Skip to main content

Dietary nitrate supplementation prevents radiotherapy-induced xerostomia

Cite this dataset

Wang, Songlin et al. (2021). Dietary nitrate supplementation prevents radiotherapy-induced xerostomia [Dataset]. Dryad.


Management of salivary gland hypofunction caused by irradiation (IR) therapy for head and neck cancer remains lack of effective treatments. Salivary glands, especially the parotid gland, actively uptake dietary nitrate and secrete it into saliva. Here, we investigated the effect of dietary nitrate on the prevention and treatment of IR-induced parotid gland hypofunction in miniature pigs, and elucidated the underlying mechanism in human parotid gland cells (hPGCs). We found that nitrate administration prevented IR-induced parotid gland damage in a dose-dependent manner, by maintaining the function of irradiated parotid gland tissue. Mechanically, Nitrate could increase sialin expression, a nitrate transporter expressed in the parotid gland, making the nitrate-sialin feedback loop that facilitates nitrate influx into cells for maintaining cell proliferation and inhibiting apoptosis. Nitrate enhanced cell proliferation via the epidermal growth factor receptor (EGFR)–protein kinase B (AKT)–mitogen-activated protein kinase (MAPK) signaling pathway in irradiated parotid gland tissue. Collectively, nitrate effectively prevented IR-induced xerostomia via the EGFR–AKT–MAPK signaling pathway. Dietary nitrate supplementation may provide a novel, safe, and effective way to resolve IR-induce xerostomia.


Animals were sacrificed at the end points of our experimental designs (Fig. 4a, d; Fig. 5a). Parotid glands were collected, fixed in 4% paraformaldehyde, dehydrated in gradient ethanol solutions, embedded in paraffin, and sectioned at 4-μm thickness. Hematoxylin and eosin (H&E) staining was performed to analyze morphological changes. Immunohistochemical staining was employed to determine expression of CD31 (Abcam, Cambridge, UK), Ki67 (Abcam), AQP5 (Thermo Fisher Scientific, Waltham, MA, USA), and sialin (Thermo Fisher Scientific). CD31 and Ki67 were used to analyze the microvessel density (MVD) and cell proliferation, respectively. AQP5 is expressed in apical membranes of acinar cells and plays a key role in saliva secretion. Thus, AQP5 expression was assessed to determine the saliva secretory function of the parotid gland on a histological level.

Total protein was extracted by radioimmunoprecipitation assay (RIPA) lysis buffer (Applygen Technologies, Beijing, China), and the protein concentration was measured with a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific). Total protein samples (20 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Beijing, China). Membranes were blocked by 5% skim milk and incubated with the following primary antibodies: rabbit anti-sialin (Thermo Fisher Scientific), rabbit anti-phospho-EGF Receptor (Tyr1068) (Cell Signaling Technology, CST, Shanghai, China), rabbit anti-EGF Receptor (D38B1) (CST), rabbit anti-phospho-AKT (Ser473) (CST), rabbit anti-AKT (CST), rabbit anti-phospho-ERK1/2 (Thr202/Tyr204) (CST), rabbit anti-ERK1/2 (CST), and mouse anti-β-actin (Abcam). Thereafter, membranes were incubated with goat anti-rabbit or anti-mouse secondary antibodies (both Abcam) and visualized by enhanced chemiluminescence (BD Biosciences, Franklin Lakes, NJ, USA).

Usage notes

Figure 1g, Figure 2e, Figure 3a-d and Figure 6


National Natural Science Foundation of China, Award: 91649124

National Natural Science Foundation of China, Award: 81600883

Beijing Municipal Science and Technology Commission, Award: No. Z181100001718208