| BackgroundHypertrophic scars(HTS)are a type of scar tissue that grows and thickens excessively due to various harmful external factors that stimulate granulation tissue proliferation and fibrosis after deep skin injury.The characteristic is that the scope is limited to the original wound surface and is commonly seen in the repair process after deep partial thickness burns.The pathological process includes fibroblast transformation,proliferation,and excessive deposition of extracellular matrix(ECM)proteins,such as type I collagen(Col I)and type III collagen(Col III).However,the specific pathogenesis of HTS has not yet been fully elucidated.The currently recognized risk factors for HTS include wound tension,inflammation,and infection.Although multiple methods have been applied in clinical practice to prevent and treat HTS,there is not a single treatment method applicable to all lesions,let alone a completely effective treatment method.Peroxisome proliferators activated receptors(PPARs)are a class of nuclear receptors that can regulate sugar,fat metabolism and fibrosis.Three subtypes of PPARs:PPAR α,PPAR β/δ And PPAR γ It is expressed to varying degrees in the skin of both humans and rodents.Research on a systemic sclerosis mouse model suggests that PPARs may regulate skin fibrosis by regulating local inflammation,fibroblast function,and other means.In addition,PPARs activity disorder is related to a variety of pathological conditions,such as diabetes,cancer,and cardiovascular disease,which may also lead to the formation of HTS.Therefore,PPARs are expected to become new therapeutic targets for managing the formation of HTS,and further research in this field may bring new treatment options to patients with this disease.The literature has reported that the use of high affinity PPAR single subtype agonists such as rosiglitazone increases the risk of adverse reactions such as fractures.IVA-337(Lanifibranor)is a novel pan PPARs agonist that has shown certain efficacy and safety in clinical trials and has broad prospects for clinical application.It has moderate agonistic activity against all three PPAR subtypes,which may give it the potential to excite the three PPARs and achieve a synergistic therapeutic effect while also avoiding adverse reactions caused by specific subtype agonists.Based on the above research results and reasonable assumptions,this study intends to design experiments through in vitro experiments,wound and scar animal models,to explore the potential possibility and mechanism of action of IVA-337 as an optional drug for treating HTS.ObjectiveIn this study,we intend to explore the cytotoxicity and anti-fibrosis of IVA-337 on HFBs by constructing an in vitro cell model of HTS,explore the potential therapeutic effect of IVA-337 on wound fibrosis and HTS by constructing a mouse wound model and a rabbit ear scar model,and analyze the possible mechanism of action of IVA-337 on the therapeutic effect of HTS by constructing a mouse wound model and a rabbit ear scar model.Part Ⅰ: To explore the difference in expression of PPARs in normal HFBs and HFBs after TGF-β1 induction;To investigate the effect of IVA-337 treatment on the expression of fiber-related genes and proteins in HFBs after TGF-β1 induction.Part Ⅱ: To observe the effect of topical application of different concentrations of IVA-337 on the normal wound repair results of mice,detect and compare the expression differences of related genes and proteins with the control group,and explore the potential for promoting wound healing and regulating local inflammation to reduce scarring.Part Ⅲ: To explore the role of topical application of IVA-337 in improving scarring in rabbit ear HTS model and related histopathological and protein expression differences;Preliminary elucidating of the possible mechanism of IVA-337 to improve HTS formation after wound healing.MethodsPart Ⅰ:(1)The expression of PPARs in human tissues was observed by sectioning,embedding and immunohistochemical staining of HTS tissues without underlying chronic diseases and immune deficiencies obtained in previous clinical trials and adjacent healthy human skin tissue samples;(2)Normal skin tissues were obtained,primary HFBs were isolated,two cell models of HFBs and TGF-β1 induced HFBs were established,and q PCR and Western Blot were used to verify the differences in fibrotic markers such as type I collagen and α-SMA and PPARs expression between the two;(3)The effect of experimental concentration of IVA-337 on cell proliferation,migration and cycle was evaluated by CCK-8 experiment,Ed U labeling,scratch experiment,flow cytometry apoptosis,cycle analysis and other methods.Through the above experiments,the specific effects of IVA-337 on TGF-β1-induced HFBs proliferation capacity,motor function,collagen and actin synthesis were clarified.Part Ⅱ: Establish a normal full-thickness skin defect wound model of mice,local injection of IVA-337 at low and high concentrations around the wound,compare the control group injected with excipient solution,observe the difference in wound healing,and evaluate the effects of IVA-337 on wound-related gene expression and inflammation regulation by q PCR,Western Blot and histopathology at POD 7 and 14.Part Ⅲ: Establish a model of proliferative scar in rabbit ear,inject IVA-337 locally after complete wound re-epithelialization,compare the control group injected with excipient solution,observe the change of scar thickness,POD 42 was taken from histopathology to evaluate the effect of IVA-337 on the distribution,arrangement and local inflammation regulation of collagen in the scar,and Western Blot detected the difference in the expression of related proteins.ResultsPart Ⅰ:(1)PPARs were expressed in a certain amount in normal skin HFBs,and the transcription and protein expression of PPARs were inhibited to varying degrees in healthy human HTS tissues and HFBs after TGF-β1-induced differentiation;(2)Cell experiments found that 10μM low concentration of IVA-337 did not affect myofibroblast proliferation,cycle,apoptosis and other functions,but could significantly hinder myofibroblast migration;The high concentration of IIA-337 at 20μM significantly inhibited myofibroblast proliferation,cycling,migration,and promoted apoptosis.(3)q PCR and Western Blot results confirmed that the application of different concentrations of IVA-337 to TGF-β1-induced HFBs could significantly inhibit the synthesis and secretion of extracellular collagen and actin in myofibroblasts,and the effect was more obvious at higher concentrations,but the effect on normal cultured fibroblasts was not obvious.Part Ⅱ:(1)Topical application of two experimental concentrations of IVA-337 did not significantly promote the normal wound repair and healing of mice,but the degree of shrinkage in appearance of wound healing in the experimental group was slightly lighter than that of the control group.(2)The results of q PCR and Western Blot experiments in POD14 wound tissue samples confirmed that the high-concentration injection group(300μM)had a more significant effect on the transcription and expression of PPARs genes in wounds,and significantly inhibited TGF-β1 transcription and inhibited collagen and actin synthesis and secretion.(3)The results of immunohistochemical staining of POD 7 tissue samples showed that IVA-337 had an inhibitory effect on wound inflammation and was known for inhibiting macrophage maturation and activation.Part Ⅲ:(1)IVA-337 injection into rabbit ear hypertrophic scars can significantly improve the appearance of scars in the experimental group;(2)Western Blot and histopathological results showed that the application of IVA-337 in scars could significantly improve scar thickness,collagen arrangement and deposition,and regulate local inflammation of the wound.ConclusionIVA-337 can effectively improve and treat hypertrophic scars in a variety of ways without affecting the normal healing of wounds.It can stimulate the expression of PPARs protein in wounds,inhibit myofibroblast migration,promote fibroblast apoptosis in a concentration-dependent manner,and reduce the expression of collagen type I andα-SMA.At the same time,IVA-337 can also modulate the local inflammatory response within the wound,thereby improving the scarring event induced by excessive inflammation. |
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