Mangiferin Protects Human Dermal Fibroblasts From UVB Stress-induced Premature Senescence

BackgroundAs the sun's ultraviolet rays are the most important environmental stimulin on the skin,chronic ultraviolet irradiation accelerates skin senescence and even skin tumors,which is the so-called skin photoaging.Ultraviolet irradiation on skin makes human dermal fibroblasts(HDFs)reduce collagen synthesis and induce a variety of skin cells to produce matrix metalloproteinases(MMP)that can degrade collagen,resulting in characteristic manifestation of dermal connective tissues in skin photoaging process.In vitro studies of skin photoaging need to build cellular senescence model,the stress-induced premature senescence(SIPS)model is popularly applicated in the present studies.HDFs by repeated subtoxic doses of UVB irradiation without entering the state of death or apoptosis,exhibit some characteristics of replicative senescence.Mangiferin is a kind of flavonoid glycosides.Recent studies have found not only oral mangiferin inhibited skin thickening,wrinkles formation and collagen declining after hairless mouses were irradiated by chronic UVB,but also reduce human keratinocytes by mangiferin culturing secrete MMP9 and MMP1 after UVB irradiation and remove UV-induced oxygen free radicals,mangiferin proved to be an effective light protective agent.However,whether the intervention effects of mangiferin in UVB induced HDFs premature senescence process have not been reported.ObjectTo clarify whether mangiferin inhibits SIPS in HDFs induced by repeated exposure to a subcytotoxic dose of UVB,and to investigate the mechanism underlying the effect of UVB.Methods1.Cell culture Removal of the foreskin from the healthy young and middle-aged man circumcision,separate the epidermis and dermis to get the HDFs.Take 6-12 substituting cells in logarithmic growth phase experiment.2.Select the appropriate concentration of mangiferin stimulates HDFs 0,0.5,1.0,2.0,4.0,8.0 and 16mg/L mangiferin after incubation for 24,48 and 72 hours,cell viability and cytotoxicity was observed by CCK-8 assay.3.Ultraviolet B radiation According to the experimental design time quantitative UVB radiation,selecting the appropriate concentration of mangiferin added to the culture to the point in time with a total dose of UVB 50 mJ/cm2(5 times 10mJ/cm2 · d UVB irradiation).4.The effect on the expressions of senescence-associated signals of mangiferin on the UVB-induced premature senescence CCK-8 assay was employed to measure cell viability,SA-?-Gal histochemical staining to detect senescent cells,flow cytometry to count G1 phase arrest cells proportion.5.The impact of COLlal,COL3a1,MMP1 and MMP3 gene expression under mangiferin treatment after UVB irradiation on HDFs RT-PCR was employed to measure MMP1,MMP3,COLlal,COL3al gene expression.6.The impact of senescence-related genes and proteins p53,p21,p16 expression and synthesis under mangiferin treatment after UVB irradiation on HDFs RT-PCR and western blot were used to detect senescence-related genes and proteins p53,p21,p16 expression and synthesis.Result1.Concentration of 1.0 mg/L,2.0 mg/L,4.0 mg/L of mangiferin on HDFs had no cytotoxicityCompared with control group,the concentration of 1.0,2.0 and 4.0mg/L mangiferin didn't significantly decrease cell proliferation(P<0.05)and had no cytotoxicity on HDFs after 24,48 and 72h.2.Mangiferin reduced the UVB irradiation-induced HDFs premature senescenceCompared with UVB-SIPS group,the irradiated fibroblasts treated with three different concentration of mangiferin showed an increased activity of cell proliferation(all P<0.05),a decreased ?-galactosidase expression(P<0.05,except SIPS plus mangiferin 1.0mg/L group),a decreased G1 phase arrest cells proportion(all P<0.05)in a concentration-dependent manner.3.Mangiferin increased COLlal,COL3a1 gene expression and decreased MMP1,MMP3 gene expression after UVB irradiation on HDFsC.ompared with UVB-SIPS group,the irradiated fibroblasts treated with three different concentation of mangiferin showed a decreased MMP1,MMP3 mRNA expression(all P<0.05),an increased COLlal and COL3al mRNA expression(P<0.05,except SIPS plus mangiferin 1.0mg/L group)in a concentration-dependent manner.4.Mangiferin decreased senescence-related genes and proteins p53,p21,p16 expression and synthesis after UVB irradiation on HDFsCompared with UVB-SIPS group,the irradiated fibroblasts treated with three different concentration of mangiferin showed a decreased senescence-related genes p53,p21,p16 mRNA expression(all P<0.05),a decreased senescence-related proteins p53,p21,p16 synthesis(all P<0.05)in a concentration-dependent manner.ConclusionMangiferin can inhibit UVB-SIPS in HDFs and has anti-photoaging effects.