| ObjectivesEmerging clinical evidence suggests that the adjunctive use of5-aminolevulinic acid (ALA) with intense pulsed light (IPL), known as5-ALA-IPL-photodynamic therapy (PDT), may exert more beneficial effects on photoaged skin than intense pulsed light (IPL) treatment. The mechanisms and effect underlying5-ALA-IPL-PDT have not been fully elucidated. Firstly, we observe the impacts of IPL on the collagen synthesis of mouse skin, and on the cell proliferation of human fibroblasts in vitro. The experiment conditions were prepared for the subsequent studies.The mitogen activated protein kinase (MAPK) pathway is at the heart of ignaling networks that govern proliferation, differentiation and cell survival. Our continuing efforts were to define the molecular signaling pathway regulating IPL-induced proliferation in vitro. Therefore, this study was conducted to determine the contribution of IPL to the phosphorylation of MAPK family members (ERK, P38and JNK) in fibroblasts.Transforming growth factor (TGF)-beta is a central mediator in the progression of accumulation of ECM proteins. While MMP-1promotes the degradation of the collagens in the extracellular matrix. Both TGF-β1 and MMP-1could be secreted by fibroblast cells, but the relative importance of these factors is the subject of controversy. We asked whether IPL can regulate the balance between TGF-β1and MMP-1, which increases pericellular ECM.Cytokine (such as MMP1and TGF-(31) play important roles in the therapy of IPL. However the secretion of cytokine is rigorously regulated by signaling pathway. Here, we aimed to evaluate the activity of MAPK signaling pathway on MMP-1and TGF-β secretion of fibroblasts in vitro.The study was conducted to determine the effects of5-ALA-IPL-PDT on expression of MMP-1and TGF-β1, and to investigate roles of MAPK family members (ERK, P38and JNK) singaling pathway of fibroblasts in response to5-aminolevulinic acid-based photodynamic therapies, which will provide evidences for treatment and mechanism of action of5-ALA-IPL-PDT for photoaging.Methodsthe depilated mice were exposed to different doses of Intense Pulsed Light (IPL), At the time of1,3,7,14, and28d, the skin specimen were collected, the col1agen synthesis were determined by immunohistochem-ical method. The human fibroblasts cultured in vitro were exposed to IPL. The proliferation of fibroblasts was measured with MTT assay at48h following exposing. Human skin fibroblasts cultured in12-well plates were irradiated with IPL with fluences of0,10,18,36and72J/cm2. One hour after the irradiation, the protein of cells were collected. The level of MAPK phosphorylation was examined using western blotting.Human skin fibroblasts cultured in12-well plates were irradiated with IPL with fluences of0,10,18,36and72J/cm2.24hour after the irradiation, the culture medium of cells were collected. The level of TGF and MMP-1was examined with enzyme-linked immunosorbent assay(ELISA).The fibroblasts were plated onto the plates. Before IPL radiation PD98059, SP600125and SB203580were used to block ERK, JNK and P38, respectively.24hour after the irradiation, the culture medium of cells were collected. The level of TGF-β1and MMP-1was examined with enzyme-linked immunosorbent assay (ELISA).The fibroblasts were plated onto the plates. Two hours before IPL radiation5-aminolevulinic acid (5-ALA) were added. Survival rate of cells was detected by MTT. One hour after the irradiation, the protein of cells were collected. The level of MAPK phosphorylation was examined using western blotting.24hour after the irradiation, the culture medium of cells were collected. The level of TGF-β1and MMP-1was examined with enzyme-linked immunosorbent assay (ELISA). Results(1) Exposure to32J/cm2doses of IPL only leads a slight hyperdermis, but induces overexpression of collagen.(2) IPL could stimulate the expression of PCNA in vivo.(3) Exposure of cultured human dermal fibroblasts to a lower does of IPL can not increase the proliferation in vitro.(2) IPL (ranging from18to72J/cm2) could decrease the level of MAPK phosphorylation in a does-dependent manner. While, A dose of10J IPL radiation could increase the phosphorylation in all of three MAPK family members.(3) IPL (ranging from0to36J/cm2) have no stimulatory effect on the level of TGF-β. A significant increase could be observed at a does of72J radiation. IPL could influence the level of MMP-1in a different manner. A dose of10J/cm2IPL radiation could increase the secretion of MMP-1slightly. However, the radiation of IPL at a does larger than18J/cm2could not stimulate the secretion of MMP-1in fibroblasts.Treatment with PD98059and SP600125significantly prevented ERK and JNK phosphorylation in fibroblasts, SB203580has a slight effect on the phosphorylation of p38induced by IPL. Compared with control groups, the SP200125groups was markedly enhanced expression of MMP-1by254%. The pretreatment with PD98059and SB203580had no dramatical effect on the level of MMP-1.3) The pretreatment with SB203580had329.27%,567%and358%(P<0.05) more stimulatory effect on the secretion of TGF-B1than control groups, PD98059groups and SP600125groups, respectively.(4)1) Treatment with PD98059and SP600125significantly prevented ERK and JNK phosphorylation in fibroblasts, SB203580has a slight effect on the phosphorylation of p38induced by IPL.2) Compared with control groups, the IPL (10J)+ALA(0.5mM) groups and IPL (10J)+ALA(1.0mM) groups were markedly enhanced expression of MMP-1by94.7%and111.4%, While TGF-β1dropped34.8%and39.1%, respectively.3) IPL (10J)+ALA(0.5mM) groups had the most stimulatory effect on the phosphorylation of MAPK(ERK,P38and JNK) up to113.1%,36.1%and67.3%than IPL groups, respectively.CONCLUSIONSThe analytical results presented here provide a potential mechanistic explanation for the mechanism of clinical photorejuvenation effects of IPL that involves the increase of extracellular matrix construction by accelareting the proliferation of fibroblasts and upregulating the gene expressions of collagen Ⅲ.Higher does IPL radiation seems a negative stimulus to cause activation of MAPK family members. This may have important implications for the regulation of cell growth and secretion phenotype. IPL radiation seems to play a two-side role on the expression of MMP-1and TGF-β1. Higher does IPL radiation stimulates the secretion of TGF-β1, the lower stimulates the MMP-1. Together, these data provide new insight regarding the mechanism by which IPL regulate the cell growth and secretion phenotype of the fibroblasts.The members of MAPK have different effects on the IPL-induced secretion of TGF-β1, which is obviously down-regulated by P38, up-regulated by JNK and no significant effects by ERK. MAPK pathways were also involved in IPL-induced MMP1expression, which appear to be increased by JNK, but no ERK or P38. Besides the MAPK pathway, other salvage pathways appear to involve in IPL-induced MMP-1expression. Together, these data provide new evidence that IPL induces the expression of MMP*1and TGF-β1via MAPK pathway, which play key roles in IPL Rejuvenation.5-ALA-IPL-PDT can excert a cytotoxic effects on fibroblasts cells at higher does, which suggests that5-ALA-IPL-PDT is a stress reaction. At subcytotoxic does,5-ALA-IPL-PDT induced MAPK(ERK, P38and JNK) phosphorylation of fibroblasts, a potential mechanism involved in downregulating the expressions of TGF-β1proteins, and upregulating the expressions of MMP-1proteins. |
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