The Role Of Nrf1 Against UVB-induced Sunburn Reaction In Keratinocytes

Objective:Solar ultraviolet radiation can cause acute and chronic damage to human skin.Ultraviolet B?UVB?,as the most dangerous ultraviolet rays reaching the surface of the earth,can cause skin inflammation,aging,and apoptosis,thereby promoting the occurrence and development of skin cancer.DNA break directly caused by DNA damage and indirectly caused by excessive generation of reactive oxygen species?ROS?are important pathophysiological process of UVB damage effects.The body has defense mechanisms against oxidative stress,especially the alkaline leucine zipper family.Among them,nuclear factor erythroid 2-related factor 2?Nrf2?can protect skin cells from UVB-induced apoptosis through glutathione?GSH?-mediated pathways.However,nuclear factor erythroid 2-related factor 3?Nrf3?,which is highly similar in structure to Nrf2,can enhance UVB-induced keratinocyte apoptosis by inhibiting cell adhesion.As another member of the basic leucine zipper family,nuclear factor erythroid 2-related factor 1?Nrf1?,is still unclear in its role in UVB-induced skin injury.In this study,using keratinocyte Nrf1 specific knockout mice and lentivirus stably transfected NRF1 gene silenced HaCaT cell lines to study the role and mechanism of Nrf1 in UVB-induced skin keratinocyte cells injury from both in vivo and in vitro.Methods:1.Detection of the expression and distribution of Nrf1 in the skin under basic conditions:The skin of the back of the mice was collected,and the skin structure and keratinocyte morphology of Nrf1-specific knockout mice,namely Nrf1?K?-KO mice and Nrf1-KI mice,was observed by H&E staining,and the expression of keratin K14 and K6 were detected by immunofluorescence staining,and the expression of macrophages F4/80 and neutrophil Ly6G⁺were detected by immunohistochemistry.2.Establishment of UVB-induced skin acute sunburn model:Mice were not subjected to any treatment under anesthesia is the control group.Mice were irradiated with 300 mJ/cm²UVB irradiation is the UVB experimental group;The skin tissues of the ears and back of the mice were collected 24 h after irradiation.H&E and TUNEL staining were used to observe the morphology of keratinocytes after UVB irradiation.3.Using pathological staining to detect the role of Nrf1 in UVB-induced keratinocyte injury:8-week-old female mice were divided into 6 groups,namely K14-Cre^+/-control group,Nrf1-KI control group,Nrf1?K?-KO control group,K14-Cre^+/-experimental group,Nrf1-KI experimental group,Nrf1?K?-KO experimental group.The skin of the backs of mice was harvested to analyze the effect of Nrf1 in keratinocytes by H&E staining and TUNEL staining.4.Detection of DNA damage related indicators:8-week-old female mice were divided into6 groups,namely K14-Cre^+/-control group,Nrf1-KI control group,Nrf1?K?-KO control group K14-Cre^+/-experimental group,Nrf1-KI experimental group,Nrf1?K?-KO experimental group.The skin of the back of the mice was collected and the expression of the DNA damage indicator H2AX phosphorylated protein?-H2AX and Cyclobutane pyrimidine dimer?CPD?in the epidermal layer was measured by immunohistochemistry and the number of positive cells in the epidermal layer of mice were measured by GraphPad Prism 6 software.5.NRF1 gene silencing and verification:Stable-infected cells were screened with 1?g/mL puromycin using lentivirus NRF1-KD and Scramble cells carrying targeted NRF1 shRNA.qPCR method was used to detect the transcription level of NRF1.Scramble cells and NRF1-KD cells were treated with 10 nM Eponemycin?EPO?for 6 h,and the expression of NRF1 protein was detected by Western Blot method.6.UVB treatment of HaCaT cells:HaCaT cells were treated with non-target negative control lentivirus?Scramble?.After 2 J UVB irradiation,samples of HaCaT cells were collected at 1 h,3 h,6 h,12 h,and 24 h,and NRF1 mRNA levels were detected.Eponemycin?10 nM,6 h?and high-dose UVB?8 J/cm²,6 h?were combined to detect the expression of NRF1 protein.7.Detection of apoptosis-related indicators:Scramble and NRF1-KD HaCaT cells were irradiated with UVB irradiation at 1 J,2 J,5 J,and 8 J for 24 h,and then were treated with trypan blue staining.Apoptosis was detected by flow cytometry Annexin/V-PI double staining,and expression levels of Cleaved Caspase-3 and Cleaved PARP were detected by Western Blot method.8.Detection of DNA damage repair related indicators:Scramble and NRF1-KD HaCaT cells were given 1 J UVB irradiation treatment,and were detected by full-field cytometry at 0,12 h,24 h,36 h,and 48 h.After Scramble and NRF1-KD HaCaT cells were irradiated with 2 J UVB,respectively,samples were collected at 0,1 h,3 h,6 h,12 h,and 24 h to detect the mRNA of DNA damage recognition factor Xeroderma pigmentosum C?XPC?.9.Statistical analysis:All data in this study were analyzed using GraphPad Prism 6software.Results:1.Effect of Nrf1 knockout on the structure and function of mouse skin:H&E staining,immunofluorescence?K14 and K6?staining,F4/80 and Ly6G⁺immunohistochemical staining of back skin sections showed that the thickness of epidermal layer,dermal layer,and subcutaneous fat layer,epidermal keratinocyte morphology,keratin differentiation,and inflammatory response of Nrf1?K?-KO mice were not significantly different from those of Nrf1-KI mice.2.Nrf1 knockout promotes UVB-induced keratinocyte apoptosis:300 mJ/cm² UVB irradiation can induce keratinocyte apoptosis.The numbers of sunburned cells and TUNEL positive cells in Nrf1?K?-KO mice were significantly higher than those in K14-Cre^+/-and Nrf1-KI mice,which was about 3 times,and the difference was statistically significant?P<0.05?.3.Nrf1 deletion aggravates UVB-induced photoproduct formation and promotes DNA damage:Compared with the control group,?-H2AX positive cells and CPD positive cells appeared in the epidermal layer of the UVB experimental group,and compared with Nrf1-KI mice,Nrf1?K?-KO mice showed a significant increase in?-H2AX positive cells and CPD positive cells,which was about 2.5 times,with statistical differences?P<0.05?.4.NRF1 gene silencing and stable transfection cells were successfully established:The target gene mRNA and protein levels of HaCaT cells in the NRF1-KD group were significantly lower than those in the Scramble group,and the differences were statistically significant?P<0.05?.5.Acute UVB exposure induced NRF1 up-regulation:After 6 hours of UVB irradiation,the expression of NRF1 mRNA in the Scramble group cells was up-regulated.The NRF1level of the cells in the NRF1-KD group was lower than that in the Scramble group.UVB treatment can induce the up-regulation of NRF1 expression in Scramble group cells.Compared with EPO group,the expression of NRF1 in Scramble group cells was significantly up-regulated after UVB and EPO combined treatment.6.NRF1 gene silencing promotes UVB-induced apoptosis:After 24 h of UVB 1 J,2 J,5J,and 8 J treatment of cells,the apoptosis rates in the Scramble and NRF1-KD groups were significantly higher than those in the corresponding control group,and the apoptosis was in a dose-response relationship with UVB dose?P<0.05?;Compared with the Scramble group,the apoptosis rate of the NRF1-KD group was significantly increased?P<0.05?;meanwhile,the levels of Cleaved Capspase-3 and Cleaved PARP proteins in the NRF1-KD group were significantly higher than those in the Scramble group.7.NRF1 may play a protective role in UVB-induced apoptosis by affecting DNA damage repair:After 1 J UVB treatment,the cell proliferation rate in the NRF1-KD group was significantly lower than that in the Scramble group,and the difference was statistically significant?P<0.05?.After 2 J UVB treatment,the mRNA levels of XPC in NRF1-KD cells were not significantly different from those in the Scramble group.Conclusion:1.Nrf1 keratinocyte-specific knockout mice have no significant changes in skin structure and function under basal conditions.2.Nrf1 knockout promotes UVB-induced keratinocyte apoptosis.3.Nrf1 may play a role in DNA damage repair by reducing the formation of UVB-induced photoproducts.