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. 2016 May 17;7(20):29143-53.
doi: 10.18632/oncotarget.8661.

Protective effects of alpha lipoic acid on radiation-induced salivary gland injury in rats

Jin Hyun Kim  1   2 , Kyung Mi Kim  3 , Myeong Hee Jung  1 , Jung Hwa Jung  2   4 , Ki Mun Kang  2   5 , Bae Kwon Jeong  2   5 , Jin Pyeong Kim  2   3 , Jung Je Park  2   3 , Seung Hoon Woo  2   3
Affiliations

Protective effects of alpha lipoic acid on radiation-induced salivary gland injury in rats

Jin Hyun Kim et al. Oncotarget. .

Abstract

Purpose: Radiation therapy is a treatment for patients with head and neck (HN) cancer. However, radiation exposure to the HN often induces salivary gland (SG) dysfunction. We investigated the effect of α-lipoic acid (ALA) on radiation-induced SG injury in rats.

Results: ALA preserved acinoductal integrity and acinar cell secretary function following irradiation. These results are related to the mechanisms by which ALA inhibits oxidative stress by inhibiting gp91 mRNA and 8-OHdG expression and apoptosis of acinar cells and ductal cells by inactivating MAPKs in the early period and expression of inflammation-related factors including NF-κB, IκB-α, and TGF-β1 and fibrosis in late irradiated SG. ALA effects began in the acute phase and persisted for at least 56 days after irradiation.

Materials and methods: Rats were assigned to followings: control, ALA only (100 mg/kg, i.p.), irradiated, and ALA administered 24 h and 30 min prior to irradiation. The neck area including the SG was evenly irradiated with 2 Gy per minute (total dose, 18 Gy) using a photon 6-MV linear accelerator. Rats were killed at 4, 7, 28, and 56 days after radiation.

Conclusions: Our results show that ALA could be used to ameliorate radiation-induced SG injury in patients with HN cancer.

Keywords: Nox-2; alpha lipoic acid; complication; radiation; salivary gland.

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Conflict of interest statement

The authors declare no conflicts of interest. The funding sponsors had no role in the design of the study; collection, analyses, or interpretation of data; writing of the manuscript; or in deciding to publish the results.

Figures

Figure 1
Figure 1. ALA decreases pathological changes in the salivary gland after irradiation
Histopathological changes of the submandibular gland 4, 7, 28, and 56 days after irradiation. All salivary gland tissue were sectioned and stained with H & E. Exposing the salivary gland to irradiation resulted in the vacuolization of acinar cells (arrows) and the vacuolization decreased in the ALA treatment group before irradiation (A). Injury scoring was based on the number of injured acinar cells with cytoplasmic vacuoles in x 200 magnification field. Injury grading is described in the “Materials and Methods” section (B). Con, control. ALA, ALA only. RT, irradiated. ALA + RT, received ALA before irradiation. Scale bar, 50 μm. Each bar shows the mean ± SEM; *p < 0.05 indicates differences between RT and ALA + RT groups. Con (n = 3), ALA (n = 3), RT (n = 10), ALA + RT (n = 10).
Figure 2
Figure 2. ALA ameliorates radiation-induced salivary dysfunction
The function of acini was evaluated by amylase staining in the submandibular gland at 4, 7, 28, and 56 days after radiation (A). Amylase content decreased significantly in the irradiated gland and ALA-treated SG showed much denser signals than that of the irradiated SG at all days (A and B). Saliva lag time was examined as the first time to secret the saliva from pilocarpine stimulation and secretion is expressed as the total output of saliva collected normalized to body weight after pilocarpine stimulation. The ALA-treated group showed improved lag time and increased saliva secretion relative to the irradiated group (C). Con, control. ALA, ALA only. RT, irradiated. ALA + RT, received ALA before irradiation. Scale bar, 50 μm. Each bar shows the mean ± SEM; *p < 0.05 indicates differences between RT and ALA + RT groups. Con (n = 5), ALA (n = 5), RT (n = 15), ALA + RT (n = 15).
Figure 3
Figure 3. ALA reduces radiation-induced oxidative stress via Nox2 and 8-OHdG
gp91 mRNA expression was determined by RT-PCR. gp91 mRNA expression decreased in the ALA-treated irradiated group compared to that in the irradiated group on 4 and 7 days after radiation (A). 8-OHdG-positive signals increased significantly in the salivary gland after irradiation and less intense signals were detected in the ALA-treated irradiated group (ALA + RT) than those in the irradiated group 4 days after radiation (B). Con, control. ALA, ALA only. RT, irradiated. ALA + RT, received ALA before irradiation. Scale bar, 50 μm. Each bar shows the mean ± SEM; *p < 0.05 and **p < 0.01 indicates differences between RT and ALA + RT groups. The fold changes are calculated as the ratio of the final value in the each group to the value in control group at day 4 (set as “1”). Con (n = 3), ALA (n = 3), RT (n = 10), ALA + RT (n = 10).
Figure 4
Figure 4. ALA decreases radiation-induced cell death in the SG
Cell death was analyzed with the TUNEL assay. Microscopic image shows increased cell death in the RT group compared to the control and ALA-treated irradiated groups (ALA+RT) 4 and 7 days after irradiation. Scale bar, 50 μm (A). Western blotting was performed using anti-p-p38, anti-p-JNK. Expression levels of p-p38 and p-JNK were induced in the RT group compared to that in the control and ALA-treated irradiated groups (ALA + RT) 4 and 7 days after irradiation. β-actin was used as the loading control (B). Con, control. ALA, ALA only. RT, irradiated. ALA + RT, received ALA before irradiation. Representative blots are derived from three separate experiments. Values are represented as the mean ± SEM. *P < 0.05 and **p < 0.01 indicates differences between RT and ALA + RT groups. The fold changes are calculated as the ratio of the final value in the each group to the value in control group at day 4 (set as “1”). Con (n = 3), ALA (n = 3), RT (n = 10), ALA + RT (n = 10).
Figure 5
Figure 5. ALA inhibits radiation-induced inflammation and fibrotic changes in the salivary gland
Expression of phospho-NF-κB, phospho-IκB-α, and TGF-β1 was induced 7, 28, and 56 days after irradiation. phospho-NF-κB and phospho-IκB-α expression levels peaked 7 days after irradiation, whereas TGF-β1 expression peaked 28 days after irradiation. These levels decreased on each day in the ALA-treated irradiated group. β-actin was used as the loading control (A). All salivary gland tissue were stained with Masson's trichrome (B). Fibrosis signals (blue) were significantly enhanced in irradiated salivary glands, and ALA treatment attenuated the fibrotic changes 28 and 56 days after radiation and lysis of acinar and granular convoluted tubules 7, 14, 28 and 56 days after radiation (B and C). Con, control. ALA, ALA only. RT, irradiated. ALA + RT, received ALA before irradiation. Scale bar, 50 μm (C). Representative blots are derived from three separate experiments. Values are represented as the mean ± SEM. *P < 0.05 and **p < 0.01 indicates differences between RT and ALA + RT groups. The fold changes are calculated as the ratio of the final value in the each group to the value in control group at day 4 (set as “1”). Con (n = 3), ALA (n = 3), RT (n = 10), ALA + RT (n = 10).
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