The Role And Mechanism Of ERK/MAPK In The Low-dose ALA-PDT For Rejuvanation Of Photoaging Fibrooblasts

Backgound and ObjectivePhotoaging is the result of skin caused by long-term irritation of ultraviolet rays.Through complex biological mechanisms,chronic skin damage can lead to abnormal skin antioxidant functions,skin barrier damage,and histological abnormalities.Photoaging skin caused by long-term ultraviolet radiation manifests as skin darkening and graying,irregular pigmentation,rough skin,looseness and loss of elasticity,which is somewhat different from the acute effects of ultraviolet radiation on the skin,mainly inflammation and erythema.After acute ultraviolet radiation,the skin epidermis,dermis,and especially the thickening of the stratum corneum can be observed;while in photoaing skin,the skin tissue,especially the dermis,becomes thinner and atrophied.This suggests that the acute and chronic effects of ultraviolet radiation on skin tissue are different,not only in their performance,but also in their mechanisms,but the research on this is not clear enough.The chronic effect of ultraviolet radiation on skin tissue causes photoaging,which not only produces wrinkles and stains,but also brings a bad aesthetic sense,but also causes abnormalities in skin defense mechanisms such as antioxidant pathways and barrier functions,which may even cause benign,Nausea tumors have very adverse effects on health.At present,there are many ways to prevent and treat light aging,including applying sunscreen,antioxidant products,topical use of vitamin A derivatives,phototherapy,Filling injection therapy,and so on.Photodynamic therapy is also considered as an effective treatment to effectively treat photoaging and improve skin condition.Photodynamic therapy(PDT)is a therapy that uses photosensitizers to irradiate target cells at specific wavelengths to produce reactive oxygen species(ROS)such as oxygen free radicals and singlet oxygen,and then oxidizes stress to achieve therapeutic goals.Previous photodynamics have been successfully used to treat tumors,genital warts,and acne.High-concentration,high-dose photodynamic energy is enriched by photosensitizers in proliferative target tissues and generates reactive oxygen species(ROS)under the action of light energy.High concentrations of ROS are cytotoxic and can make tumors Cells and virus-infected cells undergo apoptosis,thereby achieving the effect of treating tumors and viral warts.After reducing a certain concentration and dose,photodynamic therapy has also achieved very satisfactory results in the treatment of acne.Not only can it treat the acute symptoms of acne,but it can also inhibit the excessively strong sebaceous glands,reduce the chance of acne recurrence,and the prognosis of skin color and The fineness has improved.In recent years,people have realized that low-dose photodynamic therapy can also make skin younger,but the specific mechanism needs to be further explored.After low-dose photodynamic therapy,the face is younger,the skin tissue barrier function is restored,the antioxidant capacity is enhanced,and the dermal tissue is thickened.According to previous research,nuclear factor NF-E2 related factor 2(Nrf2)in skin tissue is one of the key factors regulating oxidative stress response.Nrf2 is a nuclear transcription factor that binds to KEAP1(Kelch-like ECH-associated protein1)in the resting state.When nrf2 wa activated,it dissociates into the nucleus and combines with the antioxidant response element(ARE)It can regulate a series of enzymes such as glutathione-S-transferses(GSTs)to participate in oxidative stress response.In skin tissues,Nrf2 is considered to be involved in the mechanism of resisting UV damage,so we speculate that low-dose photodynamics may achieve facial rejuvenation by regulating Nrf2 related pathways.Our laboratory has received low-dose photodynamic therapy and silenced the Nrf2 gene through an in vitro fibroblast photoaging cell model to verify that Nrf2 does indeed play an important role in low-dose photodynamic therapy,but how low-dose ALA-PDT excites Nrf2 and makes it The mechanism of separation from KEAP1 into the nucleus to regulate downstream transcription is unknown.This article compares the gene expression changes of normal fibroblasts,photoaing fibroblasts,and photoaing fibroblasts after low-dose photodynamic treatment,and explores how low-dose ALA-PDT activates Nrf2 to rejuvante the skin.Method1.Human fibroblasts were separated and cultured,and the cells was treated with long-wave ultraviolet rays to make a photoaing fibroblast model.2.Fibroblasts were treated with high-dose and low-dose ALA-PDT.3.Sensence-associated ?-galactosidase(SA-?-Gal)staining kit was used to detect fibroblasts without UVA induction,UVA induced photoaging fibroblasts,photoaing fibroblasts treated with low-dose ALA-PDT and photoaing fibroblasts treated with high-dose ALA-PDT;detect the proportion of senescent cells in each group of cells by inverted microscope.4.CellTiter-Lumi ? Luminescence was used to detect Cell viability changes of fibroblasts without UVA induction,UVA-induced photoaging fibroblasts,photoaging fibroblasts treated with low-dose ALA-PDT and high-dose ALA-PDT.5.According to methods 1-2,fibroblasts were prepared without UVA induction,photoaing fibroblasts induced by UVA,and photoaing fibroblasts were treated with low-dose ALA-PDT;RNA was extracted by trizol for transcriptome sequencing test.6.According to the results of transcriptome sequencing,the gene expression was analyzed for differences of the three groups of cells without UVA-induced fibroblasts,UVA-induced photoaing fibroblasts,and photoaing fibroblasts treated with low-dose ALA-PDT.7.The total protein was extracted of non-UVA-induced fibroblasts,photoaging fibroblasts,and photoaing fibroblasts after low-dose ALA-PDT treatment,and detected by Western blot the changes in protein expression.8.The expression levels of p-ERK was detected in three groups of cells by immunofluorescence staining.9.Fibroblasts were treated with UVA induction at different time intervals,total protein of fibroblasts induced by UVA at different accumulation times was extracted,and immunoblot was used to detect changes in protein expression such as p-ERK in cells under different UVA treatment durations.10.Photoaing fibroblasts was treated with ERK inhibitors and low-dose ALA-PDT.11.SA-?-Gal staining kit was used to detect the proportion of senescent cell in the UVA-induced photoaing fibroblasts,photoaing fibroblasts after low-dose ALA-PDT and photoaing fibrfoblasts treated with p-ERK inhibitor and low-dose ALA-PDT.12.The total protein was extracted of untreated fibroblasts,UVA-induced photoaing fibroblasts,photoaing fibroblasts treated with low-dose ALA-PDT,and photoaing fibroblasts treated with low-dose ALA-PDT ERK inhibitors and low-dose ALA-PDT,and showed the changes of protein expression in four groups by western blot.Result:1.The therapeutic effect of low-dose ALA-PDT for rejuvenation was verified on a previously established photoaging cell model: it could be seen that after UVA induction,the proportion of senescent cells of fibroblasts was obvious increased,cell viability was significantly inhibited.After low-dose photodynamic treatment,the positive rate of senescent cells decreased.2.According to the results of transcriptome sequencing,in the similarity of gene expression,after ALA-PDT treatment,the gene transcription of photoaing fibroblasts approached the level of normal photofibroblasts without photoaging.There were 52 genes whose transcription changed by photoaging restored to normal fibroblast were related to MAPK.3.After UVA-induced photoaging,Nrf2 and p-ERK protein were down-regulated,while after ALA-PDT treatment,Nrf2 and p-ERK protein levels were significantly up-regulated in photoaing fibroblasts,and the trends of the two were consistent.P-P38 expression was down-regulated after low-dose photodynamic treatment;p-JNK did not change significantly.4.The results of immunofluorescence staining were consistent with the results of WB.Compared with normal fibroblasts,the p-ERK fluorescence intensity of photoaing fibroblasts is lower,and the fluorescence intensity was significantly increased after ALA-PDT treatment.5.In the initial stage of UVA induction,the expressions of Nrf2 and p-ERK in fibroblasts were significantly up-regulated.With the increase of UVA treatment time,the expressions of Nrf2 and p-ERK were gradually suppressed,and the protein level was gradually decreased.6.SA-?-Gal staining results showed that low-dose ALA-PDT treatment could reduce the proportion of senescent cells of photo-aging fibroblasts.The proportion of senescent cells in photoaing fibroblasts did not decrease significantly.7.When UVA induced photoaging,the synthesis of COL1 and COL3 was significantly reduced in fibroblasts,and after treated with low-dose ALA-PDT,the synthesis of COL1 and COL3 increased.But when photoaging fibroblasts were treated with ERK inhibitor and low-dose ALA-PDT,the expression of COL1,COL3 and Nrf2 did not increase significantly.Conclusion:1.In the in vitro photoaing fibroblast model,low-dose ALA-PDT treatment with 0.1mM ALA 21 J / cm2 red light could rejunevante photoaing fibroblasts.2.Through transcriptome sequencing,it was found that MAPK-related genes were involved in the mechanism of UVA-induced photoaging and low-dose ALA-PDT treatment of photoaging.3.In acute light damage,UVA could stimulate the expression of Nrf2 and p-ERK,while in chronic photoaging,with the increase of UVA cumulative action time and dose,antioxidant dysfunction was obstacled,proliferation was inhibited,and expression of Nrf2 and p-ERK levels were down-regulated.4.When ERK was inhibited,low-dose ALA-PDT could not up-regulate Nrf2 and rejuvenate photoaging fibroblasts;it suggested that low-dose ALA-PDT might rejuvenate photoaging fibroblasts by activating ERK / MAPK.