A Study On The Antioxidant Effect And Mechanism Of Puerarin In UVA-induced HaCaT Cells

OBJECTIVES Ultraviolet light is one of the main factors that cause the skin cancer and the eye cataract.UVA,which is the most abundant and accounts for almost 90%in the solar UV light,induces long-term deterioration and photoaging in skin.The skin photoaging induced by UVA includes patchy pigmentation,wrinkles,sagging,dryness and roughness.Nowadays the common method which protects the skin from UVA damage is to use physical sunscreens,whereas drugs capable of preventing UVA damage have not been marketed yet.Therefore,it is essential to seek safe and effective antioxidants to protect skin from UVA damage.Puerarin?Pue?,which derives from the root of the Pueraria lobata?Willd.?Ohwi and is an isoflavone derivative,is widely used in cardiovascular and cerebrovascular diseases.However,the effect of it on UVA-irradiated skin is unreported.This study is to observe the anti-oxidative effect and mechanism of puerarin on human keratinocytes?HaCaT cells?damaged by UVA,which provides puerarin antioxidation with theoretical evidences and brings a new idea to R&D of sunscreen antioxidants.METHODS In this study,The HaCaT cells were used as experimental subjects.HaCaT cells were routinely cultured at 37?,5%CO₂ and high glucose DMED medium containing 10%fetal bovine serum.After the cells were adherently cultured,the cells were irradiated with UVA after pretreated by puerarin for 24 h.The suitable puerarin concentration and the dose of UVA on HaCaT cells and cell viabilities were measured with CCK8 assay.Next,SOD,GSH-Px,MDA,CAT and T-AOC were detected according to each kit instructions.Then,flow cytometry was performed to detect cell death rates which included both apoptosis and necrosis.Finally,Western blot was used to detect protein expressions of p38MAPK,keap1,Nrf2,NQO1,HO-1,IL-1?and Fas.RESULTS In the present study,0.025 mmol/L-0.1 mmol/L puerarin slightly promoted the proliferation of HaCaT cells.It was toxic to cells at a concentration of0.2 mmol/L and above.0.025 mmol/L-0.1 mmol/L Puerarin had protective effects against 5 J/cm² UVA-induced HaCaT cells;Moreover,pretreatment of puerarin significantly increased the survival rate of UVA-induced HaCaT cells?P<0.01?in a dose-dependent manner.It was observed by light microscopy that the UVA-induced HaCaT cells became round,the intercellular space increased,the shape was irregular and there was cracking and dissolution,while the puerarin-pretreated cells was significantly reduced this phenomenon,and the necrosis was effectively alleviated.After UVA irradiation,the level of ROS increased significantly,and the average optical density value was about 0.370±0.094.The levels of SOD,GSH-Px,CAT and T-AOC decreased,the level of MDA and cell death rates significantly increased.Puerarin-pretreated cells had lower cell death rates than control cells after UVA irradiation.Puerarin increased SOD,GSH-Px,CAT,T-AOC,and decreased MDA in UVA-induced HaCaT cells.The results of Western blot showed that the expression levels of p38 in the control group,UVA model and each puerarin-pretreated group did not change much?P>0.05?.After UVA irradiation,the expressions of phospho-p38,HO-1,NQO1,IL-1?and Fas were significantly up-regulated,the expressions of keap1 and cytosol Nrf2 were significantly decreased?P<0.01?,while that of nuclear Nrf2 increased significantly?P<0.01?.Compared with the UVA model group,the expressions of phospho-p38,IL-1?and Fas were significantly down-regulated after pretreatment of puerarin,and the effects of puerarin at 0.1mmol/L were the most significant and dose-dependent.In addition,puerarin promoted the expression of keap1/Nrf2 signaling pathway related factors.Puerarin down-regulated expressions of cytosol Nrf2.nuclear Nrf2 expression,which did not show obvious change at low concentration of puerarin,was significantly increased at high concentration of it.Pretreatment of puerarin also up-regulated the expression of its downstream antioxidant enzymes HO-1 and NQO1,and enhanced the down-regulation of keap1 expression induced by UVA.CONCLUSION 0.025 mmol/L⁰.1 mmol/L Puerarin mitigates keratinocyte damage induced by UVA through antioxidative activity.Its antioxidative and protective effects on UVA-irradiated cells are associated with activation of the keap1/Nrf2 signaling pathway,which bolsters expressions of antioxidative enzymes and inhibits those of IL-1?and Fas.