Study On The Effect And Mechanism Of Keloid Inhibition By Sunitinib

Objective:Keloid is a benign skin lesion,usually a proliferative pathological change caused by abnormal healing of skin trauma,where normal skin structure and appearance are disrupted along with symptoms such as pain,itching and localized adhesions.The main feature of keloids is tumor-like growth,which is due to the abnormal proliferation of keloid-derived fibroblasts(KFs)and excessive deposition of extracellular matrix components in the dermis.So far,the problem of difficult-to-cure and easy-to-recur keloid scar has not been fundamentally solved,and secondly,the pathogenesis of keloid scar is not well understood.Therefore,effective improvement of the treatment of keloids is an urgent problem that needs to be solved nowadays.As an oral small molecule inhibitor of multiple tyrosine kinase receptors,sunitinib has been used as a promising antitumor candidate,showing significant therapeutic efficacy in diseases such as renal cell carcinoma and gastrointestinal mesenchymal tumors.The use of sunitinib in keloids has not been reported.Due to the tumor-like growth of keloids,and based on the function of sunitinib found in other tissues and cells,we hypothesized that it may be promising as a targeting agent to treat keloids.In this study,we intend to explore the cell proliferation,migration,invasion,apoptosis and collagen expression of KFs under the action of sunitinib in vivo and in vitro,to investigate the effects of the PI3K/AKT/mTOR signaling pathway on the cellular function of KFs,and to construct an animal model of keloid scarring followed by in vivo validation.The aim is to explore the role of sunitinib in the treatment and pathogenesis of keloid,and to provide a theoretical basis for the clinical targeted treatment of keloid.Methods:1.Eight cases of keloid tissues and normal skin tissues from humans were collected in the clinic and used to extract primary fibroblasts,and the expression of collagen type Ⅰ(Collagen Ⅰ,COL-Ⅰ)and collagen type Ⅲ(Collagen Ⅲ,COL-Ⅲ)was determined by Western blotting,and the two kinds of tissues were observed by H&E staining.We compared the growth and proliferation potentials of Normal Skin-derived Fibroblasts(NFs)and KFs in vitro by counting the cells and examining the expression of Ki67 protein by CCK-8 assay and immunofluorescence.2.The CCK-8 assay was utilized to determine the optimal concentration of sunitinib and the biosafety against multiple cell lines.3.KFs were treated with different concentrations of sunitinib,and the migration ability of the drug on KFs was detected by cell scratch;the invasion ability was detected by Transwell;the cell cycle and apoptosis of KFs were detected by flow cytometry;and the cytoskeleton was detected by immunofluorescence.4.After sunitinib treatment of KFs,COL-Ⅰ and COL-Ⅲ expression was determined by qPCR,immunofluorescence,and Western blotting;the effect of sunitinib on the PI3K/AKT/mTOR signaling pathway was detected by Western blotting,and then the expression level of autophagy LC3 protein was detected.5.Via surgically excised human keloid tissue to construct a nude mouse keloid animal model in vivo by tissue block transplantation method,after successful modeling,it was divided into the control group,sunitinib drug group,and triamcinolone acetonide(TAC)group,and the volume of the keloid was quantitatively injected and measured regularly after surgery.Tissue specimens of keloid tissue were obtained after intervention treatment,and the expression of Ki67,TUNEL,type Ⅰ collagen,type Ⅲ collagen,and CD31 were detected by H&E staining and immunohistochemistry,respectively.Results:1.Western blotting results suggested that the expression levels of COL-Ⅰ and COL-Ⅲ in primary KFs were higher than those in NFs;compared with NFs,KFs proliferated faster as suggested by CCK-8 assay,and the high expression of Ki67 in the latter in immunofluorescence assay further suggested that KFs were more active in cell proliferation.2.Sunitinib shows no cytotoxicity in cell lines including NFs,adipose and bone marrow-derived mesenchymal stem cells,etc.The optimal concentration of Sunitinib for the action of KFs was set at 6.0μM.3.Sunitinib effectively inhibited cell proliferation,migration and invasion of KFs;reduced the percentage of G1/G0 cells in the cell cycle,and induced an increase in the S phase;immunofluorescence results suggest that Sunitinib destroys the cytoskeleton of KFs.4.Sunitinib induced apoptosis in over 60%of KFs cells,immunofluorescence results suggested that the fluorescence intensity of type Ⅰ and type Ⅲ collagen was weakened after drug treatment of KFs,qPCR and Western blotting results suggested that sunitinib effectively reduced the expression level of COL-Ⅰ and COL-Ⅲ.5.Sunitinib intervention in KFs enhanced the ratio of autophagy proteins LC3B-Ⅱ/LC3B-Ⅰ,decreased p-AKT,p-mTOR and p-PI3K protein expression.6.In vitro,we found that sunitinib could significantly inhibit the proliferation of KFs,etc.After successfully constructing an in vivo mice model of keloid,sunitinib also inhibited the proliferation of KFs,reduced the expression of COL-Ⅰ and COL-Ⅲ,CD31,and induced the regression of keloid in vivo.Conclusion:Sunitinib inhibits KFs cell proliferation,migration,invasion,and promotes apoptosis and autophagy in Vitro.Then,sunitinib could mediate KFs cell function and reduce Collagen protein expression through inhibition of the PI3K/AKT/mTOR signaling pathway.In Vivo,sunitinib could significantly inhibit the proliferation of KFs,reduce Collagen in keloid tissues,and induce keloid regression.