include hormone therapy, bisphosphonates, calcium and vitamin D, while none of these therapies have been found to decrease the oxidation induced by high glucose. Given the prevalence of diabetic osteoporosis and the lack of effective therapies, there is a need to develop harmless and affordable alternative therapies for preventing osteoporosis. Natural products and dietary components have positive effects on bone remodeling, particularly by inhibiting bone resorption [10,11]. Silibinin, the active component of silymarin, has been shown to have a broad spectrum of pharmacological activities such as
682
T. Wang et al. / Biomedicine & Pharmacotherapy 89 (2017) 681–688
hepatoprotective activity [12], anti-cancer [13], antioxidant [12,14], neuroprotective [15] and cardioprotective effects [16]. Milic et al. have summarized the effects of silibinin and suggested that the milk thistle may be a possible agent in the prevention or treatment of cancer, atherosclerosis, hepatitis, and cirrhosis [17]. Further, It has been reported that silibinin had the potential to prevent bone loss in vitro by suppressing osteoclastogenesis and enhancing osteoblastogenesis in murine MC3T3-E1 pre-osteoblastic cells [18–20]. In addition, we found that silibinin could increase osteogenic effect by stimulating osteogenic biomarkers of alkaline phosphatase, type I collagen and osteocalcin expression in bone marrow stem cells (hBMSCs) [21]. Further we found that silibinin can attenuate high glucose-mediated hBMSCs dysfunction through antioxidant effect and modulation of PI3K/Akt pathway [9]. However, the action mechanisms of silibinin for promoting bone formation process in the state of high glucose in vivo still remain unclear. Hence, we investigate whether silibinin supplementation can be protective against bone loss induced by diabetes mellitus or not. To investigate the effects of silibinin in DO, we focused on the streptozotocin (STZ)-induced diabetic rat, which is one of the most common animal models of type 2 diabetes mellitus. In the current study, we attempted to determine whether silibinin could prevent diabetic osteoporosis, assessed the bone microarchitecture and evaluated oxidative stress in STZ-induced diabetic rats. 2. Materials and methods 2.1. Animal feeding study All animal procedures were approved by the Animal Care and Use Committee at Wenzhou Medical University and were conducted in accordance with the policies of the Ethics Committee for Animal Research. This study employed STZ injection technique for inducing diabetes mellitus and diabetic osteoporosis. Female Wistar rats, purchased from the Shanghai Animal Experimental Center (Shanghai, China), 180 g–190 g, 7–8 weeks of age, were used for the experiment. Upon arrival in the animal care facilities, rats were individually caged and kept in rooms maintained at 22 2 C with a 12 h light – 12 h dark cycle (lights on at 06:00, lights off at 18:00). The animals were allowed to acclimatize for a week before beginning experiments. Following acclimation, rats were randomly assigned as follows: (1) control group (CG) (n = 10); (2) diabetic group (DG) (n = 10); (3) diabetic group with 50 mg kg 1day 1 of silibinin (sigma) orally (DG-50) (n = 10); and (4) diabetic group with 100 mg kg 1day 1 of silibinin orally (DG-100) (n = 10). Rats in diabetic and silibinin-treated diabetic groups were fed with high fat diet (Diet #MD45%fat; Mediscience Ltd, China) for a period of 4 weeks to induce insulin resistance. After the 4 weeks of dietary manipulation, the animals were intraperitoneally injected with STZ (Sigma-Aldrich, St Louis, MO, USA) (35 mg/kg body weight in 100 mL of sterile citrate buffer, pH 4.5) on two consecutive days to induce the type II diabetes mellitus [22,23]. Rats in the CG group were fed with standard chow and water ad libitum and injected with citrate vehicle alone. After 72 h of STZ injection, animals with venous blood glucose levels of over 16.7 mmol/L were considered diabetic and selected for further studies. Venous blood glucose was measured every week to ensure the blood glucose level more than 16.7 mmol/L. All rats had free access to standard chow and tap water throughout the experiment. All rats were provided a vehicle control or silibinin for 12 weeks after STZ injection. Due to its low solubility in water, silibinin was suspended in 0.5% sodium carboxymethyl cellulose. Silibinin was administered at a daily dose of 50 mg kg 1day 1 in the DG-50 group and 100 mg kg 1day 1 in the DG-100 group by oral gavage while the controls were administered 1 mL of deionized distill water (ddH2O) in 0.5%
CMC-Na by oral gavage for 12weeks. The dose of silibinin was according to previous published study [24]. At 12 weeks after oral gavage administration of silibinin, rats were anesthetized by administration of 2% (w/v) pentobarbital sodium (35 mg/kg, Solarbio Science & Technology, Beijing, China) via intraperitoneal injection. The bones were collected from each animal and dissected with care being taken to protect the periosteum. Each bone was individually wrapped in ddH2Osoaked gauze and stored at 20 C until analyzed. 2.2. Bone morphometry and microarchitecture Bone morphometry measurements of length, width and height were determined using a vernier caliper. Bones were dried at 110 C for 48 h and then weighed. The femoral length was measured from the medial condyle to greater trochanter. The femoral width was measured from the lateral condyle to medial condyle and the femoral height was measured from the top to the bottom of the lateral condyle in this study. Morphometry measurements were averaged after no bilateral differences were determined using a paired t-test with significance level set at P < 0.05. Bone radiographs of excised femur were taken with Dualenergy X-ray absorptiometry (DXA) scans (Kubetic) on the right femora. For microarchitecture, trabecular bone architecture was determined on the left femora using microcomputed tomography (mCT) (MicroCTm100, SCANCO Medical, Switzerland). The trabecular bone within the distal femur metaphysis was scanned and 100 images (14.8 mm/slice or 1.48 mm) were analyzed with semiautomatically drawn contours beginning 50 slices (740 mm) away from the growth plate within the volume of interest (VOI). The femora were scanned at 70KV and 200 mA and 300 ms exposure time to obtain image resolution of 14.8 mm. Next, the reconstructed images were analyzed using the software (Ray V4.0-4, Switzerland). The VOI was assessed for structural parameters including trabecular bone volume fraction (BV/TV), trabecular number (TbN), trabecular thickness (TbTh), trabecular separation (TbSp) and structure model index. 2.3. Histological examination For Harris hematoxylin and Shandon Instant eosin (H&E) staining, the right femur were excised, fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.2) for 3 days at room temperature, decalcified with decalcifying solution (SigmaAldrich, St Louis, MO, USA) for 4 weeks. The tissue samples were then dehydrated in a graded series of ethanol solutions for 18 h, embedded in paraffin and cut into 5 mm sections in thickness. The specimens were subjected to histomorphometric analysis under a light microscope with a micrometer, using an image analyzer (Olympus, Japan). 2.4. Serum and urine biochemical analyses After 12 weeks of silibinin treatment, rats were anesthetized by administration of 2% (w/v) pentobarbital sodium (35 mg/kg) via intraperitoneal injection, blood was obtained using cardiac puncture, and serum samples obtained by centrifugation (3000 rpm, 10 min) were stored at 80 C until analysis. Plasma osteocalcin, a marker of bone formation, was measured by a commercially available rat-specific enzyme immunoassay (EIA) (Shanghai Haling biological technology, PR China). Serum concentrations of parathyroid hormone (PTH) were determined using a rat PTH ELISA kit (Immutopics Inc., San Clemente, CA, USA). Serum samples were analyzed for their calcium and phosphorus contents by the arsenazo-3 dye and ammonium molybdate colorimetric methods respectively. Plasma C-reactive protein
T. Wang et al. / Biomedicine & Pharmacotherapy 89 (2017) 681–688
Fig. 1. Body weight and serum glucose of rats in each group. CG control group, DG diabetic group, DG-50 diabetic group with 50 mg kg group with 100 mg kg 1day 1 of silibinin. Means standard deviations were indicated at each week and each group.
683
1
day
1
of silibinin, DG-100 diabetic
Shanghai, China). In this study, serum GOT and GPT levels were not influenced by the administration of silibinin in the DG-50 and DG-100 group, indicating that they did not induce liver toxicity.
(CRP) was determined using a commercially available rat CRP enzyme-linked immunosorbent assay kit purchased from the Shanghai Langka Company (Shanghai, China). All samples were performed in duplicate for the assays. To evaluate the extent of oxidative stress, urinary 8-OHdG, a sensitive indicator of oxidative DNA damage, was measured. After 12 weeks of silibinin treatment, a 24 h-urine collection from each rat using metabolic cages was conducted. The urine was kept at 20 C until the concentrations of 8-OHdG were measured. Urinary 8-OHdG was determined using a sandwich enzyme-linked immunosorbent assay kit (Uscnlife Life Sciences, Inc). Serum levels of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) were measured by using enzymatic kits (Shanghai Kehua Bio-engineering Co., Ltd.,
2.5. Statistical analysis Results are expressed as means standard error of the mean. The statistical significant of difference between groups was obtained by the student’s t-test using SPSS software (ver. 19.0; SPSS Inc., Chicago, IL). Differences were considered significant at P < 0.05. The relationship between plasma osteocalcin and urinary excretion of 8-OHdG was determined by linear regression of individual animals in the DG-50 and DG-100 group. All experiments were repeated at least three times.
Table 1 Femoral morphometry, femoral trabecular bone microstructure and biochemistry in diabetic rats administered silibinin or vehicle control. parameters
Control group (n = 10)
1
Diabetic group + 50 mg kg
0.61 0.08b 4.89 0.07b 2.93 0.05b 2.71 0.05b
0.63 0.06b 4.91 0.07b 2.95 0.07b 2.73 0.06b
0.72 0.05a 5.06 0.05a,b 3.08 0.05a,b 2.81 0.05a,b
Femoral trabecular bone microstructure BV/TV (%) 0.41 0.02 0.30 0.01b TbN (mm) 3.53 0.11 3.07 0.13b TbTH (mm) 0.098 0.005 0.096 0.004 TbSp (mm) 0.25 0.02 0.32 0.02b SMI 1.39 0.05 1.85 0.09b
0.29 0.01b 3.03 0.17b 0.097 0.002 0.32 0.01b 1.78 0.05b
0.36 0.02a,b 3.46 0.07a 0.105 0.007 0.28 0.01a 1.46 0.07a
biochemistry Osteocalcin (nmol/L) ALP (IU/L) Ca (mmol/L) P (mmol/L) PTH(pg/ml) CRP (mg/mL) 8-OHdG (ng/day) GOT (IU/L) GPT (IU/L)
21.4 4.7b 153 15b 8.8 1.1 5.8 0.9 43.8 6.8 44.8 4.8b 41.60 18.65b 45.44 5.56 25.00 4.53
27.3 4.5a 231 14a,b 8.7 0.85 6.1 1.1 40.4 5.3 36.4 4.6a,b 20.10 7.88a,b 46.36 5.25 24.42 4.36
Femoral morphometry Dry mass (g) 0.79 0.07 Width (mm) 5.24 0.06 Depth (mm) 3.21 0.05 Length (cm) 2.95 0.04
31.3 4.7 182 14 8.7 0.5 5.7 0.6 40.1 4.8 10.8 2.0 2.65 0.33 49.36 5.40 25.82 4.98
18.3 2.5b 135 13b 9.1 0.5 6.0 0.8 42.5 8.5 47.2 3.9b 72.98 23.10b 48.34 5.75 25.10 4.59
day
1
Diabetic group (n = 10)
of silibinin (n = 10)
Diabetic group + 100 mg kg
1
day
1
of silibinin (n = 10)
Values are expressed as the mean SEM of n = 10 groups. The statistical significant of difference between groups was obtained by the student’s t-test comparison using SPSS software. BV/TV trabecular bone volume per unit of total volume, TbN trabecular number, TbTh trabecular thickness, TbSp trabecular separation, SMI structure model index. ALP alkaline phosphatase, Ca calcium, P phosphorus, PTH parathyroid, CRP C-reactive protein, 8-OHdG 8-hydroxydeoxyguanosine, GOT glutamic oxaloacetic transaminase, GPT glutamic pyruvic transaminase. a P < 0.05 vs. the DG group. b P < 0.05 vs. the CG group.
684
T. Wang et al. / Biomedicine & Pharmacotherapy 89 (2017) 681–688
Fig. 2. Femoral X-ray and bone mineral density (BMD). CG control group, DG diabetic group, DG-50 diabetic group with 50 mg kg with 100 mg kg 1day 1 of silibinin. a P < 0.05 vs. the DG group. b P < 0.05 vs. the CG group.
3. Results 3.1. General features Body weights and serum glucose of experimental rats were showed in Fig. 1. The data of body weight and serum glucose from week 1 to week 16 were present as mean SEM. Body weights in all groups increased until administration of STZ. After injection of STZ, body weights of diabetic and silibinin-treated diabetic groups decreased, while body weights in the CG group kept on growing. There were no significant differences in body weights among the three diabetic groups after STZ injection (P > 0.05). Serum glucose suddenly exceeded the normal level in diabetic and silibinin-treated diabetic groups after STZ injection. Comparing with the DG and DG-50 groups, the serum glucose decreased 5 weeks later after the administration of silibinin in the DG-100 group (Fig. 1(B)), which meant hyperglycemia in diabetic rats could be partly reversed by silibinin. 3.2. Bone morphometry and bone mineral density The dry mass, length, width and height of femur were showed in Table 1. The length, width and height of femur in the DG group and DG-50 group decreased significantly compared with that in the CG group. Comparing with the DG and DG-50 groups, the length, width and height of femur increased significantly in the DG-100 group (P < 0.05). 12 weeks after silibinin administration, rats showed severe osteopenia in the femur by X-ray (Fig. 2(A)) and femoral BMD in the DG and DG-50 group was 35% and 32% lower respectively than that in the CG group (P < 0.05). In the DG-100 group, the femoral BMD was 25% lower compared with the CG group (P < 0.05) and was 19% higher compared with the DG group (P < 0.05) (Fig. 2(B)). 3.3. Bone microarchitecture The effects of diabetes mellitus with or without silibinin were evaluated using microarchitectural parameters in the distal femoral metaphysic (Table 1). The reconstructed images of the femur from rats in the four groups were constructed using micro-CT (Fig. 3(A), (B)). There were no significant effects between the DG and DG-50 rats
1
day
1
of silibinin, DG-100 diabetic group
on femoral trabecular bone microarchitectures. Similarly, there were no significant effects between the CG and DG-100 rats. The femoral trabecular bone microarchitectures in the DG and DG-50 rats had significant differences compared with that in the CG and DG-100 group. BV/TV and TbN, which decreased with diabetes mellitus, was significantly increased in the DG-100 group (P < 0.05). On the other hand, increased TbSp and SMI in femur were significantly reduced by administration of sibilinin at 100 mg kg 1day 1 (P < 0.05 or less). TbTh of the CG group was slightly greater than that of the DG and DG50 group and was slightly smaller than that of the DG-100 group; however, there was no significant difference in TbTh between the four groups (P > 0.05). 3.4. Histological examination Morphologic changes of the femoral metaphysis stained with the hematoxylin and eosin’s method were observed under light microscopy. In the twelve weeks after administration of silibinin, the histological cross sections of the femur showed that the bony meshwork appeared in the metaphysis of each group (Fig. 4). There was an apparent reduction in trabecular (cancellous) bones in the DG and DG-50 group. In contrast, the cancellous bones appeared to be added in the metaphyseal area of diabetic group treated with silibinin. Oral administration of 100 mg kg 1day 1 silibinin might protect from bone loss led by diabetes mellitus. 3.5. Serum biochemistry The effects of diabetes mellitus with or without silibinin were evaluated using biochemical parameters in serum (Table 1). The concentrations of serum osteocalcin, markers of bone formation, was significantly increased in diabetes mellitus (p < 0.01), which were suppressed by the administration of silibinin (P < 0.05). Since ALP is a biomarker for the matrix maturation of osteoblasts, this study investigated the osteoblastic activity of silibinin. The ALP concentration was significantly decreased in the DG and DG-50 group (p < 0.01), which was positively influenced by the administration with 100 mg kg 1day 1 silibinin (p < 0.01). No significant differences in serum Ca, P and PTH were found among the groups. Moreover, there was no significant difference in serum GOT and GPT indicating that silibinin did not induce liver toxicity.
T. Wang et al. / Biomedicine & Pharmacotherapy 89 (2017) 681–688
685
Fig. 3. The longitudinal Micro-CT images (a) and three-dimensional Micro-CT images (b) of the distal femur in each treatment group. Compared with the CG group, changes of trabecular bones microarchitecture diabetic group were confirmed. In contrast, the changes in diabetic condition were partly restored in the DG-100 group.
3.6. Assessment of oxidative and inflammatory stress
3.7. Association between oxidative stress and bone formation
To evaluate the extent of oxidative stress in the animals, urinary 8- OHdG was determined. Urinary 8-OHdG was markedly (p < 0.01) higher in the DG and DG-50 group than in the CG group. However, silibinin-treated diabetic rats in the DG-100 group showed suppressed excretion of the marker, resulting in an absence of any significant difference with the control rats. Serum CRP, a sensitive marker of systemic inflammation, was significantly higher in the DG and DG-50 group than in the CG group (p < 0.01). Whereas it was positively influenced by the treatment with 100 mg kg 1day 1 silibinin (p < 0.01). Taken together, these results demonstrate that oxidative stress and inflammatory stress both existed in diabetes mellitus. Moreover, the results demonstrate that silibinin treatment rescues both increased oxidative stress and increased inflammatory stress.
Linear regression was performed to assess the association between the parameters of bone formation (osteocalcin) and the markers of oxidative stress (urinary excretion of 8-OHdG) in silibinin-treated group (Fig. 5). There were a negative relationship (R2 = 0.827, P < 0.01), which means that 8-OHdG were inversely associated with plasma osteocalcin. 4. Discussion Diabetes is the theme of 2016 World Health Day and WHO has published the Global Report on Diabetes to raise awareness and spark momentum for action at the necessary scale. People with all types of diabetes are at risk of developing a range of complications and the high costs of care increase the risk of catastrophic medical
686
T. Wang et al. / Biomedicine & Pharmacotherapy 89 (2017) 681–688
Fig. 4. Hematoxylin and eosin stain. Femurs were removed after 12 weeks of silibinin or vehicle treatment. Compared with control rats and 100 mg kg 1day 1 silibinintreated diabetic rats, decreases of trabecular bones in diabetic rats and 50 mg kg 1day 1 silibinin-treated diabetic rats were confirmed. Magnification is 200 and the scale bar is 200 mm.
expenditure [25]. Sustained hyperglycemia and long-term metabolic disorders can lead to the damage, dysfunction and failure in body tissues and organs. DO, characterized by low bone mass, is a complication of diabetes mellitus [2–4] and frequent in diabetic patients but is asymptomatic until the fracture. Oei et al., found that fracture risk in type II diabetes patients with poor glycemic control increased by 47%–62% compared with non-diabetic patients and those with good glycemic control [26]. Bone width increases by tissue mineralization of the periosteal bone surface by osteoblasts. Increased bone formation by
Fig. 5. Relationship between serum osteocalcin and urinary 8-OHdG. There were 20 individual rats in the DG-50 and DG-100 group. A negative relationship could be found between urinary excretion of 8-OHdG and plasma osteocalcin.
osteoblasts was indicated by higher plasma osteocalcin concentration. In our study, the width of femur was greater in the DG-100 group than in the DG and DG-50 groups and silibinin supplementation in dose of 100 mg kg 1day 1 could significantly increase plasma osteocalcin (P < 0.05), a marker of bone formation. These results suggest that silibinin have the capability to promote bone formation resulting in prevention of bone loss following diabetic osteoporosis. Currently, mCT is considered the gold standard for assessing BMD and trabecular bone microarchitecture. Bouxsein et al. asserted that at least four parameters (i.e., BV/TV, TbTh, TbSp, and TbN) must be used to describe trabecular bone microarchitecture [27]. Therefore, these indicators were used as the assessment indicators in our study. In previous studies of osteoporosis, trabecular bone microarchitecture at the distal femoral metaphysic was the VOI [28]. Therefore, this anatomical region was selected as the VOI in the study. Bone volume (BV) was calculated using tetrahedrons corresponding to the enclosed volume of the triangulated surface [29]. Total tissue volume (TV) was the volume of entire scanned sample. BV/TV was then calculated from these values. TbTh, TbN and TbSp were estimated [30,31]. SMI is an index for trabecular structure and expresses the ratio of plate-like to rod-like structures. An SMI of 0 represents a perfect plate-like structure, and an SMI of 3 represents a perfect rod-like structure. Except for TbTh, diabetes accelerated loss of bone microarchitecture indicated by significantly decreased BV/TV and TbN, and increased TbSp and SMI in the DG group. These results indicated that bone formation was suppressed in STZ-induced diabetic rats, which simulated diabetic osteoporosis. To exclude potential effects of silibinin treatment, we also examined two silibinin-treated diabetic groups. Compared with diabetic rats in the DG group, the changes in BV/TV, TbN, TbSp, and SMI of rats in the DG-100 group were 20.0%, 12.7%, 12.5%, and 21.1%, respectively.
T. Wang et al. / Biomedicine & Pharmacotherapy 89 (2017) 681–688
However, the changes in BV/TV, TbN, TbSp, and SMI of rats in the DG-50 group were 0.03%, 0.01%, 0%, and 0.04%, respectively. This indicated that silibinin supplementation of diabetic rats had significant effects on bone microarchitecture and the significant effects may rely on the dose of silibinin. Unfortunately, this study observed no significant difference in TbTh between the DG and DG100 groups (p = 0.053). The main reason may be that the number of the rats used in this study was small. Few studies related to diabetic osteoporosis have been conducted; thus, no comparison was made between previous studies and this study. As to the mechanisms of diabetic osteoporosis, in vitro studies have reported that high glucose influences osteoblast differentiation [32], impairs bone formation [33], and inhibits bone mineralization [34]. In addition, experimental and clinical studies have demonstrated that osteoblast function is impaired under diabetic conditions [35,36]. Our previous studies have reported that high glucose suppressed osteogenic differentiation of human bone marrow stem cells, manifested by an increase of oxidative damage markers including reactive oxygen species and all of the observed oxidative damage can be inhibited by silibinin [9]. Concerning the pathogenesis of diabetic osteoporosis, oxidative stress may be an important factor. In the present study, urinary excretion of 8-OHdG was elevated in STZ-induced diabetic rats, while the marker was significantly suppressed to control levels in silibinin-treated diabetic rats. Our animal study demonstrated a negative relationship (R2 = 0.827, P < 0.01) between urinary 8OHdG and bone formation (plasma osteocalcin), which means the deleterious effects of skeletal unloading on bone may be suggested to be due, in part, to increased oxidation. These findings indicate that oxidative stress in diabetic conditions was suppressed in silibinin-treated diabetic rat. In vitro studies have shown that oxidative stress inhibits osteoblastic differentiation [37,38] and induces osteoblast apoptosis [39,40], which is consistent with our previous study [9]. In vivo study has shown that the increase in oxidative stress may at least partly contribute to the development of diabetic osteoporosis [7]. It is in accordance with our study that the deleterious effects of diabetes on bone may be suggested to increased oxidation and silibinin supplementation to female rats had in part bone protective effects. This study had several limitations. Specifically, we measured only the trabecular bone microarchitecture in the femur but did not explore osteoporosis of the spine or wrist. Another limitation of our study is the relatively small number of rats. This limits the power of the study, particularly due to the fact that the group is divided into four groups for analyses. Finally, we mainly used micro-CT to analyze bone mass but did not perform a biomechanical test to measure bone strength. In the future, we will carry out more research to overcome these limitations and understand the mechanism of diabetic osteoporosis. Based on our study findings, diabetes can cause osteoporosis in diabetic rats. Large-dose silibinin supplementation can attenuate demineralization and microstructure deterioration. The bone protective effects of silibinin appeared to be mediated by decreased oxidation and increased bone formation. Therefore, silibinin supplementation may have potential use as a possible therapy for maintaining skeletal health and the study may enhance the understanding of pathophysiology for diabetic osteoporosis. Conflict of interest The authors declare no competing financial interest. Acknowledgments The authors thank all the staff in the Laboratory of Orthopedic Research Institute and Scientific Research Center of Second
687
Affiliated Hospital of Wenzhou Medical University. This work was supported by grants from the National Natural Science Foundation of China (81402980) and the Wenzhou Technology Project (Y20140154). References [1] L.R. McCabe, Understanding the pathology and mechanisms of type I diabetic bone loss, J. Cell. Biochem. 102 (6) (2007) 1343–1357. [2] M.J. Kayath, S.A. Dib, J.G. Vieira, Prevalence and magnitude of osteopenia associated with insulin-dependent diabetes mellitus, J. Diabetes Complications 8 (2) (1994) 97–104. [3] S.A. Kemink, A.R. Hermus, L.M. Swinkels, J.A. Lutterman, A.G. Smals, Osteopenia in insulin-dependent diabetes mellitus; prevalence and aspects of pathophysiology, J. Endocrinol. Invest. 23 (5) (2000) 295–303. [4] P.J. Lopez-Ibarra, M.M. Pastor, F. Escobar-Jimenez, M.D. Pardo, A.G. Gonzalez, J. D. Luna, M.E. Requena, M.A. Diosdado, Bone mineral density at time of clinical diagnosis of adult-onset type 1 diabetes mellitus, Endocr. Pract. 7 (5) (2001) 346–351. [5] M.E. Levin, V.C. Boisseau, L.V. Avioli, Effects of diabetes mellitus on bone mass in juvenile and adult-onset diabetes, N. Engl. J. Med. 294 (5) (1976) 241–245. [6] Y. Seino, H. Ishida, Diabetic osteopenia: pathophysiology and clinical aspects, Diabetes Metab. Rev. 11 (1) (1995) 21–35. [7] Y. Hamada, S. Kitazawa, R. Kitazawa, H. Fujii, M. Kasuga, M. Fukagawa, Histomorphometric analysis of diabetic osteopenia in streptozotocin-induced diabetic mice: a possible role of oxidative stress, Bone 40 (5) (2007) 1408– 1414. [8] J. Liu, J. Yang, Uncarboxylated osteocalcin inhibits high glucose-induced ROS production and stimulates osteoblastic differentiation by preventing the activation of PI3K/Akt in MC3T3-E1 cells, Int. J. Mol. Med. 37 (1) (2016) 173– 181. [9] X. Ying, X. Chen, H. Liu, P. Nie, X. Shui, Y. Shen, K. Yu, S. Cheng, Silibinin alleviates high glucose-suppressed osteogenic differentiation of human bone marrow stromal cells via antioxidant effect and PI3K/Akt signaling, Eur. J. Pharmacol. 765 (2015) 394–401. [10] A. Shirwaikar, S. Khan, S. Malini, Antiosteoporotic effect of ethanol extract of Cissus quadrangularis Linn. on ovariectomized rat, J. Ethnopharmacol. 89 (2– 3) (2003) 245–250. [11] C.L. Shen, J.K. Yeh, J.J. Cao, O.L. Tatum, R.Y. Dagda, J.S. Wang, Green tea polyphenols mitigate bone loss of female rats in a chronic inflammationinduced bone loss model, J. Nutr. Biochem. 21 (10) (2010) 968–974. [12] L. Al-Anati, E. Essid, R. Reinehr, E. Petzinger, Silibinin protects OTA-mediated TNF-alpha release from perfused rat livers and isolated rat Kupffer cells, Mol. Nutr. Food Res. 53 (4) (2009) 460–466. [13] R.P. Singh, R. Agarwal, Mechanisms and preclinical efficacy of silibinin in preventing skin cancer, Eur. J. Cancer 41 (13) (2005) 1969–1979. [14] H.L. Ha, H.J. Shin, M.A. Feitelson, D.Y. Yu, Oxidative stress and antioxidants in hepatic pathogenesis, World J. Gastroenterol. 16 (48) (2010) 6035–6043. [15] S. Kittur, S. Wilasrusmee, W.A. Pedersen, M.P. Mattson, K. Straube-West, C. Wilasrusmee, B. Lubelt, D.S. Kittur, Neurotrophic and neuroprotective effects of milk thistle (Silybum marianum) on neurons in culture, J. Mol. Neurosci. 18 (3) (2002) 265–269. [16] M. Agoston, R.G. Cabello, A. Blazovics, J. Feher, A. Vereckei, The effect of amiodarone and/or antioxidant treatment on splenocyte blast transformation, Clin. Chim. Acta 303 (1–2) (2001) 87–94. [17] N. Milic, N. Milosevic, L. Suvajdzic, M. Zarkov, L. Abenavoli, New therapeutic potentials of milk thistle (Silybum marianum), Nat. Prod. Commun. 8 (12) (2013) 1801–1810. [18] C.V. Kavitha, G. Deep, S.C. Gangar, A.K. Jain, C. Agarwal, R. Agarwal, Silibinin inhibits prostate cancer cells- and RANKL-induced osteoclastogenesis by targeting NFATc1, NF-kappaB, and AP-1 activation in RAW264.7 cells, Mol. Carcinog. 53 (3) (2014) 169–180. [19] J.H. Kim, K. Kim, H.M. Jin, I. Song, B.U. Youn, J. Lee, N. Kim, Silibinin inhibits osteoclast differentiation mediated by TNF family members, Mol. Cells 28 (3) (2009) 201–207. [20] J.L. Kim, S.H. Park, D. Jeong, J.S. Nam, Y.H. Kang, Osteogenic activity of silymarin through enhancement of alkaline phosphatase and osteocalcin in osteoblasts and tibia-fractured mice, Exp. Biol. Med. (Maywood) 237 (4) (2012) 417–428. [21] X. Ying, L. Sun, X. Chen, H. Xu, X. Guo, H. Chen, J. Hong, S. Cheng, L. Peng, Silibinin promotes osteoblast differentiation of human bone marrow stromal cells via bone morphogenetic protein signaling, Eur. J. Pharmacol. 721 (1–3) (2013) 225–230. [22] M.S. Islam, T. Loots du, Experimental rodent models of type 2 diabetes: a review, Methods Find Exp. Clin. Pharmacol. 31 (4) (2009) 249–261. [23] E.J. Henriksen, Invited review: effects of acute exercise and exercise training on insulin resistance, J. Appl. Physiol. (1985) 93 (2) (2002) 788–796. [24] J.L. Kim, Y.H. Kim, M.K. Kang, J.H. Gong, S.J. Han, Y.H. Kang, Antiosteoclastic activity of milk thistle extract after ovariectomy to suppress estrogen deficiency-induced osteoporosis, Biomed. Res. Int. 2013 (2013) 919374. [25] C.M. Smith-Spangler, J. Bhattacharya, J.D. Goldhaber-Fiebert, Diabetes, its treatment, and catastrophic medical spending in 35 developing countries, Diabetes Care 35 (2) (2012) 319–326. [26] L. Oei, M.C. Zillikens, A. Dehghan, G.H. Buitendijk, M.C. Castano-Betancourt, K. Estrada, L. Stolk, E.H. Oei, J.B. van Meurs, J.A. Janssen, A. Hofman, J.P. van
688
[27]
[28]
[29] [30]
[31]
[32]
[33]
T. Wang et al. / Biomedicine & Pharmacotherapy 89 (2017) 681–688 Leeuwen, J.C. Witteman, H.A. Pols, A.G. Uitterlinden, C.C. Klaver, O.H. Franco, F. Rivadeneira, High bone mineral density and fracture risk in type 2 diabetes as skeletal complications of inadequate glucose control: the Rotterdam Study, Diabetes Care 36 (6) (2013) 1619–1628. M.L. Bouxsein, S.K. Boyd, B.A. Christiansen, R.E. Guldberg, K.J. Jepsen, R. Muller, Guidelines for assessment of bone microstructure in rodents using microcomputed tomography, J. Bone. Miner. Res. 25 (7) (2010) 1468–1486. A. Xiang, M. Kanematsu, S. Kumar, D. Yamashita, T. Kaise, H. Kikkawa, S. Asano, M. Kinoshita, Changes in micro-CT 3D bone parameters reflect effects of a potent cathepsin K inhibitor (SB-553484) on bone resorption and cortical bone formation in ovariectomized mice, Bone 40 (5) (2007) 1231–1237. W.E. Lorensen, H.E. Cline, Marching cubes a high resolution 3D surface construction algorithm, Comput. Graph. 21 (1987) 163–169. T. Hildebrand, P. Rüegsegger, A new method for the model-independent assessment of thickness in three-dimensional images, J. Microsc. 185 (1) (1997) 67–75. A.M. Parfitt, C.H.E. Mathews, A.R. Villanueva, Relationships between surface, volume, and thickness of iliac trabecular bone in aging and in osteoporosis. Implications for the microanatomic and cellular mechanisms of bone loss, J. Clin. Invest. 72 (4) (1983) 1396–1409. M. Zayzafoon, C. Stell, R. Irwin, L.R. McCabe, Extracellular glucose influences osteoblast differentiation and c-Jun expression, J. Cell. Biochem. 79 (2) (2000) 301–310. M. Terada, M. Inaba, Y. Yano, T. Hasuma, Y. Nishizawa, H. Morii, S. Otani, Growth-inhibitory effect of a high glucose concentration on osteoblast-like cells, Bone 22 (1) (1998) 17–23.
[34] E. Balint, P. Szabo, C.F. Marshall, S.M. Sprague, Glucose-induced inhibition of in vitro bone mineralization, Bone 28 (1) (2001) 21–28. [35] K.M. Thrailkill, L. Liu, E.C. Wahl, R.C. Bunn, D.S. Perrien, G.E. Cockrell, R.A. Skinner, W.R. Hogue, A.A. Carver, J.L. Fowlkes, J. Aronson, C.K. Lumpkin Jr., Bone formation is impaired in a model of type 1 diabetes, Diabetes 54 (10) (2005) 2875–2881. [36] R. Bouillon, M. Bex, E. Van Herck, J. Laureys, L. Dooms, E. Lesaffre, E. Ravussin, Influence of age, sex, and insulin on osteoblast function: osteoblast dysfunction in diabetes mellitus, J. Clin. Endocrinol. Metab. 80 (4) (1995) 1194– 1202. [37] N. Mody, F. Parhami, T.A. Sarafian, L.L. Demer, Oxidative stress modulates osteoblastic differentiation of vascular and bone cells, Free Radic. Biol. Med. 31 (4) (2001) 509–519. [38] X.C. Bai, D. Lu, J. Bai, H. Zheng, Z.Y. Ke, X.M. Li, S.Q. Luo, Oxidative stress inhibits osteoblastic differentiation of bone cells by ERK and NF-kappaB, Biochem. Biophys. Res. Commun. 314 (1) (2004) 197–207. [39] R.M. Chen, G.J. Wu, H.C. Chang, J.T. Chen, T.F. Chen, Y.L. Lin, T.L. Chen, 2,6Diisopropylphenol protects osteoblasts from oxidative stress-induced apoptosis through suppression of caspase-3 activation, Ann. N. Y. Acad. Sci. 1042 (2005) 448–459. [40] A.A. Fatokun, T.W. Stone, R.A. Smith, Hydrogen peroxide-induced oxidative stress in MC3T3-E1 cells: the effects of glutamate and protection by purines, Bone 39 (3) (2006) 542–551.