Quantitative Proteomic Analysis Based On Mass Spectrometry Reveals The Effects Of Negative-Pressure Wound Therapy On The Healing Of Diabetic Foot Ulcers

Objectives1.Explore the changes in proteomics of the wounds of patients with diabetic foot ulcers(DFUs)after negative-pressure wound therapy(NPWT)and further understand the impact of negative-pressure wound therapy on the healing of diabetic foot ulcers.2.To explore the relationship between cathepsin S(CTSS)expression and diabetic foot ulcer(DFU)wound healing and its value as a biomarker in diagnosing the prognosis of patients with diabetic foot ulcers.Methods1.Three patients with diabetic foot ulcers were hospitalized with Wagner grade 3 foot ulcers.The patient received negative pressure wound treatment for 1 week.Collect the granulation tissue samples of the three patients before and after NPWT.The pre-NPWT group was used before treatment,and the post-NPWT group was used after treatment.Then,label-free quantitative mass spectrometry(label-free quantitative mass spectrometry)was used to analyze the protein expression profile and compare it with the previous NPWT to identify differentially expressed proteins(DEPs).Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)were analyzed to annotate the related signaling pathways of DEPs and DEPs.The differentially expressed proteins cathepsin S(CTSS),peroxidase 2(PRDX2),α-trypsin inhibitor heavy chain 4(ITIH4),and protein S1(PROS1)were selected to pass the enzyme-linked immunosorbent assay(enzyme-linked immunosorbent).assay,ELISA)to detect the levels of differentially expressed proteins in the serum of the NPWT pre-NPWT and post-NPWT groups.Western blotting(WB)was used to detect 7 the expression of the abovementioned differentially expressed proteins in the wound granulation tissues of the NPWT pre-and post-NPWT groups.2.From September 2018 to December 2020,a total of 39 DFU patients who were hospitalized and received NPWT in the Endocrinology Department of the First Affiliated Hospital of Anhui Medical University were recruited,and 54 patients with newly diagnosed type 2 diabetes(T2DM)were recruited.Eighty healthy people who were examined at the Physical Examination Center of the First Affiliated Hospital of Anhui Medical University,according to whether the subjects had a history of type 2diabetes and whether they were undergoing NPWT due to DFU,were set as the prenegative pressure treatment group and the postnegative pressure treatment group,T2 DM group and control group.We collected clinical data,including the subjects’ measurement characteristics,blood pressure and morning fasting blood samples,and detected the expression levels of CTSS in the serum of the above four groups of subjects by enzyme-linked immunosorbent assay(ELISA).Immunohistochemistry was used to observe the expression of CTSS protein in the granulation tissues of DFU patients before and after NPWT treatment.Receiver operating characteristic(ROC)curve analysis was used to evaluate the diagnostic efficacy of CTSS level on DFU healing time.Results1.By comparing the differences in protein profiles of granulation tissue samples before and after NPWT for one week,36 proteins with significant differences were identified(p <0.05);33 of these proteins were upregulated,and 3 were downregulated.NPWT altered proteins mainly associated with antioxidation & detoxification,cytoskeleton,regulation of inflammatory response,complement and coagulation cascades and lipid metabolism.Functional validation of DEPs showed that the levels of cathepsin S(CTSS)in peripheral blood and granulation tissue were significantly lower than those before NPWT(P < 0.05),while the levels of protein S isoform 1(PROS1),inter-α-trypsin inhibitor heavy chain H4(ITIH4)and peroxiredoxin-2(PRDX2)in peripheral blood and granulation tissue were significantly higher than those before NPWT(P < 0.05).Both WB and ELISA methods confirmed that the expression of differentially expressed proteins in granulation tissue and blood after NPWT was consistent with the results of protein profiling experiments.2.We conducted a further study on the expression of CTSS in the serum of the four groups of subjects by ELISA.The results showed that compared with the control group,the expression of CTSS in the serum of the T2 DM group and the group before negative pressure treatment increased significantly(P <0.001).Compared with the T2 DM group,the serum CTSS expression level of the prenegative pressure treatment group was significantly higher(P <0.001).Compared with the prenegative pressure treatment group,the serum CTSS expression level of the negative pressure treatment group was reduced(P <0.05).3.The results of immunohistochemistry showed that the expression level of CTSS in the wound granulation tissue decreased after negative pressure suction treatment.4.Spearman correlation analysis showed that the level of CTSS was positively correlated with fasting plasma glucose(FPG),total cholesterol(TCH)and albumin(ALB)(FPG: r = 0.3446,P <0.0001;TCH: r = 0.2874,P = 0.0002;ALB:r=0.2725,P =0.0004)and negatively correlated with high-density lipoprotein(HDL-C)(HDL-C: r =-0.4759,P <0.0001).The level of CTSS is related to age,sex,body mass index(BMI),low-density lipoprotein(LDL-C),systolic blood pressure(SBP)and diastolic blood pressure(DBP).No significant correlation.ConclusionsNPWT improves microcirculation by reducing thrombosis.Furthermore,the extracellular matrix may increase after NPWT.The decrease in CTSS protein levels in granulation tissue and blood may be a potential biomarker for effective healing of diabetic foot wounds after NPWT.We identified a variety of new proteins with NPWT changes,which laid a foundation for further study of the mechanism of NPWT action.