| Background and objective:Scarring after skin injury is a challenging clinical problem.Fibroplasia in adult skin due to a variety of causes,including trauma,burns,infection,or surgery,often results in multiple types of scarring,among which the most difficult pathological types are hypertrophic scars and keloids.Scars can cause discomfort,such as itching or reduced mobility,and can also have adverse effects on mental health.This psychological pressure makes patients seek effective means to beautify or reduce scarring.However,the current scar treatment methods are still limited.Studies have shown that the skin in the early embryo will regenerate perfectly after injury,but this ability is lost in the late embryo,resulting in two completely different outcomes due to embryonic development.Early studies on embryonic trauma were mostly based on large mammalian models,while mice were gradually included in the scope of research due to their short gestation period,low cost,and high homology to humans.However,due to the high difficulty of surgery,the scope of current research is still limited.Unlike near-normal healing after age-injury in mice in early gestation(embryonic day of 16,E16),wound healing in late gestation(embryonic day of 18,E18)and adulthood is accompanied by fibrosis.And the mechanisms that lead to this transition are largely unknown.Therefore,we aimed to explore the causes of scarring from the perspective of fetal development,find the key genes that lead to scarring in the late embryo and combine them with adult scar treatment to provide new ideas for clinical treatment of pathological scars.Methods:1.In the first part of this study,C57B1/6J mice were used as the research object,and the mouse fetal full-thickness skin wound model was stably constructed at different embryonic stages(E16 and E18),and the generalized repaired images of E16and E18 were photographed.The photo shows the difference between embryonic E16and E18 healing after injury.The tissue after injury was also obtained for H&E(hematoxylin-eosin)staining to analyze the difference in the tissue structure of the repaired area between the E16 and E18 groups.The collagen structure of the two groups was analyzed by Sirius red staining,and the difference in collagen structure was evaluated by F_D(fractal dimension)and L(lacunarity).At the same time,immunohistochemical staining of TGF-β3(transforming growth factor-β3)was performed on the skin sections of the E18 scarring group to evaluate the scars.2.The second part of this study is based on the results found in the first part.24 hours after injury,the skin tissue of mice on day E18 and their self-controls were selected for transcriptomic sequencing(n=3).The results were subjected to bioinformatics analysis,including GO(Gene Ontology)analysis and KEGG(Kyoto Encyclopedia of Genes and Genomes)analysis,involving the biological process,molecular function and cellular components of differentially expressed genes as well as enrichment of related functional signaling pathways.q RT-PCR(quantitative real-time PCR)was used to verify the expression of the screened differential genes between the sample24h after E18 injury and the normal E18 controls.3.In the third part of this study,q RT-PCR,Western Blot and immunohistochemical staining experiments were used to examine the changes of CTSS m RNA and protein levels in different embryonic stages after injury.The mechanism of CTSS in vitro was explored in human skin fibroblasts.After synthesizing CTSS knockdown plasmids,cells were transfected and the knockdown effect was confirmed by q RT-PCR and Western Blot experiments.Protein expression levels of collagen type I,collagen type III and fibronectin were detected.Then,the expression of IRF7 in the damaged tissues of embryos at different stages was detected by q RT-PCR,Western Blot and immunohistochemical experiments.The possible binding sites of IRF7 and CTSS were predicted using bioinformatics analysis tools,and the IRF7 overexpression plasmid and blank control were synthesized.Combined with dual luciferase reporter experiments and ch IP experiments,the binding of IRF7 to the CTSS promoter region was confirmed.Meanwhile,IRF7 was overexpressed in fibroblasts,and the changes of CTSS levels were detected by q RT-PCR and Western blot experiments.The m RNA and protein levels of scarring-related genes(collagen type I,collagen type III and fibronectin)were tested to verify the mechanism of the IRF7/CTSS regulatory axis in scarring,and the reverse experiment was used to further clarify the regulatory role.Finally,q RT-PCR,Western blot,and immunohistochemical experiments were used to detect the expression levels of IRF7 and CTSS in skin tissue of human keloid patients.The adult mouse hypertrophic scar model was used to study the in vivo mechanism of CTSS,and the CTSS inhibitor LY30003283 was administered to the injured skin to observe the curative effect.The scar area and the expression of myofibroblast markerα-SMA(alpha smooth muscle actin)were detected by H&E staining and immunohistochemical experiments.Results:1.In the embryonic stage E16 and E18,the lesions healed within 48 hours,but the skin was completely regenerated as normal on E16 and the skin was scarred on E18.The results of H&E staining and Sirius red staining showed that the tissue structure on E16 and E18 days was significantly different,and the healed tissue after E18 day was similar to the scar structure.The expression of TGF-β3 in the scar area after injury at E18 was less than that in the control group,demonstrating the activation of the scar-producing effect in the injury area.2.Through transcriptomic sequencing of the skin tissue of mice 24 hours after injury on day E18 and their own normal controls,283 differentially expressed genes were found,including 258up-regulated genes and 25 down-regulated genes.Biological process included dysregulated genes that were increased in responses to stimuli,metabolic processes,and intercellular communication.Molecular functions are more reflected in protein binding and nucleic acid binding.The top ten most relevant signaling pathways were analyzed by GO and KEGG enrichment,and the most significant common pathway in the two results was found—TLR signaling pathway.The differential genes of the TLR signaling pathway were verified to be consistent with the sequencing results.3.The CTSS in the TLR pathway was selected according to the significance and difference fold of the differential genes,and it was verified that it showed differences in temporal expression after E16 and E18 injury.The E18 day group that produced scarring was significantly up-regulated in the injured tissue.In vitro experiments confirmed that inhibiting the expression of CTSS can significantly reduce the expression of collagen type I,collagen type III and fibronectin in human skin fibroblasts.At the same time,the only transcription factor IRF7 in the TLR pathway was found to exhibit consistent temporal expression differences in CTSS.Dual-luciferase reporter assays and ch IP assays confirmed the binding of IRF7 to the CTSS promoter region;overexpression of IRF7 demonstrated the up-regulation of CTSS expression by IRF7.Up-regulation of the IRF7/CTSS regulatory axis can promote the expression of collagen type I,collagen type III,and fibronectin in fibroblasts,and the up-regulation of downstream proteins by overexpression of IRF7can be reversed by knockdown of CTSS.IRF7 and CTSS were also significantly up-regulated in the skin tissue of patients with keloids;CTSS inhibitors in vivo could significantly reduce the hyperplasia of scars and inhibit the activation of myofibroblasts.Conclusion:1.The skin of E16 and E18 days of embryonic development can be repaired after 48 hours.E16 wounds can be healed without scars,but scars appear after E18 wounds in the late embryonic stage.Day E18 is a critical developmental time point for the transition from scarless repair to scarring in embryonic wounds.2.At 24h after trauma on day E18 in the late embryonic stage of mice,it is in the transitional stage from the inflammatory response period to the proliferative remodeling period after trauma,in which the activation of TLR signaling pathway plays an important role.3.The differential expression of the IRF7/CTSS regulatory axis in late embryos promotes the formation of post-traumatic scars,and IRF7 acts as a transcription factor to activate the transcription of CTSS.4.IRF7 and CTSS have different expressions after trauma in different embryonic stages.The differential expression can last from late embryonic stage to adulthood,which is closely related to the formation of pathological keloids in adulthood.5.Targeted inhibition of CTSS expression can effectively inhibit scar formation and myofibroblast activation after trauma in adult mice. |
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