补阳还五汤激活PDGF信号通路促进小鼠后肢缺血后动脉生成的作用机制

Buyang Huanwu Decoction promotes arteriogenesis after hindlimb ischemia in mice by activating PDGF signaling pathway

基于血小板衍生生长因子(platelet-derived growth factor, PDGF)信号通路探讨补阳还五汤对缺血组织血流恢复和动脉生成的影响及机制。40只C57BL/6小鼠随机分为模型组、阳性对照药贝前列素钠组和补阳还五汤低、中、高剂量组,每组8只。行股动脉结扎建立后肢缺血模型,模型组给予纯净水10 mL·kg^(-1)·d^(-1)灌胃,贝前列素钠组给予贝前列素钠18μg·kg^(-1)·d^(-1)灌胃,补阳还五汤低、中、高剂量组分别给予补阳还五汤10、20、40 g·kg^(-1)·d^(-1)灌胃,连续14 d。采用激光散斑血流灌注成像仪观察小鼠缺血后肢血液灌注,缺血腓肠肌冰冻切片染CD31、α-SMA抗体标记毛细血管和小动脉,蛋白免疫印迹法检测缺血腓肠肌PDGFB和PDGF下游信号分子(p-MEK、MEK、p-ERK、ERK)的表达,real-time PCR检测缺血腓肠肌PDGFB mRNA表达。小鼠原代血管平滑肌细胞(vascular smooth muscle cells, VSMCs)低氧培养建立缺氧模型,按体积比分别加入10%和20%补阳还五汤含药血清,应用增殖、迁移实验观察VSMCs促动脉生成的功能。结果显示,与模型组比较,贝前列素钠组和补阳还五汤低、中、高剂量组小鼠后肢缺血后血流恢复增加,毛细血管、小动脉生成增多,其中补阳还五汤中剂量组效果最显著,PDGFB、p-MEK、p-ERK蛋白及PDGFB mRNA表达明显升高;10%补阳还五汤含药血清处理的VSMCs增殖、迁移功能显著增强。结果表明,补阳还五汤促进缺血后PDGFB转录,激活PDGF信号通路(p-MEK/p-ERK表达升高),进而推动VSMCs增殖、迁移形成小动脉,从而恢复血液灌注,发挥保护缺血组织的作用。

This study aims to investigate the effect of Buyang Huanwu Decoction on blood flow recovery and arteriogenesis after hindlimb ischemia in mice via the platelet-derived growth factor(PDGF) signaling pathway. Forty C57BL/6 mice were randomized into model(clean water, 10 mL·kg^(-1)·d^(-1)), beraprost sodium(positive control, 18 μg·kg^(-1)·d^(-1)), and low-, medium-, and high-dose(10, 20, and 40 g·kg^(-1)·d^(-1), respectively) Buyang Huanwu Decoction groups(n=8). The hindlimb ischemia model was established by femoral artery ligation. The mice were administrated with corresponding agents by gavage daily for 14 days after ligation. For laser Doppler perfusion imaging, the mice were anesthetized and measured under a Periscan PSI imager. The density of capillary and arterio-le in the ischemic gastrocnemius was measured using immunofluorescence staining of the frozen tissue sections. Western blot was employed to determine the expression of PDGF subunit B(PDGFB), phosphorylated mitogen extracellular kinase(p-MEK), MEK, phosphorylated extracellular signal-regulated kinase(p-ERK), and ERK. Real-time PCR was employed to determine the mRNA level of PDGFB. The Buyang Huanwu Decoction-containing serum was used to treat the vascular smooth muscle cells(VSMCs) in hypoxia at doses of 10% and 20%. The proliferation and migration of VSMCs was assessed in vitro. The results showed that compared with the model group, beraprost sodium and Buyang Huanwu Decoction enhanced the blood flow recovery, increased the capillary and arteriole density, and up-regulated the protein levels of PDGFB, p-MEK, p-ERK, and mRNA levels of PDGFB, with the medium-dose Buyang Huanwu Decoction demonstrating the most significant effect. The 10% Buyang Huanwu Decoction-containing serum enhanced the proliferation and migration of VSMCs. Our findings demonstrate that Buyang Huanwu Decoction up-regulates PDGFB transcription and activates PDGF signaling pathway to promote arteriogenesis and blood flow recovery in ischemic gastrocnemius.