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 the sham and preventive nitrate groups, but not in the IR control group (n=4 animals per group); the sham group received 0 Gy, and the nitrate and IR control groups received 20 Gy. (c) Change in local blood flow rate (%) in the parotid gland in the sham, preventive nitrate, and IR control groups (n=4 animals per group). (d–f) Therapeutic nitrate administration to miniature pigs. (d) Schematic of experimental design. (e) SFR (ml/10 min) of the parotid gland in the sham, therapeutic nitrate, and IR control groups (n=4 animals per group); the sham group received 0 Gy, and the nitrate and IR control groups received 20 Gy. (f) Change in local blood flow rate (%) in the parotid gland in the sham, therapeutic nitrate, and IR control groups (n=4 per group). Data are expressed as the mean ± standard error of the mean (SEM). *p<0.05, **p<0.01, nitrate group versus IR control group at different time points. The sham group served as baseline for reference (b), (c), (e), and (f). (g) Hematoxylin and eosin staining of parotid gland sections in the sham, preventive nitrate, therapeutic nitrate, and IR control groups at 4 months post-IR. Scale bar: 100 µm.
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 experiment at different time points. No significant differences were observed in saliva constituents (data not shown), or hematological or serum chemical parameters (data not shown). Data are expressed as the mean ± SEM, n=4 animals per group; *p<0.05, **p<0.01 for the nitrate group versus the IR control group. (d) Tissue apoptosis in the sham, nitrate (2 mmol/kg day), and IR control groups 4 months after fractionated IR, measured using terminal deoxynucleotidyl transferase dUTP nick-end labeling in situ staining. Red cells indicate positive apoptotic cells. Scale bar: 100 µm (n=4 animals per group). The proportion of positive cells was detected using fluorescence microscopy, and quantified using ImagePro. Few cells underwent apoptosis, and there were no significant differences among the experimental groups. Data are expressed as the mean ± SEM. SEM, standard error of the mean.
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 the parotid gland (ml/10 min) in the sham group, the four nitrate groups, and the IR control group (n=4 animals per group; data are expressed as mean ± SEM); the sham group received 0 Gy, and the nitrate groups and the IR control group received 7.5 Gy for five consecutive days. (b’) SFR at 4 months post-IR compared with pre-IR SFR in the sham group, the nitrate groups, and the IR control group. (c) Change in local blood flow rate (%) in the parotid gland in the sham group, the nitrate groups, and the IR control group (n=4 animals per group; data are expressed as the mean ± SEM). Treatment groups correspond to those shown in (b). (d) Weight of the parotid gland in the sham group, the nitrate groups, and the IR control group (n=4 animals per group); data are expressed as the mean ± SEM, *p<0.05, **p<0.01, ***p<0.005 (each nitrate group compared with the sham group); Δ<0.05, ΔΔ<0.01 (each nitrate group compared with the IR control group). (e) Hematoxylin and eosin, and Masson trichrome staining of parotid gland sections in the sham group, the nitrate groups, and the IR control group (n=4 animals per group). Scale bar: 100 μm. SEM, standard error of the mean.
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 representative time point. In the IR control group, the amylase and calcium concentrations were significantly lower than those in the sham group, and the potassium concentration was significantly higher. Data are expressed as the mean ± SEM, n=4 animals per group. *p<0.05, **p<0.01, ***p<0.005 for the IR control group or each nitrate group versus the sham group. SEM, standard error of the mean.
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 experimental design). Scale bar: 50 μm. Because the results for all nitrate doses showed similar trends (data not shown), we used data from the highest dose (2 mmol/kg∙day) as representative of all doses. Nitrate group versus the sham group or the IR control group (a–c). (a) Cell proliferation rate, measured by Ki67 expression. (b) Microvessel density, measured by CD31 expression. (c) Aquaporin 5 (AQP5) expression; the areas of AQP5-positive cells among the groups are compared in the graph. (d) Immunohistochemical staining and western blot analysis of sialin expression.(e) Diagram of nitrate administration to human parotid gland cells (hPGCs) (in vitro, under physiological circumstances). (f) Sialin expression in hPGCs at 72 h after addition of different nitrate doses to culture medium (0.1, 0.2, or 0.5 mmol/L), measured by RT-PCR (left) and western blot (right); sham: blank control. n=5 per group. Data are expressed as the mean ± SEM. *p<0.05, **p<0.01, ***p<0.005, ns, no significance. RT-PCR, reverse transcription PCR; SEM, standard error of the mean.
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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 (b) or EdU staining (c). (d) Cell apoptotic rate, determined using flow cytometry. (e) Sialin expression in the IR control group and the nitrate groups at 72 h post-IR, determined using RT-PCR and western blot (representative time point; all remaining time points exhibited similar trends). (a–d) Addition of different nitrate doses (0.1, 0.2, or 0.5 mmol/L) to hPGCs culture medium prior to IR. 500 µM nitrate was used as the representative dose. All remaining doses exhibited similar trends. (a–e) Data are expressed as the mean ± standard error of the mean (SEM), n=5 culture plate replicates per group. *p<0.05, **p<0.01 for each nitrate group versus the IR control group. (f–i) Effects of sialin on hPGCs. (f) Schematic of experimental design for sialin siRNA or sialin plasmid delivery to hPGCs. (g) Cell proliferation rate determined using CCK-8 assay at 24, 48, and 72 h after siRNA or plasmid transfection. The 12 h time point was used as baseline, and scrambled siRNA and empty plasmid served as the control groups. (h) Cell proliferation rate determined by number of EdU+/Hoechst + cells at 24, 48, and 72 h after siRNA or plasmid transfection. Because plasmid transfection required 70% cell confluence, cell proliferation was determined at 24 and 48 h after plasmid transfection. (i) Cell cycle distribution analysis at 72 h (representative time point) after siRNA transfection. (j) Cell apoptotic rate, determined by flow cytometry, at 72 h after siRNA transfection (representative time point). Slc17a5 overexpression did not affect apoptosis when hPGCs were cultured under normal conditions (data not shown). (f–j) Data are expressed as the mean ± SEM, n=5 culture plate replicates per group. *p<0.05, **p<0.01. (k, l) Submandibular gland cell proliferation compared between sgRNA two-cell embryo and wild-type (WT) mice. The expression level of sialin in sgRNA two-cell embryos was about 60% of that in WT mice. (k) Cell proliferation rate determined using CCK-8 assay. (l) Cell proliferation rate, determined by number of EdU+/Hoechst + cells. (k, l) Data are expressed as the mean ± SEM, n=5 animals per group; *p<0.05, **p<0.01 for the sgRNA two-cell embryo group versus the WT group. (m, n) Nitrate or sialin has an effect on cell proliferation. (k) The experimental design scheme (sialinH183R is defective in nitrate transportation). (l) Cell proliferation rate, determined using CCK-8. Data are expressed as the mean ± SEM, n=5 culture plate replicates per group. *p<0.05, **p<0.01 for the sialin plasmid group versus the empty plasmid group. RT-PCR, reverse transcription PCR.
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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, determined using CCK-8 assay, at 24, 48, 72, and 96 h. The 12 h time point was used as the baseline. (d) Cell apoptosis rate, determined using flow cytometry, at 72 h (representative time point). N=5 culture plate replicates per group. (a–d) Data are expressed as the mean ± SEM. *p<0.05, **p<0.01, ***p<0.005. (e–h) Transfection efficiencies of sialin siRNA and plasmids to hPGCs. (e, f) Messenger RNA (e) and protein levels (f) of sialin at 24, 48, and 72 h after siRNA transfection. Sialin was effectively knocked down at each time point. (g, h) Messenger RNA (g) and protein levels (h) of sialin at 24, 48, and 72 h after plasmid transfection. Sialin was effectively overexpressed at each time point. (e–h) Data are expressed as the mean ± SEM, n=5 culture plate replicates per group. ***p<0.001. SEM, standard error of the mean.
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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 CCK-8 (b) or EdU assay (c). (d) Cell apoptotic rate, determined using flow cytometry. (e) Sialin expression, measured using reverse transcription PCR (RT-PCR; left) and western blot (right). (b–e) Sham group: hPGCs received 0 Gy; IR group: hPGCs received 5 Gy, n=5 culture plate replicates per group. Data are expressed as the mean ± SEM. *p<0.05, **p<0.01, ***p<0.005. (f–j) Effects of sialin overexpression on cell proliferation and apoptosis. (f) Schematic of experimental design. (g) Sialin expression in the sialin plasmid and empty plasmid groups, determined using RT-PCR and western blot. (h), (i) Cell proliferation rate, detected using CCK-8 assay (h) or EdU staining (i). (j) Cell apoptotic rate, determined using flow cytometry assay, at 72 h post-IR. (g–j) Data are expressed as the mean ± SEM, n=5 culture plate replicates per group. *p<0.05, **p<0.01, ***p<0.005 for the sialin plasmid group versus the empty plasmid group. SEM, standard error of the mean.
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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 western blot (right). Scrambled siRNA was used as the control. (c) Cell proliferation, determined by number of EdU+/Hoechst + cells, at 24, 48, and 72 h post-IR. (d) Cell proliferation rate, determined using CCK-8 assay, at 24, 48, 72, and 96 h post-IR. The 12 h time point was used as baseline. (e) Cell apoptosis rate, determined using flow cytometry, at 72 h post-IR. Data are expressed as the mean ± SEM, n=5 cell culture plates per group. *p<0.05, **p<0.01, ***p<0.005 for each siRNA group or nitrate plus siRNA group versus the scrambled siRNA control group (b–e). RT-PCR, reverse transcription PCR; SEM, standard error of the mean.
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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 replicates per group. (b) Cell proliferation rate after nitrate administration plus blockade of EGFR, determined using CCK-8 assay at 24, 48, 72, and 96 h. The 12 h time point was used as baseline. Data are expressed as the mean ± SEM, n=5 sample replicates per group. *p<0.05 for the nitrate plus gefitinib group versus the nitrate group. (c) Phosphorylation of EGFR, AKT, and ERK following EGFR blockade or nitrate administration plus EGFR blockade for 24 h, n=5 sample replicates per group. (d) Sialin and phosphorylation of EGFR expression after blocking nitrate-nitrite-nitric oxide (NO) pathway by PTIO. (a–d) Nitrate administration dose: 0.5 mmol/L. (e) Phosphorylation of EGFR, AKT, and ERK in parotid gland tissues of sham, nitrate, and IR control groups, in vivo study. (f) Schematic of the proposed nitrate–sialin feedback loop in radioprotection of salivary glands (a: activation; pho: phosphorylation). SEM, standard error of the mean.
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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
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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
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Dietary nitrate supplementation prevents radiotherapy-induced xerostomia
eLife 10:e70710.
https://doi.org/10.7554/eLife.70710
