Experimental Study For Protective Effects Of TGF-β1 On UVA Irradiated Human Skin Fibroblasts In Vitro

BackgroundSkin aging can be divided into two basic categories:intrinsic aging and photoaging.The factors of intrinsic aging are inevitable and the specific mechanism is undiscovered.In photoaging,the ultraviolet light plays a important role.Thus skin aging caused by being repeated exposure to sunlight is named photoaging.It is characterized with coarse,skin laxitas,deep,severe wrinkling,freckles,telangiectasia and pigmentary changes or even cancer on exposed areas such as the face,neck and forearm.Fibroblasts are the most important components of the cells in the dermal. Ultraviolet(UV)leads fibroblasts to earlier aging,decreases the synthesis of collagen on the one hand,and it increases the expression of matrix metalloproteinases and degrades extracellular matrix on the other hand.In sum,it leads to skin photoaging. According to its wavelength,ultraviolet light is classified into UVC(200 to 280 nm), UVB(280 to 320 nm)and UVA(320 to 400 nm).About 99 percent the UVA reaches the Earth's surface,whereas more than 50 percent of UVA penetrates the epidermis and arrived at fibroblasts.Therefore,UVA is closely related to skin-ageing and has attracted worldwide attention in the public health domain.As to skin-aging mechanisms,the theory of UV irradiation producing ROS' is universally accepted at the present.As a result,in recent years domestic and foreign scholars have done more antioxidant research against UVA irradiation's oxygen free radicals,but so far definitive anti-aging methods have not been identified.With development of studying on the anti-aging mechanism,researchers have found that the cytokine plays a very important role in regulating skin-ageing,and have devoted more attention to cytokine's study.However,papers and reports about cytokines' anti-skin aging are inadequate.ObjectiveIn this study,we investigateⅠ,Ⅲcollagen synthesis and expression of TGF-β1 after UVA radiate fibroblasts in vitro,and the effects of TGF-β1 to collagen synthesis. We discuss whether or not TGF-β1 inhibits the expression of MMP-1,MMP-3 and protects mitochondrial DNA(mtDNA)deletion,and analyze the effects of TGF-β1 to heat shock protein 70's expression.Briefly we study comprehensivly the protective effects of TGF-β1 to fibroblasts in vitro being irradiated by UVA and the effects of TGF-β1 to the secretion of IGF-1,KGF,VEGF when UVA irradiates dermal fibroblasts in vitro.Methods1.We conduct experiments by culturing fibroblasts of foreskin from young adults and using the five dynasties cells.Specifically,by dividing cells into goups and using different doses of UVA(5 J/cm~2,10J/cm~2,20J/cm~2)irradiated cells,we establish control group(0 J/cm~2)and fibroblast photo-aging model.Then we test cell proliferation activity,Ⅰ,Ⅲcollagen content in supematant by MTT, enzyme-linked immunosorbent assay(ELISA)after UVA irradiation skin fibroblasts, and TGF-β1 treatment.We also testⅠ,Ⅲcollagen mRNA and TGF-β1 mRNA expression by using semi -quantitative(RT-PCR)method.2.By adding different doses of TGF-β1 to the culture medium,small dose(0.1 ng/ ml),medium dose(1.0 ng/ml),high dose(10.0ng/ml)and with UVA15J / cm2 irradiation,we test frstlyⅠ,Ⅲcollagen content and HSP70 expression changes in supematan using immunosorbent assay(ELISA),and secondarlyⅠ,Ⅲcollagen mRNA and MMP-1,MMP-3 mRNA,Smad3 mRNA expression changes,and finally the Mitochondrial DNA(mtDNA)4977bp deletion by RT-PCR.We use enzyme-linked immunosorbent assay(ELISA)to determine the content of IGF-1, KGF,VEGF in the supernatant.Results1.Using different doses of UVA(5J/cm~2,10J/cm~2,20J/cm~2)irradiate cultured skin fibroblasts,we have found that when UVA irradiation dose increased,the fibroblast OD values decrease.Compared with the control group,UVA 10J/cm~2, UVA 20J/cm~2 irradiated group's OD values are significantly decreased.There is a very significant difference(p＜0.01).While increasing the dose of UVA irradiation,Ⅰcollagen's content and mRNA expression decrease in a dose-dependent manner. Being compared with the control group,UVA 10J/cm~2,UVA 20J/cm~2 irradiated group decrease significantly(p＜0.05,P＜0.01),whereas UVA 20J/cm~2 group inhibits collagen synthesis more obviously while comparing with the control group. Comparing the two groups,there is a significant difference(P＜0.01).As toⅢ collagen's content and mRNA expression,UVA 20J/cm~2 irradiated group is significantly reduced,comparing with the control group.There is a very significant difference(P＜0.01).TGF-β1 protein content and mRNA expression decrease when the dose of UVA irradiation is increased in a dose-dependent manner.Comparing with the control group,TGF-β1 protein content and mRNA expression in UVA 10J/ cm~2,UVA 20J/cm~2 group is significantly reduced,hence a very significant difference(P＜0.01).2.Using different doses of TGF-β1 to UVA15J/cm~2 to irradiate the fibroblasts. The experiment shows that:when increasing the dose of TGF-β1,large,medium dose group OD values are significantly higher than that of the UVA irradiation group. There is a very significant difference(P＜0.01).Ⅰ,Ⅲcollagen protein content increases with the dose of TGF-β1 increased and there is a dose-dependent manner. Comparing large,medium dose of TGF-β1 group with UVA irradiation group,there are significant differences(P＜0.01,P＜0.05).High dose group promoting collagen synthesis is more obvious and there is a very significant difference(P＜0.01).With the dose of TGF-β1 increased,Ⅰ,Ⅲcollagen mRNA expression also increase in a dose-dependent manner.Ⅰcollagen mRNA expression in the large,medium-dose group is significantly increased.Comparing with UVA irradiation group,there is a very significant difference(P＜0.01).Comparing large dose group with UVA irradiation group,Ⅲcollagen mRNA expression in the large dose group is significantly increased.There is a very significant difference(P＜0.01).After adopting different doses of UVA irradiation and comparing UVA 10J/ cm~2,UVA 20J/cm~2 irradiated group with control group,MMP-1,MMP-3 mRNA expression are significantly increased.There are significant differences(P＜0.05, P＜0.01).In UVA 20 J/cm~2 irradiated group,MMP-1,MMP-3mRNA expression increase more obviously and there is a very significant difference(P＜0.01).After TGF-β1 treatment by different doses,UVA irradiates fibroblast.MMP-1,MMP-3 mRNA expression decrease with the dose of TGF-β1 increases.There is a dose-dependent manner.Large,medium dose group's MMP-1 mRNA expression are lower than that of the control group.There is a very significant difference(P＜0.01). Large dose group's MMP-3 mRNA expression is lower than that of the control group. There is a significant difference(P＜0.05).After different doses of UVA irradiation, Smad3 mRNA expression is lower than that of the control group.Being compared UVA 10J/cm~2,UVA 20J/cm~2 group with the control group,there is a very significant difference(P＜0.01),while being compared 5J/cm~2 irradiation dose group with the control group,there is signifcant differences(P＜0.05).Treatment with different doses of TGF-β1 to UVA irradiated fibroblasts.With increased dose of TGF-β1,Smad3 mRNA expression increases in a dose-dependent manner.In the high and medium dose group,Smad3 mRNA expression increases more obviously,while comparing with UVA irradiation group there is a very significant difference(P＜0.01). Comparing small dose group with UVA irradiation group,the difference is significant (P＜0.05).3.When the cumulative dose of UVA arrives 60J/cm~2,fibroblasts mitochondria DNA 4977 bp deletion occures.When the cumulative dose of UVA arrives 90J/cm~2, deletion is very significant(P＜0.01).After TGF-β1 treatment,the high dose group mtDNA 4977 bp deletion reduces.While being compared medium,small dose group with that of the UVA irradiation group,there is no significant difference(P＞0.05).4.Different doses of UVA irradiation have shown a dose-dependent inhibition to the HSP70 expression.Comparing UVA 10J/cm~2,UVA 20J/cm~2 irradiated group with the control group,HSP70 are significantly reduced and there are significant differences(P＜0.05,P＜0.01).Comparing UVA 20J/cm~2 irradiated group with that of the control group,the inhibition affects are more obvious.There is a very significant difference(P＜0.01).Prior to irradiation of TGF-β1 treaments,HSP70 content increases with the dose of TGF-β1 and there is in a dose-dependent manner. High dose group HSP70 is significantly higher than that of the UVA irradiation group, there is a very significant difference(P＜0.01).Medium dose group HSP70 is higher than that of the UVA irradiation group,there is significant differences(P＜0.05).5.UVA irradiation in cultured skin fibroblasts resulted IGF-1,KGF decreased secretion,UVA 10J/cm~2,UVA 20J/cm~2 irradiated group decreases significantly, comparing with the control group.There is a significant difference(P＜0.01).VEGF secretion increases in UVA 20J/cm~2 irradiated group,comparing with the control group,hence a significant difference(P＜0.01).After TGF-β1,TGF-β1 dose-dependent increases UVA irradiation in vitro skin fibroblasts secreting three factor level.Large dose group IGF-1,KGF,VEGF are significantly higher than the UVA irradiation group whereby there is a significant difference((P＜0.01). Conclusions1.UVA irradiating on cultured skin fibroblasts leads to cell aging,inhibits proliferation activity and the typeⅠ,Ⅲcollagen synthesis.Inhibiting TGF-β1 protein content and mRNA expression increase with the dose of UVA irradiation.It suggests that UVA irradiation on cultured skin fibroblasts causing collagen synthesis decreases It is also related to lower expression of TGF-β1.Low expression of TGF-β1 and TGF-β/Smad signal transduction pathway plays an important role in the skin photoaging process.2.TGF-β1 treatment before UVA irradiation can improve skin fibroblasts proliferation activity in vitro.It is in a dose-dependent manner to promote the synthesis of collagenⅠ,Ⅲand mRNA expression on UVA irradiated fibroblasts. UVA irradiating fibroblasts can induce MMP-1,MMP-3 mRNA high expression and Smad3 mRNA low expression.TGF-β1 can lower the MMP-1,MMP-3mRNA expression and enhance the expression of Smad3 mRNA.It has protective effects onto UVA irradiation fibroblast-ageing.The mechanism maybe TGF-β1 promotes fibroblast proliferation and enhances the binding ability between TβR-Ⅱand TGF-β1. It could promote fibroblast collagen synthesis.Moreover,it inhibites MMP-1, MMP-3mRNA expression through TGF-β/ Smad pathway.3.UVA(360nm)irradiating skin fibroblasts,when the cumulative dose of UVA arrives UVA60J/cm2,mtDNA 4977 bp deletion occures,and when UVA arrives 90J/cm~2 deletion is obvious.After certain dose(10ng/ml)of TGF-β1 treatment,skin fibroblast mtDNA4977 bp deletion is reduced.4.UVA irradiating fibroblast can inhibit HSP70 expression and TGF-β1 can increase it's expression.TGF-β1 induces the HSP70 to high expression and protects fibroblast from UVA irradiation.5.TGF-β1 can improve IGF-1,KGF,VEGF levels on UVA irradiated dermal fibroblasts.