| Objective:To study the protective effect and its regulatory mechanism of ginseng oligosaccharide(GO)on photoaging of human permanent biochemical keratinocytes(HaCaT)induced by UVB.Methods:(1)Cell viability and the content of matrix metalloproteinase(MMP-1)in HaCaT cells treated with different doses of UVB were detected by an MTT assay and an ELISA kit to determine the optional dose of UVB as a model condition.(2)The survival rate of HaCaT cells treated with different doses of GO was detected by an MTT assay to screen out the effective concentration of GO.(3)Aftertreatment with UVB and GO,cell survival rate was detected by an MTT assay.The contents of MMP-1 and MMP-9 and the expression of MMP-1 protein were determined by ELISA kits and Western blot(WB),respectively.(4)The contents of inflammatory factors IL-1β,IL-8,IL-6,TNF-αand PGE-2 were determined by ELISA kits,and the expression of COX-2protein was detected by WB.(5)The content of intracellular reactive oxygen species(ROS)was measured by Flow cytometry(FCM).(6)The phosphorylated protein of ERK,JNK and p38 in the MAPK signaling pathway and the levels ofc-Jun and c-Fos in AP-1 signaling pathway,as well as the expression of P65 and IκB-αin NF-κB signaling pathway were detected by WB.Results:(1)The irradiation with 40 mJ/cm~2 UVB reduced,the survival rate of HaCaT cells to 76%of the blank group,and increased the content of MMP-1 in culture medium to 143.32±2.01%of the blank group.Based on above findings,we confirm the dosage of UVB was40mJ/cm~2 in HaCaT cells as a model condition of photoaging for further study.(2)GO has no effect on the survival rate of HaCaT cells.(3)GO at the concentration of 10,20 or 40μg/m L increased the cell survival rate of HaCaT cells damaged by UVB,and 40μg/mL GO increased the cell survival rate from 67%to 105%.The contents of MMP 1 and MMP-9 were 143.32±2.01%and129.2±1.27%of the blank group in HaCaT cells treated with UVB.When GO was added to HaCaT cells,the secretion of MMP-1 and MMP-9 were reduced to 103.47±1.86%and 102.1±1.97%,respectively.Moreover,WB analysis showed that the expression of MMP-1 was also reduced by GO.(4)UVB prompted the contents of IL-1β,IL-6,IL-8,TNF-α,and PGE-2,which was reversed by the different concentrations of GO in a dose-dependent manner.GO treatment reduced the expression of COX-2from 2.27 fold to 1.13 fold.(5)The content of intracellular ROS induced by UVB was increased to 160.51±1.63%of the blank group,which was decreased to 103.42±1.96%by GO treatment.(6)After incubation with UVB,the phosphorylated protein levels of ERK,JNK,and p38 were increased to 1.44,1.42,and 1.70 folds,and the levels of c-Jun and c-Fos were increased to 3.71 and 1.53 folds,as well as the levels of p-p65 and p-IκB-αwere to 1.57 and 3.87 folds.GO treatment-reduced the phosphorylated ERK,JNK,and p38 of MAPK signaling pathway to 1.17,0.92,and 1.02 folds,inhibited the phosphorylation of c-Jun to 1.42 folds and had no change on the phosphorylation of c-Fos protein.Meanwhile,the expression of p-p65 and p-IκB-αwere reduced to1.01 and 1.78 folds by GO treatment.Conclusion:Ginseng oligosaccharides could have the anti-photoaging effect on UVB-treated HaCaT cells by inhibiting the secretion of MMPs and inflammatory factors through the ROS-mediated MAPK and NF-κB signal pathways. |
PDF Full Text Request