Exosomes Derived From Mesenchymal Stem Cells Repair Thermal Damage Of HaCaT Cells By Regulating Key Genes Involved In Acute Skin Injury

Objective: Acute skin injuries are by various factors such as mechanical injury,thermal damage,resulting in epidermis,mucous,or tissue defects.If not properly treated in early stage,it can cause serious infections,and even progress into chronic injuries,affecting the quality of life of patients and increasing the socio-economic burden.It has been widely recognized that Mesenchymal stem cells(MSCs)can repair the skin damage.Exosomes derived from MSCs(MSCs-Ex)are the main components of MSCs paracrine secretion,but the mechanism of their promotion of skin damage repair is unclear.Therefore,this study aims to investigate the therapeutic effect of MSCs-Ex on the repair of acute skin injury,and to explore its possible mechanisms involved in the repair process of acute skin injury.Methods: 1.The morphology of MSCs was observed using a microscope to determine if the MSCs was used in this study.MSCs-Ex was isolated from MSCs supernatant by ultracentrifugation.The expression of MSCs-Ex surface markers such as CD63 and CD81 were identified by flow cytometry,and the expression levels of CD63 and CD9 on MSCs-Ex are detected by Western blotting.2.A thermal damage epidermal cell model was established by incubating the HaCaT cells in a water bath at 43℃ for 40 min.Morphology changes were observed by microscope.CCK8 assay was employed to determine the cell viability for evaluating the repair efficacy of MSCs-Ex treatment in thermal-induced HaCaT cell model.3.GSE174661,GSE28914,and GSE8056 datasets related to acute skin injuries were screened and the data were downloaded from the Gene Expression Omnibus(GEO)database.GSE174661 and GSE28914 datasets were applied as training data.The differential expressed genes(DEGs)related to acute skin injury were identified using the "limma" R 4.2.1 software package,with |Fold change|>1.5 and FDR<0.05 as selection criteria.The online analysis tool Evenn was used for further analysis.4.The DEGs were subjected to gene ontology(GO)annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis using the enrichment analysis on Sanger Box online database with a view to investigating the biological processes and signal pathways involved in DEGs.5.Protein-Protein Interaction(PPI)analysis was performed on DEGs using the STRING database,and core genes related to acute skin injuries were screened using the Cyto Hubba plugin in Cytoscape 3.9.1 software.The top 10 DEGs ranked by DEGREE-score were considered as core genes.6.GSE28914 was used as validation dataset,and Graph Pad Prism 8.0.2 software was employed to analyze the expression of core genes in acute skin injury microarray.DEGs expressed consistently with the training datasets were ultimately identified as key genes related to acute skin injury.7.Real-time PCR was used to verify that the m RNAs expression of key genes in the control,thermal damage,and MSCs-Ex(100 μg)treatment groups.Results: 1.Under the microscope observation,MSCs were found to grow in a typical long spindle shape,which conforms the typical growth status of MSCs.Furthermore,the MSCs-Ex were successfully extracted and separated using ultracentrifugation.The positive rates of CD81 and CD63,the surface markers of exosome,were approximately 85.3±1.1% and 95.8±5.5%,respectively,identified by flow cytometry.CD9 and CD63,another surface markers,were detected in three parallel MSCs-Ex samples by Western blotting,indicating that the obtained MSCs-Ex can be used for subsequent studies.2.Morphological results showed that compared with the control group,HaCaT cells treated by thermal injury had a certain degree of shrinkage,and their growth status was restored and the cell number was increased after MSCs-Ex treatment.The CCK8 experimental results showed that thermal damage significantly reduced the cell viability of HaCaT cells compared with control group(P<0.001),while compared with the thermal damage group,the cell viability of thermal-damaged HaCaT cells were significantly increased after the treating with MSCs-Ex at the dosed of 50 μg,100 μg and 150 μg(P<0.001),indicating that MSCs-Ex can repair cell damage induced by thermal injury in HaCaT cells..3.To further explore the mechanisms underlying MSCs-Ex-mediated acute skin injury repair,the GSE174661 dataset was analyzed as training dataset.A total of 3280 DEGs were obtained between the acute wound at day 1(wound 1)and normal skin groups,including 898 upregulated genes and 658 downregulated genes.A total of2858 DEGs were identified between the wound at day 7(wound 7)and normal skin groups,with 688 were upregulated genes and 591 were downregulated genes.The intersect of upregulated and downregulated DEGs was taken separately from the wound 1 and wound 7 groups,resulting in 452 continuously upregulated DEGs and378 continuously downregulated DEGs.Furthermore,we also analyzed another training dataset GSE8056 which was obtained after thermal injury.Compared with the normal group,834 DEGs were identified between the skin tissue samples within 0-3days after thermal injury(0-3 days)and normal skin,including 471 upregulated genes and 363 downregulated genes.For skin tissue samples within 4-7 days after thermal injury(4-7 days),573 DEGs were identified,including 427 upregulated genes and146 downregulated genes,compared with normal skin tissue.The intersect of upregulated and downregulated DEGs was taken separately from these two groups,resulting in 149 continuously upregulated DEGs and 105 continuously downregulated DEGs.Finally,the intersect of continuously upregulated and downregulated DEGs identified from GSE174661 and GSE8056 were analyzed,resulting in 64 genes that were upregulated and 15 genes that were downregulated in both datasets.4.By conducting GO and KEGG enrichment analyses on 64 continuously upregulated genes and 15 continuously downregulated genes,it was found that the continuously upregulated genes mainly participate in biological processes(BP)related to defense response,myeloid leukocyte activation,and regulation of leukocyte immune response.The enriched cellular component(CC)entries were related to granules secretion,exosomes secretion,and other substances.The co-participating molecular functions(MF)were significantly enriched in entries such as Toll-like receptor binding.However,the continuously down-regulated genes enriched in BP are mainly related to biological processes in the development of urogenital system,enriched in the central module of fibril in CC,and the down-regulated genes are involved in calcium binding in MF.Further analysis on KEGG pathway enrichment showed that continuously upregulated genes were mainly enriched in pathways such as the IL17 signaling pathway and the NF-Kappa B signaling pathway,while continuously downregulated genes were mainly enriched in pathways such as sulfur metabolism,Hippo signaling pathway,and WNT signaling pathway.5.Based on the differential expression of upregulated DEGs and downregulated DEGs in acute injury,a PPI network diagram was constructed.The upregulated genes included 64 nodes and 178 edges in the PPI network diagram,and the down-regulated gene obtained a PPI network diagram with 14 nodes and 0 edges.The nodes contained in the two PPI network diagrams were ranked by weight score.The top 10 ranked genes were considered as PPI core genes according to DEGREE-score.The sustained upregulated genes were IL1 B,IL6,CXCL8,TLR2,CCL2,SELL,PTGS2,FGR,MMP3,TIMP1.6.Further validation was conducted using the validation set GSE28914,and six sustained upregulated DEGs were detected consistent with the result of training set.These sustain upregulated DEGs were IL1 B,TLR2,SELL,FGR,MMP3,and TIMP1.Therefore,these genes were suggested to be key genes involved in the repair of acute skin injury and may be used for the follow-up mechanism research.7.Real-time PCR results showed that the m RNA levels of IL1B(P<0.05),TLR2(P<0.001),FGR(P<0.01),MMP3(P<0.001),and TIMP1(P<0.001)were significantly increased compared with the control group.However,when treated with100 μg MSCs-Ex,m RNA levels of IL1B(P<0.001),TLR2(P<0.001),FGR(P<0.001),MMP3(P<0.001),and TIMP1(P<0.01)were significantly reduced compared with the thermal injury group,suggesting that MSCs-Ex might repair the thermal injury-induced damage of HaCaT cells by downregulating the key genes involved in acute skin injury repair.Conclusion: MSCs-Ex promotes the repair of thermal damaged epidermal HaCaT cells induced by thermal injury,which may be related to the downregulation of TLR2,FGR,IL-1B,MMP3,and TIMP1.This study provides intervention targets for the repair of acute skin injury,and experimental basis for MSCs-Ex treatment of acute skin injuries.