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Year : 2018  |  Volume : 14  |  Issue : 12  |  Page : 1098-1104

Evaluation of the effect of hesperidin on vascular endothelial growth factor gene expression in rat skin animal models following cobalt-60 gamma irradiation

1 Department of Radiology and Radiobiology, Faculty of Paramedicine, Shiraz University of Medical Sciences, Shiraz, Iran
2 Department of Radiotherapy, Physics Unit, Namazi Hospital, Shiraz University of Medical Sciences, Shiraz, Iran
3 Laboratory Sciences Research Center, Faculty of Paramedicine, Shiraz University of Medical Sciences, Shiraz, Iran

Date of Web Publication11-Dec-2018

Correspondence Address:
Akbar Abbaszadeh
Department of Radiology and Radiobiology, Faculty of Paramedicine, Shiraz University of Medical Sciences, Shiraz
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0973-1482.202892

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 > Abstract 

Introduction: Skin is highly prone to radiation damage. Radiation burn is defined as damage to the skin or other biological tissues induced by radiofrequency or ionizing radiation. Vascular endothelial growth factor (VEGF) is a heparin-binded pro-angiogenic factor. Flavonoids belong to a family of polyphenol chemical compounds that are frequently present in fruits and vegetables. Hesperidin is an agent belonging to the flavonoid family. The aim of this study is to investigate whether hesperidin can affect the VEGF gene expression in rat skin following gamma irradiation or not.
Materials and Methods: A total number of 36 male Sprague-Dawley rats were divided into three groups. First group: radiation group (n = 12), second group: radiation + hesperidin-treated group (n = 12), and third group: untreated control group (n = 12). The hesperidin administration dose was 100 mg/kg body weight. The rats received a 22 Gy single dose at a dose rate of about 0.3 Gy/min using a cobalt-60 external beam radiotherapy unit. The animals were euthanized 24 h postirradiation. VEGF gene expression data were analyzed using the equation 2–ΔΔCT, where ΔΔCT = (Threshold cycle [CT], of target gene – CT of housekeeping gene)treated group– (CT of target gene – CT of housekeeping gene)untreated control group. Glyceraldehyde-3-phosphate dehydrogenase gene was used as a housekeeping gene.
Results: VEGF gene in the radiation + hesperidin group overexpressed 25-fold relative to the control group. In addition, VEGF gene in the radiation group underexpressed 0.15-fold relative to the control group. When the three groups were compared relative to each other using the Kruskal–Wallis test, P < 0.001 was obtained. Based on the Mann–Whitney U-test, when all groups were compared to each in a binary model, P = 0.001 was achieved. These tests all showed statistically significant changes in VEGF gene expression.
Conclusions: We can conclude that hesperidin is a potent angiogenic factor. Hesperidin as a radioprotector can initiate angiogenesis by VEGF gene induction. It may stimulate epithelialization, collagen deposition, and enhanced cellular proliferation. These changes can together accelerate wound healing, in particular, radiation-induced skin damage.

Keywords: Gamma irradiation, hesperidin, rat skin, skin burn, vascular endothelial growth factor, wound healing

How to cite this article:
Haddadi G, Abbaszadeh A, Mosleh-Shirazi MA, Okhovat MA, Salajeghe A, Ghorbani Z. Evaluation of the effect of hesperidin on vascular endothelial growth factor gene expression in rat skin animal models following cobalt-60 gamma irradiation. J Can Res Ther 2018;14, Suppl S5:1098-104

How to cite this URL:
Haddadi G, Abbaszadeh A, Mosleh-Shirazi MA, Okhovat MA, Salajeghe A, Ghorbani Z. Evaluation of the effect of hesperidin on vascular endothelial growth factor gene expression in rat skin animal models following cobalt-60 gamma irradiation. J Can Res Ther [serial online] 2018 [cited 2020 May 27];14:1098-104. Available from: http://www.cancerjournal.net/text.asp?2018/14/12/1098/202892

 > Introduction Top

Skin is composed of two different layers including epidermis and dermis.[1] It is very prone to the radiation damage.[2],[3] The reason behind this great susceptibility is continuous self-renewal, fast growth, and reproduction capabilities of the skin.[3]

Radiotherapy is widely used for the treatment of many malignant tumors. It is estimated that over half of cancer patients receive radiotherapy as a part of their treatments.[4] Although lots of progresses have been made in the treatment planning and dose-delivering techniques, considerable radiotoxicity to the normal organs still remains as a serious problem.[5] Radiation burn is defined as damage to the skin or other biological tissues induced by radiofrequency waves or ionizing radiation. Radiation burn can be caused by high-dose X-rays delivered during repeated diagnostic imaging techniques, interventional radiology, and radiotherapy.[6] Despite all the care that is exercised, unintentional radiation damages could happen due to the mechanical problems, electrical instability, and human errors.[6]

Based on published papers, 90%–95% of patients who received radiotherapy showed different grades of radiation-induced skin reactions.[3],[7] According to the majority of articles, radiotherapy is identified as the main reason for radiation burn.[6],[7],[8]

Skin reactions to the different sources of ionizing and nonionizing radiation can be categorized as a wide range from erythema and dry desquamation to the moist desquamation and in more severe cases, ulcers.[1],[2],[8]

Angiogenesis is defined as creation of new vessels from preexisting blood vessels. In the process of angiogenesis, high blood vessel permeability, extracellular matrix molecules degradation, and endothelial cells migration may happen.[9],[10],[11]

Vascular endothelial growth factor (VEGF) is a glycoprotein homodimeric.[11],[12],[13] Its molecular weight is 36–46 kDa. It is a heparin-binded angiogenic growth factor which is specific to endothelial cells.[14] Hypoxia is a great stimulator of its induction. However, in many tumors, there is a probability for VEGF gene overexpression in the cells which are located marginally to the tumor origin, even in the presence of oxygen. In this case, VEGF production by tumoral cells is following a hypoxia-independent pathway. In this path, p53 as a tumor suppressor gene, extrinsic factors such as different hormones and growth factors are inactivated.[14]

Group of agents which are administered before irradiation and reduce radiation-induced damages to different organs are called radioprotectors.[15] These compounds can be applied before the irradiation of cancer patients and radiation workers as well as members of the public in the case of a radiation accident.[16] Since 1949 mercaptoethylamine, cistamine, and WR2721 have been reported as the most effective radioprotectors. These drugs had some side effects including drowsiness, hypotension, nausea, vomiting, and toxicity that limited their clinical use in practice. Hence, many attempts have been made to find new radioprotectors with low levels of toxicity.[17]

Because epidemiological studies have shown that fruit and vegetable consumption can prevent cancer, flavonoid compounds have gained great interest among researchers.[18] Flavonoids belong to a family of polyphenol chemical compounds which are frequently present in fruits and vegetables. Flavonoids have widespread biological features such as antibacterial, antivirus, antitumor, antioxidative, immune functions, and angiogenic effects.[19] Hesperidin belongs to the flavonoid family. This compound is ample in fruits. Hesperidin molecular formula is C28H34O15. Its molecular weight is about 57.61 kDa.[20] Surface layer and membrane parts of fruits have the highest hesperidin concentrations. It has been reported that hesperidin has a wide range of pharmaceutical properties such as antioxidant, anti-inflammation, antitumor, antisensitivity, and hypolipidemic effects.[21],[22]

The aim of this study is to investigate whether hesperidin is effect on the VEGF gene expression in the rat skin animal model following gamma irradiation.

 > Materials and Methods Top


A total number of 36, 8–10-week-old male Sprague-Dawley rats of approximately 250–300 g in weight were supplied by the Shiraz University of Medical Sciences animal laboratory and were kept according to the guidelines for care and use of laboratory animals as adopted by the University's Ethics Committee. The animals were housed on a 12 h/12 h light-dark cycle with food and water provided ad libitum. The rats were housed in temperature and humidity controlled conditions. Before the irradiation of the buttocks skin, the hair was shaved using electric clippers. The rats were divided into three groups. The first group received no hesperidin but underwent irradiation, the second (radiation + hesperidin) group received oral hesperidin 30 min before irradiation, and the third group was an untreated control group that received neither hesperidin nor radiation. There were 12 rats in each group.


Hesperidin was dissolved and diluted in 1 ml phosphate-buffered saline (PBS). The administration dose of hesperidin was 100 mg/kg body weight according to the previous studies.[23] Hesperidin was administered orally 30 min before irradiation. In the untreated control group, PBS was prepared at the same volume with hesperidin + PBS and administered orally. The rats were anesthetized by intraperitoneal injection of 300 microliter ketamine/xylazine (90/10 mg/kg body weight) mixture.

Radiation treatment and tissue sample preparation

Rat skin irradiation was performed using a cobalt-60 external beam radiotherapy unit. Eight rats at a time were given general anesthesia, and each group of four rats was placed side by side in a row next to each. The other four were positioned in the same way but facing the opposite way so that their buttocks were in contact with the first row of four rats, and therefore, the buttocks regions of all eight could be irradiated simultaneously using a long and narrow (10 cm × 35 cm) field while minimizing the irradiation of other organs. They received a 22 Gy single dose (localized to their buttocks region) at a dose rate of approximately 0.3 Gy/min according to the previous studies.[24] A 0.5 cm thick perspex plate placed near their skin was used to act as a “beam spoiler” and counteract the dose build-up effect at the surface, thereby increasing the skin dose to the stated value. The previous dosimetric measurements had been performed using a farmer ionization chamber (PTW Freiburg, Germany) to assess the dose nonuniformity of the off-axis regions of this radiation field with respect to the on-axis. As a result, the positions of the outer rats were interchanged with the inner ones halfway through irradiation to ensure that all of them received the same dose.

After the irradiation and the drug treatment, the animals were returned to their home cages and euthanized by rapid cervical dislocation 24 h postirradiation. Skin samples were rapidly removed and immediately frozen in liquid nitrogen. Then, the skin flap samples in 2 ml microtubes were transfered to a-80°C freezer until further analysis.

Quantitative real-time polymerase chain reaction

Rat skin samples were homogenized with 1 ml trizol reagent (Sinaclon, Bioscience) in a rotor-stator tissue homogenizer. Then, total RNA was isolated from tissue homogenates according to the manufacturer's protocol. One microgram of total RNA was reverse-transcribed at 25°C for 5 min, 42°C for 60 min, and 70°C for 5 min. The materials used in the mixture included 4 μL reaction buffer, 2 μL deoxynucleotide triphosphate, 1 μL oligo-dT, 1 μL random hexamer, 1 μL riboLock, 1 μL revert aid enzyme, and 10 μL DNase-added RNA suspension.

The primers used for the detection of VEGF messenger RNA (mRNA) levels were forward 5'-CACCACCACACCACCATC-3', Reverse 5'-GCGAATCCAGTTCCACGAG-3', yielding a product of 190 bp. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were forward5'-GCAAGTTCAACGGCACAGTCAAGG-3', reverse 5'-CCACGACATACTCAGCACCAGCAT-3', yielding a product of 120 bp. GAPDH were used as a control to normalize gene expression.

Amplification of individual genes was performed on a BioSystem 7300 real-time polymerase chain reaction (PCR) unit. For quantification process, real Q plus 2x Master Mix Green without ROX, Ampliqon Denmark, and a standard thermal cycler protocol (95°C, 15 min, one cycle and 95°C: 15 s, 60°C: 30 s, 72°C: 30 s, forty cycles each) were used. All PCR reactions were performed in 20 μL total reaction volume.

The threshold cycle (CT) CT, which indicates the fractional cycle number at which the amount of amplified target gene reaches a fixed threshold, was determined for each well using the applied BioSystem Sequence Detection Software v1.2.3 (Thermo fisher scientific corporation, Waltham, Massachusetts, USA). The data were analyzed using the equation 2−ΔΔCT, where ΔΔ CT = (CT of target gene – CT of housekeeping gene)treatedgroup− (CT of target gene − CT of housekeeping gene)untreatedcontrolgroup. For the treated samples, 2−ΔΔCT indicates the relative changes in gene expression normalized to a housekeeping gene, compared to the untreated control.[25]

Statistical analysis

Statistical analysis of data was performed using SPSS V20.0 software (IBM corporation, Armonk, New York, USA). The Kruskal–Wallis test was used to compare mean responses among the treatments. For relative comparisons between each two groups, the Mann–Whitney U-test as a nonparametric test was used. The results were presented as mean ± standard deviation (SD). P < 0.05 was considered statistically significant.

 > Results Top

In this study, PCR efficiency was calculated and found to be 100%. We placed CT values of VEGF gene as a main gene and GAPDH gene as a housekeeping gene in the related formula. CT numbers and melt curves for different groups was provided in [Figure 1] and [Figure 2]. According to this formula, it was determined that VEGF gene in radiation + hesperidin group (where hesperidin was administered 30 min before the irradiation) overexpressed 25-fold relative to the control group. In addition, VEGF gene in radiation group (in which rats received 22 Gy single dose gamma rays) underexpressed 0.15-fold relative to the control group. Fold induction in different groups were shown in [Figure 3]. Mean ± SD values of 0.15 ± 0.018 in the radiation group, 25.81 ± 1.14 in the radiation + hesperidin group, and 1 ± 0.15 in the control group were obtained. Mean ± SD values for different groups were shown in [Figure 4]. When the three groups were compared relative to each other using the Kruskal–Wallis test, P < 0.001 was obtained. Based on the Mann–Whitney nonparametric test, when different groups were compared in a binary model, P = 0.001 was reported. These tests all showed statistically significant changes in VEGF gene expression.
Figure 1: Threshold cycle number in different groups

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Figure 2: Melt curve in different groups

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Figure 3: Fold induction in different groups[25]

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Figure 4: Mean ± standard deviation in different groups

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 > Discussion Top

According to the results of this study, radiation attenuated VEGF gene expression level 0.15-fold relative to the control group. This is a considerably significant change in VEGF gene expression level relative to the control and in line with some other experiments, which will be described as follows. Polytarchou et al. studied X-ray effects on the expression of some genes involved in the process of angiogenesis in chorioallantoic membrane of chicken embryos. In this experiment, a 10 Gy dose was administered. Results showed that VEGF 165, 190 PCR levels decreased significantly 6 h postirradiation.[26] Gille et al. studied VEGF gene expression level postultraviolet (UVA) irradiation in HaCaT keratinocyte cell lines. VEGF gene level underexpressed significantly 16 h and 24 h postirradiation.[27] Yano et al. studied the effect of thrombospondin 1 overexpression on the epidermis layer of transgenic mice in the prevention of angiogenesis and UVB-induced skin photodamage. In situ hybridization determined that VEGF gene expression level in the dermis layer was significantly lower than the control group.[28] Lee et al. studied the effects of gamma rays on VEGF, ang1, ang2, tie2 genes expression levels. VEGF gene level underexpressed significantly 8 h and 24 h postirradiation. VEGF protein levels also decreased significantly 4, 8, and 24 h postirradiation relative to the control group in rat brain tissue.[29] Zorkina et al. exposed whole brain of albino rats with a total dose of 36 Gy according to a fractionated regimen using a linear accelerator. Results showed that expression of genes of neurotrophic factors such as brain-derived neurotrophic factor, VEGF was decreased at 4th and 8th weeks following irradiation.[30] Joki et al. found that mRNA levels of growth factors such as VEGF and platelet-derived growth factor receptor B were decreased in postradiation recurrent tumors, as compared with primary tumors, in 75% of patients with glioblastoma who were treated by 60 Gy fractionated radiotherapy using a conventional X-ray linear accelerator. Microvessel counts also demonstrated that blood vessel growth was decreased significantly in postradiation recurrent tumor specimens.[31]

On the other hand, the findings of our experiments disagree with some of the published reports, too.[9],[10],[13],[24],[32],[33] Haddadi et al. reported that VEGF gene levels overexpressed 3-fold; 8 weeks, 4-fold; 20 weeks, and 14-fold; 22 weeks postgamma irradiation in rat spinal cord tissue.[24] In addition, Ando et al. showed that VEGF gene levels overexpressed significantly 16 and 24 h following 15 Gy X-ray dose in squamous carcinoma cell line.[32]

It should be emphasized that, when it comes to interpreting VEGF gene underexpression following gamma-irradiation, different experimental conditions such as timing (animals irradiation and scarification time), tissue portions irradiated and the radiation dose delivered, should be taken into the account. These conditions can considerably change results of the study.

We found that VEGF gene in radiation + hesperidin group overexpressed 25-fold relative to the control group. Based on our knowledge, we assume that hesperidin-mediated VEGF gene induction in rats could be attributed to the hypoxia-inducible factor-1 alpha (HIF1α) overexpression or an increase in the level of some cytokines such as tumor necrosis factor-alpha, platelet-derived growth factor, epidermal growth factor, and basic fibroblast growth factor. Mechanisms by which hesperidin can affect HIF1α or prementioned cytokines mRNA or protein levels still remain unclear and requires further studies.

Recently, it has been shown that hesperidin has some radioprotective properties.[23],[34] There is a large body of evidence which indicates the role of angiogenesis in the process of wound healing (specially skin damage) induced by different stimulators.

During wound healing, angiogenic capillary sprouts invade the places which have high concentration of fibrin/fibronectin flexible filaments and within a few days, converts into a microvascular network through the granulation of tissues.[35],[36] Experimental data have shown that VEGF may stimulate epithelialization and collagen deposition in a wound. In addition, VEGF stimulates wound healing through angiogenesis.[36] With regard to the angiogenesis clear mechanisms, therapeutic options can be accessible to improve disorders which are still the main causes of mortality and morbidity, including cardiovascular diseases, cancer, chronic inflammatory disorders, diabetic retinopathy, excessive tissue defects, and chronic nonhealing wounds. Restoring blood flow to the injured tissue is necessary for a successful repair response to happen. In particular, repair of skin defects is a highly suitable model for analyzing angiogenesis, because it can be easily reached to control and manipulate this process.[37]

Galiano et al. showed significantly accelerated repair in VEGF-treated wounds in genetically diabetic mice. In VEGF-treated wounds, increased epithelialization, matrix deposition, and enhanced cellular proliferation were detectable. They concluded that topical VEGF can repair wounds by locally upregulating growth factors, systemically exciting bone marrow-derived cells, and applying these cells to the damaged area.[38] Brown et al. showed evidences to support that VEGF may be responsible for the hypermeable state and angiogenesis, which are characteristics of the wound repair process. Hyperpermeable blood vessels were reported to be effective in healing guinea pig and rat biopsy skin wounds. These data revealed that vascular permeability factor is an important cytokine in wound healing.[39] Wu et al. showed that administration of bone marrow-derived mesenchymal stem cells (BM-MSCs) around the wound significantly reinforces wound healing in diabetic mice relative to the controls. BM-MSC treated wounds exhibited significantly accelerated wound closure, with increased reepithelialization, cellularity, and angiogenesis. Real-time PCR and Western blot analysis revealed that VEGF and angiopoietin-1 were overexpressed in BM-MSCs treated wounds.[40]

 > Conclusions Top

We can conclude that the hesperidin is a potent angiogenic factor. In addition, angiogenesis is an important element in the process of wound healing. Hence, in the case of radiation-induced skin burns, hesperidin as a radioprotector can initiate formation of new vessels and a microvascular network by VEGF gene induction. VEGF may stimulate epithelialization, collagen deposition, and enhanced cellular proliferation. These changes can together accelerate wound healing, in particular, radiation-induced skin damage.


We would like to thanks, Ms. Dehbozorgi from Radiotherapy Department, Namazi Hospital who was in charge for animals' irradiation. We really appreciate all the help from the Laboratory Sciences Research Center and the animal laboratory of Shiraz University of Medical Sciences.

Financial support and sponsorship

This study was funded by grant number 8643 from the Vice-Chancellor of Research at Shiraz University of Medical Sciences.

Conflicts of interest

There are no conflicts of interest.

 > References Top

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