Paeonol Suppresses Solar Ultraviolet-induced Skin Inflammation By Targeting T-LAK Cell-originated Protein Kinase

Objective:Excessive exposure to solar UV(SUV)is related with numerous human skin disorders,such as skin inflammation,photoaging and carcinogenesis.P38 mitogen-activated protein kinase(p38)and c-Jun N-terminal kinases(JNKs),two major members of MAPK family which can be activated by SUV irridation,are involved in the initiation and the development of inflammation.T-LAK cell originated protein kinase(TOPK),an upstream of p38 andJNKs,plays an important role in SUV-induced skin inflammation,and targeting TOPK has already been a strategy to prevent skin inflammation.We want to find a new TOPK inhibitor to prevent SUV-induced skin inflammation.Methods:(1)H&E staining was used to detect the pathological changes and IHC method to detect the level of TOPK,phospho-p38,phospho-JNK in human solar dermatitis.(2)Western blot was used to detect the level of phospho-p38,phospho-JNK in HaCat and JB6 cells after SUV irritation.(3)MTS assay was used to detect the toxicity of paeonol toHaCat and JB6 cells.(4)MST assay was used to detect the affinity between paeonol and TOPK in vitro.(5)In vitro kinase assay was used to detect if paeonol can inhibit the activity of TOPK.(6)Western blot was used to detect the effect of paeonol onthe downstream of TOPK in HaCat and JB6 cells.(7)ELISA was used to detect the effect of paeonol on inflammation-associated-cytokines such as IL-6 and TNF-a secreted byHaCat and JB6 cells.(8)Western blot was used to detect the effect of paeonol on the downstream of TOPKin Babl/c mice.(9)ELIS A was used to detect the effect of paeonol on inflammation-associated-cytokines such as IL-6 and TNF-a secretedin the skin of Babl/c mice.RESULTS:(1)The clinical data indicated that the levels of TOPK,p-p38,p-JNKs were highly increased in human dermatitis.(2)The phosphorylation level of p38 and JNKs were increased in a dose and time dependent manner in JB6 C141 and HaCat cells after SUV irradiation.(3)MTS assay showed that paeonol has no toxicity to both HaCat and JB6 cells.(4)MST assay showed that paeonol has a high affinity with TOPK.(5)In vitro kinase assay showed that paeonol could inhibit the activity of TOPK.(6)Paeonol down-regulates SUV-induced downstream TOPK signaling pathway in a dose-and time-dependent manner in the JB6 Cl41 and HaCat cells.(7)Paeonol caninhibit the secretion of cytokines irritated by SUV in the JB6 C141 and HaCat cells.(8)Paeonol can inhibit the increase of p-p38,p-JNKs andγ-H2AX irritated by SUV in Babl/c mice.(9)Paeonol can inhibit the secretion of IL-6 and TNF-a irritated by SUV in Babl/c mice.Conclusion:Our data indicated a protective role of paeonol against SUV-induced inflammation by targeting TOPK,and paeonol could be a promising agent for the treatment of SUV-induced skin inflammation.