TSP1 Promotes Fibroblast Proliferation And Extracellular Matrix Deposition Through IL6/JAK2/STAT3 Signaling Pathway In Keloid

Keloid,which only occurs in humans,is a benign fibroproliferative lesion of unknown pathogenesis characterized by fibroblast proliferation and excessive collagen deposition secondary to skin injury(eg,trauma,burn,or surgery),scarring The tissue proliferates and protrudes beyond the edge of the skin lesion.Excessive scar tissue not only affects the appearance,causing pain and itching,but also causes physical and mental illness in the patient.Although keloids are not malignant in nature,they are fibrous tumors of the dermis that form during a lengthy wound healing process,and to date,there is no satisfactory treatment for this fibroproliferative disorder.TSP1 is a multifunctional protein that exists both as a secreted protein and as an insoluble extracellular matrix molecule.It can regulate the process of wound healing and promote the occurrence of many fibrotic diseases.Therefore,elucidating the role of TSP1 in keloids and related molecular mechanisms will help to clarify the pathogenesis of keloids and find better methods for the clinical treatment of keloids.Objective:1.Clarify the differential expression of TSP1 in keloids and normal skin;2.Explore the effect of TSP1 on the proliferation of keloid fibroblasts and the production of extracellular matrix;3.Explore the mechanism of TSP1 on the proliferation of keloid fibroblasts and the production of extracellular matrix.Methods:1.Collect keloid tissue and normal skin tissue,make paraffin sections and culture primary fibroblasts.The differential expression of TSP1 in normal tissue and scar tissue was detected by immunohistochemical staining and RT-PCR,and the differential expression of TSP1 in normal skin fibroblasts and keloid fibroblasts was detected by RT-PCR and Western blot;2.Construct TSP1-knockdown keloid fibroblasts and normal skin fibroblasts treated with exogenous TSP1 molecules,respectively,and detect the changes of fibroblast proliferation and collagen I expression by EDU,Western blot and other detection methods;3.Perform RNA-seq on TSP1-knockdown keloid fibroblasts,and analyze the effect of TSP1 knockdown on keloid fibroblasts by differential gene enrichment and cluster analysis.Changes in the gene set of keloid fibroblasts;4.Detect the activation of IL6/JAK2/STAT3 signal transduction pathway in keloid fibroblasts with low TSP1 expression and normal skin fibroblasts treated with exogenous TSP1 molecules by RT-PCR,Elisa,Western blot and other methods;Exogenous IL6 molecule stimulated keloid fibroblasts with low expression of TSP1 to detect the recovery of JAK2/STAT3 pathway;normal skin fibroblasts were treated with exogenous TSP1 molecule and JAK2 inhibitor at the same time,and the effect of JAK2 inhibitor on TSP1-induced fibroblasts was detected.Whether IL6/JAK2/STAT3 activation has inhibitory effects.To evaluate the role of IL6/JAK2/STAT3 signaling pathway in TSP1 affecting keloid fibroblast proliferation and collagen secretion;5.Detect the expression of IL6 in keloids and normal skin by immunohistochemistry,RT-PCR and other detection methods,detect the expression of p-STAT3 in keloids and normal skin by immunohistochemistry,analyze the relation between IL6,p-STAT3 and TSP1.Results:1.The results of histochemical staining and RTqPCR experiments showed that compared with normal skin,the expression of TSP1 was up-regulated in keloids;the protein expression of TSP1 was up-regulated in KF.2.Knockdown of TSP1 can significantly reduce the proliferation of KF and the expression of collagen I;exogenous TSP1 molecules can cause the upregulation of the proliferation of NF and the expression of collagen I.3.RNA seq analysis of KF gene expression after TSP1 knockdown Volcano plot and differential gene cluster analysis found that KF survival genes were differentially expressed after TSP1 knockdown,and GSEA was enriched in the IL6/JAK2/STAT3 gene set;4.RT-PCR,Elisa and Western blot results showed that knockdown of TSP1 significantly reduced the expression of IL6 in KF and the activation of the downstream JAK2/STAT3 pathway,while exogenous TSP1 molecules could increase the expression of IL6 in NF and the activation of this pathway.activation;exogenous IL6 was used to stimulate KF with knockdown of TSP1,and it was found that IL6 could restore cell proliferation and collagen I reduction caused by TSP1 knockdown,and reactivate the JAK2/STAT3 pathway;at the same time,simultaneous stimulation with JAK2 inhibitor and TSP1 NF,it was found that JAK2 inhibitors can inhibit the effects of TSP1,including cell proliferation,increased collagen I and STAT3 signaling;5.The results of immunohistochemistry and RT-PCR showed that the expression of IL6 was increased in keloids,and the results of immunohistochemistry showed that the expression of p-STAT3 was increased in keloids.Correlation analysis showed the expressions of IL6,p-STAT3 and TSP1.has a positive correlation.Conclusion:The abnormally high expression of TSP1 in keloids can promote the proliferation and collagen secretion of fibroblasts through the IL6/JAK/STAT3 signal transduction pathway,thereby this may promote the formation of keloids.