| With the continuous progress of the society and the improvement of people's life,the problem of skin aging has been paid more and more attention.Many manifestations of skin aging,such as skin relaxation,thinning,wrinkles,uneven pigmentation,large pores,telangiectasia,can not only affect the appearance,even aggravate the psychological burden of patients and reduce the self-confidence,then may further have a impact on the normal social life and work.Laser treatment of skin aging has shown its effectiveness and advantages,and has become the focus of research.The results show that the increase of antioxidant capacity,the decrease of matrix metalloproteinase activity and the increase of collagen synthesis are the key steps in the laser treatment of skin aging.However,the specific mechanism is not clear.Recent studies have shown that down-regulation of S100A8 protein can inhibit the activation of RAGE receptor,then inhibit MAPK,NFkappa B signaling pathway and downstream transcription factor AP-1,which may be an important way to contact the key links.Although the clinical effectiveness of laser treatment of skin aging has gradually been recognized by all parties,but its specific molecular biological mechanisms have not been fully clarified.Therefore,there is a certain degree of experience dependence and not easy to quantify in the laser treatment.To explore the related molecular mechanisms will provide a theoretical basis for the more rational use of laser treatment of skin aging.In this study,1064 nm Q-switched Nd:YAG laser was used as the light source,Ha Ca T cells and mouse fibroblasts were used as the experimental object.The cell culture,cell transfection,RNA interference,Real-time PCR,Western blot and other methods were used to study the role of S100A8 protein,RAGE receptor and its mediated MAPK,NF-kappa B signal pathway in the treatment of skin aging by 1064 nm Q-switched Nd:YAG laser.All of these were aimed to clarify the signal mechanism and the target of laser action in order to provide a solid theoretical basis for the laser treatment of skin aging.Part ? Effects of 1064 nm Q-switched Nd:YAG laser irradiation on the proliferation and antioxidant capacity of Ha Ca T cells and mouse fibroblastsObjective To explore the effects of 1064 nm Q-switched Nd:YAG laser irradiation on the proliferation and antioxidant capacity of Ha Ca T cells and mouse fibroblastsMethods The CCK8 kits were used to detect cell proliferation changes of HaCaT cells and mouse fibroblasts in the laser irradiation group and the control group.Cell ROS levels of both groups were measured by flow cytometry and the SOD activity of cells were determinated by superoxide dismutase activity assay kits.Results Cellular morphology and proliferation ability of Ha Ca T cells and mouse fibroblasts did not change significantly after 1064 nm Q-switched Nd:YAG laser irradiation,but the SOD activity of cells increased remarkably.Meanwhile the higher the laser voltage,the higher the SOD level.Conclusions 1064 nm Q-switched laser irradiation does not cause damage to Ha Ca T cells and mouse fibroblasts,which hinting that it is safe to use laser to treat skin aging.At the same time,1064 nm Q-switched Nd:YAG laser irradiation can not promote the oxidative stress response,but can enhance the antioxidant capacity of cells,suggesting that the mechanism of skin aging treated by laser may be related to the increase of the expression of antioxidants.Part ? Effects of 1064 nm Q-switched Nd:YAG laser irradiation on collagen synthesis and degradation in mouse fibroblastsObjective To explore the effects of 1064 nm Q-switched Nd:YAG laser irradiation on type I and III collagen synthesis and degradation in mouse fibroblasts.Methods Real-time PCR technique was used to detect the expression difference of type I and III collagen,MMP-1,MMP-2,MMP-9,TIMP-1 and TIMP-2 in the laser irradiation group and the control group at the transcriptional level.Results The expressions of type I and type III collagen m RNA were significantly higher after 1064 nm Q-switched Nd:YAG laser irradiation for 24 hours in the laser irradiation group than that of the control group.The expression of MMP-1 and MMP-2 m RNA decreased markedly,but there was no noteble changes in the expression of MMP-9 m RNA.However the TIMP-1 and TIMP-2 m RNA expression were observably increased.Conclusions 1064 nm Q-switched Nd:YAG laser irradiation can promote the synthesis of collagen in mouse fibroblasts,which may be related to the inhibition of MMP-1 and MMP-2 expression and the expression of TIMP-1 and TIMP-2.Part ? Effects of 1064 nm Q-switched Nd:YAG laser irradiation on the inflammatory response of Ha Ca T cells and mouse fibroblastsObjective To explore the effects of 1064 nm Q-switched Nd:YAG laser irradiation on the inflammatory response of Ha Ca T cells and mouse fibroblasts.Methods The expression difference of RAGE,S100A8,TGF-beta,TNF-alpha and IL-6 m RNA in the laser irradiation group and the control group were detected by using Real-time PCR technology.And Western blot test was used to determine the expression difference of RAGE and S100A8 protein of Ha Ca T cells and mouse fibroblasts.Results The expressions of S100A8,RAGE,TGF-beta,TNF-alpha and IL-6 m RNA in Ha Ca T cells were significantly increased after 1064 nm Q-switched Nd:YAG laser irradiation.Meanwhile,the expressions of S100A8,RAGE protein in Ha Ca T cells and mouse fibroblasts were strikingly increased after 1064 nm Q-switched Nd:YAG laser irradiation.Conclusions The expressions of S100A8 and RAGE protein can be upregulated by 1064nm Q-switched Nd:YAG laser irradiation,which can cause the inflammatory reaction,then promote the expressions of inflammatory cytokines and activate the signal pathways involved in the collagen synthesis and decomposition.Part ? Effects of 1064 nm Q-switched Nd:YAG laser irradiation on the expessions of MAPK and NF-?B signal pathways in Ha Cat cells and mouse fibroblastsObjective To explore the Effects of 1064 nm Q-switched Nd:YAG laser irradiation on the expessions of MAPK and NF-?B signal pathways in Ha Ca T cells and mouse fibroblasts.Methods RNA interference and Real-time PCR technology were applied to detect the effects of S100A8 and si S100A8 on the expression of TGF-beta,TNF-alpha,IL-6,IL-8 and other inflammatory factors in Ha Ca T cells.And the effects of S100A8 and si S100A8 on the synthesis of collagen in mouse fibroblasts also were derminated by Real-time PCR technology.Western blot were used to examine the expressions of ERK1/2,JNK,p38 proteins in MAPK signaling pathway and p65 protein in NF-?B signaling pathway in both Ha Ca T cells and mouse fibroblasts after 1064 nm Q-switched Nd:YAG laser irradiation.Results After HaCaT cells were treated with si S100A8 for 48 hours,the expression of inflammatory factors IL-6,IL-8,TNF-alpha and TGF-beta were significantly decreased.However,after Ha Ca T cells were treated with S100A8 for 24 hours,the expression of inflammatory factors IL-6,IL-8,TNF-alpha and TGF-beta were significantly increased.The expression of type I and type III collagen m RNA decreased markably in mouse fibroblasts after treatment with si S100A8 and significantly increased after treated with S100A8 for 24 hours.The expressions of p-p65,p-p38 and p-JNK proteins were significantly increased in Ha Ca T cells after 1064 nm Q-switched Nd:YAG laser irradiation for 1 hours and 3 hours.In addition,the expression of p-p38 protein was remarkably increased in mouse fibroblasts after being irradiated by 1064 nm Q-switched Nd:YAG laser for 1 hour and 3 hours.Conclusions 1064 nm Q-switched Nd:YAG laser irradiation can activate the activity of the p-p65,p-p38,p-JNK proteins in Ha Ca T cells and the activity of the p-p38 in mouse fibroblasts.All of these signaling factors are initiated by S100A8 protein and they are together involved in the regulation of skin collagen synthesis induced by 1064 nm Q-switched Nd:YAG laser. |
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