Inhibition of Notch signaling pathway reduces angiogenesis in hypertrophic scar.

Hypertrophic scar (HS) is the most common pathological scar in clinical practice. During its formation, angiogenesis-related factors show dynamic expression. Modern studies have found that Notch signaling pathway has an extremely important role in maintaining the construction and remodeling of vascular endothelial cells and vascular network. The correlation between Notch signaling pathway and angiogenesis in hypertrophic scar has been rarely reported. This study aims to investigate correlation between Notch signaling pathway and the expression of angiogenic factors in a proliferative scar model.A total of 81 Sprague Dawley rats (SPF grade) were randomly assigned into a blank control group, a model group, and a blocker group. In the blocker group, a 2 cm diameter circular scald head was placed on the back of the rats for 10 s at 75 ℃ by using a constant temperature and pressure electrothermal scalding apparatus to form a rat deep II° burn model, and a hyperplastic scar model rat was obtained after natural healing of the wound skin (21 to 23 day epithelialization). A syringe was used to inject a needle from the normal skin around the scar at the 1st, 3rd, 5th, 7th, and 14th days after modeling. The γ-secretase inhibitor was injected locally at 2 mg/kg in a dilution of 0.1 mL at the base of the scar. The rats in the model group was injected with the same amount of saline after modeling; the rats in the blank control group was injected with the same amount of saline. Nine rats in each group was randomly killed by air embolization at the 21st, 28th, and 35th days, respectively. The protein expressions of collagen type I (COL-I) and collagen type III (COL-III) were detected by immunohistochemistry. The protein expressions of vascular endothelial growth factor (VEGF), angiopoietin 1 (Ang1), transforming growth factor-β1 (TGF-β1), and matrix metalloproteinase-2 (MMP-2) were detected by Western blotting.Immunohistochemical results showed that, at the 21st,28th, and 35th days, the protein expressions of COL-I and COL-III in the model group were up-regulated compared with the blank control group (all P<0.05) and the protein expressions of COL-I and COL-III in the blocker group were decreased compared with the model group (all P<0.05). Western blotting showed that, at the 21st, 28th, and 35th days, the protein expressions of VEGF, Ang1, TGF-β1, and MMP-2 in the model group were significantly higher than those in the blank control group (all P<0.05). Except for the 21st day, the protein expressions of VEGF, Ang1, TGF-β1, and MMP-2 in the blocker group were lower than those in the model group at the 28th and 35th days (all P<0.05).In the Sprague Dawley rat proliferative scar model, inhibition of Notch signaling pathway could attenuate the expressions of COL-I and COL-III, reduce traumatic scar proliferation, down-regulate the expressions of VEGF, Ang1, TGF-β1, and MMP-2, and inhibit angiogenesis. The expressions of angiogenesis-related factors appeare to be up-regulated during the formation of proliferative scar. When the Notch signaling pathway is inhibited, the up-regulated angiogenic factors show a decreasing trend and the proliferative scar is alleviated, which suggests that Notch signaling pathway may affect the formation of hyperplastic scar by regulating the expression of angiogenic factors.目的: 增生性瘢痕是临床上最常见的病理性瘢痕，在其形成过程中，血管生成相关因子呈现出动态表达。现代研究发现Notch信号通路对于维持血管内皮细胞和血管网的构建与重塑有着极其重要的作用，而Notch信号通路与血管生成的相关性在增生性瘢痕中的研究鲜有报道。本研究旨在探讨增生性瘢痕模型中Notch信号通路与血管生成因子表达的相关性。方法: 将81只Sprague Dawley大鼠(SPF级)随机分成空白对照组、模型组和阻滞剂组。其中阻滞剂组采用恒温恒压电热烫伤仪，在75 ℃条件下，将一个直径为2 cm的圆形烫伤头置于大鼠背部10 s，造成大鼠深II°烧伤，待烧伤皮肤自然愈合(21~23 d上皮化)后得到增生性瘢痕模型大鼠，在造模后第1、3、5、7和14天5个时间点用注射器从瘢痕周围正常皮肤进针，在瘢痕基底局部按2 mg/kg注射0.1 mL γ-分泌酶阻滞剂的稀释液。模型组在造模后同法注射等量生理盐水；空白对照组不造模，用同法注射等量生理盐水。每组分别在第21、28、35天3个时间点随机选取9只大鼠采用空气栓塞法处死。采用免疫组织化学法检测I型胶原蛋白(collagen type I，COL-I)、III型胶原蛋白(collagen type III，COL-III)的蛋白质表达；采用蛋白质印迹法检测血管内皮生长因子(vascular endothelial growth factor，VEGF)、血管生成素1(angiopoietin 1，Ang1)、转化生长因子-β1(transforming growth factor-β1，TGF-β1)、基质金属蛋白酶-2(matrix metalloproteinase-2，MMP-2)的蛋白质表达。结果: 免疫组织化学结果显示：在第21、28、35天3个时间点，与空白对照组比较，模型组COL-I、COL-III的蛋白质表达上调(均P<0.05)；与模型组相比，阻滞剂组COL-I、COL-III的蛋白质表达下降(均P<0.05)。蛋白质印迹法结果显示：在第21、28、35天3个时间点，与空白对照组相比，模型组VEGF、Ang1、TGF-β1、MMP-2的蛋白质表达量均显著升高(均P<0.05)；除第21天外，在第28、35天阻滞剂组VEGF、Ang1、TGF-β1及MMP-2的蛋白质表达量低于模型组(均P<0.05)。结论: 在Sprague Dawley大鼠增生性瘢痕模型中，抑制Notch信号通路可以减弱COL-I、COL-III的表达，减轻创面瘢痕增生，下调VEGF、Ang1、TGF-β1、MMP-2的表达，抑制血管新生。可见在增生性瘢痕的形成过程中，血管生成相关因子的表达出现上调；当抑制Notch信号通路后，上调的血管生成因子出现下降趋势，增生性瘢痕的情况也得到缓解。说明Notch信号通路可能通过调控血管生成因子的表达来影响增生性瘢痕的形成。.