| Objective:Ultraviolet(UV)irradiation promotes the expression of matrix metalloproteinases(MMPs)by activating mitogen-activated protein kinase(MAPK),thereby degrading collagen and inhibiting collagen synthesis leading to skin photoaging.Recent studies have shown that MAPK and MMPs are also involved in the treatment of skin photoaging by intense pulsed light(IPL)irradiation of human skin keratinocytes,but the exact mechanism is unclear.Based on previous studies,this study further studied the role of MAPKs in regulating the expression of MMPs,collagen remodeling and proliferation of human skin keratinocytes during the treatment of skin photoaging by IPL and explored its molecular mechanism.Methods:(1)In this study,human skin keratinocytes were extracted from healthy adolescent foreskin tissue.Human skin keratinocytes were irradiated with different doses of UV/IPL,and p-ERK,p-P38 and p-JNK protein levels were detected by WB method at 15mins to screen out the optimal irradiation dose;human skin keratinocytes were irradiated with UV100m J/cm~2and 17J/cm~2IPL respectively,and p-ERK,p-P38 and p-JNK protein levels were detected by WB method at different time points after irradiation to screen out the optimal time point for detection;human skin keratinocytes were irradiated with different doses of UV/IPL,and p-c-fos and p-c-jun protein levels were detected by WB method at 15mins.(2)Human skin keratinocytes were irradiated with different doses of UV/IPL,and the expressions of Cyclin D1 and MMP-1,MMP-2,MMP-3and MMP-9 were detected by WB at 24h.On day 1,3 and 5,cell proliferation was detected by CCK-8 method.The activity changes of MMP-2 and MMP-9 were detected by gelatin enzyme spectrometry at 24h.The ultrastructure of cells was detected by transmission electron microscopy after 24h.(3)Human skin keratinocytes were irradiated with UV100m J/cm~2to establish photoaging models,followed by 17J/cm~2IPL and WB for protein levels of p-ERK,p-c-fos and p-c-jun at 15mins;The protein expressions of Cyclin D1,MMP-1,MMP-2,MMP-3 and MMP-9 were detected by WB at24h.Human skin keratinocytes incubated with 5,10 and 20μM PD98059were UV100m J/cm~2irradiated for 24h,and WB was used to detect p-ERK protein levels after 15mins.Human skin keratinocytes were incubated with20μM PD98059 for 24h,irradiated with UV100m J/cm~2and detected with WB at 15mins.(4)The back skin of guinea pigs was irradiated by UV 100m J/cm~2once a day for 10 weeks to establish the animal photoaging model.Immediately after the end of the irradiation,17J/cm~2IPL was irradiated once every 3 weeks for 3 consecutive times.After the end of the IPL irradiation,the skin tissues were trepaned at the 1st,7th and 14d,respectively.HE staining was used to detect the change of guinea pig epidermal thickness.The changes of skin collagen in guinea pigs were detected by Masson and Ceravine scarlet staining.The ultrastructure of collagen in guinea pigs was detected by TEM.The protein expressions of p-c-fos,p-c-jun,MMP-1,MMP-3 and MMP-9 in guinea pig skin were detected by immunohistochemical staining.Results:(1)Compared with control group,there was no significant difference in p-P38 protein level in UV dose group(P>0.05).The protein levels of p-ERK and p-JNK were significantly up-regulated(P<0.05).Compared with the control group,there was no significant difference in p-P38 protein level in UV time group(P>0.05).The protein levels of p-ERK and p-JNK peaked in 15mins(P<0.05)and returned to baseline level in 120mins.Compared with control group,there were no significant changes in p-P38protein level in IPL dose group(P>0.05).The levels of P-JNK protein were significantly up-regulated in a dose-dependent manner by IPL17 and23J/cm~2irradiation(P<0.05).P-ERK protein level was significantly down-regulated at 17J/cm~2IPL and p-ERK protein level was significantly up-regulated at 23J/cm~2IPL(P<0.05).Compared with the control group,there were no significant changes in the expression level of P-P38 in IPL time group(P>0.05).The p-ERK protein level was down-regulated immediately after IPL irradiation and the P-JNK protein level peaked at15mins(P<0.05)and all of them returned to baseline level at 120mins.Compared with control group,UV dose group significantly down-regulated p-c-fos protein level and promoted up-regulated p-c-jun protein level(P<0.05).Compared with control group,there was no significant difference in p-c-fos protein level in IPL dose group(P>0.05).P-c-jun protein level was significantly up-regulated at 17J/cm~2IPL(P<0.05).(2)Compared with the control group,the expression of Cyclin D1protein in UV dose group was significantly down-regulated(P<0.05).Compared with control group,the expression of Cyclin D1 protein in IPL dose group was significantly up-regulated(P<0.05).Compared with control group,UV100m J/cm~2significantly inhibited cell proliferation;IPL17J/cm~2significantly promoted cell proliferation(P<0.05).Compared with the control group,the protein expressions of MMP-1,MMP-2,MMP-3 and MMP-9 in UV dose group were significantly up-regulated,the17J/cm~2IPL dose group significantly inhibited the expression of MMP-1and MMP-3 protein(P<0.05),while there were no significant changes in the expression of MMP-2 and MMP-9 protein(P>0.05).Compared with the control group,the activities of MMP-2 and MMP-9 increased in the UV dose group,while the activities of MMP-2 and MMP-9 did not change significantly in the IPL dose group.Compared with controls,UV100m J/cm~2and 17J/cm~2IPL promoted the increase of cellular microvilli.(3)Compared with the UV group,the p-ERK protein level was significantly down-regulated,p-c-fos and p-c-jun protein levels were significantly up-regulated,Cyclin D1 protein expression was significantly increased,MMP-1,MMP-2 and MMP-3 protein expression were all down-regulated in the UV+IPL group(P<0.05),while MMP-9 was unchanged(P>0.05).In the PD98059 intervention group,the expression level of p-ERK decreased and the expression level of p-c-fos increased significantly(P>0.05),the expression level of p-c-jun did not change significantly(P>0.05).(4)Compared with the control group,the back epidermis thickness of guinea pigs in UV group was significantly thinner,compared with UV group,skin thickness in UV+IPL group increased significantly from day 7(P<0.05).Compared with the control group,the collagen content in the back skin tissue of guinea pigs in UV group was reduced and the arrangement was sparse.Compared with UV group,the content of dermal collagen fiber in UV+IPL group was increased and evenly distributed from day 7(P<0.05).Compared with the control group,the ultrastructure of collagen fibers was significantly changed,the collagen fibers were disordered,broken,and the average diameter became thinner.Compared with the UV group,the collagen fibers in the UV+IPL group were rearranged,densely distributed,and the average diameter of the fibers became thicker.(5)Compared with the control group,the expression of p-c-fos protein in the back skin tissue of guinea pigs in UV group was significantly decreased,and the level of p-c-fos protein in UV+IPL group was significantly increased after treatment(P<0.05).Compared with the control group,the p-c-jun protein level in the back skin tissue of guinea pigs in UV group was significantly up-regulated;compared with UV group,the p-c-jun protein level in UV+IPL group was significantly up-regulated at the 7th and 14d after treatment(P<0.05).Compared with the control group,the protein expressions of MMP-1,MMP-3 and MMP-9 in UV group were significantly increased;compared with UV group,the protein expressions of MMP-1 and MMP-3 in UV+IPL group were significantly decreased(P<0.05),while the protein expression of MMP-9 was not changed(P>0.05).Conclusions:(1)At the cellular level:IPL irradiates aged human skin keratinocytes,inhibits ERK,activates JNK,then activates c-jun and c-fos,inhibits MMPs and up-regulates the expression of Cyclin D1,the former reduces collagen degradation,the latter promotes the proliferation of keratinocytes,thus achieving the effect of treating skin photoaging.The expression pattern of MAPK/AP-1/MMP was different from that of UV-induced MAPK/AP-1/MMP.(2)After IPL irradiation,the epidermal thickness of photoaged guinea pig skin increased,c-fos and c-jun were activated,and the expressions of MMP-1,MMP-3 and MMP-9 were inhibited,which was consistent with the cell experiment.The results of electron microscopy and special stain indicated that collagen deposition increased.(3)The different expression patterns of MAPK,AP-1,Cyclin D1 and MMPs induced by IPL and UV may play a role in promoting the proliferation of keratinocytes and reducing the degradation of collagen to treat skin photoaging.(4)The results of this study showed that 17J/cm~2IPL irradiation promoted the proliferation of human keratinocytes and the epidermal thickening of guinea pigs and restored damaged dermal collagen by inhibiting the ERK/AP-1 signaling pathway in photoaged human keratinocytes and guinea pig skin.It is suggested that epidermis,keratinocytes and dermal collagen play an important role in IPL treatment of skin photoaging,which is one of the possible mechanisms of IPL treatment of skin photoaging,and provides a theoretical basis for clinical IPL treatment of skin photoaging.. |
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