Cefradine Blocks Solar-ultraviolet Induced Skin Inflammation Through Direct Inhibition Of T-LAK Cell-originated Protein Kinase

Objective:Skin inflammation and skin cancer induced by excessive solar ultraviolet (SUV) is a great threat to human health. SUV induces skin inflammation through activating p38 mitogen-activated protein kinase (p38) and c-Jun N-termeinal kinases (JNKs). T-LAK cell-originated protein kinase (TOPK) plays an important role in this process. We want to search for TOPK inhibitor to alleviate SUV-induced skin inflammation.Methods:(1) Clinical data to detect the change of pathologic and the level of TOPK, phospho-p38, phospho-JNKs in human solar dermatitis. (2). Western blot to detect the level of SUV-induced phospho-p38, phospho-JNKs and γ-H2AX in HaCaT cells and JB6 cells. (3). Knock down TOPK to detect the level of SUV-induced phospho-p38, phospho-JNKs and γ-H2AX in HaCaT cells. (4). Molecule docking model to detect wheather cefradine can bind with TOPK. (5). the in vitro binding assay further verified the releationship between cefradine and TOPK. (6). In vitro kinase assay to detect wheather cefradine can inhibit TOPK activity. (7). In HaCaT and JB6 cells, MTS detect the effect of cefradine to the vibility of cells; Western blot to detect the effect of cefradine on SUV-induced the level of TOPK down-stream signling pathway; ELSIA to detect the effect of cefradine on SUV-induced the change of inflammatory foctor. (8). In Babl/c mice, HE to detect cefradine effect on SUV-induced the pathological change of skin; IHC to detect cefradine effect on SUV-induced the level of phospho-p38, phospho-JNKs and γ-H2AX; ELISA to detect cefradine effect on SUV-induced the secretion of inflammatory factor in Babl/c mouse.Result:(1). The clinical data showed TOPK, phospho-p38, phospho-JNKs were highly expressed in human solar dermatitis. (2). Western blot results studies showed that SUV induced the phosphorylation of p38, JNKs and H2AX in HaCaT cells and JB6 cells in a dose and time dependent manner. (3). Knock down TOPK inhibit SUV-induced phospho-p38, phospho-JNKs and γ-H2AX in HaCat cells. (4) Molecule docking model indicated cefradine, an FDA-approved cephalosporin antibiotic, directly binds with TOPK. (5). The result of in vitro binding assay verified cefradine can directly bind with TOPK. (6). In vitro kinase results showed cefradine can inhibit TOPK. activity. (7). In HaCaT and JB6 cells, MTS results showed cefradine has no effect to the viability of cells; Western blot results studies further showed cefradine inhibited SUV-induced the protein level of p-TOPK, p-p38, p-JNKs and p-ERK1/2, P-NF-kB p65, NF-kB p65 and γ-H2AX in a dose and time dependent manner; ELISA results showed that cefradine inhibited SUV-induced the secretion of IL6 and TNFa. (8). In Babl/c mice, HE results showed that cefradine inhibited SUV-induced the pathological change of skin in Babl/c mice; IHC results showed that cefradine down-regulated SUV-induced the phosphorylation of p38, JNKs and H2AX; ELISA results showed cefradine inhibited SUV-induced the secretion of IL6 and TNFa.Conclution. These results indicated that cefradine can inhibit SUV-induced skin inflammation by blocking TOPK signaling pathway, and TOPK is an effective target for suppressing inflammation induced by SUV irradiation.