The Effect And Mechanism Of Negative Pressure Wound Therapy For Infected Burn Wound In Mice Explored By Transcriptome Sequencing

ObjectiveTo investigate the effect of negative pressure wound therapy?NPWT?on the wound healing of burned infectious wounds in mice,and to explore the underlying mechanisms of the pro-healing effect based on transcriptome resequencing.Methods1.Third-degree burned injury with fluorescent labelled pseudomonas aeruginosa infection model of mice was established and randomly divided into NPWT treatment group and control group.Eschar shaving was performed after 24 hours.Then the wound in NPWT group were covered by medical polyurethane foam dressing and sealed with biosemipermeable membrane and then received NPWT.while the control group received same procedure but with no NPWT.Observed indicators are as follows:?1?Observe and record the state of wound infection and healing in mice;?2?Measure the blood flow status of wounds on days 1,3,5,and 7 using Laser Doppler Perfusion Imager;?3?Samples of wound tissues in day 1,3,5,and 7 were collected and histopathologic changes were observed by HE staning;Expressions of CD31,Ki67,VEGF,and TGF-?1 were detected by immunohistochemistry;?4?The levels of IL-1?,TNF-?,VEGF,and TGF-?1 were evaluated by ElISA.2.Third-degree burned injury with fluorescent labelled pseudomonas aeruginosa infection model of mice was established and NPWT was performed.24 hours after negative treatment,tissues of wound area were collected and total RNA was extracted.After quality analysis transcriptome sequencing and bioinformatics analysis were performed.The basic steps are as follows:?1?Using the Illumina HiseqXten to sequence the samples entire genome,localize the whole gene structure,and analyze the gene structure and chromosomes to ensure the reliability of the results,and then calculate the gene expression levels,and screen differentially expressed genes between the NPWT and control groups;?2?Predict the circRNA that may exist in the samples,calculate the expression quantity and screen the differentially expressed circRNA according to the structural characteristics of circRNA;?3?Differential gene screening?DifGeneFinder?was performed using the DESeq algorithm to obtain differentially expressed lncRNAs based on mRNA differential gene screening results.?4?miRNA within 18-30 nt were separated and purified from total RNA.Then the linker sequence of 3' and 5' ends of each miRNA were connected by T4 ligase followed by amplification by RT-PCR.The resulting miRNA library was sequenced and analyzed to screen differentially expressed genes.?5?Bioinformatics tools and methods were used to classify differentially expressed genes in infected wound after NPWT treatment,construct gene regulation network,analysis signal pathway,and predict target circRNAs/lncRNAs-miRNAs-mRNAs relationship to screen circRNAs,lncRNAs,miRNAs,and mRNAs that may play a key role in NPWT-promoted wound healing and preliminary validation of the screening results.Results1.The fluorescence intensity of NPWT group on the first,third,and fifth days was significantly decreased compared with that in the control group?P<0.01?;In the mouse wound,the area of the NPWT group was significantly reduced compared with the control group on the third day?P <0.05?,the difference reached the peak on the 7th day;On the 3rd,5th and 7th day of the experiment,the blood perfusion of NPWT group was significantly increased compared with control group?P<0.01?.HE staining showed that the infiltration of inflammatory cells was significantly decreased compared with control group?p<0.05?.IHC res?lts showed that NPWT group contained significantly higher expression of CD31,Ki67,TGF-?1 and VEGF?p<0.05?.ELISA results showed that the level of TNF-? in NPWT group was significantly decreased compared with control group?P<0.01?at 1,3,and 5 days after injury.The level of IL-1? in NPWT group showed no change during the whole process,while in control group,the level of IL-1? increased 3 days before the start of the experiment?P<0.01?.The level of VEGF in NPWT group was increased in day 3?P<0.01?and maintained the high level until to the end of the experiment,and the control group also showed increased level in day 3,but was still significantly lower than that of the NPWT group.The TGF-?1 level in NPWT group was significantly increased than control group?P<0.01?on the first day after experiment and reached peak on the third day of the experiment.The above results show that NPWT can promote wound healing and can cause a wide range of biological effects.2.The transcriptomes of each sample were successfully constructed using RNA-seq technology.The specific analysis results are as follows:?1?864 mRNAs with significant difference were obtained,of which 610 mRNAs were up-regulated and 254 mRNAs were down-regulated;37 circRNAs with significant differences were predicted,of which 12 circRNAs were up-regulated and 25 circRNAs were down-regulated;107 lncRNAs significant expression differences were obtained,of which 68 lncRNAs were up-regulated,39 lncRNAs were down-regulated;67 miRNAs with significant differences were obtained,of which 37 were up-regulated and 30 were down-regulated.?2?Through bioinformatics analysis,5 exon-type circRNAs were screened with significant differences related to miRNAs and 4 miRNAs were screened with significant differences related to ceRNAs,including mmu-miR-877-3p,mmu-miR-669m-5p,mmu-miR-1224-3p,and mmu-miR-497a-5p,which regulate 52 mRNAs with significantly difference.Analysis showed that these gene functions enriched on immune response,inflammatory response,and virus defense.107 lncRNAs with a length of more than 200 bp were screened and the GO analysis showed that the lncRNAs were consistent with the circRNAs.Three of the miRNAs with ceRNAs relationship with these lncRNAs also had a ceRNA relationship with cricRNAs,ie,mmu-miR-669m-5p,mmu-miR-1224-3p and mmu-miR-497a-5p.Therefore,there are 44 mRNAs that have functional intersection and are regulated by circRNAs and lncRNAs.In summary,after bioinformatics analysis,our research team selected three circRNAs,five lncRNAs,three miRNAs,and 44 mRNAs.Which might play a key role in promoting wound healing in NPWT.Its function is focused on immune response,inflammatory response,and virus defense.The qPCR and western blotting experiments were performed to verify the above-mentioned cricRNAs,lncRNAs,miRNAs,and mRNAs.The qPCR verification results were consistent with the sequencing results.Western blotting confirmed that the TIFA,GLA,PLCB2,CD84,and SLFN9 molecules were significantly up-regulated in the experimental group,which was consistent with the sequencing results.Conclusion1.Burn wounds can cause bacterial proliferation in local tissues of mice wounds,increased inflammatory response,reduced blood perfusion and impede wound healing.NPWT treatment can reduce local wound infection,increase blood perfusion,inhibit the expression of anti-inflammatory factors,and promote growth factors express and speed up wound healing process.2.Using transcriptome sequencing technology,it was found that NPWT may regulate miR-669m-5p,miR-1224-3p,and miR-497a-5p through mmu_circ₀004796,mmu_circ₀011322,mmu_circRNA_Hnrnpll,LOC102634528,LOC102639837,LOC102639918,4930431P03 Rik,Gm10782 based on the construction of mouse transcriptome expression profiles,through biological information analysis,which in turn affect the expression of TIFA,GLA,CD300 a,PLCB2,EIF2AK2,CD84,SLFN9,Lasp1 and GDF11 to promote wound healing.