补肾活血中药激活MMP-9信号通路动员大鼠骨髓EPCs的分子机制研究

Chinese Herbs for Shen Invigorating and Blood Activating Activated MMP- 9 Signaling Pathway to Mobilize Rats′ Bone Marrow EPCs: a Molecular Mechanism Research

目的探讨补肾活血中药对心肌梗死(myocardial infarction,MI)模型大鼠骨髓、外周血内皮祖细胞(endothelial progenitor cells,EPCs)数量及骨髓基质金属蛋白酶9(matrix metalloproteinase 9,MMP-9)信号通路的影响。方法结扎法建立大鼠MI模型,30只造模成功大鼠随机分为补肾活血中药高剂量组、补肾活血中药低剂量组和模型组,每组10只,另取10只正常大鼠为空白组。补肾活血中药高、低剂量组分别用补肾活血中药按3g/kg、1.5g/kg加生理盐水4mL灌胃,每天1次。空白组及模型组每天单用生理盐水4mL灌胃1次。4周后骨髓、外周血EPC培养,7天后收集贴壁细胞运用流式细胞仪鉴定CD34/CD133表型,Western blot法检测MMP-9和水溶性Kit配体(solouble Kit ligand,sKitL),ELISA法检测血管内皮生长因子(vascular endothelial growth factor,VEGF)和基质细胞衍生因子-1α(stromal cell-derived factor-1α,SDF-1α)。结果补肾活血中药高、低剂量组骨髓和外周血表达CD34/CD133阳性率、EPC数量均高于模型组(P<0.05,P<0.01);且两组骨髓和外周血VEGF、SDF-1α、MMP-9和sKitL表达均高于模型组(P<0.05,P<0.01)。结论补肾活血中药能够激活MMP-9信号通路,增加其上游和下游信号表达水平,动员骨髓EPC进入血液循环。

Objective To explore the effect of Chinese herbs for Shen invigorating and blood ac- tivating （CHSIBA） on the number of endothelial progenitor cells （EPCs） in the bone marrow and the pe- ripheral blood and the signaling pathway of bone marrow matrix metalloproteinase 9 （MMP-9） of the myo- cardial infarction （MI） model rats. Methods The MI rat model was established by ligation. Thirty suc- cessfully modeled rats were randomly divided into the high dose CHSIBA group, the low dose CHSIBA group, and the model group, 10 in each group. Besides, another 10 normal rats were recruited as the blank group. Rats in the high dose CHSIBA group and the low dose CHSIBA group were administered with CHSIBA at 3 g/kg and 1.5 g/kg body weight by gastrogavage （by adding them in 4 mL physiological sa- line）, once daily. Rats in the model group and the blank group were administered with 4 mL physiological saline once daily. The EPCs were collected from the bone marrow and the peripheral blood 4 weeks later. Seven days later the CD34/CD133 phenotype was identified in collected sticking wall cells using flow cy- tometry. The MMP-9 and water soluble Kit ligand （sKitL） were detected using Western blot. The expres- sions of vascular endothelial growth factor （VEGF） and stromal cell-derived factor-1α （SDF-1 α） were de-tected using ELISA. Results The CD34/CD133 positive rate and the EPC quantity in the bone marrow and the peripheral blood were higher in the high dose CHSIBA group and the low dose CHSIBA group than in the model group （P 〈0.05, P 〈0.01）. Besides, the expressions of VEGF, SDF-1α, MMP-9, and sKitL in the bone marrow and the peripheral blood were also higher in the high dose CHSIBA group and the low dose CHSIBA group than in the model group （P 〈0.05, P 〈0.01 ）. Conclusion CHSIBA could activate MMP-9 signaling pathway, increase its upstream and downstream signal expression levels, and mobilize EPCs in the bone marrow to enter the blood circulation.