| ObjectiveTo explore the potential targets and mechanisms of combination therapy of TSA and SAB in the treatment of HS;TSA and SAB co-loaded liposomes（TSA-SAB Lips）were prepared and investigated for their preliminary efficiency in inhibiting HS.On this basis,a transdermal formulation of TSA-SAB Lips-oxidized hyaluronic acid/N-succinyl chitosan hydrogel（TSA-SAB Lips/Gel）was further constructed with a view to improve the percutaneous permeation efficiency,providing a new idea for Salvia miltiorrhiza based transdermal formulation to prevent and treat HS.Methods（1）Potential targets and mechanism prediction of TSA and SAB in the treatment of HS.TSA and SAB relevant targets were identified using pharmacophore mapping,database mining,and literature search.HS-related targets were collected and screened on the basis of skin wound hypertrophic scars caused by burn,trauma,or operation.Cytoscape 3.8.2was used to map the“TSA/SAB-target-HS”network,and the STRING database was used to construct the PPI network.In combination with Cyto Hubba plug-in,six algorithms of DEG,MNC,MCC,CLO,BC and EPC were selected to screen out the important targets in the network.Cell composition,molecular function,and biological process of potential targets were analyzed using Clue GO plug-in;Reactome pathway enrichment analysis of potential targets was performed using the Clusterprofiler 3.8 toolkit.（2）Preparation and quality evaluation of TSA-SAB Lips.CCK-8method was used to investigate the effects of combination of TSA and SAB on HSF cell proliferation.The simultaneous determination of TSA and SAB was established by HPLC.The TSA-SAB Lips were prepared by thin film dispersion method combined with p H gradient active drug loading method,and its physical and chemical properties including morphology,particle size,potential,p H value,initial stability and lyophilized stability were investigated.（3）The effect of TSA-SAB Lips on the biological behavior of HSF.In vitro culture of HSF was established and CCK-8 method was used to determine the effect of TSA-SAB Lips on the proliferation of HSF.Cell scratch assay and Transwell chamber assay were used to evaluate the effects of TSA-SAB Lips treatment on the migration and invasion of HSF.Western blotting was used to investigate the effects of TSA-SAB Lips on the protein expression levels of MMP2 and COL I in HSF.（4）The construction and the quality assessment of TSA-SAB Lips/Gel.A series of oxidized hyaluronic acid/N-succinyl chitosan hydrogel（OHA/NSC）systems with different oxidation degrees were prepared,followed by structural characterization and basic performance characterization to verify the possibility of forming and drug loading of the hydrogel system,which were further comprehensively analyzed and screened out the suitable OHA/NSC system for carrying liposomes.The TSA-SAB Lips were further loaded into the hydrogel to prepare the liposome composite hydrogel（TSA-SAB Lips/Gel）and its characterization was investigated.The dynamic dialysis method was employed to study the drug release regularity of TSA-SAB Lips/Gel in vitro.The modified Franz diffusion cell method was used to analyze the permeation and dermal layer retention performance of TSA-SAB Lips/Gel in vitro;the animal skin irritation studies was conducted to preliminarily evaluate the safety of TSA-SAB Lips/Gel for transdermal administration.Results（1）A total of 82 potential targets were screened,and the"TSA/SAB-targets-HS"network and PPI network were constructed,and 24important targets including VEGFA,TNF-ɑ,TIMP1 and TGF-β1were identified.The results of GO and Reactome enrichment analysis showed that these targets involved in many aspects of molecular functions and biological processes,and played an anti HS role by regulating 259pathways such as immune system,extracellular matrix tissue,signal transduction,apoptosis,and cell response to external stimuli.（2）The combination of TSA-SAB had a synergistic effect on the inhibition of HSF proliferation.Meanwhile,TSA-SAB Lips were successfully prepared with round shape,uniform particle size distribution,the average particle size was（189.50±1.57）nm,the dispersion coefficient of PDI was 0.246±0.03,the Zeta potential was（-18.73±1.41）m V,the encapsulation efficiency of TSA and SAB were（87.93±0.97）%and（91.20±0.47）%,respectively,and the p H range was（6.85±0.03）,and its quality was good and stable.（3）The TSA-SAB Lips in different dosage groups had certain inhibitory effects on the proliferation,migration and invasion of HSF in a dose-dependent manner,and could increase protein synthesis of MMP2and reduce the protein expression level of COL I.（4）The OHA/NSC hydrogels with different oxidation degrees were successfully prepared and the OHA50/NSC was selected as the carrier of TSA-SAB Lips.The formed composite hydrogels were stable in quality,uniform and delicate in texture,no agglomerated agglomerates,suitable in gelation time,good in swelling,moisturizing and adhesion,and no skin p H irritation.Both TSA and SAB in TSA-SAB lips/gel showed sustained slow drug release behavior without burst release effect,which were consistent with the Hixson-Crowell equation model.TSA-SAB Lips exhibited good transdermal performance and dermal retention effect.It had no obvious irritation to animal skin,which means that TSA-SAB Lips/Gel had certain safety of skin medication.ConclusionThis study reflects that TSA-SAB has the characteristics of multi-target and multi-pathway synergistic inhibition of HS.At the same time,stable quality co-carrier TSA-SAB Lips was prepared,and the synergistic inhibition of proliferation,migration and invasion of HSF was obvious in vitro,and the expression of MMP2 was significantly up-regulated while the synthesis of COL I was down-regulated.The TSA-SAB Lips/Gel drug loading system has a certain sustained release performance,can improve the drug transdermal performance and dermal retention,without skin irritation,and can lay a certain foundation for the follow-up research on the prevention and treatment of HS in vivo. |
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