| Objective The whole layer skin defection rat was used and to evaluate the therapeuticeffect of the Chinese herbal compound prescription-Yuhonggao in wound healing. Toobserve the effect of Yuhonggao on the expression of integrinβ1 and p63 to investigate the mechanism of promoting wound healing.Methods The skin in the rat back was cut as model, which was round and diameter was 1．8 cent. 108 healthy male rat were randomly divided into 3 groups: Yuhonggao group, Jingwanhong group, model group, 36 rats per group. With 6 normal rats as control and Jingwanhong as positive compare medicine. The rats in Yuhonggao group were external applied with Yuhonggao, 0．1 g each wound surface and 2 times a day. The application of Jingwanhong groups was same to Yuhonggao. The wound surface and healing time were observed and recorded, the diameter of wound surface and count healing rate was calculated, and then draw the wound skin. The sample was handled and embed with Paraffin, HE stained to observe the skin regrowth state under optical microscope. The integrinβ1 and p63 expression, the marker of epithemal stem cells, were measured and compared by immunohistochemistry stain. To select 3 visual field in edge and central of wound each sample of integrin'β1 staining and 3 visual field in cuticular layer of p63' staining under 20 multiple objective. To analyze the optical density (the fig of OD) of integrin'β1 staining and to count the positive cell rate of p63 by Image-Pro-Plus image analysis software. All counted data was expressed in the form of (?)±s, analysis of variance was used to interblock count data by SPSS 13．0 statistics software.Results①By gross observing, there were more and thick exudates in the wound surface of Yuhonggao group at the first day and the 3th day after wounding. While there less and thin exudates in Yuhonggao group and model group. The growth of granulation tissue and reepithelization hadn't significant difference between the Yuhonggao group and Jingwanhong group, which were better than model group.②Average wound healing time had't significant difference between the Yuhonggao group [(13．5±0．9) d] and Jingwanhong group [(12．6±0．9) d], which were better than model group [(19．1±2．6) d] (P0．05) . The rate of woud healing had't significant difference between the Yuhonggao group and Jingwanhong group, which were better than model group in the same observation time (P<0．05) .③From the HE stain, the time which signed the feature of inflammation, proliferation and remodle in wound healing process showed up earlier in Yuhonggao group and Jingwanhong group than model group and had't significant difference between the Yuhonggao group and Jingwanhong group. According to the score of histopathology there were no significant difference in epidermal structure, collagen fiber bundle and PMN infiltration of dermis structure between the Yuhonggao group and Jingwanhong group, which were better than model group (P<0．01) .④The image analysis result of immunohistochemistry staining of integrinβ1: the fig of OD increased from the first day of wound. The fig of OD of Yuhonggao group, Jingwanhong group and model group were different than normal group in the 1th, 3th, 5th, 7th, 14th day after wound (P<0．05) . Until the wound healing completely there were no different between the 4 groups (P>0．05) . The integrinβ1 has good specificity as the marker of epithemal stem cells, the result indicated that the wound was the order of proliferation and differentiation of epidermal stem cells. The fig of OD of Yuhonggao group and Jingwanhong group achieved peak at the 7th day, while the peak of model group came at the 14th day. All these indicated that the Yuhonggao could promote proliferation and differentiation of epidermal stem cells in edge of skin wound surface. Furthermore, the fig of OD of Yuhonggao group was obviously higher than Jingwanhong group at the 7th day and the 14th day (P<0．05) .⑤The image analysis result of immunohistochemistry stain of p63: The total number of p63 positive cell were more in wound skin than in the normal skin (P<0．05) . The difference were little between the Jingwanhong group, model group and normal group. The total number of p63 positive cell in the Yuhonggao group was more than in the normal group significantly. The total number of p63 positive cell of Yuhonggao group and Jingwanhong group achieved peak at the the 7th day, while the peak of model group came at the 14th day. Until the wound healing completely at the 21th day, there were difference between Yuhonggao group, Jingwanhong group and normal group (P<0．05) , and near to normal group. However, the fig of model group was still in high level.⑥Position of positive express of the integrinβ1 and p63 in immunohistochemistry stain: In normal rat skin the positive cells of the integrinβ1 distribute scarcely in basal layer of epidermis and outer root sheath of hair follicle, the positive cells of p63 distribute widespreadly in basal layer of epidermis and the epithelial lamina of hair follicle and sebaceous glands. In wound skin, from the first day of wound and with the time going, the position of positive cells distributed not only in the above districts but also in the spinous layer and granular layer of epidermis. The positive cells distributed from monolayer to double layer and multilayer and became denser in the wound edge, more and more with the diminution of wound surface. It still exist when the wound healing and epithelization completely. The model group was inferiorer than Yuhonggao group and Jingwanhong group in volume,amount,distribution and staining extent of positive cells.Discussion The integrinβ1 play a important role in signal transduction and is the mark of epidermal stem cells and transit complifying cells. Yuhonggao could promote the signal transduction between cells and the proliferation and differentiation of epidermal stem cells in the edge of skin wound surface. p63 is the mark of cell in the powerful proliferation state, so the result of statistics indicated the cell in cuticular layer of Yuhonggao group had the powerful capability of proliferation, which demonstrated that Yuhonggao could promote proliferation of cells.Conclusion①Yuhonggao can accelerate skin wound healing and improve the quality of wound healing.②The mechanism of promoting wound healing possibly relate with promoting the expression of integrinβ1 and p63 in edge of skin wound surface. |
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