The Mechanism Of Gremlin On UV Induced-Skin Cells Damage

Objective:1.To study the effect of gremlin on Ultra Violet(UV)induced skin cells damage and the potential medicinal value of Gremlin against UV radiation.2.To investigate the mechanism of Gremlin in skin cells damage caused by UV irradiation.Method:1.The primary skin keratinocytes,HaCaT keratinocytes and human skin fibroblasts were maintained in medium.The above-mentioned cells were pretreated with gremlin,cells were subjected to UV radiation,cell viability was tested by MTT assay and trypan blue staining assay.To investigate the efficacy of Gremlin on UV induced skin cells damage and estimate the decent concentration.2.Gremlin at 25 ng/mL showed decent anti-UV activity,and this concentration was chosen for following experiments.The primary skin keratinocytes,HaCaT keratinocytes and human skin fibroblasts were pretreated with gremlin(25 ng/mL),cells were subjected to UV radiation,cell apoptosis was then tested by the Caspase-3 activity assay,TUNEL assay and Histone-DNA ELISA assay.3.The primary skin keratinocytes were blocked by the VEGFR2 inhibitors SU5416,Axitinib and ZD6474.The shRNA(VEGFR2)method was applied to knockdown VEGFR2 in keratinocytes.Primary skin keratinocytes were treated with gremlin(25 ng/mL).The expression of proteins(VEGFR2,p-VEGFR2,Akt,p-Akt)were tested by Western blot assay.Then,the cells were also subjected to UV radiation(20 mJ/cm~2),cell viability(MTT assay)and cell apoptosis(Histone DNA ELISA assay)were tested.To investigate the activation of VEGFR2-Akt signal pathway on UV induced skin cells damage with gremlin inhibition.4.Primary skin keratinocytes or the primary skin fibroblasts were pretreated with gremlin(25 ng/mL)were subjected to UV(20 mJ/cm~2)radiation,ROS production and DNA single strand breaks(SSBs)were tested.Result:1.UV decreased skin cells(skin keratinocytes,HaCaT keratinocytes and human skin fibroblasts)survival,which was largely inhibited with gremlin pretreatment.Gremlin demonstrated a dose-dependent response against UV in skin cells.2.UV induced skin cells(skin keratinocytes,HaCaT keratinocytes and human skin fibroblasts)apoptosis,which was inhibited with gremlin pretreatment.Gremlin inhibits UV-provoked apoptosis in skin keratinocytes and fibroblasts.3.Treatment with gremlin in primary skin keratinocytes induced significant VEGFR2 and Akt,the major downstream of VEGFR2,phosphorylation,which was blocked by the VEGFR2 inhibitors SU5416,Axitinib and ZD6474.Co-treatment with the VEGFR2 inhibitors in skin keratinocytes almost abolished gremlin-mediated cytoprotection against UV.The shRNA method was applied to knockdown VEGFR2 in keratinocytes.Gremlin-induced VEGFR2-Akt phosphorylation was also largely inhibited in VEGFR2-silenced cells.UV decreased skin cells death rate and apoptosis rate in VEGFR2-silenced cells.Gremlin-mediated cytoprotection against UV was almost nullified in VEGFR2-silenced keratinocytes.VEGFR2-Akt activation mediates gremlin-induced cytoprotection against UV.4.UV radiation to skin cells will induce ROS production and DNA single strand breaks(SSBs)in primary skin keratinocytes and human skin fibroblasts.Gremlin inhibits UV-induced ROS production and DNA SSBs in above-mentioned cells.Conclusion:1.Gremlin protects skin cells from UV damages2.Gremlin protects skin cells from UV damages via activating VEGFR2-Nrf2signaling.Gremlin could be further tested as a novel anti-UV skin protectant.