肉桂醛通过PINK1/Parkin信号通路介导的线粒体自噬促进糖尿病大鼠创面愈合机制

Mechanism of Cinnamaldehyde in Promoting Wound Healing in Diabetes Rats via PINK1/Parkin-mediated Mitochondrial Autophagy

目的:采用高糖高脂饲料喂养联合腹腔注射链尿佐菌素(STZ)及手术制备全层皮肤缺损的方法制备糖尿病大鼠创面模型,观察肉桂醛对糖尿病大鼠创面愈合情况的影响,并基于PTEN诱导假定激酶(PINK)1/帕金森病蛋白(Parkin)信号通路介导的线粒体自噬探讨肉桂醛对糖尿病大鼠创面的治疗机制。方法:48只雄性SD大鼠随机分为空白组12只,糖尿病组36只,糖尿病组随机分为模型组、肉桂醛组和贝复新组,每组12只,空白组与模型组创面常规消毒后予生理盐水,肉桂醛组创面局部外敷含4μmol·L^(-1)肉桂醛的聚乙二醇400(PEG 400)凝胶,贝复新组创面外敷贝复新凝胶,每日换药1次。观察各组创面愈合率,取干预后7、14 d大鼠创面组织,苏木素-伊红(HE)染色观察局部组织病理变化,免疫组化(IHC)检测白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、血管内皮生长因子(VEGF)、胶原纤维的表达情况,免疫荧光(IF)、实时荧光定量聚合酶链式反应(Real-time PCR)检测PINK1、Parkin、微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)蛋白和mRNA的表达情况。结果:腹腔注射STZ后,与空白组比较,糖尿病组大鼠随机血糖值显著升高(P<0.01),均高于16.7 mmol·L^(-1),且在造模后一段时间持续保持高血糖状态。与空白组比较,模型组大鼠创面肉芽组织生长、愈合情况较差,创面愈合率显著降低(P<0.01);干预后7 d,空白组创面已有鳞状上皮覆盖,与空白组比较,模型组大鼠创面仅创缘少量结痂,创面中大量炎细胞浸润,组织IL-6、TNF-α蛋白表达水平显著上升(P<0.01),PINK1、Parkin、LC3Ⅱ蛋白表达及mRNA水平显著降低(P<0.01);干预后14 d,空白组创面肉芽组织成熟,愈合良好,与空白组比较,模型组创面炎细胞浸润和红细胞渗出尚未完全消退,组织VEGF、胶原纤维蛋白表达水平显著降低(P<0.01),PINK1、Parkin、LC3Ⅱ蛋白表达及mRNA水平显著升高(P<0.01)。与模型组比较,肉桂醛组和贝复新组大鼠创面愈合情况较好,创面愈合率显著升高(P<0.01),干预后7 d组织IL-6、TNF-α蛋白表达水平显著降低(P<0.01),PINK1、Parkin及LC3Ⅱ蛋白表达及mRNA水平显著升高(P<0.01),干预后14 d组织VEGF、胶原纤维蛋白表达水平显著上升(P<0.01),PINK1、Parkin、LC3Ⅱ蛋白表达及mRNA水平显著降低(P<0.01)。结论:肉桂醛能促进糖尿病创面愈合,上调创面愈合率,降低炎症因子IL-6、TNF-α的水平,增加VEGF、胶原纤维蛋白的水平,其机制可能是通过调节PINK1/Parkin信号通路表达,激活线粒体自噬,抑制炎症反应,增加血管新生和胶原合成,从而达到促进糖尿病大鼠创面愈合的目的。

Objective:To establish a rat model of diabetic wound by feeding on a high-fat and highsugar diet combined with intraperitoneal injection of streptozotocin(STZ) and surgical preparation of fullthickness skin defects,observe the effect of cinnamaldehyde on the wound healing of diabetes rats,and explore the therapeutic mechanism of cinnamaldehyde in improving wound healing of diabetes rats based on the PTENinduced putative kinase(PINK1)/Parkin pathway-mediated mitochondrial autophagy.Method:Forty-eight male SD rats were randomly divided into blank group(n=12) and diabetes group(n=36).The diabetes group was further randomly divided into model group,cinnamaldehyde group,and Beifuxin group,with 12 rats in each group.The blank group and the model group received routine disinfection with physiological saline after creating the wounds,while the cinnamaldehyde group received topical application of polyethylene glycol 400(PEG 400) gel containing 4 μmol·L^(-1) cinnamaldehyde,and the Beifuxin group received topical application of Beifuxin gel.Dressings were changed once daily.The wound healing rate of each group was observed.On the 7th and 14th days after intervention,the wound tissues of the rats were collected.Hematoxylin and eosin(HE) staining was performed to observe the pathological changes in the local tissues.Immunohistochemistry(IHC) was used to detect the expression of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),vascular endothelial growth factor(VEGF),and collagen fibers.Immunofluorescence(IF) and Real-time polymerase chain reaction(Real-time PCR) were used to detect the protein,and mRNA expression of PINK1,Parkin,microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ ).Result:After intraperitoneal injection of STZ,compared with the blank group,the random blood glucose values of rats in the diabetic group increased significantly(P<0.01),all higher than 16.7 mmol·L^(-1),and persistently hyperglycemic for some time after modeling.Compared with the blank group,the model group showed poor growth and healing of granulation tissue in the wounds,and the wound healing rate decreased(P<0.01).On the 7th day after intervention,the blank group had squamous epithelial coverage on the wounds.Compared with the blank group,the model group only had a small amount of scab at the wound edges,with a large number of infiltrating inflammatory cells in the wounds.The protein expression levels of IL-6 and TNF-α in the tissues increased(P<0.01),and the protein and mRNA levels of PINK1,Parkin,and LC3 Ⅱ decreased(P<0.01).On the 14th day after the intervention,the granulation tissue in the wounds of the blank group was mature and well-healed.Compared with the blank group,the model group still had infiltrating inflammatory cells and red blood cell exudation.The protein expression levels of VEGF and collagen fibers in the tissues decreased(P<0.01),and the protein and mRNA expression levels of PINK1,Parkin,and LC3 Ⅱ increased(P<0.01).Compared with the model group,the cinnamaldehyde group and the Beifuxin group showed better wound healing,with increased wound healing rates(P0.01).On the 7th day after intervention,the protein expression levels of IL-6 and TNF-α in the tissues decreased(P<0.01),and the protein and mRNA expression levels of PINK1,Parkin,and LC3 Ⅱ increased(P0.01).On the 14th day after intervention,the protein expression levels of VEGF and collagen fibers in the tissues increased(P0.01),and the protein and mRNA expression levels of PINK1,Parkin,and LC3 Ⅱdecreased(P0.01).Conclusion:Cinnamaldehyde can promote the wound healing of diabetes rats by increasing the wound healing rate,reducing the levels of inflammatory factors IL-6 and TNF-α,and increasing the levels of VEGF and collagen fibers.Its mechanism may be related to the regulation of the PINK1/Parkin signaling pathway,activation of mitochondrial autophagy,inhibition of inflammatory responses,and promotion of angiogenesis and collagen synthesis,thereby promoting the wound healing of diabetes rats.