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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

ELISA results regarding the amounts of IL-6, IL-1, and TNF-α in HCECs. Pretreatment with 17-β-estradiol inhibited the hyperosmolarity- induced increases in IL-6, IL-1, and TNF-α production. Data are the mean±SEM of the results from three independent experiments. With pretreatment with 17-β-estradiol, IL-6, IL-1, and TNF-α production was greatly inhibited (black box) as compared with cells cultured in hyperosmolar media without pretreated 17-β-estradiol (white box). *p<0.05, **p<0.01.
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f3: ELISA results regarding the amounts of IL-6, IL-1, and TNF-α in HCECs. Pretreatment with 17-β-estradiol inhibited the hyperosmolarity- induced increases in IL-6, IL-1, and TNF-α production. Data are the mean±SEM of the results from three independent experiments. With pretreatment with 17-β-estradiol, IL-6, IL-1, and TNF-α production was greatly inhibited (black box) as compared with cells cultured in hyperosmolar media without pretreated 17-β-estradiol (white box). *p<0.05, **p<0.01.

Mentions: ELISA was used to determine the effects of the 40-min pretreatment with 10−10 M 17-β-estradiol on the hyperosmolarity-induced increases in IL-6, IL-1, and TNF-α production. At 24 h, IL-6, IL-1, and TNF-α production was about 2–4 fold greater than that of the control group, without the 17-β-estradiol pretreatment. At 24 h, IL-6, IL-1, and TNF-α protein in pretreated cells were as much as 68.65%, 43.68%, and 62.73% less, respectively, than in cells that had not been pretreated with 17-β-estradiol (Figure 3). That is, comparing the two groups, these proteins were present in a smaller amount in the pretreated cells than in the non-pretreated cells. These results indicated that IL-6, IL-1, and TNF-α production was greatly inhibited in the 17-β-estradiol-pretreated cells.


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)

ELISA results regarding the amounts of IL-6, IL-1, and TNF-α in HCECs. Pretreatment with 17-β-estradiol inhibited the hyperosmolarity- induced increases in IL-6, IL-1, and TNF-α production. Data are the mean±SEM of the results from three independent experiments. With pretreatment with 17-β-estradiol, IL-6, IL-1, and TNF-α production was greatly inhibited (black box) as compared with cells cultured in hyperosmolar media without pretreated 17-β-estradiol (white box). *p<0.05, **p<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: ELISA results regarding the amounts of IL-6, IL-1, and TNF-α in HCECs. Pretreatment with 17-β-estradiol inhibited the hyperosmolarity- induced increases in IL-6, IL-1, and TNF-α production. Data are the mean±SEM of the results from three independent experiments. With pretreatment with 17-β-estradiol, IL-6, IL-1, and TNF-α production was greatly inhibited (black box) as compared with cells cultured in hyperosmolar media without pretreated 17-β-estradiol (white box). *p<0.05, **p<0.01.
Mentions: ELISA was used to determine the effects of the 40-min pretreatment with 10−10 M 17-β-estradiol on the hyperosmolarity-induced increases in IL-6, IL-1, and TNF-α production. At 24 h, IL-6, IL-1, and TNF-α production was about 2–4 fold greater than that of the control group, without the 17-β-estradiol pretreatment. At 24 h, IL-6, IL-1, and TNF-α protein in pretreated cells were as much as 68.65%, 43.68%, and 62.73% less, respectively, than in cells that had not been pretreated with 17-β-estradiol (Figure 3). That is, comparing the two groups, these proteins were present in a smaller amount in the pretreated cells than in the non-pretreated cells. These results indicated that IL-6, IL-1, and TNF-α production was greatly inhibited in the 17-β-estradiol-pretreated cells.

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