从P38MAPK/cyclinD1-CDK4信号转导通路探讨四君子合引经药川牛膝促进下肢创面愈合的机制

The recovery of ulcer surface is a continuous and complicated process. The key step of ulcer surface to recover is the damaged tissues filled by granulomatous tissues, among which the fibroblast is a kind of the most important function cell. The signal pathway, TGF-β1 /PDGF- P38MAPK-- CDK4/cyclinD1-- fibroblasts proliferation and differentiation , plays a crucial regulative role on the recovery of ulcer surface. The theory of Traditional Chinese Medicine(TCM) affords the opinion of " the spleen govern muscles". In details, all muscles of whole body are needed to be nourished by qi and blood from essences of water and food, which are transformed by the spleen and stomach. Then granulomatous tissues of ulcer surface are able to grow up and defective tissues can be repaired. And the TCM guide theory affords the opinion that the Cyathula as medical guide can lead other drugs downward. Our previous studies showed that SiJunZi plus Cyathula decoction was able to promote the recovery of ulcer surface, which increased the amount of the fibroblast cells,the weight of the granulomatous tissues and the express of VEGF, but the mechanism was still unclear. In present research, the methods, such as immunohistochemical stain,western blot,image analysis and so on, will be performed. As results, the cell cycle of fibroblast will be investigated; the expression of CyclinD1、CDK4、TGF-β1、PDGF、P38MAPK will be observed. Based on above results, the mechanism and crucial steps of SiJUnZi Tang promoting the recovery of ulcer surface will be explored from P38MAPK/cyclinD1-CDK4 signal pathway; the strengthening effect and the mechanism between Cyathula and SiJunZi Tang will be discussed.

创面修复是一个连续复杂过程，肉芽组织填充是创面修复的关键环节，成纤维细胞是其最主要的功能细胞。而TGF-β1、 PDGF--P38MAPK--CDK4、cyclin D1--成纤维细胞增殖分化，对创面修复起关键调控的作用。 基于"脾主肌肉"、"脾为气血生化之源"理论，项目组提出了"补虚生肌、引药下行"治疗原则。项目组前期研究证实，"四君子合引经药川牛膝"可以促进下肢创面愈合，增加创面成纤维细胞数量、肉芽组织湿重，上调VEGF表达，但其作用机制有待于进一步加强。 本研究运用免疫组化等研究方法，通过观察成纤维细胞S期细胞比例、TGF-β1、 PDGF、p38MAPK、cyclinD1-CDK4等关键指标，进一步从P38MAPK/cyclinD1-CDK4角度探讨四君子汤调控细胞周期、促进肉芽组织生长、加速创面愈合的作用机制；以及川牛膝对于四君子汤治疗病位在下溃疡的增效作用及作用机制。

创面修复是一个连续复杂过程，肉芽组织填充是创面修复的关键环节，成纤维细胞是其最主要的功能细胞。而TGF-β1、PDGF—P38MAPK—CDK4、cyclinD1—成纤维细胞增殖分化，对创面修复起关键调控的作用。该项目运用免疫组化等研究方法，通过观察上述关键指标，进一步从P38MAPK/cyclinD1-CDK4角度探讨四君子汤调控细胞周期、促进肉芽组织生长、加速创面愈合的作用；初步探讨川牛膝引经作用的机制。.造模后第7天，四君子汤组及四君子加川牛膝方组bFGF、TGF-β1、PDGF、P38MAPK浓度明显高于生理盐水组（P<0.05,P<0.01）。造模后第7天，四君子汤组、四君子加川牛膝方组、川牛膝组、生理盐水组，CyclinD1、CDK4组间比较无明显差异（P>0.05）。对成纤维细胞S期细胞比例的表达，在造模后第7天，四君子汤组、四君子加川牛膝方组、川牛膝方组均明显高于生理盐水组（P<0.01）；在造模后第14天，四君子加川牛膝方组明显高于生理盐水组（P<0.01），亦明显高于四君子汤组（P<0.01）。.项目基于“脾主肌肉”以及“脾为气血生化之源”的理论，确立了“补虚生肌”的治疗原则，进一步完善与发展创面修复理论。将中药引经理论引入皮肤溃疡研究领域，明确了川牛膝对于四君子汤治疗病位在下的溃疡增效作用，其引经作用的机制可能是通过调节成纤维细胞S期细胞比例，促进成纤维增殖而发挥的协同作用。首次明确补虚生肌类中药四君子汤及四君子加川牛膝方，通过上调TGF-β1、PDGFB、bFGF等细胞因子的合成和分泌，共同调控P38MAPK-cyclinD1/CDK4信号转导通路，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，促进创面肉芽组织增生，填充组织缺损，从而促进创面愈合。