| objectiveTo study the effects and mechanism of ferulic acid(FA) on chronic wound healing of type 2 diabetic rats in vivo. Respectively in normoxic and hypoxic conditions, to research the effects of ferulic acid with different concentrations on angiogenesis of human umbilical vein endothelial cells(HUVECs) in vitro; the effects of ferulic acid on protein expression of hypoxia inducible factor-1 alpha(HIF-1 alpha) and vascular endothelial growth factor(VEGF) and platelet-derived growth factor(PDGF) of HUVECs;and the effects of ferulic acid on regulating VEGF and PDGF expression of HUVECs through the extracellular regulated protein kinases 1/2(ERK1 / 2) signaling pathway.MethodsWe made the ferulic acid ointment with the matrix of Vaseline. In order to study the dissolution rate of ferulic acid from the ointment in water, we placed 150 mg ferulic acid ointment of 2%, 5% and 10% in 50 m L of water respectively, and collected samples at different time points, then the absorbance values were detected by colorimetric method and the dissolution rates of ferulic acid ointment in water were calculated.We fed SD rats with high glucose and fat food for 4 weeks, then injected 45mg/kg streptozotocin(STZ) in the abdominal cavity, and established the model of type 2 diabetic rats. Established chronic wound model of diabetic rats, set diabetic control group(DC),Vaseline control group(VC), normal saline control group(NSC), 10% ferulic acid ointment treatment group(10%FA) and 40mg/kg ferulic acid irrigation treatment group(40mg FA).Analyzed the effects of different treatments on % wound closure and healing time, and the effects of ferulic acid on vascularization of chronic wound were analyzed by CD34 immunohistochemical staining.Cultured primary HUVECs, respectively in normoxic and hypoxic conditions, treated with different concentrations of ferulic acid(1×10-8, 1×10-7, 1×10-6, 1×10-5, 1×10-4 and1×10-3 mol/L). The effects of different concentrations of ferulic acid on the proliferation,the migration and the formation of lumen like structure of human umbilical vein endothelial cells were analyzed by MTS method, the wound healing assay and the tube formation assay on Matrige respectively. In normoxic and hypoxic conditions, HUVECs were treated with ferulic acid of 1×10-5 mol / L, respectively,and at 0h, 3h, 6h, 24 h the protein expression of the factors closely related to angiogenesis, such as VEGF, PDGF and HIF-1alpha were detected by western blot. In normoxic and hypoxic conditions,HUVECs were pretreated by PD98059,which was the specific MEK1 inhibitor, for 30 minute, with blocked the ERK1/2 signaling pathway, then were treated with ferulic acid of1×10-5 mol/L for 6 hours.At the last, changes of the expression of VEGF and PDGF were detected by western blot.ResultsThe dissolution rate of ferulic acid ointment in water was satisfactory. After 12 hours,the dissolution rates of ferulic acid ointment of 2%, 5% and 10% were 8.6%, 11.2% and16.2% respectively, and which of after 24 hours were 10.4%,13.2% and 21.8%.After 4 weeks of SD rats fed with high glucose and fat food, the level of blood glucose was above 16.7 mmol/L,and lasted for at least 4 weeks after the intraperitoneal injection. They could be used for the study of chronic wounds.After the treatment of sixteenth days, the rate of wound healing of 10% FA group was94.01±6.17%, higher than that was 87.24±6.40% in VC group. The wound healing rate was 91.27±6.93% in 40 mg FA group, and was significantly higher than that was82.16±6.85% in NSC group. The healing time of 10%FA group was 20.29±1.67 days,compared with the VC group(22.86±2.23 days), was significantly shorter.The healing time of 40 mg FA group was 20.57±1.84 days,compared with the NSC group(23.29±1.98 days),the wound healing rate was significantly accelerated. Eleven days after treatment, CD34 immunohistochemical staining could find that, numbers of positive cells of 10% FA group and 40 mg FA group were significantly higher than that of in control groups.The Micro-vessel Density of 10% FA group and 40 mg FA group was statisticsed, compared with the control groups, and there were statistically significant difference.Under normoxia and hypoxia, HUVECs were treated with different concentrations of ferulic acid,and the proliferative activity of the cells in each group was detected by MTS method; treatment with the wound healing assay for 24 hours, the migration activity of the cells was detected;treaedt for 7 hours, the ability of tube like structure formation were detected respectively. Found that in normoxic and hypoxic conditions, when the concentration was between 1×10-8 and 1×10-5mol/L, ferulic acid could promote the proliferation, the migration and the tube like structures formation in a concentration dependent manner; when the concentration was between 1×10-5 and 1×10-3 mol/L, the effects were gradually weakened. In another word,then, when the concentration of 1×10-5mol / L, ferulic acid had the best effects, compared to the control group of the condition of itself, there was statistically significant difference.In normoxic and hypoxic conditions, HUVECs was treated with 1×10-5 mol /L ferulic acid, at the time of 0h, 3h, 6h, 24 h, respectively, protein expression of VEGF, PDGF and HIF-1alpha was detected by western blot. The expression of the proteins peaked at 6h,compared to that was at 0h, had statistical differences.In normoxia and hypoxia, one group of HUVECs were treated with 1×10-5 mol / L ferulic acid for 6h, and western blot test found that the expression of VEGF and PDGF protein were raised. The another group were pretreated by ERK1/2 signaling specificity blocked PD98059(5×10-5 mol / L) for 30 min, and then were treated by 1×10-5 mol /L ferulic acid for 6h, western blot test found that the VEGF and PDGF protein expression did not increase, even less than the control group. This can be inferred that the up-regulation of VEGF and PDGF protein expression by ferulic acid was through the ERK1/2 signaling pathway.ConclusionIn water, ferulic acid could release from the ointment of Vaseline matrix.Ferulic acid could accelerate chronic wounds healing of diabetic rats model, and could increase vascular density in the tissue of wounds. In normoxic and hypoxic conditions, ferulic acid could promote the proliferation, migration and lumen like structure formation of HUVECs.In normoxia and hypoxia, ferulic acid could up-regulate the protein expression of HIF-1alpha, VEGF and PDGF, and its affects of up-regulation of VEGF and PDGF protein expression were through ERK1/2 signaling pathway. |
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