| Refractory diabetic ulcer is a major cause of death and disability in patients with diabetes mellitus (DM) and there is still no efficient therapy available. The proposed mechanisms involve sustained inflammation, excess oxidative stress and compromised agiogenesis. Epoxyeicosatrienoic acids (EETs) are epoxidation products of arachidonic acid catalyzed by CYP epoxygenases. Previous studies showed that EETs have anti-inflammatory and pro-angiogenetic activities. In this study, we explored the role and effects of CYP epoxygenases/EETs on wound healing in ob/ob mice.Aim:To investigate the effectsof Cytochrome P450 epoxygenase pathway on diabetic impaired wound healing, and to explore the possible mechanisms underlyingMethods:The Full-thickness punch biopsy wounds were created on mice, then, ob/ob mice were randomly divided into 4 groups, including saline, insulin,11,12-EET and EET inhibitor for treatment, C57BL/6 mice were treated with saline as control. mRNA and protein expression of epoxygenase were detected by RT-PCR and western blotting, respectively; expression of MMP-9 were measured by western blotting; neutrophil and monocyte/microphage infiltration was analyzed by immunohistochemistry; the levels of tumor necrosis factor (TNF-a), interleukin (IL)-1β and IL-6 were measured by enzyme-linked immuno-sorbent assay (ELISA). Vascular-like structures was detected by immunofluorescence.Results:1. Compared to control mice, CYP2C65 and CYP2J6 mRNA expression levels in granulation tissues were significantly decreased in ob/ob mice compared with control mice (P<0.05, respectively). Similarly, CYP2C65 and CYP2J6 protein expression levels in ob/ob mice were significantly decreased on the 3 and 6 day of wound healing course (P<0.05, respectively).2. The wound healing rate in ob/ob mice was slower than C57BL/6 control mice; 11,12-EET significantly accelerated wound healing as compared with vehicle-treated in ob/ob mice. In contrast, the EETs inhibitor decreased wound healing rate in ob/ob mice compared to vehicle-treated ob/ob mice. There was no significant difference in wound healing rate between insulin-treated and vehicle-treated groups in ob/ob mice.3. Three or Six days after wound, neutrophil and macrophage cell numbers in the granulation tissues of vehicle-treated ob/ob mice were significantly more than C57BL/6 control mice.11, 12-EET treatment significantly decreased neutrophil and monocyte/macrophage infiltration compared to vehicle-treated ob/ob mice.4. At day 6 after wound, the levels of IL-1β, IL-6 and TNF-a in the granulation tissues of ob/ob mice were far higher than C57BL/6 mice.11,12-EET treatment significantly reduced the levels of these pro-inflammatory compared to vehicle-treated ob/ob mice (P<0.05). The levels of these pro-inflammatory cytokines were not significantly different between vehicle-treated ob/ob mice and insulin-treated ob/ob mice.5. Compared with C57BL/6 control mice, MMP-9 protein expression in the granulation tissues of vehicle-treated ob/ob mice was significantly increased (P<0.05) on the third or sixth day after wound.11,12-EET treatment significantly reduced MMP-9 protein expression in ob/ob mice when copmpared with vehicle-treated ob/ob mice (P<0.05). In contrast, EETs inhibitor significantly increased MMP-9 protein expression (P<0.05).6. Compared with vehicle-treated ob/ob mice, EET treatment significantly increased collagen deposit, whereas, insulin showed no obvious effect on collagen deposit. EET inhibitor significantly reduced collagen deposit in ob/ob mice.7. At day 6 after wound, immunostaining of fresh granulation tissue from vehicle-treated ob/ob mice showed a striking decrease in vascular-like structures (CD31-positive) compared with C57BL/6 control mice(P<0.05). EET treatment significantly increased the newly formed vascular-like structures in ob/ob mice when copmpared with vehicle-treated ob/ob mice (P<0.05). Insulin alone also increased the capillary number to some extent when copmpared with vehicle-treated ob/ob mice, but with no statistical significance.8. CYP2J2 and CYP2C11 protein levels were significantly decreased (P<0.05) but MMP-9 protein level was significantly increased in the granulation tissues in patients with diabetic foot ulcers compared with non-diabetic controls.Conclusions:1. The mRNA and protein expression levels of CYP2C65 and CYP2J6 in the granulation of wounds in ob/ob mice were significantly reduced compared to C57BL/6 mice.2. Treatment with EETs significantly improved wound healing in ob/ob mice.3. Further investigation of mechanisms underlying the protective effects of EETs demonstrated that exogenous EETs attenuated neutrophil and monocyte/macrophage infiltration,4. Administration of exogenous EETs resulted in reduced production of inflammatory cytokines in ob/ob mice wound healing.5. EET decreased MMP-9 expression in ob/ob mice.6. EETs increased collagen accumulation in the granulation of wounds in ob/ob mice.7. EETs increased angiogenesis in the granulation of wounds in ob/ob mice.8. Compared to healthy controls, protein expression of CYP2C11 and CYP2J2 were reduced and MMP-9 protein expression was increased in the granulation of wounds in patients with diabetic foot.Taken together, these findings indicated that reduced expression of CYP epoxygenases may play an important role in the pathogenesis of impaired diabetic wound healing and exogenous EETs improves wound healing possibly via mechanisms of anti-inflammation and pro-angiogenesis.Significance:In this study, we demonstrated reduced expression of CYP epoxygenases may play an important role in the pathogenesis of impaired diabetic wound healing, raising the possibility of the use of EETs as a novel therapy for refractory diabetic ulcers... |
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