This study presents that DHT suppressed the expression of proinflammatory cytokines in HConjECs under hyperosmolar condition. The DHT pretreatment caused a significant decrease in the levels of TNF-α, IL-1β, and IL-8 mRNA as compared with hyperosmolar media-exposed control cells. DHT pretreated cells inhibited phosphorylated p38 and ERK for 30 minutes after hyperosmolar stress but their expression upregulated in 1 hour. DHT increased the cell viability ratio compared to hyperosmolar media-exposed control cells.
DED is a disorder characterized by tear deficiency or excessive evaporation. In DED, the ocular surface under the hyperosmolar condition leads to oxidative stress and disruption of DNA. [21] Previous studies demonstrated the role of pro-inflammatory cytokines such as IL-1, TNF-α, IL-8 and matrix metalloproteinases (MMPs) in the pathogenesis of dry eye. [22–24] The conjunctival epithelial cells appear to be one source of the increased concentration of proinflammatory cytokines in the tear fluid and impression cytology samples of patients with DED. [25, 26]
Sex hormones affect the ocular structures involved in the production, regulation and maintenance of the normal tear film. [27] Their imbalance, especially reduction in androgen levels, may predispose individuals to the development of dry eye and autoimmune conditions affecting the ocular surface. [28] Androgens showed an anti-inflammatory effect on the expression and activity of inflammatory parameters in both health and disease. [29] Decreased serum androgens are associated with DED and add important evidence to the growing research that links androgens to DED. [30] Our previous study demonstrated that DHT has an immunoregulatory role in human corneal epithelial cells under hyperosmolar conditions. DHT decreased the inflammatory cytokine expressions while increased cell viability. [31] Present study showed that DHT suppressed TNF-α and IL-1β expression after hyperosmolar stress, and this effect persisted for up to 3 hours in human conjunctiva cell line. The expression of IL-8 was low after 30 minutes of hyperosmolar stress, but it was up-regulated after 1 hour in DHT treated HConjECs. While the 10− 8 M DHT suppressed the IL-8 mRNA level for up to 30 minutes, it lost suppressive effect in 1 hour. We considered that the administered dose is insufficient to suppress the IL-8 response over the long term, and higher doses may be required to suppress the IL-8 response. In vitro studies showed that testosterone treatment reduced the production of TNF-α, IL-1β, and IL-6 in human macrophages and human monocytes. [32] Sullivan et al. demonstrated that testosterone manage expression of thousands of genes in meibomian gland such as downregulation of IL-1β. [33] Marangoz et al. reported that DHT pretreatment HConjECs suppressed TNF-α, IL-6, IL-1β, VEGF-A cytokines when exposed to lipopolysaccharide. [34] Our data is in accordance with previous results and supports the regulatory effect of sex hormones especially androgens in conjunctival epithelial cells in inflammatory conditions.
Sahin et al. showed that HConjECs which was pretreated with 10nM DHT significantly increased 33 gene ontologies linked to the immune system. However, they did not indicate which inflammatory markers spesificly were increased in association with inflammation. [35] According to our results, TNF-α and IL-1β were downregulated in pretereated groups with DHT. We think that the difference between two studies originated from different stress model and time point of the stress application.
The purpose of our study was to investigate the specific pathway involved in the elevation of proinflammatory cytokines. The MAPKs are activated by diverse extracellular stimuli, such as growth factors, cytokines, mitogens, hormones, and other various cellular stresses including oxidative stress, heat shock, hypoxia, ischemia, ultraviolet irradiation, and DNA-damaging agents. The MAPK p38 which regulates the expression of several cytokines is activated in immune cells by inflammatory cytokines, and plays an important role in the activation of host immune response. The ERK is widely expressed and involved in the regulation of meiosis, mitosis and post-mitotic functions in a variety of cells. Cytokines are among the many different stimuli capable of activating the ERK pathways. [36, 37] Luo et al. reported the 500 mOsM hyperosmolar condition stimulates the secretion of proinflammatory cytokines, such as IL-1β, TNF-α and MMP-9 from ocular surface epithelia and increased levels of phosphorylated JNK1/2, ERK1/2, and p38 MAPKs in the corneal and conjunctival epithelia in experimental mice model. [38] Li et al. demonstrated that JNK and ERK MAPK mediate induction of TNF-α, IL-1β and IL-8 depend on hyperosmolar stress in human limbal epithelial cells [13]. Our results showed that the phosphorylated p38 and ERK were dramatically activated by hyperosmolar stress and the phosphorylation of p38 and ERK was greatly suppressed by pretreatment with DHT. To the best of our knowledge, there is no published study about the effect of DHT on the MAPK pathway and related proteins in HConjECs.
Apoptosis can be induced by different signals, including activation of Fas or TNF receptors, deprivation of growth factors, excessive DNA damage, treatment with chemotherapeutic drugs or stresses such as heat shock, osmotic shock, or UV irradiation. [39] Experimentally induced dry eye in mice causes apoptosis of cells in ocular surface tissues including the central and peripheral corneal epithelium, bulbar and tarsal conjunctival epithelia, tarsal conjunctival stroma, and lid margin. [40] Brignole et al showed that there is strong correlation between apoptotic and inflammatory pathways in the ocular surface. [41] According to our results, DHT decreased the percentage of apoptosis in HConjECs compared to high osmolarity groups without treatment. There is no published study about the protective effect of DHT against apoptosis in HconjECs under hyperosmolar conditions.
The Limitation of our study is using Phenol-Red media in our in-vitro experiments. Phenol red as a pH indicator is available in most tissue culture media and acts as a weak estrogen that can trigger some estrogen-sensitive cells. However, the relative impact of phenol red on various cell lines is controversial. [42] Estrogens and androgens are well known to exert opposing effects in several tissues. The previous studies demonstrated that estrogen reduced androgen receptor levels in human mammary cancer cells. [43, 44] We should notice that the source of the phenol red and the sensitivity of different cell types to the indicator show its effect on the response of cells in tissue culture. However, we use different cell types and have a control group in the experiment.
In conclusion, these findings support our hypothesis DHT showed an anti-inflammatory effect on HConjECs under hyperosmolar conditions and this effect is associated with the MAPK pathway.