The Research On Role Of Hydrogen In The Trauma Healing Of DM Mice

Diabetes (Diabetes Mellitus, DM) is a chronic metabolic disorder. Refractory diabetic skin wounds are the most common chronic complications of diabetes. The diabetic foot is the typical example of such diseases, for which many patients have to suffer amputation. The reason for why hyperglycemia causes the skin damage and develops into a refractory wound remains inconclusive. Many theories have been raised. Now many researchers agree that oxidative stress (Oxidative Stress, OS) induced by hyperglycemia is an important common mechanism.OS can produce large amounts of free radicals, among which hydroxyl radical (?OH) and peroxynitrite anion (ONOO-) have stronger oxidation, react with biological macromolecules (such as DNA, proteins, lipids) and result in destruction of their function and structure. Therefore, measures against free radicals can reduce the OS induced damage. Traditional antioxidants such as vitamin C neutralize free radicals non-selectively and may disturb the physiological function of free radicals. Developing some specific radical scavenger may be more effective in diabetic treatment. There have been some specific anti-oxidants used in clinical treatment of examples, such as edaravone, can remove ?OH selectively. Edaravone has been used to improve the neurological symptoms caused by acute cerebral infarction. Although there are beneficial effects, more side effects were observed.Hydrogen (H₂) is the simplest gas in nature. In the past, it has been considered an inert gas biologically. No roles were observed in the organism. Recently some studies have shown that H₂ inhalation or hydrogen water consumption exerted protective roles in heart, brain and liver after ischemia-reperfusion injury. In a cell-free chemical system in vitro, H₂ selectively reacted with ?OH and ONOO- which eventually reduced the OS. As a newly discovered gas, the effects of hydrogen on diabetic wounds remain unknown. Since it selectively reduced the ?OH and ONOO- levels, we hypothesized H₂ could benefit the recovery of diabetic wounds. Therefore, we designed experiments to explore the roles of H₂ in diabetic wound healing process and the corresponding mechanisms. Hopefully it will set up a new clinical approach to treat diabetic wounds in the future.In diabetic wounds, the persistent inflammation is detrimental to their healing. Infiltration of inflammatory cells caused by OS produced a large number of free radicals, and lead to inflammatory injury. Besides, OS activated the key inflammation transcription factor nuclear factor-κB (NF-κB), generated large amounts of inflammatory cytokines, caused cell damage and interfered wound healing. H₂ has been proved to reduce the inflammatory cytokines such as IL-6, IL-1βin the small intestine transplantation and alleviated small bowel injury induced by inflammation. Whether H₂ inhibits excessive local inflammatory reaction in diabetic wound is worth studying.Mitogen-activated protein kinase (MAPK) signaling pathway is involved in cell proliferation and apoptosis. Several signal molecules are involved in this pathway. including several pathways as follows: extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and P38. In diabetic wounds, OS activates MAPK, subsequently affect fibroblasts biological behaviors, and delays tissue regeneration. P38 increases vascular permeability, resulting in tissue edema, which is harmful to healing. Previous studies also have shown that ERK, JNK and P38 activation was likely to be the mechanism of diabetic peripheral nerve abnormalities. With its ability to neutralize free radicals, hydrogen may play a protective role in the process of the diabetic wound healing by inactivating MAPK pathway.Based on the above data, this study aimed to: (1) develop mice diabetic wound model, injection of hydrogen-rich saline intraperitoneally, observe the morphological and histological aspects of the role of hydrogen on wound healing, and detect oxidative indexes.(2) culture normal human skin fibroblasts, induce cellular oxidative stress with high glucose, treat the cells with hydrogen-rich medium, reveal whether hydrogen could protect high-glucose caused oxidative damage to fibroblasts, and determine the effects of hydrogen on the cell oxidation products and antioxidant system, as well as MAPK and NF-κB pathway.Methods: At first, streptozotocin(STZ) was used to establish type I diabetes mice model(DM mice)and full thickness round skin wound was made by perforex. Hydrogen-rich saline was prepared and intraperitoneal injected to the mice. Tissue from healing wound was then examined by HE, Masson staining and electron microscope to observe its ultrastructure. Immunohistochemical staining was also been proceeded to measure the changes of TGF-β1, PCNA and NF-κB during wound healing. The levels of O2- and oxidation products (malondialdehyde, MDA) were finally detected. Secondly, fibroblasts were separated from human skin and cultured in hydrogen-rich medium. Then cell proliferation was measured by CCK-8 kit and the change of mitochondrial membrane potential was analyzed by JC-1 staining. Intracellular O2-, MDA, 8-OHdG, SOD and GSH levels were also been examined. Furthermore, MAPK, NF-κB, and TGF-β1 expressions were detected by western blotting.Results:1. Establishment of STZ-DM wound healing impairment model.2. STZ-DM wound healing was significantly speeded up after intraperitoneal injection of hydrogen-rich saline.3. Intraperitoneal injection of hydrogen-rich saline could significantly reduce infiltrating time of inflammatory cell and promote generation of granulation tissue, fibroblasts, microvasculation and collagen in healing wound of STZ-DM.4. Observed under electron microscope, there were more inflammatory cells infiltrating in wounds of DM and less fibroblasts which were in poor shape. However, hydrogen could promote the proliferation of fibroblasts, increase their nuclear size; enlarge their rough endoplasmic reticulum.5. Analyzed by immunohistochemical staining, TGF-β1 and PCNA expression were upregulated by intraperitoneal injection of hydrogen-rich saline, while NF-κB expression was downregulated.6. Hydrogen could significantly inhibit cutaneous O2- level and serum MDA level.7. Hydrogen-rich cell culture medium could resist the inhibiting effect of high glucose treatment on cell proliferation and improve the living state of human fibroblasts.8. Hydrogen-rich cell culture medium could inhibit the decreasing of mitochondrial membrane potential, observed under confocal laser scanning microscope.9. Hydrogen-rich cell cultivation could decrease expressions of cellular oxidation products (MDA and 8-OHdG), and increase expressions of anti-oxidation products (SOD and GSH).10. After high glucose treatment, TGF-β1 expression of fibroblast was downregulated, and the MAPK and NF-κB signal transduction pathways were excessively activated. Hydrogen-rich cell cultivation could reverse these effects.Conclusions:1. Intraperitoneal injection of hydrogen-rich saline could significantly promote the wound healing of DM, improve the biological activity of fibroblast, and increase collagen synthesis.2. Hydrogen could downregulate the O2- level and oxidation products generation, reduce the excessive inflammation, promote the growth of granulation tissue, make the fibroblasts thriving, increase TGF-β1 and PCNA expression, and inhibit the excessive activation of NF-κB in wound healing.3. Hydrogen could ease the oxidative damage to human cutaneous fibroblast after high glucose treatment, raise cellular systemic ability of anti-oxidation, and protect mitochondrial membrane potential. The increasing level of SOD may be one of the reasons why hydrogen could reduce extravagant O2- caused by high glucose.4. The effects of hydrogen to promote wound healing and resist oxidative damage caused by high glucose maybe related to the downregulation of activation of MAPK and NF-κB.