Study On The Active Ingredients Screening That Promote Macrophages-Mediated Elimination Of Senescent Skin Cells And Its Mechanism

The accumulation of senescent skin cells contributes to the progression of skin aging.Immune cells such as macrophages can selectively recognize and eliminate senescent cells.This function is termed as “senescence immunosurveillance”.However,this function of immunocytes become compromised with age,resulting in an excessive accumulation of senescent cells in skin,leading to the skin aging eventually.Therefore,enhancing the senescence immunosurveillance function of macrophages may be a safe and effective strategy for delaying skin aging.The present study is designed to establish a macrophages-senescent cells co-culture system to screen the active ingredients that can promote macrophage-mediated elimination of senescent skin cells,and further explore the underlying mechanism to provide scientific basis for the development of new senescent skin cells-targeting strategy for delaying skin aging.The main work includes the following three parts:(1)Established a co-culture model of macrophages and senescent skin cells: The THP-1 derived macrophages and tert-Butyl hydroperoxide(t-BHP)-induced senescent Ha Ca T keratinocytes or HFF-1 fibroblasts were used to establish the co-culture model.The senescent skin cells were detected after fluorescence staining with SPi DER-βgal,and the expression of senesence-associated secretory phenotype(SASP)factors were assayed by q PCR.The experimental conditions were optimized in regard to optimal dose and treatment time of t-BHP,as well as the suitable ratio between macrophages and senescent skin cells(effector:target ratio)in the co-culture model.To induce the cellular senescence,Ha Ca T cells were treated with t-BHP at 250 μM for 2 h,and HFF-1 cells were treated with t-BHP at 300 μM for 2 h.In the co-culture model,ratio between macrophages and skin cells at 5:1 was found suitable for screening the enhancer of macrophage’s senescence immunosurveillance function.(2)Screened active ingredients that can potentiate the senescence immunosurveillance function of macrophages: The effects of several candidate ingredients on senescent skin cells were compared in the macrophages-senescent skin cells co-culture model and the senescent skin cells single-culture model.It is found that centella asiatica extracts and carnosine can promote the macrophages-mediated elimination of senescent skin cells in the co-culture system.Other candidate ingredients,including Ganoderma extracts,Palmitoylpentapeptide-4,Acetylhexapeptide-8 and Hexapeptide-3,do not possess the activity to enhance the supervision function of macrophages,but they can directly eliminate senescent skin cells and inhibit the expression of SASP on their own.(3)Investigated the mechanism which carnosine enhance the senescence immunosurveillance function of macrophages: The fluorescent microsphere phagocytosis assay demonstrated that carnosine can improve the phagocytosis function of macrophages.q PCR results indicated that carnosine can elevate the m RNA expression levels of phagocytic receptor CD36 and RAGE in macrophages.Furthermore,CD36 and RAGE antagonists abrogate the enhancing effect of carnosine on macrophages-mediated clearance of senescent skin cells in co-culture of macrophage and senescent skin cells.Western blot results demonstrated that carnosine stimulates the activation of AKT2 signaling pathway.AKT2 inhibitor abrogates the enhancing effect of carnosine on the m RNA expression of phagocytosis receptor CD36 and RAGE in macrophages as well as its enhancing effect on the clearance of senescent skin cells by macrophages in the co-culture macrophages and senescent skin cells.Collectively,the present study established the co-culture model of macrophages and senescent skin cells,and found that carnosine can effectively promote macrophages to eliminate senescent skin cells.The further mechanism research revealed that carnosine can up-regulate the expression of CD36 and RAGE via AKT2 signaling pathway and elevate the phagocytic ability of macrophages,further promote the activity of macrophages to eliminate senescent skin cells.