Effects Of The Ultraviolet On Hrd1 Expression In Skin Fibroblasts And The Intervention Of Astragaloside

BackgroundThe skin is the body's largest organ,but also a natural protective barrier of the body.As prolonged skin exposure to the external environment,skin is the most vulnerable organ to ultraviolet radiation.The ultraviolet(UV)radiation in sunlight is the major environmental cause of skin damage.UVB or UVA radiation may couse chronic cutaneous changes as erythema,immunosuppression,skin cancers and photoaging.UVB affects the superficial dermis and UVA affects the middle dermis,fibroblasts are the main target of its role.UV-induced premature skin aging seriously affect human health,and discover how to delay skin aging has been a research hotspot.Skin aging is a complex biological phenomena,including two factors:the one is natural aging(mainly caused by genetic factors);the other is influenced by environmental factors called exogenous aging(UV,dust,weather,etc).The ultraviolet(UV)is the particularly important effects,which commonly defined as photoaging.Photoaging refers to the accumulation UV damage in process of skin aging,and it is the interaction result of natural aging and ultraviolet radiation.The major histological changes of photoaging are collagen decrease and denaturation of elastic fibers,resulting in clinical manifestations such as wrinkles and skin elasticity.Endoplasmic reticulum is a place of eukaryotic cells to translate and synthesize protein.Unfolded or misfolded protein occurs when cells suffer physical,chemical irritation or inflammatory state.These non-functional protein accumulation causes environmental imbalance and dysfunction,resulting in cell apoptosis.This phenomenon is called ERS(endoplasmic reticulum stress).Hrd1(HmgCoA reductase degradation 1)is a important family member of E3 ligase,which can break down the non-functional protein via ERAD(ER Associated Degradation),and protect cells from ERS to avoid apoptosis.There is no literature to explain the Hrdl expression under ultraviolet radiation in skin cells.Exploring an efficient and safe natural product to protect human skin from UV damage is really important.Protective effect of traditional Chinese medicine on the skin has gradually been recognized.As a astragalus saponin monomer components,AST is the main active ingredients of astragalus.Research suggests that AST is able to regulate immune function,resist oxidation,inflammation,virus and aging.The emerging evidence suggest that astragalus extract has a protective effect on UV-induced photoaging.But whether the astragaloside changes skin fibroblasts E3 ligase Hrd1 has not been reported.In this study,we explore the effects of the ultraviolet on Hrdl expression in skin fibroblasts and the intervention of astragaloside by using primary cultures of human skin fibroblasts at the cellular level.ObjectiveTo investigate the effects of ultraviolet on E3 ligase Hrdl expression in human skin fibroblasts and the intervention of Astragaloside.Proving that astragaloside may protect human skin fibroblasts irradiated under UV light and further clarify its role in photoprotection mechanism.Materials and methods1.Cell culture:Human skin primary fibroblasts(FB)were prepared "from prepuce biopsy of healthy adult normal human.The cells were incubated in Dulbecco's modiWed Eagle medium(DMEM),supplemented with 20%fetal bovine serum(FBS)at 37? in humidified atmosphere containing 5%CO2,and cells were plated in 24 wells?96 wells and 10cm dish with an equal number of cells.2.Ultraviolet irradiation and AST intervention:Primary human skin fibroblasts were irradiated by 10 J/cm UVA or 30mJ/cm2 UVB respectively and treated with astragaloside.3.Group:FB was divided into nine groups,they are Group A(control group),Group B(low dose AST),Group C(high dose AST),Group D(UVA irradiation),Group E(UVA irradiation + low dose AST),Group F(UVA irradiation + high dose AST),Group G(UVB irradiation),Group H(UVB irradiation + low dose AST),Group I(UVB irradiation + high dose AST).4.Inverted microscope:To observe the morphology changes of fibroblast.5.MTT assay:To detect cellular activity in fibroblasts.6.Real Time-PCR:To detect Hrdl mRNA expression in fibroblasts.7.Inmmunohistochemical staining:To determine Hrdl protein expression in fibroblasts.Results1.Effects of different concentrations of AST on the cellular activity in human skin fibroblasts.Astragaloside treatment promoted the fibroblasts proliferation remarkably in a dose-dependent manner.Detected by MTT assay,the proliferation rates reached high values at 10,20,30?g/ml dosages(P<0.001);Determining 10,30 mg/L for the low and high final experimental drug concentration.2.Effects of ultraviolet light on Hrdl mRNA expression in skin fibroblastsCompaerd with the control group,HrdlmRNA expression in ultraviolet irradiation(UVA or UVB)group was significantly increased.The result suggested that the Hrdl mRNA expression in human skin fibroblasts was up-regulated after ultraviolet radiation(P<0.001).3.Effects of AST on Hrdl mRNA expression in skin fibroblastsHrdl mRNA expression in high dose AST group was increased compared with the control group(P<0.05),but the difference was not statistically significant in low dose AST group(P>0.05).Compared with the ultraviolet radiation(UVA or UVB)group,Hrdl mRNA expression was decreased after the intervention of astragaloside.The result suggested that astragaloside can significantly inhibited Hrdl mRNA expression after UV radiation(P<0.001).4.Effects of ultraviolet light on Hrdl protein expression in skin fibroblastsCompaerd with the control group,Hrdl protein expression in ultraviolet irradiation(UVA or UVB)group was significantly increased.The result suggested that the Hrdl protein expression in human skin fibroblasts was up-regulated after ultraviolet radiation(P<0.05).5.Effects of AST on Hrdl mRNA expression in skin fibroblastsHrdl protein expression in low or high dose AST alone group was not changed compared with the control group(P>0.05).Compared with the ultraviolet radiation(UVA or UVB)group,Hrdl protein expression was decreased after the intervention of astragaloside.The result suggested that astragaloside can significantly inhibited Hrdl protein expression after UV radiation(P<0.05).ConclusionThe E3 ligase Hrdl expression in human skin fibroblasts was increased after ultraviolet radiation and the astragaloside may have protective effect on it.