| Backgroud:Skin aging involves intrinsic and extrinsic processes.Environmental factors,primarily ultraviolet(UV)light,cause extrinsic skin aging.Although there are various etiologies of skin photoaging,common points include less dermal type Ⅰ and Ⅲcollagen expression.The predominant form of collagen in dermis is type Ⅰ,followed by small amounts of type Ⅲ.Type Ⅰ collagen is characterized by thick fiber that confer stiffness and resistance to perform a crucial function in maintaining the structure of dermis.Whereas collagen type Ⅲ is characterized by thin fiber that present the resiliency of skin.Collagen fibers arrange parallel to skin surface and are responsible for the high tensile strength and resiliency of skin.The degradation of type Ⅰ and Ⅲ fibrillar collagens is initiated by matrix metalloproteinases-1(MMP-1)and MMP-3 respectively,which belongs to the matrix metalloproteinases(MMPs),a large family of zinc-dependent endo-proteases with a broad range of substrate specificities and the capacity of degrading all extracellular matrix proteins.Fibroblasts regulate production and degradation of the extracellular matrix and make multiple cytokines and.glycoproteins.Fibroblasts exposed to UV reduce collagen by both promoting its degradation and interfering with its production.Other studies have reported early senescent changes that were confirmed by measuring β-galactosidase activity,p53,p21 and p16 expressions were detected when human fibroblasts were exposed properly to UVB.In previous reports,fibroblasts could be photoaged by UVB in vitro and in vivo and these experimental models were proven to be applicable in various skin aging studies.Baicalin is the predominant flavonoid isolated from the roots of Scutellaria lateriflora Georgi(Huang Qin).It has been reported that this compound exhibits many different pharmacological activities.Baicalin has displayed beneficial effects on several diseases model such as hypoxia/reoxygenation caused cardiomyocytes injury,hepatic cytotoxicity,iron-overloaded mouse,rheumatoid arthritis and so on.Furthermore,recent studies have confirmed the photoprotective effect of baicalin against acute and multiple UVB-induced photodamage,and this effect is thought to be associated with reduction of oxidative stress.However,whether baicalin can protect human dermal fibroblasts form UVB irradiation induced premature senescence remains to be clarified.In this study,the anti-photoaging effects of baicalin were evaluated in isolated dermal skin fibroblasts and the skin of hairless mice.For in vitro studies,the effects of baicalin on expressions of β-galactosidase activity,protein levels of p53,p21,p16 and y-H2AX,production in skin fibroblasts after UVB irradiation were investigated.For in vivo studies,young mice were pretreated with UVB irradiation to induce photoaging.The expression of for MMP-1,MMP-3,collagen-1 and collagen-3 were quantitatively measured from the nontreated and baicalin-treated mouse skin using immpunostaining and real-time RT-PCR methods.Methods:1.Normal human skin samples were obtained from circumcisions in accordance with the ethical committee approval process of Jiangsu Provincial People’s Hospital,Nanjing,Jiangsu,China.Specimens were sterilized in 70%ethanol,minced,and incubated in Dulbecco’s modified Eagle medium(DMEM)supplemented with 10%fetal bovine serum and 1%penicillin-streptomycin in an atmosphere of 5%CO2 at 370C.Dermal HDFs normally grew from the explants after 5-7 days.The cells from passages 8 to 11 were used in this study.2.Female C57BL/6 mice were divided into following six groups.Five mice in the first group did not receive any treatment and served as a control.Five mice in the second group receivd a topical treatment of baicalin(1 mg/cm2 skin area/mouse/100μL acetone)on their dorsal skin.Five mice in the third group received UVB treatment.The mice in the fourth group received a topical application of 100μL acetone followed by UVB irradiation(2 hours following acetone treatment).The mice in the fifth group received a topical application of baicalin(0.5 mg/cm2 skin area/mouse/100 μL acetone)followed by UVB irradiation(2 hours following baicalin treatment).The mice in the sixth group received a topical application of baicalin(1 mg/cm2 skin area/mouse/100 μL acetone)followed by UVB(2 hours following baicalin treatment).3.UVB-stressed cells were irradiated at a subcytotoxic dose of 10 mJ/cm2 twice a day for 5 days.The irradiation intensity represented as the minimal erythemal dose(MED)was set at 1 MED during the first 2 weeks(60 mJ/cm2),and was elevated to.2 MED(120 mJ/cm2)in the 3rd week,to 3 MED(180 mJ/cm2)in the 4th week and to 4 MED(240 mJ/cm2)during the 5th-8th weeks of the experiment.Studies were performed at 24 h after the last UVB exposure.4.Cell proliferation was assayed using a CCK-8 Kit.5.β-galactosidase staining for detection of senescent cells.6.To determine whether UVB-stressed HDFs exhibit cell growth arrest,cell-cycle analysis with flow cytometry was performed.7.To investigate the level of senescence-related proteins(p16INK-4a,p21WAF-1,p53 and γ-H2AX proteins),Western Blotting analysis was performed.8.After treatment with polyester wax,the skin samples were sliced into 6-mm thicknesses.The sliced sections were treated with haematoxylin and eosin(H&E)and9.To investigate the levels of senescence-related proteins(COL-1,COL-3,MMP-1,MMP-3),Immunohistochemical analysis was performed.10.To investigate the levels of senescence-related mRNA(COL-1,COL-3,MMP-1,MMP-3),Real-time RT-PCR analysis was performed.Results:1.Baicalin suppresses UVB-induced epidermal thickening in mouse skin Because epidermal thickening is a major biomarker of photoaging,we evaluated the effect of baicalin on UVB-induced epidermal thickening.In a quantitative analysis,hematoxylin and eosin staining demonstrated that UVB irradiation induced 4.23 folds increase in epidermal thickness(p<0.05 vs.the non-irradiated control group;n=5).Topically applied baicalin(0.5 or 1 mg/cm2 skin area/mouse/100 μL acetone)decreased the amount of UVB-induced epidermal thickening respectively(p<0.05 vs.the irradiated group;n=5)2.Baicalin protects against UVB-induced dermal collagen fiber lossWe found that collagen levels were decreased significantly in UVB-radiated mice by about 68.17%(p<0.05,N=5),compared with control mice,but baicalin alone had no effect.However,in baicalin-radiated mice skin,baicalin at two different dosages augmented the UVB-induced collagen compared with UVB-radiated mice.Baicalin prevented UVB-induced decrease in type Ⅰ and Ⅲ collagen in dorsal mice skin3.Baicalin protects against UVB irradiation-induced type Ⅰ collagen fiber loss.Immunohistochemistry revealed that UVB decreased type Ⅰ and Ⅲ collagen expression throughout the dermis.Baicalin-pretreated skin demonstrated greater intracellular procollagen staining in dermal fibroblasts,compared with vehicle-pretreated skin,after UV irradiation,indicating that the Baicalin pretreatment counteracted the downregulating effects of UV on type Ⅰ and Ⅲ collagen.However,in unirradiated skin,baicalin-treated skin demonstrated no obviously impaction on type Ⅰ and Ⅲ collagen expression compared with the vehicle-treated control.For real-time RT-PCR results of type Ⅰ and type Ⅲ procollagens,the similar modulation tendency to those in the histological and immunochemistry pictures was observed i.e.there was a significant statistical difference of mRNA expressions between UVB treated and UVB+baicalin treated groups(p<0.05),which implied Baicalin could prevented UVB-induced decrease mRNA expression of type Ⅰ and Ⅲ procollagen.4.Baicalin prevented UVB-induced induction of MMP-1 and 3 in dorsal mice skinImmunohistochemistry revealed that UVB induced amplification of MMP-1 and 3 expression throughout the skin,especially in epidermis.Baicalin-pretreated skin demonstrated decreased MMP-1 and 3 staining in skin,compared with vehicle-pretreated skin,after UV irradiation,indicating that the Baicalin pretreatment counteracted the upregulating effects of UVB on MMP-1 and 3.For real-time RT-PCR results of MMP-1 and 3,the similar modulation tendency to those in the histological and immunochemistry pictures was observed i.e.there was a significant statistical difference of mRNA expressions between UVB treated and UVB+baicalin treated groups(p<0.05),which implied baicalin could prevented UVB-induced mRNA expression of MMP-1 and 3.5.Baicalin protects HDFs against UVB-SIPS induced impaired cell viabilityCompared with the control group,the cell viability of the UVB-SIPS group was significantly decreased(P<0.05).The cell viability in the UVB-irradiated groups treated with 6.25μg/ml,12.5 μg/ml,and 25μg/ml of baicalin compared to that of the UVB-SIPS group showed significant increases in a dose-dependent manner,whilst there seemed to no obvious reduction in the cell viabilit of the baicalin group compared to the control group.6.Baicalin decreased the percentage of SA-β-gal positive cells in UVB-SIPS fibroblastsThe cells in the UVB-SIPS group became obviously enlarged,flattened,and irregular compared to the control group.There was a 10.2-fold increase of positive cells in the UVB-SIPS group compared to the control group.The percentage of positive cells in UVB-SIPS group was 90.52%,while it was 8.91%in the control group.The percentage of SA-β-gal positive cells in the UVB-irradiated groups treated with 6.25,12.5 and 25 μg/ml baicalin was significantly reduced compared to that in the UVB-SIPS group in a dose-dependent manner.The percentage of positive cells in the UVB+6.25μg/ml,12.5 μg/ml,and 25μg/ml of baicalin were 80.13%,62.44%,and 32.88%,respectively.There was no significant difference in the percentage of positive cells between control group and baicalin group.However,the positive expression of SA-β-gal showed no difference in normal fibroblasts treated with or without baicalin,even for a long term(Fig.8c and 8d).7.Baicalin decreased G1 phase cell proportion in UVB-SIPS fibroblastsThe results showed that UVB-stressed HDFs were blocked mostly in the G1 phase of the cell cycle.Compared with the control group(from 38.1%to 81.5%),the G1 phase cell proportion of the UVB-SIPS group was significantly higher(P<0.05).The G1 phase cell proportions in the UVB-irradiated groups treated with 6.25,12.5 and 25μg/ml baicalin compared to that of the UVB-SIPS group showed significant decreases in a dose-dependent manner(from 81.5%to 57.97%,55.2%and 54.12%,respectively).There seemed to be no obvious reduction in the G1 phase cell proportion of the baicalin group compared to the control group(from 42.64%to 38.1%).8.Baicalin decreased the level of p16INK-4a,p21WAF-1,and p53 proteins in the UVB-SIPS fibroblasts The levels of p16INK-4a,p21WAF-1,and p53 proteins in the UVB-SIPS group showed 5.2-,14.2-,and 12.3-fold increase,respectively,compared to the control group.The levels of p16INK-4a,p21WAF-1 and p53 proteins in the UVB-irradiated groups treated with 6.25,12.5 and 25 μg/ml baicalin decreased significantly,respectively,compared to those of the UVB-SIPS group.There were no significant changes in the level of p16INK-4a,p21WAF-1,and p53 protein in the baicalin group compared to that in the control group.9.Baicalin caused significant a dose-dependent decrease in the level of γ-H2AX proteins in the UVB-SIPS fibroblastsThere was a 7.5-fold increase in the levels of y-H2AX proteins in the UVB-SIPS group compared to the control group.The levels of y-H2AX in the UVB-SIPS groups treated with 6.25,12.5 and 25 μg/ml baicalin was significantly decreased compared to that of the UVB-SIPS group.There was no significant difference in the levels of y-H2AX in the baicalin group compared to that in the control group.10.Long-term Baicalin incubation of UVB-SIPS fibroblasts gave no effects on the cell proliferationWith a long term culture of fibroblasts over 8 weeks,the cell proliferation in the UVB-SIPS group treated with 6.25μg/ml,12.5 μg/ml,and 25μg/ml of baicalin showed no difference with the UVB-SIPS group,whilst there seemed to no obvious reduction in the cell proliferation of the baicalin group compared to the control group.Conclusion:In summary,We observed that baicalin significantly antagonizes photoaging induced by UVB in vivo and in vitro,indicating the potential of baicalin application for anti-photoaging treatment.Cellular senescence is a state of permanent proliferation inhibition,a lot of factors such as UVB radiation can induce the premature skin aging,leading to a variety of biological abnormalities.Ex vivo experiments have shown that the repeated exposures of human skin fibroblasts to UVB or 8-methoxypsoralen plus ultraviolet-A irradiation(PUVA)at subcytotoxic levels triggers ultraviolet stress-induced premature senescence(SIPS).Under these conditions,fibroblasts cease to divide,and instead undergo a series of dramatic morphological and metabolic changes.In vitro studies have demonstrated that cell senescence can occur due to a variety of processes including genetically programmed pathways,telomere shortening,and the accumulation of DNA damage.Autophagy,the dynamic process of degrading unnecessary or dysfunctional cell components,has also been linked to aging.Autophagy is a cellular catabolic mechanism that is activated in response to stress conditions,including ultraviolet(UV)irradiation,starvation,and misfolded protein accumulation.Studies have shown that reduction in autophagy can accelerate the aging process,while the stimulation of autophagy may have potent anti-aging effects.However,the role of autophagy specifically in photoaging has not been thoroughly studied.And the underlying molecular mechanism linking autophagy to photoaging is still not known.Furthermore,miRNAs have also been linked to the process of aging and senescence.MiRNAs are endogenously expressed small RNA molecules that mediate posttranscriptional gene silencing and have the capacity to simultaneously regulate tens to hundreds of target genes.As a result,they are potential targets for anti-aging,and more specifically anti-photoaging therapy.Furthermore,miRNAs have also been shown to regulate autophagy pathways.While autophagic activity is regulated by a variety of factors,including insulin receptor-signaling pathway,the TOR pathway,Sirtl,and caloric restriction.While the role of miRNAs in autophagy has been established,and the role of autophagy in aging,it has not yet been demonstrated whether miRNAs have any role in photoaging.However,miR-23 a serves as a promising target as the link between miRNA expression and photoaging,as it has been reported to be up-regulated in several in vitro and in vivo aging models.But the role of miR-23a in PUVA-and UVB-induced premature senescence and autophagy has yet to be established.Therefore,the aim of the current study is to identify this role.Furthermore,the molecular target of miR-23a was also identified via a bioinformatics approach in an effort to elucidate the mechanism of regulation of miR-23a.Methods:1.Cell Cultrue This part is the same as Part I.2.UVB irradiation For the UVBA-SIPS model,it is the same as Part I.For the PUVA-SIPS model,The fibroblasts were pre-incubated with 8-methoxypsoralen(100 ng/ml)for 24 hours and subsequently received UVA irraiation at a dosage of 9 J/cm2.3.UVB-stressed cells were irradiated at a subcytotoxic dose of 10 mJ/cm2 twice a day for 5 days.The irradiation intensity represented as the minimal erythemal dose(MED)was set at 1 MED during the first 2 weeks(60 mJ/cm2),and was elevated to 2 MED(120 mJ/cm2)in the 3rd week,to 3 MED(180 mJ/cm2)in the 4th week and to 4 MED(240 mJ/cm2)during the 5th-8th weeks of the experiment.Studies were performed at 24 h after the last UVB exposure.4.β-galactosidase staining for detection of senescent cells5.Bioinformatic analysis of miR-23a target genes The putative miR-23a targets were predicted using several different algorithms,including TargetScan(http://www.targetscan.org/),PicTar(http://pictar.bio.nyu.edu/)and miRanda(http://microrna.sanger.ac.uk/).An interaction between miR-23a and the 3’-UTR of its target gene was predicted using RNAhybrid(http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/).6.Western Blotting analysis was performed as Part I.7.Immunoprecipitation assay was performed to determine the interaction between vps34 and Beclin 1.Results:1.Decreased autophagy flux in PUVA-and UVB-SIPS fibroblastsConfocal microscopy revealed that PUVA and UVB irradiation could repress GFP-LC3 puncta formation in fibroblasts,indicating that autophagy is inhibited under these conditions.The lipid conjugation of free LC3-I to the autophagic membrane-associated LC3-II was attenuated in the extracts of the cells following subcytotoxic ultraviolet irradiation,and the degradation of the autophagic cargo receptor protein p62/SQSTM1 was reduced in sham-irradiated cell extracts.2.Increased senescence in PUVA-and UVB-SIPS fibroblastsWe also demonstrated increases in senescence-related expressions of SA-β-gal,p16,p53,and p21,as well as an increase in G1 cell cycle arrest and a decrease in the percentage of EdU-positive cells in the PUVA-SIPS and UVB-SIPS fibroblasts.3.Expression and effects of miR-23a-27a-24-2 cluster in PUVA-and UVB-SIPS fibroblastsThe expression level of miR-23a was increased in PUVA-and UVB-SIPS fibroblasts.Meanwhile,miR-24-2 showed no obvious changes in PUVA-and UVB-SIPS fibroblasts,while miR-27a expression was up-regulated only in UVB-SIPS fibroblasts.4.MiR-23a-specific antagomirs(Ant-23a)stimulated autophagy in PUVA-and UVB-SIPS fibroblastsAfter ultraviolet irradiation,down-expression of miR-23a significantly increased GFP-LC3 dot formation.Meanwhile,lipid conjugation of free LC3-Ⅰ to the autophagic membrane-associated LC3-Ⅱ was stimulated following Ant-23 a transfection)Hence,autophagy was accelerated in UV-SIPS cells transfected with Ant-23a,but not with control antagomirs.Moreover,SQSTM1 degradation was more prominent after Ant-23a transfection compared with Ant-CNT groups.5.MiR-23a-specific antagomirs(Ant-23a)suppressed senescence in PUVA-and UVB-SIPS fibroblastsUnder-expression of miR-23a may decrease SA-β-gal-positive cells and increase EdU-positive cells in PUVA-and UVB-SIPS fibroblasts.The UV-irradiated cells transfected with Ant-23a showed a significant decrease in G1 phase cell proportions compared to those UV-irradiated cells transfected with Ant-CNT.Similar changes could be found in the levels of p53,p16,and p21 proteins.6.MiR-23a inhibits AMBRA1 expression by targeting its 3’-UTRMiR-23a suppressed the luciferase activity of the pmiR-AMBRA1-wt compared with the negative control,while the mutation of the miR-23a binding site blocked this suppressive effect.We further performed an immunoblot analysis in control and Ant-23a-transfected cell extracts using an AMBRA1-specific antibody.AMBRA1 protein levels were increased when miR-23a was underexpressed in PUVA-and UVB-SIPS fibroblasts.Similarly,the introduction of the Ant-23a but not Ant-CNT resulted in an increase in AMBRA1 mRNA levels in the PUVA-and UVB-SIPS fibroblasts.7.miR-23a mitigated rapamycin-induced autophagy and anti-senescence in PUVA-and UVB-SIPS fibroblasts.Rapamycin can increase autophagy,as evidenced by increased accumulation of GFP-LC3 puncta and LC3-Ⅱ accumulation,and decreased SQSTM1/p62 level in PUVA-and UVB-SIPS fibroblasts transfected by Ago-CNT.Moreover,overexpression of miR-23a led to the attenuation of GFP-LC3 puncta formation,decrease in protein levels of LC3 Ⅱ,and prevention of SQSTM1/p62 degradation,confirming the inhibitory effect of this miRNA on autophagy.AMBRA1 protein level was increased in Rapamycin-treated cells,and endogenous miR-23a level was decreased following rapamycin treatment.Furthermore,Rapamycin increased EdU-positive cells and decreased SA-β-gal positive cell percentages and G1 phase-arrested cell percentages,as well as p16,p53,and p21 protein levels in PUVA-and UVB-SIPS fibroblasts transfected by Ago-CNT.However,these effects were suppressed in fibroblasts transfected by miR-23a agomirs.8.Upregulation of AMBRA1 activated autophagic flux and restrained senescence in PUVA-and UVB-SIPS fibroblasts,and its effects can be mitigated by miR-23a agomirsTo confirm that AMBRA1 activated autophagic flux,similar autophagy tests were performed in cells transfected with either Ad-AMBRA1 or Ad-CNT.Indeed,transfection with Ad-AMBRA1 led to a prominent accumulation of GFP-LC3 dots and LC3-Ⅱ protein and a significant decrease in SQSTM1.However,these effects of Ad-AMBRA1 were found to be suppressed in fibroblasts transfected by miR-23 a agomirs.We also found that overexpression of AMBRA1 can result in an increase in the percentage of EdU-positive cells and a decrease in SA-β-gal positive cell percentages and G1 phase-arrested cell percentages,as well as an increase in p16,p53,and p21 protein levels in PUVA-and UVB-SIPS fibroblasts transfected by Ago-CNT.However,again these effects of AMBRA1were found to be suppressed in fibroblasts transfected by miR-23a agomirs.Conclutions:This study concludes that miR-23a-regulated autophagy is a novel and important regulator of ultraviolet-induced premature senescence,and AMBRA1 is a rate-limiting miRNA target in this pathway. |
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