摘要
目的:以PINK1/Parkin通路介导的线粒体自噬为靶向,探讨丹黄散对糖尿病溃疡创面的促愈机制。方法:选择雄性无特定病原体(SPF)级db/db小鼠18只,同遗传背景的雄性SPF级db/m小鼠6只,按随机数字表法将18只db/db小鼠分为模型组、生长因子组、丹黄散组,每组6只;6只db/m小鼠作为空白组。使用4%水合氯醛麻醉,用无菌打孔器在小鼠背部建立直径为0.8 cm的皮肤缺损,制备糖尿病溃疡模型。空白组和模型组不予药物干预,对照组和丹黄散组分别给予重组人表皮生长因子凝胶和丹黄散外敷,每天换药1次,连续21 d。于造模后第7天、第14天取创面组织分析。通过苏木精-伊红染色法(HE)、Masson染色观察创面病理情况;透射电镜观察创面组织线粒体及自噬小体情况;Western-Blot检测创面组织LC3-Ⅰ和LC3-Ⅱ、PINK1、Parkin蛋白表达。结果:造模后的第7天、第14天、第21天空白组、生长因子组、丹黄散组创面愈合率均高于模型组(P<0.05);第7天、第14天的小鼠创面组织HE染色、Masson染色后发现,与模型组相比,空白组、生长因子组、丹黄散组的炎症反应较轻,新生血管数量明显增加,创面中细胞排列紧密,创面基质形成丰富胶原纤维,创面表皮组织恢复佳;第7天创面组织透射电镜观察发现,空白组、生长因子组和丹黄散组创面组织中线粒体肿胀情况较模型组轻,可见自噬小体数量高于模型组;Western-Blot检测发现,空白组、生长因子组、丹黄散组小鼠创面组织LC3-Ⅱ/LC3-Ⅰ、PINK1、Parkin蛋白表达均高于模型组(P<0.05)。结论:丹黄散通过上调PINK1/Parkin通路,激活线粒体自噬,保护创面细胞线粒体正常结构和功能,促进血管新生和肉芽组织的生长,有效地促进糖尿病小鼠溃疡创面愈合。
Objective:To explore the promotion mechanism of Danhuang Powder on the wound of diabetic ulcers by taking the mitophagy through the PINK1/Parkin pathway as a target. Method:18 specific pathogen free(SPF)grade male db/db mice and 6SPF-grade male db/m mice with the same genetic background were selected. The 18 db/db mice were divided into the model group,growth factor group,and Danhuang Powder group,6 in each,according to the random digital tables method;6 db/m mice as a blank group. Use4% chloral hydrate for anesthesia,and establish a skin defect with a diameter of 0. 8 cm on the back of mice with a sterile pitch to prepare the diabetic ulcer model. The blank group and the model group received non-drug intervention. The control group and the Danhuang Powder group received the restructured human epidermal growth factor gel and Danhuang Powder for external application,respectively.The medicine was changed once a day for 21 d. On the 7th and 14th days after the model creation,the facade organization analysis was taken to analyze. The pathological situation of wounds was oberved through HE and Masson staining. The condition of wound tissue mitochondria and spontaneous bodies through transmitted electron microscope;detect wound tissue LC3-Ⅰ and LC3-Ⅱ,PINK1,and Parkin protein expression were observed by Western-Blot. Results:The healing rate of the traumatic surface of the blank group,growth factor group,and Danhuang San group was higher than that of the model group on the 7th,14th,and 21st days after modeling(P<0. 05).HE and Masson staining of the traumatic surface of the mice on the 7th and 14th days showed that compared with the model group,the inflammatory reaction of the blank group,growth factor group,and Danhuang Powder group was lighter;the number of neovascularization increased significantly;the cells in the traumatic surface were closely arranged;the traumatic stroma formed abundant collagen fibers,and the traumatic epidermal tissue recovered well. Transmission electron microscopic observation of the traumatic tissue on day 7 revealed that the mitochondrial swelling condition in the blank group,the growth factor group,and the Danhuang Powder group were lighter than the model group,which showed that the quantity of autophagosome was higher than the model group;the Western-Blot test showed that LC3-Ⅱ/LC3-Ⅰ,PINK1,and Parkin protein expression of the blank group,growth factor group,and Danhuang Powder group were higher than the model group(P<0. 05). Conclusions:Danhuang Powder could raise the PINK1/Parkin pathway and active mitophagy to protect the normal structure and functions of mitochondria in wound cells,promote the growth of vascular and granulite tissue,and effectively promote ulcer wound healing in diabetic mice.
作者
王庚新
张春玲
赵伟
陈露
邸铁涛
张云
王艳辉
刘海艳
WANG Gengxin;ZHANG Chunling;ZHAO Wei;CHEN Lu;DI Tietao;ZHANG Yun;WANG Yanhui;LIU Haiyan(Guizhou University of Traditional Chinese Medicine,Guizhou 550002 China)
出处
《护理研究》
北大核心
2022年第24期4370-4375,共6页
Chinese Nursing Research
基金
国家自然科学基金资助项目,编号:81960805
贵州省研究生教育创新计划项目,编号:黔教合YJSCXJH(2020)161。
