Dietary nitrate supplementation prevents radiotherapy-induced xerostomia
- Beijing Laboratory of Oral Health, Capital Medical University, China
- Salivary Gland Disease Center and Molecular Laboratory for Gene Therapy & Tooth Regeneration, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, School of Stomatology, Capital Medical University, China
- Department of Pediatric Dentistry, Capital Medical University School of Stomatology, China
- Biochemistry and Molecular Biology, School of Basic Medical Sciences, China
Figures
Figure 1 with 1 supplement
Preventive and therapeutic effects of nitrate administration on the parotid gland in miniature pigs subjected to single-dose irradiation (IR).
(a–c) Preventive nitrate administration to miniature pigs. (a) Schematic of experimental design. (b) Nitrate administration maintained salivary flow rate (SFR) (ml/10 min) of the parotid gland in …
Figure 1—figure supplement 1
Parotid gland irradiation (IR) range diagrams, blood vessel distribution, salivary chemistry, and tissue apoptosis at 4 months after IR.
(a) Diagram of unilateral parotid gland IR range. (b) Blood vessel distribution model of the normal miniature pig parotid gland. (c) Clinical chemistry of saliva obtained from the single-dose IR …
Figure 2 with 1 supplement
Effects of different preventive nitrate doses on the parotid gland in miniature pigs subjected to fractionated irradiation (IR).
(a) Schematic of the experimental design for preventive nitrate administration of four different doses of nitrate (0.25, 0.5, 1, or 2 mmol/kg∙day) to miniature pigs. (b) Salivary flow rate (SFR) of …
Figure 2—figure supplement 1
Nitrate concentrations in serum (a) and parotid saliva (b), and changes in salivary constituents at 4 months after fractionated irradiation (IR).
(a) Nitrate concentrations in serum. (b) Nitrate concentrations in parotid saliva. (c–e) Salivary constituents changes. (c) Potassium; (d) Calcium; (e) Amylase. Four months post-IR was used as the …
Figure 3
Changes in the parotid gland following irradiation (IR).
(a–d) Immunohistochemical staining of parotid gland sections from miniature pigs in the sham, nitrate (2 mmol/kg·day), and IR control groups (n=4 animals per group; see Figure 2a for in vivo …
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Figure 3—source data 1
Western blot data for Figure 3d.
- https://cdn.elifesciences.org/articles/70710/elife-70710-fig3-data1-v2.zip
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Figure 3—source data 2
Western blot data for Figure 3e.
- https://cdn.elifesciences.org/articles/70710/elife-70710-fig3-data2-v2.zip
Figure 4 with 1 supplement
Effects of nitrate and sialin on salivary gland cell proliferation and apoptosis.
(a–e) Effects of nitrate on human parotid gland cells (hPGCs). (a) Schematic of experimental design for nitrate administration to hPGCs. (b, c) Cell proliferation rate, determined using CCK-8 assay …
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Figure 4—source data 1
Western blot data for Figure 4e.
- https://cdn.elifesciences.org/articles/70710/elife-70710-fig4-data1-v2.zip
Figure 4—figure supplement 1
Effects of nitrate on human parotid gland cells (hPGCs) under physiological conditions and transfection efficiencies of sialin siRNA and plasmids to hPGCs.
(a–d) Effects of nitrate on hPGCs. (a) Schematic of experimental design. (b) Cell proliferation, determined by number of EdU+/Hoechst + cells, at 24, 48, and 72 h. (c) Cell proliferation rate, …
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Figure 4—figure supplement 1—source data 1
Western blot for Figure 4—figure supplement 1f.
- https://cdn.elifesciences.org/articles/70710/elife-70710-fig4-figsupp1-data1-v2.zip
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Figure 4—figure supplement 1—source data 2
Western blot for Figure 4—figure supplement 1h.
- https://cdn.elifesciences.org/articles/70710/elife-70710-fig4-figsupp1-data2-v2.zip
Figure 5 with 1 supplement
Sialin overexpression promoted cell proliferation and reduced cell apoptosis after irradiation (IR) in vitro.
(a–e) Proliferation, apoptosis, and sialin expression of hPGCs cultured under physiological and IR conditions. (a) Schematic of experimental design. (b, c) Cell proliferation rate, determined using …
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Figure 5—source data 1
Western blot for Figure 5e.
- https://cdn.elifesciences.org/articles/70710/elife-70710-fig5-data1-v2.zip
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Figure 5—source data 2
Western blot for Figure 5g.
- https://cdn.elifesciences.org/articles/70710/elife-70710-fig5-data2-v2.zip
Figure 5—figure supplement 1
Reduction of sialin expression impaired cell proliferation and increased apoptosis after irradiation (IR).
(a) Schematic of experimental design for nitrate administration to sialin siRNA±human parotid gland cells (hPGCs) subjected to 5 Gy IR. (b) Sialin expression, determined using RT-PCR (left) and …
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Figure 5—figure supplement 1—source data 1
Western blot for Figure 5—figure supplement 1b.
- https://cdn.elifesciences.org/articles/70710/elife-70710-fig5-figsupp1-data1-v2.zip
Figure 6
Mechanism of nitrate regulation of cell proliferation.
(a) Phosphorylation of EGFR, AKT, and ERK following nitrate administration for 0.5, 1, 2, and 4 h, and phosphorylation of EGFR, AKT, and ERK following sialin knockdown for 24 h, n=5 sample …
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Figure 6—source data 1
Western blot data for Figure 6a (the left part).
- https://cdn.elifesciences.org/articles/70710/elife-70710-fig6-data1-v2.zip
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Figure 6—source data 2
Western blot data for Figure 6c.
- https://cdn.elifesciences.org/articles/70710/elife-70710-fig6-data2-v2.zip
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Figure 6—source data 3
Western blot for Figure 6a (the right part).
- https://cdn.elifesciences.org/articles/70710/elife-70710-fig6-data3-v2.zip
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Figure 6—source data 4
Western blot data for Figure 6d.
- https://cdn.elifesciences.org/articles/70710/elife-70710-fig6-data4-v2.zip
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Figure 6—source data 5
Western blot data for Figure 6e.
- https://cdn.elifesciences.org/articles/70710/elife-70710-fig6-data5-v2.rar
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
| Gene (Sus scrofa) | Sialin | NCBI | NC-010443.5 | |
| Gene (Homo sapiens) | Sialin | NCBI | NC_000006.12 | |
| Strain, strain background (S. scrofa) | Wild-type | Chinese Agricultural University | RRID:Addgene_66988 | 8–12 months, male |
| Transfected construct (H. sapiens) | siRNA to sialin | Thermo Fisher Scientific | 5′-TCCTGGAGGATATGTTGCCAGCAAA-3′5′-CATCACAAATACATTTGCCACTATT-3′ | |
| Antibody | (Rabbit polyclonal) anti-ki67 | Abcam | ab15580RRID:AB_443209 | IHC(1:200) |
| Antibody | (Rabbit polyclonal) anti-CD31 | Abcam | ab28364RRID:AB_726362 | IHC (1:50) |
| Antibody | (Rabbit polyclonal) anti-AQP5 | Thermo Fisher Scientific | PA5-36529RRID:AB_2553573 | IHC (1:50) |
| Antibody | (Rabbit polyclonal) anti-sialin | Thermo Fisher Scientific | PA5-42456RRID:AB_2577049 | IHC (1:200)WB (1:500) |
| Antibody | (Rabbit monoclonal) anti-pEGFR | Cell Signaling Technology | #3777 SRRID:AB_2096270 | WB (1:1000) |
| Antibody | (Rabbit monoclonal) anti-EGFR | Cell Signaling Technology | #4267 SRRID:AB_2246311 | WB (1:1000) |
| Antibody | (Rabbit monoclonal) anti-pAKT | Cell Signaling Technology | #4060 SRRID:AB_2315049 | WB (1:1000) |
| Antibody | (Rabbit monoclonal) anti-AKT | Cell Signaling Technology | #9272 SRRID:AB_2246311 | WB (1:1000) |
| Antibody | (Rabbit monoclonal) anti-pERK | Cell Signaling Technology | #4370 SRRID:AB_2315112 | WB (1:1000) |
| Antibody | (Rabbit monoclonal) anti-ERK | Cell Signaling Technology | #4695 SRRID:AB_390779 | WB (1:1000) |
| Recombinant DNA reagent | Sialin plasmid (pHS-LW066 vector) | Obio Technology | Wild-type | |
| Recombinant DNA reagent | SialinH183R plasmid (pHS-LW066 vector) | Obio Technology | Mutant type does not transport nitrate | |
| Commercial assay or kit | Total Nitric Oxide and Nitrate/Nitrite Parameter Assay Kit | R&D Systems | PKGE001 | |
| Software, algorithm | Prism 6 | GraphPad | https://www.graphpad.com/; RRID:SCR_002798 |
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/70710/elife-70710-transrepform1-v2.docx
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Supplementary file 1
Table 1 Reverse transcription- PCR primer sequences.
- https://cdn.elifesciences.org/articles/70710/elife-70710-supp1-v2.docx
