Objective: To explore the effects of human erythropoietin (hEPO) on healing related transforming growth factor β1 (TGF-β1)/Smad3 signal transduction pathway in acute wounds of rats. Methods: Seventy-two healthy Sprague Dawley rats were divided into normal saline control group, low dose group, middle dose group, and high dose group according to the random number table, with 18 rats in each group, after round acute wounds with diameter of 2.5 cm were inflicted on the back of rats. Rats in the 4 groups had debridement routinely. Wounds of rats in normal saline control group were covered by gauzes infiltrated with 1 mL normal saline, while wounds of rats in low dose group, middle dose group, and high dose group were respectively covered by gauze infiltrated with 1 mL hEPO in doses of 50, 100, and 150 U every day, and then the wounds were bandaged with 6 layers of dry gauze. Dressing change was performed once every day. On treatment day (TD) 3, 7, and 14, 6 rats from each group were taken for general observation and calculation of wound healing rate. Then the wound tissue samples were harvested after the rats were sacrificed for observation of expressions of CD31 and transforming growth factor β1 (TGF-β1) with immunohistochemical method. Protein expression of phosphorylated Smad3 of the wound tissue of 3 rats were detected by Western blotting. Data were processed with analysis of variance of factorial design, one-way analysis of variance, least-significant difference test, and Bonferroni correction. Results: (1) On TD 3, obvious exudation and scab were observed in the wounds of rats in the 4 groups. On TD 7, the wounds of rats in low dose group, middle dose group, and high dose group were reduced compared with those in normal saline control group. On TD 14, all wounds of rats in the 4 groups were healed. On TD 7, the wound healing rates of rats in middle dose group and high dose group were significantly higher than the rate in normal saline control group (P<0.01). At the other time points, the wound healing rates of rats in the 4 groups were close (P>0.05). (2) CD31 mainly expressed in blood vessels. Except for those in low dose group on TD 3 and 7 (P>0.05), the expressions of CD31 in wound tissue of rats in low dose group on TD 14 and in middle dose group and high dose group on TD 3, 7, and 14 were significantly higher than those in normal saline control group (P<0.01). Except for those on TD 3 (P>0.05), the expressions of CD31 in wound tissue of rats in middle dose group and high dose group on TD 7 and 14 were significantly higher than those in low dose group (P<0.01). Except for that on TD 3 (P>0.05), the expressions of CD31 in wound tissue of rats in high dose group on TD 7 and 14 were significantly higher than those in middle dose group (P<0.01). (3) Except for that in low dose group on TD 3 (1.9±0.7, P>0.05), the expressions of TGF-β1 in wound tissue of rats in low dose group on TD 7 and 14 (3.3±1.0, 3.7±0.7), and in middle dose group and high dose group on TD 3, 7, and 14 (3.3±1.0, 3.6±1.0, 3.9±0.9, 3.4±0.7, 3.8±0.8, 4.2±0.4) were significantly higher than those in normal saline control group (1.7±0.5, 2.7±1.0, 3.0±0.9, P<0.01). Except for those on TD 7 (P>0.05), the expressions of TGF-β1 in wound tissue of rats in middle dose group and high dose group on TD 3 and 14 were significantly higher than those in low dose group (P<0.01). Except for that on TD 14 (P<0.01), the expressions of TGF-β1 in wound tissue of rats in high dose group on TD 3 and 7 were close to those in middle dose group (P>0.05). (4) Except for those in low dose group on TD 3 and 14 and in middle dose group and high dose group on TD 14 (P>0.05), the protein expressions of phosphorylated Smad3 in wound tissue of rats in the 3 groups at the other time points were significantly higher than those in normal saline control group (P<0.01). Except for those on TD 14 (P>0.05), the protein expressions of phosphorylated Smad3 in wound tissue of rats in middle dose group and high dose group on TD 3 and 7 were significantly higher than those in low dose group (P<0.01). Except for that on TD 14 (P>0.05), the protein expressions of phosphorylated Smad3 in wound tissue of rats in high dose group on TD 3 and 7 were significantly lower than those in middle dose group (P<0.01). Conclusions: Exogenous hEPO can increase the expressions of CD31, TGF-β1, and phosphorylated Smad3 in acute wounds of rats, promote angiogenesis of wounds, and activate TGF-β1/Smad3 signal transduction pathway to promote wound healing.
目的： 探究人促红细胞生成素(hEPO)对大鼠急性创面愈合相关转化生长因子β1(TGF－β1)/Smad3信号转导通路的影响。 方法： 选取72只健康SD大鼠，在大鼠背部制成直径约2.5 cm的圆形急性创面，按随机数字表法将大鼠分为生理盐水对照组、低剂量组、中剂量组、高剂量组，每组18只。4组大鼠每日常规清创后，生理盐水对照组大鼠创面予1 mL生理盐水浸润的纱布外敷，低剂量组、中剂量组、高剂量组大鼠创面每天分别予1 mL 50、100、150 U的hEPO浸润的纱布湿敷，再用6层干纱布包扎固定，每天换药1次。治疗3、7、14 d每组取6只大鼠行大体观察并计算创面愈合率，处死后取创面组织采用免疫组织化学法观察CD31和TGF－β1的表达，蛋白质印迹法检测3只大鼠磷酸化Smad3蛋白的表达。对数据行析因设计方差分析、单因素方差分析、LSD检验、Bonferroni校正。 结果： (1)治疗3 d，4组大鼠创面均有明显的渗出并有结痂。治疗7 d，低剂量组、中剂量组、高剂量组大鼠创面均较生理盐水对照组明显缩小。治疗14 d，4组大鼠创面均全部愈合。治疗7 d，与生理盐水对照组相比，中剂量组、高剂量组大鼠创面愈合率明显增加(P<0.01)；余时间点，各组大鼠创面愈合率相近(P>0.05)。(2)CD31主要定位在血管上。除低剂量组治疗3、7 d(P>0.05)外，低剂量组大鼠治疗14 d和中剂量组、高剂量组大鼠治疗3～14 d创面组织中CD31表达均明显高于生理盐水对照组(P<0.01)；除治疗3 d(P>0.05)外，中剂量组和高剂量组大鼠治疗7、14 d创面组织中CD31表达均明显高于低剂量组(P<0.01)；除治疗3 d(P>0.05)外，高剂量组大鼠治疗7、14 d创面组织中CD31表达明显高于中剂量组(P<0.01)。(3)除低剂量组治疗3 d(1.9±0.7，P>0.05)外，低剂量组大鼠治疗7、14 d(3.3±1.0、3.7±0.7)和中剂量组、高剂量组大鼠治疗3～14 d创面组织中TGF－β1表达(3.3±1.0、3.6±1.0、3.9±0.9，3.4±0.7、3.8±0.8、4.2±0.4)均明显高于生理盐水对照组(1.7±0.5、2.7±1.0、3.0±0.9，P<0.01)；除治疗7 d(P>0.05)外，中剂量组和高剂量组大鼠治疗3、14 d创面组织中TGF－β1表达均明显高于低剂量组(P<0.01)；除治疗14 d(P<0.01)外，高剂量组大鼠治疗3、7 d创面组织中TGF－β1表达与中剂量组相近(P>0.05)。(4)除低剂量组治疗3、14 d及中剂量组和高剂量组治疗14 d(P>0.05)外，3组大鼠余时间点创面组织中磷酸化Smad3蛋白表达明显高于生理盐水对照组(P<0.01)；除治疗14 d(P>0.05)外，中剂量组和高剂量组大鼠治疗3、7 d创面组织中磷酸化Smad3蛋白表达明显高于低剂量组(P<0.01)；除治疗14 d(P>0.05)外，高剂量组大鼠治疗3、7 d创面组织中磷酸化Smad3蛋白表达明显低于中剂量组(P<0.01)。 结论： 外源性hEPO可提高大鼠急性创面CD31、TGF－β1、磷酸化Smad3的表达，促进创面血管新生，激活TGF－β1/Smad3信号转导通路促进创面愈合。.
Keywords: Antigens, CD31; Human erythropoietin; Smad3 protein; Transforming growth factor beta1; Wound healing.