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VEGFR-2 signaling pathway in osteoporotic rats
Accepted Manuscript Naringin promotes fracture healing through stimulation of angiogenesis by regulating the VEGF/VEGFR-2 signaling pathway in osteoporotic rats Nan Song, Zhihu Zhao, Xinlong Ma, Xiaolei Sun, Jianxiong Ma, Fengbo Li, Lei Sun, Jianwei Lv PII:
S0009-2797(16)30501-4
DOI:
10.1016/j.cbi.2016.10.020
Reference:
CBI 7841
To appear in:
Chemico-Biological Interactions
Received Date: 2 August 2016 Revised Date:
19 September 2016
Accepted Date: 25 October 2016
Please cite this article as: N. Song, Z. Zhao, X. Ma, X. Sun, J. Ma, F. Li, L. Sun, J. Lv, Naringin promotes fracture healing through stimulation of angiogenesis by regulating the VEGF/VEGFR-2 signaling pathway in osteoporotic rats, Chemico-Biological Interactions (2016), doi: 10.1016/j.cbi.2016.10.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Naringin promotes fracture healing through stimulation of angiogenesis by regulating the VEGF/VEGFR-2 signaling pathway in osteoporotic rats Nan Song a,1, Zhihu Zhaob,c,1, Xinlong Ma b *, Xiaolei Sun, Jianxiong Ma b, Fengbo Li b, Lei Sun b
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Jianwei Lvb a
Tianjin Huanhu Hospital, No.122, Qixiangtai Road, Hexi District, Tianjin, China.
b
Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 122, Munan Road,
Tianjin, 300050, China Graduate School of Tianjin Medical University, Tianjin, China
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c
Correspondence to: Prof. Xinlong Ma, Tianjin Institute of Orthopedics in Traditional Chinese and Western
Medicine,
Tianjin
Hospital,
NO.122
Munan
Road,
Tianjin,
300050,
China.
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Tex:+8613516118843, Fax: (+ 022) 28313173, E-mail: [email protected] 1These authors contributed equally to this work. Abstract
Postmenopausal osteoporosis is characterized by a reduction in the number of sinusoidal and arterial capillaries in the bone marrow and reduced bone perfusion. Thus, osteogenesis and
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angiogenesis are coupled in the process of osteoporosis formation and fracture healing. Naringin is the main ingredient of the root Rhizoma Drynariae, a traditional Chinese medicine, and it has potential effects on promoting fracture healing. However, whether naringin stimulates angiogenesis in the process of bone healing is unclear. Here, we show that naringin promotes fracture healing through
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stimulating angiogenesis by regulating the VEGF/VEGFR-2 signaling pathway in osteoporotic rats. Key words: Angiogenesis; naringin; VEGF; VEGFR-2
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Introduction
Osteoporosis (OP) is an imbalance of bone resorption and bone formation characterized by low
bone mineral density (BMD) and microarchitectural deterioration, resulting in bone fragility and, subsequently, a high risk of fracture1. Patients with osteoporosis may experience hip and vertebral
fracture because of microarchitecture damage; therefore, the risk of morbidity and mortality is increased, especially in postmenopausal women. Although the main reason for postmenopausal osteoporosis is the lack of estrogen, reduction in the number of sinusoidal and arterial capillaries in the
bone marrow and reduced bone perfusion suggest a vascular component in the pathogenesis of postmenopausal osteoporosis2-4. Moreover, a major angiogenic factor, VEGF, which plays important
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roles in the cartilaginous callus that bridges the fracture gap and in mineralized bone formation, was found to be significantly reduced in the bones of ovariectomized mice5-7. Similarly, Araldi E et al suggested that blood vessels contribute to the process of osteogenesis, both in development and bone
fracture union and decrease the rate of nonunion.
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repair8. Therefore, increasing the number of blood vessels in the early phase of fracture may promote
Naringin, chemically 4’,5,7-trihydroxy flavanone-7-rhamnoglucoside, is a kind of flavanone glycoside, and it possesses a variety of biological and pharmacological effects9,10. Naringin is the major
active ingredient in the Chinese herb Drynaria fortune, which can be obtained from grape, tomato, and
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citrus fruit species. It has been documented that naringin has antioxidant and anti-inflammatory properties, and even antiapoptotic activity11. Studies have reported that naringin can promote osteoblast
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differentiation and even effectively reverse ovariectomized-induced osteoporosis12. Kandhare et al9 have reported that naringin can promote the healing of diabetic foot ulcers through modulation of inflammatory and VEGF expression in rats 13.
However, we assessed whether naringin could promote fracture healing in osteoporotic rats and the underlying mechanisms by which naringin promote osteoporotic angiogenesis during bone fracture healing. Therefore, we hypothesized that naringin may improve the capacity of angiogenesis, thus
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improving the strength of bone fracture. The purpose of this study is to investigate the angiogenesis effect of narigin in rats with osteoporotic fractures. Material and methods
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Rats and reagents
Naringin was obtained from Sigma Company (St. Louis, MO, USA) and stored at -4°C. Female 3-month-old Sprague-Dawley rats (body weight, 240 ± 12 g) were purchased from the Experimental
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Animal Center of Tianjin Hospital (Tianjin, China). The rats were housed in an air-conditioned environment (22 ± 2°C) with a 12-hour light/dark cycle (7:00-19:00) and were allowed free access to food pellets and water throughout the experiment. Seventy-two rats were randomly divided into four groups (18/group), in accordance with the random number table. Under anesthesia with chloral hydrate (400 mg/kg, IP), ovariectomy was performed by removing the bilateral ovaries together with their capsules and part of the oviduct through a dorsal approach. Each incision was then closed by one stitch performed with a 5-0 synthetic absorbable suture14. Thirty days after surgery, a fracture of the upper middle one-third of the tibia was performed in all animals in the four groups. Three groups were treated
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with oral naringin (40, 100, and 300 mg/kg/day) by a nasogastric tube, and the control group received an equal volume of normal saline. The rats were euthanized at 2, 4, and 8 weeks after bone fracture. The experimental animals received humane care, and the study protocols were in accordance with the
Bone mineral density, weight, and blood urea
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guidelines of the Ethics Committee of Tianjin Hospital.
Thirty days after the ovariectomy, the BMD of distal femoral metaphysis in vivo was measured by dual-energy x-ray absorptiometry (DXA, Lunar-Prodigy, GE), as previously described15. After receiving anesthesia with chloral hydrate (300 mg/kg, IP), the rats were placed in a supine position,
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with the right limb externally rotated and hip, knee, and ankle joints at 90° flexion. The weight of the rats in the four groups was measured before they were euthanized. Meanwhile, the serum was obtained
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for the measurement of blood urea. Vascular perfusion
To analyze the situation of blood vessels within the fracture, we perfused the rats with a radiopaque silicone compound containing lead chromate (Microfil MV-122, Flow Tech, Carver, MA). First, the thoracic cavity was opened immediately after the animals were euthanized (chloral hydrate 5 mL/kg). An incision was made at the right auricle. The vasculature was flushed with 0.9% normal
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saline containing heparin sodium (100 U/mL) via a needle inserted into the left ventricle. The specimen was then perfused with 4% neutral buffered paraformaldehyde. The specimen was flushed from vessels using heparinized saline, until the paraformaldehyde was flushed out. Microfil was then injected from
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the needle with a pressure of 100 mm Hg and at a speed of 3 mL/min. The intact specimen was stored at 4°C overnight. After overnight storage, the entire right leg of the rat, containing the tibia, tibialis anterior, and gastrocnemius, was dissected for decalcification for 3 weeks in 15% ethylene diamine
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tetra-acetic acid (EDTA). Subsequently, the specimen received the first micro-CT scan using an Inveon PET.SPECT.CT system (Siemens) and COBRA Exxim (licensed to Siemens) to reconstruct it. Scans were performed at an effective pixel size of 8.52 µm, with voltage, current, and integration time of 80 kVp, 500 µA, and 2000 ms, respectively. The VV/TV, VN, Vt, Vs, and VSp were counted by Ineon
Research Workplace software. We chose 750 continuous images ranging from the upper to the lower fracture line. Because the fracture line cannot be judged in the 3D image after the decalcification procedure, we drew a line on the platform. The fracture line was directly fixed as the standard line. While in the reconstruction view, the middle of the image was the site at which the fracture line was
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located. For the qualitative and morphologic analyses, eight digital images of each section were taken randomly and under a lightscope (Nikon Corporation, Japan) at 20× magnification. Images were analyzed by Image Software Pro-Plus 6.0 (Media Cybernetics, USA). The vessel area and vessel number of each image was counted for statistical analysis.
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Histological test
The specimens were taken at the 2nd, 4th, and 8th weeks after surgery. The entire legs on the
fracture side were removed, and the muscles were excised carefully. The tissues were soaked in the 4%
paraformaldehyde for 24 hours at 4°C, and 10% EDTA was used for decalcification. After
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decalcification for 2 months, the specimens were dehydrated, embedded in paraffin, and then cut into slices in the sagittal plane. The sections were stained with hematoxylin and eosin (H&E) stain.
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Polymerase Chain Reaction
After the rats were euthanized, the calluses were immersed in RNA store reagent (Com Win Biotech, Beijing, China) immediately and frozen with liquid nitrogen until mRNA analysis by quantitative real-time polymerase chain reaction (PCR). Total RNA was extracted from each callus using
Trizol
reagent
(Invitrogen,
USA)
following
manufacturer's
instructions
and
then
reverse-transcribed to cDNA using ReverTra Ace qPCR RT Kit (Toyobo Co. Ltd., Osaka, Japan).
β-actin
Reverse
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Primer sequences used were as follows: β-actin Forward 5'-AGA TCC TGA CCG AGC GTG GC-3', 5'-CCA
GGG
AGG
AAG
AGG
ATG
CG-3,
VEGF:
Forward
GAGCCTTGCCTTGCTGCTCTAC, reverse CACCAGGGTCTCGATTGGATG, VEGFR-2 Forward
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5' TGA CAC GGA AAC TGA AGA CC 3', Reverse 5' TTG GAG TTT CAG AGG CAG GT 3'. RT-PCR was performed using Recombinant Taq DNA Polymerase TaKaRa Taq™ (Takara Bio Inc., Shiga, Japan) according to manufacturer's instructions. QRT-PCR was performed using SYBR® Green
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Real-time PCR Master Mix (Toyobo Co. Ltd., Osaka, Japan). β-Actin served as a normalizer for correction. Relative mRNA levels of VEGF and VEGFR-2 were expressed as fold changes normalized to β-actin and were analyzed according to a comparative method with the equation expressed as 2−∆∆Ct. Immunohistochemistry (IHC) Calluses were fixed in 4% neutral buffered formalin for at least 24 hours and decalcified with 10% EDTA for at least 3 months; they were embedded in paraffin after decalcification. Before the immunohistochemistry analysis, the slides were deparaffinized at 56°C for 30 minutes and then bathed in alcohol for 5 minutes. Endogenous peroxidase activity was inactivated by 3% H2O2 treatment for 10
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minutes, and 5% bovine serum albumin (BSA) blocking solution was used for antigen blocking. Then, all the slides were incubated with rabbit anti-rat VEGFR-2 antibody (Abcam, CA, UK) in 1:200 dilution at 4°C overnight. After thawing to 37°C for 30 minutes with 1:150 diluted biotin-coupled secondary antibody (Boster) and streptavidin-biotin complex (SABC), the color reaction was
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developed with a 3,3’-diaminobenzidine-tetrachloride kit (DAB kit; Boster). Sections were washed and
counterstained with hematoxylin and observed with an Olympus BX51 microscope (Olympus Optical, Tokyo, Japan). Sections for VEGFR-2 density counting were stained with claybank and hematoxylin and imaged at 100× magnification with the microscope.
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Statistical Analysis
All values are expressed as mean ± SD. The Graphpad Prism version 5.0 (GraphPad Software, La
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Jolla, CA, USA) was used for statistical analyses, and intergroup variance was evaluated by one-way ANOVA; the comparison between the two groups was analyzed by Dunnett’s test. When data were not normally distributed, significant differences were identified by the non-parametric Kruskal-Wallis test. Differences with a P value of less than 0.05 were considered statistically significant. Results The general condition of the rats
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Thirty days after the bilateral ovaries were removed, the BMD was decreased, and the difference was statistically significant (P<0.05). There was no significant difference between the blood urea and the weight among the four groups (P>0.05, Figure 1 A). These findings indicate that there is no sign of
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toxicity for naringin between the doses of 40 mg/kg and 300 mg/kg (Figure 1 B). Naringin can promote angiogenesis of the callus To evaluate the angiogenesis of the callus, the results of Microfil and subsequent micro-CT scan
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were analyzed. At 2 weeks, 4 weeks, and 8 weeks after fracture, the number of vessels, vessel volume, and vessel thickness around the fracture sites in the naringin group is higher than in the control group, and the vessel spacing in the naringin group is lower than in the control group. However, there was a statistically significant difference between the high dose of naringin and the control group (P<0.05, Figure 2). Effects of different doses of naringin on the vascular area and vascular number at 2 weeks tested by HE
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The 2-week HE staining slices show that the high-dose naringin group (300 mg/kg) has the most significant effect on the vascular area, as well as the vascular number. When compared with the control group, the low-dose group, and the middle-dose group, the difference was statistically significant (P<0.05, Figure 3). Results also indicated that the vascular area and the vascular number in the
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low-dose and middle-dose naringin group are higher than in the control group. The differences were statistically significant (P<0.05, Figure 3).
Effects of different doses of naringin on the relative gene expression of VEGF and VEGFR-2 at 2, 4, and 8 weeks
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As illustrated in Figure 3, VEGF mRNA expression in the high-dose naringin group increased by 4.52-fold (P<0.05), 7.25-fold (P<0.001), and 6.58-fold (P<0.001), respectively, compared with the
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control group at 2 weeks, 4 weeks, and 8 weeks, respectively. In addition, VEGFR-2, the VEGFR-2 mRNA expression in the high-dose naringin group, increased by 4.14-fold (P<0.05), 7.12-fold (P<0.001), and 5.23-fold (P<0.001), respectively, compared with the control group at 2 weeks, 4 weeks, and 8 weeks, respectively (Figure 4).
Results of different doses of naringin for the protein expression of VEGFR-2 tested by IHC The results of the protein expression of VEGFR-2 can be seen in Figure 4. Compared with the
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control group, naringin in the high-dose group (300 mg/kg) can effectively promote the expression of VRGFR-2 in callus at 2 weeks, 4 weeks, and 8 weeks. Another result is that, from 2 weeks to 8 weeks, the VEGFR-2 expression in each group decreased compared with the corresponding group(Figure 5).
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Discussion
This research investigated for the first time the effect and mechanism of naringin in the treatment of osteoporotic fracture in rats from the perspective of angiogenesis. We found that the administration
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of naringin at clinically relevant times induced more neovascularization, and thus accelerated dose-dependent bone healing at 2, 4, and 8 weeks post-fracture. This was also shown with increased vessel numbers and larger vessel area shown by H&E staining. A high rate of vessel volume/total volume, an increased on
vascular number, increased vessel thickness, and less vessel space were shown
Microfil scan. In addition, we found that naringin can up-regulate VEGF and VEGFR-2 activities
in osteoporotic fracture callus tissue. These results indicate that naringin has a higher capacity for treating osteoporotic fracture through angiogenesis, and its specific mechanism may be through the regulation of the signaling pathway of VEGF/VEGFR-2.
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Naringin, a major ingredient of the Chinese herbal medicine Rhizoma Drynariae possesses many pharmacological effects, and has been shown to increase BMP-2 expression, induce bone formation, and inhibit bone resorption.16 Our findings are in accordance with the former finding that naringin-mediated VEGF-c, IFG-1, and TGF-b synthesis improved capillary density, enhanced diabetic foot ulcers in the rat model9.
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angiogenesis, and, thus, increased wound breaking strength in
Estrogen and nutritional deficiency, heredity, deficiencies, chronic diseases, and aging may contribute to osteoporosis and fracture. The main reason that osteoporosis is frequently found in elderly women is
the lack of estrogen and the decreased blood supply in this population. Burkhardt et al17 found that the
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number of blood capillaries and blood sinuses are reduced when osteoporosis occurs. Ding et al6 also found that the VEGF level, which plays a pivotal role in angiogenesis, will be markedly lower in the
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bone of ovariectomized rats. Therefore, increasing the level of VEGF may promote angiogenesis, increasing bone mass and treating osteoporosis. This study first demonstrated that naringin has an effect in promoting osteoporotic fracture healing through increasing the VEGF level by interacting with its receptor VEGFR-2.
Because there are many complications associated with estrogen that surpass its clinical outcome, research into naringin is a positive step to finding a new treatment for osteoporosis. BMP-2 has been
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identified to possess indirect angiogenic effects18. As shown in the present study, naringin can promote the increase in the amounts of VEGF and VEGFR-2. Microfil results show that naringin can increase the vessel number, vessel volume/total volume, vessel separation factor, vessel thickness, and decrease the vessel space. Local vasculature rupture at the end of the fracture results in the formation of a
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hematoma, and therefore, a relatively regional hypoxic microenvironment will be formed19. HIF-1α is an important factor that regulates cellular responses to hypoxia20. Meanwhile, Komatsu et al21 and
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Maes et al22 have identified that HIF-1α is expressed in the bone-healing process, indicating that activation of the HIF-signaling pathway to induce VEGF-mediated response can accelerate bone healing. The VEGF/VEGFR-2 signaling pathway is also important for angiogenesis and fracture healing, and there are numerous studies that have reported that inhibited VEGF activity through VEGF antagonist and a soluble VEGFR resulted in impaired healing of femoral fracture and cortical bone defects in mice, whereas local administration of VEGF leads to improved bone repair in both models23. Naringin can bind to the estrogen receptor and promote the expression of VEGF, the critical factor that promotes angiogenesis, and thus improves proliferation and adhesion in the matrix.
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Osteoporosis and osteoporotic-related fractures have been a major cause of morbidity and mortality in the elderly population. Thus, the economic burden of patients with osteoporosis is increasing24. Johnell et al25 have reported that the number of osteoporotic fractures is as high as 9 million. It is well known that a timely and coordinated angionetic response is of vital importance for
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successful bone healing26. Neovascularization at the fracture site requires not only angiogenesis but
also circulating EPCs during vasculogenesis. Circulating EPCs are part of osteogenesis27. Rong28 reported that naringin can promote the expression of VEGF and brain-derived neurotrophic factor (BDNF) after spinal cord injury.
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Conclusion
To the best of our knowledge, this is the first article on the use of naringin for the treatment of
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osteoporotic fracture from the prospective of vascularization. The main outcome is that naringin can promote the vascularization of the callus in the fracture, by promoting the expression of VEGF and VEGFR-2. Acknowledgments
This study was funded by the National Natural Science Foundation of China (No.81401792 and
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Figure 1 A. The bone mineral density before the ovary was removed and 30 days after the ovary was
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removed; B, the blood urea and the weight among the four groups at 8 weeks.
Figure 2 1. Morphological analysis of vasculature within the tibia from Microfil-perfused analysis of different naringin groups and the control group rats at 2 weeks after fracture. 2. Morphological analysis of vasculature within the tibia from Microfil-perfused analysis of different naringin groups and the control group rats at 4 weeks after fracture. 3. Morphological analysis of vasculature within the tibia from Microfil-perfused analysis of different naringin groups and the control group rats at 2 weeks after
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fracture. A: Control group, B: Low dose: 40 mg/kg, C: Middle dose: 100 mg/kg, D: High dose: 300 mg/kg. Vessel number, vessel spacing, vessel separation factor, vessel thickness, and vessel volume/total volume are compared among different groups.
* P was a significant difference (<0.05),
compared with control group.
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Figure 3 Hematoxylin and eosin staining for the callus of different groups; red circles are the new vessels in the callus. Low dose: 40 mg/kg, Middle dose: 100 mg/kg, High dose: 300 mg/kg. Compared
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with control group, * P was a significant difference (<0.05). Red arrows: the blood vessels in the fracture site.
Figure 4 Effects of naringin on VEGF mRNA expression in each group at 2 weeks (Figure 2 A), 4 weeks (Figure 2 B), and 8 weeks (Figure 2 C) and VEGFR-2 mRNA in each group at 2 weeks (Figure 2 D), 4 weeks (Figure 2 E), and 8 weeks (Figure 2 F) analyzed by QRT-PCR, respectively. A p < 0.05, vs. control group; n = 6. VEGF: gene of vessel endothelial growth factor; VEGFR-2: gene of vessel endothelial growth factor receptor-2. Figure 5 VEGFR-2-positive osteocytes distributed in fracture sites in each group indicated by immunohistochemical staining. All of the panel magnification ×100. A, B, C, D represent the control
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group, low dose, middle dose, and high dose at 2 weeks, respectively. E, F, G, H represent the control group, low dose, middle dose, and high dose at 4 weeks, respectively. I, J, K, L represent the control group, low dose, middle dose, and high dose at 8 weeks, respectively. Red arrows: the
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Naringin promote angiogenesis of fracture healing in osteoporotic rats. Naringin exerts its angiogenetic role via stimulating VEGF/VEGFR-2 signaling.
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High dose naringin with 300 mg/kg is better than the low dose and middle dose.
S0009-2797(16)30501-4
DOI:
10.1016/j.cbi.2016.10.020
Reference:
CBI 7841
To appear in:
Chemico-Biological Interactions
Received Date: 2 August 2016 Revised Date:
19 September 2016
Accepted Date: 25 October 2016
Please cite this article as: N. Song, Z. Zhao, X. Ma, X. Sun, J. Ma, F. Li, L. Sun, J. Lv, Naringin promotes fracture healing through stimulation of angiogenesis by regulating the VEGF/VEGFR-2 signaling pathway in osteoporotic rats, Chemico-Biological Interactions (2016), doi: 10.1016/j.cbi.2016.10.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Naringin promotes fracture healing through stimulation of angiogenesis by regulating the VEGF/VEGFR-2 signaling pathway in osteoporotic rats Nan Song a,1, Zhihu Zhaob,c,1, Xinlong Ma b *, Xiaolei Sun, Jianxiong Ma b, Fengbo Li b, Lei Sun b
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Jianwei Lvb a
Tianjin Huanhu Hospital, No.122, Qixiangtai Road, Hexi District, Tianjin, China.
b
Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 122, Munan Road,
Tianjin, 300050, China Graduate School of Tianjin Medical University, Tianjin, China
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c
Correspondence to: Prof. Xinlong Ma, Tianjin Institute of Orthopedics in Traditional Chinese and Western
Medicine,
Tianjin
Hospital,
NO.122
Munan
Road,
Tianjin,
300050,
China.
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Tex:+8613516118843, Fax: (+ 022) 28313173, E-mail: [email protected] 1These authors contributed equally to this work. Abstract
Postmenopausal osteoporosis is characterized by a reduction in the number of sinusoidal and arterial capillaries in the bone marrow and reduced bone perfusion. Thus, osteogenesis and
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angiogenesis are coupled in the process of osteoporosis formation and fracture healing. Naringin is the main ingredient of the root Rhizoma Drynariae, a traditional Chinese medicine, and it has potential effects on promoting fracture healing. However, whether naringin stimulates angiogenesis in the process of bone healing is unclear. Here, we show that naringin promotes fracture healing through
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stimulating angiogenesis by regulating the VEGF/VEGFR-2 signaling pathway in osteoporotic rats. Key words: Angiogenesis; naringin; VEGF; VEGFR-2
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Introduction
Osteoporosis (OP) is an imbalance of bone resorption and bone formation characterized by low
bone mineral density (BMD) and microarchitectural deterioration, resulting in bone fragility and, subsequently, a high risk of fracture1. Patients with osteoporosis may experience hip and vertebral
fracture because of microarchitecture damage; therefore, the risk of morbidity and mortality is increased, especially in postmenopausal women. Although the main reason for postmenopausal osteoporosis is the lack of estrogen, reduction in the number of sinusoidal and arterial capillaries in the
bone marrow and reduced bone perfusion suggest a vascular component in the pathogenesis of postmenopausal osteoporosis2-4. Moreover, a major angiogenic factor, VEGF, which plays important
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roles in the cartilaginous callus that bridges the fracture gap and in mineralized bone formation, was found to be significantly reduced in the bones of ovariectomized mice5-7. Similarly, Araldi E et al suggested that blood vessels contribute to the process of osteogenesis, both in development and bone
fracture union and decrease the rate of nonunion.
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repair8. Therefore, increasing the number of blood vessels in the early phase of fracture may promote
Naringin, chemically 4’,5,7-trihydroxy flavanone-7-rhamnoglucoside, is a kind of flavanone glycoside, and it possesses a variety of biological and pharmacological effects9,10. Naringin is the major
active ingredient in the Chinese herb Drynaria fortune, which can be obtained from grape, tomato, and
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citrus fruit species. It has been documented that naringin has antioxidant and anti-inflammatory properties, and even antiapoptotic activity11. Studies have reported that naringin can promote osteoblast
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differentiation and even effectively reverse ovariectomized-induced osteoporosis12. Kandhare et al9 have reported that naringin can promote the healing of diabetic foot ulcers through modulation of inflammatory and VEGF expression in rats 13.
However, we assessed whether naringin could promote fracture healing in osteoporotic rats and the underlying mechanisms by which naringin promote osteoporotic angiogenesis during bone fracture healing. Therefore, we hypothesized that naringin may improve the capacity of angiogenesis, thus
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improving the strength of bone fracture. The purpose of this study is to investigate the angiogenesis effect of narigin in rats with osteoporotic fractures. Material and methods
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Rats and reagents
Naringin was obtained from Sigma Company (St. Louis, MO, USA) and stored at -4°C. Female 3-month-old Sprague-Dawley rats (body weight, 240 ± 12 g) were purchased from the Experimental
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Animal Center of Tianjin Hospital (Tianjin, China). The rats were housed in an air-conditioned environment (22 ± 2°C) with a 12-hour light/dark cycle (7:00-19:00) and were allowed free access to food pellets and water throughout the experiment. Seventy-two rats were randomly divided into four groups (18/group), in accordance with the random number table. Under anesthesia with chloral hydrate (400 mg/kg, IP), ovariectomy was performed by removing the bilateral ovaries together with their capsules and part of the oviduct through a dorsal approach. Each incision was then closed by one stitch performed with a 5-0 synthetic absorbable suture14. Thirty days after surgery, a fracture of the upper middle one-third of the tibia was performed in all animals in the four groups. Three groups were treated
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with oral naringin (40, 100, and 300 mg/kg/day) by a nasogastric tube, and the control group received an equal volume of normal saline. The rats were euthanized at 2, 4, and 8 weeks after bone fracture. The experimental animals received humane care, and the study protocols were in accordance with the
Bone mineral density, weight, and blood urea
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guidelines of the Ethics Committee of Tianjin Hospital.
Thirty days after the ovariectomy, the BMD of distal femoral metaphysis in vivo was measured by dual-energy x-ray absorptiometry (DXA, Lunar-Prodigy, GE), as previously described15. After receiving anesthesia with chloral hydrate (300 mg/kg, IP), the rats were placed in a supine position,
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with the right limb externally rotated and hip, knee, and ankle joints at 90° flexion. The weight of the rats in the four groups was measured before they were euthanized. Meanwhile, the serum was obtained
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for the measurement of blood urea. Vascular perfusion
To analyze the situation of blood vessels within the fracture, we perfused the rats with a radiopaque silicone compound containing lead chromate (Microfil MV-122, Flow Tech, Carver, MA). First, the thoracic cavity was opened immediately after the animals were euthanized (chloral hydrate 5 mL/kg). An incision was made at the right auricle. The vasculature was flushed with 0.9% normal
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saline containing heparin sodium (100 U/mL) via a needle inserted into the left ventricle. The specimen was then perfused with 4% neutral buffered paraformaldehyde. The specimen was flushed from vessels using heparinized saline, until the paraformaldehyde was flushed out. Microfil was then injected from
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the needle with a pressure of 100 mm Hg and at a speed of 3 mL/min. The intact specimen was stored at 4°C overnight. After overnight storage, the entire right leg of the rat, containing the tibia, tibialis anterior, and gastrocnemius, was dissected for decalcification for 3 weeks in 15% ethylene diamine
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tetra-acetic acid (EDTA). Subsequently, the specimen received the first micro-CT scan using an Inveon PET.SPECT.CT system (Siemens) and COBRA Exxim (licensed to Siemens) to reconstruct it. Scans were performed at an effective pixel size of 8.52 µm, with voltage, current, and integration time of 80 kVp, 500 µA, and 2000 ms, respectively. The VV/TV, VN, Vt, Vs, and VSp were counted by Ineon
Research Workplace software. We chose 750 continuous images ranging from the upper to the lower fracture line. Because the fracture line cannot be judged in the 3D image after the decalcification procedure, we drew a line on the platform. The fracture line was directly fixed as the standard line. While in the reconstruction view, the middle of the image was the site at which the fracture line was
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located. For the qualitative and morphologic analyses, eight digital images of each section were taken randomly and under a lightscope (Nikon Corporation, Japan) at 20× magnification. Images were analyzed by Image Software Pro-Plus 6.0 (Media Cybernetics, USA). The vessel area and vessel number of each image was counted for statistical analysis.
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Histological test
The specimens were taken at the 2nd, 4th, and 8th weeks after surgery. The entire legs on the
fracture side were removed, and the muscles were excised carefully. The tissues were soaked in the 4%
paraformaldehyde for 24 hours at 4°C, and 10% EDTA was used for decalcification. After
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decalcification for 2 months, the specimens were dehydrated, embedded in paraffin, and then cut into slices in the sagittal plane. The sections were stained with hematoxylin and eosin (H&E) stain.
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Polymerase Chain Reaction
After the rats were euthanized, the calluses were immersed in RNA store reagent (Com Win Biotech, Beijing, China) immediately and frozen with liquid nitrogen until mRNA analysis by quantitative real-time polymerase chain reaction (PCR). Total RNA was extracted from each callus using
Trizol
reagent
(Invitrogen,
USA)
following
manufacturer's
instructions
and
then
reverse-transcribed to cDNA using ReverTra Ace qPCR RT Kit (Toyobo Co. Ltd., Osaka, Japan).
β-actin
Reverse
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Primer sequences used were as follows: β-actin Forward 5'-AGA TCC TGA CCG AGC GTG GC-3', 5'-CCA
GGG
AGG
AAG
AGG
ATG
CG-3,
VEGF:
Forward
GAGCCTTGCCTTGCTGCTCTAC, reverse CACCAGGGTCTCGATTGGATG, VEGFR-2 Forward
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5' TGA CAC GGA AAC TGA AGA CC 3', Reverse 5' TTG GAG TTT CAG AGG CAG GT 3'. RT-PCR was performed using Recombinant Taq DNA Polymerase TaKaRa Taq™ (Takara Bio Inc., Shiga, Japan) according to manufacturer's instructions. QRT-PCR was performed using SYBR® Green
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Real-time PCR Master Mix (Toyobo Co. Ltd., Osaka, Japan). β-Actin served as a normalizer for correction. Relative mRNA levels of VEGF and VEGFR-2 were expressed as fold changes normalized to β-actin and were analyzed according to a comparative method with the equation expressed as 2−∆∆Ct. Immunohistochemistry (IHC) Calluses were fixed in 4% neutral buffered formalin for at least 24 hours and decalcified with 10% EDTA for at least 3 months; they were embedded in paraffin after decalcification. Before the immunohistochemistry analysis, the slides were deparaffinized at 56°C for 30 minutes and then bathed in alcohol for 5 minutes. Endogenous peroxidase activity was inactivated by 3% H2O2 treatment for 10
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minutes, and 5% bovine serum albumin (BSA) blocking solution was used for antigen blocking. Then, all the slides were incubated with rabbit anti-rat VEGFR-2 antibody (Abcam, CA, UK) in 1:200 dilution at 4°C overnight. After thawing to 37°C for 30 minutes with 1:150 diluted biotin-coupled secondary antibody (Boster) and streptavidin-biotin complex (SABC), the color reaction was
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developed with a 3,3’-diaminobenzidine-tetrachloride kit (DAB kit; Boster). Sections were washed and
counterstained with hematoxylin and observed with an Olympus BX51 microscope (Olympus Optical, Tokyo, Japan). Sections for VEGFR-2 density counting were stained with claybank and hematoxylin and imaged at 100× magnification with the microscope.
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Statistical Analysis
All values are expressed as mean ± SD. The Graphpad Prism version 5.0 (GraphPad Software, La
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Jolla, CA, USA) was used for statistical analyses, and intergroup variance was evaluated by one-way ANOVA; the comparison between the two groups was analyzed by Dunnett’s test. When data were not normally distributed, significant differences were identified by the non-parametric Kruskal-Wallis test. Differences with a P value of less than 0.05 were considered statistically significant. Results The general condition of the rats
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Thirty days after the bilateral ovaries were removed, the BMD was decreased, and the difference was statistically significant (P<0.05). There was no significant difference between the blood urea and the weight among the four groups (P>0.05, Figure 1 A). These findings indicate that there is no sign of
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toxicity for naringin between the doses of 40 mg/kg and 300 mg/kg (Figure 1 B). Naringin can promote angiogenesis of the callus To evaluate the angiogenesis of the callus, the results of Microfil and subsequent micro-CT scan
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were analyzed. At 2 weeks, 4 weeks, and 8 weeks after fracture, the number of vessels, vessel volume, and vessel thickness around the fracture sites in the naringin group is higher than in the control group, and the vessel spacing in the naringin group is lower than in the control group. However, there was a statistically significant difference between the high dose of naringin and the control group (P<0.05, Figure 2). Effects of different doses of naringin on the vascular area and vascular number at 2 weeks tested by HE
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The 2-week HE staining slices show that the high-dose naringin group (300 mg/kg) has the most significant effect on the vascular area, as well as the vascular number. When compared with the control group, the low-dose group, and the middle-dose group, the difference was statistically significant (P<0.05, Figure 3). Results also indicated that the vascular area and the vascular number in the
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low-dose and middle-dose naringin group are higher than in the control group. The differences were statistically significant (P<0.05, Figure 3).
Effects of different doses of naringin on the relative gene expression of VEGF and VEGFR-2 at 2, 4, and 8 weeks
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As illustrated in Figure 3, VEGF mRNA expression in the high-dose naringin group increased by 4.52-fold (P<0.05), 7.25-fold (P<0.001), and 6.58-fold (P<0.001), respectively, compared with the
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control group at 2 weeks, 4 weeks, and 8 weeks, respectively. In addition, VEGFR-2, the VEGFR-2 mRNA expression in the high-dose naringin group, increased by 4.14-fold (P<0.05), 7.12-fold (P<0.001), and 5.23-fold (P<0.001), respectively, compared with the control group at 2 weeks, 4 weeks, and 8 weeks, respectively (Figure 4).
Results of different doses of naringin for the protein expression of VEGFR-2 tested by IHC The results of the protein expression of VEGFR-2 can be seen in Figure 4. Compared with the
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control group, naringin in the high-dose group (300 mg/kg) can effectively promote the expression of VRGFR-2 in callus at 2 weeks, 4 weeks, and 8 weeks. Another result is that, from 2 weeks to 8 weeks, the VEGFR-2 expression in each group decreased compared with the corresponding group(Figure 5).
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Discussion
This research investigated for the first time the effect and mechanism of naringin in the treatment of osteoporotic fracture in rats from the perspective of angiogenesis. We found that the administration
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of naringin at clinically relevant times induced more neovascularization, and thus accelerated dose-dependent bone healing at 2, 4, and 8 weeks post-fracture. This was also shown with increased vessel numbers and larger vessel area shown by H&E staining. A high rate of vessel volume/total volume, an increased on
vascular number, increased vessel thickness, and less vessel space were shown
Microfil scan. In addition, we found that naringin can up-regulate VEGF and VEGFR-2 activities
in osteoporotic fracture callus tissue. These results indicate that naringin has a higher capacity for treating osteoporotic fracture through angiogenesis, and its specific mechanism may be through the regulation of the signaling pathway of VEGF/VEGFR-2.
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Naringin, a major ingredient of the Chinese herbal medicine Rhizoma Drynariae possesses many pharmacological effects, and has been shown to increase BMP-2 expression, induce bone formation, and inhibit bone resorption.16 Our findings are in accordance with the former finding that naringin-mediated VEGF-c, IFG-1, and TGF-b synthesis improved capillary density, enhanced diabetic foot ulcers in the rat model9.
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angiogenesis, and, thus, increased wound breaking strength in
Estrogen and nutritional deficiency, heredity, deficiencies, chronic diseases, and aging may contribute to osteoporosis and fracture. The main reason that osteoporosis is frequently found in elderly women is
the lack of estrogen and the decreased blood supply in this population. Burkhardt et al17 found that the
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number of blood capillaries and blood sinuses are reduced when osteoporosis occurs. Ding et al6 also found that the VEGF level, which plays a pivotal role in angiogenesis, will be markedly lower in the
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bone of ovariectomized rats. Therefore, increasing the level of VEGF may promote angiogenesis, increasing bone mass and treating osteoporosis. This study first demonstrated that naringin has an effect in promoting osteoporotic fracture healing through increasing the VEGF level by interacting with its receptor VEGFR-2.
Because there are many complications associated with estrogen that surpass its clinical outcome, research into naringin is a positive step to finding a new treatment for osteoporosis. BMP-2 has been
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identified to possess indirect angiogenic effects18. As shown in the present study, naringin can promote the increase in the amounts of VEGF and VEGFR-2. Microfil results show that naringin can increase the vessel number, vessel volume/total volume, vessel separation factor, vessel thickness, and decrease the vessel space. Local vasculature rupture at the end of the fracture results in the formation of a
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hematoma, and therefore, a relatively regional hypoxic microenvironment will be formed19. HIF-1α is an important factor that regulates cellular responses to hypoxia20. Meanwhile, Komatsu et al21 and
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Maes et al22 have identified that HIF-1α is expressed in the bone-healing process, indicating that activation of the HIF-signaling pathway to induce VEGF-mediated response can accelerate bone healing. The VEGF/VEGFR-2 signaling pathway is also important for angiogenesis and fracture healing, and there are numerous studies that have reported that inhibited VEGF activity through VEGF antagonist and a soluble VEGFR resulted in impaired healing of femoral fracture and cortical bone defects in mice, whereas local administration of VEGF leads to improved bone repair in both models23. Naringin can bind to the estrogen receptor and promote the expression of VEGF, the critical factor that promotes angiogenesis, and thus improves proliferation and adhesion in the matrix.
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Osteoporosis and osteoporotic-related fractures have been a major cause of morbidity and mortality in the elderly population. Thus, the economic burden of patients with osteoporosis is increasing24. Johnell et al25 have reported that the number of osteoporotic fractures is as high as 9 million. It is well known that a timely and coordinated angionetic response is of vital importance for
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successful bone healing26. Neovascularization at the fracture site requires not only angiogenesis but
also circulating EPCs during vasculogenesis. Circulating EPCs are part of osteogenesis27. Rong28 reported that naringin can promote the expression of VEGF and brain-derived neurotrophic factor (BDNF) after spinal cord injury.
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Conclusion
To the best of our knowledge, this is the first article on the use of naringin for the treatment of
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osteoporotic fracture from the prospective of vascularization. The main outcome is that naringin can promote the vascularization of the callus in the fracture, by promoting the expression of VEGF and VEGFR-2. Acknowledgments
This study was funded by the National Natural Science Foundation of China (No.81401792 and
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Figure 1 A. The bone mineral density before the ovary was removed and 30 days after the ovary was
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removed; B, the blood urea and the weight among the four groups at 8 weeks.
Figure 2 1. Morphological analysis of vasculature within the tibia from Microfil-perfused analysis of different naringin groups and the control group rats at 2 weeks after fracture. 2. Morphological analysis of vasculature within the tibia from Microfil-perfused analysis of different naringin groups and the control group rats at 4 weeks after fracture. 3. Morphological analysis of vasculature within the tibia from Microfil-perfused analysis of different naringin groups and the control group rats at 2 weeks after
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fracture. A: Control group, B: Low dose: 40 mg/kg, C: Middle dose: 100 mg/kg, D: High dose: 300 mg/kg. Vessel number, vessel spacing, vessel separation factor, vessel thickness, and vessel volume/total volume are compared among different groups.
* P was a significant difference (<0.05),
compared with control group.
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Figure 3 Hematoxylin and eosin staining for the callus of different groups; red circles are the new vessels in the callus. Low dose: 40 mg/kg, Middle dose: 100 mg/kg, High dose: 300 mg/kg. Compared
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with control group, * P was a significant difference (<0.05). Red arrows: the blood vessels in the fracture site.
Figure 4 Effects of naringin on VEGF mRNA expression in each group at 2 weeks (Figure 2 A), 4 weeks (Figure 2 B), and 8 weeks (Figure 2 C) and VEGFR-2 mRNA in each group at 2 weeks (Figure 2 D), 4 weeks (Figure 2 E), and 8 weeks (Figure 2 F) analyzed by QRT-PCR, respectively. A p < 0.05, vs. control group; n = 6. VEGF: gene of vessel endothelial growth factor; VEGFR-2: gene of vessel endothelial growth factor receptor-2. Figure 5 VEGFR-2-positive osteocytes distributed in fracture sites in each group indicated by immunohistochemical staining. All of the panel magnification ×100. A, B, C, D represent the control
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group, low dose, middle dose, and high dose at 2 weeks, respectively. E, F, G, H represent the control group, low dose, middle dose, and high dose at 4 weeks, respectively. I, J, K, L represent the control group, low dose, middle dose, and high dose at 8 weeks, respectively. Red arrows: the
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VEGFR-2-positive cells.
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Naringin promote angiogenesis of fracture healing in osteoporotic rats. Naringin exerts its angiogenetic role via stimulating VEGF/VEGFR-2 signaling.
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High dose naringin with 300 mg/kg is better than the low dose and middle dose.