Abstract: First author：YU Jie，Doctor Medicinae，Attending doctor，lecturer，Master's Supervisor；E-mail: hecafei@sina.com
Background   Throughout the general situation of research on moxibustion in the treatment of pressure injury，there are the following issues including“the clinical effect of moxibustion in the treatment of pressure injury is exact without clear molecular mechanism” and “while the molecular mechanism of angiogenesis in tissue with pressure injury in Modern Medicine is more in-depth，but lack of effective treatment means”.It is required to attach great importance to the focus as well as the issues，integrating the modern medical angiogenesis theory with the original features of moxibustion therapy of TCM.It is expected to make up for the unclear molecular mechanism of moxibustion in the treatment of pressure injury and explore the molecular regulatory mechanism of moxibustion intervention on promoting angiogenesis in tissue with pressure injury.Objective　To investigate the molecular regulation mechanism of moxibustion on promoting angiogenesis in tissue with pressure injury，so as to provide theoretical and experimental basis for moxibustion in clinical treatment of pressure injury and skin ulcers.Methods　A total of 120 female healthy clean SD rats were selected in the experiment from December 2015 to March 2017，85 of which were given modeling process and 35 of which were treated as blank group.The models of rats with pressure ulcers in stage 2 to 3 were established by using self-made devices of ischemia reperfusion injury.Successful models of 70 SD female rats with skin ischemia reperfusion injury were randomly divided into model group and moxibustion group，each with 35 SD rats.There were 5 SD rats in the model group and the moxibustion group respectively falling off from the model to the intervention process. In the blank group， 5 SD rats had negative lesions during skin preparation and were removed.The blank group was only given iodophor intervention to simulated model position.The model group was only given conventional iodophor intervention as well.Moxibustion group was given iodophor intervention at first and then direct moxibustion intervention to the local tissue of pressure ulcer with cyclotron operation at the radius of 10 mm，with moxibustion treatment once a day and 15 minutes each time.The protein expression levels of vascular endothelial growth factor（VEGF），RAS and Phospho-ERK1/2（P-ERK1/2） in wound tissues of rats were detected by immunohistochemistry at the first，third，fifth，seventh and tenth day after intervention or treatment.Results　The immunohistochemistry results showed that the positive expression of VEGF protein in skin tissues with pressure injuries was in the cytoplasm which expressed in epithelial cells and macrophages.The positive expression position of both RAS and P-ERK1/2 protein in the skin was located in the cytoplasm.When pressure injury occurred，the expression levels of VEGF and P-ERK1/2 protein in wound tissues gradually increased and then decreased to normal levels.The expression levels of VEGF and P-ERK1/2 protein in moxibustion group and model group both increased at first and then decreased.However，there were significant differences between two groups in the time-point when high peaks of expression levels emerged.The highest peak in moxibustion group appeared in the third day after treatment，while that of model group occurred in the fifth day after iodophor intervention.When pressure injury occured，the expression of RAS protein in wound tissue decreased at first and then increased.The expression of RAS protein in moxibustion group and model group both showed a rising trend.The expression level of VEGF in wound tissues with pressure injuries in model group was higher than that of blank group at the first，third，fifth，seventh and tenth day after intervention（P<0.05）．The expression level of VEGF in wound tissues with pressure ulcers in moxibustion group was significantly higher than that of model group at the third and fifth day after intervention or treatment（P<0.05）．The expression level of RAS in wound tissues with pressure injuries in model group was lower than that of blank group at the first，third，fifth，seventh and tenth day after iodophor intervention（P<0.05）．The expression level of RAS in wound tissues with pressure ulcers in moxibustion group was higher than that of model group at the third，fifth，seventh and tenth day after treatment（P<0.05）．The expression level of P-ERK1/2 in wound tissues with pressure injuries in model group was higher than that of blank group at the third，fifth，seventh and tenth day after iodophor intervention（P<0.05）．The expression level of P-ERK1/2 in wound tissues with pressure ulcers in moxibustion group was higher than that of  model group at the third and fifth day after treatment（P<0.05）．Conclusion　Moxibustion intervention can increase the expression level of VEGF protein in wound tissues with pressure injuries，which may increase the expression level of RAS protein and promote the phosphorylated expression level of ERK1/2 protein by activating RAS/RAF/ERK signaling pathway.Hence，moxibustion intervention might enhance the activity of the RAS/RAF/ERK signaling pathway and contribute to the proliferation of vascular endothelial cells as well.

Key words: Pressure ulcer, Moxa stick moxibustion, Neovascularization, RAS/RAF/ERK signaling pathway, Vascular endothelial growth factors

摘要： 背景  纵观艾灸治疗压力性损伤的研究概况，基于“艾灸治疗压力性损伤的临床效果确切、但其分子机制不清，而恰恰现代医学对压力性损伤组织血管新生的分子机制研究较为深入，但缺乏有效的治疗手段”的问题现状。本课题组认为其研究重点可以将问题作为切入点，融合现代医学血管新生理论，同时体现中医学艾灸疗法的原创特色，有望弥补艾灸治疗压力性损伤分子机制不清的研究断层，探讨艾灸促进压力性损伤组织血管新生的分子调控机制。 目的　探讨艾灸促进压力性损伤组织血管新生的分子调控机制，为临床艾灸治疗压力性损伤，乃至皮肤溃疡提供理论和实验依据。方法　2015年12月—2017年3月选取清洁级雌性健康SD大鼠120只，85只给予造模处理，另35只作为空白组。采用自制模型装置通过缺血-再灌注损伤方式建立2~3期压力性损伤动物模型，将造模成功的70只SD雌性大鼠随机分为模型组与艾灸组，35只/组；造模后至干预过程中模型组和艾灸组各有5只大鼠脱落。空白组有5只大鼠于备皮时出现负损伤给予剔除。模型组仅对压力性损伤创面进行碘伏常规处理；艾灸组先对压力性损伤创面予碘伏处理，继予艾灸回旋灸操作，以压力性损伤创面为中心，10 mm为半径，1次/d，15 min/次；空白组仅对模拟造模部位进行碘伏处理。通过免疫组织化学法检测各组大鼠治疗第1、3、5、7、10天压力性损伤创面组织中血管内皮生长因子（VEGF）、RAS、磷酸化细胞外调节蛋白激酶（P-ERK1/2）表达水平。结果　VEGF蛋白在压力性损伤皮肤组织中的阳性表达部位位于胞质中，在上皮细胞、巨噬细胞中均有表达。RAS蛋白、P-ERK1/2蛋白在压力性损伤皮肤组织中的阳性表达部位均位于胞质中。当压力性损伤出现后，创面组织中VEGF及P-ERK1/2蛋白表达均逐渐升高，再降低，向正常皮肤组织表达水平回落。艾灸组与模型组两组组内均呈现先升高后降低的表达趋势，但两组组内表达高峰值存在区别，艾灸组组内高峰值出现在治疗3 d后，而模型组组内高峰值出现在碘伏处理5 d后。当损伤出现后，创面组织中RAS蛋白表达较正常组织呈现先降低，而后升高的表达状态。艾灸组与模型组两组组内均呈现逐渐升高的趋势。第1、3、5、7、10天模型组压力性损伤创面组织中VEGF表达水平高于空白组（P<0.05）；第3、5天艾灸组压力性损伤创面组织中VEGF表达水平高于模型组（P<0.05）。第1、3、5、7、10天模型组压力性损伤创面组织中RAS表达水平低于空白组（P<0.05）；第3、5、7、10天艾灸组压力性损伤创面组织中RAS表达水平高于模型组（P<0.05）。第3、5、7、10天模型组压力性损伤创面组织中P-ERK1/2表达水平高于空白组（P<0.05）；第3、5天艾灸组压力性损伤创面组织中P-ERK1/2表达水平高于模型组（P<0.05）。结论　艾灸干预从蛋白层面上调压力性损伤创面组织中VEGF表达水平，其可能通过激活RAS/RAF/ERK信号通路，上调RAS蛋白表达水平、促进ERK1/2蛋白的磷酸化水平，增强RAS/RAF/ERK信号通路的活性，发挥促进血管内皮细胞增殖的作用。

关键词: 压力性溃疡, 艾条灸, 血管生成, RAS/RAF/ERK信号通路, 血管内皮生长因子类