Alpha lipoic acid attenuates radiation-induced thyroid injury in rats

Abstract

Exposure of the thyroid to radiation during radiotherapy of the head and neck is often unavoidable. The present study aimed to investigate the protective effect of α-lipoic acid (ALA) on radiation-induced thyroid injury in rats. Rats were randomly assigned to four groups: healthy controls (CTL), irradiated (RT), received ALA before irradiation (ALA + RT), and received ALA only (ALA, 100 mg/kg, i.p.). ALA was treated at 24 h and 30 minutes prior to irradiation. The neck area including the thyroid gland was evenly irradiated with 2 Gy per minute (total dose of 18 Gy) using a photon 6-MV linear accelerator. Greater numbers of abnormal and unusually small follicles in the irradiated thyroid tissues were observed compared to the controls and the ALA group on days 4 and 7 after irradiation. However, all pathologies were decreased by ALA pretreatment. The quantity of small follicles in the irradiated rats was greater on day 7 than day 4 after irradiation. However, in the ALA-treated irradiated rats, the numbers of small and medium follicles were significantly decreased to a similar degree as in the control and ALA-only groups. The PAS-positive density of the colloid in RT group was decreased significantly compared with all other groups and reversed by ALA pretreatment. The high activity index in the irradiated rats was lowered by ALA treatment. TGF-ß1 immunoreactivity was enhanced in irradiated rats and was more severe on the day 7 after radiation exposure than on day 4. Expression of TGF-ß1 was reduced in the thyroid that had undergone ALA pretreatment. Levels of serum pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6) did not differ significantly between the all groups. This study provides that pretreatment with ALA decreased the severity of radiation-induced thyroid injury by reducing inflammation and fibrotic infiltration and lowering the activity index. Thus, ALA could be used to ameliorate radiation-induced thyroid injury.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Representative photomicrographs of thyroid histology in the control, ALA, RT and ALA + RT groups after radiation.

Hematoxylin-eosin staining was performed on cross sections of the thyroid glands. Irregular-sized, smaller follicles than in the control group are noted in addition to inflammatory cell infiltration in the interfollicular area in the irradiated thyroid. Follicles of the ALA + RT group are more normal in size than those of the irradiated rats. CTL, control. RT, irradiated. ALA, ALA only. ALA + RT, received ALA before irradiation. Scale bar, 100 µm.

Figure 2. Representative photomicrographs of thyroid histology in the control, ALA, RT and ALA + RT groups after radiation.

Masson's trichrome staining was performed on cross sections of the thyroid glands. There was severe fibrosis in the interfollicular space and interfollicular edema was present in the irradiated rat thyroid on days 4 and 7 post-radiation (RT). Irradiation-induced histological changes were attenuated by ALA (ALA + RT). CTL, control. RT, irradiated. ALA, ALA only. ALA + RT, received ALA before irradiation. Scale bar, 100 µm.

Figure 3. The percentage of three size-classes of the follicles.

Follicles were divided into three categories based on their size: small (18–30 µm in diameter), medium (31–60 µm), and large (>60 µm). In the irradiated rats (RT), there occurred a statistically significant increase in the quantity of small and medium follicles compared to in the control and ALA-treated irradiated rats (ALA + RT). Small follicles were more common in the irradiated rats (RT), and greater numbers were observed on day 7 post-radiation compared to day 4. Small follicles in ALA-treated irradiated rats (ALA + RT) were less frequent than in irradiated rats (RT). (CTL, control; ALA, ALA only; RT-D4, Irradiated rats on day 4; RT-D7, Irradiated rats on day 7; ALA + RT-D4, ALA-irradiated rats on day 4; ALA + RT-D7, ALA-irradiated rats on day 7) (*P

Figure 4. A. Representative photomicrographs of thyroid histology in the control, ALA, RT and ALA + RT groups after radiation.

PAS staining was performed on cross sections of thyroid gland tissue. Follicles in the control rats were of regular size and had intensely stained colloid, indicating a low activity index. Follicles in the ALA-treated rats (ALA) without irradiation show similar degrees of colloid to the control group. The follicles were filled with less intensely stained colloid and were smaller than those in the control group, indicating a higher activity index. The follicles were filled with dense colloid similar to the controls in the ALA-treated irradiated rats. CTL, control. RT, irradiated. ALA, ALA only. The ALA + RT group received ALA before irradiation. Scale bar, 100 µm. B. PAS-positive colloid density. The PAS-positive density of the colloid in irradiated rats (RT) was decreased significantly compared with in all other groups. ALA-treated rats (ALA) showed a similar density of PAS-positive colloid to control rats (*P<0.05, §P<0.01).

Figure 5. TGF-ß1 immunohistochemical staining of cross sections of rat thyroid gland tissue.

The immunohistochemical localization of TGF-ß1 appears as dark-brown staining. TGF-ß1 expression was more intense in the follicular epithelium of the irradiated rats (RT) (thyrocytes strongly express TGF-ß1). The signals were stronger on day 7 than day 4. TGF-ß1 expression was less intense in the ALA group compared to the RT group. CTL, control. RT, irradiated. ALA, ALA only. ALA + RT, received ALA before irradiation. Scale bar, 100 µm.

Figure 6. Immunohistochemical staining for 8-OHdG and MDA.

8-OHdG-positive signals were much dense at day 4 than day 7 after radiation (A). Expression of MDA was localized at the stromal area in radiation groups (B). Both signals were decreased in ALA-treated groups. Scale bar, 100 µm.