Yang HS et al. (2020), Alpha-tocopherol exerts protective function aga...

XB-ART-61847

Sci Rep 2020 Apr 10;101:6224. doi: 10.1038/s41598-020-63085-6.

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Alpha-tocopherol exerts protective function against the mucotoxicity of particulate matter in amphibian and human goblet cells.

Yang HS, Sim HJ, Cho H, Bang WY, Kim HE, Kwon TK, Kwon T, Park TJ.

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Exposure to particulate matter (PM) in ambient air is known to increase the risk of cardiovascular disorders and mortality. The cytotoxicity of PM is mainly due to the abnormal increase of reactive oxygen species (ROS), which damage cellular components such as DNA, RNA, and proteins. The correlation between PM exposure and human disorders, including mortality, is based on long-term exposure. In this study we have investigated acute responses of mucus-secreting goblet cells upon exposure to PM derived from a heavy diesel engine. To this end, we employed the mucociliary epithelium of amphibian embryos and human Calu-3 cells to examine PM mucotoxicity. Our data suggest that acute exposure to PM significantly impairs mucus secretion and results in the accumulation of mucus vesicles in the cytoplasm of goblet cells. RNA-seq analysis revealed that acute responses to PM exposure significantly altered gene expression patterns; however, known regulators of mucus production and the secretory pathway were not significantly altered. Interestingly, pretreatment with α-tocopherol nearly recovered the hyposecretion of mucus from both amphibian and human goblet cells. We believe this study demonstrates the mucotoxicity of PM and the protective function of α-tocopherol on mucotoxicity caused by acute PM exposure from heavy diesel engines.

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Species referenced: Xenopus laevis
Genes referenced: gapdh muc5ac otogl2 tubb2b tubb4a tubb4b
GO keywords: cellular response to toxic substance
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	Figure 1. The mucociliary epithelium of amphibian embryos is structurally and physiologically similar to human airway epithelium. (A) The embryonic epithelium of Xenopus laevis was visualized by fluorescent imaging analysis. Goblet cells were stained with WGA-Alexa 488 and multi-cilia were stained with anti-acetylated tubulin antibody. (B) The embryonic epithelium of Bombina orientalis was visualized by the same protocol. (C) Bicuculline reversibly inhibited mucus secretion from the goblet cells of X. laevis and B. orientalis embryonic epithelium. Statistical analysis was performed using one-way ANOVA. From left to right, n=57, 42, 39, 17, 5, 8. (D) Phorbol 12,13-dibutyrate increased mucus secretion from the goblet cells of X. laevis and B. orientalis embryonic epithelium. Statistical analysis was performed using Students t-test. From left to right, n=57, 42, 17, 18. (E) Narasin inhibited mucus secretion from X. laevis and B. orientalis embryonic epithelium. Statistical analysis was performed using Students t-test. From left to right, n=57, 42, 17, 18. Asterisks represent: *(p<0.001), (0.001<p<0.01), *(0.01<p<0.05), ns (0.05<p).
	Figure 2. Particulate matter from heavy diesel engine disturbs mucus secretion from the mucociliary epithelium in B. orientalis and X. laevis embryos. (A) Increasing doses of PM were administered to B. orientalis embryos and secreted mucus levels were analyzed by ELLA using WGA-HRP. 50g/mL or 100g/mL of PM significantly disturbed mucus secretion from the embryonic epidermis. Statistical analysis was performed using one-way ANOVA. From left to right, n=21, 16, 20. (B) The WGA positive mucus vesicles were visualized by fluorescent microscopy. PM exposure significantly accumulated mucus vesicles in the cytoplasm. Scale bar = 10 m. (C) Quantification of WGA positive mucus vesicles in (B). Statistical analysis was performed using one-way ANOVA. From left to right, n=4, 4, 4. (D) Particulate matter-induced hyposecretion of mucus was recovered by -tocopherol in Xenopus embryos. Statistical analysis was performed using one-way ANOVA. From left to right, n=24, 22, 22. (E) The WGA positive mucus vesicles were visualized by fluorescent microscopy. The exposure to the particulate matter significantly accumulated the mucus vesicles in the cytosol. Scale bar = 20 m. (F,G) Quantification of WGA positive mucus vesicles in (E). For (F), statistical analysis was performed using one-way ANOVA. From left to right, n=4, 4, 4. For (G), statistical analysis was performed using one-way ANOVA. From left to right, n=30, 30, 30. Asterisks represent *(p<0.001), (0.001<p<0.01), *(0.01<p<0.05), ns (0.05<p). PM, particulate matter; ELLA, enzyme-linked lectin assay.
	Figure 3. Acute exposure to particulate matter changes gene expression, but did not significant alter expression of mucus-related genes.(A) RNA-seq and differentially expressed gene (DEG) analysis revealed that PM exposure significantly changed the gene expression profile of X. laevis embryos. (B) Otogelin expression was slightly reduced by PM treatment without statistical significance. Statistical analysis was performed using Student’s t-test. From left to right, n = 3, 3, 3. Asterisks represent *(p < 0.001), (0.001 < p < 0.01), *(0.01 < p < 0.05), ns (0.05 < p). PM, particulate matter.
	Figure 4. Schematic of experimental procedure to analyze protective functions of antioxidants on particulate matter mucotoxicity. X. laevis embryos were pretreated with the indicated antioxidants trolox, NAC (N-acetyl cysteine), or α-tocopherol. After 1 h incubation in the antioxidant-containing media, embryos were transferred to PM treatment media. The level of secreted mucus was measured by ELLA assay using WGA-HRP. PM, particulate matter; ELLA, enzyme-linked lectin assay.
	Figure 5. α-tocopherol treatment protected X. laevis mucociliary epithelium from particulate matter mucotoxicity. (A) Indicated doses of antioxidants administered to X. laevis embryos followed by PM. Secreted mucus from X. laevis embryonic mucociliary epithelium was measured by ELLA using WGA-HRP. Pretreatment with antioxidants trolox and NAC (N-acetyl cysteine) did not affect PM-induced mucus hyposecretion. However, pretreatment with α-tocopherol significantly recovered the reduction in mucus secretion induced by PM. Statistical analysis was performed using one-way ANOVA. From left to right, n = 52, 14, 12, 7, 6, 7, 7, 6, 4. (B) WGA positive mucus vesicles accumulated in embryonic mucociliary epithelium were visualized by fluorescent microscopy. Scale bar = 20 μm. (C,D) Quantification of WGA positive mucus vesicles in (B). Statistical analysis was performed using one-way ANOVA. For (C), from left to right, n = 6, 4, 6, 4, 6. For (D), from left to right, n = 12, 13, 14, 11, 14. Pretreatment with α-tocopherol ameliorated the accumulation of mucus vesicles induced by PM. Asterisks represent *(p < 0.001), (0.001 < p < 0.01), *(0.01 < p < 0.05), ns (0.05 < p). PM, particulate matter; ELLA, enzyme-linked lectin assay.
	Figure 6. α-tocopherol treatment protects B. orientalis mucociliary epithelium from PM mucotoxicity. (A) Indicated doses of antioxidants were administered to B. orientalis embryos followed by PM exposure, and secreted mucus from B. orientalis embryonic mucociliary epithelium was measured by ELLA using WGA-HRP. Pretreatment with antioxidants trolox and NAC (N-acetyl cysteine) did not affect PM-induced mucus hyposecretion. However, pretreatment with α-tocopherol significantly recovered the reduction in mucus secretion induced by PM. Statistical analysis was performed using one-way ANOVA. From left to right, n = 36, 32, 36, 34, 31, 36. (B) WGA positive mucus vesicles accumulated in embryonic mucociliary epithelium were visualized by fluorescent microscopy. Scale bar = 10 μm. (C) Quantification of WGA positive mucus vesicles in (B). Pretreatment with α-tocopherol ameliorated the accumulation of mucus vesicles induced by PM. Statistical analysis was performed using one-way ANOVA. From left to right, n = 7, 6, 7, 4, 6. Asterisks represent *(p < 0.001), (0.001 < p < 0.01), *(0.01 < p < 0.05), ns (0.05 < p). PM, particulate matter; ELLA, enzyme-linked lectin assay.
	Figure 7.Particulate matter induced the accumulation of mucus vesicles in human Calu-3 goblet cells. (A) PM treatment induced the accumulation of WGA positive mucus vesicles in Calu-3 cells. Mucus vesicles were stained with WGA-Alexa 488. Scale bar = 10 μm. (B) PM treatment induced the accumulation of MUC5AC positive mucus vesicles in Calu-3 cells. MUC5AC vesicles were stained with an anti-MUC5AC antibody. Consistent with the amphibian mucociliary epithelium, pretreatment with α-tocopherol significantly reduced the accumulation of mucus vesicles induced by PM in Calu-3 cells. Scale bar = 10 μm. (C,D) Quantification of WGA and MUC5AC positive vesicles from (A) and (B), respectively. Statistical analysis was performed using one-way ANOVA. For (C), from left to right, n = 6, 4, 6, 8, 8. For (D), n = 6, 5, 7, 4, 5. Asterisks represent *(p < 0.001), (0.001 < p < 0.01), *(0.01 < p < 0.05), ns (0.05 < p). PM, particulate matter.
	Figure 8. Schematic of the protective effect of α-tocopherol on particulate matter mucotoxicity. Our data suggest that acute exposure to PM causes mucus hyposecretion, likely through interference with secretory and exocytic processes in goblet cells. α-tocopherol offers a protective function over PM mucotoxicity. PM, particulate matter.

	Figure 1. The mucociliary epithelium of amphibian embryos is structurally and physiologically similar to human airway epithelium. (A) The embryonic epithelium of Xenopus laevis was visualized by fluorescent imaging analysis. Goblet cells were stained with WGA-Alexa 488 and multi-cilia were stained with anti-acetylated tubulin antibody. (B) The embryonic epithelium of Bombina orientalis was visualized by the same protocol. (C) Bicuculline reversibly inhibited mucus secretion from the goblet cells of X. laevis and B. orientalis embryonic epithelium. Statistical analysis was performed using one-way ANOVA. From left to right, n=57, 42, 39, 17, 5, 8. (D) Phorbol 12,13-dibutyrate increased mucus secretion from the goblet cells of X. laevis and B. orientalis embryonic epithelium. Statistical analysis was performed using Students t-test. From left to right, n=57, 42, 17, 18. (E) Narasin inhibited mucus secretion from X. laevis and B. orientalis embryonic epithelium. Statistical analysis was performed using Students t-test. From left to right, n=57, 42, 17, 18. Asterisks represent: *(p<0.001), (0.001<p<0.01), *(0.01<p<0.05), ns (0.05<p).
	Figure 2. Particulate matter from heavy diesel engine disturbs mucus secretion from the mucociliary epithelium in B. orientalis and X. laevis embryos. (A) Increasing doses of PM were administered to B. orientalis embryos and secreted mucus levels were analyzed by ELLA using WGA-HRP. 50g/mL or 100g/mL of PM significantly disturbed mucus secretion from the embryonic epidermis. Statistical analysis was performed using one-way ANOVA. From left to right, n=21, 16, 20. (B) The WGA positive mucus vesicles were visualized by fluorescent microscopy. PM exposure significantly accumulated mucus vesicles in the cytoplasm. Scale bar = 10 m. (C) Quantification of WGA positive mucus vesicles in (B). Statistical analysis was performed using one-way ANOVA. From left to right, n=4, 4, 4. (D) Particulate matter-induced hyposecretion of mucus was recovered by -tocopherol in Xenopus embryos. Statistical analysis was performed using one-way ANOVA. From left to right, n=24, 22, 22. (E) The WGA positive mucus vesicles were visualized by fluorescent microscopy. The exposure to the particulate matter significantly accumulated the mucus vesicles in the cytosol. Scale bar = 20 m. (F,G) Quantification of WGA positive mucus vesicles in (E). For (F), statistical analysis was performed using one-way ANOVA. From left to right, n=4, 4, 4. For (G), statistical analysis was performed using one-way ANOVA. From left to right, n=30, 30, 30. Asterisks represent *(p<0.001), (0.001<p<0.01), *(0.01<p<0.05), ns (0.05<p). PM, particulate matter; ELLA, enzyme-linked lectin assay.
	Figure 3. Acute exposure to particulate matter changes gene expression, but did not significant alter expression of mucus-related genes.(A) RNA-seq and differentially expressed gene (DEG) analysis revealed that PM exposure significantly changed the gene expression profile of X. laevis embryos. (B) Otogelin expression was slightly reduced by PM treatment without statistical significance. Statistical analysis was performed using Student’s t-test. From left to right, n = 3, 3, 3. Asterisks represent *(p < 0.001), (0.001 < p < 0.01), *(0.01 < p < 0.05), ns (0.05 < p). PM, particulate matter.
	Figure 4. Schematic of experimental procedure to analyze protective functions of antioxidants on particulate matter mucotoxicity. X. laevis embryos were pretreated with the indicated antioxidants trolox, NAC (N-acetyl cysteine), or α-tocopherol. After 1 h incubation in the antioxidant-containing media, embryos were transferred to PM treatment media. The level of secreted mucus was measured by ELLA assay using WGA-HRP. PM, particulate matter; ELLA, enzyme-linked lectin assay.
	Figure 5. α-tocopherol treatment protected X. laevis mucociliary epithelium from particulate matter mucotoxicity. (A) Indicated doses of antioxidants administered to X. laevis embryos followed by PM. Secreted mucus from X. laevis embryonic mucociliary epithelium was measured by ELLA using WGA-HRP. Pretreatment with antioxidants trolox and NAC (N-acetyl cysteine) did not affect PM-induced mucus hyposecretion. However, pretreatment with α-tocopherol significantly recovered the reduction in mucus secretion induced by PM. Statistical analysis was performed using one-way ANOVA. From left to right, n = 52, 14, 12, 7, 6, 7, 7, 6, 4. (B) WGA positive mucus vesicles accumulated in embryonic mucociliary epithelium were visualized by fluorescent microscopy. Scale bar = 20 μm. (C,D) Quantification of WGA positive mucus vesicles in (B). Statistical analysis was performed using one-way ANOVA. For (C), from left to right, n = 6, 4, 6, 4, 6. For (D), from left to right, n = 12, 13, 14, 11, 14. Pretreatment with α-tocopherol ameliorated the accumulation of mucus vesicles induced by PM. Asterisks represent *(p < 0.001), (0.001 < p < 0.01), *(0.01 < p < 0.05), ns (0.05 < p). PM, particulate matter; ELLA, enzyme-linked lectin assay.
	Figure 6. α-tocopherol treatment protects B. orientalis mucociliary epithelium from PM mucotoxicity. (A) Indicated doses of antioxidants were administered to B. orientalis embryos followed by PM exposure, and secreted mucus from B. orientalis embryonic mucociliary epithelium was measured by ELLA using WGA-HRP. Pretreatment with antioxidants trolox and NAC (N-acetyl cysteine) did not affect PM-induced mucus hyposecretion. However, pretreatment with α-tocopherol significantly recovered the reduction in mucus secretion induced by PM. Statistical analysis was performed using one-way ANOVA. From left to right, n = 36, 32, 36, 34, 31, 36. (B) WGA positive mucus vesicles accumulated in embryonic mucociliary epithelium were visualized by fluorescent microscopy. Scale bar = 10 μm. (C) Quantification of WGA positive mucus vesicles in (B). Pretreatment with α-tocopherol ameliorated the accumulation of mucus vesicles induced by PM. Statistical analysis was performed using one-way ANOVA. From left to right, n = 7, 6, 7, 4, 6. Asterisks represent *(p < 0.001), (0.001 < p < 0.01), *(0.01 < p < 0.05), ns (0.05 < p). PM, particulate matter; ELLA, enzyme-linked lectin assay.
	Figure 7.Particulate matter induced the accumulation of mucus vesicles in human Calu-3 goblet cells. (A) PM treatment induced the accumulation of WGA positive mucus vesicles in Calu-3 cells. Mucus vesicles were stained with WGA-Alexa 488. Scale bar = 10 μm. (B) PM treatment induced the accumulation of MUC5AC positive mucus vesicles in Calu-3 cells. MUC5AC vesicles were stained with an anti-MUC5AC antibody. Consistent with the amphibian mucociliary epithelium, pretreatment with α-tocopherol significantly reduced the accumulation of mucus vesicles induced by PM in Calu-3 cells. Scale bar = 10 μm. (C,D) Quantification of WGA and MUC5AC positive vesicles from (A) and (B), respectively. Statistical analysis was performed using one-way ANOVA. For (C), from left to right, n = 6, 4, 6, 8, 8. For (D), n = 6, 5, 7, 4, 5. Asterisks represent *(p < 0.001), (0.001 < p < 0.01), *(0.01 < p < 0.05), ns (0.05 < p). PM, particulate matter.
	Figure 8. Schematic of the protective effect of α-tocopherol on particulate matter mucotoxicity. Our data suggest that acute exposure to PM causes mucus hyposecretion, likely through interference with secretory and exocytic processes in goblet cells. α-tocopherol offers a protective function over PM mucotoxicity. PM, particulate matter.