Andrographolide Sodium Bisulfite Alleviates UV-induced Photo-damage Based On The Balance System Between Nrf2 And NF-κB Signaling Pathways In HaCaT Keratinocytes

ObjectiveExperiments were designed to explore the underlying mechanisms of ASB,which was a kind of soluble derivative composed of andrographolide and sodium bisulfatein,in relieving UV-induced(UV,97%UVA+3%UVB)photo-damage.Methods1.The effect of ASB on the viability of UV-induced HaCaT cells.HaCaT cells were irradiated with different doses of UV.MTT assay was used to screen out the appropriate dose of UV.HaCaT cells were pretreated with different concentrations of ASB for 24 h.Cell viability was detected with MTT assay,and the safe concentrations of ASB were screened out.2.The effect of ASB on viability,apoptosis and necrosis in UV-induced HaCaT cells.HaCaT cells were pretreated with ASB(10,30 and 100 μM)for 24 h and then were irradiated with UV(90 mJ/cm2=300 μW/cm2·s×300 s).Fluorescence intensity was determined with Hoechst 33342 and propidium iodide(PI)double staining 12 h later.MTT assay was used to test the cell viability 24 h later.3.The impact of UV on NF-κB and Nrf2 signaling pathways.HaCaT cells were irradiated with UV(90 mJ/cm2).RT-qPCR was used to assay the mRNA expressions of antioxidant genes(HO-1,GCLC and NQO1)and inflammatory genes(IL-1β,IL-6 and TNF-α)2 h later.After UV irradiation(90 mJ/cm2),the cytoplasmic and nuclear protein expressions of p65,IκBα,Nrf2 and keap1 at different time point(0,0.5,1,2,3,4,5 and 6 h)were detected by Western blot.HaCaT cells were pretreated with ML385(5 μM)for 12 h and irradiated with UV(90 mJ/cm2).The mRNA expression of IL-6 and the protein expression of nuclear p65 were assayed by RT-qPCR and Western blot separately 2 h later.MTT assay was used to detect the cell viability at 24 h after irradiation.4.The effect of ASB on inflammation and oxidative stress in UV-induced HaCaT cells.HaCaT cells were pretreated with ASB(10,30 and 100 μM)for 24 h and irradiated with UV(90 mJ/cm2).DCFH-DA staining was used to detect the fluorescence intensity of ROS 1 h later.Antioxidant genes GCLC and NQO1 were assessed by RT-qPCR 2 h later.The levels of TNF-α,IL-1β and IL-6 in the supernatant of the cultured HaCaT cells were detected by ELISA 12 h and 24 h later.5.The effect of ASB on Nrf2 and NF-κB singaling pathways.HaCaT cells were pretreated with ASB(10,30 and 100 μM)for 24 h and irradiated with UV(90 mJ/cm2).The cells were respectively collected at 2 h and 6 h after UV irradiation.Then the cytoplasmic and nuclear protein expressions of p65,IκBα,Nrf2 and keapl were detected by Western blot.The protein expressions and localization of p65 and Nrf2 were assayed by immunofluorescence.Results1.The cell viability was gradually decreased in a dose-dependent manner(0 mJ/cm2,10 mJ/cm2,30 mJ/cm2,60 mJ/cm2 and 90 mJ/cm2).Compared with NC group,when the UV dose was higher or equal to 60 mJ/cm2,the cell viability was significantly decreased(P<0.05).When the UV dose was higher or equal to 90mJ/cm2,the cell viability was lower than 50%(P<0.01,vs.NC group).ASB at the concentration of 0.01～2000 μM had no cytotoxicity on HaCaT cells.90 mJ/cm2 of UV and 10,30 and 100 μM of ASB were used in the following experiments.2.Compared with control group,the cell viability was markedly reduced in UV-induced HaCaT cells(P<0.01).And it was significantly increased by ASB(P<0.01).Observation with ordinary light showed that,compared with the NC group,the number of the HaCaT cells was significantly decreased and the cell refraction and adhesion abilities were weakened;Compared with the UV group,pretreatment with ASB obviously increased cell number,restored cell morphology,and enhanced cell refraction and adhesion abilities.Moreover,observation with fluorescent light showed that,compared with the NC group,apoptotic cells in UV group was significantly increased,which was manifested by cell volume reduction and nuclear fragmentation.At the same time some necrotic cells were appeared;Compared with the UV group,ASB dose-dependently reduced the apoptosis and necrosis of HaCaT cells.3.Compared with the NC group,the mRNA expressions of HO-1,GCLC and NQO1 were markedly decreased(P<0.01),that of IL-1β,IL-6 and TNF-α were markedly increased(P<0.01).The time-effect experiment results demonstrated that,after UV irradiation(0,0.5,1,2,3,4,5 and 6 h),the protein expressions of Nrf2 and NF-κB singaling pathways were continuously changed.The protein expression of cytoplasmic Nrf2 and keapl were continuously increased and peaked at 3 h,then gradually decreased.The protein expression of nuclear Nrf2 was peaked at 1 h and lower than 0 h after 5 h.The protein expressions of cytoplasmic p65 and IκBα were continually increased from 0.5 to 6 h,while the protein expression of nuclear p65 was peaked at 3 h,and then gradually decreased.Compared with the NC group,the cell viability were markedly decreased(P<0.01),the protein expression of nuclear p65 and mRNA expression of IL-6 were markedly increased in UV and UV+ML385 group(P<0.01);Compared with the UV group,the cell viability were markedly decreased(P<0.01),the protein expression of nuclear p65 and mRNA expression of IL-6 were markedly increased in UV+ML385 group(P<0.05 or P<0.01).4.Compared with the NC group,the production of ROS in UV group was significantly increased,the expressions of antioxidant genes GCLC and NQO1 were significantly down-regulated(P<0.05),and the productions of IL-1β,IL-6 and TNF-α were significantly increased(P<0.01).Compared with the UV group,ASB pretreatment inhibited the production of UV-induced ROS and inflammatory factors(P<0.01),and high concentration of ASB significantly upregulated GCLC and NQO1 mRNA expressions in UV-induced HaCaT cells(P<0.05).5.Compared with the NC group,the protein expressions of keapl and nuclear Nrf2 were significantly downregulated in UV-induced group(P<0.05);Compared with the UV group,ASB pretreatment markedly increased the protein expressions of keap1,cytoplasmic and nuclear Nrf2(P<0.01 or P<0.05).Immunofluorescence results showed that,compared with NC group,the expression of nuclear Nrf2 was decreased in UV group.The expression of nuclear Nrf2 was increased in ASB group(vs.UV group).Compared with the NC group,the expressions of IκBα,cytoplasmic and nuclear p65 were significantly upregulated in UV-induced group(P<0.01 or P<0.05).Compared with the UV group,ASB pretreatment markedly decreased the protein expressions of IκBα,cytoplasmic and nuclear p65(P<0.01 or P<0.05).Immunofluorescence results showed that,compared with NC group,the expression of nuclear p65 was increased in UV group.The expression of nuclear p65 was decreased in ASB group(vs.UV group).Conclusion1.ASB increases the cell viability,decreases the apoptotic cells,thus alleviates UV-induced photo-damage.2.ASB relieves UV-induced inflammation and oxidative stress in HaCaT keratinocytes.3.Andrographolide sodium bisulfite alleviates UV-induced photo-damage based on the balance system between Nrf2 and NF-κB signaling pathways in HaCaT keratinocytes.