The Study Of Protective Mechanism Of Myricetin Against Ultraviolet-induced Skin Photoaging

BackgroundSkin photoaging is a chronic inflammatory process caused by ultraviolet radiation.Skin photoaging has become a serious social problem.Myricetin(Myr)belongs to Myricaceae.It has been used for food therapy for a long time.Myricetin has many biological activities such as anti-cancer,anti-bacterial,anti-virus,anti-inflammatory,anti-oxidation and so on.Studies have shown that Myricetin can be used as skin photoprotective agents.However,the protective effect and potential mechanism of Myricetin on ultraviolet-induced skin photoaging remain unclear.ObjectTo explore the protective effect of Myricetin on ultraviolet-induced skin photoaging,and its potential mechanism.MethodsPart Ⅰ:Protective effect of myricetin on primary fibroblasts injured by ultraviolet radiationFibroblasts were isolated from epithelium and dermis of fresh skin tissue.Dermal fibroblasts were cultured by tissue block adherence method.Dermal fibroblasts then treated with different doses of UVA+UVB(5,15,30,60mJ/cm2),respectively.Then cell viability,disintegration,disordered arrangement and degranulation were observed.And we established the photodamage model of fibroblasts after ultraviolet irradiation.The cells were pretreated with different concentrations of myricetin,and irradiated with UVA+UVB(15mJ/cm2)24 hours later.After irradiation,the cells were incubated with corresponding concentration of myricetin for 24 hours.Cell morphology and cell viability was calculated,while cell proliferation activity was measured by MTT,and hydroxyproline(Hydroxyproline)in fibroblast culture supernatant was detected by colorimetry.The level of MMP-1 secretion was detected by ELISA.Part Ⅱ:Myricetin inhibited the damage of keratinocytes induced by UVB radiation through IKB/NF-B pathwayHuman immortalized keratinocytes HaCaT were irradiated with different doses of UVB(0,15,20,25,30 mJ/cm2).The viability of HaCaT cells was detected by MTT.After UVB irradiation,Myricetin and saline(as control group)were used to treat the cells.MTT assay was used to explore the viability of HaCaT cells treated with Myricetin.Flow cytometry was used to analyze the ROS content of cells irradiated by UVB.Western Blot and RT-PCR were used to detect the expression levels of p IkB and IkB pathway markers COX2,respectively.Part Ⅲ:Protective effect of myricetin on skin photodamage induced by ultraviolet radiation in miceFifty white male mice were randomly divided into five groups:group A:blank control group;group B:UVA+UVB irradiation;group C:UVA+UVB irradiation+Myrica flavone(5mg/ml);group D:UVA+UVB irradiation+Myrica flavone(20mg/ml);group E:UVA+UVB irradiation+Myrica flavone(50mg/ml).After 30 minutes of uniform application of saline(control group)or Myricetin,each group was irradiated with UVA+UVB every other day.The irradiation intensity was 0.18 mW/cm2,UVA was 1.25 mW/cm2,and the irradiation time was 30 minutes from the first week to the seventh week.The irradiation time was prolonged by 10 minutes,80 minutes at the twelfth week,and the UVA exposure was terminated at 14 weeks(the cumulative dose of 148.5 J/cm2 and UVB irradiation time was 30 minutes later).The cumulative measurement is 21.38J/cm2.During the course of ultraviolet irradiation,the back skin of mice in each group was measured by Glogau method.And the back of mice were stained with HE and collagen fibers,and the changes of skin were observed under the microscope.The levels of SOD,HYP and T-AOC were measured in the irradiated skin.Total RNA was extracted from skin tissue of irradiated area and the expression levels of MMP-1 and IkB were determined by RT-PCR.ResultsPart Ⅰ:Protective effect of myricetin on primary fibroblast injury induced by ultraviolet irradiationCompared with the control group,after the formation of fibroblasts by UVA/UVB radiation,the number of fibroblasts decreased significantly with the increase of the dose,resulting in disintegration,disordered arrangement and degranulation.With the increase of radiation dose,the survival rate of fibroblast(FB)decreased significantly(P<0.05).Ultraviolet radiation can attenuate the viability of FB cells and indue apoptosis and necrosis.Compared with the irradiation group,the viability of FB cells in Myricetin group increased with the increase of Myricetin concentration.Compared with the normal control group,collagen secretion decreased after UVA/UVB irradiation.After treatment with myricetin,it was found that myricetin concentration below 50 ug/ml could increase the total collagen secretion in fibroblast culture supernatant after ultraviolet irradiation.When the concentration was 50 ug/ml,the total collagen secretion increased significantly(P<0.05).It was also found that Myricetin concentration of 50 ug/ml could significantly inhibit the secretion of MMP-1 in fibroblasts(P<0.05).When Myricetin concentration was more than 100 ug/ml,the secretion of MMP-1 was higher than that in irradiation group(P<0.05).Part Ⅱ:Myricetin inhibit the damage of keratinocytes induced by UVB radiation through IkB/NF-kB pathwayWith the increase of UVB dosage,the survival rate of HaCaT cells decreased significantly(P<0.05).Ultraviolet B radiation can reduce the viability of HaCaT cells and induce apoptosis and necrosis.When the radiation dose reached 20 mJ/cm2,the activity of HaCaT cells decreased by nearly 50%(P<0.05).We irradiated HaCaT cells with 20 mJ/cm2 UVB and treated them with different Myrica flavonoids(0,5,15,25,35,50 μM).The results showed that compared with saline control group,the viability of HaCaT cells increased with the increase of Myrica flavonoids concentration.The protective effect of low concentration of myricetin was not significant(P>0.05),and the survival rate of HaCaT cells was about 80%when the concentration of myricetin reached 50 u M.After UVB irradiation,the ROS level of HaCaT cells increased,and different concentrations of myricetin could gradually reduce the ROS level of HaCaT cells,indicating that myricetin could effectively inhibit the ROS level of HaCaT cells induced by UVB radiation.After ultraviolet irradiation,the levels of COX2 gene and protein in HaCaT cells were significantly increased(P<0.05).Myricetin could effectively inhibit the levels of COX2 gene and protein induced by ultraviolet B radiation.With the increase of flavone content in Myrica rubra,the levels of COX2 gene and protein gradually decreased.After UVB irradiation,the expression of NFkB signaling pathway marker protein pIkB was up-regulated(P<0.05),but the expression of IkB did not change significantly(P>0.05).When Myricetin were used to treat cells with 50 μM,the expression of pIkB was inhibited and IkB did not change significantly.Part Ⅲ:Protective effect of myricetin on skin photodamage induced by ultraviolet radiation in miceAfter 14 weeks of continuous irradiation with UVA and UVB on the back skin of white mice,there were obvious changes of photoaging in the back skin:fibroblasts were damaged or even broken,collagen fibers were broken,arranged disorderly,collagen fibers content was decreased,accompanied by infiltration of mononuclear inflammatory cells.Before irradiation,Myricetin with external concentration of 5 mg/ml,20 mg/ml and 50 mg/ml were smeared respectively.The results showed that Myricetin could alleviate the degeneration of collagen fibers in varying degrees,reduce the damage of collagen fibers,arrange neatly,increase the content,and decrease the infiltration of inflammatory cells(P<0.05).The activities of antioxidant enzymes SOD and T-AOC in the skin of mice after ultraviolet irradiation decreased,and the activities of antioxidant enzymes in each group increased after applying myricetin(P<0.05).There was significant difference in SOD of superoxide dismutase among the groups(P<0.05).When the concentration of flavonoids in Bayberry was 5 mg/ml,the activities of T-AOC and the content of HYP increased significantly.When the concentration of MMP-1 was 5 mg/ml and 20 mg/ml,the level of MMP-1 was significantly increased(P<0.05).ConclusionMyricetin have a protective effect on skin photodamage induced by ultraviolet irradiation in a certain concentration range.Myricetin can indue cell proliferation and oxidase activity after irradiation,and inhibit the secretion of MMP-1 through IkB/NFkB pathway.