Effects of moxibustion at different temperature on wound healing and apoptosis in rats with pressure ulcer based on PI3K/AKT/mTOR signal pathway
基于PI3K/AKT/mTOR信号通路探究不同温度艾灸对压疮大鼠创面愈合及细胞凋亡的影响

Si-an PAN（潘思安）^a,b, Shao-hua WANG（汪少华）^b, Han-yu YUAN（袁菡钰）^b, Juan LI（李娟）^b, Xiao XUE（薛晓）^b, Zeng-hui YUE（岳增辉）^b, Yu LIU（刘余）^b

^a School of Chinese Medicine, Hunan University of Chinese Medicine, Changsha 410208, China（湖南中医药大学中医学院，长沙410208，中国）

^b School of Acupuncture, moxibustion&Tuina, Hunan University of Chinese Medicine, Changsha 410208, China（湖南中医药大学针灸推拿与康复学院，长沙410208，中国）

Abstract

Objective: To investigate the effects of moxibustion on wound healing and evaluate whether the efficacy of moxibustion at different temperatures varies. Additionally, to explore the mechanism by which moxibustion regulates the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian rapamycin target protein (mTOR) signaling pathway on wound healing and cell apoptosis.

Methods: A total of 90 SPF-grade SD rats were randomly divided into six groups (15 rats per group): control group, model group, recombinant bovine basic fibroblast growth factor gel (rb-bFGF) group, moxibustion group A [temperature (42 ± 1) ℃], moxibustion group B [temperature (45 ± 1) ℃], and moxibustion group C [ temperature (48 ± 1) ℃]. Except for the control group, the interventions were conducted in the other groups with pressure ulcer induced by ischemia/reperfusion injury. In the control group, povidone-iodine was administered at the knee joint. In the model group, after povidone-iodine applied at the pressure ulcer, petroleum jelly gauze was bandaged. In the rb-bFGF group, after povidone-iodine applied at the pressure ulcer, rb-bFGF was smeared and petroleum jelly gauze was bandaged. In the moxibustion group A, the suspending moxibustion with moxa stick was delivered 3 cm to 5 cm above the ulcer. The paperless temperature recorder probe was placed on the wound surface where moxibustion was delivered, and the real-time temperature was monitored and adjusted, kept at (42 ± 1) ℃. The moxibustion temperature was kept at (45 ± 1) ℃ in the moxibustion group B, and (48 ± 1) ℃ in the moxibustion group C. The intervention lasted 14 days in each group. The wound healing rates were compared on the 3, 5, 7 and 14 days after the first intervention among the 6 groups. After 14-day intervention respectively, the histopathological changes were observed with HE staining, apoptosis was detected with TUNEL method and flow cytometry; VEGF-A protein expression determined with immunohistochemistry; the contents of Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) in serum with ELISA, and PI3K/AKT/mTOR related protein and mRNA expression was with Western blotting and qRT-PCR in wound tissues of each group.

Results: Regarding the intervention effects, (1) compared with the control group, the model group showed significant inflammatory infiltration in wound tissues, higher levels of cell apoptosis, and elevated serum levels of IL-6, TNF-α and TGF-β (P<0.05); (2) compared with the model group, the rb-bFGF group and 3 moxibustion groups showed higher wound healing rates (P<0.05), reduced inflammatory infiltration in the wound tissues, lower levels of cell apoptosis, and decreased serum levels of IL-6, TNF-α and TGF-β (P<0.05). Among the moxibustion groups, group C showed significantly better results than group A (P<0.01). Regarding the mechanism, (1) compared with the control group, the model group exhibited significantly higher protein and mRNA expression levels of PI3K/AKT/mTOR signal pathway components (P<0.05); (2) compared with the model group, the rb-bFGF group and 3 moxibustion groups showed lower protein and mRNA expression levels of PI3K/AKT/mTOR signal pathway components (P<0.05). Among the moxibustion groups, group C showed significantly higher expression levels than group A (P<0.01).

Conclusion: Moxibustion significantly promotes wound healing, with high-temperature moxibustion being more effective than low-temperature moxibustion in reducing the expression of inflammatory factors and cell apoptosis, thereby improving wound healing rates. It is presented that the higher moxibustion temperature, the higher wound healing rate, which may be associated with the regulation of PI3K/AKT/mTOR signal pathway with moxibustion.

【摘要】

目的：探究艾灸对创面愈合的影响及不同温度艾灸对创面愈合的有效性是否具有差异，并借此探究艾灸干预磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（AKT ）/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路影响创面愈合及细胞凋亡的机制。

方法：将90只SPF级SD大鼠随机分为空白组、模型组、贝复新组、艾灸[灸温（42±1）℃A组、艾灸[灸温（45±1）℃B组、艾灸[灸温（48±1）℃C组，每组各15只。除空白组外，其他组制备缺血/再灌注损伤压疮大鼠模型后接受相应干预。空白组：于大鼠在膝关节处涂抹碘伏。模型组：在压疮位置涂抹碘伏后外敷凡士林纱布并包扎。贝复新组：在压疮位置涂抹碘伏后，创面处涂抹贝复新，再用凡士林纱布包扎。艾灸A组：在压疮位置上方3 cm至5 cm处悬挂艾灸条，采用无纸温度记录仪探头置于施灸创面皮肤表面以监测并调节实时温度，温度保持在（42±1）℃；艾灸B组：温度保持在（45±1）℃；艾灸C组：温度保持在（48±1）℃。所有组别均共干预14天。比较6组大鼠在接受干预后3 d、5 d、7 d和14 d的创面愈合率。接受干预后14 d，分别采用HE染色观察创面组织病理学改变，TUNEL法及流式细胞仪检测细胞凋亡情况，免疫组化法分析VEGF-A蛋白表达，ELISA法检测血清中IL-6、TNF-α及TGF-β含量变化，Western Blot及qRT-PCR法检测各组创面组织中PI3K/AKT/mTOR相关蛋白及mRNA表达水平。

结果：与干预效果相关：1.与空白组比较，模型组创面组织出现炎性浸润，细胞凋亡程度较高，血清中IL-6、TNF-α及TGF-β含量较高（P<0.05）。2.与模型组比较，贝复新组及3个艾灸组创面愈合率均较高（P<0.05），创面组织炎性浸润较轻，细胞凋亡程度较低，血清中IL-6、TNF-α及TGF-β含量均较低（P<0.05），其中艾灸C组各项指标均高于艾灸A组（P<0.01）。与机制相关：1.与空白组比较，模型组PI3K/AKT/mTOR信号通路蛋白及mRNA表达水平明显较高（P<0.05）。2.与模型组比较，贝复新组及3个艾灸组PI3K/AKT/mTOR信号通路蛋白及mRNA表达水平均较低（P<0.05），其中艾灸C组PI3K/AKT/mTOR信号通路蛋白及mRNA表达水平高于艾灸A组（P<0.01）。

结论：艾灸能够明显促进创面愈合，且高温艾灸比低温艾灸更能够有效降低压疮大鼠炎症因子表达和细胞凋亡，提高创面愈合率，呈现出创面愈合率随着艾灸温度升高而升高的趋势，其机制可能与艾灸调控PI3K/AKT/mTOR信号通路有关。