The Role And Mechanism Of Nrf2 In The Low-dose ALA-PDT For The Treatment Of Photoaging Fibroblasts

Background and Objective:Skin aging is a complex biological process that includes Extrinsic and intrinsic skin ageing.About 80%of facial skin ageing is attributed to UV irradiation.Due to at least 95%of ultraviolet A?UVA?can reach the ground and penetrates deep into the epidermis and dermis,long-term UV radiation can lead to rough skin,fine lines,pigmentation,telangiectasia,dermis thinning,deep wrinkles,and even skin tumors.UVA radiation is one of the most important environmental factors that cause skin light damage and aging.At present,there are many treatments to prevent or treat skin aging,including sunscreen,skin care products,laser,injection,surgery,photodynamic therapy and so on.Photodynamic therapy?PDT?refers to a method using specific light source to irradiate the damaged area with the participation of oxygen and photosensitiser,stimulating redox reaction to achieve the purpose of treatment in skin disease.Photodynamic effect has two kinds of mechanisms,including?and?reaction.Hydrogen atom or electronic transfer to produce other than singlet oxygen free radicals or ion,these free radicals reacts with the biological molecules and oxygen molecules generated oxidation product,called type?reaction.Through energy transfer mainly produce highly active antioxidants with singlet oxygen?¹O₂?,¹O₂ quickly react with neighboring biological molecules and cause damage,a process known as type?reaction.Both types of reactions can occur simultaneously,eventually achieving damage to tissue or cells.At present,photodynamic therapy has been widely used for the treatment of skin diseases,such as solar keratosis,condyloma,acne,bowen disease and so on.In recent years,the low-dose photodynamic rejuvenation has gradually been recognized as a new non-invasive,safe,economical,simple and repeatable photoaging treatment.It has been found that high-dose photodynamic therapy can kill tumor and microorganisms by generating a large number of cytotoxic reactive oxygen species?ROS?in the target tissues and inducing apoptosis in many clinical studies.While low-dose photodynamic therapy can improve the skin texture and luster,but the mechanism is not very clear.It's possible that the production of a small amount of ROS can cause cell damage and repair,stimulate fibroblast proliferation,accelerate skin healing,increase dermal collagen and reduce elastic fiber degeneration,so as to achieve the effect of dermal remodeling and collagen regenerationWhen long-term ultraviolet radiation causes excessive accumulation of reactive oxygen species in the body,it exceeds the body's ability to resist oxidative stress and results in photoaging.The Nrf2/ARE signaling pathways plays a key regulatory role in controlling a series of antioxidant and detoxifying enzymes.The pathway has protective and therapeutic effect in photoaging.In this paper,the skin photoaging model in vitro was established to investigate whether low-dose ALA-PDT can activate Nrf2 in photoaging cells,generate anti-oxidative stress reaction,achieve the role of reversing photoaging and study the mechanism.Methods:The main methods used in this study are as follows:Real-time Cell Analyzer?RTCA?for detecting cell proliferation;Senescence-associated-galactosidase kit for detecting the proportion of senescent cells;The SOD kit for detecting the total SOD level of cells;Build siNrf2 adenovirus vector,the efficiency of infection by fluorescence microscope observation,through Quantitative polymerase chain reaction method?qPCR?and Western blot?WB?method detection of Nrf2 in mRNA and protein level of silent efficiency;WB for detecting Nrf2,TGF-?1,HO-1,COL1,COL3,ERK,p-ERK and MMP-9 protein expression.Results:1.Establishment of the skin photoaging model:as UVA dose increases,the form of fibroblasts that we saw by the microscope becomes wider and flater and the degranulation phenomenon,even apoptosis appears;Real-time analyzer detect the slow proliferation of fibroblasts after UVA irradiation,when UVA dose of 10 J/cm²,the cells proliferate and then stagnate,when the UVA dose of 20 J/cm² cell proliferation becomes significantly inhibited;The positive rate of senescent cells detected by SA-?-GAL is dependent on the dose of UVA.2.Expression changes of Nrf2 and related factors after treatment with ALA-PDT on photoaging fibroblasts:?1?the total SOD activity in cells increased,but the increase was most obvious in the low-dose photodynamic group.?2?the nucleoprotein separation experiment indicated that the nuclear expression of Nrf2 was significantly increased in low-dose ALA-PDT treatment photoaging group.?3?according to the results of low-dose ALA-PDT treatment after photoaging fibroblasts,the protein expression of Nrf2,COL1,COL3,TGF-?1,HO-1 and p-ERK has increased;while MMP-9 express declined.After the photoaging cells were successfully intervented by Ad-siNrf2,the protein expression of Nrf2,COL1,COL3,TGF-?1,HO-1 declined,and MMP-9 has increased.Conclusion:1.The photoaging cells model can be successfully prepared by irradiation with UVA dose of 10J/cm² for 5 consecutive days;2.In the treatment of photoaging fibroblasts with photodynamic therapy,low-dose photodynamic therapy enhances the antioxidant capacity by activating nrf2-related pathways,promotes the generation of dermal collagen and reduces its degradation;Knockdown of Nrf2 expression reduced collagen synthesis and antioxidant level,suggesting that the transcription factor Nrf2 plays an important regulatory role in anti-oxidation and promoting collagen proliferation.