Proteomic Analysis of Peri-Wounding Tissue Expressions in Extracorporeal Shock Wave Enhanced Diabetic Wound Healing in a Streptozotocin-Induced Diabetes Model

Our former studies have demonstrated that extracorporeal shock wave therapy (ESWT) could enhance diabetic wound healing but the bio-mechanisms remain elusive. This study investigated the changes of topical peri-wounding tissue expressions after ESWT in a rodent streptozotocin-induced diabetic wounding model by using the proteomic analysis and elucidated the molecular mechanism. Diabetic rats receiving ESWT, normal control, and diabetic rats receiving no therapy were analyzed. The spots of interest in proteome analysis were subjected to mass spectrometry to elucidate the peptide mass fingerprints. Protein expression was validated using immunohistochemical staining and related expression of genes were analyzed using real-time RT-PCR. The proteomic data showed a significantly higher abundance of hemopexin at day 3 of therapy but down-regulation at day 10 as compared to diabetic control. In contrast, the level of serine proteinase inhibitor (serpin) A3N expression was significantly decreased at day 3 therapy but expression was upregulated at day 10. Using real-time RT-PCR revealed that serpin-related EGFR-MAPK pathway was involved in ESWT enhanced diabetic wound healing. In summary, proteome analyses demonstrated the expression change of hemopexin and serpin with related MAPK signaling involved in ESWT-enhanced diabetic wound healing. Modulation of hemopexin and serpin related pathways are good strategies to promote wound healing.


Introduction
The non-healing wound ulcer is a major morbidity in diabetic patients which is usually accompanied by infection, inflammation, or even amputations. Various treatment modalities for

Analysis of Two-Dimensional Electrophoresis Profiles
Two-dimensional electrophoresis was assessed using peri-wounding skin tissue samples received from animals in the normal control group, diabetic group, and the group that received ESWT at days 3 and 10 after treatment. Results showed that approximately 218 spots (218.2 ± 36.5 spots in the normal control group, 215.6 ± 13.1 spots in the diabetic control group, and 219.8 ± 43.4 spots in the ESWT group) (p > 0.8) could be found in the gels (Figures 1 and 2). Protein spots in each rat of the ESWT group were further imaged and matched with those in the normal group and diabetic control group, and the relative thickness of individual spot in each matched gel was computed. On day 3 after ESWT, the densities of nine protein spots were significantly distinct, as shown in Figure 1. Two of the nine spot intensities were increased and seven were decreased in ESWT rats, as compared to that in diabetic controls ( Figure 1). In contrast, the intensities of nine protein spots were the significant difference on day 10 post-ESWT ( Figure 2). All of the intensities of nine protein spots were increased in ESWT except one which was decreased, as reflated with the diabetic controls ( Figure 2).
ESWT treatment, when contrasted with the diabetic controls (Figure 1, below). At day 10 after ESWT treatment, the outcome uncovered a noteworthy upregulation of serpin A3N (p < 0.05), tektin-4 (p < 0.01), tropomyosin alpha-1 chain (p < 0.05), plectin-1 (p < 0.05), lamin-A (p < 0.05), tropomyosin alpha-4 chain (p < 0.01), ferritin heavy chain (p < 0.05), and myosin regulatory light chain 2, skeletal muscle isoform (p < 0.05) and downregulation of hemopexin (p < 0.05) when contrasted with that in diabetic controls (Figure 2, below). Protein spots of taking into consideration in 2D gel electrophoretograms of peri-wounding skin tissue proteins acquired from and a diabetic rodent on day 3 after two sessions of ESWT (ESW2), and that long period of normal control (NC) and diabetic control (DM). (Above) Representative 2D gel electrophoretograms of peri-wounding skin tissue proteins. The protein samples (150 µg) were exposed to isoelectric focusing (pH 4 to 7), SDS-PAGE, and silver staining. The numbers on the left demonstrate the molecular weight (M W ), in kilodaltons. (Center) Enlarged fields of the 9 spots of interest in the sliver-stained SDS-PAGE gels. The spots in the gels of NC, DM, and ESW2 were coordinated utilizing Bio-Rad Proteoweaver 2-D Analysis Software Version 4.0. The arrows prevail upon the spots of interest. (Below) Relative densities of the emphatically distinguished proteins. Relative intensity was determined by separating the thickness of coordinated spots by the thickness of all the coordinated spots in the individual gel. Abbreviation: ESWT, extracorporeal shock wave therapy.
interest in the sliver-stained SDS-PAGE gels. The spots in the gels of NC, DM, and ESW2 were coordinated utilizing Bio-Rad Proteoweaver 2-D Analysis Software Version 4.0. The arrows prevail upon the spots of interest. (Below) Relative densities of the emphatically distinguished proteins. Relative intensity was determined by separating the thickness of coordinated spots by the thickness of all the coordinated spots in the individual gel. Abbreviation: ESWT, extracorporeal shock wave therapy.

Figure 2.
Protein spots of taking into consideration in 2D gel electrophoretograms of peri-wounding skin tissue proteins acquired from day 10 after two sessions of ESWT (ESW2), normal controls (NC), a diabetes controls (DM) at the same days. (Above) Representative 2D gel electrophoretograms of peri-wounding skin tissue proteins. The skin tissue protein samples (150 µ g) were exposed to isoelectric focusing (pH 4 to 7), SDS-PAGE, and silver staining. The numbers on the left demonstrate the molecular weight (MW), in kilodaltons. (Center) Enlarged fields of the 9 spots of interest in the sliver-stained SDS-PAGE gels. The spots in the gels of NC, DM, and ESW2 were coordinated utilizing Bio-Rad Proteoweaver 2-D Analysis Software Version 4.0. The arrows prevail upon the spots of interest. (Below) Relative densities of the emphatically distinguished proteins. Relative intensity was Figure 2. Protein spots of taking into consideration in 2D gel electrophoretograms of peri-wounding skin tissue proteins acquired from day 10 after two sessions of ESWT (ESW2), normal controls (NC), a diabetes controls (DM) at the same days. (Above) Representative 2D gel electrophoretograms of peri-wounding skin tissue proteins. The skin tissue protein samples (150 µg) were exposed to isoelectric focusing (pH 4 to 7), SDS-PAGE, and silver staining. The numbers on the left demonstrate the molecular weight (MW), in kilodaltons. (Center) Enlarged fields of the 9 spots of interest in the sliver-stained SDS-PAGE gels. The spots in the gels of NC, DM, and ESW2 were coordinated utilizing Bio-Rad Proteoweaver 2-D Analysis Software Version 4.0. The arrows prevail upon the spots of interest. (Below) Relative densities of the emphatically distinguished proteins. Relative intensity was determined by separating the thickness of coordinated spots by the thickness of all the coordinated spots in the individual gel. Abbreviation: ESWT, extracorporeal shock wave therapy.

Detection of Hemopexin and Serpin A3N Expression Using Immunohistochemical Staining
Since the proteome analysis revealed the changes of specific proteins after ESWT, we further confirmed protein expression by IHC staining. As shown in Figure 3, the IHC staining assessment displayed that hemopexin expression was statistically increased and serpin A3N expression was statistically decreased in the ESWT group at day 3 after treatment when contrasted with that in diabetic controls (p < 0.001). These outcomes were steady with our finding observed in 2D gel electrophoresis of peri-wounding tissue samples.

Detection of Hemopexin and Serpin A3N Expression Using Immunohistochemical Staining
Since the proteome analysis revealed the changes of specific proteins after ESWT, we further confirmed protein expression by IHC staining. As shown in Figure 3, the IHC staining assessment displayed that hemopexin expression was statistically increased and serpin A3N expression was statistically decreased in the ESWT group at day 3 after treatment when contrasted with that in diabetic controls (p < 0.001). These outcomes were steady with our finding observed in 2D gel electrophoresis of peri-wounding tissue samples.

ESWT-Enhanced Wound Healing Is Associated with Early Activation of EGFR Pathway
The study showed serpin as an epithelial barrier involved in epidermal growth factor receptor (EGFR) activation [12]. Cell proliferation and angiogenesis are important roles via regulating the EGFR-MAPK pathway [13,14]. We recognized EGFR-MAPK signal cascade related genes' mRNA levels of a peri-wounding tissue test by quantitative RT-PCR rather than traditional Western blotting at the beginning time (three days) and late-stage (10 days) after ESWT.
The results showed higher gene levels of Egfr, Kras, Mek1, Elk3, Jun, Jnk, and Jnkk at the beginning time (three days) in the ESWT group, when contrasted with that in diabetic rats ( Figure  4a). However, the upregulation of Kras, Mek1, Elk3, Jnkk, Jnk, and Jun was strongly suppressed at the late stage (10 days) in ESWT rats, particularly the expression of Elk3 was reversed as compared to that in diabetic rats (Figure 4b). These results indicated EGF initiation of the Raf-MEK-ERK signaling is engaged with ESWT enhanced diabetic wound healing.

ESWT-Enhanced Wound Healing Is Associated with Early Activation of EGFR Pathway
The study showed serpin as an epithelial barrier involved in epidermal growth factor receptor (EGFR) activation [12]. Cell proliferation and angiogenesis are important roles via regulating the EGFR-MAPK pathway [13,14]. We recognized EGFR-MAPK signal cascade related genes' mRNA levels of a peri-wounding tissue test by quantitative RT-PCR rather than traditional Western blotting at the beginning time (three days) and late-stage (10 days) after ESWT.
The results showed higher gene levels of Egfr, Kras, Mek1, Elk3, Jun, Jnk, and Jnkk at the beginning time (three days) in the ESWT group, when contrasted with that in diabetic rats (Figure 4a). However, the upregulation of Kras, Mek1, Elk3, Jnkk, Jnk, and Jun was strongly suppressed at the late stage (10 days) in ESWT rats, particularly the expression of Elk3 was reversed as compared to that in diabetic rats (Figure 4b). These results indicated EGF initiation of the Raf-MEK-ERK signaling is engaged with ESWT enhanced diabetic wound healing.

Discussion
Studies have indicated that ESWT speaks to a doable strategy for enhancing wound healing related to expanded epithelization and neo-angiogenesis, tissue recovery, and topical mitigating reaction [8,15]. In our former study utilized proteomic technology to distinguish the progressions in foundational serum protein profiling in diabetic wound healing after ESWT. Our results revealed that numerous proteins were involved in ESWT-treatment enhanced diabetic wound healing such as increased haptoglobin protein levels and downregulation of serpin A3N and vitamin D-binding protein on day 3 after treatment [11]. In this study, we extended our study and examine the protein expressions of topical per-wounding tissue in these diabetic rats using proteomic study and MALDI-TOF mass spectrometry.
Recent studies have indicated that hemopexin and alpha-2-HS-glycoprotein had cytoprotective action against oxidative damage in cells [16]. The expression of skin tissue hemopexin was up-and

Discussion
Studies have indicated that ESWT speaks to a doable strategy for enhancing wound healing related to expanded epithelization and neo-angiogenesis, tissue recovery, and topical mitigating reaction [8,15]. In our former study utilized proteomic technology to distinguish the progressions in foundational serum protein profiling in diabetic wound healing after ESWT. Our results revealed that numerous proteins were involved in ESWT-treatment enhanced diabetic wound healing such as increased haptoglobin protein levels and downregulation of serpin A3N and vitamin D-binding protein on day 3 after treatment [11]. In this study, we extended our study and examine the protein expressions of topical per-wounding tissue in these diabetic rats using proteomic study and MALDI-TOF mass spectrometry.
Recent studies have indicated that hemopexin and alpha-2-HS-glycoprotein had cytoprotective action against oxidative damage in cells [16]. The expression of skin tissue hemopexin was up-and downregulated, respectively, at three and 10 days after ESWT. In contrast, the expression of skin tissue serpin A3N was down-and upregulated, respectively, at three and 10 days after ESWT treatment. IHC analysis also revealed consistent results of hemopexin and serpin A3N expressions after ESWT. These indicated that hemopexin and serpin A3N both are involved in ESWT-accelerated diabetic wound healing.
Hemopexin is a heme-restricting plasma glycoprotein which, after haptoglobin, shapes the second line of barrier against hemoglobin-mediated oxidative injury [17]. Haptoglobin is the essential Hb-restricting protein in human plasma. Hemopexin is another plasma glycoprotein ready to tie heme with high partiality. Hemopexin and haptoglobin prevent heme's pro-inflammatory and pro-oxidant effects and enhance its detoxification. Results of tissue proteomic analysis revealed significantly higher levels of hemopexin three days after ESWT and verified by IHC staining. This result is consistent with our former study that the serum of proteomic analysis in the ESWT-treated group showed upregulated haptoglobin protein levels [11]. These indicate ESWT modulation of hemopexin expression is involved in an earlier anti-inflammatory response and the suppression of oxidative stress to accelerate diabetic wound healing.
In addition to hemopexin, differential expressions of several proteins were identified in peri-wounding tissues that likewise assume a significant job in wound healing. Serpin A3N is an inhibitor of a few proteases, for example, cathepsin G, elastase, and chymase, made from neutrophils and mast cells, etc. [18]. Serpin A3 seems to have a multifaceted job and is related to inflammatory responses [19]. A study showed serpin A3N advances granulation tissue development and collagen deposition using a diabetic murine model [20]. In this study, our results revealed the down-regulatory effect of serpin A3N found in the peri-wounding tissue of the ESWT-treated group on day 3 but up-regulated at day 10 after ESWT. This might be serpin A3N related inflammatory response was suppressed in the early phase (day 3) after ESWT, but upregulation due to involved in enhancing the angiogenesis and tissue regeneration at later phase (day 10) after ESWT, as compared to the diabetic controls. These indicated modulation of serpin A3N expression could be an important factor in ESWT promotion of the wound healing process.
Studies showed the serpin involved in EGFR activation and epithelial barrier [12,21]. EGFR is an important role in angiogenesis and cell proliferation via regulating the Raf-MEK-ERK signaling [13,14]. In this investigation, real-time quantitative RT-PCR was performed on mRNA extracted from biopsy samples. Our results revealed that ESWT treatment could increase epithelial cell migration and cell multiplication and correlated with serpin-EGF initiates the Raf-MEK-ERK cascades to enhance diabetic wound healing ( Figure 5).
Other factors have also been detected in this tissue proteomic study. Leukocyte elastase inhibitor A also called serpin B1A is an individual from the clade B of serpins [22]. It is an intracellular protein and acts fundamentally to shield the cell from proteases released into the cytoplasm during oxidative stress. Ongoing information demonstrates that it has likewise a job in cell migration proposing that it could be involved in dissimilar processes such as malignant metastases and wound healing [22]. Our results revealed that wounded diabetic rats that received ESWT had significantly lower levels of serpin A3N and serpin B1A on day 3 after treatment. More research has uncovered that serpins work in inflammatory response and infection, by regulating serine and cysteine proteases activities [23]. It might be due to the anti-inflammatory effects of ESWT, peri-wounding tissue samples had lower levels of serpin A3N and leukocyte elastase inhibitor A. Besides inflammation and proliferation, connective tissue regeneration is also an important process in wound healing. Tropomyosin α-1 chain and tropomyosin α-4 chain showed an increased expression in ESWT peri-wounding tissue samples at day 10 after treatment. It is noteworthy that tropomyosin is the archetypal-coiled coil, yet investigations of its structure and capacity have demonstrated it to be a dynamic controller of actin filament function in non-muscle and muscle cells [24]. This is an important finding in the understanding of the tropomyosin may be involved in the angiogenesis even remodeling in the enhancing diabetic wound healing by ESWT. Together, the present findings suggest that serpin and tropomyosin needed further study in detail.
In summary, our proteomic study of topical wound edge protein expressions reveals ESWT improving chronic wound healing involves in neo-angiogenesis and anti-inflammation. The modulation of hemopexin and serpin-related pathways will be a good strategy to promote wound healing ( Figure 5). cells, etc. [18]. Serpin A3 seems to have a multifaceted job and is related to inflammatory responses [19]. A study showed serpin A3N advances granulation tissue development and collagen deposition using a diabetic murine model [20]. In this study, our results revealed the down-regulatory effect of serpin A3N found in the peri-wounding tissue of the ESWT-treated group on day 3 but up-regulated at day 10 after ESWT. This might be serpin A3N related inflammatory response was suppressed in the early phase (day 3) after ESWT, but upregulation due to involved in enhancing the angiogenesis and tissue regeneration at later phase (day 10) after ESWT, as compared to the diabetic controls. These indicated modulation of serpin A3N expression could be an important factor in ESWT promotion of the wound healing process.
Studies showed the serpin involved in EGFR activation and epithelial barrier [12,21]. EGFR is an important role in angiogenesis and cell proliferation via regulating the Raf-MEK-ERK signaling [13,14]. In this investigation, real-time quantitative RT-PCR was performed on mRNA extracted from biopsy samples. Our results revealed that ESWT treatment could increase epithelial cell migration and cell multiplication and correlated with serpin-EGF initiates the Raf-MEK-ERK cascades to enhance diabetic wound healing ( Figure 5).

Animal Investigations
The consideration and lodging states of the animals conformed to the guidelines of the Institutional Animal Care and Use Committee on the assurance of animals utilized for scientific purposes (IACUCA Animal use protocol approval number: 2007111902).

Streptozotocin (STZ)-Induced Diabetes Mellitus in a Rodent Wounding Model
Wistar rodents with diabetes were actuated by a single intraperitoneal injection of STZ (50 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) following our previous report [9,10,25]. Briefly, rodents with a glucose level more prominent than 300 mg/dl one week after injection were characterized as having effective acceptance of diabetes and were then utilized for subsequent experiments. To adjust the glucose level at 200 mg/dL, diabetic rodents were subcutaneously administered continual-acting insulin (1 to 2 unit/kg; Montards Novo Nordisk A/S, Bagsvaerd, Demark) until the animals were immolated. The wounding model was assessed a month after the STZ injection. The dorsum skin tissue of the Wistar rodents was excised to make a wounding area of 6 × 5 cm 2 . The whole skin was disfigured underneath the level of the dorsal fascia, and the edge of the wound defect was stitched set up with 4-0 silk stitches to forestall wound contracture. The wound was incidentally secured with lucent Tegaderm (3M HealthCare, Borken, Germany) till ESWT was initiated.

Experimental Design and Tissue Samples Collection
Eighteen 4-month-old male Wistar rodents (National Experimental Animals Production Center, Taipei, Taiwan) with STZ-prompted diabetes were separated into three groups (six rodents in each group): normal group, diabetic group, and ESWT group. The dorsal wounding model was made on all rodents. ESWT group was treated with two sessions of defocused shock waves (Reflector Type CP155; MTS, GmbH, Konstanz, Germany) utilizing 800 driving forces at 10 kV, proportionate to an energy flux density of 0.09 mJ/mm2 on days 3 and 7 after wounding following our former protocol [9,11]. Peri-wounding tissue tests were gathered at 3 and 10 days after ESWT treatment or those days in the normal control (NC) and control diabetes (DM) group. Immediately after excision, the skin tissue samples were snap-solidified in fluid nitrogen and put away until use.

Isoelectric Focusing, Gel Electrophoresis, Silver Staining, and Gel Imaging
Peri-wounding skin tissue protein was extricated utilizing PRO-PREPTM protein extraction solution (iNtRON Biotechnology, Gyeonggi-do, Korea) followed by a 2-D Clean-Up Kit (GE Healthcare Life Sciences, Uppsala, Sweden). Two-dimensional electrophoresis including isoelectric focusing and SDS-PAGE were executed on an Ettan IPGphor II/3 IEF system and SE 600 Ruby gel apparatus (GE Healthcare Life Sciences). Every sample was exposed to isoelectric focusing and SDS-PAGE in copy. The silver-recolored polyacrylamide gels were examined utilizing an ImageScanner (GE Healthcare Life Sciences). The gel pictures and spot patterns were coordinated and dissected utilizing Bio-Rad Proteoweaver 2-D Analysis Software ver. 4.0 (Bio-Rad Laboratories, Hercules, CA, USA).

Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry
The interesting spots were physically extracted, washed with deionized water, destained, rehydrated, reduced, and following trypsin digested. The extraction products were harvested with 1% trifluoroacetate in acetonitrile. Aliquots of the extracted digestion products were then stacked onto AnchorChip (Bruker Detection, Leipzig, Germany) trailed by MALDI-TOF evaluation utilizing an Ultraflex TOF/TOF mass spectrometer (Bruker Detection). The peptide mass information was submitted to the National Center for Biotechnology Information and Swiss-Port database utilizing Mascot (Matrix Science, Boston, MA, USA.) web search tools for peptide coordinating. The coordinated peptides that were considered as potential up-and-comers had the most noteworthy Mascot score (≥65) and a peptide sequence coverage of 20% of the coordinated peptide.

Immunohistochemical Staining
Specific proteins of proteome analyses were confirmed by IHC staining. Polyclonal antibodies against serine protease inhibitor (serpin) A3N (LSbio, Irvine, CA, USA) and Hemopexin (MyBioSource, San Diego, CA, USA) were utilized as the first antibodies. The slides were incubated with first antibodies (1:500 dilutions) for 1 h and afterward incubated with goat biotinylated anti-rabbit antibodies for another 30 min. Perception of the particular binding was developed by an enzymatic transformation of the chromogenic substrate 3,3 -diaminobenzidine into a brown precipitate by a horseradish peroxidase-diaminobenzidine staining kit (Thermo Systems, Minneapolis, MN, USA). In the wake of recoloring, the sections were mounted, cleared, cover-slipped, and inspected utilizing a Zeiss magnifying instrument (Zeiss, Gottingen, Germany).

Real-Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Analysis
All-out RNA was extracted from skin tissues utilizing TRIzol reagent (Invitrogen Life Technologies; Carlsbad, CA, USA). The 2 µg RNA contribution for cDNA synthesis was calculated by spectrophotometric optical density 260 estimation and cDNA was created with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) depending on the manufacturer's protocols. The expression of interesting genes was examined utilizing the TaqMan ® Gene Expression Assays bought from Applied Biosystems (Applied Biosystems, Foster City, CA, USA). The names of the analyzed gene, GenBank accession numbers, amplicon sizes, and assay ID of gene expression assays are list in Table 3. Expression of rat housekeeping genes, Actb (β-actin) was utilized for equalizing analyzed target genes expression in real-time quantitative RT-PCR. All reactions were completed in a 10 µL last volume containing 20 ng of cDNA, 5 µL of 2× TaqMan ® Universal PCR Master Mix, and 0.5 µL of 20× TaqMan ® Gene Expression Assay (Applied Biosystems). Real-time quantitative PCR was assessed using an ABI 7500 Fast Real-Time System (Applied Biosystems) and the thermal cycling programs were set as follows: 95 • C for 10 min followed by 40 repeats of PCR at 95 • C for 20 s and then 60 • C for another 1 min. The expression level of the gene of interest was equaled to the house-keeping control Actβ to calculate the relative threshold cycle (∆Ct) and the relative expression between two groups was determined by the comparative Ct (∆∆Ct) method [10].