Alpha-Lipoic Acid Ameliorates Radiation-Induced Salivary Gland Injury by Preserving Parasympathetic Innervation in Rats

Abstract

Radiation therapy is a standard treatment for patients with head and neck cancer. However, radiation exposure to the head and neck induces salivary gland (SG) dysfunction. Alpha lipoic acid (ALA) has been reported to reduce radiation-induced toxicity in normal tissues. In this study, we investigated the effect of ALA on radiation-induced SG dysfunction. Male Sprague-Dawley rats were assigned to the following treatment groups: control, ALA only (100 mg/kg, intraperitoneally), irradiation only, and ALA administration 24 h or 30 min prior to irradiation. The neck area, including SGs, was irradiated evenly at 2 Gy/min (total dose, 18 Gy) using a photon 6 MV linear accelerator. The rats were sacrificed at 2, 6, 8, and 12 weeks after irradiation. Radiation decreased SG weight, saliva secretion, AQP5 expression, parasympathetic innervation (GFRα2 and AchE expression), regeneration potentials (Shh and Ptch expression), salivary trophic factor levels (brain-derived neurotrophic factor and neurturin), and stem cell expression (Sca-1). These features were restored by treatment with ALA. This study demonstrated that ALA can rescue radiation-induced hyposalivation by preserving parasympathetic innervation and regenerative potentials.

Keywords: Alpha lipoic acid; parasympathetic innervation; radiation therapy; salivary gland; xerostomia.

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1

ALA ameliorates the SG weight loss and impaired saliva secretion induced by irradiation. Sprague–Dawley rats were subjected to 18 Gy irradiation in the head and neck region. (A) Body weight, (B) SG weight, and (C) saliva levels were measured at each time points after irradiation. N = 3-4 SGs/group. a, control vs. irradiation group. b, ALA vs. irradiation groups. c, irradiation vs. ALA plus irradiation groups. Values are represented as the mean ± SEM. a,b,cp < 0.05.

Figure 2

The administration of ALA improves radiation-induced AQP5 expression. Sprague–Dawley rats were subjected to 18 Gy radiation in the head and neck region. (A) SGs were collected at each time points after irradiation and subjected to Western blotting. β-actin was used as the loading control. Western blot for the expression of AQP5 is normalized to β-actin, the expression was shown as signal intensity, and the expression in each group was represented as fold change. The fold change is calculated as the ratio of the final value in each group to the value in the control group at 2 weeks (set as “1”). Size markers mean 25 and 50 kDa from upper. Con; control (n = 3). RT; irradiation only group (n = 4). ALA+RT; ALA plus irradiation groups (n = 3). (B) immunohistochemical staining of AQP5 was shown in representative images. Positive signals were calculated as signal density. The fold change is calculated as the ratio of the final value in each group to the value in control group at 2 weeks (set as “1”). Scale bar, 50 μm. N = 3-4 SGs/group. Values are represented as the mean ± SEM. *p < 0.05.

Figure 3

ALA rescues parasympathetic innervation in SGs. Sprague–Dawley rats were subjected to 18 Gy radiation in the head and neck region. (A) SGs were collected at each time points after radiation and subjected to Western blotting of GFRα2. β-actin was used as the loading control. The expression of GFRa2 is normalized to β-actin, was shown as signal intensity, and the expression in each group was represented as fold change. The fold change is calculated as the ratio of the final value in each group to the value in control group at 2 weeks (set as “1”). Size markers mean 50 kDa. Con; control (n = 3). RT; irradiation only group (n = 4). ALA+RT; ALA plus irradiation groups (n = 3). The immunohistochemical staining of AchE (brown in (B) and neurofilaments (C) was shown in representative images. * indicate the ducts in SGs in C. Scale bar, 50 μm. N = 3–4 SGs/group. (D) Western blot and positive signals for each target were calculated as signal density. Values are represented as the mean ± SEM. *p < 0.05.

Figure 4

ALA maintains neurotrophic factor levels in SGs. Sprague–Dawley rats were subjected to 18 Gy irradiation in the head and neck region. SGs and serum were collected at each time points after irradiation and subjected to ELISA to measure BDNF (left in A and B) and neurturin (right in A and B) levels. N = 12–16 SGs/group. Values are represented as the mean ± SEM. * p < 0.05.

Figure 5

ALA rescues Hh signaling in SGs after irradiation. Sprague–Dawley rats were subjected to 18 Gy radiation in the head and neck region. SGs were collected at each time points, and RNA was extracted and subjected to qPCR analysis of Shh and Ptch expression. N = 12–16 SGs/group. Values are represented as the mean ± SEM. *p < 0.05.

Figure 6

ALA ameliorates radiation-induced impairment of stem cells. Sprague–Dawley rats were subjected to 18 Gy radiation in the head and neck region. (A and C) SGs were collected at each time points, and RNA was extracted and subjected to reverse-transcription PCR analysis of Sca-1. GAPDH was used as the loading control. The expression level of Sca-1 is normalized to GAPDH. The expression in each group was represented as fold change. Con; control (n = 3). ALA; ALA only treated group (n = 3). RT; irradiation only group (n = 3). ALA+RT; ALA plus irradiation groups (n = 3). (B and D) The sections were immunostained with anti-c-Kit. Figures are representative images from each group. c-Kit positive signals on SG sections (B) and the quantification of staining intensity (D). Scale bar, 50 μm. N = 3–4 SGs/group. The fold change is calculated as the ratio of the final value in each group to the value in control group at 2 weeks (set as “1”). Values are represented as the mean ± SEM. * p < 0.05.