| Objective: To investigate the repair effect of moist exposed burn therapy(MEBT)/moist exposed burn ointment(MEBO)on chronic refractory wound in rats.The expression of matrix metalloproteinase 2(MMP-2)and matrix metalloproteinase 9(MMP-9)in chronic refractory wound tissue was affected by phosphatase and tension homology deleted on chromosome ten(PTEN)/ phosphorylated protein kinase B(p-AKT)pathway,which provides a reliable theoretical basis for MEBT/MEBO clinical popularization.Methods:(1)The model of chronic refractory wound is based on the improved plastic ring granuloma quantitative method by Fu Xiaobing and Shen Daoxiu.Rats of grade SPF Wistar were divided into 5 groups according to the random digital table method,namely,MEBO group(30),rb-b FGF group(30),model group(30),acute wound group(30),blank group(30).The wound healing rate of rats in each group was observed 3,7 and 14 d,and the morphological changes of wound tissue were observed by HE and Masson staining.(2)The expression of TNF-α and IL-6 protein in granulation tissue of chronic refractory wound was detected by ELISA,and the inflammatory reaction was observed.(3)The changes of PTEN and p-AKT protein expression were detected by Western Blot,and the effects of wound tissue intervention on PTEN/AKT signaling pathway were observed.(4)The changes of MMP-2 and MMP-9 protein expression and the transcription of MMP-2 m RNA and MMP-9m RNA genes in in granulation tissue of chronic refractory wound and the transcription of MMP-2 m RNA and MMP-9 m RNA genes.Results:(1)The results of HE staining and Masson staining were as follows: the wound healing of acute wound group was better than that of model group,while that of MEBO group and rb-b FGF group was better than that of model group(P<0.05).(2)Changes of TNF-α and IL-6 protein expression in wound tissues of rats in each group:(1)The expression of TNF-αprotein in tissue homogenates of acute wound group,MEBO group and rb-b FGF group increased first and then decreased,while that of model group increased(P <0.05)on the 3rdand 7th decreased on day 14(P <0.05).(2)Expression of IL-6 protein in tissue homogenates of MEBO group,rb-b FGF group and model group increased first and then decreased,while acute wound group decreased.Compared with the model group,the expression of MEBO group and rb-b FGF group increased(P <0.05)on the 3rd day,and the expression of acute group decreased(P <0.05)on the 7th day and 14 th day.(3)Changes of PTEN/AKT signaling pathway in wound tissues of rats in each group: Western Blot:(1)The expression of PTEN protein in acute wound group,MEBO group and rb-b FGF group showed a tendency of decreasing first and then increasing,while the expression in model group showed a significant time dependence,that is,decreasing with time.On the 3rd and 7th day of modeling,acute wound group,MEBO group and rb-b FGF group were lower than model group(P <0.05).On the 14 th day,MEBO group and rb-b FGF group were higher than model group(P <0.05).(2)The expression of p-AKT in acute wound group,MEBO group and rb-b FGF group increased first and then decreased,while the expression in model group increased gradually.On the 3rd day,the acute wound group,MEBO group and rb-b FGF group were higher than the model group(P <0.05).On the 7th day,MEBO group and rb-b FGF group were higher than the model group(P<0.05).On the 14 th day,the acute wound group,MEBO group and rb-b FGF group were lower than the model group(P<0.05).(4)Changes of MMP-2 and MMP-9 protein expression and gene transcription in wound tissues of rats in each group:(1)ELISA: The expression of MMP-2 and MMP-9 protein in the tissue homogenate of acute wound group,MEBO group and rb-b FGF group showed the trend of first increasing and then decreasing,the model group showed the trend of increasing;compared with the model group,the expression of MMP-2 and MMP-9 protein in the acute wound group,MEBO group and rb-b FGF group was increased on the 3rd and 7th day of modeling(P <0.05).On the 14 th day,the expression of acute wound group,MEBO group and rb-b FGF group was lower than that in the model group(P <0.05);(2)Western Blot: the expression of MMP-2 and MMP-9 in acute wound group,MEBO group and rb-b FGF group showed a tendency to increase first and then decrease,while in model group,the expression of MMP-2 and MMP-9 protein in acute wound group,MEBO group and rb-b FGF group was increased on 3rd and 7th day compared with model group(P <0.05).At the 14 th day,the expression of acute wound group,MEBO group and rb-b FGF group was lower than that of model group(P <0.05).(3)RT-PCR: The expression of MMP-2 and MMP-9 in acute wound group,MEBO group and rb-b FGF group all showed the trend of first increasing and then decreasing,while the model group showedthe trend of gradually increasing.Compared with the model group,the transcript levels of MMP-2 and MMP-9 genes in the acute wound group,MEBO group and rb-b FGF group were increased on the 3rd and 7th day(P <0.05).On the 14 th day,the transcription levels of genes in the acute wound group,MEBO group and rb-b FGF group were lower than those in the model group(P <0.05).Conclusion:(1)MEBT/MEBO significantly shorten the healing time of chronic refractory wound,improve the healing rate of wound,effectively improve the morphological changes of wound tissue,and promote the repair of chronic refractory wound tissue.(2)MEBT/MEBO can inhibit the release of inflammatory cells and reduce the inflammatory response of wound by down-regulating the protein expression of TNF-α and IL-6 in chronic refractory wound.(3)MEBT/MEBO can up-regulate the expression of PTEN protein,then down-regulate the expression of p-AKT protein,regulate the signaling pathway in chronic refractory of chronic refractory wound.(4)MEBT/MEBO can downregulate MMP-2 and MMP-9 protein expression and gene transcription,regulate homeostasis of basement membrane(BM)and accelerate wound healing. |
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