压力对增生性瘢痕形成及TGF-β1/Smad信号通路的影响

The effect of pressure therapy on the formation of hypertrophic scar and TGF-β1/Smad signaling pathway

目的研究压力对增生性瘢痕(HTS)形成及转化生长因子β1(TGF-β1)/Sma和Mad同源物蛋白(Smad)信号通路的影响。方法健康成年新西兰大白兔12只(空军军医大学动物实验中心提供),用直径为1 cm的圆形打孔器在每侧兔耳腹侧标记出4个圆形创面,用手术小圆刀沿标记线切开皮肤全层至兔耳软骨膜,分离软骨膜及全层皮肤,刮除创面残留组织,暴露创面,术后第28天观察瘢痕形成情况。兔耳HTS模型构建成功后进行分组,采用自身对照研究,兔左耳设为实验组,右耳为对照组,用抽签法于每侧兔耳选取2个HTS模型作为研究对象,每组24个。实验组:使用4-0尼龙丝线将直径为1.5 cm的圆形钕铁硼磁铁垫上压力垫片缝合至兔耳软骨上,使用Flexiforce压力传感器测量压力大小并每周进行调整,将压力控制在20~25 mmHg(1 mmHg=0.133 kPa),每天持续时间≥23 h;对照组:不做处理。在施加压力后第40天观察兔耳瘢痕大体形态,并切取组织行HE、Masson染色进行组织学研究,计算瘢痕增生指数(SEI)、成纤维细胞数、角质层厚度;采用荧光定量PCR检测TGF-β1、Smad3、Ⅰ型胶原(CollagenⅠ)、Ⅲ型胶原(CollagenⅢ)、α-平滑肌肌动蛋白(α-SMA)mRNA相对表达量;采用蛋白质印迹法检测TGF-β1、CollagenⅠ、CollagenⅢ、α-SMA蛋白相对表达量及Smad3磷酸化蛋白(p-Smad3)相对表达量(p-Smad3与Smad3蛋白表达量的比值)。使用Excel 2019、GraphPad Prim 8.0软件进行数据统计分析,计量资料均符合正态分布,以x±s表示,2组间比较采用配对样本t检验,P<0.05为差异有统计学意义。结果12只兔共形成96个创面,有27个创面无明显增生,其余69个创面形成突出皮肤表面、质地坚硬、暗红色的瘢痕增生样组织块,瘢痕形成率为71.9%(69/96)。施加压力后第40天,与对照组相比,实验组瘢痕外观凸起明显降低,硬度变软,颜色稍浅;HE染色和Masson染色结果显示,实验组中角质层厚度、SEI和成纤维细胞数分别为(69.33±6.03)μm、1.30±0.08、(236.30±14.64)个/视野,均显著低于对照组的(114.00±10.15)μm、1.72±0.05、(320.30±14.57)个/视野(均P<0.01),真皮层未见丰富的毛细血管、炎性细胞和成纤维细胞,胶原纤维排列有序,且较稀疏。荧光定量PCR检测结果表明,实验组TGF-β1、Smad3、CollagenⅠ、CollagenⅢ、α-SMA mRNA相对表达量分别为0.48±0.08、0.58±0.05、0.04±0.01、0.15±0.02、0.31±0.03,均低于对照组的1.00±0.07、1.00±0.05、1.00±0.08、1.00±0.10、1.00±0.06(均P<0.01)。蛋白质印迹法检测结果显示,实验组TGF-β1、CollagenⅠ、CollagenⅢ、α-SMA蛋白相对表达量及Smad3磷酸化蛋白相对表达量分别为0.65±0.03、0.07±0.01、0.43±0.03、0.53±0.03、0.54±0.03,均低于对照组的1.02±0.06、0.93±0.05、0.92±0.03、0.82±0.03、0.92±0.03(均P<0.01)。结论压力疗法可明显抑制瘢痕的增生,改善HTS组织结构,有利于胶原纤维蛋白的正常排列,减少胶原蛋白的过度沉积;压力疗法可能通过调控TGF-β1/Smad信号通路来抑制瘢痕的增生。

Objective To observe the effect of pressure therapy on the formation of hypertrophic scars(HTS)and transforming growth factor beta 1(TGF-β1)/Sma and Mad homolog proteins(Smad)signaling pathway.Methods Twelve adult healthy New Zealand white rabbits(provided by the Animal Experiment Center of the Air Force Military Medical University)were wounded with 1 cm round punch on 4 sites of the ventral side of each ear.Round scalpels were used to make incisions along the marked lines,dissect the skin and perichondrium.The remaining tissue was scraped off to expose the wound surface.Scar formation was observed on the 28th day after surgery.After the establishment of rabbit ear HTS models,the right ears were used as self-controls,while the left ears were set as the experimental group.Two hypertrophic scars were randomly selected from each rabbit ear,24 per group.Experimental group:4-0 nylon silk thread was used to sew the pressure pad on the circular NdFeB magnets pad with a diameter of 1.5 cm to the rabbit ear cartilage.Flexiforce pressure sensor was used to measure the pressure,and the pads were adjusted to maintain a pressure of 20-25 mmHg(1 mmHg=0.133 kPa)for more than 23 h per day.Control group:no treatment.On the 40th day of pressure therapy,the general morphology of rabbit ear scars were observed,and the tissues were harvested for hematoxylin and eosin(HE)staining and Masson’s Trichrome staining for histological study.The scar elevation index(SEI),the number of fibroblasts,and the thickness of the stratum corneum were calculated.The relative mRNA expression levels of TGF-β1,Smad3,collagen type(Collagen)Ⅰ,Collagen Ⅲ,α-smooth muscle actin(α-SMA)were measured with qPCR;Western blotting was used to detect the relative protein expression levels of TGF-β1,Collagen Ⅰ,Collagen Ⅲ,α-SMA and the phosphorylation level of Smad3(the ratio of p-Smad3 and Smad3 proteins).Statistical analysis was performed with Excel 2019 and GraphPad Prism 8.0.The measurement data conformed to normal distribution and was expressed as Mean±SD.Student’s t-test was used for the comparison between two groups.P<0.05 was considered statistically significant.Results A total of 96 wounds were formed in 12 rabbits,27 wounds had no obvious hyperplasia,and the remaining 69 wounds formed hypertrophic scar tissue blocks with a prominent skin surface,firm texture,and dark red appearance.The scars formation rate was 71.9%(69/96).On the 40th day after the application of pressure,the scars in the experimental group were significantly reduced,softer,and the color was slightly lighter compared with the control group.The results of HE staining and Masson’s Trichrome staining showed that the thickness of the stratum corneum,SEI,and the number of fibroblasts were(69.33±6.03)μm,1.30±0.08,and(236.30±14.64)cells/field,respectively,which were significantly lower than those in the control group[(114.00±10.15)μm,1.72±0.05,(320.30±14.57)cells/field](all P<0.01).Abundance in capillaries,inflammatory cells,and fibroblasts were not observed in the dermal layer.The collagen fibers were orderly arranged and sparse.The results of fluorescence quantitative PCR showed that the relative expression levels of TGF-β1,Smad3,Collagen Ⅰ,Collagen Ⅲ,and α-SMA mRNA in the experimental group were 0.48±0.08,0.58±0.05,0.04±0.01,0.15±0.02,0.31±0.03,respectively,lower than those of the control group(1.00±0.07,1.00±0.05,1.00±0.08,1.00±0.10,1.00±0.06)(all P<0.01).The results of Western blotting showed that the relative protein expression of TGF-β1,Collagen Ⅰ,Collagen Ⅲ,α-SMA and the phosphorylation level of Smad3 in the experimental group were 0.65±0.03,0.07±0.01,0.43±0.03,0.53±0.03,0.54±0.03,all lower than the control group’s 1.02±0.06,0.93±0.05,0.92±0.03,0.82±0.03,0.92±0.03(all P<0.01).Conclusions Pressure therapy can significantly inhibit the hyperplasia of scars,improve the structure of HTS tissue,facilitate the normal arrangement of collagen fiber,and reduce the excessive deposition of collagen.Pressure therapy may inhibit scar proliferation by regulating the TGF-β1/Smad signaling pathway.