The Role Of Nrf2 In Keratinocytes In Skin Fibrosis In Mice And Its Underlying Mechanisms

Objective:The skin protects from external factors as the first barrier of human body.The occurrence of skin fibrosis is closely related to people's quality of life.Skin fibrosis is usually a beneficial tissue response,while increased fibrosis can destroy the appearance,lead to organ dysfunction,and even cause the loss of labor.The skin fibrosis can greatly reduce people's quality of life because of organ dysfunction and psychological sequel.Despite such serious effects,mechanisms of fibrosis are still not understood.Skin fibrosis is the result of abnormal repair of tissue damage that always accompanied with chronic or severe dermal damage and inflammatory response.As we all know,NF-E2-related factor 2(Nrf2)is a nuclear transcription factor.The antioxidant response mediated by Nrf2 is the potential protection of normal cells to various intrinsic and external stresses.Wound healing is associated with the occurrence of skin fibrosis.The reactive oxygen species(ROS)produced in this process can not only prevent against bacterial invasion,but also induce skin damage.Nrf2 may play a particularly important role in wound healing,but the specific role needs further analysis.The expression of various key players involved in wound healing was significantly reduced in early wounds of the Nrf2 knockout mice.The late phase of repair was characterized by prolonged inflammation,and then the fibrosis would be aggravated.A study found that Nrf2 in fibroblasts attenuates fibrotic responses and mediates the antifibrotic effects of CDDO,one of a Nrf2 agonist.The skin external medication directly acted on the epidermis,but the role of Nrf2 in keratinocytes in skin fibrosis has not been reported.Therefore,this study explores the role of Nrf2 in keratinocytes in skin fibrosis using mice as a research model,and provides new ideas for the prevention skin fibrosis and the application of Nrf2 regulators.Methods: 1.The establishment of the Nrf2(K)-KO mouse model via the Cre-loxP system: We used K14-Cre mice crossed with Nrf2-KI mice in order to obtain Nrf2(K)-KO mouse.Cre is an enzyme that acts as a site-specific recombinase to recognize a loxP site.Keratin 14(K14)is the specific promoter-driven Cre recombinase in keratinocytes,and the K14-Cre excision Nrf2 DNA sequence which surrounded by loxP site.We used PCR to amplify DNA,and the DNA bands of mice were obtained by agarose gel imaging system.Then we identify the genotypes of the pups.2.The confirmation of Nrf2(K)-KO mice model: We used 1.2 U/mL concentration of dispase II to separate the epidermal from the dermal.The mRNA levels of Nrf2,Nrf1,Nrf3 in the epidermal of Nrf2(K)-KO mice and Nrf2-KI mice were detected by Real-time Fluorescence Quantitative PCR(RT-qPCR).The mRNA levels of Nrf2 in other control organs(liver,kidney and bladder)were also detected by RT-qPCR.The protein level of NRF2 was detected by Western Blot.3.The establishment of BLM-induced mouse skin fibrosis model: In the experimental group,the mice were subcutaneously BLM given for three weeks(0.5 mg/mL,100 ?L/time,every other day).In the control group,the mice were given PBS(100 ?L/time,every other day).This mouse model was applying on WT mice,and the success of skin fibrosis or not was analyzed by HE staining.The protein level of NRF2 was detected by Western Blot.4.The role of Nrf2 in keratinocytes in skin fibrosis in mice: Six-week-old male mice were divided into four groups(Nrf2-KI PBS group,Nrf2-KI BLM group,K14-Cre PBS group,K14-Cre BLM group,Nrf2(K)-KO PBS group,and Nrf2(K)-KO BLM group).We analyze the role of Nrf2 in keratinocytes via HE staining,and the thickness of the dermis was measured by Image J.The fibrosis marker COL III was determined by immunofluorescence.And the fibrosis marker ?-SMA was determined by immunohistochemistry.The number of positive cells in the mouse skin was determined by Image J,respectively.5.The role of Nrf2 deficiency in keratinocytes on inflammation: The mice were subcutaneously BLM given(0.5 mg/mL,100 ?L)or PBS(100 ?L).The skin tissues were obtained at 6 h after the first injection.The inflammatory infiltration of the skin was observed via HE staining.The expression of LY6 G and F4/80 was detected by immunohistochemistry in order to determine the infiltrating cell types,and the number of them was determined by Image J.They were neutrophils and macrophage markers,respectively.6.The effect of BLM on the expression of cytokines and chemokines in HaCaT cells: HaCaT cells were treated with 1 ?M and 10 ?M BLM for 6 h.The mRNA levels of chemokines(IL-8,MCP-1,CCL-3,CCL-4,CCL-5,CXCL-10),inflammatory factors(IL-6,IL-1?)and cytokines(TNF-?,TGF-?1)were detected by RT-qPCR.Transduction of HaCaT cells was conducted with lentiviral-based shRNAs targeting NRF2 or scrambled nontarget negative control.Transducted cells were maintained in DMEM medium containing 1.0 ?g/mL of puromycin.7.NRF2 silencing in HaCaT cells: HaCaT Scr cells and NRF2-KD cells were treated with 10 ?M As for 6 h,and the expression of NRF2 protein was detected by Western Blot.8.The effect of NRF2 silencing on the expression of chemokines and cytokines in HaCaT cells: HaCaT Scr cells and NRF2-KD cells were treated with 1 ?M and 10 ?M BLM for 6 h.The mRNA levels of chemokines(IL-6,IL-8,MCP-1)and cytokines(CCL-3,CCL-5,CXCL-10)were detected by RT-PCR.Results: 1.The establishment of the Nrf2(K)-KO mouse model: Agarose gel imaging showed that the 174 bp band represented the exist of the loxP site.When the 324 bp and 494 bp bands all existed,it represented that the mice carried K14-Cre.We called them the Nrf2(K)-KO mice.When the 324 bp bands existed alone,we called them the Nrf2-KI mouse.Compared with Nrf2-KI mice,Nrf2(K)-KO mice showed that the mRNA levels and protein levels of Nrf2 were decreased significantly(P < 0.05),but not Nrf1 and Nrf3;the mRNA levels of Nrf2 in liver,kidney and bladder showed no significantly different.2.The establishment of BLM-induced mouse skin fibrosis model: We found that BLM-induced skin fibrosis showed increased thickness of the dermis,attrition of dermal white adipose tissue,disordered arrangement of the fiber bundles,and the Stratum corneum showed tightly.The protein level of NRF2 in the epidermal were decreased obviously(P < 0.05).3.The protective role of Nrf2 in keratinocytes in BLM-induced skin fibrosis: Compared with PBS group,mice in BLM group showed skin fibrosis and showed increased thickness of the dermis(P < 0.05).The skin fibrosis marker ?-SMA and COL III in the dermis showed more positive cells(P < 0.05).There was no significant difference between Nrf2(K)-KO and Nrf2-KI mice or K14-Cre mice in PBS group.However,in the BLM group,Nrf2(K)-KO mice showed increased thickness of the dermis compared with Nrf2-KI mice and K14-Cre mice(P < 0.05).The positive cells ?-SMA and collagen III were significantly increased(P < 0.05).4.The deficiency of Nrf2 in keratinocytes promotes inflammatory response: Compared with PBS group,HE staining of the BLM group showed significant inflammatory infiltration and increased inflammatory cells(400 ×,P < 0.05).In the BLM group,compare with Nrf2-KI mice,the inflammatory infiltration of Nrf2(K)-KO mice was more severely.Immunohistochemical staining showed that the BLM group had more neutrophil and macrophage compared with the PBS group(P < 0.05);and in the BLM group,compared with Nrf2-KI mice,the number of neutrophils and macrophages in Nrf2(K)-KO mice was significantly increased(P < 0.05).5.BLM promoted the expression of chemokines in HaCaT cells: The best doses of BLM that induced the synthesis and release of chemokines,inflammatory factors and cytokines in HaCaT cells were 1 ?M and 10 ?M.The mRNA levels of TNF-?,CCL-3,CCL-4,IL-6,IL-8 and MCP-1 was increased(P < 0.05),and there had a dose-response relationship between IL-6,IL-8 and MCP-1(P < 0.05).6.The silence of NRF2 in HaCaT cells promoted the expression of chemokines and cytokines: NRF2 was been successfully knockdown in HaCaT cells.The RT-qPCR results showed that the mRNA levels of IL-6,IL-8 and MCP-1 in NRF2-KD cells were higher compared with Scr cells(P < 0.05).Although the mRNA levels of CCL-3,CCL-5 and CXCL-10 also showed increased,the too low expression of them had no sense.Conclusion: 1.Nrf2(K)-KO mouse model has been successfully established.Nrf2(K)-KO mice are more sensitive to bleomycin-induced skin fibrosis.2.Deficiency of Nrf2 in keratinocytes leads to increase expression of cytokines and chemokines,which promotes the inflammation of skin and probably aggravates BLM-induced skin fibrosis.