The Effect And Mechanisms Of Adipose-derived Stem Cells Repair UVB-induced Skin Aging

Background:Skin aging is not only a physiological change,but also a pathological state caused by the external environment.Photoaging caused by ultraviolet B(UVB)is the main contributors.Adipose-derived stem cells(ASCs)is one kind of multipotent mesenchymal stem cells with multiple differentiation potentials.At the same time,it has the ability to secrete a variety of cytokines.ASCs therapy has become a promising treatment due to its abundant sources,easy access and low immune rejection.At present,some clinical studies and animal experiments have shown that ASCs can effectively improve the aging skin,but the mechanism of ASCs improving aging skin has not been fully clarified.Objective:1.To investigate whether ASCs can improve the aging skin by impairing normal and UVB-induced damaged human dermal fibroblasts(HDFs).2.To study the effects of ASCs on extracellular matrix remodeling related cytokines(including MMP1,TGF-?,EGF and IGF-1)and the changes of skin extracellular matrix via animal experiments.3.To clarify the protective effect of ASCs on skin oxidative stress injury and its mechanism.4.To evaluate the effect of ASCs on angiogenesis and local tissue water(LTW)in the aging skin of nude mice.Methods:1.ASCs were isolated and cultured.The phenotype and multipotential differentiation potential were also identified.2.HDFs was divided into four groups:normal HDFs group(blank control),UVB irradiated photoaging HDFs group,ASCs and normal HDFs co-culture group and ASCs and photoaging HDFs co-culture group.We detected the effects of ASCs on HDFs senescence,proliferation,apoptosis and migration.3.The effect of ASCs on expression of extracellular matrix remodeling related cytokines(including MMP1,TGF-?1,EGF and IGF-1)was detected using Western blotting and qRT-PCR.The expression of these cytokines in cell culture medium were detected by ELISA.4.Photoaging mice and natural aging mice models were established.After the mouse models were established after 8 weeks,either PBS(control)or ASCs in PBS were administered bilaterally on the mouse dorsum.For each group,5 mice were sacrificed at weeks 3,4,5,6 and 7 post-injection.We analysis the histological structure of mice skin,the thickness of skin and the collagen changes.At the same time,PCR,ELISA and Immunohistochemical staining were used to detect the changes of cytokines involved in extracellular matrix remodeling.5.The level of reactive oxygen species(ROS)and the expression of Nrf2 and IL-6 in oxidative stress related pathways were detected.Furthermore,the role of oxidative stress related factors and ROS expression in skin rejuvenation by ASCs was further verified in animal experiments.6.A laser Doppler flowmeter and MoistureMeter-D were used to detect blood flow perfusion and skin LTW in natural aging mice and photoaging mice.Results:1.UVB irradiation significantly increased the number of senescent HDFs,reduced the proliferation ability of HDFs and promoted its apoptosis,but UVB irradiation had no effect on HDFs migration.Co-culture with ASCs can reduce the number of senescent HDFs after UVB irradiation,but cannot significantly affect the number of senescent HDFs without UVB irradiation.For both UVB-irradiated HDFs and nonirradiated HDFs,ASCs significantly increase their ability of proliferation and migration,while decrease the cell apoptosis rate.2.ASCs can reduce HDFs secretion of MMP1 to protect against collagen degradation and increase HDFs secretion of TGF-?1 to promote collagen synthesis.Analysis of the expression of MMP1 and TGF-?1 showed that in the UVB-irradiated photoaging group,the reduction in MMP-1 make a significant contribution to thickening the dermis in the side with ASCs-injection,mainly via decreasing the collagen degradation and increasing the amount of collagen.In the natural aging group,the increase in TGF-?1 plays an important role in the side with ASCs-injection,mainly by promoting collagen synthesis.3.ASCs can decrease the expression of ROS to reduce the damage caused by oxidative stress.The antioxidant stress factor Nrf-2 showed no significant difference between natural aging mice and photoaging mice.Our results showed,the level of IL-6 was increased in UVB-irradiated photoaging mice and control mice.4.After 7 weeks,blood perfusion was significantly higher on the side with ASCs-injection than on the side with PBS-injection in the UVB-irradiated photoaging group,while blood perfusion showed no significant difference on both side in the natural aging group.ASCs therapy significantly increased the thickness of the dermis,the number of capillaries,and the expression of some angiogenic growth factors(VEGF,IGF-1,and EGF).5.In natural aging mice,there was significant no differences in LTW between the ASCs-injection side and the PBS-injection side within 7 weeks after injection.However,in UVB irradiated photoaging mice,the LTW increased on the PBS-injection side and showed no significant changes on the ASCs-injection side at 7 weeks after injection.Conclusion:1.In general,the paracrine effect of ASCs can significantly improve the senescence,proliferation,apoptosis and migration of HDFs in both UVB irradiated and non-irradiated groups.Compared with UVB-irradiated photoaging HDFs,nonirradiated HDFs showed stronger proliferation and migration ability,lower apoptosis rate and fewer senescent cells when co-cultured with ASCs.These outcomes suggested that under the paracrine effect of ASCs,the number of nonirradiated HDFs was greater and the cell status was better than those of irradiated HDFs.2.ASCs changes the gene expression and protein level of ECM related cytokines in HDFs through paracrine action,thus remodeling the structure of extracellular matrix and improving aging skin.3.By reducing ROS and interfering with the expression of oxidative stress pathway related factors,ASCs effectively reduce skin damage caused by oxidative stress.4.ASC may mainly affect the relatively healthy HDFs in the lower dermis and make it competitively replace the aging HDFs in the upper dermis,so as to improve skin aging.5.Seven weeks after ASCs treatment,the skin blood perfusion,the number of dermal capillaries and the expression of angiogenic growth factors were increased in the skin with ASCs-injection.6.In the photoaging mice group,the LTW on the PBS-injection side increased significantly and was significantly higher than that on the ASCs-injection side at 7 weeks after injection.Because there is an inverse relationship between the number of red blood cells in the skin and the tissue water content.Our study indicated that with the decrease in capillaries in photoaging mice,the LWT in the skin increased.