Proteomic Analysis of Peri-Wounding Tissue Expressions in Extracorporeal Shock Wave Enhanced Diabetic Wound Healing in a Streptozotocin-Induced Diabetes Model
Abstract
:1. Introduction
2. Results
2.1. Analysis of Two-Dimensional Electrophoresis Profiles
2.2. Protein Analysis and Identification
2.3. Detection of Hemopexin and Serpin A3N Expression Using Immunohistochemical Staining
2.4. ESWT-Enhanced Wound Healing Is Associated with Early Activation of EGFR Pathway
3. Discussion
4. Materials and Methods
4.1. Animal Investigations
4.2. Streptozotocin (STZ)-Induced Diabetes Mellitus in a Rodent Wounding Model
4.3. Experimental Design and Tissue Samples Collection
4.4. Isoelectric Focusing, Gel Electrophoresis, Silver Staining, and Gel Imaging
4.5. Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry
4.6. Immunohistochemical Staining
4.7. Real-Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Analysis
4.8. Statistical Analysis
Author Contributions
Funding
Conflicts of Interest
Abbreviations
DM | Diabetes Mellitus; in this study mean “control diabetes” |
EGFR | Epidermal growth factor receptor |
ESWT | Extracorporeal shock wave therapy |
HRP-DAB | Horseradish peroxidase-diaminobenzidine |
IHC | Immunohistochemical |
MALDI-TOF | Matrix-Assisted Laser Desorption Ionization Time-of-Flight |
NC | Normal control |
Serpin | Serine protease inhibitor |
STZ | Streptozotocin |
References
- Cavanagh, P.R.; Lipsky, B.A.; Bradbury, A.W.; Botek, G. Treatment for diabetic foot ulcers. Lancet 2005, 366, 1725–1735. [Google Scholar] [CrossRef]
- Orneholm, H.; Apelqvist, J.; Larsson, J.; Eneroth, M. Heel ulcers do heal in patients with diabetes. Int. Wound J. 2017, 14, 629–635. [Google Scholar] [CrossRef] [PubMed]
- Dalton, S.J.; Whiting, C.V.; Bailey, J.R.; Mitchell, D.C.; Tarlton, J.F. Mechanisms of chronic skin ulceration linking lactate, transforming growth factor-beta, vascular endothelial growth factor, collagen remodeling, collagen stability, and defective angiogenesis. J. Investig. Dermatol. 2007, 127, 958–968. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Antonic, V.; Mittermayr, R.; Schaden, W.; Stojadinovic, A. Evidence supporting extracorporeal shock wave therapy for acute and chronic soft tissue wounds. Wounds 2011, 23, 204–215. [Google Scholar] [PubMed]
- Tinazzi, E.; Amelio, E.; Marangoni, E.; Guerra, C.; Puccetti, A.; Codella, O.M.; Simeoni, S.; Cavalieri, E.; Montagnana, M.; Adani, R.; et al. Effects of shock wave therapy in the skin of patients with progressive systemic sclerosis: A pilot study. Rheumatol. Int. 2011, 31, 651–656. [Google Scholar] [CrossRef] [Green Version]
- Kuo, Y.R.; Wu, W.S.; Hsieh, Y.L.; Wang, F.S.; Wang, C.T.; Chiang, Y.C.; Wang, C.J. Extracorporeal shock wave enhanced extended skin flap tissue survival via increase of topical blood perfusion and associated with suppression of tissue pro-inflammation. J. Surg. Res. 2007, 143, 385–392. [Google Scholar] [CrossRef]
- Wang, C.J.; Wang, F.S.; Yang, K.D.; Weng, L.H.; Hsu, C.C.; Huang, C.S.; Yang, L.C. Shock wave therapy induces neovascularization at the tendon-bone junction. A study in rabbits. J. Orthop. Res. 2003, 21, 984–989. [Google Scholar] [CrossRef]
- Kuo, Y.R.; Wang, C.T.; Wang, F.S.; Yang, K.D.; Chiang, Y.C.; Wang, C.J. Extracorporeal shock wave treatment modulates skin fibroblast recruitment and leukocyte infiltration for enhancing extended skin-flap survival. Wound Repair Regen. 2009, 17, 80–87. [Google Scholar] [CrossRef]
- Kuo, Y.R.; Wang, C.T.; Wang, F.S.; Chiang, Y.C.; Wang, C.J. Extracorporeal shock-wave therapy enhanced wound healing via increasing topical blood perfusion and tissue regeneration in a rat model of stz-induced diabetes. Wound Repair Regen. 2009, 17, 522–530. [Google Scholar] [CrossRef]
- Chen, R.F.; Chang, C.H.; Wang, C.T.; Yang, M.Y.; Wang, C.J.; Kuo, Y.R. Modulation of vascular endothelial growth factor and mitogen-activated protein kinase-related pathway involved in extracorporeal shockwave therapy accelerate diabetic wound healing. Wound Repair Regen. 2019, 27, 69–79. [Google Scholar] [CrossRef] [Green Version]
- Yang, M.Y.; Chiang, Y.C.; Huang, Y.T.; Chen, C.C.; Wang, F.S.; Wang, C.J.; Kuo, Y.R. Serum proteomic analysis of extracorporeal shock wave therapy-enhanced diabetic wound healing in a streptozotocin-induced diabetes model. Plast. Reconstr. Surg. 2014, 133, 59–68. [Google Scholar] [CrossRef] [PubMed]
- Lahey, K.A.; Ronaghan, N.J.; Shang, J.; Dion, S.P.; Desilets, A.; Leduc, R.; MacNaughton, W.K. Signaling pathways induced by serine proteases to increase intestinal epithelial barrier function. PLoS ONE 2017, 12, e0180259. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Shen, K.; Sheng, Y.; Ji, L.; Wang, Z. Involvement of c-jun n-terminal kinase and extracellular signal-regulated kinase 1/2 in egf-induced angiogenesis. Cell Biol. Int. 2010, 34, 1213–1218. [Google Scholar] [CrossRef] [PubMed]
- Wang, H.; Gambosova, K.; Cooper, Z.A.; Holloway, M.P.; Kassai, A.; Izquierdo, D.; Cleveland, K.; Boney, C.M.; Altura, R.A. Egf regulates survivin stability through the raf-1/erk pathway in insulin-secreting pancreatic beta-cells. BMC Mol. Biol. 2010, 11, 66. [Google Scholar] [CrossRef] [Green Version]
- Mittermayr, R.; Antonic, V.; Hartinger, J.; Kaufmann, H.; Redl, H.; Teot, L.; Stojadinovic, A.; Schaden, W. Extracorporeal shock wave therapy (eswt) for wound healing: Technology, mechanisms, and clinical efficacy. Wound Repair Regen. 2012, 20, 456–465. [Google Scholar] [CrossRef]
- Kanno, T.; Yasutake, K.; Tanaka, K.; Hadano, S.; Ikeda, J.E. A novel function of n-linked glycoproteins, alpha-2-hs-glycoprotein and hemopexin: Implications for small molecule compound-mediated neuroprotection. PLoS ONE 2017, 12, e0186227. [Google Scholar] [CrossRef] [Green Version]
- Delanghe, J.R.; Langlois, M.R. Hemopexin: A review of biological aspects and the role in laboratory medicine. Clin. Chim. Acta 2001, 312, 13–23. [Google Scholar] [CrossRef]
- Wagsater, D.; Johansson, D.; Fontaine, V.; Vorkapic, E.; Backlund, A.; Razuvaev, A.; Mayranpaa, M.I.; Hjerpe, C.; Caidahl, K.; Hamsten, A.; et al. Serine protease inhibitor a3 in atherosclerosis and aneurysm disease. Int. J. Mol. Med. 2012, 30, 288–294. [Google Scholar] [CrossRef] [Green Version]
- Dimberg, J.; Strom, K.; Lofgren, S.; Zar, N.; Hugander, A.; Matussek, A. Expression of the serine protease inhibitor serpina3 in human colorectal adenocarcinomas. Oncol. Lett. 2011, 2, 413–418. [Google Scholar] [CrossRef]
- Hsu, I.; Parkinson, L.G.; Shen, Y.; Toro, A.; Brown, T.; Zhao, H.; Bleackley, R.C.; Granville, D.J. Serpina3n accelerates tissue repair in a diabetic mouse model of delayed wound healing. Cell Death Dis. 2014, 5, e1458. [Google Scholar] [CrossRef]
- Chen, M.; Chen, L.M.; Lin, C.Y.; Chai, K.X. The epidermal growth factor receptor (egfr) is proteolytically modified by the matriptase-prostasin serine protease cascade in cultured epithelial cells. Biochim. Biophys. Acta 2008, 1783, 896–903. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Torriglia, A.; Martin, E.; Jaadane, I. The hidden side of serpinb1/leukocyte elastase inhibitor. Semin. Cell Dev. Biol. 2017, 62, 178–186. [Google Scholar] [CrossRef] [PubMed]
- Bao, J.; Pan, G.; Poncz, M.; Wei, J.; Ran, M.; Zhou, Z. Serpin functions in host-pathogen interactions. PeerJ 2018, 6, e4557. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Hitchcock-DeGregori, S.E.; Barua, B. Tropomyosin structure, function, and interactions: A dynamic regulator. Subcell Biochem. 2017, 82, 253–284. [Google Scholar] [PubMed]
- Kuo, Y.R.; Wang, C.T.; Cheng, J.T.; Wang, F.S.; Chiang, Y.C.; Wang, C.J. Bone marrow-derived mesenchymal stem cells enhanced diabetic wound healing through recruitment of tissue regeneration in a rat model of streptozotocin-induced diabetes. Plast. Reconstr. Surg. 2011, 128, 872–880. [Google Scholar] [CrossRef] [PubMed]
Relative Expression | ||||||||
---|---|---|---|---|---|---|---|---|
Spot | Identified Protein | Score | Molecular Weight (kilodalton) | Theoretical Isoelectric Point | Sequence Coverage (%) | Matched Peptides | in DM (Compared with NC) | in ESWT (Compared with DM) |
1 | Hemopexin | 138 | 52.1 | 8.6 | 18.7 | 6 | ↓ | ↑ |
2 | Alpha-2-HS-glycoprotein | 112 | 38.8 | 6.1 | 15.6 | 4 | ↓ | ↑ |
3 | Serine protease inhibitor A3N | 270 | 46.8 | 5.2 | 22.2 | 8 | ↑ | ↓ |
4 | Serine protease inhibitor A3N | 335 | 46.8 | 5.2 | 29.9 | 9 | ↑ | ↓ |
5 | Serine protease inhibitor A3N | 442 | 46.8 | 5.2 | 28.2 | 13 | ↑ | ↓ |
6 | Serine protease inhibitor A3N | 419 | 46.8 | 5.2 | 26.8 | 9 | ↑ | ↓ |
7 | Serine protease inhibitor A3N | 304 | 46.8 | 5.2 | 27.0 | 10 | ↑ | ↓ |
8 | Leukocyte elastase inhibitor A | 245 | 42.9 | 5.9 | 24.0 | 11 | ↑ | ↓ |
9 | Catechol O-methyltransferase | 222 | 29.8 | 5.3 | 37.9 | 18 | ↑ | ↓ |
Relative Expression | ||||||||
---|---|---|---|---|---|---|---|---|
Spot | Identified Protein | Score | Molecular Weight (kilodalton) | Theoretical Isoelectric Point | Sequence Coverage (%) | Matched Peptides | in DM (Compared with NC) | in ESWT (Compared with DM) |
1 | Serine protease inhibitor A3N | 74 | 46.8 | 5.33 | 29 | 16 | ↓ | ↑ |
2 | Tektin-4 | 69 | 52.7 | 58.3 | 34 | 17 | ↓ | ↑ |
3 | Tropomyosin alpha-1 chain | 71 | 32.7 | 4.69 | 33 | 10 | ↓ | ↑ |
4 | Plectin-1 | 74 | 53.5 | 5.71 | 20 | 85 | ↓ | ↑ |
5 | Lamin-A | 53 | 74.6 | 6.54 | 25 | 13 | ↓ | ↑ |
6 | Tropomyosin alpha-4 chain | 147 | 28.5 | 4.66 | 50 | 15 | ↓ | ↑ |
7 | Ferritin heavy chain | 59 | 21.3 | 5.85 | 53 | 7 | ↓ | ↑ |
8 | Myosin regulatory light chain 2, skeletal muscle isoform | 102 | 19.1 | 4.82 | 58 | 11 | ↓ | ↑ |
9 | Hemopexin | 172 | 52.1 | 7.58 | 50 | 26 | ↑ | ↓ |
Gene | GenBank Accession No. | Amplicon Size (bp) | Assay Location | Assay ID (Applied Biosystems) |
---|---|---|---|---|
Egfr | NM_031507.1 | 104 | 241 | Rn00580398_m1 |
Kras | NM_031515.3 | 88 | 448 | Rn01463171_m1 |
Raf1 | NM_012639.2 | 88 | 973 | Rn00466507_m1 |
Braf | XM_231692.5 | 97 | 1843 | Rn01500557_m1 |
Mek1 (Map2k1) | NM_031643.4 | 70 | 130 | Rn00581264_m1 |
Erk (Ephb1) | NM_001104528.1 | 100 | 2611 | Rn00557962_m1 |
Elk3 (Kcnh8) | NM_145095.1 | 87 | 1232 | Rn00595436_m1 |
Mekk1 (Map3k1) | NM_053887.1 | 90 | 4577 | Rn01490142_m1 |
Jnkk (Map2k7) | NM_001025425.1 | 104 | 122 | Rn01403106_m1 |
Jnk (Mapk8) | XM_341399.5 | 85 | 247 | Rn01218952_m1 |
Jun | NM_021835.3 | 106 | 1644 | Rn99999045_s1 |
Actb | NM_031144.2 | 884 | 91 | Rn00667869-m1 |
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Chen, R.-F.; Yang, M.-Y.; Wang, C.-J.; Wang, C.-T.; Kuo, Y.-R. Proteomic Analysis of Peri-Wounding Tissue Expressions in Extracorporeal Shock Wave Enhanced Diabetic Wound Healing in a Streptozotocin-Induced Diabetes Model. Int. J. Mol. Sci. 2020, 21, 5445. https://doi.org/10.3390/ijms21155445
Chen R-F, Yang M-Y, Wang C-J, Wang C-T, Kuo Y-R. Proteomic Analysis of Peri-Wounding Tissue Expressions in Extracorporeal Shock Wave Enhanced Diabetic Wound Healing in a Streptozotocin-Induced Diabetes Model. International Journal of Molecular Sciences. 2020; 21(15):5445. https://doi.org/10.3390/ijms21155445
Chicago/Turabian StyleChen, Rong-Fu, Ming-Yu Yang, Ching-Jen Wang, Chun-Ting Wang, and Yur-Ren Kuo. 2020. "Proteomic Analysis of Peri-Wounding Tissue Expressions in Extracorporeal Shock Wave Enhanced Diabetic Wound Healing in a Streptozotocin-Induced Diabetes Model" International Journal of Molecular Sciences 21, no. 15: 5445. https://doi.org/10.3390/ijms21155445