四君子加川牛膝方干预P38MAPK/CyclinD1-CDK4通路促进大鼠下肢难愈性创面愈合的机制研究

Study on the mechanism of Sijunzi plus Radix Cyathulae Decoction intervention on the P38MAPK/CyclinD1-CDK4 pathway in promoting the healing of the refractory wound in rats'lower limbs

目的:探讨四君子加川牛膝方干预P38MAPK/CyclinD1-CDK4通路促进大鼠下肢难愈性创面愈合的机制。方法:64只雄性SD大鼠右下肢制造全层皮肤缺损型难愈性创面模型,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。在造模后的第7天、第14天,取创面新生肉芽组织及血液标本,应用免疫组化及图像分析法检测标本中细胞周期蛋白D1(cyclinD1)、细胞周期素依赖性激酶4(CDK4)含量,应用Western blot法观察P38丝裂素活化蛋白激酶(p38MAPK)水平变化。结果:造模后第7天,四君子汤组、四君子加川牛膝方组P38MAPK的表达明显高于生理盐水组,差异有统计学意义(P<0.01);而川牛膝方组与生理盐水组组间比较无明显差异(P>0.05),四君子汤组与四君子加川牛膝方组组间比较无明显差异(P>0.05);造模后第14天,4组间P38MAPK浓度比较均无明显差异(P>0.05)。造模后第7天、第14天,4组间CyclinD1、CDK4的表达无明显差异(P>0.05)。结论:四君子加川牛膝方可调控P38MAPK/CyclinD1-CDK4信号转导通路,促进成纤维细胞的增殖,这可能是其促进肉芽组织增生及创面愈合的作用机制.

Objective To explore the mechanism of how“Sijunzi plus Radix Cyathulae Decoction”interfere the P38MAPK/CyclinD1-CDK4 pathway in promoting the healing of the refractory wound in Rats’lower limbs.Methods The models of full-layer skin defect and refractory wound on the right lower limb were established,and 64 male SD rats were randomly divided into Normal Saline group,“Sijunzi plus Radix Cyathulae Decoction”group,“Sijunzi Decoction”group and Radix Cyathulae group.Each group was given normal saline and corresponding traditional Chinese medicine intragastric treatment.Wound granulation tissue and new blood of rats in each group were respectively extracted on the 7th day and on the 14th day.The contents of cyclinD1 and CDK4 in granulation tissue were detected by immunohistochemistry and image analysis.The p38MAPK protein level in granulation tissue was detected by Western blot.Results On day 7 after modeling,the p38MAPK in“Sijunzi Decoction”group and“Sijunzi plus Radix Cyathulae Decoction”group were markedly higher than those in Normal Saline group,there was significant difference(P<0.01);there was no significant differences between“Radix Cyathulae”group and Normal Saline group(P>0.05);there was no significant difference between Sijunzi decoction group and Sijunzi Jiachuan Niushifang group(P>0.05).On day 14 after modeling,there was no significant differences of P38MAPK concentration among the four groups(P>0.05).On day 7 and 14 after modeling:there was no significant differences about expression of CyclinD1 and CDK4 among the four groups(P>0.05).Conclusion The prescription of“Sijunzi plus Radix Cyathulae Decoction”can regulate the P38MAPK/CyclinD1-CDK4 signal transduction pathway and promote the proliferation of fibroblasts,which may be the mechanism of promoting granulation tissue hyperplasia and wound healing.