Diabetic ulcers are one of the most serious and costly chronic complications for diabetic patients. Hyperglycemia-induced oxidative stress may play an important role in diabetes and its complications. The aim of the study was to explore the effect of heme oxygenase-1 on wound closure in diabetic rats. Diabetic wound model was prepared by making an incision with full thickness in STZ-induced diabetic rats. Wounds from diabetic rats were treated with 10% hemin ointment for 21 days. Increase of HO-1 protein expression enhanced anti-inflammation and antioxidant in diabetic rats. Furthermore, HO-1 increased the levels of VEGF and ICAM-1 and expressions of CBS and CSE protein. In summary, HO-1 promoted the wound closure by augmenting anti-inflammation, antioxidant, and angiogenesis in diabetic rats.
Patients with diabetes customarily suffer from chronic nonhealing ulcers, which is one of the most serious and costly complications of diabetes [
Heme oxygenase-1, an inducible enzyme, degrades heme into biliverdin, carbon monoxide (CO), and ferrous iron. HO-1 protein expression can be induced by ubiquitous stimuli not only cytokines and growth factors but also heme (its substrate) and antioxidant. HO-1 and the heme catabolism play pleiotropic effects in preventing injury caused by many diseases [
Hydrogen sulfide (H2S) is known for its odor and poison, but massive evidences have indicated that it possesses various important roles in physiology and pathophysiology. The same as nitric oxide (NO) and carbon monoxide (CO), hydrogen sulfide is regarded as a gaseous signaling molecule. Increasing studies indicate that hydrogen sulfide executes various biological functions such as reducing oxidative stress [
In the present study, we aimed to evaluate the effects of increased expression of HO-1 protein on delayed wound healing via examining the change of inflammatory cytokines such as TNF-
Chloral hydrate, streptozotocin, hemin, and a horseradish peroxidase-conjugated secondary anti-rabbit antibody were purchased from Sigma (Sigma Chemical Co., St. Louis, MO, USA). Rabbit polyclonal antibodies
Male Sprague-Dawley (SD) rats weighing 240–280 g were provided by the Animal Experimental Center in Wannan Medical College. All animal experiments were in compliance with Chinese Community guidelines for the use of experimental animals. Animals were raised in temperature-controlled animal laboratory with
Two weeks after STZ treatment, the animals were randomly assigned to three groups: nondiabetic control rats (NDC); diabetic rats treated with vehicle (DTV); diabetic rats treated with hemin (DTH). Rats were housed individually with standard pellet diet and water ad libitum. Animals were anaesthetized with 10% chloral hydrate by intraperitoneal injection at a dose of 300 mg/kg. The hind dorsum was shaved and disinfected with 75% ethanol. A circular incision with full thickness was created on the dorsum skin from each rat. The wounds from NDC and DTV rats were treated with control cream, and rats from DTH group received 10% hemin ointment on wound. The sizes of the wounds were measured on days 5, 10, 15, and 20, respectively. Twenty-one days after ointment treatment, the rats were anaesthetized with chloral hydrate (300 mg/kg i.p.) and sacrificed, and fasting blood samples and granulation tissues from wound were collected for biochemical analyses.
The wounds from each individual rat were digitally snapped. The wound closure in this experiment was quantified with Image-Pro Plus 5 software. The rate of wound closure was expressed as the ratio of wound closure: the wound closure rate (%) = (initial area − detected area)/initial area × 100%.
Fasting blood samples were centrifuged at 1300 ×g for separation of serum. Serum was used to determine MDA content and SOD activity by commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Serum VEGF and ICAM-1 levels were determined with rat VEGF and ICAM-1 specific ELISA kit (Hefei Bomei Biotechnology Co., Ltd., China) according to kit instruction. The concentrations of VEGF and ICAM-1 in serum were expressed as ng/L and pg/mL, respectively.
Prothrombin time, thrombin time, and fibrinogen were determined on semiautoanalyzer by the enzymatic colorimetric methods (STAGO, France) according to kit instruction.
Wound samples were collected for histological study. Fixed wound tissues in 4% neutral formalin were embedded in paraffin and then sectioned at the thickness of five microns for Hematoxylin-Eosin (H-E) staining. Stained sections were used to evaluate reepithelialization, granulation tissue under a light microscope. The small blood vessels in wounds were counted in high power field.
Wound tissues (0.2 g) were harvested and lysed in 2 mL of ice-cold lysis buffer with 2 mmol/L PMSF, 100 mmol/L Na4P2O7, 50 mmol/L HEPES, 10 mmol/L EDTA, 100 mmol/L sodium fluoride, 10 mmpl/L sodium orthovanadate, and protease inhibitor mixture for 15 min. Lysate was centrifuged at 13,000 g for 20 min at 4°C. Proteins denatured in supernatants were electrophoretically separated on an 8% stacking gel and following 12% sodium dodecyl sulfate-polyacrylamide gel and then transferred to 0.2-
Data were presented as mean ± SE. Differences between the groups were analyzed with one-way analysis of variance (ANOVA) using least significance difference and
After injection of STZ, rats showed an increased concentration of fasting blood glucose and diabetic signs such as polyuria, increased water consumption, and weight loss.
The wound closure was observed by measuring the change of the wound area and checking macroscopic differences of the wounds. The wounds were swelling and purulent and the closure rate of the wound was not significantly different in the next two days. Compared with DTV rats, heme oxygenase-1 significantly accelerated the wound closure rates from DTH rats after 5 days (
Effects of HO-1 on closure rate of wound.
NDC | DTV | DTH | |
---|---|---|---|
Day 5 | 23.0 ± 3.3 | 13.3 ± 3.3 |
20.6 ± 2.8## |
Day 10 | 54.3 ± 4.0 | 33.2 ± 5.8 |
56.1 ± 6.6## |
Day 15 | 81.7 ± 3.8 | 50.6 ± 8.0 |
84.6 ± 4.1## |
Day 20 | 95.9 ± 3.1 | 74.8 ± 7.6 |
96.3 ± 3.9## |
Photomicrographs of H-E staining in the wound tissue of each group (×100). (a) Nondiabetic control rats showed reepithelialization around the wound. (b) Inflammatory cell infiltration was observed in the wound from diabetic rats treated with vehicle. (c) Reepithelialization was observed in the wound from diabetic rats treated with hemin.
The change of coagulation activity is one of the factors on microcirculation, which play an important role in wound healing. The results from the study indicated that sustained hyperglycemia significantly shortened prothrombin time (PT) and thrombin time (TT) and increased level of fibrinogen in serum (
Effects of HO-1 on coagulation activity.
NDC | DTV | DTH | |
---|---|---|---|
PT (s) | 12.3 ± 0.7 | 10.1 ± 0.6 |
11.1 ± 0.5## |
TT (s) | 29.4 ± 2.1 | 20.8 ± 2.7 |
27.1 ± 2.1## |
Fibrinogen (mg/mL) | 1.85 ± 0.16 | 2.96 ± 0.25 |
1.19 ± 0.18## |
HO-1 protein expression was reduced in wound tissue from diabetic rats when compared with NDC rats. Hemin treatment significantly induced HO-1 protein expression. Decreased activity of SOD and increased content of MDA were observed in diabetic rats (
HO-1 enhanced antioxidant effects in diabetic rats. (a) Activity of SOD in serum was significantly corrected by induction of HO-1 in diabetic rats (
The levels of TNF-
Induction of HO-1 elevated anti-inflammation in diabetic rats. HO-1 decreased expressions and relative levels of TNF-
Thromboxane synthase (TXS) is an enzyme which catalyzes prostaglandin endoperoxide into thromboxanes. Our result indicated that expression of TXS was increased in wound tissues from diabetic rats when compared with control rats (
To estimate microcirculation, we checked the number of the small vessels in the wound under microscope. The results showed that fewer vessels were observed in the granulation tissues from DTV rats compared with NDC rats (
HO-1 ameliorated angiogenesis in granulation tissues from diabetic wounds. (a) The number of vessels was increased in granulation tissues from diabetic wounds treated with hemin (
To explore the beneficial effect of heme oxygenase-1 on wound healing in diabetic rats and its possible mechanism, CSE and CBS protein expressions were determined. The results revealed that diabetes reduced the expressions of CSE and CBS protein (
HO-1 improved the expression of hydrogen sulfide synthase. HO-1 increased expressions and relative levels of CBS ((a) and (c)) and CSE ((b) and (d)) (
In the present study, we evaluated the effect of induction of HO-1 on wound healing in diabetic rats. Our results showed that hyperglycemia impaired cutaneous wound closure from diabetic rats. Induction of HO-1 accelerated delayed wound closure by reducing inflammatory cytokines such as TNF-
Poor wound healing represents one of the most serious diabetic complications. Sustained hyperglycemia impairs endothelial cells and leads to vascular dysfunction in wounds. Ischemia results in a serious infection and impairs formation of granulation tissue in diabetic wounds [
Considerable evidence from both animal models and clinic indicates that oxide stress plays an important role in the occurrence and development of disease. Oxide stress results from excessive ROS and reduced antioxidant, which impairs vascular function [
Wound healing is a complex pathophysiological process involved with physiological events such as inflammation, angiogenesis, and reepithelialization. However, wound healing in diabetes was impaired by many factors including infection, hypoxia, and excess inflammation. Delayed wound healing is associated with increased inflammatory cytokines in diabetes [
Blood vessels were impaired by ROS and inflammatory cytokine in diabetes, which led to disorder of coagulation and anticoagulation, and peripheral circulation failure [
Hydrogen sulfide (H2S) has been known as a physiologically and pathophysiologically active gasotransmitter [
In conclusion, our results reveal the beneficial effects of HO-1 on diabetic wound healing. HO-1 showed its anti-inflammatory response and antioxidant activity. Furthermore, the protective effects of HO-1 may be associated with its angiogenesis in granulation tissues from diabetic wounds. In addition, production of hydrogen sulfide may be involved in the effect of HO-1 on the wound healing in diabetes. Our results suggest that it is significant to further study the precise mechanism of HO-1 to heal diabetic wound for clinical application.
All authors declare that there is no conflict of interests regarding the publication of this paper.
Qing-Ying Chen and Guo-Guang Wang contributed equally to this work; they both are the first authors.
This study was supported by The Army Key Project (no. BJN14C001), the National Natural Science Foundation of China (no. 81172790), and initial funding of Wannan Medical College (no. 06020204).