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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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Real-time PCR results of IL-6, IL-1, and TNF-α mRNAs. Real-time PCR results of IL-6, IL-1, and TNF-α mRNAs in SV40-immortalized HCECs pretreated with 17-β-estradiol and exposed to hyperosmolar media for 24 h. Controls were cells cultured in hyperosmolar media without pretreatment with 17-β-estradiol. IL-6, IL-1, and TNF-α mRNA expression was gradually upregulated in a 450 mOsM hyperosmotic medium from 3 to 24 h. Pretreatment with 17-β-estradiol greatly inhibited IL-6, IL-1, and TNF-α mRNA expression and the suppression effect lasted as long as 24 h. Similar results were obtained in three independent experiments. *p<0.05, **p<0.01.
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f2: Real-time PCR results of IL-6, IL-1, and TNF-α mRNAs. Real-time PCR results of IL-6, IL-1, and TNF-α mRNAs in SV40-immortalized HCECs pretreated with 17-β-estradiol and exposed to hyperosmolar media for 24 h. Controls were cells cultured in hyperosmolar media without pretreatment with 17-β-estradiol. IL-6, IL-1, and TNF-α mRNA expression was gradually upregulated in a 450 mOsM hyperosmotic medium from 3 to 24 h. Pretreatment with 17-β-estradiol greatly inhibited IL-6, IL-1, and TNF-α mRNA expression and the suppression effect lasted as long as 24 h. Similar results were obtained in three independent experiments. *p<0.05, **p<0.01.

Mentions: Real-time PCR analysis revealed that IL-6, IL-1, and TNF-α mRNA expression was gradually upregulated in a 450 mOsM hyperosmotic medium as exposure time was prolonged (3−24 h) (Figure 2). The pretreatment with 17-β-estradiol for 40 min greatly inhibited IL-6, IL-1, and TNF-α mRNA expression in the early stage of the hyperosmotic stimulation, and the suppression effect lasted as long as 24 h. The inhibitory effects of 17-β-estradiol on IL-6, IL-1, and TNF-α mRNA expression increased with time and at 24 h, the suppressive effects reached 90.1%, 60.9%, and 68.5%, respectively.


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)

Real-time PCR results of IL-6, IL-1, and TNF-α mRNAs. Real-time PCR results of IL-6, IL-1, and TNF-α mRNAs in SV40-immortalized HCECs pretreated with 17-β-estradiol and exposed to hyperosmolar media for 24 h. Controls were cells cultured in hyperosmolar media without pretreatment with 17-β-estradiol. IL-6, IL-1, and TNF-α mRNA expression was gradually upregulated in a 450 mOsM hyperosmotic medium from 3 to 24 h. Pretreatment with 17-β-estradiol greatly inhibited IL-6, IL-1, and TNF-α mRNA expression and the suppression effect lasted as long as 24 h. Similar results were obtained in three independent experiments. *p<0.05, **p<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Real-time PCR results of IL-6, IL-1, and TNF-α mRNAs. Real-time PCR results of IL-6, IL-1, and TNF-α mRNAs in SV40-immortalized HCECs pretreated with 17-β-estradiol and exposed to hyperosmolar media for 24 h. Controls were cells cultured in hyperosmolar media without pretreatment with 17-β-estradiol. IL-6, IL-1, and TNF-α mRNA expression was gradually upregulated in a 450 mOsM hyperosmotic medium from 3 to 24 h. Pretreatment with 17-β-estradiol greatly inhibited IL-6, IL-1, and TNF-α mRNA expression and the suppression effect lasted as long as 24 h. Similar results were obtained in three independent experiments. *p<0.05, **p<0.01.
Mentions: Real-time PCR analysis revealed that IL-6, IL-1, and TNF-α mRNA expression was gradually upregulated in a 450 mOsM hyperosmotic medium as exposure time was prolonged (3−24 h) (Figure 2). The pretreatment with 17-β-estradiol for 40 min greatly inhibited IL-6, IL-1, and TNF-α mRNA expression in the early stage of the hyperosmotic stimulation, and the suppression effect lasted as long as 24 h. The inhibitory effects of 17-β-estradiol on IL-6, IL-1, and TNF-α mRNA expression increased with time and at 24 h, the suppressive effects reached 90.1%, 60.9%, and 68.5%, respectively.

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