Study On The Regulation And Mechanism Of AngiotensinⅡ On Skin Elastic Fiber

Background The gradual atrophy of elastic fiber network or abnormal protein deposition in skin dermis is an important feature of skin aging.Reducing the degradation of elastic fiber and promoting its regeneration are important ideas for delaying skin aging.Elastin is the main component of elastic fiber,with a content of90%.It is synthesized only in the mid-late embryonic period and the neonatal period and stably exists in tissues.However,with the influence of various internal and external factors,damage and degeneration will gradually occur.At present,there are many researches on cardiovascular elastic fibers,which can be used as a reference for the regulation of skin elastic fibers.The cardiovascular system’s response to certain physiological and pathological stimuli shows structural remodeling,with changes in cells,matrix and fiber components.Renin-angiotensin system（RAS）not only changes hemodynamics,but also participates in remodeling process as endocrine factor.Experiments show that RAS blocker can up-regulate the content of elastic fibers in vascular wall.There are two kinds of RAS in organisms.One is systemic and mainly regulates blood pressure,body fluid volume and electrolyte homeostasis.The other is local RAS,which exists in different organs and tissues and plays a certain role in tissue repair and reconstruction.All components of RAS exist in the skin,and angiotensin II（AngⅡ）,the main effector,can be locally formed,so AngⅡ may have certain regulatory effects on the synthesis of skin elastin and elastic fibers.Objectives To explore the regulation effect and possible mechanism of angiotensin II and its different receptor antagonists on the synthesis of elastin in human skin fibroblasts,and to find a new method to promote the synthesis of elastin and elastin fibers,and to verify it in the skin of living animals,providing a new direction for clinically solving the skin aging problem.Methods Primary human dermal fibroblasts were cultured by enzyme digestion,and the AngⅡ receptors AT1R and AT2R were detected by RT-PCR,Western blot and immunofluorescence.AngⅡ,AT1R antagonist losartan and AT2R antagonist PD123319 with different concentrations(10^-8M¹0^-5M)were used to interfere with fibroblasts for 24 and 48 hours respectively,and CCK-8 method was used to detect their effects on cell proliferation.Different concentrations of AngⅡ(10^-8M¹0^-6M)with or without losartan(10^-5M)and PD123319(10^-5M)were applied to fibroblasts to extract total RNA and total protein.Total RNA was reverse transcribed into cDNA,and the changes of elastin mRNA content were detected by fluorescence quantitative PCR.The total protein was quantified and Western Blot was used to detect the content of soluble tropoelastin.After determining the effect of AngⅡ on elastin and its receptor,the corresponding receptor antagonist and AngⅡ were used to interfere with fibroblasts.Western Blot was used to detect the phosphorylation levels of EGFR and ERK1/2.EGFR phosphorylation inhibitor AG1478 and MEK1/2 inhibitor U0126were used to interfere with fibroblasts.Western Blot was used to detect elastin content to verify the effect of EGFR/ERK1/2 pathway.8-week-old female Balb/c mice were selected,and the fractional CO₂ laser was performed after the hair removal treatment.Immunohistochemistry was used to detect the expression changes of AngⅡ,AT1R and AT2R in local skin from 1 day to 8weeks after treatment to verify whether skin RAS was activated after fractional CO₂laser.Losartan,captopril or vaseline were applied to the skin of mice after fractional CO₂ laser treatment,and a control group was set up which only added external medicine but no fractional laser.After the mice were killed at the 2nd,4th and 8th weeks after the treatment,the skin tissue on the back was taken for resorcinol basic fuchsin staining,the thickness of dermis was measured,and the elastic fiber content was analyzed.Results Primary human skin fibroblasts were successfully cultured,and the AngⅡ receptors AT1R and AT2R were stably expressed through mRNA,protein and cell level verification.After AngⅡ acts on fibroblasts,the expression of elastin mRNA and soluble tropoelastin decreases,and the phosphorylation levels of EGFR and ERK1/2 increase.AT1R antagonist losartan,EGFR inhibitor AG1478 and MEK1/2inhibitor U0126 can antagonize the inhibitory effect of AngⅡ on elastin expression.After fractional CO₂ laser was applied to Balb/c mouse skin,local AngⅡ,AT1R,AT2R expressions were enhanced,RAS was activated,and the thickness of mouse dermis was increased.Losartan or captopril can obviously increase the content of elastic fibers in mouse skin after fractional CO₂ laser.Conclusions AngⅡ inhibits the expression of elastin in human skin fibroblasts via receptor AT1R via EGFR/ERK1/2 pathway.Exfoliative carbon dioxide lattice laser can activate RAS in BALB/c mice skin,and fractional CO₂ laser plus external RAS blocker can increase the content of elastic fibers in mice skin.