Boric Acid Modified Evodiamine Derivative Targeting Myofibroblasts Reduces Hypertrophic Scarring

Background:It is widely acknowledged that myofibroblasts in hypertrophic scars possess the characteristics of hyperproliferation,inadequate apoptosis,extracellular matrix overgeneration,and excessive contraction and migration.Evodiamine is a bioactive ingredient extracted from the traditional herb Evodiae Fructus and has been proved its leading power of anti-tumour effects by inhibiting cell proliferation and promoting cell apoptosis.However,whether it hinders the development of hypertrophic scars remains unclear.The non-selective inhibition of normal skin fibroblasts（HFB）and scar muscle fibroblasts（HSB）is prone to cause excessive inflammatory responses,resulting in tissue atrophy and scar recurrence.Therefore,the ideal anti-scar therapy should selectively target the function of myofibroblasts.Accumulating evidence indicated that the differentiation of quiescent fibroblasts into myofibroblasts during wound healing is often accompanied by an increase in endogenous ROS production.Meanwhile,boric acid possesses high ROS scavenging ability,thus is widely implicated in ROS-responsive drug delivery systems.Based on this specificity,a novel evodiamine derivative BED 2-185 was designed and synthesized by modifying evodiamine with boric acid to improve the anti-myofibroblasts specificity and achieve high efficiency and minimal adverse effects.Methods:Part I:After chemical synthesis of the BED 2-185,the compound was characterized by¹H-NMR,¹³C-NMR,and HRMS spectrum and relative drug release rate by H₂O₂ and elution conditions with HPLC.Part II:HFB and HSB isolation and Culture,CCK8 assessed the proliferation inhibition of Evodiamine and BED 2-185 on HFB and HSB,ROS detection compared the ROS production levels of HFB and HSB,flow cytometry determined the effect of different concentrations of BED 2-185 on apoptosis and cell cycle,scratch experiment and collagen gel contraction experiment compared the effect of different concentrations of BED 2-185 on the migration and contractility of HSB,The collagen andα-SMA expression of HSB treated with different concentrations of BED 2-185 were tested by q PCR and Western blotting.Part III:Rabbit ear hypertrophic scar model establishment and treatment,HE and Masson staining,Real-time PCR and Western blotting to validate the therapeutic effects of BED 2-185 in vivo.Part IV:Analyze the gene expression profiles of BED 2-185-treated HSB by RNA-Sequencing and further invest cell skeleton structure of HSB by immunofluorescence staining.Findings:The compound characterization tests showed that BED 2-185 could release the active ingredient evodiamine under the catalysis of ROS.Cell experiments claimed that there was no difference in evodiamine’s inhibitory activity on HFB and HSB.ROS detection showed HSB with higher endogenous ROS levels than HFB.CCK8 and flow cytometry results indicated BED 2-185 triggered by ROS to selectively target HFB proliferation inhibition and apoptosis and significantly hinder contractility and migration,reducingα-SMA and collagen gene expression.Moreover,through building the rabbit ear hypertrophic scar model,BED2-185 was further verified to prevent scar formation via decreasing collagen deposition andα-SMA expression.Finally,RNA-Sequencing and immunofluorescence staining demonstrated that BED 2-185 exerted its anti-scarring effect by interfering with the structure of microtubules and microfilaments.Discussion:It is the first study to explore the effect and mechanism of evodiamine in hypertrophic scar and use the high expression of ROS in myofibroblasts as a stimulus to design and synthesize anti-scar drugs that selectively inhibit myofibroblasts to maximize the therapeutic efficiency and reduce adverse reactions.This study used boric acid as a ROS capturing agent to modify evodiamine.The evodiamine derivative BED 2-185 was designed and synthesized with highly selective inhibition of myofibroblasts to inhibit the hypertrophic scar formation with minimal side effects on normal skin fibroblasts.This study provides a new perspective for the treatment of hypertrophic scar and other fibrotic diseases in the future.