| Background: The human epidermis has no blood circulation,and the form of energy metabolism in tissue cells is mainly anaerobic enzymes,with the end metabolite being lactic acid.Skin is the first barrier for human body to deal with harmful environment.It can be divided into epidermis and dermis from outside to inside.Keratinocytes are the main cells in epidermis and fibroblasts are the main cells in dermis.Previous studies have found that lactic acid content in skin after sunlight exposure or aging is significantly increased.Ultraviolet radiation B(UVB)in sunlight is the key environmental factor causing skin aging.Whether UVB irradiation can change the content of lactic acid in skin cells and the effect of lactic acid on skin cells are still unclear and need to be studied urgently.Purpose:1.It is clear that UVB irradiation can promote lactic acid production by changing metabolic pattern of skin cells.2.To explore the physiological effects of lactic acid on two types of skin cells,namely epidermal keratinocytes and dermal fibroblasts,including the effects of proliferation activity,apoptosis and aging molecular characteristics.3.To explore the possible mechanisms of lactic acid involved in the aging of two types of skin cells,including regulatory pathways and key molecules.It provides theoretical basis for the prevention and treatment of skin photoinjury,photoaging and photoinduced dermatosis from metabonomics level.Research methods: 1.UV 801 KL irradiator light source was used in this study,and10 m J/cm was selected2,20 m J/cm2,30 m J/cm2 Human immortalized keratinocyte line(Ha Cat)was irradiated by three irradiation gradients for 48 hours.The changes of cell number and morphology were observed to select the appropriate irradiation dose.The changes of lactic acid level in culture medium were detected by lactic acid kit,and the changes of LDHA gene expression before and after lactic acid stimulation were detected by q-PCR.In this study,C57BL/6 mice were selected for UVB irradiation experiment,and the skin pathology of mice was detected.2.Lactic acid stimulating cells: Ha Cat cells and human primary dermal fibroblasts(HDF)were used to stimulate the two kinds of cells with L(+)-lactic acid in different concentrations of lactic acid culture medium,and the effects of lactic acid stimulation on the two kinds of cells were detected.3.In this study,RNA-seq technique was used to screen the differential genes of Ha Cat cells before and after lactic acid stimulation,and the functional enrichment of GO and KEGG was studied.4.Cell proliferation experiment:Celltiter cell viability test kit was used to detect the effect of lactic acid on cell proliferation,and the luminescence intensity of cells was detected at 0h,24 h,48h and72 h after lactic acid stimulation.5.Apoptosis experiment: After lactic acid stimulation for 48 h,the cells were collected and stained with Annexin V-PI kit,and the fluorescence intensity was detected by flow cytometry.6.Study on cell aging: In this study,the changes of ROS levels in Ha Cat cells and HDF cells stimulated by lactic acid were detected by reactive oxygen species kit,and the cells were treated with ROS inhibitors to analyze whether ROS is one of the reasons for lactic acid-induced cell phenotype.The effects of lactic acid stimulation on COL1,MMP1,p-ERK1/2 and TGF β R2 protein were detected by Western Blot,and the changes of cell aging level were detected by β-galactosidase staining kit.Research results:1.The number of Ha Cat cells decreased after UVB irradiation,20 m J/cm2 For the appropriate irradiation dose.20 m J/cm2 The lactic acid secretion of Ha Cat cells increased significantly after UVB irradiation(p < 0.01);At 48 h and 72 h after UVB irradiation,LDHA gene in Ha Cat cells was activated(p < 0.05).In vivo experiments,after UVB irradiation,the skin of mice appeared rough,desquamation,erythema and other light damage,and HE staining of mouse skin showed thickening of epidermis and dermal structure disorder.In the immunohistochemical staining of GLUT1 and LDHA in the skin of irradiated mice,the expression of GLUT1 and LDHA in epidermis increased.2.A total of 33 differential genes were identified by transcriptomic analysis before and after lactic acid stimulation in Ha Cat cells,of which 11 genes were up-regulated and 22 genes were down-regulated after lactic acid stimulation.Differential genes are enriched in interleukin-6 secretion and metallopeptidase activity.3.Lactic acid at 15 m M and above inhibited the proliferation of Ha Cat cells(P < 0.05).The proliferative activity of HDF cells stimulated by lactic acid of 10 m M or above decreased(P < 0.05).4.After lactic acid stimulated Ha Cat cells,the proportion of late apoptosis cells increased in a dose-dependent manner.The proportion of premature apoptosis cells in HDF was increased by lactic acid stimulation,and ROS inhibitor could inhibit the increase of premature apoptosis cells in HDF.After lactic acid of 5.5 m M or above stimulated Ha Cat and HDF cells,their ROS levels increased significantly,and adding ROS inhibitor NAC could inhibit the increase of ROS levels in Ha Cat and HDF cells induced by lactic acid.6.After lactic acid stimulated Ha Cat cells,the expression of MMP1 gene increased,the level of COL1 decreased and p-ERK was activated.The addition of ROS inhibitor can inhibit the degradation of COL1 and the activation of p-ERK in Ha Cat cells induced by lactic acid.After lactic acid stimulated HDF cells,the expression of MMP1 protein increased,the expression of COL1 protein decreased,and p-ERK1/2 was activated.TGF β R2 was inhibited.Beta The results of galactosidase staining showed that the aging level of HDF cells increased obviously after lactic acid stimulation,and the aging level of HDF cells was partially inhibited by NAC pretreatment before lactic acid stimulation.Conclusions:1.Ultraviolet radiation induced lactic acid accumulation in epidermal cells,indicating that anaerobic glycolysis was enhanced,which not only reflected a stress state of cells,but also suggested that lactic acid,as a metabolite,participated in the aging process of cells induced by light radiation.2.The increase of lactic acid in epidermal cells induced by ultraviolet radiation suggests that ROS may cause damage to aerobic respiratory chain and compensatory enhancement of metabolic bypass(anaerobic glycolysis pathway).3.Lactic acid accumulation interferes with the metabolic activity of keratinocytes and fibroblasts,affects the phenotype of cell proliferation and apoptosis,and directly affects the synthesis of collagen fibers through ROS-independent forms,resulting in pathological changes of skin photoaging.4.Lactic acid can promote the production of MMP1 by activating ERK pathway.Lactic acid can inhibit COL1 synthesis by inhibiting TGF β pathway.5.Cell aging is the basis of skin tissue aging.The results showed that lactic acid participated in the process of skin photoaging through its influence on cell metabolic biology. |
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