Lycium Barbarum Polysaccharide Protects Against UV-induced Acute Photo-damage Through Nrf2 Pathway In Human Skin Fibroblasts Cells

[Background]Solar radiation includes ultraviolet(UV),visible and infrared light.UV radiation is the most biologically effective part which has the most impact on human health and disease.Owing to the different biological function,UV can be divided into three bands according to wavelength:UVA(320–400 nm),UVB(280–320 nm),else UVC(200–280 nm).Due to the refraction and scattering effect of the atmospheric ozone layer,most UVB and almost the whole of UVC are absorbed,they can have not yet reached the surface of the earth.Accordingly,human beings on the earth are mainly radiated to UVA and a small amount of UVB radiation.Skin is the most important organ in our body that is the first line of defense against external mechanical,biological,chemical,physical stimuli.Ultraviolet radiation is the major factor that causes skin damage and destruction of the skin barrier.Long-term,cumulative or a large number of ultraviolet expose to the earth’s surface affect human health seriously,which can lead to a variety of photodermatosis,for instance,acute sunburn,chronic actinic dermatitis,solar keratosis,polymorphous light eruption,photo aging and skin cancer.Notable is,as the depletion of the global ozone layer and the increase of ultraviolet radiation and the change of human lifestyle,therefore,the incidence of photodermatosis has increased year by year,which has become a hot issue of global concern.So far,Nuclear factor erythroid 2-related factor 2(Nrf2)signaling pathway is the most important anti-oxidative stress pathway.Ultraviolet radiation caused excess intra-cellular ROS.At the moment the transcription factor BTB-CNC homology-1(Bach1)in the nucleus has been uncoupled from Maf recognition element(MAREs),which was desorbed and degradated.Now Nrf2 in the cytoplasm uncoupling with kelch-like-Ech-associated-protein 1 entered the nuclear combines with the Maf recognition elements.After forming a heterodimer,which further recognizes and binds to the antioxidant response element,thereby activating the expression of downstream II phase detoxification enzyme,antioxidant enzyme and anti-inflammatory protein.For instance,superoxide dismutase and glutathione peroxidase,quinone oxidoreductase 1 and heme oxygenase,epoxide hydrolase,acetaldehyde reductase.Therefore,it can scavenger ROS and alleviate oxidative stress damage,keeping the structure and the function of the normal cells.Accordingly,it is a new trend to find natural ultraviolet protective product,which activates Nrf2 pathway to protect skin from ultraviolet.In recent years,Lycium barbarum polysaccharide extracted from Chinese wolfberry.As is known to all,which is a famous traditional Chinese medicine.Lycium barbarum polysaccharide has anti-oxidant,anti-aging,irradiation properties.But it is not clear whether Lycium barbarum polysaccharide can play a strong antioxidant activity to protect photo damage by regulating the Nrf2 pathway.Furthermore,its specific mechanism is not clear.Therefore,this study probes on the protective effect of Lycium barbarum polysaccharide and the regulation of Nrf2 on UV induced by human skin fibroblasts,providing a scientific basis for the development of late-model photo damage resistance.[Object]1.To probe into the protective effect of Lycium barbarum polysaccharide from UVA/UVB induced photo damage in HSF cells.2.To investigate to potential underlying mechanisms of Lycium barbarum polysaccharide against UVA/UVB caused photo damage in HSF cells.3.To explore the regulatory role of Lycium barbarum polysaccharide on Nrf2signaling pathway,further illuminate its protective mechanism for defending light damage.[Method]1.Cultured HSF cells with different concentration of Lycium barbarum polysaccharide(100μg/ml,200μg/ml,300μg/ml,400μg/ml,500μg/ml,600μg/ml,1000μg/ml,1500μg/ml,2000μg/ml)screening the suitable and nontoxic concentration by inverted optical microscope with MTT colorimetry to observe the cells morphological changes and cells proliferation.2.The UV spectrophotometric method to detect the UVA and UVB absorption of Lycium barbarum polysaccharide.3.Pretreated HSF cells with the optimal Lycium barbarum polysaccharide concentration for 24 h,exposed to UVA(25 J/cm~2)and UVB(300 mJ/cm~2)respectively.And MTT method was carried out to detect the cellular proliferative activity.ROS was detected by dichlorodihydrofluorescein diacetate fluorescent.Determining the extent of lipid peroxide used by the Nile red staining.Comet assay was used for evaluating the DNA strand breakage damage.Enzymatic-biochemical method was employed to estimate the activities of SOD and GSH-Px with LDH.4.The expression of nrf2 mRNA,bach1 mRNA,sod mRNA,cat mRNA,nqo1mRNA,gclc mRNA,and gclm mRNA in HSF cells processed by Lycium barbarum polysaccharide were detected by qRT-PCR.The expression of Nrf2 protein and the cellular distribution was verified by Western blotting.5.To acquire Nrf2 knockdown cells(Nrf2~KDD HSF cells),Nrf2 was silenced by constructing lentivirus vector to transduce HSF cells with Nrf2-targeted small hairpin RNA.6.Nrf2~KDD HSF cell proliferation activity was measured by MTT and ROS was detected by DCFH-DA fluorescence.The lipid peroxide was evaluated by the Nile red staining.Enzymatic-biochemical method to test the activities of SOD,GSH-Px in the Nrf2~KDD HSF cytoplasm.[Results]1.Incubated HSF cells with different concentration of Lycium barbarum polysaccharide(100μg/ml,200μg/ml,300μg/ml,400μg/ml,500μg/ml,600μg/ml,1000μg/ml,1500μg/ml,2000μg/ml).And we found that each group had no significant difference in morphology.MTT assay suggested that compared with the control group,when the concentration of Lycium barbarum polysaccharide is less than or equal to 300μg/ml,the proliferative activity of HSF cell was unaffected(P>0.05).However,Lycium barbarum polysaccharide inhibited the cell relative growth rate when it was more than 300μg/ml(P<0.05).So 300μg/ml was the suitable concentration for our study.2.The absorbance values of Lycium barbarum polysaccharide are all under 0.3.3.Pretreated HSF cells with Lycium barbarum polysaccharide selected to be 300μg/ml and exposed under UV irradiation.MTT assay showed that compared to control cells UV radiation group inhibit the cell proliferating(P<0.05).Pretreating with Lycium barbarum polysaccharide of UV irradiation group,the inhibitory amplitude of cell proliferation was markedly decreased(P<0.05).The results showed that compared with single UV radiation group,pretreating with Lycium barbarum polysaccharide of UV irradiation group,LDH,ROS and lipid peroxidation of cells decreased significantly(P<0.05).Comet assay results displayed that compared to the single UV radiation group,tail length decreased after pretreatment of Lycium barbarum polysaccharide.And the DNA fluorescence intensity was weak.Moreover,the degree of DNA damage was significantly reduced(P<0.05).4.Pretreated HSF cells with LBP,qRT-PCR results demonstrated that the expression of nrf2,sod,cat,nqo1,gclc,gclm mRNA were increased in drug group,and bach1mRNA expression decreased(all P<0.05).Compared to the blank group,western blotting results showed that 300μg/ml LBP can promoted protein expression of Nrf2in nucleus.Moreover,the protein abundance of 300μg/ml drug group was significantly higher than that of the 100μg/ml and the 600μg/ml Lycium barbarum polysaccharide.5.Constructing lentivirus vector to nrf2-shrna to HSF cells,the control rate of Nrf2in the stable cell is 70%of>.6.Silencing of Nrf2 decreased the cell viability.Similarly,the activity of SOD and GSH-Px obviously decreased after the radiation treatment.This decrease may be incapable of reversing by Lycium barbarum polysaccharide.Conversely,the ROS level and the level of LPO increased significantly.Lycium barbarum polysaccharide could help to protect HSF cells against UV,its functions were obviously diminished when Nrf2 was silenced(all P<0.05).[Conclusion]1.Lycium barbarum polysaccharide can effectively attenuate the acute photo damage induced by UV in HSF cells.2.Lycium barbarum polysaccharide could have a regulatory activity in HSF cells to prevent UV damage.Lycium barbarum polysaccharide could regulate activate the nuclear translocation of Nrf2 and increase the expression of nrf2 and phase II enzyme mRNA through regulating the Nrf2 signal pathway.Therefore,Lycium barbarum polysaccharide is able to scavenge ROS and suppress lipid peroxide and delay DNA damage,resulting in improvement of their viability and antagonizing oxidative stress injury.