四君子加川牛膝方对大鼠下肢难愈性创面组织成纤维细胞及相关细胞因子的影响

Effects of Sijunzi plus Radix Cyathulae Decoction on Fibroblasts and Related Cytokines in Refractory Wounds of Rat Lower Limbs

目的:探讨四君子加川牛膝方促进大鼠下肢难愈性创面愈合可能的作用机制,以及引经药川牛膝的作用。方法:建立SD大鼠右下肢难愈性创面模型,随机分为生理盐水组、四君子加川牛膝方组、四君子汤组、川牛膝方组,各组分别给予生理盐水及相应中药灌胃处理。在创面愈合过程中(造模后的第7天、第14天),取创面新生肉芽组织及血液,检测血清碱性成纤维生长因子(b FGF)、转化生长因子-β1(TGF-β1)水平的变化,并应用流式细胞仪检测成纤维细胞的S期细胞比例。结果:(1)造模后第7天,四君子汤组及四君子加川牛膝方组TGF-β1、b FGF浓度明显高于生理盐水组(P<0.05),而川牛膝方组与生理盐水组无明显变化(P>0.05),其中四君子汤组与四君子加川牛膝方组间比较无明显差异(P>0.05);造模后第14天,各组间TGF-β1、b FGF浓度比较均无明显差异(P>0.05)。(2)造模后第7天,各药物组成纤维细胞G0/G1期细胞比例的表达均明显低于生理盐水组(P<0.01或P<0.05)、S期细胞比例的表达均明显高于生理盐水组(P<0.01),而各组间G2/M期细胞比例的表达均无显著性差异(P>0.05),其中四君子加川牛膝方组G0/G1期细胞比例明显低于四君子汤组(P<0.05),S期细胞比例明显高于四君子汤组(P<0.05);在造模后第14天,各组间G0/G1期及G2/M细胞比例的表达均无显著性差异(P>0.05),而四君子加川牛膝方组S期细胞比例明显高于四君子汤组与川牛膝方组(P<0.01)。结论:四君子加川牛膝方可上调TGF-β1、bFGF的合成和分泌,加速成纤维细胞由G0/G1期进入S期,促进成纤维细胞的增殖,这可能为其促进肉芽组织增生及创面愈合的作用机制,其中引经药川牛膝有提高成纤维细胞S期细胞比例的协同作用。

Objective： To investigate the possible mechanism of Sijunzi plus Radix Cyathulae Decoction on promoting the healing of refractory wounds in rat lower limbs, and the effect of Radix Cyathulae as channel ushering drug. Methods： The model of refractory wounds was established in right lower limb of SD rats, and the rats were randomly divided into the normal saline group, Sijunzi plus Radix Cyathulae Decoction （combination）group, Sijunzi Decoction group and Radix Cyathulae Decoction group. Each group was treated with normal saline and the corresponding drugs for intragastric administration. In the course of wound healing （7th and 14th day after modeling）, the granulation tissue in wound and the blood sample were collected, the serum levels of basic fibroblast growth factor （bFGF） and transforming growth factor-β1 （TGF-β1 ） were detected and compared, and the cellular proportion of S phase in fibroblasts was detected by flow cytometry. Results： （1）On 7th day after modeling,the concentrations of TGF-β1 and bFGF in the Sijunzi Decoction group and combination group were significantly higher than those in the normal saline group （P 〈 0. 05 ）, but no obvious changes were found in the Radix Cyathulae Decoction group and normal saline group（ P 〉 0.05 ）. There were no significant differences on the concentrations of TGF-13~ and bFGF between the Sijunzi Decoction group and combination group（ P 〉 0. 05 ）. On 14th day after modeling, there were no significant differences on the concentrations of TGF-β1 and bFGF among groups （P 〉0. 05 ）. （2）On 7th day after modeling, the fibroblasts cellular proportion of GO/G1 phase in the drug groups was significantly lower than that in the normal saline group（ P 〈0.01 or P 〈 0. 05 ） ,the cellular proportion of S phase in the drug groups was significantly higher than that in the normal saline group （ P 〈 0. 01 ）, but there were no significant differences on the cellular proportion of G2/M phase among groups （ P 〉 0.05 ）. The cellular proportion of GO/G1 phase in the combination group was significantly lower than that in the Sijunzi Decoction group （ P 〈 0.05 ） , the cellular proportion of S phase in the combination group was significantly higher than that in the Sijunzi Decoction group（ P 〈 0. 05 ）. On 14th day after modeling, there were no significant differences on the cellular proportion of GO/G1 and G2/M phase among groups（ P 〉0.05）, and the cellular proportion of S phase in the combination group was significantly higher than that in the Sijunzi Decoction group and Radix Cyathulae Decoction group （ P 〈 0. 01 ）. Conclusion ： Sijunzi plus Radix Cyathulae Decoction can up-regulate the synthesis and secretion of TGF-β1 and bFGF, accelerate the fibroblast differentiation from G0/G1 phase to S phase, and promote the proliferation of fibroblasts, which may be its mechanism on promoting granulation tissue proliferation and wound healing. Radix Cyathulae as channel ushering drug shows the synergistic effect on increasing the cellular proportion of S phase in fibroblasts.