Purpose  To evaluate the effects of estrogen on hyperosmolar stress-induced proinflammatory cytokine production of interleukin (IL)-6 IL-1 and TNF-α in SV40 immortalized human corneal epithelial cells (SV40 HCECs) and the regulatory effects of MAPK signaling pathways on this process.

Methods  SV40 HCECs cultured in normal osmolar media were switched to a higher osmolarity (450 mOsM) achieved by adding NaCl with or without pretreatment of 17-β-estradiol. Real-time polymerase chain reaction (PCR) and ELISA characterized IL-6, IL-1, and TNF-α gene and protein expression, respectively. Cells treated for 15−60 min were lysed in RIPA buffer and subjected to Western blot with phospho (p)-specific antibodies against ERK1/2, P38 and JNK1/2.
Results  17-β-estradiol at concentrations of 10^-10 and 10^-11 M did not affect the cell viability of cultured corneal epithelial cells. 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 administration of SB203580 (10 μM), an inhibitor of the p38 pathway, the inhibiting effect of 17-β-estradiol disappeared. 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 stimulated by hyperosmolarity via the p38 MAPK signaling pathway in SV40 immortalized HCECs. These findings may contribute to the further understanding of the etiologic roles and therapeutic implications of the hormone estrogen in dry eye disease.