A Preliminary Study On The Protective Effect And Mechanism Of Ergothioneine On Aging Skin In Mice

Objective:1.The transdermal penetration test in vitro was used to detect whether EGT could permeate through the skin and observe the local absorption.2.The protective activity of EGT on aging skin tissue and skin cells was observed by UV irradiation mice skin photoaging model and Etoposide（EPEG）induced aging cell model.3.To explore the effect of anti-aging activity of EGT on oxidative stress signaling pathway PI3K/Akt-Nrf2 and endogenous natural senescence signal p38MAPK,explore the possible mechanism of anti-skin aging of EGT,so as to provide a basis for the application of EGT.Methods:1.Diffusion experiment of EGT cream in vitro:the skin of isolated SD rats was divided into two groups:cream matrix group and EGT cream group.The dosage was 0.2 m L/cm².The receiving solution were collected by the TPY-2 transdermal absorber for 2 h,4 h,8 h,12 h and 24 h.The content of EGT at different time point was detected by high-performance liquid chromatography（HPLC）and calculated the cumulative permeation amount of EGT.2.The protective effect of EGT on skin photoaging model of mice irradiated by UV and its mechanism:mice were divided into control group（CTRL）,UV radiation group（UV）,cream matrix group（UV+Matrix）,quercetin group（UV+Que）,low-dose ergothioneine group（UV+0.1 m M EGT）,medium-dose ergothioneine group（UV+1 m M EGT）,high-dose ergothioneine group（UV+10 m M EGT）,The skin of mice was treated with cream before UV radiation.At the end of the experiment,the skin tissue was taken for pathological examination to observe the changes of skin thickness.The Hyp content,MDA content and SOD activity in the skin tissue were detected by kit.The expression levels of collagen and matrix metalloproteinases（MMPs）,PI3K,p-PI3K,Akt,p-Akt,Keap1,Nrf2,HO-1,p38MAPK and p-p38MAPK in UV mice skin were detected by Western Blot assay.Transcriptional levels of Nrf2 and its downstream signal molecules were detected by RT-PCR.3.Study on the effect and mechanism of EGT on etoposide-induced cell senescence:the cells were divided into control group（CTRL）,etoposide-induced group（10μM EPEG）,quercetin group（10μM Que）,low-dose,medium-dose and high-dose ergothioneine group（250,500,1000 n M EGT）.The cells were induced by 10μM EPEG for 48 hours and treated with different doses of drugs for 48 hours.CCK-8 assay was used to detect the effect of different concentrations of drugs on the activity of HaCaT cell.Senescence-associatedβ-galactosidase（SA-β-Gal）kit was used to detect the number of SA-β-Gal positive cells.Immunofluorescence was used to detect the expression of p16 andγ-H2A.X.RT-PCR was used to detect the m RNA levels of IL-6 and TNFα.The protein expressions of PI3K,p-PI3K,Akt,p-Akt,Keap1,Nrf2,p38MAPK and p-p38MAPK were detected by Western Blot assay.Results:1.In vitro diffusion experiments showed that EGT could permeate through the skin.The transdermal rate constant of EGT was 0.3864μg·cm^-2·h^-1,the permeability coefficient was 1.683 cm^-2·h^-1,the retardation time was 0.222 h,and the cumulative permeation percentage was19.87±8.184%.2.HE staining showed that EGT could reduce the epidermis thickness of mice skin induced by UV radiation.The kit test showed that EGT significantly increased the content of Hyp and the activity of SOD,with decreased the content of MDA in the skin.Western Blot assay showed that EGT significantly increased the expression of Collagen I and Collagen III protein and decreased the expression of matrix metalloproteinase（MMPs）in the skin,up-regulated phosphorylated PI3K and Akt protein levels,inhibited p38MAPK protein expression,and activated Nrf2 into the nucleus.RT-PCR showed that EGT up-regulated the levels of Nrf2 and its target genes HO-1,Txnrd1,Nqo1 and Gpx2.3.CCK-8 assay showed that EGT could inhibit the activity of HaCaT cell at the concentration of 1000 n M.Kit staining showed that EGT could significantly down-regulate the number of SA-β-Gal positive cells induced by EPEG.Immunofluorescence staining showed that EGT could significantly down-regulate the protein expression of p16 andγ-H2A.X.RT-PCR showed that EGT could significantly reduce the transcription levels of inflammatory cytokines IL-6 and TNFα.Western Blot results showed that EGT up-regulated the expression of phosphorylated PI3K and Akt,then activated Nrf2.PI3K inhibitor LY294002 increased the expression of p16 andγ-H2A.X in HaCaT cells,while p38MAPK inhibitor SB203580 reduced the expression of p16 andγ-H2A.X in HaCaT cells.Conclusion:1.EGT can permeate through skin and the cumulative permeation percentage of EGT at 24 h was 19.87±8.184%.2.EGT alleviates UV radiation-induced skin photoaging in mice by activating PI3K/Akt-Nrf2 signaling pathway and inhibiting p38MAPK protein expression,enhances skin antioxidant capacity,increases collagen content in skin and reduces MMPs expression,thus exerting antioxidant capacity and anti-aging effect.3.EGT activates the PI3K/Akt-Nrf2 signaling pathway to inhibit the expression of senescence related biomarkers in EPEG-induced senescence cells.