目的 探讨Rho激酶抑制剂GSK429286A促进下肢缺血大鼠侧支血管生长及运动功能恢复效果。 方法 分离、结扎并截断SD大鼠或ICR小鼠右侧股动脉，随机分入GSK429286A组和生理盐水组，分别注射10 mg/Kg的GSK429286A或等量生理盐水。X线、MicroCT建立下肢动脉3D可视化模型；CD31、α-SMA染色评估侧支血管管径及密度；Western blot检测腓肠肌内VEGF、HO-1蛋白表达；激光散斑血流成像仪观察患侧足底血流量；HE染色评估腓肠肌纤维萎缩情况；足印、足底功能指数评估下肢运动功能恢复；EdU细胞增殖实验和划痕愈合实验评估GSK429286A对人脐静脉内皮细胞（HUVEC）增殖与迁移的影响。 结果 术后14 d，大鼠下肢血管3D扫描图像和免疫荧光染色均显示GSK429286A组内收肌群内平均血管管径显著高于生理盐水组（P<0.01）。术后3 d，与生理盐水组相比，GSK429286A组小鼠患侧腓肠肌内VEGF含量显著降低，而HO-1含量显著升高；激光散斑血流成像图显示，GSK429286A组小鼠患足的血流灌注恢复程度显著优于生理盐水组，且在术后第7 d和第14 d两组数据出现统计学差异（P<0.01）；术后30 d，GSK429286A组肌肉萎缩程度显著低于生理盐水组（P<0.01），而生理盐水组的腓肠肌内血管密度显著高于GSK429286A组（P<0.05）；足底功能指数测量显示GSK429286A组患肢运动功能显著优于生理盐水组，在术后30 d出现统计学差异（P<0.05）。体外细胞实验的结果表明，GSK429286A能够促进HUVEC的增殖和迁移。 结论 GSK429286A能够促进大、小鼠严重下肢缺血后侧支循环的建立及其运动功能的恢复。
Abstract
Objective To evaluate the effects of Rho kinase inhibitor GSK429286A on collateral vessel growth and functional recovery after lower limb ischemia. Methods The right femoral artery was isolated, ligated, and transected. Animals were randomly assigned to receive 10 mg/kg GSK429286A or saline. 3D modelings of lower limb arteries were established via X-ray and Micro CT, and collateral vessel diameter was assessed using CD31 and α-SMA staining. VEGF and HO-1 protein levels in mouse gastrocnemius muscle were measured by Western blot. Blood flow in the affected footpad was monitored by laser speckle flowmetry. Muscle atrophy and vascular density in the gastrocnemius were evaluated by H&E staining and immunostaining. The foot function index was analyzed. Additionally, the effects of GSK429286A on HUVEC proliferation and migration were assessed using EdU and scratch assays. Results On postoperative 14 days, rats in the GSK429286A group had significantly larger vessel diameter (P<0.01). On postoperative 3 days, VEGF levels were lower but HO-1 levels were higher in the GSK429286A group compared to the saline group. Blood perfusion recovery was significantly better in the GSK429286A group, with differences on postoperative 7 and 14 days (P<0.01). On postoperative 30 days, muscle atrophy was less severe (P<0.01) and vascular density was lower (P<0.05) in the GSK429286A group. The foot function index was significantly better in the GSK429286A group, with differences on postoperative 30 days (P<0.05). In vitro, GSK429286A promoted HUVEC proliferation and migration. Conclusions GSK429286A promotes collateral vessel formation and functional recovery in rodent models of lower limb ischemia.
关键词
GSK429286A； /
下肢缺血； /
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侧支循环； /
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功能恢复
Key words
GSK429286A; /
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Lower limb ischemia; /
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Collateral circulation; /
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Functional recovery
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基金
国家级大学生创新训练项目（202310392013）
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