title: 17-β-estradiol inhibits hyperosmolarity-induced proinflammatory cytokine elevation via the p38 MAPK pathway in human corneal epithelial cells.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Ophthalmology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Jiefang Road 88, Hangzhou, China.
authors.forename: Changjun
authors.initials: C
authors.lastname: Wang
authors.forename: Xin
authors.initials: X
authors.lastname: Shi
authors.forename: Xiaoyi
authors.initials: X
authors.lastname: Chen
authors.forename: Han
authors.initials: H
authors.lastname: Wu
authors.forename: Huina
authors.initials: H
authors.lastname: Zhang
authors.forename: Jiajun
authors.initials: J
authors.lastname: Xie
authors.forename: Xianyan
authors.initials: X
authors.lastname: Yang
authors.forename: Zhongru
authors.initials: Z
authors.lastname: Gou
authors.forename: Juan
authors.initials: J
authors.lastname: Ye
mesh_terms: D000952:Antigens, Polyomavirus Transforming; D002461:Cell Line, Transformed; D003315:Cornea / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004958:Estradiol / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007375:Interleukin-1 / Q000096:biosynthesis / Q000235:genetics; D015850:Interleukin-6 / Q000096:biosynthesis / Q000235:genetics; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009994:Osmolar Concentration; D009997:Osmotic Pressure; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000952:Antigens, Polyomavirus Transforming; C508600:IL6 protein, human; D007093:Imidazoles; D007375:Interleukin-1; D015850:Interleukin-6; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D014409:Tumor Necrosis Factor-alpha; D012965:Sodium Chloride; D004958:Estradiol; D020928:Mitogen-Activated Protein Kinases; C093642:SB 203580