The Protective Mechanism Of Lycium Barbarum Extraction On UV-induced Skin Photodamage

Ultraviole（Ultraviolet,UV）is the main factor causing skin injury and damage of skin barrier function.According to epidemiological studies,about80%of exposed skin diseases are caused by skin photodamage caused by UV rays,therefore,the study of effective substances that can resist UV rays has a certain scietific value and practical significance.As one of the significantly plant resources in the aird and high ultraviolet areas western China,Lycium barbarum fruit has the functions of nourishing the blood,enriching the yin,replenishing qi and clear vision,and is often used in eye care,prevention of pigmentation and resistance to UV rays.There are many active substances in Lycium barbarum,including Lycium barbarum polysaccharides,carotenoids,Lycium barbarum flavonoids and Lycium barbarum polyphenols.Lycium barbarum polysaccharides have the effects of immune regulation,antioxidation and anti-radiation,while Lycium barbarum flavonoids and polyphenols have the effects of obviously scavenging free radicals.In summary,in view of the fact that there are many active substances in Lycium barbarum have a certain effect on photodamage and immuno regulation,so in this paper,on the basis of the antioxidation of LBE,this paper proposes to repair the skin barrier by regulating the skin barrier functional protein.While maintaining good skin barrier function,it can enhance the inherent antioxidant capacity of the skin and maintain the integrity of skin structure and function.The research content of this paper is arranged as follows:Firstly,the contents of total flavonoids and total polyphenols in LBE were determined by spectrophotometry.The results showed that the contents of total flavonoids and total polyphenols were 4.65±0.02μg/mg and 86.03±3.06ug/mg respectively.The antioxidant effect of LBE was evaluated by DPPH radical scavenging experiment in vitro.The DPPH radical scavenging rate IC₅₀of LBE was 0.1484 mg/m L,which showed good antioxidant activity.In order to test the pre-protective effect of LBE on keratinocyte（Hacat）and fibroblast（NIH-3T3）UVB-induced cell damage,concentration 50μg/m L and 100μg/m L of LBE were given respectively before UVB injury,and survival rates of Hacat and NIH-3T3 cells determined by MTT method,under theconcentration 50μg/m Lof LBE,the survival rates of Hacat and NIH-3T3 cells were59.11±1.69%and 59.57±1.26%,respectively,the survival rates of Hacat and NIH-3T3 cells were67.47±2.44%and 72.29±5.27%,respectively,under the intervention of 50μg/m L LBE.The results showed that LBE had protective effect on UVB damage of Hacat and NIH-3T3 cells.Then in order to explore the protective effect of LBE on UV-induced skin photodamage,UVA combined with UVB to simulate the sun ultraviolet radiationi in mice model was established.The LBE was apply locally on the back skin of KM mice before 2h irradiate the back skin of mice,and lasted for9 weeks.The TEWL was tested and the skin light damage wrinkles were scored experimental session.The results showed that LBE could significantly inhibit skin injury,restore skin structural integrity and inhibit TEWL to maintain skin hydration integrity.H&E staining method was used to analyze the thickness of the epidermis and Masson staining was used to analyze the collagen fibers in the dermis.The results showed that LBE could inhibit the thickening of epidermis,promoted the increase of collagen density and restore the elastic fiber structure,and especially the concentration of LBE was 50 mg/m L.Finally,from the mechanism of oxidative stress and the expression of proteins related to skin barrier function discussed the possible mechanism of LBE protective effect on skin injury.The protein expression levels of MMP-3and MMP-9 were determined by immunohistochemical（IHC）method,which confirmed that LBE could effectively inhibit the expression of MMP-3 and MMP-9;The activity of ROS in skin tissue by ELISA assay,contents of SOD,CAT,GSH-Px and MDA in skin tissue were determined by colorimetry.It was proved that the extract of LBE significantly increased the activities of antioxidant enzymes SOD,CAT and GSH-Px in the skin of mice induced by UV,while decreased the content of MDA and scavenged the content of ROS.Nrf2m RNA gene transcription and its protein expression were detected by Real-time quantitative polymerase chain reaction（RT-q PCR）and Western blotting（WB）assay respectively,the RT-q PCR assay was used to verify the transcription level of heme oxygenase-1（HO-1）m RNA.Results showed LBE could activate the expression of Nrf2 and regulate the transcription of HO-1m RNA.The differentiation proteins of skin barrier contents of filaggrin（FLG）and Loricrin（LOR）were determined by ELISA assay,FLG and involucrin（INV）were investigated by IHC assay,RT-q PCR and WB methods to test both aquaporins-3（AQP3）m RNA and its protein expression.Result was found that LBE could enhance the expression levels of FLG,INV and LOR protein to maintain the integrity of skin barrier function,up-regulate the AQP3m RNA transcription level and AQP3 protein expression to maintained hydration integrity of epidermis.