| UVB could cause DNA damage thereby resulting in skin diseases.Overexposure to UVB results in skin inflammation,cell death,even cancer.Physical protection such as sunscreen is primarily used for protection against UVB,but it does not exert biological effect against UVB damage.Therefore,effective drugs are wanted.We have been focusing on revealing the pharmacological effects of silibinin,a flavonoid compound,against UVB damage.It inhibits UVB-induced skin inflammation and apoptosis,but the underlying mechanism has still remained obscure.It is difficult to identify the precise target that is responsible for the function of silibinin,because that UVB brings versatile and subtle effects on skin.Toll-like receptor 3(TLR3)is a member of pattern recognition receptors that recognizes double-stranded RNA(dsRNA)derived from virus or self-damaged cells.It was most recently discovered that TLR3 initiates skin immune responses and induces severe skin inflammation by detecting UVBdamaged dsRNA released by necrotic cells.Most of the previous studies established UVB irradiation model with high doses to try to understand the cause of UVB-induced skin damage,low dose of UVB irradiation may better represent radiation received by skin cells,even though its effect is rather subtle.In addition,previous studies did not take the role played by TLR3 into account.In this study,I combine two factors,TLR3 activation and UVB irradiation,to investigate how they work on skin cells,to find out a possible target(s)of silibinin.Poly I:C,a synthetic TLR3 ligand,alone does not cause cell death in human epidermal HaCaT cells.However,it works synergistically with sublethal UVB irradiation(UVB-Poly I:C)to induce cell death.Occurrence of apoptosis was examined by Annexin V/PI staining,and the alternation of apoptosis-related protein was detected by western blot analysis.Results show that UVB-Poly I:C induces apoptosis.Z-VAD,a caspase inhibitor,significantly blocked cell death.Silencing TLR3 with siRNA significantly inhibited apoptosis.NF-κB lies in downstream of TLR3 pathway.This study indicates that UVB-Poly I:C promotes nuclear translocation of NF-κB and up-regulates the expression of TNFα.BAY,an inhibitor of NF-κB,attenuated UVB-Poly I:Cinduced apoptosis.Further studies focusing on the activation of NF-κB signaling pathway revealed that any of the three members in NF-κB family with transcription activity,RelA,RelB and RelC,was not responsible for UVB-Poly I:C-induced apoptosis.IκBα is the major inhibitory protein of NF-κB,which also down-regulates apoptosis by protecting mitochondrial membrane integrity.Expression of IκBα decreased in UVB-Poly I:C co-treatment,and silencing IKBα slightly enhanced UVB-Poly I:C-induced cell death.Staining mitochondria with MitoTracker Deep Red indicated that silencing IκBα causes reduction of mitochondrial membrane potential.Furthermore,MG132,a proteasome inhibitor that prevents IκBαdegradation,rescues mitochondrial dysfunction in UVB-Poly I:C-treated HaCaT cells.However,retaining IκBα with MG 132 did not inhibit UVB-Poly I:C-induced apoptosis.These imply that IκBα may only partly contribute to suppression of apoptosis,or reduction of IκBαis not the central inducer of apoptosis.IKK complex,an upstream molecule in NF-κB pathway,containing two catalytic units IKKα/IKKβ and one regulatory unit NEMO,phosphorylates IκBα and triggers activation of NF-κB.UVB-Poly I:C induces sustained phosphorylation of IKKα/β.Using IKK inhibitor,IKK-16,or silencing IKK complex with siRNA revealed that IKK-16 reduces apoptosis of UVB-Poly I:C,and silencing of IKKα and NEMO inhibits UVBPoly I:C-induced apoptosis.IKKα induces apoptosis directly through p53 family,independently of transcriptional activity of NF-κB.Silencing p53 showed no effect on UVBPoly I:C-induced apoptosis,but silencing of another member of p53 family,p73,significantly inhibited UVB-Poly I:C-induced apoptosis.EGFR is one of the receptor tyrosine kinases,which plays an important role in skin immune responses and closely related to several skin diseases including psoriasis.Moreover,EGFR is essential for the activation of TLR3.In this study,it was found that UVB-Poly I:C-induced apoptosis is also dependent on the kinase activity of EGFR.Phosphorylation of IKKα and apoptosis induction by UVB-Poly I:C were significantly inhibited by AG 1478,an EGFR kinase inhibitor.Silibinin has a significant inhibitory effect on cell death and TNFα production induced by UVB-Poly I:C.Silibinin inhibited the phosphorylation of IKKα caused by UVB-Poly I:C and down-regulated NF-κB nuclear translocation and TNFα production.In summary,combining sublethal UVB irradiation with relatively low dose of Poly I:C triggers severe apoptosis and induces upregulation of TNFαin HaCaT cells,demonstrating that UVB-Poly I:C is an innovative model for study on skin damage caused by UVB.The model illustrates a plausible mechanism accounting for the loss of skin cells under low doses of UVB irradiation.EGFR disorder per se is not the direct cause of skin disease,but the findings that it interacts with TLR3 in skin cells should be taken into consideration in the pathology of some skin diseases.Silibinin is not just a promising drug that prevents against the skin damage caused by high doses of UVB irradiation but the skin damage caused by sublethal doses of UVB irradiation as shown in the present study using UVB-Poly I:C model.Silibinin with its protective effect on UVB-Poly I:C model against apoptosis induction and TNFα expression.EGFR,a key factor in skin health,may be another principal target of silibinin... |
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