| Objective:Hypertrophic scar(HS)is the most common pathological scar.A range of plant-derived substances have been identified as potential treatment medicines for HS.Nevertheless,the restricted water solubility and stability of these substances,together with the epidermal barrier,hinder their efficient penetration and limit their application in clinical settings.In this study,bioinformatics analysis was employed to identify target genes and signaling pathways closely related to HS.Based on this,a list of small molecule compounds with potential therapeutic effects was obtained,included the plant-derived substance Resveratrol(Res).Network pharmacology was used to further investigate the core target genes and signaling pathways of Res in the treatment of HS.Subsequently,mesoporous silica nanoparticle(MSN)and 8%FC/AHA thermosensitive hydrogel were employed as drug carriers to treat HS in situ with encapsulation of free Res and Res-laden MSN respectively.The effects of these two drug-loaded materials on the regression and maturation of HS were compared in vitro and in vivo.Additionally,the mechanism was confirmed.Methods:Part Ⅰ:(1)Differentially expressed genes(DEGs)were extracted from the GEO dataset GSE188952 associated with HS;(2)DEGs were used to construct a protein-protein interaction(PPI)network to identify the key(Hub)genes;(3)Gene Ontology(GO)biological function and KEGG signaling pathway enrichment analysis were conducted on the DEGs;(4)Res,a plant compound,was predicted and selected for its potential therapeutic effects on HS based on the Hub genes;(5)The common targets of Res and HS were obtained by network pharmacology,the core targets were then determined by creating a PPI.GO and KEGG enrichment analysis were conducted on the common targets to investigate the potential mechanism of Res in treating HS.Part II:(1)Mesoporous silica nanoparticles laden with resveratrol(MSN@Res)was prepared and their properties were characterized,including particles size,water solubility,accelerated stability,x-ray diffraction spectroscopy(XRD),fourier transform infrared spectroscopy(FTRI),and in vitro drug release testing;(2)Human hypertrophic scar fibroblasts(HSFs)were cultured under conditions of hypoxia and nutrient deprivation to mimic the formation of hypertrophic scars.The cells were exposed to MSN@Res,MSN and Res at equivalent concentrations,then cell viability,apoptosis,reactive oxygen species(ROS)generation,and autophagosome formation were detected by CCK-8,Calcein/PI staining,flow cytometry,JC-1,DCFH-DA and MDC fluorescence staining,respectively.(3)After pre-treatment with a p38-MAPK inhibitor/activator or a ROS scavenger/generation agent,the effects of MSN@Res on autophagy,apoptosis and the protein expression of HIF-1α,p53,LC3-II,Beclin-1,Bax and Cleaved caspase3 were detected by immunofluorescence and western blotting,respectively.(4)The ultrastructural alterations of HSFs after treatment with MSN@Res were observed by a transmission electron microscope(TEM).Part III:(1)An injectable thermosensitive hydrogel containing MSN@Res(8%FC/AHA-MSN@Res)was prepared,and its properties were characterized,including scanning electron microscope(SEM),~1H-NMR,FTRI,rheological properties,swelling rate,compression,in vitro degradation,and drug release tests.(2)The cell viability of HSFs treated with MSN@Res,8%FC/AHA and 8%FC/AHA-MSN@Res was assessed by CCK-8 and Calcein/PI staining.(3)The HS model of rat tail was established and treated by in situ injection based on the aforementioned groups.The area,prominence,texture,and color of scars were recorded at baseline(0 week),2 and 6 weeks post-treatments,and the clinical efficacy was evaluated.The epidermal and dermal thickness,as well as the collagen deposition and organization in normal skin and scar tissue,were evaluated by HE and Masson staining.The protein expressions of Col I,Col III,andα-SMA in scar or normal skin were evaluated by immunohistochemical staining.The ROS activity and protein expression of phosphorylated p38-MAPK,HIF-1α,p53,LC3-II,and Cleaved caspase3 in scar or normal skin were detected by Elisa and immunofluorescence staining.(4)The impact of each material on the vital organs of rats were assessed by HE staining to evaluate the system safety.Results:Part Ⅰ:(1)8 Hub genes associated with HS,including HMGCS1,DHCR7 and MSMO1,were identified from the screened 804 DEGs.(2)DEGs were significantly enriched in the tight junction pathway,arachidonic acid metabolism,and ECM-receptor interaction pathway.Additionally,they were associated with epidermal related functions such as epidermal/keratinocyte development and differentiation.(3)The predicted drugs based on Hub genes included plant compounds such as Res andβ-sitosterol;(4)10 core targets including MAPK3,TNF,TP53 and CASP3 were identified among the 121common targets of Res and HS.The common targets were significantly enriched in signaling pathways associated with lipid and atherosclerosis,reactive oxygen species,apoptosis,MAPK and HIF-1,as well as in the biological function of responding to hypoxia and nutrition level.Part II:(1)The average particle size of MSN@Res was154.4±25.1nm,with a drug encapsulation efficiency of 90.38%and a drug loading efficiency of 45.19%.These results indicate a high level of dispersion and stability.The FTIR and XRD spectra provided evidence that Res was present in an amorphous state when encapsulated in MSN.The water solubility of MSN@Res was increased by approximately 2.1-fold in comparison to free Res.The release of Res from MSN@Res initially followed a linear trend in the first 6hours,resulting in a cumulative release of(51.6±0.43)%.After that,it was gradually released over a period of 6 days.(2)Both MSN@Res and Res have the ability to decrease the mitochondrial membrane potential,ROS activity and the formation of autolysosome.At the same time,they can increase the intracellular calcium concentration and apoptosis rate of HSFs.MSN@Res exhibited more significant effects on calcium concentration,apoptotic rate,and cell survival in comparison to Res.(3)The CCK8 assay demonstrated that MSN@Res decreased cell viability by suppressing the phosphorylation of p38-MAPK.Furthermore,pre-treatment with inhibitors of apoptosis and autophagy partially reversed the decline in cell viability induced by MSN@Res.Flow cytometry analysis demonstrated that pre-treatment with p38-MAPK and autophagy inhibitors both increased cell apoptosis triggered by MSN@Res.(4)Immunofluorescence analysis revealed that MSN@Res inhibited physiological autophagy and triggered apoptosis in HSFs via reducing ROS activity and blocking the phosphorylation of p38-MAPK.Western blot analysis indicated that MSN@Res suppressed the phosphorylation of p38-MAPK by reducing ROS activity.This resulted in a decrease in the expression of HIF-1αand p53 protein.In addition,it down-regulated the expression of LC3-II and Beclin-1 protein,while up-regulating the expression of Cleaved caspase3 and Bax protein.(5)TEM revealed the cells in the control group displayed physiological autophagy morphology,which was defined by the presence of autophagosomes.In contrast,the cells in the MSN@Res group exhibited distinct morphology associated with apoptosis and autosis.Part III:(1)The composite hydrogel’s components were effectively modified to aldehyde-pronic F127(F127-CHO)and adipic dihydrazide-modified hyaluronic acid(AHA),as confirmed by both ~1H NMR and FTIR.The rheological test demonstrated that 8%FC/AHA-MSN@Res has desirable injectability and temperature sensitivity,along with excellent mechanical strength,swelling,degradation properties.Furthermore,it exhibits a greater level of controlled drug release performance compared to MSN@Res.(2)The CCK8 analysis indicated that8%FC/AHA-MSN@Res had a lower inhibitory effect on cell viability of HSFs compared to MSN@Res during the initial stage in vitro.Nevertheless,the hydrogel’s controlled release of Res efficiently suppressed cell viability for an extended duration throughout the later stage.(3)The scar area and clinical score were considerably decreased in the8%FC/AHA-MSN@Res group compared to the control group after 6 weeks of treatment.HE and Masson staining confirmed that both MSN@Res and 8%FC/AHA-MSN@Res effectively decreased dermal thickness and collagen deposition in HS.However,8%FC/AHA-MSN@Res showed a stronger impact compared to MSN@Res,whereas MSN@Res did not have a noticeable effect on scar epidermis.(4)Immunohistochemistry analysis revealed that 8%FC/AHA-MSN@Res inhibited the protein expression of Col I,Col III andα-SMA in HS effectively.(5)Elisa and Immunofluorescence assays demonstrated that 8%FC/AHA-MSN@Res consistently suppressed the activity of ROS,facilitated the down-regulation of phosphorylated p38-MAPK,p53,LC3-II protein expression,and promoted the up-regulation of Cleaved caspase3 protein expression in HS.While both MSN@Res and 8%FC/AHA-MSN@Res were able to down-regulate the expression of HIF-1αprotein in HS,the latter exhibited a stronger inhibitory impact.(6)The administration of 8%FC/AHA,MSN@Res,and 8%FC/AHA-MSN@Res through intralocal injection did not have any detrimental or negative effects on the vital organs of rats.Conclusions:(1)Impaired skin barrier and abnormal lipid metabolism may be involved in the formation and progression of HS.Plant compounds such as Res andβ-sitosterol were included in the drug list based on Hub gene.Res may modulate the MAPK and HIF-1/p53signaling pathways by impacting the anti-oxidative stress response.This,in turn,might influence the processes of autophagy and apoptosis in HSFs and contribute to the regression of HS.Res may also impact the epidermal cells by modulating lipid metabolism for the treatment of HS.(2)In comparison to free Res,MSN@Res demonstrated a more potent ability to suppress cell viability,which may be related to its superior water solubility,stability,and controlled drug release performance.It hinders physiological autophagy of HSFs under hypoxia condition by scavenging ROS and down-regulating the p38-MAPK/HIF-1α/p53 axis.This leads to cell death by apoptosis and autosis.Furthermore,autosis may have a more significant impact on cell viability compared to apoptosis.(3)8%FC/AHA-MSN@Res is a drug-loaded hydrogel with excellent injectability,thermal responsiveness,degradability and dual sustained drug release properties.Continuous release of Res may down-regulate the ROS/p38-MAPK/HIF-1α/p53 axis,hence affecting the autophagy and apoptosis of HSFs.The reduction in cell viability and promotion of cell death subsequently slows collagen deposition and accelerates HS regression.Its bioavailability was also superior to MSN@Res. |
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