富血小板血浆凝胶联合象皮生肌膏对糖尿病大鼠创面愈合及TGF-β1/Smad3信号通路的影响

Effects of platelet-rich plasma gel combined with Xiangpi Shengji Ointment on wound healing and the TGF-β1/Smad3 signaling pathway in diabetic rats

摘要:

目的 观察富血小板血浆（PRP）凝胶联合象皮生肌膏对糖尿病大鼠创面愈合的影响，并从转化生长因子β1（TGF-β1）/Smad家族成员3（Smad3）信号通路的角度探讨其可能的机制。方法 将60只SPF级大鼠按抽签法随机分为正常对照组（涂抹无菌氯化钠溶液）、PRP组、生肌膏组和联合组，每组15只。复制糖尿病大鼠创面模型，PRP组、生肌膏组和联合组分别给予PRP凝胶、象皮生肌膏、PRP与象皮生肌膏治疗，连续21 d。第7、14、21天治疗结束后，观察4组大鼠创面面积变化，计算愈合率；第21天治疗结束后经大鼠腹主动脉取血，检测大鼠血清血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）、TGF-β1和白细胞介素-6（IL-6）的表达。采用CD34免疫组织化学染色检测大鼠创面组织微血管密度。采用Western blotting法检测大鼠创面组织TGF-β1/Smad3通路相关蛋白表达。结果 联合组、生肌膏组、PRP组、正常对照组治疗后第7、14、21天大鼠创面愈合率比较，结果 ①不同时间点大鼠创面愈合率比较，差异有统计学意义（P <0.05）；②4组大鼠创面愈合率比较，差异有统计学意义（P <0.05）；③4组大鼠不同时间点创面愈合率变化趋势比较，差异有统计学意义（P <0.05）。治疗7、14、21天后，PRP组、生肌膏组和联合组的大鼠创面愈合率均高于正常对照组（P <0.05）。正常对照组、PRP组、生肌膏组和联合组的VEGF、bFGF、TGF-β1和IL-6水平，创面组织微血管密度，TGF-β1、Smad3和pSmad3蛋白相对表达量比较，差异均有统计学意义（P <0.05）；与正常对照组比较，PRP组、生肌膏组和联合组大鼠血清VEGF、bFGF、TGF-β1水平均上升，IL-6水平下降（P <0.05）；PRP组、生肌膏组和联合组大鼠的创面组织微血管密度均上升（P <0.05）；PRP组、生肌膏组和联合组TGF-β1、Smad3和pSmad3蛋白相对表达量均升高（P <0.05）。结论 PRP和象皮生肌膏可通过调控TGF-β1/Smad3的表达，促进新血管生成，减轻炎症反应而促进大鼠创面的愈合。

Abstract:

Objective To observe the effect of platelet-rich plasma (PRP) gel combined with Xiangpi Shengji Ointment on wound healing in diabetic rats and explore its possible mechanism from the perspective of the transforming growth factor β1 (TGF-β1)/Smad family member 3 (Smad3) signaling pathway.Methods Sixty rats were randomly divided into four groups (15 rats in each group): the normal control group (topical application of sterile sodium chloride solution), the PRP group, the Xiangpi Shengji Ointment group, and the combined treatment group. A diabetic wound model was established in all rats. The PRP group received PRP gel, the Xiangpi Shengji Ointment group received Xiangpi Shengji Ointment, and the combined treatment group received both PRP gel and Xiangpi Shengji Ointment. All treatments lasted for 21 consecutive days. Changes in wound area were observed and healing rates were calculated on day 7, day 14, and day 21 after the initiation of treatment. On day 21, blood samples were collected from the abdominal aorta of rats to measure serum levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), TGF-β1 and interleukin-6 (IL-6). CD34 immunohistochemical staining was used to detect microvessel density in wound tissues. Western blotting was used to determine the expression of TGF-β1/Smad3 signaling pathway-related proteins in wound tissues.Results Repeated-measures analysis of variance showed significant time, group, and time-by-group interaction effects on wound healing rates (F = 4 028.000, 387.200, and 84.130, respectively; all P < 0.05). After 7, 14 and 21 days of treatment, wound healing rates in the PRP gel group, the Xiangpi Shengji Ointment group and the combined treatment group were significantly higher than those in the normal control group (P < 0.05). Analysis of variance found statistically significant differences in levels of VEGF, bFGF, TGF-β1 and IL-6, microvessel density in wound tissues, and protein expression levels of TGF-β1, Smad3 and p-Smad3 among the normal control group, the PRP group, the Xiangpi Shengji Ointment group, and the combined treatment group (P < 0.05). Compared with the normal control group, the three treatment groups showed significantly increased serum VEGF, bFGF and TGF-β1 levels, and significantly decreased IL-6 levels (P < 0.05). Microvessel density in wound tissues was significantly higher in the PRP, Xiangpi Shengji Ointment, and combined treatment groups than in the normal control group (P < 0.05). Compared with the normal control group, the expression levels of TGF-β1, Smad3 and p-Smad3 proteins were higher in the three treatment groups (P < 0.05).Conclusion Platelet-rich plasma gel combined with Xiangpi Shengji Ointment may regulate the TGF-β1/Smad3 pathway, promote angiogenesis, reduce inflammation, and accelerate wound healing in diabetic rats.