Study On The Repairing Effect And Mechanism Of Acanthopanax Senticosus Glycoprotein On Skin Photoaging Induced By Ultraviolet Rays

Objective:In this paper,the glycoprotein of Acanthopanax senticosus was extracted from the traditional genuine medicinal material Acanthopanax senticosus.The glycoprotein was isolated and purified,and the physicochemical properties,composition and structure-activity relationship of Acanthopanax senticosus glycoprotein were systematically studied;To explore the protective effect of Acanthopanax senticosus glycoprotein on skin and its repair effect and mechanism in skin light damage caused by ultraviolet light through in vivo and in vitro experiments,and further explore its starting protein with antioxidant activity from the perspective of proteomics,find the target protein,and then synthesize the polypeptide in vitro,so as to open up a new field for the research of Acanthopanax senticosus glycoprotein,To provide theoretical support for the better use of Acanthopanax senticosus glycoprotein and the development of related products for skin protection and repair of light damage.Methods:（1）Using response surface method,the glycoprotein of Acanthopanax senticosus was extracted by constant temperature and constant speed shaking method.Taking the protein yield of Acanthopanax senticosus as the single factor evaluation index,the best extraction process of Acanthopanax senticosus glycoprotein was screened.The purity and molecular weight of the protein were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）.The anti-o-glcnac modification sites of Acanthopanax senticosus glycoprotein were investigated by Western blot（WB）;In addition,the structure of Acanthopanax senticosus glycoprotein was analyzed by measuring the content of neutral sugar and acidic sugar and investigating the UV spectrum,infrared spectrum,monosaccharide composition and amino acid composition.（2）MTT method and cell scratch test were used to investigate the promoting effect of Acanthopanax senticosus glycoprotein on the proliferation and migration of hhuman keratinocytes Cell（Ha Ca T）;At the same time,the rat skin photoaging model was established.The therapeutic effect of Acanthopanax senticosus glycoprotein on rat photoaging skin was evaluated from the appearance,skin tissue morphology and ulultra-weak photon emission（UPE）.The rat skin tissue was stained with hematoxylin eosin（HE）and aldehyde fuchsin,and then the morphological changes of rat skin were observed,The levels of malondialdehyde（MDA）,Glutathione peroxidase（GSH-Px）,superoxide dismutase（SOD）and hydroxyproline（HYP）,matrix metalloproteinases-1（MMP-1）,matrix metalloproteinases-3（MMP-3）and interleukin-1β（IL-1β）、Interleukin-6（IL-6）and tumor necrosis factor-α（TNF-α）To explore the therapeutic effect and mechanism of Acanthopanax senticosus glycoprotein on skin photoaging in rats.（3）Proteomic identification of the target protein of Acanthopanax senticosus glycoprotein and in vitro synthesis of peptides were carried out,and then relevant in vitro antioxidant verification was carried out to find the effective protein in Acanthopanax senticosus glycoprotein.Results:（1）The optimum extraction process of Acanthopanax senticosus glycoprotein was obtained by single factor and response surface method.The extraction time was 12h/time,a total of 3 times,the liquid-material ratio was 1:16,the p H value of buffer was 8,the extraction temperature was 70℃,and the shaking speed was 180rpm.After purification,the content of Acanthopanax senticosus glycoprotein was 32.12%,the content of neutral sugar was 16.27%,and the content of acidic sugar was 24%.SDS-PAGE results show that the protein molecular weight of Acanthopanax senticosus glycoprotein is mainly about 25 k D,indicating that the protein content of this size is high,which can be further used as a key research object.UV spectrum showed that the type of glycopeptide bond was o-glycopeptide bond,and there was an ultraviolet absorption peak of protein at 240 nm;Acanthopanax senticosus glycoprotein had anti-o-glcnac modification.The results showed that the band position was the same as that of Acanthopanax senticosus glycoprotein,about25 k D;Infrared spectrum showed that the glycoprotein sample of Acanthopanax senticosus had absorption peaks at about 3300 cm^-1,2900 cm^-1,1000-1075 cm^-1,and there were polysaccharides and protein molecules;The main monosaccharides in Acanthopanax senticosus glycoprotein were glucose,rhamnose and xylose;The results of amino acid composition analysis show that Acanthopanax senticosus glycoprotein contains 18 kinds of amino acids,of which 8 are essential amino acids for human body.（2）Acanthopanax senticosus glycoprotein can promote the proliferation and migration of human skin keratinocytes.Acanthopanax senticosus glycoprotein can effectively improve the skin cuticle thickening,elastic fiber breakage,aggregation,obvious back oxidative stress response and enhanced UPE photon signal of photoaging skin in rats;Compared with the model group,the content of MDA in skin tissue of Acanthopanax senticosus glycoprotein group decreased significantly,the activities of GSH-Px,SOD and HYP increased significantly,and the inflammatory factor IL-1β、IL-6 and tumor necrosis factor TNF-αThe contents of matrix metalloproteinase MMP-1 and MMP-3 decreased significantly.（3）It was identified that Acanthopanax senticosus glycoprotein had four main proteins and five peptide segments and synthesized.The results showed that VFDKDQNGFISAAELR-（Cam）had excellent antioxidant activity.It was speculated that it was the main effective protein in Acanthopanax senticosus glycoprotein.Conclusion:The extraction and purification process of Acanthopanax senticosus glycoprotein was established;The composition and structure of glycoprotein were studied.The monosaccharide composition and amino acid composition were determined by UV and IR scanning.Finally,the best extraction process and its configuration were determined;Through in vivo and in vitro experiments,using UPE technology,HE and aldehyde fuchsin tissue section staining and ELISA kit,it is concluded that Acanthopanax senticosus glycoprotein can effectively repair the damage of skin photoaging in rats;Through proteomic sequencing of Acanthopanax senticosus glycoprotein,the peptide results were synthesized after identification.After synthesis,it was verified by antioxidant experiment in vitro.It is speculated that VFDKDQNGFISAAELR-（Cam）is the effective protein in Acanthopanax senticosus glycoprotein.