Study On The Role Of ACTL6a In Psoriasis And Wound

BackgroundActin-6a(ACTL6a),also known as BAF53a or Arp4,is an important member of the ATP-dependent SWI/SNF-like BAF chromatin remodeling complex.It was first identified as an actin-related protein in the T lymphocyte BAF complex and localized to nuclear chromosomes,especially in region which is rich of euchromatin.Studies have shown that ACTL6a is involved in a number of cellular biological processes including transcriptional regulation of genes,staining Quality remodeling and nuclear translocation.In 2017,Srinivas Vinod Saladi found that ACTL6a and P63 of HNSCC had physical interaction that co-amplified and synergistically regulated key genes including WWC1,activated Hippo-YAP pathway,so that can promot proliferation,and inhibit differentiation.In 2016,Xiao Shuai found that ACTL6a promotes hepatocellular carcinoma(HCC)metastasis and epithelial-mesenchymal transition(EMT)through SOX2/Notchl signaling.In 2013,Bao Xiaomin found that ACTL6a can inhibit SWI/SNF-induced KLF4 to maintain the stem character of epidermal stem cells.In recent years,articles had been published that ACTL6a inhibits cell differentiation,maintains the function of self-renewal and pluripotency(including embryonic stem cells,epidermal stem cells and progenitor cells in the hematopoietic system).ACTL6a is associated with osteosarcoma and cervical squamous cell carcinoma.Psoriasis is a chronic inflammatory proliferative skin disease affecting about 2-3%of the world's population,about 125 million people,with some complications such as arthritis,cardiovascular disease,and metabolic syndrome(Obesity,hypertension,dyslipidemia and diabetes),chronic kidney disease,gastrointestinal disorders,shingles,infections,mood disorders,malignancies and lung diseases.Typical clinical manifestations are erythema and scale,the common lesion sites include the scalp,knees,extensor side of the elbow,trunk,calf anterior and nails.Nearly 10 billion U.S.dollars are spent globally each year on psoriasis care.Psoriasis patients generally bear a huge psychological burden,while physical activity,cognitive function and quality of life significantly reduced.A meta-analysis reported that 28%of psoriasis patients in the top hospitals had depressive symptoms.The pathogenesis of psoriasis involves genetic and environmental factors.Up to 30%of psoriasis patients can find relatives with similar medical history.Abnormal proliferation of epidermal keratinocytes is thought to have a close relationship with the development of psoriasis.Generally epidermal keratinocytes from the basal layer to the stratum corneum takes about 28 days,while in patients with psoriasis,epidermal transit time is only 3-5 days.From histopathology,it can be clearly observed hyperkeratosis,keratosis,accompanied by clubbing hyperplasia hypertrophy and reduction or deletion of the granular layer.Studies have shown that psoriasis lesions contain both epithelial and mesenchymal markers,belong to the EMT type 2.Wound repair is a time-space highly regulated process.The basis of the cellular biology of wound repair relies on rapid proliferation,migration and differentiation of epidermal cells,and requires a large number of cytokines to maintain cell survival and proliferation,thereby promoting wound repair.Keratinocytes constitute the epidermal structure mainly.Ivor J.Lim et al.had shown that the interaction of epithelial and interstitial cells in a paracrine and autocrine.Daniel N et al.reported that the epidermis may play a role in regulating fibroblasts during wound healing.ACTL6a plays an important role in the maintenance of epidermal stem cells(proliferation,migration)and EMT.It has a certain degree of overlap with the pathogenesis of psoriasis and the repair mechanism of wound,prompting us to consider whether ACTL6a is involved in the occurrence of psoriasis,what role played?What is its role in predicting the progression and prognosis of psoriasis?What is its function in the skin wound repair process?Based on the literature review,there is no in vivo experimental study of ACTL6a.We constructed ACTL6a transgenic mice in order to explore further.ObjectiveTo elucidate the location of ACTL6a in normal and psoriatic skin;to clarify the expression of ACTL6a in keratinocytes cultured in normal and psoriatic skin in vitro;to construct ACTL6a transgenic mice;to study the functional changes of epidermis of ACTL6a transgenic mice;induced psoriasis and wound models to explore the biological role of ACTL6a.MethodsObtained skin samples of normal people and psoriasis patients and cultured keratinocytes.To explore the tissue localization of ACTL6a in normal and psoriatic skin by immunohistochemistry and immunofluorescence.The normal and psoriatic keratinocytes were isolated and the RNA and protein were extracted.The mRNA and protein expression of ACTL6a were detected by qRT-PCR and western blot seperately.Constructed ACTL6a transgenic mouse model,extracted the DNA of toe and verified by PCR amplification and western blot.Epidermal function changes in ACTL6a transgenic mice were studied by qRT-PCR.C57BL/6 mice and ACTL6a mice were used as research objects to construct imiquimod-induced mice model of psoriasis.PASI score was applied to the skin lesions.The changes of skin thickness were compared between the two groups.The lesions were observed by H&E And Masson staining about the pathological changes such as epidermal thickness and vascular area.The expression of inflammatory cytokines was detected by qRT-PCR,and the lymph nodes and spleens were obtained for flow cytometry to study the role of ACTL6a in psoriasis.C57BL/6 mice and ACTL6a mice were used as research objects to establish the model of mouse wound.The area of trauma and the rate of wound healing were calculated.HE and Masson staining were used to observe the histopathology of skin lesion,and the ECM of skin samples were detected by qRT-PCR.Results1Localization and expression of ACTL6a in normal and psoriatic skin tissue and cultured keratinocytes.1.1 Immunohistochemistry and immunofluorescence experiments showed that expression of ACTL6a in normal skin and psoriatic lesions were high,and the expression in psoriasis patients was higher than normal persons.1.2.At mRNA and protein levels,the expression of ACTL6a in keratinocytes in psoriatic lesions was up-regulated compared with normal keratinocytes.2.Construction and Identification of ACTL6a Transgenic Mouse Model and Its Epidermal Function2.1.ACTL6a transgenic mice which were constructed by Sai Yee Co.,Ltd.were verified on the level of toe DNA and epidermal protein.The results indicated that ACTL6a transgenic mice were successfully constructed.2.2.Compared with the C57BL/6 mouse epidermis,the expression of proliferation related gene KRT16 of ACTL6a transgenic mice was significantly increased at mRNA level,but there was no significant statistical difference in KRT6 and KRT17;senescence related gene p53,p21 was significantly reduced,while no significant difference in p27;cycle related gene CyclinBl significantly increased,while no significant difference in CyclinD1 and cdk2.The results suggested that ACTL6a can promote mouse epidermal proliferation,while inhibiting the senescence.3.Study on imiquimod(IMQ)-induced psoriasiform(IPI)model and wound model of ACTL6a transgenic mice3.1.Compared with C57BL/6 mice,ACTL6a transgenic mice showed a relatively severe psoriatic lesion in IPI model,histopathology showed that epidermal thickening and vascular proliferation were increased.Compared with C57BL/6 mice,the percentage of CD3+CD8+ T cells and CD4 + CD25 + FoxP3 + Treg cells in lymph nodes and spleens of ACTL6a transgenic mice were decreased.There was no significant difference in CD3 + CD4 + T cells,MHC + DCIR2 + DCs,and CD11c + CD207 + DCs.The mRNA expression levels of IL-23,IL-17A and TNF-a in the inflammatory tissue of ACTL6a transgenic mice were significantly increased.3.2.Compared with C57BL/mice,ACTL6a transgenic mice exhibited a faster repair progress in the wound repair model,and histopathology showed that there were more epidermal layers around the wound and a large number of cells migrated to the wound;The level of mRNA expressions of ECM(Collagen 1a,Collagen 1a and MMP-3)were increased in the lesions.Conclusion1.Expression of ACTL6a were both in normal skin and psoriatic lesions,and the expression in psoriasis patients was higher than normal persons.2.The expression of ACTL6a in keratinocytes of psoriatic lesions was up-regulated compared with normal keratinocytes.3.ACTL6a transgenic mice were successfully constructed.4.ACTL6a overexpression in epidermis affected epidermal proliferation and senescence.5.ACTL6a transgenic mice had a relatively severe psoriatic lesion in IPI model and had a faster repair progressThe disadvantages of this study was the lack of the specific mechanisms involved in the role of ACTL6a in psoriasis and wound in-depth.