MEBT/MEBO对慢性难愈合创面组织内Nrf2/HO-1/NQO1信号通路的影响

Effect of MEBT/MEBO on the Nrf2/HO⁃1/NQO1 Signaling Pathway in Tissues of Chronical Non⁃healing Wounds

目的探讨皮肤再生医疗技术(MEBT/MEBO)对大鼠慢性难愈合创面组织内核转录因子红系2相关因子2(Nrf2)/血红素氧合酶1(HO⁃1)/醌氧化还原酶1(NQO1)信号通路的影响。方法选取90只SPF级Wistar雄性大鼠适应性饲养1周后,采用随机数表法将其随机分为空白组、对照组、模型组、MEBO组和贝复新组,每组18只。其中空白组大鼠只做备皮处理,对照组大鼠建立急性创面模型,模型组、MEBO组和贝复新组大鼠建立慢性难愈合创面模型。模型建立后,空白组大鼠备皮处皮肤及对照组、模型组大鼠创面予以生理盐水纱布换药处理,MEBO组大鼠创面予以湿润烧伤膏(MEBO)药纱换药处理,贝复新组大鼠创面予以重组牛碱性成纤维细胞生长因子凝胶药纱换药处理,对比观察各组大鼠创面愈合率、组织病理学变化情况以及皮肤/创面组织内丙二醛(MDA)、超氧化物歧化酶(SOD)及Nrf2、HO⁃1、NQO1蛋白与HO⁃1、NQO1 mRNA表达水平。结果干预第3天,各组大鼠创面愈合率尤以对照组最高、贝复新组次之(P均<0.05);干预第7、14天,各组大鼠创面愈合率尤以MEBO组最高、贝复新组次之(P均<0.05)。HE染色结果显示,干预第3、7、14天,对照组、MEBO组和贝复新组大鼠创面组织内坏死细胞及炎性细胞逐渐减少,成纤维细胞与毛细血管逐渐增多,而模型组大鼠创面组织内坏死细胞和炎性细胞减少不明显;Masson染色结果显示,干预第3、7、14天,对照组、MEBO组和贝复新组大鼠创面组织内胶原纤维由细少且紊乱逐渐趋于粗大且平整,而模型组大鼠创面组织内胶原纤维虽逐渐增粗、增多,但排列仍较紊乱。干预第7天,对照组、MEBO组和贝复新组大鼠创面组织内MDA表达水平均明显低于模型组(P均<0.05);干预第3、7天,对照组、MEBO组和贝复新组大鼠创面组织内SOD表达水平均明显高于模型组(P均<0.05)。干预第3、7天,MEBO组和贝复新组大鼠创面组织内Nrf2、HO⁃1、NQO1蛋白以及HO⁃1、NQO1 mRNA表达水平均明显高于空白组、对照组和模型组(P均<0.05),且干预第7天,MEBO组大鼠创面组织内Nrf2、HO⁃1蛋白以及HO⁃1 mRNA表达水平均明显低于贝复新组(P均<0.05)。结论MEBT/MEBO可能能够通过激活Nrf2/HO⁃1/NQO1信号通路减轻大鼠慢性难愈合创面氧化应激损伤,促进创面再生修复。

Objective To investigate the effect of MEBT/MEBO on the nuclear factor⁃erythroid 2⁃related factor 2(Nrf2)/heme oxygenase⁃1(HO⁃1)/quinone oxidoreductase 1(NQO1)signaling pathway in tissues of rats with chronical non⁃healing wounds.Methods 90 Wistar male rats of SPF grade were selected,and divided,after one week of adaptive feeding,into five groups using the random number table:blank group(n=18),control group(n=18),model group(n=18),MEBO group(n=18)and rb⁃bFGF group(n=18).Only skin preparation was performed on the rats in the blank group,acute wound models were established on the rats in the control group,and chronic non⁃healing wound models were established on the rats respectively in the model group,MEBO group and rb⁃bFGF group.After the models were estab⁃lished,the prepared skin of rats in the blank group and the wounds of rats both in the control group and model group were treated with dressing change of normal saline gauze,while the wounds of rats in the MEBO group were treated with dressing change of MEBO gauze,and the wounds in the rb⁃bFGF group were treated with dressing change of recombinant bovine basic fibroblast growth factor(rb⁃bFGF)gel gauze.The wound healing rate,histopathological changes,and expression levels of malondialdehyde(MDA),superoxide dismutase(SOD),Nrf2,HO⁃1,and NQO1 proteins,as well as HO⁃1 mRNA and NQO1 mRNA in the skin/wound tissues of each group were compared among the groups.Results On day 3 of intervention,the rats in the control group presented the highest wound healing rate,followed by that in the rb⁃bFGF group(all P<0.05).On day 7 and 14 of intervention,the rats in the MEBO group presented the highest wound healing rate,followed by that in the rb⁃bFGF group(all P<0.05).HE staining showed that on day 3,7 and 14 of intervention,the number of necrotic cells and inflammatory cells gradually decreased,and the number of fibroblasts and capillaries gradually increased in the wound tissues of rats respectively in the control group,MEBO group,and rb⁃bFGF group,whereas the number of necrotic cells and inflammatory cells didn’t show obvious decrease in the wound tissues of rats in the model group.Masson staining showed that on day 3,7 and 14 of intervention,collagen fibers in the wound tissues of rats respec⁃tively in the control group,MEBO group,and rb⁃bFGF group gradually became thicker and more well⁃ordered from tenuous and disordered,while collagen fibers in the wound tissues of rats in the model group,though became thicker and more in number,were still in disorganized manner.On day 7 of intervention,the expression levels of MDA in the wound tissues of rats were significantly lower respectively in the control group,MEBO group,and rb⁃bFGF group compared with the model group(all P<0.05);on day 3 and 7 of intervention,the expression levels of SOD in the wound tissues of rats respectively in the control group,MEBO group,and rb⁃bFGF group were significantly higher than that in the model group(all P<0.05),and the expression levels of Nrf2,HO⁃1,and NQO1 proteins,as well as HO⁃1 mRNA and NQO1 mRNA in the wound tissues of rats in the MEBO group and rb⁃bFGF group were obviously higher than that respectively in the blank group,control group,and model group(all P<0.05).Moreover,on day 7 of intervention,the expression levels of Nrf2 protein,HO⁃1 protein and HO⁃1 mRNA in the wound tissues of rats were significantly lower in the MEBO group compared with the rb⁃bFGF group(all P<0.05).Conclusion MEBT/MEBO may alleviate oxidative stress damage in rats with chronic non⁃healing wounds and promote wound regenerative restoration by activating the Nrf2/HO⁃1/NQO1 signaling pathway.