This study aims to explore the radioprotective effects of recombinant human erythropoietin (rhEPO) on rats' submandibular gland hypofunction induced by irradiation (IR).
Materials and Methods
Thirty rats were divided into 3 groups: 1) control group, 2) IR group, and 3) IR + rhEPO group. The IR group and IR + rhEPO group received a single dose of 15 Grays (Gy) (0.98 Gy/min), plus, the IR + rhEPO group also received subcutaneous administration of rhEPO at a dose of 3,000 IU/kg body weight 3 days before irradiation and then repeated every 24 hours for the first 2 weeks after irradiation. Immunohistochemistry analysis to erythropoietin receptor was performed to detect the levels of erythropoietin receptor in submandibular glands with or without radiation. Ninety days after irradiation, the salivary flow rates were assessed, and the submandibular gland of every rat was subjected to hematoxylin and eosin staining and immunohistochemical staining with antiaquaporin 5 and anti–proliferating cell nuclear antigen antibodies. Apoptosis was examined by the terminal deoxynucleotidyl transferase biotin-dUDP nick end-labeling assay. In addition, to examine the protective role of rhEPO on human submandibular gland cells, the apoptotic and proliferation rate of cells under a radiation dose of 8 Gy was detected. One-way analysis of variance was carried out to analyze the results of each group, and the P value was set at 0.05.
Erythropoietin receptor was expressed in the submandibular glands at a low level under normal conditions but upregulated after irradiation. rhEPO administration remarkably alleviated gland atrophy, increased salivary flow rates with upregulation of aquaporin-5 compared with the IR group. In addition, fewer apoptotic cells and more proliferative cells were observed in the IR + rhEPO group compared with the IR group, both in vivo and in vitro.
rhEPO administration may be a useful countermeasure to mitigate submandibular gland hypofunction after therapeutic radiation exposure.
Salivary gland hypofunction, characterized by a remarked diminishing of salivary flow, is 1 of the most common and deleterious side effects during and after head and neck cancer treatments. It has been reported that two-thirds of patients with oral cavity and pharyngeal cancer who had received radiotherapy would develop salivary gland hypofunction, , which severely worsens patients' life quality owing to its subsequent sequelae such as pain, mucositis, taste loss, dysphagia, and dental caries resulted from hyposalivation and xerostomia.
Conventional radiotherapy is given a total dose of about 70 Grays (Gy) in 6 to 7 weeks as a primary or adjuvant treatment of head and neck cancers. Although fluid-producing acinar cells of salivary glands are well-differentiated, they have unexpected high radiosensitivity and are prone to be collaterally destroyed when part or whole glands lie within the field of radiation. A dose-response relationship has also been elicited, with the loss of quantitative salivary output shown to be associated with its radiation dose exposure. , The submandibular glands are the major contributors to moistening of oral tissues because they produce most, approximately 60 to 70%, of the unstimulated saliva in healthy individuals. It is reported that a cumulative mean dose to submandibular glands exceeding 39 Gy could cause permanent ablation of both stimulated and unstimulated salivary flow. Another report states that the average dose of 46.4 Gy of radiation could lead to continuous increased shrinkage and deformation of submandibular glands for about 2 years. , Hence, submandibular glands often receive high radiation doses beyond the threshold of tissue recovery in patients during radiotherapy for head neck cancers.
Cell apoptosis of the acini and oxidative stress play vital roles in postirradiation damage of salivary glands. The irradiated glands are predisposed to varying degrees of salivary flow rate reduction owing to loss of saliva-producing acinar cells and depletion of stem cell. , , To minimize the adverse radioactive impacts on submandibular glands, scholars have been pursuing effective drugs either protecting these cells from radical-induced DNA damage or maintaining or stimulating cell proliferation. Erythropoietin (EPO) is 1 of the most potent free radical scavengers because EPO stimulates proliferation and antioxidative effects, and EPO and EPO receptor (EPOR) play an essential role in the balance of superoxide dismutase and reactive oxygen species, which is the key in the oxidative stress.
EPO is a hormone characterized by regulating red blood cell production. Circulating EPO, mainly produced by the kidney, plays a vital role in red blood cell mass maintenance. Based on the role of hematopoiesis, recombinant human erythropoietin (rhEPO) is clinically used for anemia. Under circumstances such as hypoxic or physical stress, many tissues also locally produce and release EPO and subsequently promotes healing in a paracrine or autocrine manner. EPO commences the biological response through and acts only in cells with EPOR, the latter is widely expressed in organs such as the brain, heart, and kidney. ,
However, no expression information about EPOR in submandibular glands has been reported in the literature. In our study, we found that EPOR expressed in the rat submandibular glands at a low level. Intriguingly, its expression increased dramatically after exposure to therapeutic irradiation. Thus, we hypothesize that EPO targets EPOR and protects the submandibular glands during and after irradiation. The aim of the present study was to explore the radioprotective effects of rhEPO on rats' submandibular gland hypofunction induced by irradiation.
Materials and Methods
Thirty male Wistar rats weighing from 180 to 220g were included in this experimental study. They were kept in cages under standard laboratory conditions with an alternating 12:12 light and dark cycle. The animals' protocol was approved by the Institutional Animal Care and Use Committee of Jilin University (Changchun, Jilin Province, China).
Human submandibular gland (HSG) cells (obtained from Dr. M. Sato, Tokushima University, Japan) were cultured in Dulbecco's modified Eagle’s medium (Gibco) with 5% fetal bovine serum (Gibco) and antibiotics at 37°C in the presence of 5% CO 2 .
Rats were divided into 3 groups: sham irradiation group (n = 9) (referred to as control group); irradiation group (n = 15) (referred to as IR group); irradiation + rhEPO group (n = 6) (referred to as IR + rhEPO group). Before irradiation, all rats were anesthetized by intraperitoneal injection of 0.3 ml/100 g chloral hydrate. Then, rats of the IR and IR + rhEPO groups were immobilized in a box shielded with 5-mm thickness of lead, only the head and neck regions were exposed. The rats were locally irradiated in the region of the head and neck with a single dose of 15 Gy (0.98 Gy/min), which could cause a sensible impairment to the glands, as confirmed in the previous studies. , Irradiation was carried out by a self-contained x-ray system (X-RAD 320; Precision X-Ray). The control group was anesthetized at the same time and placed the same way as the other groups but did not receive radiation.
rhEPO (3SBIO.INC, Shenyang, China) was diluted with saline to 1000IU/ml and stored at 4°C. The IR + rhEPO group received subcutaneous administration of rhEPO at a dose of 3,000 IU/kg body weight as per previous studies, , and the control group and IR group were injected with equivalent saline at the same time point. The treatment was started 3 days before irradiation and then repeated every 24 hours for the first 2 weeks after irradiation. The rhEPO administration method and rhythm followed that of our previous studies. All animals survived in this period.
Hematoxylin and Eosin Staining
As stated previously, after weighting, the submandibular glands were fixed in 4% cold paraformaldehyde in 0.1 mol/L phosphate buffer saline for 24 hours. Then, the submandibular glands were processed for paraffin embedding and sectioned at 4 μm thickness. Histopathologic structures of the glands were analyzed with hematoxylin and eosin staining.
To detect the expression levels of EPOR, proliferation marker, and functional marker of submandibular glands, the following antibodies were used: rabbit polyclonal antibody against EPOR (1:100, Novus), proliferating cell nuclear antigen (1:100; Novus) and aquaporin 5 (AQP-5) (1:150; Abcam, UK). During immunohistochemical staining, the antigens were retrieved by microwave, and the kit of polink-2 plus polymer horseradish peroxidase system (PV-9001) (ZSGB-BIO, China) was used for detection. Diaminobenzidine working solution was added for color development. The nucleus was counterstained with hematoxylin. The areas of positively stained zones were measured with ImageJ 1.52a (Wayne Rasband, National Institutes of Health).
Gland Weighing and Collection of Saliva
At day 90 after irradiation, the rats were injected with pilocarpine chloride (2 mg/kg) intraperitoneally to stimulate saliva secretion. Three minutes after injection, the whole saliva was collected by 10 small preweighed cotton balls placed under the tongue for 10 minutes. Methodologically, 1 cotton ball was soaked with saliva in the mouth for 1 minute, then the cotton ball was taken out and replaced by a new preweighed cotton ball, and so forth. Ten balls were weighed together for each rat.
After saliva collection, the rats were sacrificed, and the submandibular glands of each rat were dissected and weighed. The salivary flow rate was estimated in the way of Xmg/10 min, following Zhang et al. The gland weights of the 3 groups were statistically quantitated.
Terminal Deoxyribonucleotide Transferase-Mediated dUTP Nick End-Labeling Assay
A one-step terminal deoxyribonucleotide transferase-mediated dUTP nick end-labeling (TUNEL) apoptosis assay kit (Beyotime, China) was used to detect the cell apoptosis following the manufacturer's instructions. Nuclei were signed with 4′, 6-diamidino-2-phenylindole dihydrochloride (Beyotime, China). The percentage of TUNEL-positive cells was evaluated in 3 randomly selected fields in every slide and quantified.
To test the effects of rhEPO on HSG cells without irradiation, HSG cells were plated in 96 wells (100 μL/well) and treated with rhEPO (10IU/mL, 50IU/mL, 100 IU/mL) or saline for 3 days. After that, all the cells were transferred into standard medium, and the proliferation of all groups was detected by Cell Counting Kit-8 (Sino Biological, China) for 5 consecutive days. To investigate the protective role of rhEPO, the cells were plated in the same method mentioned previously and treated with a low dose of rhEPO (10IU/mL) for 3 days, and then, the cells were irradiated with a single dose of 8.0 Gy (1.0 Gy/min). After that, the cells were transferred into standard medium, and its proliferation was detected by Cell Counting Kit-8 for 3 consecutive days after irradiation.
Flow Cytometry Assay
The apoptotic rate of cells was detected by flow cytometry with the Annexin V FITC/PI (fluorescein isothiocyanate/propidium iodide) Apoptosis Detection Kit (7sea biotech, China). Cells were digested and centrifuged at 1,000 rpm for 5 minutes, then the supernatant was removed, and 195 μL Annexin V binding buffer was added. Then, 5 μL Annexin V–fluorescein isothiocyanate was added into every tube. After incubating for 15 minutes, 10 μL of propidium iodide was added.
The data are presented as the mean ± standard deviation. Statistical analysis was performed using 1-way analysis of variance. Values of P < .05 were considered statistically significant. All analyses were estimated using SPSS software, version 21.