Ionizing Radiation Promoting CCL27 Secretion By Crosstalk Of TNF-alpha Autocrine And ROS Excitation

Objective:Radiation skin injury is one of skin inflammations by physical factor causing; meanwhile, the acute radiation-induced skin reaction is the most common complication in the patients with cancer radiotherapy. The alterations of skin, including erythema, moist desquamation, and more serious skin reaction, were found in about 90% patients, Severe radiation-induced skin injury can cause the reduction of radiotherapy efficiency for the sake of limitation of radiation dose. To immune reaction of skin, chemokines plays an important role on the multi-steps homing pattern of T-cells,including Chemokine 27 (CCL27). CCL27 is one small molecular protein and secreted from keratinocytes of epiderm, which distributes in the special tissue and cells. In present study, the human keratinocyte HaCaT cell line was used to investigate the change of CCL27 secretion from irradiated cell.Methods:ROS levels in keratinocytes exposure to 2Gy and 5Gy irradiation were firstly detected by DCFH-DA probe. Simultaneously, the concentrations of CCL27 and tumor necrosis factor-α (TNF-α) in medium were measured by ELISA. To certifying the alteration of CCL27 secretion via Oxygen reactive species ROS inducing,5mM N-Acetylcysteine, one ROS scavenger, was used to treat keratinocytes prior to exposure to irradiation. The intracellular ROS level and the concentrations of CCL27 and TNF-α were then detected respectively. Additionally, CCL27 concentration was assayed with H2O2 (400μM) and TNF-α (10ng/ml) pre-treatment respectively. After HaCaT cells were translated with TNF-α siRNA. The ROS levels in keratinocytes exposure to 2Gy and 5Gy irradiation were detected by DCFH-DA probe. Subquestly TNF-α concentration was assayed after H2O2 treatment alone. The ROS levels in keratinocytes pre-treated with TNF-α (10ng/ml).Results:The results showed that the concentrations of CCL27 and TNF-a in medium were increased after HaCaT cells were exposed to X-rays in different doses, concomitance with the elevation of ROS level. On other hand, the concentrations of CCL27 and TNF-a were secreated from irradiated keratinocytes down-regulated by NAC pretreatment. CCL27 concentration in the medium of keratinocytes was increased by H2O2 or TNF-a treatment alone. The ROS levels in the keratinocytes translated with siRNA was reduced after these cells exposure to 2Gy and 5Gy. Interestingly TNF-a secreated from HaCaT cells was increased by H2O2 (400μM) treatment alone. Moreover intracellular ROS levels were was increased by TNF-α pre-treatment.Conclusion:It is therefore concluded that ionizing radiaiton can promote CCL27 secretion from keratinocytes. Such increased levels may be correlation with the crosstalk of TNF-a autocrine and ROS excitation.