鹿茸通过PI3K/AKT信号通路促进下肢动脉硬化性闭塞症大鼠血管新生的作用机理

The mechanism of deer antler promoting angiogenesis in rats with lower limb arteriosclerotic obliterans through PI3K/AKT signaling pathway

目的探索鹿茸通过磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)信号通路促进下肢动脉硬化性闭塞症大鼠血管新生的作用机理。方法肢体缺血手术、高脂饲料喂养制作大鼠下肢动脉硬化闭塞症模型。多普勒检测各个时期大鼠肢体血流灌注恢复程度;TUNEL法检测给药后骨髓中内皮祖细胞(EPCs)的凋亡率;流式细胞术检测给药后外周血、骨髓中CD34^(+)细胞比例;Western blot法检测给药后患侧腓肠肌PI3K、AKT、VEGF的相对表达水平;免疫荧光法检测给药后患侧腓肠肌VEGFR的阳性表达比例。结果模型组EPCs凋亡率显著高于鹿茸处理组(P<0.01);LY294002组凋亡率显著高于模型组(P<0.05);鹿茸高剂量组与空白组及假手术组无明显统计学意义(P>0.05),其余鹿茸高剂量+LY294002组、鹿茸中剂量、鹿茸中剂量+LY294002组、鹿茸低剂量组、鹿茸低剂量+LY294002组凋亡率依次增高,之间均有显著差异(P<0.05);CD34^(+)细胞比例模型组及给药组均显著高于空白组及给药组(P<0.05);其中LY294002组、模型组、鹿茸低剂量+LY294002组、鹿茸低剂量组、鹿茸中剂量+LY294002组、鹿茸中剂量组、鹿茸高剂量+LY294002组、鹿茸高剂量组CD34^(+)含量依次上升,其之间均有显著差异(P<0.05);PI3K、AKT、VEGF的相对表水平以及VEGFR的阳性表达比例均为模型组及给药组均显著高于空白组及假手术组(P<0.05),其中LY294002组、模型组、鹿茸低剂量+LY294002组、鹿茸低剂量组、鹿茸中剂量+LY294002组、鹿茸中剂量组、鹿茸高剂量+LY294002组、鹿茸高剂量组表达量依次增高,之间均有显著差异(P<0.05)。结论鹿茸通过PI3K/AKT信号通路促进了EPCs的增殖及分化,从而促进了下肢动脉硬化闭塞症大鼠的血管新生。

Objective To explore the mechanism of deer antler promoting angiogenesis in rats with lower extremity arteriosclerotic obliterans through the phosphatidylinositol 3-kinase(PI3 K)/protein kinase B(AKT)signaling pathway.Methods Limb ischemia surgery and high-fat diet were used to make a rat model of lower limb arteriosclerosis obliterans.Doppler was used to detect the recovery degree of blood perfusion in the limbs of rats in each period;TUNEL method was used to detect the apoptotic rate of EPCs in bone marrow;flow cytometry was used to detect the proportion of CD34^(+)cells in peripheral blood and bone marrow;Western blot was used to detect the relative expression levels of PI3 K,AKT and VEGF in the affected gastrocnemius muscle.Immunofluorescence assay was used to detect the positive expression rate of VEGFR in the affected gastrocemius muscle after administration.Results The apoptosis rate of EPCs in the model group was significantly higher than that of the velvet antler treatment group(P<0.01);the apoptosis rate of the LY294002 group was significantly higher than that of the model group(P<0.05);there was no significant statistics in the velvet high-dose group,the blank group and the sham operation group Significance(P>0.05).The apoptotic rate of other antler high dose+LY294002 group,antler medium dose,antler medium dose+LY294002 group,antler low dose group,antler low dose+LY294002 group increased sequentially,and there were significant differences among them(P<0.05);The ratio of CD34^(+)cells in the model group and the administration group was significantly higher than the blank group and the administration group(P<0.05);among them,the LY294002 group,the model group,the antler low-dose+LY294002 group,the antler low-dose group,The CD34 content of antler medium dose+LY294002 group,antler medium dose group,antler high dose+LY294002 group,and antler high dose group increased successively,and there were significant differences among them(P<0.05);PI3 K,and AKT,positive expression of VEGF and VEGFR relative levels of table proportion of model group and drug group were significantly higher than that of blank group and control group(P<0.05),with LY294002 low dose group,model group,velvet+LY294002,deer antler,low dose group,velvet antler dose+LY294002 group,velvet antler,deer antler,high dose group dose+LY294002 group,velvet antler increased in high dose group,between all have significant difference(P<0.05).Conclusion Velvet antler promotes the proliferation and differentiation of EPCs through the PI3 K/AKT signaling pathway,thus promoting angiogenesis in rats with lower extremity arteriosclerosis obliteration.