普萘洛尔通过负调控VEGF和PI3K/Akt信号通路抑制大鼠烧伤后的瘢痕形成

Inhibition of Propranolol on Burn-induced Scar in Rats by Negatively-regulating VEGF and PI3K/Akt Signal Pathway

目的探讨普萘洛尔对大鼠烧伤后瘢痕形成的影响及机制。方法将30只SD大鼠随机分为对照组、模型组、普萘洛尔低剂量组、普萘洛尔中剂量组、普萘洛尔高剂量组,每组各6只。对照组大鼠正常饲养;恒温恒压烫伤仪烫伤其它各组大鼠背部皮肤构建模型组。普萘洛尔低剂量组、普萘洛尔中剂量组、普萘洛尔高剂量组大鼠分别使用剂量为12.5 mg/kg、25 mg/kg、50 mg/kg的普萘洛尔进行灌胃处理,持续灌胃3周。每周观察记录大鼠状态和瘢痕状况。利用免疫荧光法检测各组瘢痕细胞中血管标志物CD31和CD34的表达。采用实时荧光逆转录聚合酶链反应(reverse transcription quantitative polymerase chain reaction,RT-qPCR)检测各组大鼠瘢痕组织内血管内皮生长因子(vascular endothelial growth factor,VEGF)、磷脂酰肌醇-3激酶(phosphatidylinositol 3-kinase,PI3K)和蛋白激酶B(protein kinase B,Akt)的mRNA表达;采用免疫组织化学法检测VEGF、磷酸化蛋白激酶B(phosphorylated protein kinase B,p-Akt)、磷酸化磷脂酰肌醇-3激酶(phosphorylated phosphatidylinositol 3-kinase,p-PI3K)的蛋白表达情况。结果经过治疗后,与模型组比较,普萘洛尔低、中、高剂量组的瘢痕组织厚度、血管分布、柔软性评分均明显降低(P均<0.05)。与模型组比较,普萘洛尔低、中、高剂量组大鼠瘢痕组织内CD31和CD34表达显著降低(P均<0.05)。与对照组比较,模型组和普萘洛尔低、中、高剂量组大鼠瘢痕组织内VEGF、PI3K和Akt的mRNA表达,p-PI3K、p-Akt和VEGF的蛋白表达显著增加(P均<0.01);与模型组比较,普萘洛尔低、中、高剂量组大鼠瘢痕组织内VEGF、PI3K和Akt的mRNA表达,p-PI3K、p-Akt和VEGF的蛋白表达显著下调(P均<0.05);与普萘洛尔低剂量组相比,普萘洛尔中剂量组和普萘洛尔高剂量组大鼠瘢痕组织内VEGF、PI3K和Akt的mRNA表达,p-PI3K、p-Akt和VEGF蛋白表达显著下调(P均<0.05);与普萘洛尔中剂量组相比,普萘洛尔高剂量组大鼠瘢痕组织内VEGF、PI3K和Akt的mRNA表达,p-PI3K、p-Akt和VEGF蛋白表达显著下调(P均<0.05)。结论普萘洛尔通过负调控VEGF和PI3K/Akt信号通路抑制血管生成,从而有效治疗瘢痕。

Objective To investigate the effects of propranolol on burn-induced scar in rats and its mechanism.Meth-ods A total of 30 SD rats were randomly divided into control group,model group,low,medium and high-dose propranolol groups,with 6 rats in each.The control group was raised normally;the model groups were constructed by scalding the back skin of rats in other groups by scald apparatus with constant temperature and isopiestic pressure.The low-dose,medium-dose and high-dose propranolol groups were treated with 12.5 mg/kg,25 mg/kg and 50 mg/kg propranolol by gavage for 3 weeks respec-tively.The state and scar status of the rats were observed and recorded every week.The expression of vascular markers CD31 and CD34 in scar cells of each group were detected by immunofluorescence.The mRNA expressions of vascular endothelial growth factor(VEGF),phosphatidylinositol 3-kinase(PI3K)and protein kinase B(Akt)of scar tissues in each group of rats were de-tected by reverse transcription quantitative polymerase chain reaction(RT-qPCR);the expressions of VEGF,phosphorylated protein kinase B(p-Akt)and phosphorylated phosphatidylinositol 3-kinase(p-PI3K)were detected by im-munohistochemical method.Results After the treatments,compared with the model group,the thickness,vascular distribution and pliability scores of scar tissues in low-dose,medium-dose and high-dose propranolol groups significantly decreased(all P<0.05).Compared with the model group,the expressions of CD31 and CD34 in scar tissues decreased in low-dose,me-dium-dose and high-dose propranolol groups(all P<0.05).Compared with control group,and the mRNA expressions of VEGF,PI3K and Akt,and the protein expressions of p-PI3K,p-Akt and VEGF in scar tissues of rats in model group and low-dose,me-dium-dose and high-dose propranolol groups significantly increased(all P<0.01);compared with the model group,the mRNA expressions of VEGF,PI3K and Akt,and the protein expressions of p-PI3K,p-Akt and VEGF in scar tissues of rats in low-dose,medium-dose and high-dose propranolol groups were significantly down-regulated(all P<0.05);compared with the low-dose propranolol group,the mRNA expressions of VEGF,PI3K and Akt,and the protein expressions of p-PI3K,p-Akt and VEGF in scar tissues of rats in medium-dose and high-dose propranolol groups were significantly down-regulated(all P<0.05);compared with the medium-dose propranolol group,the mRNA expressions of VEGF,PI3K and Akt,and the protein expressions of p-PI3K,p-Akt and VEGF in scar tissues of rats in high-dose propranolol group were significantly down-regulated(all P<0.05).Conclusion Propranolol inhibits angiogenesis by negatively regulating VEGF and PI3K/Akt signaling pathways,which can ef-fectively treating scars.