Relationship between hyposalivation and oxidative stress in aging mice

Abstract

The increase in oxidative stress that accompanies aging has been implicated in the abnormal advance of aging and in the onset of various systemic diseases. However, the details of what effects the increase in oxidative stress that accompanies aging has on saliva secretion are not known. In this study, naturally aging mice were used to examine the stimulated whole saliva flow rate, saliva and serum oxidative stress, antioxidant level, submandibular gland H-E staining, and immunofluorescence staining to investigate the effect of aging on the volume of saliva secretion and the relationship with oxidative stress, as well as the effect of aging on the structure of salivary gland tissue. The stimulated whole saliva flow rate decreased significantly with age. Also, oxidative stress increased significantly with age. Antioxidant levels, however, decreased significantly with age. Structural changes of the submandibular gland accompanying aging included atrophy of parenchyma cells and fatty degeneration and fibrosis of stroma, and the submandibular gland weight ratio decreased. These results suggest that oxidative stress increases with age, not just systemically but also locally in the submandibular gland, and that oxidative stress causes changes in the structure of the salivary gland and is involved in hyposalivation.

Keywords: aging; hyposalivation; oxidative stress; salivary gland.

Fig. 1

Comparison of salivary flow rate and submandibular gland weight ratio. (a) Stimulated whole salivary flow rate (g/5 min). Values are expressed as mean ± SD (n = 5; *p<0.05: 7 w vs 30 w, **p<0.01: 30 w vs 72 w, ***p<0.05: 48 w vs 72 w, ANOVA with Tukey test). (b) Salivary α-amylase activity (U/ml). Values are expressed as mean ± SD (n = 5; *p<0.05: 48 w vs 72 w, ANOVA with Tukey test). (c) Correlation between the stimulated whole salivary flow rate and salivary α-amylase activity (r = 0.6382). (d) Submandibular gland weight ratio. Values are expressed as mean ± SD (n = 5; *p<0.01: 7 w vs 30 w, 48 w, 72 w, **p<0.05: 48 w vs 72 w, ANOVA with Tukey test).

Fig. 2

Comparison of saliva and serum 8-OHdG levels. (a) Saliva and serum 8-OHdG levels (ng/ml). 8-OHdG levels were measured using ELISA. Values are expressed as mean ± SD, (n = 5; *p<0.05: 30 w vs 72 w, **p<0.01: 7 w vs 72 w, ***p<0.05: 30 w vs 72 w, ANOVA with Tukey test). (b) Correlation of 8-OHdG between saliva and serum (r = 0.84676). (c) Correlation between the stimulated whole salivary flow rate and saliva 8-OHdG (r = 0.63402).

Fig. 3

Comparison of serum d-ROMs test (U.CARR). Values are expressed as mean ± SD (n = 5; *p<0.05: 7 w vs 72 w, **p<0.01: 30 w vs 72 w, ANOVA with Tukey test).

Fig. 4

Comparison of Saliva BAP test (µMs). Values are expressed as mean ± SD (n = 5; *p<0.01: 30 w vs 72 w, **p<0.01: 48 w vs 72 w, ***p<0.05: 7 w vs 72 w, ****p<0.01: 30 w vs 72 w, ANOVA with Tukey test).

Fig. 5

H-E and immunofluorescence staining of submandibular gland. (a–d) H-E staining of submandibular gland in a: 7 weeks, b: 30 weeks, c: 48 weeks, d: 72 weeks (scale bars: 100 µm). (e–h) PCNA immunofluorescence staining (red) of submandibular gland in e: 7 weeks, f: 30 weeks, g: 48 weeks, h: 72 weeks. Nuclei are counterstained with DAPI (blue) (scale bars: 100 µm). (i–l) TUNEL immunofluorescence staining (green) of submandibular gland in i: 7 weeks, j: 30 weeks, k: 48 weeks, l: 72 weeks. Nuclei are counterstained with DAPI (scale bars: 100 µm). (m–p) 8-OHdG immunofluorescence staining (green) in submandibular gland in m: 7 weeks, n: 30 weeks, o: 48 weeks, p: 72 weeks. Nuclei are counterstained with DAPI (scale bars: 100 µm).