敲除组织蛋白酶K基因改善高脂饮食小鼠缺血性血管生成

Deficiency of cathepsin K improves ischemic angiogenesis in high-fat diet fed mice

本文旨在探讨组织蛋白酶K(cathepsin K,Cat K)对高脂饮食小鼠缺血性血管生成的作用及机制。利用高脂饮食Cat K基因敲除(Cat K^(-/-))小鼠制备单侧下肢缺血模型,利用激光多普勒血流测定仪观察缺血后不同时间段血流恢复程度;免疫组织化学染色法观察缺血下肢新生毛细血管数量;免疫印迹法检测胰岛素受体底物-1(insulin receptor substrate 1,IRS-1)、p-Akt、Akt及血管内皮生长因子(vascular endothelial growth factor,VEGF)等信号蛋白表达。首先观察高脂饮食对野生型小鼠缺血性血管生成的影响。野生型小鼠随机分为对照组和高脂饮食组,分别以普通饲料或60%高脂肪饲料饲养16周。结果表明,与对照组相比,高脂饮食组小鼠体重明显增加,缺血下肢血流恢复程度明显减弱,血浆Cat K浓度及缺血侧骨骼肌中Cat K基因表达显著升高(P<0.05)。接着利用野生型和Cat K^(-/-)高脂饮食小鼠进一步观察Cat K对高脂肪负荷下缺血性血管生成的作用及机制。结果表明,Cat K^(-/-)小鼠缺血下肢的血流恢复程度明显强于野生型小鼠,缺血14天后缺血下肢的CD31阳性细胞显著增多(P<0.05);同时观察到Cat K^(-/-)小鼠缺血侧骨骼肌IRS-1、p-Akt及VEGF蛋白表达水平显著增加(P<0.05)。上述结果提示,敲除Cat K基因可能通过IRS-1-Akt-VEGF信号通路改善高脂饮食小鼠缺血性血管生成过程。

The present study aims to investigate the effect of cathepsin K(CatK)on ischemic angiogenesis in high-fat diet fed mice.The mice were subjected to unilateral hindlimb ischemic surgery,and the ischemic blood flow was measured with a laser Doppler blood flow imager.Immunohistochemical staining was used to observe the quantity of new capillaries in the ischemic lower extremity,and Western blot was used to detect the expression of insulin receptor substrate-1(IRS-1),p-Akt,Akt and vascular endothelial growth factor(VEGF).Firstly,the effect of high-fat diet on ischemic angiogenesis was observed in wild-type mice,which were randomly divided into control group and high-fat diet group and were fed with normal diet or 60%high-fat diet respectively for 16 weeks.The results showed the body weight and the plasma CatK concentration of the high-fat diet group was significantly increased compared with the control group(P<0.05),and the blood flow recovery of the high-fat diet group was significantly lower than control group(P<0.05).Then,wild-type and CatK knock out(CatK^(-/-))mice were both fed with high-fat diet to further observe the effect and mechanism of CatK on ischemic angiogenesis under high-fat diet.The results showed that the blood flow recovery in the CatK^(-/-)group was significantly greater than the wild-type group,and the number of CD31 positive cells was significantly increased(P<0.05).At the same time,the protein expression levels of IRS-1,p-Akt and VEGF in the ischemic skeletal muscle were significantly increased in the CatK^(-/-)group compared with the wild-type group(P<0.05).These results suggest that the deficiency of CatK improves ischemic angiogenesis in high-fat diet fed mice through IRS-1-Akt-VEGF signaling pathway.