Alpha-Lipoic Acid Ameliorates Radiation-Induced Lacrimal Gland Injury through NFAT5-Dependent Signaling

Hyuna Kim, Woong-Sun Yoo, Jung Hwa Jung, Bae Kwon Jeong, Seung Hoon Woo, Jin Hyun Kim, Seong Jae Kim, Hyuna Kim, Woong-Sun Yoo, Jung Hwa Jung, Bae Kwon Jeong, Seung Hoon Woo, Jin Hyun Kim, Seong Jae Kim

Abstract

Dry eye syndrome related to radiation therapy is relatively common and can severely impair a patient's daily life. The nuclear factor of activated T cells 5(NFAT5) is well known for its osmoprotective effect under hyperosmolar conditions, and it also has immune-modulating functions. We investigated the role of NFAT5 and the protective effect of α-lipoic acid(ALA) on radiation-induced lacrimal gland (LG) injuries. Rats were assigned to control, ALA only, radiation only, and ALA administered prior to irradiation groups. The head and neck area, including the LG, was evenly irradiated with 2 Gy/minute using a photon 6-MV linear accelerator. NFAT5 expression was enhanced and localized in the LG tissue after irradiation and was related to cellular apoptosis. ALA had a protective effect on radiation-induced LG injury through the inhibition of NFAT5 expression and NFAT5-dependent signaling pathways. Functional radiation-induced damage of the LG and cornea was also restored with ALA treatment. NFAT5 expression and its dependent signaling pathways were deeply related to radiation-induced dry eye, and the condition was improved by ALA treatment. Our results suggest a potential role of NFAT5 and NF-κB in the proinflammatory effect in LGs and cornea, which offers a target for new therapies to treat dry eye syndrome.

Keywords: alpha-lipoic acid; dry eye syndrome; nfat5; radiation therapy.

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
Figure 1
The nuclear factor of activated T cells (NFAT5) is differentially expressed by times in the lacrimal gland after radiation. (A) Lacrimal gland expression of NFAT5 in control and injured glands 2, 6, 8, or 12 weeks after radiation. β-actin was used as internal control. (B) Signal density of NFAT5 expression level in all groups. Data are expressed as mean ± SEM. * p < 0.05 versus each marked group. Con, control. RT, radiation.
Figure 2
Figure 2
α-lipoic acid (ALA) decreased radiation-induced structural changes and NFAT5 expression in the lacrimal gland. (A) Histopathological changes and immunohistochemical staining micrographs show NFAT5 expression. (B) Pathological scoring is examined by number of acinar cells with vacuoles. (C) Positive signal density of NFAT5 expression level in all groups. (D and E) Lacrimal gland expression of NFAT5 in all groups 2 weeks after radiation. Signal density of NFAT5 expression level in all groups. * p < 0.05 versus each marked group. Con, control. ALA, alpha-lipoic acid. RT, radiation. ALA + RT, ALA and radiation. Scale bar, 50 μm.
Figure 3
Figure 3
ALA ameliorates radiation-induced apoptotic cell death in the lacrimal gland. (A and B) Lacrimal gland expression of cleaved caspase-3 in all groups, 2 weeks after radiation. Signal density of cleaved caspase-3 expression level in all groups. (C) NFAT5 expression and apoptosis. Boxed areas are enlarged and presented in the right column. Arrows indicate positive signals. Dot lines in RT group indicate abundant positive signals. * p < 0.05 versus each marked group. Con, control. ALA, alpha-lipoic acid. RT, radiation. ALA + RT, ALA and radiation. Scale bar, 100 μm.
Figure 4
Figure 4
ALA inactivates the NFAT5-involved signaling pathway by radiation in the lacrimal gland. (A) Lacrimal gland expression of NF-κB and MAPKs (p-p38, pJNK, and pERK) in all groups 2 weeks after radiation. (B) Signal density of each expression level in all groups. * p < 0.05 versus each marked group. Con, control. ALA, alpha-lipoic acid. RT, radiation. ALA + RT, ALA and radiation.
Figure 5
Figure 5
ALA restores radiation-induced lacrimal gland dysfunction. (A) The lacrimal gland tissue lysates were examined by immunoblot with antibodies against AQP-5. (B) Quantified analysis was performed by densitometry. Data are mean ± SEM. * p < 0.05 versus each marked group. Con, control. ALA, alpha-lipoic acid. RT, radiation. ALA + RT, ALA and radiation.
Figure 6
Figure 6
ALA ameliorates radiation-induced corneal fluorescein staining. (A) Fluorescent slit-lamp photographs of the eyes of all groups. (B) Corneal epitheliums of each groups stained with H&E. Scale bar, 25 μm (C) Corneal fluorescein grading score of each group. (D) Corneal tissue injury was scored by epithelium height. The quantitative data are presented as means ± SEM. * p < 0.05 versus each marked group. Con, control. ALA, alpha-lipoic acid. RT, radiation. ALA + RT, ALA and radiation.

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