DOI: 10.5061/dryad.fn2z34ttq
title: Dietary nitrate supplementation prevents radiotherapy-induced xerostomia

description: 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.
description: 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).
description: Figure 1g, Figure 2e, Figure 3a-d and Figure 6