Effect And Mechanism Of Puerariae Lobatae Radix On UVB-induced Skin Aging

ObjectiveUVB-induced skin aging may result in skin diseases and even skin cancer,but the efficacy of current anti-aging drugs is limited or some anti-aging drugs may cause adverse reactions.Thus,there is a pressing need to discover safer and more effective anti-aging drugs.Puerariae lobatae radix（PLR）is a traditional Chinese medicine,which is known for its antioxidant and anti-aging properties.However,it remains unclear whether PLR has the effect of delaying the skin aging induced by UVB.BMAL1 and REV-ERBαare critical components of the circadian clock system.It has been found that BMAL1 and REV-ERBαnot only play an important role in regulating circadian rhythm in mammals,but also associated with aging.Nevertheless,their regulation and mechanisms in skin aging still require further investigation.In this thesis,we aim to explore the protective effect and mechanism of PLR on UVB-induced skin aging.Methods1.To construct the skin-aged mice,we irradiated the bare skin on the backs of C57BL/6J mice with UVB ultraviolet light（120 m J/cm²）.In order to investigate the effect of PLR on UVB-induced skin aging,we used HE staining,q PCR and kit assays to evaluate various skin aging-related indicators,such as skin epidermal thickness,Mmp-1,p21,p53,HYP,SOD,and MDA.Subsequently,based on mouse fibroblast L929 cells,a aged cell model exposed to UVB（100 m J/cm²）,we evaluated various indicators,such as cell proliferation,Mmp-1,p21,p53,β-galactosidase,ROS by CCK-8,q PCR,and kit assays,to explore the effects of PLR on L929 cell senescence.2.We measured the m RNA and protein levels of the clock gene Bmal1 in both aged skin of mice and L929 cells using q PCR and Western blot techniques.Furthermore,the expression of Bmal1 target genes,such as Cry1,Cry2,Per1,and Per2,was detected to investigate the regulatory effect of PLR on BMAL1.3.Luciferase reporter gene technology and L929 cells were used to investigate the regulatory effect of PLR on REV-ERBα.We verified Rev-erbα^-/-mice using q PCR.Then wild-type and Rev-erbα^-/-mice were used as experimental subjects and skin aging-related indicators were detected through HE staining,q PCR and kit assays to explore the crucial role of REV-ERBαin the protective effect of PLR against skin aging.4.In order to explore the regulatory effect of PLR on NRF2 antioxidant pathway.we measured the m RNA levels of Nrf2,Prdx6,Sod1 and Sod2,which are downstream genes of Bmal1 by q PCR.Results1.PLR has a protective effect against UVB-induced skin agingWe successfully constructed the UVB-induced skin aging mouse model and L929cells aging model.By measuring skin aging-related indicators,we found that compared to the model group,PLR decreased the thickness of skin epidermis and the content of MDA,but increased the levels of HYP and SOD.Meanwhile,the expression of Mmp-1,p21,and p53 was down-regulated.At the cellular level,PLR enhanced the viability of L929 cells,decreased the activity of intracellularβ-galactosidase,and reduced ROS levels.These findings suggested that PLR has a protective effect on UVB-induced skin aging.2.PLR promotes the expression of the clock factor BMAL1PLR up-regulated both the m RNA and protein levels of Bmal1 in aging skin tissue and L929 cells.In addition,PLR enhanced the expression of downstream genes of Bmal1,including Cry1,Cry2,Per1,and Per2.These results suggested that the anti-photoaging effect of PLR may be related to BMAL1.3.PLR counteracts the clock factor REV-ERBαto delay UVB-induced skin agingBased on the results of the luciferase reporter assay,we observed that PLR antagonized REV-ERBαand significantly increase the expression of the Bmal1.Furthermore,we compared several skin aging-related indicators such as skin epidermal thickness,HYP,SOD,MDA,Mmp-1,p21 and p53 in both wild-type and Rev-erbα^-/-mice under UVB-induced conditions.Our results showed that the knockout of the Rev-erbαimproved the skin aging in mice.However,the protective effect of PLR on UVB-induced skin aging disappeared in Rev-erbα^-/-mice,suggesting that REV-ERBαplayed a significant role in anti-skin photoaging of PLR.4.PLR delays UVB-induced skin aging through the REV-ERBα-BMAL1-NRF2antioxidant pathwayThe results of q PCR assay revealed that PLR activated the NRF2 antioxidant pathway by antagonizing REV-ERBα.Upon comparing the expression levels of Nrf2,Prdx6,Sod1,and Sod2 in the skin of wild-type and Rev-erbα^-/-mice under UVB-induced conditions,we observed that the knockout of the Rev-erbαactivated the NRF2 antioxidant pathway,which was characterized by increased gene expression of Nrf2,Prdx6,Sod1 and Sod2.However,PLR had no effect on the NRF2 antioxidant pathway in the skin of Rev-erbα^-/-mice,indicating that PLR delayed UVB-induced skin aging through the REV-ERBα-BMAL1-NRF2 antioxidant pathway.ConclusionPLR promotes the expression of BMAL1 by antagonizing REV-ERBα,and then activates NRF2 antioxidant pathway to delay UVB-induced skin aging.The research findings provide theoretical and scientific basis for further exploration,utilization and clinical application of PLR.