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17-β-estradiol inhibits hyperosmolarity-induced proinflammatory cytokine elevation via the p38 MAPK pathway in human corneal epithelial cells.

Wang C, Shi X, Chen X, Wu H, Zhang H, Xie J, Yang X, Gou Z, Ye J - Mol. Vis. (2012)

Bottom Line: Pretreatment with 10(-10) M 17-β-estradiol greatly inhibited the increased expression and production of IL-6, IL-1, and TNF-α induced by hyperosmolarity, whereas with the administration of SB203580 (10 μM), an inhibitor of the p38 pathway, the inhibiting effect of 17-β-estradiol disappeared.The western blot results showed that the increased phosphorylation level of p38 caused by hyperosmolarity was greatly inhibited by 17-β-estradiol. 17-β-estradiol greatly inhibited the expression and production of proinflammatory cytokines IL-6, IL-1, and TNF-α, which were stimulated by hyperosmolarity in SV40-immortalized hCECs.The results also suggested that the p38 MAPK signaling pathway was involved in the regulatory effects of estrogen on hCECs.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Jiefang Road 88, Hangzhou, China.

ABSTRACT

Purpose: To evaluate the effects of 17-β-estradiol on hyperosmolar stress-induced proinflammatory cytokine production of interleukin (IL)-6, IL-1, and tumor necrosis factor-alpha (TNF-α) in SV40-immortalized human corneal epithelial cells (hCECs) and the regulatory effects of the mitogen-activated protein kinase (MAPK) signaling pathways in this process.

Methods: SV40 hCECs cultured in normal osmolar media were switched to a higher osmolarity (450 mOsM) by adding NaCl with or without pretreatment with 17-β-estradiol. Real-time polymerase chain reaction and ELISA were applied to characterize IL-6, IL-1, and TNF-α gene and protein expression. Cells were treated for 15-60 min, lysed in radioimmunoprecipitation assay (RIPA) buffer and subjected to a western blot with phospho (p)-specific antibodies against extracellular signal-regulated protein kinase 1/2 (ERK1/2), P38 kinase, and c-Jun N-terminal kinase 1/2 (JNK1/2).

Results: The expression and production of IL-6, IL-1, and TNF-α in SV40 hCECs increased when the media osmolarity was switched to 450 mOsM. Pretreatment with 10(-10) M 17-β-estradiol greatly inhibited the increased expression and production of IL-6, IL-1, and TNF-α induced by hyperosmolarity, whereas with the administration of SB203580 (10 μM), an inhibitor of the p38 pathway, the inhibiting effect of 17-β-estradiol disappeared. The western blot results showed that the increased phosphorylation level of p38 caused by hyperosmolarity was greatly inhibited by 17-β-estradiol.

Conclusions: 17-β-estradiol greatly inhibited the expression and production of proinflammatory cytokines IL-6, IL-1, and TNF-α, which were stimulated by hyperosmolarity in SV40-immortalized hCECs. The results also suggested that the p38 MAPK signaling pathway was involved in the regulatory effects of estrogen on hCECs. These findings may contribute to an understanding of the etiologic roles and therapeutic implications of the hormone estrogen in dry eye disease.

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Related in: MedlinePlus

Effects of 17-β-estradiol on cell viability. Cells were cultured with 17-β-estradiol at different concentrations for 24 h. The percentage of apoptotic cells were calculated (10−8 M 14.24%; 10−9 M 11.27%; 10−10 M 5.11%; 10−11 M 5.38%, respectively). Statistical analysis showed that 17-β-estradiol at concentrations of 10−10 and 10−11 M did not show a significant difference (p>0.05) as compared with the controls, cells cultured without 17-β-estradiol.
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f1: Effects of 17-β-estradiol on cell viability. Cells were cultured with 17-β-estradiol at different concentrations for 24 h. The percentage of apoptotic cells were calculated (10−8 M 14.24%; 10−9 M 11.27%; 10−10 M 5.11%; 10−11 M 5.38%, respectively). Statistical analysis showed that 17-β-estradiol at concentrations of 10−10 and 10−11 M did not show a significant difference (p>0.05) as compared with the controls, cells cultured without 17-β-estradiol.

Mentions: Flow cytometry (Figure 1) showed that cultured hCECs treated with 17-β-estradiol at concentrations of 10−10 M and 10−11 M for 24 h did not show a significant difference in cell viability as compared with cells cultured without 17-β-estradiol (Figure 1; control). With the use of 17-β-estradiol at concentrations of 10−8 M and 10−9 M, the cell apoptosis percentages were increased to 14.24% and 11.27%, respectively, which suggested that treatment with 17-β-estradiol at concentrations higher than 10−10 M may have adverse effects on cell survival. Therefore, we used 10−10 M 17-β-estradiol in all of the following experiments in this study.


17-β-estradiol inhibits hyperosmolarity-induced proinflammatory cytokine elevation via the p38 MAPK pathway in human corneal epithelial cells.

Wang C, Shi X, Chen X, Wu H, Zhang H, Xie J, Yang X, Gou Z, Ye J - Mol. Vis. (2012)

Effects of 17-β-estradiol on cell viability. Cells were cultured with 17-β-estradiol at different concentrations for 24 h. The percentage of apoptotic cells were calculated (10−8 M 14.24%; 10−9 M 11.27%; 10−10 M 5.11%; 10−11 M 5.38%, respectively). Statistical analysis showed that 17-β-estradiol at concentrations of 10−10 and 10−11 M did not show a significant difference (p>0.05) as compared with the controls, cells cultured without 17-β-estradiol.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351403&req=5

f1: Effects of 17-β-estradiol on cell viability. Cells were cultured with 17-β-estradiol at different concentrations for 24 h. The percentage of apoptotic cells were calculated (10−8 M 14.24%; 10−9 M 11.27%; 10−10 M 5.11%; 10−11 M 5.38%, respectively). Statistical analysis showed that 17-β-estradiol at concentrations of 10−10 and 10−11 M did not show a significant difference (p>0.05) as compared with the controls, cells cultured without 17-β-estradiol.
Mentions: Flow cytometry (Figure 1) showed that cultured hCECs treated with 17-β-estradiol at concentrations of 10−10 M and 10−11 M for 24 h did not show a significant difference in cell viability as compared with cells cultured without 17-β-estradiol (Figure 1; control). With the use of 17-β-estradiol at concentrations of 10−8 M and 10−9 M, the cell apoptosis percentages were increased to 14.24% and 11.27%, respectively, which suggested that treatment with 17-β-estradiol at concentrations higher than 10−10 M may have adverse effects on cell survival. Therefore, we used 10−10 M 17-β-estradiol in all of the following experiments in this study.

Bottom Line: Pretreatment with 10(-10) M 17-β-estradiol greatly inhibited the increased expression and production of IL-6, IL-1, and TNF-α induced by hyperosmolarity, whereas with the administration of SB203580 (10 μM), an inhibitor of the p38 pathway, the inhibiting effect of 17-β-estradiol disappeared.The western blot results showed that the increased phosphorylation level of p38 caused by hyperosmolarity was greatly inhibited by 17-β-estradiol. 17-β-estradiol greatly inhibited the expression and production of proinflammatory cytokines IL-6, IL-1, and TNF-α, which were stimulated by hyperosmolarity in SV40-immortalized hCECs.The results also suggested that the p38 MAPK signaling pathway was involved in the regulatory effects of estrogen on hCECs.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Jiefang Road 88, Hangzhou, China.

ABSTRACT

Purpose: To evaluate the effects of 17-β-estradiol on hyperosmolar stress-induced proinflammatory cytokine production of interleukin (IL)-6, IL-1, and tumor necrosis factor-alpha (TNF-α) in SV40-immortalized human corneal epithelial cells (hCECs) and the regulatory effects of the mitogen-activated protein kinase (MAPK) signaling pathways in this process.

Methods: SV40 hCECs cultured in normal osmolar media were switched to a higher osmolarity (450 mOsM) by adding NaCl with or without pretreatment with 17-β-estradiol. Real-time polymerase chain reaction and ELISA were applied to characterize IL-6, IL-1, and TNF-α gene and protein expression. Cells were treated for 15-60 min, lysed in radioimmunoprecipitation assay (RIPA) buffer and subjected to a western blot with phospho (p)-specific antibodies against extracellular signal-regulated protein kinase 1/2 (ERK1/2), P38 kinase, and c-Jun N-terminal kinase 1/2 (JNK1/2).

Results: The expression and production of IL-6, IL-1, and TNF-α in SV40 hCECs increased when the media osmolarity was switched to 450 mOsM. Pretreatment with 10(-10) M 17-β-estradiol greatly inhibited the increased expression and production of IL-6, IL-1, and TNF-α induced by hyperosmolarity, whereas with the administration of SB203580 (10 μM), an inhibitor of the p38 pathway, the inhibiting effect of 17-β-estradiol disappeared. The western blot results showed that the increased phosphorylation level of p38 caused by hyperosmolarity was greatly inhibited by 17-β-estradiol.

Conclusions: 17-β-estradiol greatly inhibited the expression and production of proinflammatory cytokines IL-6, IL-1, and TNF-α, which were stimulated by hyperosmolarity in SV40-immortalized hCECs. The results also suggested that the p38 MAPK signaling pathway was involved in the regulatory effects of estrogen on hCECs. These findings may contribute to an understanding of the etiologic roles and therapeutic implications of the hormone estrogen in dry eye disease.

Show MeSH
Related in: MedlinePlus