广西医学

目的探讨红花抗硬皮病小鼠皮肤纤维化的作用及对转化生长因子β1（TGF⁃β1）/Smads信号通路的影响。方法将64只雌性BALB/c小鼠分为对照组（16只）、模型组（16只）、泼尼松组（8只）、红花前干预组（16只）和红花后干预组（8只）。除对照组外，其他4组小鼠采用皮下注射博来霉素诱导硬皮病小鼠模型。造模当天给予泼尼松组小鼠灌胃0.45 mg/mL醋酸泼尼松混悬液，给予红花前干预组小鼠灌胃0.15 g/mL红花水提液，给予对照组、模型组小鼠灌胃生理盐水，造模第15天给予红花后干预组小鼠灌胃0.15 g/mL红花水提液，以上各组小鼠均连续灌胃4周（0.2 mL/d）。干预4周后，观察各组小鼠皮肤外观变化及皮肤病理组织学改变，比较各组小鼠的真皮厚度，采用免疫组化法检测各组小鼠皮肤组织Ⅰ型胶原蛋白（COLⅠ）、α⁃平滑肌肌动蛋白（α⁃SMA）的蛋白表达水平，采用实时荧光定量PCR检测各组小鼠皮肤组织COLⅠ⁃a1、COLⅠ⁃a2、α⁃SMA、TGF⁃β1、Smad2、Smad3的mRNA表达水平，采用Western blot检测各组小鼠皮肤组织TGF⁃β1、Smad2、Smad3、磷酸化Smad2、磷酸化Smad3的蛋白表达情况。结果（1）模型组小鼠背部未见新生毛发，皮肤硬化明显、有粘连，不易被提起，真皮厚度增厚，各药物干预组皮肤症状较模型组改善，真皮厚度变薄，以泼尼松组、红花前干预组改善最明显。（2）与对照组比较，模型组小鼠皮肤组织COLⅠ、α⁃SMA的蛋白表达水平，以及COLⅠ⁃a1、COLⅠ⁃a2、α⁃SMA的mRNA表达水平升高（P<0.05）；与模型组比较，红花前干预组、红花后干预组和泼尼松组小鼠皮肤组织COLⅠ、α⁃SMA的蛋白表达水平，以及COLⅠ⁃a1、COLⅠ⁃a2、α⁃SMA的mRNA表达水平下降，且泼尼松组、红花前干预组小鼠皮肤组织COLⅠ、α⁃SMA的蛋白表达水平低于红花后干预组，红花前干预组小鼠皮肤组织COLⅠ⁃a1、COLⅠ⁃a2的mRNA表达水平低于红花后干预组（P<0.05）。（3）与对照组比较，模型组小鼠皮肤组织TGF⁃β1、Smad2、Smad3的蛋白及mRNA，以及磷酸化Smad2、磷酸化Smad3的蛋白表达水平升高，红花前干预组小鼠皮肤组织TGF⁃β1、Smad3的mRNA表达水平降低，Smad2的mRNA表达水平升高（P<0.05）；与模型组比较，红花前干预组小鼠皮肤组织TGF⁃β1、Smad2、Smad3的蛋白及mRNA，以及磷酸化Smad2、磷酸化Smad3的蛋白表达水平降低（P<0.05）。结论红花可以抑制肌成纤维细胞的激活，减少细胞外基质的生成与堆积，从而发挥抗硬皮病小鼠皮肤纤维化的作用，其机制可能与下调硬皮病小鼠皮肤组织TGF⁃β1/Smads信号通路相关蛋白的表达有关。

Objective To explore the effect of skin fibrosis in mice with Carthami flos against scleroderma and its influence on transforming growth factor β1 (TGF⁃β1)/Smads signaling pathway. Methods A total of 64 BALB/c female mice were assigned to control group (16 mice), model group (16 mice), prednisone group (8 mice), Carthami flos pre⁃intervention group (16 mice), or Carthami flos post⁃intervention group (8 mice). Except for the control group, the mice model of scleroderma in the remaining 4 groups was induced by subcutaneous injection of bleomycin. On the day of modeling, mice of the prednisone group received intragastric administration of 0.45 mg/mL prednisone acetate suspension, mice of the Carthami flos pre⁃intervention group received intragastric administration of 0.15 g/mL Carthami flos water⁃extraction, and mice of the control group and the model group received intragastric administration of normal saline. On the 15 days of modeling, mice of the Carthami flos post⁃intervention group received intragastric administration of 0.15 g/mL Carthami flos water⁃extraction. Mice of various groups as above continuously received intragastric administration of 0.2 mL/d for 4 week. After 4 weeks of intervention, skin appearance changes and skin histopathological changes of mice in various groups were observed, dermal thickness of mice were compared between various groups, protein expressions of type Ⅰ collagen (COLⅠ), α⁃smooth muscle actin (α⁃SMA) in skin tissues of mice in various groups were detected by using the immunohistochemical method, mRNA expressions of COLⅠ⁃a1, COLⅠ⁃a2, α⁃SMA, TGF⁃β1, Smad2, and Smad3 in skin tissues of mice in various groups were detected by employing the real⁃time fluorescent quantitative PCR, and protein expressions of TGF⁃β1, Smad2, Smad3, phosphorylated⁃Smad2, and phosphorylated⁃Smad3 were detected in skin tissues of mice in various groups by using the Western blot. Results (1) In mice of the model group, no new hair was found on the back, and their skin was obviously hardened and adhered, which was not easy to be lifted, and dermal thickness was thickened; in addition, skin symptoms in various drug intervention groups were ameliorated as compared with the model group, and dermal thickness became thinner, as well as the prednisone group and the Carthami flos pre⁃intervention group exhibited the most significant improvement. (2) Compared with the control group, the model group yielded elevated expressions of COLⅠ and α⁃SMA proteins, and COLⅠ⁃a1, COLⅠ⁃a2, α⁃SMA mRNAs in skin tissues (P<0.05). Compared with the model group, the Carthami flos pre⁃intervention group, the Carthami flos post⁃intervention group, and the prednisone group exhibited decreased expressions of COLⅠ and α⁃SMA proteins, and COLⅠ⁃a1, COLⅠ⁃a2, α⁃SMA mRNAs in skin tissues, as well as expressions of COLⅠ and α⁃SMA proteins in skin tissues of the prednisone group and the Carthami flos pre⁃intervention group were lower than those of the Carthami flos post⁃intervention group, mRNA expressions of COLⅠ⁃a1, COLⅠ⁃a2 in skin tissues of the Carthami flos pre⁃intervention group were lower than those of the Carthami flos post⁃intervention group (P<0.05). (3) Compared with the control group, the model group interpreted elevated expressions of TGF⁃β1, Smad2, Smad3 proteins and mRNAs, and phosphorylated⁃Smad2 and phosphorylated⁃Smad3 proteins in skin tissues; furthermore, the Carthami flos pre⁃intervention group depicted decreased TGF⁃β1 and Smad3 mRNAs, whereas an elevated Smad2 mRNA expression in skin tissues (P<0.05). Compared with the model group, the Carthami flos pre⁃intervention group expressed decreased expressions of TGF⁃β1, Smad2, and Smad3 proteins and mRNAs, and phosphorylated⁃Smad2 and phosphorylated⁃Smad3 proteins in skin tissues (P<0.05). Conclusion Carthami flos can inhibit the activation of myofibroblasts, reduce the generation and accumulation of extracellular matrix, so as to exert effects of skin fibrosis in mice with anti⁃scleroderma. Its mechanism may be related to the down⁃regulation of TGF⁃β1/Smads signaling pathway associated proteins expressions in skin tissues of mice with scleroderma.