The suppressive role of sex steroids in inflammatory response and viability of limbal mesenchymal stem cells under stress condition

Abstract

AbstractPurposeTo investigative the regulatory role of sex steroids on mitogen‐activated protein kinase pathway, autophagy pathway, and cell survival of Limbal Mesenchymal Stem Cells (LMSCs) under serum starvation induced stress conditions.MethodsThe L‐MSCs were isolated from cadaver corneal tissues and were characterized by flow cytometry and were differentiated into 3 lineages. The L‐MSCs in passage 3 were cultured with low glucose DMEM completed with fetal bovine serum (FBS) and penicillin‐streptomycin until reaching 80% confluency. After confluency, cells were trypsinized and cultured with charcoal FBS containing medium either with 10‐8 M dihydrotestosterone (DHT) or 10‐8 M 17‐β‐estradiol. After 48 hours, the medium was changed to only DMEM without any serum supplement for the purpose of serum starvation. Serum starvation treated cells for all conditions were lysed and western blot analysis was applied for phosphorylated forms of extracellular signal‐regulated kinase 1/2 (ERK1/2), p38 kinase, c‐Jun N‐terminal kinase 1/2 (JNK1/2) and as an autophagic marker, Beclin‐1 ATG‐7 and LC3I‐II. The LMSCs viability was measured with Annexin V/propidium iodide.ResultsP‐JNK and P‐ERK were inhibited only by 17‐β‐estradiol pretreated groups while pretreatment LMSCs with DHT and 17‐β‐estradiol suppressed the upregulation of p‐p38 up to 4 hours of serum starvation stress. The expression of Beclin‐1 was not significantly changed in both groups, while expression of ATG7 was significantly inhibited in the 17‐β‐estradiol group for up to 4 hours. The conversion of the LC3‐I to LC3‐II was found to be suppressed in both groups compared to the control group. DHT and 17‐β‐estradiol pretreated L‐MSCs were increased cell survival compared to untreated conditions.ConclusionAccording to our results, DHT and 17‐β‐estradiol especially 17‐β‐estradiol suppressed the mitogen‐activated protein kinase signalling pathway and autophagic pathway. These findings may help to explain the function of sex hormones in various ocular surface illnesses, as well as their implications for treatment.