丹皮酚调控p38MAPK-Nrf2信号通路减轻内皮细胞焦亡影响大鼠皮瓣缺血再灌注损伤机制

Paeonol alleviates rat skin flap ischemia-reperfusion injury by reducing endothelial cell pyroptosis via regulating p38MAPK-Nrf2 signaling pathway

关键词:  丹皮酚  P38丝裂原激活蛋白激酶  大鼠  皮瓣  缺血再灌注损伤  氧化应激

Key words:  paeonol  p38 mitogen-activated protein kinase  rat  skin flap  ischemia-reperfusion injury  oxidative stress

摘要:

[目的] 探讨丹皮酚改善大鼠皮瓣缺血再灌注损伤（I/R）的机制。[方法] 60只雄性Sprague-Dawley大鼠随机分为4组：假手术（Sham）组、I/R组、丹皮酚组、丹皮酚+ML385组，每组15只。除Sham组外，其他组建立 I/R损伤皮瓣模型。丹皮酚组、丹皮酚+ML385组给予50 mg/kg丹皮酚治疗。丹皮酚+ML385组腹腔注射30 mg/kg ML385。7 d后，分析大鼠皮瓣存活面积和皮瓣中的血流量。通过免疫荧光染色分析皮瓣中肌动蛋白α2（ACTA2）、CD31、消皮素D蛋白N端结构域（GSDMD-N）表达情况，二氢乙锭染色分析活性氧（ROS）水平，蛋白免疫印迹分析P38丝裂原激活蛋白激酶（p38MAPK）-核因子E2相关因子2（Nrf2）信号。[结果] 与Sham组相比，I/R组大鼠的皮瓣存活面积、皮瓣血流信号强度、CD31和ACTA2共定位阳性血管面积、血管内皮生长因子（VEGF）、基质金属蛋白酶9（MMP9）蛋白表达降低（P<0.05），CD31和GSDMD-N共定位阳性细胞数增加（P<0.05）。与I/R组相比，丹皮酚组大鼠的皮瓣存活面积、皮瓣血流信号强度、CD31和ACTA2共定位阳性血管面积以及VEGF、MMP9血管生成相关蛋白表达增加（P<0.05），CD31和GSDMD-N共定位阳性细胞数明低（P<0.05）。丹皮酚组大鼠皮瓣ROS的相对荧光强度、丙二醛（MDA）水平较I/R组降低（P<0.05），还原型谷胱甘肽（GSH）水平较I/R组增加（P<0.05）。与丹皮酚组相比，丹皮酚+ML385组大鼠的皮瓣中Nrf2蛋白表达减少（P<0.05），p-P38蛋白表达增加（P<0.05）。此外，ML385加入逆转了丹皮酚对皮瓣I/R大鼠模型皮瓣存活面积、皮瓣血流信号强度、血管生成、细胞焦亡、氧化应激的改善作用。[结论] 丹皮酚通过增强皮瓣I/R模型大鼠抗氧化防御系统，保护真皮的血管内皮细胞免受氧化应激损伤，减轻了细胞焦亡，其作用机制可能与调控p38MAPK-Nrf2信号通路有关。

Abstract:

[Objective] To explore the mechanism of paeonol in improving skin flap ischemia-reperfusion injury（I/R） in rats. [Methods] Sixty male Sprague-Dawley rats were randomly divided into four groups：sham group，I/R group，paeonol group，and paeonol+ML385 group，with 15 rats in each group. Except for the sham group，the other groups were used to establish flap I/R injury models. The paeonol group and paeonol+ML385 group were treated with 50 mg/kg paeonol. The paeonol+ML385 group was intraperitoneally injected with 30 mg/kg ML385. After 7 days，the survival area of the rat flaps and blood flow in the flaps were analyzed. The expressions of actin alpha cardiac muscle 2（ACTA2），CD31，and the N-terminal domain of gasdermin D（GSDMD-N） in the flaps were analyzed by immunofluorescence staining. Reactive oxygen species（ROS） levels were analyzed by dihydroethidium staining. The p38 mitogen-activated protein kinase（p38MAPK）-nuclear factor E2-related factor 2（Nrf2） signaling pathway was analyzed by Western blot. [Results] Compared with the sham group，the I/R group showed significantly decreased flap survival area，flap blood flow signal intensity，area of CD31 and ACTA2 co-localized positive vessels，and protein expressions of vascular endothelial growth factor（VEGF） and matrix metalloproteinase 9（MMP9）（P<0.05），while the number of CD31 and GSDMD-N co-localized positive cells was significantly increased（P<0.05）. Compared with the I/R group，the paeonol group showed significantly increased flap survival area，flap blood flow signal intensity，area of CD31 and ACTA2 co-localized positive vessels，and protein expressions of VEGF and MMP9（P<0.05），while the number of CD31 and GSDMD-N co-localized positive cells was significantly decreased（P<0.05）. The relative fluorescence intensity of ROS and malondialdehyde（MDA） level in the flap of the paeonol group were significantly lower than those in the I/R group（P<0.05），while the glutathione（GSH） level was significantly higher（P<0.05）. Compared with the paeonol group，the paeonol+ML385 group showed significantly decreased Nrf2 protein expression（P<0.05） and significantly increased p-p38 protein expression（P<0.05） in the flaps. Furthermore，the addition of ML385 reversed the beneficial effects of paeonol on flap survival area，blood flow signal intensity，angiogenesis，pyroptosis，and oxidative stress in the rat flap I/R model. [Conclusion] Paeonol protects dermal vascular endothelial cells from oxidative stress injury and reduces pyroptosis by enhancing the antioxidant defense system in rats with flap I/R injury. Its mechanism may be related to the regulation of the p38MAPK-Nrf2 signaling pathway.