Study On The Biological Mechanism Of Pressure Therapy For Hypertrophic Scar

Hypertrophic scar(HS)is a pathological fibrotic disease caused by excessive wound healing.HS is characterized by overproliferation of fibroblast and excessive deposition of extracellular matrix,often accompanied by itching and pain,which seriously affects the appearance and may lead to dysfunction.HS is still an important problem that remains unresolved in plastic surgery.Although domestic and foreign scholars have conducted in-depth research on the formation and development of scars,the specific pathogenesis has not been fully elucidated.According to statistics,pressure therapy is successful in 60% to 85%of patients as a non-invasive treatment,but it is based on clinical experience.Meanwhile,because of the slow,random nature of hypertrophic scars,and the sample in the human body is difficult to obtain continuously,so there is no clear scientific data on the molecular mechanism of pressure treatment.Using animal models to elucidate the pathogenesis and development of hypertrophic scars,reveal the biological mechanism of pressure treatment,not only makes the continuous research on the occurrence and development of HS to be possible,but also provides theoretical basis and guidance for clinical application of pressure therapy.Based on this,we selected Bama minipigs to establish the animal model of hypertrophic scar by making skin injury on the back and performed pressure therapy.A systematic and continuous in vivo study about the process of HS formation and development and pressure therapy was conducted combining histological analysis and molecular biology experimental methods.We analyzed the mechanism of scar formation and the development process,and the underlying molecular mechanisms of pressure therapy from the apparent,histological,transcriptologic and proteinic level.The main contents and results of this study are as follows:(1)The animal model of hypertrophic scar was successfully established.The Bama minipigs were selected as the research object,and the skin on the back were wounded deep to the dermis with an electric knife,making it naturally healed by epithelization.The healing of the wound and the formation of scar were observed on the 14 th,30th,60 th,90th and 120 th day,meanwhile,the skin tissue samples were obtained.HE staining and masson staining were performed to observe the proliferation of cells and the deposition of collagen.The results showed that on the 60 th day after skin extraction,the scar formed was significantly harder than the surrounding normal skin,and the center was dark red,all wounds had a dense,fibrotic,convex but inconspicuous appearance.Tissue section staining showed obvious cell proliferation,collagen fiberdeposition and disordered arrangement.It was confirmed that hypertrophic scar was formed after the skin injury of Bama minipigs;(2)Pressure therapy improved the appearance and slowed the development of scars.On the 60 th day after skin extraction,the self-made pressure bandage was used to treat the hypertrophic scar formed on the back of Bama minipigs.The value of pressure was 3.4 kPa(25.5 mmHg),and it was continued for 24 hours a day.The effects of pressure treatment on the apparent and histological level of the scar were observed on the 90 th and 120 th days afterwards.The results showed that pressure treatment alleviated the pigmentation,smoothed the surface and improved the appearance of the hypertrophic scar.Tissue section staining showed that the number of fibroblasts and the collagen fiber nodules decreased and the arrangement became neat,almost parallel to the skin surface.The pressure therapy was identified to have a good clinical effects;(3)Pressure therapy affected transcriptome expression levels in the animal model of HS.RNA-seq technique was used to detect the expression of transcriptome levels in skin tissues at different time points after skin extraction.Bioinformatics analysis software was used to calculate the difference in gene expression during scar formation and development and pressure therapy,and GO function enrichment analysis and KEGG signal pathway enrichment analysis for differentially expressed genes(DEGs)were performed,finally qRT-PCR was used to verify the reliability of sequencing results.The resultsshowed that the number of DEGs after 2 months of pressure therapy was less than that after 1 month of pressure therapy,but the GO function items enriched by DEGs in the two groups were similar,including extracellular matrix,growth factor binding and epidermis cell migration;the KEGG signaling pathways enriched by DEGs in the two groups were also similar,including extracellular matrix-receptor interactions,protein digestion and absorption.These results confirmed that pressure therapy affected the transcriptome expression level of the animal model;(4)Pressure therapy inhibited the expression of IGF-1/IGF-1R and its downstream PI3K/AKT signaling pathway in scar model tissues.Based on RNA-seq data,the transcriptome expression level of IGF-1/IGF-1R signaling pathway and its downstream MEK/ERK and PI3K/AKT signaling pathways in skin tissues were calculated at different time points after skin extraction,the expression levels of genes and proteins were detected by qRT-PCR and Western blot.The role of these signal pathways in the process of scar formation and development and pressure therapy was explored.Our results showed that the expression of MEK/ERK signaling pathway was increased after pressure therapy,but the expression level of IGF-1/IGF-1R signaling pathway and its downstream PI3K/AKT signaling pathway was significantly decreased after pressure therapy;(5)Pressure therapy inhibited the expression of extracellular matrix in scar model tissues.Based on RNA-seq data,the transcriptome expression levelof vascular endothelial growth factor(VEGF),type I collagen(Collagen I),type III collagen(Collagen III),fibronectin(FN)and α-smooth muscle actin(α-SMA)in skin tissues were calculated at different time points after skin extraction.The qRT-PCR method was used to verify the accuracy of RNA-seq data.We investigated the changes of VEGF and extracellular matrix expression during scar formation and development and the effect of pressure therapy on extracellular matrix secretion.The results showed that the expression of Collagen I,Collagen III,FN and α-SMA was significantly inhibited after 1month of pressure treatment,but increased after 2 months of pressure treatment.In summary,an animal model of hypertrophic scar can be successfully established by taking skin injury from the back of Bama minipigs,and IGF-1/IGF-1R and PI3K/AKT signaling pathways may play an important role in the pressure treatment of hypertrophic scars.