| ObjectiveTo establish the rat model of chronic skin ulcer and observe the influences of Huiyang Shengji Gao and its modified formulae on wound healing and related cytokines. Analyse the mechanism of Huiyang Shengji Gao on curing chronic skin ulcer.Methods1. Male SD rats (n=30) were randomly divided into3group, established skin ulcer by three different methods which were dermal deficiency (dermal deficiency group), dermal deficiency+hormone intervention (dermal deficiency+hormone group) and dermal deficiency+hormone intervention+bacterial infection (dermal deficiency+hormone+bacterial group). The general condition and the character of wound surface and the organic morphology of the granulation tissue stained by hematoxylin and eosin (H&E) were observed, wound area was recorded and wound healing rate was calculated. Compare the different impact of hormone intervention and hormone intervention+bacterial infection on wound healing.2. Male SD rats (n=68) were randomly divided into model group, group of whole formula of Huiyang Shengji Gao (whole formula group), group of modified Wenyang Yiqi formula (Wenyang Yiqi group) and group of Huoxue Shengji formula (Huoxue Shengji group). The rat model of chronic skin ulcer was established by using compound factor superposition method of dermal deficiency+hormone intervention+bacterial infection. Model group was given petrolatum gauze, and other groups were given the corresponding pharmic gauze. Some rats (n=44) were continuously dosed until all the wound epithelazation. It was last28days. The general condition and the character of wound surface were observed, and the wound area and the time of epithelization were recorded. The other rats (n=24) were dosed7days, and the granulation tissue stained by hematoxylin and eosin (H&E) was observed.3. Male SD rats (n=30) were randomly divided into control group, model group, whole formula group, Wenyang Yiqi group and Huoxue Shengji group. The rat model of chronic skin ulcer was established by using compound factor superposition method. Control group and model group were given petrolatum gauze, and other groups were given the corresponding pharmic gauze. The concentration of serum interleukin-1(IL-1) and tumor necrosis factor-a (TNF-a) were detected by enzyme linked immunosorbent assay (ELISA)7days after medication. The expression of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-2(VEGFR-2), endothelial nitic oxide synthase (eNOS) and cluster of differentiation34(CD34) in granulation tissue were calculated by immunohistochemistry-paraffin section (IHC-P)7days after medication.Results 1. The compound factor superposition method of dermal deficiency+hormone intervention+bacterial infection aggravated the wound injury and extended the wound healing time compared with the other methods. The model rats showed the basic features of syndrome of yang deficiency, such as eating less, loose bowels, weight loss, cold tail, drowsiness and activity decrease, et al. The wound surface was kermesinus, the secretion was stench, and the wound healing time was extended. The H&E staining showed that in dermal deficiency group the wound had new integrated cuticular layer, and there were fibroblasts and inflammatory cells dispersed distribution and abundant blood capillaries and collagenous fibers under the cuticular layer; in dermal deficiency+hormone group the wound had new cuticular layer but it was thin and not integrated, and there were less fibroblasts and more blood capillaries and coUagenous fibers under the cuticular layer; in dermal deficiency+hormone+bacterial group the wound had new cuticular layer which was not complete covering the wound, and above the cuticular layer there were crustas, in which were inflammatory cells aggregated distribution, and beneath the crustas there was tissue fluild, and there were less blood capillaries and collagenous fibers in corium layer.2. The wound healing rate was higher in whole formula group than that in model group (P<0.05), Wenyang Yiqi group (P<0.05) and Huoxue Shengji group (P<0.05)3days after medication; the wound healing rate was was higher in whole formula group and Wenyang Yiqi group than that in model group (P<0.01, P<0.05)7days after medication; the wound healing rate was was higher in whole formula group and Wenyang Yiqi group than that in model group (P<0.01) and Huoxue Shengji group (P<0.05)11days after medication; the wound healing rate was lower in model group than that in the other groups (P<0.01)14days after medication. The average wound healing time was longer in model group than that in whole formula group (P<0.05), Wenyang Yiqi group (P<0.05) and Huoxue Shengji group (P<0.05)28days after medication. The time that wound begun to epithelization was first in Whole formula group, second in Huoxue Shengji group, last in Wenyang Yiqi group and model group. The time that all wound had epilazation was first in whole formula group, second in Wenyang Yiqi group, third in model group and last in Huoxue Shengji group.3.7days after medication, the H&E staining showed that in model group, the wound wasn’t epithelization, in corium layer, inflammatory cells were aggregated distribution and fibroblasts were dispersed distribution and the number of blood capillaries and collagenous fibers were less; in whole formula group, the wound had new integrated cuticular layer, fibroblasts and inflammatory cells were dispersed distribution under the stratum basale, in corium layer, blood capillaries were abundant and collagenous fibers were more but thin and didn’t weave into net; in Wenyang Yiqi group, the wound had new stratum basale covered by crustas, there was tissue fluild under the crustas, and there were fibroblasts and inflammatory cells aggregated distribution under the stratum basale, collagenous fibers were more but thin; in Huoxue Shengji group, the wound was covered by crustas, in which were inflammatory cells aggregated distribution, there were fibroblasts aggregated distribution and abundant blood capillaries and less collageaous fibers under the crustas.4.7days after medication, the content of TNF-a was lower in model group than that in control group (P<0.05), and that was lower in whole formula group than that in model group (P<0.05), and the comparion between Wenyang Yiqi group and model group had no statistical significance (P>0.05), and that was lower in Huoxue Shengji group than that in model group (P<0.05), and that was higher in Wenyang Yiqi group than that in whole formula group (P<0.05) and Huoxue Shengji group (P<0.01), and the comparion between whole formula group and Huoxue Shengji group had no statistical signficance (P>0.05); the content of IL-1compared among four groups had no statistical signficance (P>0.05).5.7days after medication, the IHC-P showed that the expression of VEGF was lower in model group than that in control group (P<0.01), and that was higher in whole formula group than that in model group (P<0.05), and that was lower in Wenyang Yiqi group and Huoxue Shenji group than that in control group (P<0.01) and whole formula group (P<0.01), and the comparion between other groups had no statistical significance (P>0.05); the expression of VEGFR-2was lower in model group than that in control group (P<0.05), and that was higher in whole formula group than that in model group (P<0.05), and that was lower in Wenyang Yiqi group and Huoxue Shenji group than that in control group (P<0.05), and that was lower in Huoxue Shenji group than that in whole formula group (P<0.05), and the comparion between other groups had no statistical significance (P>0.05); the expression of eNOS was lower in model group than that in control group (P<0.01), and that was higher in whole formula group than that in model group (P<0.05), and that was lower in whole formula group than that in control group (P<0.05), and that was lower in Wenyang Yiqi group and Huoxue Shenji group than that in control group (P<0.05), and the comparion between other groups had no statistical significance (P>0.05); the expression of CD34was lower in model group than that in control group (P<0.05), and that was higher in whole formula group and Wenyang Yiqi group than that in model group (P<0.01), and that was lower in Huoxue Shengji group than that in whole formula group (P<0.05), and the comparion between other groups had no statistical significance (P>0.05).Conclusion1. The rat model that established by using compound factor superposition method showed the basic feactures of syndrome of yang deficiency, and the method could aggravate the wound injury and extent the wound healing time. The wound form was similar with chronic skin ulcer that also called as yin-syndrome skin ulcer, and the rat model would meet the demand of experience.2. Huiyang Shengji Gao and its modified formulae all could improve wound healing in rats with chronic skin ulcer, and Huiyang Shengji Gao was better than Wenyang Yiqi modified formula and Huoxue Shengji modified formula.3. Huiyang Shengji Gao could alleviate the wound inflammation by decreasing the content of serum TNF-α, and this function was closely related to Huoxue Shenji modified formula.4. Huiyang Shengji Gao could pertect endothelial cells and permote angiogenesis by increasing the expression of VEGF, VEGFR-2and eNOS within granulation tissue, and its modified formulae didn’t show apparent effect as well as Huiyang Shenji Gao. |
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