Effect And Mechanism Of Bamboo Leaf C-glycoside Flavonoids On Skin Aging

Bamboo leaf C-glycoside flavonoids(BLF)refer to the natural flavonoids with Chinese local resource characteristics and independent intellectual property rights,which are separated and purified from bamboo leaves represented by Phyllostachys Sieb.et Zucc.Existing studies have shown that BLF have skin care effects such as whitening,antioxidation and radiation protection,but there is a lack of systematic evaluation and mechanism elaboration on their anti-aging effects.This study intends to clarify the role and mechanism of BLF in delaying skin aging by establishing in vitro cellular senescence model and mouse skin photoaging model,and provide theoretical basis and technical guidance for their addition and application in anti-aging functional products.At the same time,BLF have problems such as deep color,low bioavailability,difficult to be absorbed by the skin,and easy to lose their original functional properties due to external influences,which limit their application in industrialization.In view of the above problems,this study intends to take high-precision BLF(the mass fraction of BLF is more than 80%)as the research object,and take nanoliposomes as a technological breakthrough to improve the bioavailability and transdermal absorption of BLF,and the biological efficacy of skin care.The main research contents and results are as follows:(1)A cellular senescence model induced by oxidative stress was established to analyze the protective effect of BLF on skin aging.The senescence of HaCaT cells was induced by free radical inducer 2,2’-azobis-2-methyl-propanimidamide(AAPH).Through the detection of cell proliferation,cell cycle and apoptosis indicators,it was preliminarily determined that the dosage of AAPH was 1 m M,and the action time was 48 h.The detection of multiple biomarkers of cellular senescence such as SA-β-Gal,SAHF positive cell rate,p21,p16 and K9M-H3 proteins proved that 1 m M AAPH treatment for 48 h could successfully induce the senescence of HaCaT cells.BLF at 10～40μg/m L could inhibit AAPH-induced senescence of HaCaT cells.In addition,the detection of indicators of oxidative stress such as reactive oxygen species(ROS),mitochondrial membrane potential,antioxidant enzyme activity,markers of oxidative damage and inflammatory factors such as IL-2,IL-10 and COX-2 showed that the inhibitory effect of BLF on senescence was related to their antioxidant and anti-inflammatory activities.(2)RNA-Seq was carried out to explore the molecular mechanism of BLF delaying cellular senescence of skin.The results of differentially expressed gene screening showed that there were 337 genes co-regulated between the control and AAPH group and the AAPH and BLF group.The results of go enrichment analysis were mainly in the biological process module.BLF group and AAPH group were mainly enriched in response to unfolded proteins,negative regulation of cell proliferation,response to topological error proteins,cell death,positive regulation of programmed cell death,cell cycle and cell morphology regulation.KEGG analysis showed that four signal pathways were co enriched,and the regulation of MAPK signal pathway was the most obvious.qRT-PCR validated the accuracy of RNA-Seq results.The MAPK signaling pathway mainly includes p38,JNK and ERK pathways.Quantitative detection of p38,JNK and ERK protein expression indicated that p38MAPK was mainly involved in the anti-senescence effect of BLF.The p38 MAPK-specific activator Dehydrocorydaline(DE)and specific inhibitor SB203580(SB)could attenuate and enhance the regulatory effect of BLF on aging markers,respectively.(3)Autophagy is closely related to aging,and the MAPK signaling pathway may be a potential junction between skin aging and autophagy.The detection of autophagy markers LC3 A/B,Beclin-1,p62 and the observation of autophagosomes indicated that BLF activated autophagy to inhibit AAPH-induced cellular senescence.The addition of autophagy activator rapamycin and inhibitor 3-methyladenine,and the knockdown of ULK1,a key protein that regulates autophagy,changed the biomarkers of senescence,demonstrating that autophagy was involved in anti-senescence effect of BLF.In addition,the addition of DE and SB effectively regulated the activation and inhibition of autophagy in HaCaT cells,indicating that the p38 MAPK/ULK1pathway may be the main upstream mechanism involved in BLF-induced autophagy.(4)The thin layer dispersion method combined with sonication was used to prepare BLF-Lip.Chitosan coated BLF-Lip(CH-BLF-Lip)and alginate-chitosan coated BLF-Lip(AL-CH-BLF-Lip)were further prepared by electrostatic layer-by-layer self-assembly.The increase of particle size,the positive and negative alternation of zeta potential,the increase of encapsulation efficiency(EE),the change of FTIR spectra and the increase of T_c andΔH demonstrated the successful preparation of BLF-Lip,CH-BLF-Lip and AL-CH-BLF-Lip.In addition,after encapsulating BLF in nanoliposomes,the antioxidant activity,in vitro release,skin permeation and anti-senescence ability have been significantly improved,especially CH-BLF-Lip and AL-CH-BLF-Lip.(5)UVB irradiation for 8 weeks induced skin photoaging in mice.Determination of skin moisture content,epidermal thickness,collagen fiber content,MMP-3 protein expression,superoxide dismutase,glutathione peroxidase and catalase activities demonstrated the protection of BLF and AL-CH-BLF-Lip on aging skin.In addition,through the detection of skin aging biomarkers,MAPK and autophagy signals in mice,it was proved at the animal level that the effect of BLF in delaying skin aging was related to the MAPK and autophagy signaling pathways.In conclusion,BLF has the potential to intervene in skin aging,and its anti-aging effect is related to the signaling pathways of MAPK and autophagy.In addition,encapsulating BLF in nanoliposomes can effectively improve their bioavailability and transdermal absorption.This study expands the application value of BLF,and provides theoretical basis for the development of BLF-related functional foods and cosmetics.