Molecular Mechanism Of PPAR Pathway In Regulating Aging Caused By Chronic Skin Barrier Injury

Objective(s):Skin aging can be divided into endogenous aging and exogenous aging,but no studies have shown whether barrier breakdown is the cause of aging.By establishing a model of chronic skin barrier damage in mice,we compared barrier and age-related indicators in different groups to explore the influence of chronic barrier damage on skin aging and the potential pathway,thus discovering new aging factors and revealing the regulatory mechanism of aging caused by barrier damage.Methods:Part I: Using the adhesive tape method to construct an animal model of damaged skin barrier in mice.(1)Balb/c mice were randomly divided into Control group(Control group)and tape group(Tape Stripping).Mice in control group were treated with 3M tape after anesthesia and shaved,and mice in tape group were pasted 10-20 times in each place.The taping stop index was: TEWL value increased 6-8 times,once a day,for 3months.(2)The skin appearance of mice was observed,TEWL and water content of mice were detected by CK apparatus,and barrier indexes of mice in two groups were detected by immunofluorescence staining,including tight link protein(ZO-1),filaggrin(FLG),inner coating protein(IVL),E-cadherin and β-catenin.HE,Masson,VG and weigert staining were used to detect the skin pathology of mice.ROS,MDA,CAT,SOD,GSH-PX were detected by the kit.The expression of P53,P21,P16,gamma-H2 Ax,matrix metalloproteinase 1(MMP1),collagen type I(Col-Ⅰ),collagen type III(Col-Ⅲ),Lamin B1(Lamin B1)were detected by Western Blot,immunohistochemistry and immunofluorescence.(3)Finally,RNA-seq was performed on the back skin tissues of the two groups of mice,and then KEGG enrichment analysis was performed to find the related genes that may lead to aging after the destruction of the skin barrier.Immunofluorescence and Western Blot verified the changes in their expression.Part two: To establish PPARγ overexpression animal models using difference factor(PPARγ)agonists,and to verify the mechanism of PPARγ in skin aging after skin barrier breakdown in mice.(1)Balb/c mice were randomly divided into four groups: Control group(Control),tape group(Taped),tape +DMSO solvent group(Negative control)-Tape pasted and applied normal saline with 1% DMSO concentration,tape +PPARγ agonist group(PGZ)-tape pasted and applied 30μg/kg pioglitazone(dissolved in 1%)Dmso-concentration saline)for three months of modeling.(2)The skin appearance of the four groups of mice was observed,TEWL value was detected,and the barrier index FLG was detected by immunofluorescence staining.HE and Masson staining were used to detect the skin pathological manifestations of mice,and oxidative stress indexes(ROS and MDA)were detected by kit.Western Blot was used to detect the expression of P53,P21 and P16To verify the protein expression levels of the upstream pathways(EGF,JNK,ERK)that may lead to changes in PPARγ due to chronic barrier failure.(3)The back skin of mice in the belt +PPARγ agonist group and belt +DMSO solvent group was subjected to RNA-seq to find the downstream factors of PPARγ,and the expression level was verified by Western Blot.Meanwhile,the protein interaction relationship was detected by Co-IP.Results : Part I:(1)Appearance of mice: Compared with the control group,skin erythematosis,folds increased,desquamate aggravated and cuticle thickened significantly in the duct tape group.(2)Results of the skin barrier index of mice: compared with the control group,the TEWL value of the skin of mice in the belt group was increased,while the water content was decreased.Immunofluorescence staining results showed that the expressions of core skin barrier proteins ZO-1,FLG,IVL,E-cadherin and β-catenin in the duct tape group were significantly decreased,indicating that the skin barrier was damaged.(3)Results of mice skin aging index: Compared with mice in the control group,skin pathological staining results of mice in the belt group showed that the cuticle was thickened,collagen fibers and elastic fibers were broken and disordered.In addition,oxidative stress indexes of mice in duct tape group also showed increased expressions of ROS,MDA and CAT,decreased expressions of SOD and GSH-PX,significantly increased expression levels of aging markers P53,P21,P16,gamma-H2 ax and MMP1,and significantly decreased protein expression levels of colⅰ,Col-Ⅲ and Lamin B1.(4)The PPAR signaling pathway was significantly enriched by KEGG enrichment analysis after RNA-seq detection.Part 2:(1)Appearance of mice: compared with the control group,the skin folds of mice in the duct tape group and the duct tape +DMSO solvent group increased and deepened,a large amount of desquamate and cuticle thickened significantly,and the aging phenotypes of mice such as skin folds were alleviated by PPARγ agonist(the duct tape +PPARγ agonist group).(2)Skin barrier index results of mice: Compared with duct tape group and duct tape+PPARγ agonist group,skin TEWL value decreased and FLG content expression increased.(3)Compared with the duct tape group and the duct tape +PPARγ agonist group,the oxidative stress index was increased,the MDA content was decreased,the aging index was decreased,the expression of P16,P21 and P53 was decreased,and the pathological staining results showed that the skin thickening,inflammation,collagen fiber breakage and disorder were reduced.At the same time,the upstream pathway EGF-EGFR-JNK/ERK-PPARγ was verified.(4)By analyzing the back skin transcriptomes of mice in the belt +DMSO solvent group and belt +PPARγ agonist group,NLRP3 downstream of PPARγ was found,Co-IP and Western Blot results were used to verify the protein interaction between the two groups.Conclusion(s):PPARγ is a key factor in skin barrier breakdown leading to aging.The breakdown of skin barrier may down-regulate the expression of PPARγ by stimulating the EGF-EGFR-JNK/ERK pathway.PPARγ has protein interaction with NLRP3,and down-regulates PPARγ to reduce its inhibitory effect on NLRP3.Skin aging phenomenon and degree of aggravation.