Mechanisms Study Of Wound-induced Hair Follicle Neogenesis And Effects Of IL-36? On Hair Follicle

Background and objective:Wound-induced hair follicle neogenesis is a regenerative phenomenon in adult mammals,which is defined that the full-featured hair follicle can be regenerated in center of healing area after a certain size of full-thickness skin wound.The mechanism of wound-induced hair follicle neogenesis is more similar to embryogenic hair follicle neogenesis.Epidermal stem cells,fibroblasts,keratinocytes and some immune cells are involved in the regulation of wound-induced hair follicle neogenesis.The study of wound-induced hair follicle neogenesis will help us explore the molecular mechanisms of mammals'growth and regeneration,and guide the treatment of clinical hair-related diseases.Proteomics is a new developing discipline in the post-genome era,which provides a direct material basis for explaining the nature of life.Isobaric tags for relative and absolute quantification?iTRAQ?technique is the most commonly used high-throughput screening technique for studying proteomics in recent years.It is used to quantitatively analyze the expression level of identified proteins between two groups of samples,then we can find the target protein regulated phenotype from the screened differential proteins.In the current study of wound-induced hair follicle neogenesis,the regulative effect of inflammatory factors got much attention.We screened protein molecules which predicted to regulate hair follicle growth and development by high-throughput proteomic analysis,and found that IL-36?is highly expressed in hair follicle growth areas after wound healing in mice.As a pro-inflammatory factor of IL-1 family,IL-36?can promote the expression of various inflammatory factors such as IL-1,IL-6,TNF?and the like.Therefore,this study aims to explore the regulative effect of IL-36?on wound-induced hair follicle neogenesis.Material and Methods:1.Establishment of wound mice model:A 1.5 cm×1.5 cm square full-thickness wound was made on the back of 49-day-old female C57BL/6 mice.2.The effect of hindering wound healing on hair follicle neogenesis:20 wound mice were randomly divided into 2 groups.No processing in Group 1.The wound edge was sewed together with subcutaneous muscle tissue in Group 2.Taking photos at 0,3,5,7,10,14,and 22 days after wounding and measuring the size of wound or healing area.On the 28th day after wounding,the skin tissue of the healing area was obtained.The number of new hair follicles was measured by alkaline phosphatase staining,and the characteristics of neogenetic hair follicles were observed after wounding.3.Analyzing the differences of proteomics between the area with hair follicles growth and the area without hair follicles growth:45 wound mice model were randomly divided into 3 groups.The skin of healing area on the back of mice on the 15th day after wounding was obtained and divided into two internal and external tissues.The inner tissues in the same group were mixed into a group of samples,and the outer tissues in this group were mixed into a control group of samples.A total of 3 pairs of samples were analyzed by iTRAQ technique.Bioinformatics analysis was performed on the differentially expressed proteins to predict the function of proteins related to wound-induced hair follicle neogenesis and verify the functional mechanism by subsequent study.4.Effects of IL-36?on wound-induced hair follicle neogenesis and hair growth cycle:20wound mice models were randomly divided into PBS-injected control group and murine recombinant IL-36??mr-IL-36??-injected group.On the 7-14th day after wounding,50?l PBS or mrIL-36??1 ng/?L,PBS?was injected daily under the scab.Observe and photograph the state of wound area,and count the number of neogenetic hair follicles on the 28th day after wounding.5 49-day-old C57bl/6 mice and 5 65-day-old C57bl/6 mice were used,100?l mr-IL-36?solution was injected intradermally into the left side of the back,and 100?l PBS solution was injected intradermally into the right side of the back.Observe and photograph the state of each mice.5.The regulated effect of IL-36?on epidermal and hair follicle stem cells,related inflammatory factors and Wnt signaling pathway was investigated by qPCR,flow cytometry,western blot and immunohistochemistry.Results:1.Wound mice model:The scab fell off at around the 12th-14th day after wounding.The size of wound or healing area was measured on the 0^th,3^rd,5^th,7^th,10^th,14^th,17^th and22^nd day after wounding.The extent of healing area constriction was smaller in hindering wound group than control group.On the 7^th,10^th,14^th,17^th and 22^nd day after wounding,there was a statistical difference in the size of wound area between two groups?p<0.05?.The neogenetic hair follicles growed in the center of wound healing area,and the number of neogenetic hair follicles was positively correlated with the size of healing area?rs=0.729,p<0.001?.2.Result of iTRAQ analysis:The iTRAQ analysis was performed between the hair-follicles-growth area and the non-hair-follicles-growth area on the 15th day after wounding,and total 4378 proteins were detected.There were 129 differentially expressed proteins in the outer area compared with the inner area?Fold change?1.5 or?0.667,p<0.01?,of which 46 proteins were up-regulated and 83 proteins were down-regulated.GO analysis showed that among the biological function items,the proportion of proteins involved in cellular processes was the highest?58.14%?;among the cell composition items,the proportion of proteins localized on cells?including cell membrane,cell wall,and extracellular membrane?was the highest?71.72%?;and the highest proportion among the molecular function items is binding?55.81%?.KEGG analysis showed that the number of proteins enriched in the ribosome-associated signaling pathway was the most.Multiple protein molecules were involved in the growth and development of hair follicles,and IL-36?was highly expressed in the inner area?FC=0.556,p<0.01?.3.IL-36?promotes wound-induced hair follicle neogenesis and telogen-anagen transition of mouse hair follicles:On the 15^th day after wounding,qPCR showed that the expression level of IL-36?mRNA in the inner area was 26 times higher than outer area?p<0.05?;western blot showed that IL-36?was highly expressed in the inner area of wound healing area.In mrIL-36?treated group of wound mice,the scab fell off at around PWD10,while in control group at around PWD12-14.The number of neogenetic hair follicles in IL-36?-treated group was higher than that in the control group?p<0.01?.The mice,whose hair was in the late telogen,their dorsal hair follicles in IL-36?-treated area entered the anagen earlier than that in PBS-treated area.4.IL-36?promotes the proliferation of epidermal stem cells and hair follicle stem cells:qPCR results showed that K15,CD34,Lgr5 and Lgr6 gene expression levels were higher in IL-36?-treated side than that in control side?p<0.05?.Flow cytometry showed that the proportion of CD34⁺CD49f⁺hair follicle stem cells in the IL-36?-treated area was significantly higher than that of the control area?p<0.05?.Although the proportion of CD34^-CD49f⁺Pcad^hi follicle precursor cells in the IL-36?-treated area was higher than that of the control area,but there was no significant difference between the two groups.Immunofluorescence assay showed that the expression level of K15 in the IL-36?-treated site was higher than that in the control site by using K15-EGFP transgenetic mice.The results of immunohistochemistry showed that the expression level of Lgr6 in IL-36?-treated site was higher than that in the control site.5.IL-36?has no effect on the canonical Wnt pathway but up-regulates the expression of?-catanin and related inflammatory factors:qPCR showed that the expression levels of TNF?,TCRV?3,IL-6 and?-catanin were higher in the IL-36?-treated side than that in the control side?p<0.05?,but the expression levels of other Wnt-related factors,such as LEF-1,Wnt5a,DKK-1,Lrp5,had no significantly difference between two sides?p>0.05?.Western blot analysis showed that the phosphorylation level of STAT3 in IL-36?-treated site was higher than that in the control site.Conclusion:1.Hindering wound contraction increases the final size of scar and promotes the hair follicles neogenesis induced by wound.2.The neogenetic hair follicles grow in the center of wound healing area.There are 129 differentially expressed proteins between hair-follicle-growth area and non-hair-follicle-growth area,and IL-36?is highly expressed in the hair-follicle-growth area.3.IL-36?promotes wound healing,wound-induced hair follicle neogenesis and telogen-anagen transition by inducing the proliferation of epidermal stem cells and hair follicle stem cells.4.IL-36?promotes STAT3 phosphorylation and?-catanin pathway activation by promoting the secretion of inflammatory factors,than promotes hair follicle growth.