L-精氨酸通过L-Arg-NO途径促进大鼠背部跨区皮瓣的成活

L-arginine promotes the survival of extended dorsal perforator skin flap through activating L-arginine-nitric oxide pathway

背景:有研究表明一氧化氮可有效改善皮瓣术后血供,促进皮瓣成活,但具体机制尚不清楚。目的:通过实验验证L-Arg-NO途径在L-精氨酸促进大鼠背部跨区皮瓣成活中的作用。方法:成功建立大鼠背部三穿支体跨区皮瓣模型的雄性SD大鼠81只,随机被分为3组,分别于术后即刻、术后1-7 d腹腔注射不同药物,L-Arg组注射L-精氨酸400 mg/(kg·d);L-NAME组注射一氧化氮合酶抑制剂40 mg/(kg·d);空白组注射等体积等渗氯化钠溶液。术后7 d观察皮瓣成活情况,计算皮瓣成活率;颈总动脉灌注明胶-氧化铅行血管造影,观察血管走形和分布;硝酸还原酶法测定术后即刻、1,3,5,7 d chokeⅡ区一氧化氮水平;苏木精-伊红染色观察chokeⅡ区新生血管密度和管径;蛋白印记法检测术后3,7 d chokeⅡ区血管内皮生长因子和基质金属蛋白酶2蛋白表达。实验方案经解放军联勤保障部队第九四〇医院动物实验伦理委员会批准(批准号为2015KYLL046)。结果与结论:①术后7 d,L-Arg组大鼠皮瓣成活率最高(89.47±3.17)%,3组比较差异有显著性意义(F=49.908,P<0.001);②术后7 d,L-Arg组chokeⅡ区血管结构较完整,走形清楚,扩张达到真性吻合的血管沿着皮瓣纵轴方向一直延伸到皮瓣末端,空白组和L-NAME组chokeⅡ区血管结构和走形杂乱,L-NAME组皮瓣末端血管结构消失;③L-Arg组一氧化氮水平术后开始升高,术后3 d达到高峰,之后逐渐下降;④术后7 d,L-Arg组新生血管密度和管径最高,3组比较差异有显著性意义(均P<0.001);⑤术后3 d,L-Arg组血管内皮生长因子和基质金属蛋白酶2蛋白表达量最高,3组比较差异有显著性意义(均P<0.05);术后7 d,L-Arg组血管内皮生长因子和基质金属蛋白酶2蛋白表达量最高,3组之间比较血管内皮生长因子差异有显著性意义(P<0.05),基质金属蛋白酶2蛋白差异无显著性意义;⑥结果说明,一氧化氮可有效改善大鼠背部三穿支体皮瓣chokeⅡ区血供,促进chokeⅡ区微血管扩张和增生,而L-精氨酸可通过L-Arg-NO途径提高皮瓣术后组织内一氧化氮水平,促进皮瓣术后成活。

BACKGROUND:Studies have shown that nitric oxide(NO)can effectively improve blood supply and promote flap survival,but the specific mechanism is still unclear.OBJECTIVE:To explore the effects of L-arginine(L-Arg)on the survival of extended dorsal perforator skin flap in rats by activating L-Arg-NO pathway.METHODS:Animal models of extended dorsal perforator skin flap were successfully established in 81 male Sprague Dawley rats,which were then divided into a L-Arg group,a blank group and a L-NAME group.L-Arg(400 mg/kg per day)and nitro-amino-methyl-L-arginine(L-NAME;40 mg/kg per day)were injected intraperitoneally in the L-Arg group and L-NAME group immediately and 1-7 days after operation,respectively,while the same volume of isotonic sodium chloride solution was injected intraperitoneally in the blank group at the same time points.Flap survival rate was measured.Angiography was performed to observe appearance and distribution of blood vessels at 7 days after operation.NO concentration of choke II zone was detected immediately,1,3,5,and 7 days after operation.Tissue samples were harvested from the choke II zone for detecting density and caliber of new blood vessels using hematoxylin-eosin staining and the expression of vascular endothelial growth factor and matrix metalloproteinase was detected by western blot,respectively.The study protocol was approved by the Animal Ethics Committee of the 94th Hospital of the PLA Joint Logistics Support Force(approval No.2015KYLL046).RESULTS AND CONCLUSION:The highest survival rate of flap appeared in the L-Arg group,(89.47±3.17)%,which was significantly higher than that in the other groups(F=49.908,P<0.001).At 7 days after operation,the vascular structure in the choke II zone of the L-Arg group was relatively complete with clear trajectory.The vessels that had expanded to achieve true anastomosis extended along the longitudinal axis of the flap to the end of the flap.In the blank group and L-NAME group,the vascular structure and vascular trajectory in the choke Ⅱ zone were disordered.Moreover,the vascular structure at the end of flap in L-NAME group disappeared.NO concentration in L-Arg group was increased after surgery,peaked at 3 days after operation,and then decreased gradually.Microvessel density and caliber in the L-Arg group were significantly higher than those in the other groups at 7 days after operation(P<0.001).Western blot showed that the expression of vascular endothelial growth factor and matrix metalloproteinase-2 were significantly higher in the L-Arg group than the other groups at 3 after operation(P<0.05),while at 7 days after operation,the expression of vascular endothelial growth factor in the L-Arg group was significantly higher than that in the other groups(P<0.05),but the expression of matrix metalloproteinase-2 showed no significant difference among the three groups.To conclude,NO could promote microvascular growth and dilatation in the choke II zone of extended dorsal three-vascular perforator flaps in rats;L-Arg could increase the NO concentration in tissue by activating the L-Arg-NO pathway,which is beneficial to the survival of skin flap.