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Introduction

A large proportion of patients with thoracic carcinomas receive thoracic radiotherapy (TRT) as a part of their treatment. A number of these patients exhibit radiation-induced toxicity, such as acute radiation-induced esophagitis (ARIE), which is the primary dose-limiting complication of radiotherapy. The incidence rate of ARIE in a clinical setting is relatively high and the common symptoms are non-specific, mainly comprising dysphagia, odynophagia and a substernal burning sensation (1). As demonstrated in Table I, the degrees of ARIE defined by the National Cancer Institute Common Toxicity Criteria (NCI-CTC) (2) are essentially based on the symptoms patients have experienced. Clinically, patients often complain of dysphagia, odynophagia and a substernal burning sensation 2–3 weeks after initiation of radiotherapy (2). The course of radiation treatment is delayed if complications are serious.

Table I Grade of acute radiation-induced esophagitis.

Table I

Grade of acute radiation-induced esophagitis.

Grade	Description
0	None
1	Mild dysphagia, but is able to eat regular diet
2	Dysphagia, requiring predominantly pureed, soft or liquid diet
3	Dysphagia, requiring feeding tube, i.v. hydration or hyperalimentation
4	Complete obstruction (cannot swallow saliva); ulceration with bleeding not induced by minor trauma, abrasion or perforation

Cell death and defluxion of the esophageal mucous epithelium in the radiation fields have been observed, along with bleeding or ulceration when the dose of radiation was sufficiently high (3). Inflammatory cell infiltration in the esophageal tissue is another prominent histopathological change that occurs in ARIE (3,4). These types of damage may evoke symptoms of pain, and substance P (SP) secretion may be increased, playing an important role in the inflammatory procedure. SP is an 11 amino acid polypeptide released by sensory nerves. The biological function of SP is induced by its binding to the receptor, neurokinin-1 (5), which is expressed on a limited number of cell types, including epithelial cells, fibroblasts and inflammatory cells (6). As a neurotransmitter and a neuromodulator, SP is associated with inflammatory processes and pain. It participates in the process of nociception by transmitting information regarding damage from the peripheral receptors to the central nervous system, thus resulting in the generation of symptoms of pain (7).

In the present study, we established the animal model of ARIE and demonstrated the therapeutic effect of the compound of white peony root oral liquid (cWPROL) on this model (8). We elucidated a molecular mechanism via which the symptoms of pain are relieved; cWPROL is able to relieve symptoms of pain in the ARIE animal model by decreasing the expression level of SP in esophageal tissues.

Materials and methods

Subjects

Adult male Wistar rats with an average weight of 180–220 g were used. Animals were housed with a 12-hour light/dark cycle, and were given access to food and water ad libitum. All experimental animal techniques and handling procedures were approved by the Institutional Animal Care and Use Committee of Hebei Medical University. The certification number of the animals was DK0512053.

Grouping and irradiation

Twenty-four subjects were randomly divided into four groups (n=6), including the cWPROL treatment (CT) group; the mixture of lidocaine, dexamethasone and gentamycin (mLDG) treatment (MT) group; the radiation (R) group; and the non-intervention (NI) group. In the CT group, rats were administered 0.475 g/ml cWPROL by intra-esophageal perfusion following irradiation; while in the MT group, rats were treated with mLDG, which was also administered following irradiation. By contrast, in the R group, rats were not administered any treatment following irradiation; while in the NI group, rats did not receive irradiation or treatment. Animals receiving treatment were administered 2 ml of the agent, 3 times a day, with treatment initiated on the seventh day following irradiation and continuing for 7 days. Rats were deprived of food and water for 30 min following administration.

Conscious rats received a single dose of gamma irradiation (total dose, 43 Gy) to the chest. The irradiation procedure was performed with a 60Co therapy apparatus (Xinhua Factory, Shandong, China) at a dose rate of 0.111 Gy/min. The irradiation field was 3×30 cm, with a center dose point on the back of the rats located 1 cm beneath the body surface, and an irradiation range of 3 cm on the upper esophagus; the remaining parts of the body were covered. Rats in the NI group were not irradiated, but were otherwise treated as irradiated rats.

Detection of the general condition of the rats

Before and after the experiment, rat body weights in each group were recorded, in addition to the daily weight of food and volume of water. These data were regarded as indirect evidence reflecting symptoms of pain.

Immunohistochemical staining

Rats were anesthetized with 2% pentobarbital sodium administered by intraperitoneal injection (45 mg/kg). Esophageal samples were fixed with 4% paraformaldehyde for 24 h, embedded in paraffin and sectioned at 4 μm. Tissue sections were deparaffinized with xylene, rehydrated through an ethanol series and with TBST and immersed in 3% formaldehyde hydrogen peroxide liquid to block endogenous peroxidase. Antigen retrieval was performed by microwave treatment in the presence of antigen retrieval solution. The sections were incubated with the primary antibody at 4°C overnight, and then treated with biotin-labeled secondary antibody (Zhongshan Jinqiao Bio-technology Limited Company, Beijing, China) at 37°C for 20 min. This was followed by the addition of a peroxidase-conjugated streptavidin-labeled tertiary antibody (Zhongshan Jinqiao Bio-technology Limited Company) at 37°C for 20 min. We used antibodies against SP (1:50) as the primary antibodies (Boshide Program Limited Company, Wuhan, China). The sections were counterstained with hematoxylin, dehydrated, transparentized and then sealed with neutral gum. The blank control and replacing control were treated with PBS and normal rabbit serum, respectively. The appearance of brown particles in the stained parts was regarded as positive. Five successive visual fields centered on the lesion area of each section were observed under a microscope (x400) and the average of their integral optical density (IOD) was regarded as the representative value.

Statistical analysis

Experimental results were analyzed for their statistical significance using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). Groups were compared by a one-way ANOVA. The Student-Newman-Keuls test was utilized when the variance was equal, while the Kruskal-Wallis H test was used if the variance was unequal. Results are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

Results

The effect of treatment on rat body weight

As demonstrated in Fig. 1, there was no significant difference in body weight between groups prior to the experiment (P>0.05). Following the experiment, the body weight of rats in the NI group significantly increased (P<0.05); while that of the remaining experimental groups significantly decreased (P<0.05), particularly in the MT group (P<0.01). Additionally, compared with the R group, there was no significant difference in the body weight of rats in the CT group, while the body weight significantly decreased in the MT group (P<0.05). Furthermore, the body weight of rats in the MT group was significantly lower than that of the CT group (P<0.05).

Figure 1 Change in body weight in each group. Nig, non-intervention group; rg, radiation group; ctg, the compound of white peony root oral liquid (cWPROL) treatment group; mtg, the mixture of lidocaine, dexamethasone and gentamycin (mLDG) treatment group.

Changes in the weight of food and volume of water following treatment

We detected the daily changes in the weight of food and volume of water, and these were assumed to indirectly reflect the symptoms of pain. As demonstrated in Fig. 2, the weight of food and volume of water remained stable in the NI group, while those of the remaining experimental groups gradually decreased and reached the lowest point on the seventh day following irradiation, before slowly increasing. We found that following administration of cWPROL or mLDG, the volume of water was increased to a certain extent compared with that of the R group. Notably, compared with the R group, the weight of food rats consumed in the MT group did not increase, thus demonstrating that the symptoms of pain in this group were not effectively reduced.

Figure 2 Changes in weight of food and volume of water following treatment administration. Nig, non-intervention group; rg, radiation group; ctg, the compound of white peony root oral liquid (cWPROL) treatment group; mtg, the mixture of lidocaine, dexamethasone and gentamycin (mLDG) treatment group.

The level of SP expression in each group

We observed the variation in the expression levels of SP in the esophageal tissues by immunohistochemistry, before and after irradiation and treatment administration (Fig. 3). It was revealed that the expression level of SP was increased following exposure to radiation; in the R group, the expression of SP was significantly increased compared with the NI group (P<0.01). A low level of SP expression was observed in normal rat esophageal tissues, and was mainly distributed in the mucosal epithelium and nerve fiber peripheries (Fig. 3A). Following irradiation in the R group, marked SP expression was detected in the epithelium of the area surrounding ulcers, the fibrocytes and fibroblasts of the lamina propria and the submucosa in the inflamed area (Fig. 3B). Following the administration of cWPROL, the expression of SP was lower than that of the R group (P<0.01), but higher than that of the NI group (P<0.05); while the location of SP had no clear variance (Fig. 3C). The result of the MT group was as predicted; the expression level of SP in the MT group was significantly different compared with that of the NI (P<0.01) and R (P<0.05) groups (Fig. 3D). With regard to the comparison between the CT and MT groups, although the difference in SP expression levels was not significantly different, the effect of reducing the expression level of SP by administration of cWPROL was greater than that of mLDG (P>0.05).

Figure 3 Expression level of substance P (SP) in each group (x400, immunohistochemistry). (A) Normal rat esophageal tissue in nig; (B) rat esophageal tissue in rg; (C) rat esophageal tissue in ctg; (D) rat esophageal tissue in mtg. Nig, non-intervention group; rg, radiation group; ctg, the compound of white peony root oral liquid (cWPROL) treatment group; mtg, the mixture of lidocaine, dexamethasone and gentamycin (mLDG) treatment group.

Discussion

SP is a member of the tachykinin family that is secreted by neurons and other types of cells. SP is also detected in endothelial cells, inflammatory cells, fibroblasts and other types of cells (9). It has a wide range of functions, which include inducing cancer cell proliferation, such as in malignant melanomas (10). In the present study, we focused on the mediating function of SP, which involves delivering nociceptive information to the nervous system. It is commonly known that SP acts through the tachykinin NK1 receptor, which is a member of the G-protein-coupled receptor superfamily. Additionally, Bie and Zhao (11), along with Holzer (12), have demonstrated that the tachykinin NK1 receptor may contribute to the development and maintenance of inflammatory pain as a nociceptor. It has been demonstrated that SP is upregulated in response to irradiation and is involved in the process of inflammation (13). Therefore, we hypothesized that SP may also influence the symptoms of pain caused by radiation-induced inflammation. In addition, secreted SP may promote the inflammatory procedures caused by radiation, through facilitating inflammatory cell infiltration and activation (14).

The aim of radiation treatment is to administer an effective dose of radiation to the tumor, with an acceptable dose delivered to the neighboring normal tissues; however, the radiotherapy of thoracic neoplasms is likely to expose the esophagus to a high dose of ionizing radiation. In clinical settings, ARIE is a common complication in cancer patients receiving radiotherapy. Among these patients, a large proportion suffer from serious symptoms of pain and their quality of life is reduced. Furthermore, certain patients are forced to suspend their irradiation treatment when they are not able to tolerate any further treatment, resulting in a decreased tumor control rate. As for the treatment of ARIE, there are currently no clinically effective drugs or strategies. Minimizing the extent of the esophagus that is irradiated is an effective means to prevent ARIE; however, this would reduce the control of thoracic malignancies. Adrenocorticotropic hormones and certain antibiotics are the main drugs used in the treatment of ARIE; however, few efficacies have been demonstrated in a wide range of patients (4,15). Furthermore, the adverse effects of these drugs, including an increased risk of osteoporosis and antibiotic resistance, negatively limit their usage. Studies have also investigated the effect of sucralfate on preventing ARIE; however, 58% of patients resigned from the study, due to the adverse effects of nausea and vomiting (16). Another drug, amifostine, regarded to be the most effective radioprotective compound screened by the U.S. Army, did not effectively relieve the symptoms of ARIE in a large clinical trial (RTOG 9801) (17). Therefore, there is a requirement to investigate a novel method with effective functions and without obvious side-effects.

Drugs of a herbal origin with few side effects are of high interest amongst alternative treatments. Furthermore, traditional Chinese herb medicine (tChm) may provide a novel therapy that is not only able to relieve clinical symptoms, but also improve the general condition, including eating, sleeping and immune function (18,19). We have previously investigated the effect of cWPROL in treating the animal model of ARIE, and demonstrated that this prescription formula was able to repair the damaged esophageal tissues (20). The present study was designed to demonstrate that cWPROL is able to relieve the symptoms of pain caused by irradiation, and to clarify the underlying mechanism. We observed the general conditions, including body weight, and the weight of food and volume of water, to indirectly reflect symptoms of pain. Meanwhile, the expression level of SP in tissues within the radiation field was detected using immunohistochemical staining. As demonstrated by the results, cWPROL was able to decrease the level of SP expression, which was hypothesized to be correlated with a decrease in the symptoms of pain. Correspondingly, in the CT group, the weight of food and volume of water increased to a certain extent, after the lowest point of weight of food on the seventh day and the lowest point of volume of water on the eighth day. However, in the MT group, the weight of food and volume of water did not increase, although decreased SP expression was observed. It may be concluded that the pain relieving effect of mLDG is not as strong as that of cWPROL.

According to the outcomes of the present study, a partial mechanism for the pain-relieving effect of cWPROL has been elucidated. SP plays a key role in delivering pain information to the nervous system when tissues respond to injury (9). Our study suggests that the expression level of SP may be reduced by administration of cWPROL, and that the general condition, including body weight and the intake of food or drinking water, may be improved to a certain extent. However, other detailed molecular mechanisms should also be investigated in more detail in the future. For example, the critical receptor of SP, the tachykinin NK1 receptor, may exert a simultaneous influence.

Acknowledgements

This study was supported by the Foundation Science Research Program of Hebei Province Science and Technology Office (no. 04236101D-252004-2005).

References