四妙勇安汤对糖尿病大鼠肢体缺血治疗作用及其分子机制

目的研究四妙勇安汤对糖尿病后肢缺血大鼠的治疗作用及对丝苏氨酸蛋白激酶（Akt）、p38丝裂原活化蛋白激酶（P38）蛋白水平的影响。方法40只Wistar大鼠完全随机分为4组：对照组、对照治疗组建立单纯缺血模型；模型组、模型治疗组建立糖尿病肢体缺血模型，对照治疗组、模型治疗组给予四妙勇安汤治疗。分别于术前、术后即刻、术后第3、7、14天测下肢血流变化，术后15d取腓肠肌组织免疫组织化学CD31染色血管内皮细胞观察新生血管密度，及蛋白印迹法检测内皮细胞Akt、P38蛋白变化情况。结果模型组血管损伤严重，血管重建能力明显降低，第7天对照组、对照治疗组、模型组、模型治疗组血流比值为（45±4）、（49±3）、（364-3）、（48±6）％，第14天血流比值为（63±6）、（66±4）、（45±4）、（61±4）％，与模型组比较，其余3组差异均有统计学意义（P〈0．05）。模型组术后15d新生血管密度明显减低，Akt蛋白表达明显降低，P38表达明显升高，其他3组与其差异均有统计学意义（P〈0．05）；四妙勇安汤能够明显增加新生血管数量[模型组、模型治疗组分别为（9．1±2．0）、（16．4±2．9）个，P〈0．05，模型组、模型治疗组比值分别为（0．62±0．15）、（0．86±0．18）％，P〈0．05]，增加Akt蛋白表达，降低P38表达，差异均有统计学意义[模型组与模型治疗组蛋白相对光密度比值：Akt：（0．20±0．07）、（0．28±0．56）％；P38：（1．18±0．23）、（0．99±0．04）％，P〈0．05]，对照治疗组未发现中药治疗后副作用。结论四妙勇安汤能够改善糖尿病下肢缺血血运重建过程，加快血管新生速度，其作用机制可能是通过促进Akt表达，抑制P38表达实现的。

Therapeutical effect and molecular mechanism of Simiaoyongan recipe on diabetic rats of limb ischemia

Objective To investigate the effects of Simiaoyongan recipe on angiogenesis for diabetic rats of hindlimb ischemia, and to observe the impact of Simiaoyongan recipe on Akt and P38 signal pathways. Methods Di- abetic rat models were induced with Streptozotocin, and a hindlimb ischemia models were prepared in 40 Wister rats by ligation and excision of left femoral artery and its branches. All rats were randomly divided into 4 groups ： control group, control therapeutic group, model group and model therapeutic group. Simiaoyongan recipe was given intra- gastric administration on control and model therapeutic group. Blood flow was performed with doppler scan before and after operation, at 3, 7, 14 days after operation. The pathological changes of new vascular were observed by CD31 immunohistochemistry and the expressions of Akt and P38 were measured by Western-Blot on 15 days after op- eration. Results Compared with control and control therapeutic group, the PU ratio and the number of new vascu- lar of model group were significantly decreased, as well as angiogenesis ability defect. The expression of p-Akt were obviously decreased but P38 was obviously increased（P 〈 0.05 ）. While the improvement of angiogenesis was signif- icant on model therapeutic group. The PU ratio [ model group（ % ） ： Day 7 （36 ± 3 ）, Day 14（45 ±4） ; model thera- peutic group： Day7 （48 ± 6 ）, Dayl 4 （ 61 ± 4 ）, P 〈 0.05 ]. The number of new vascular was significantly higher than that model group [ model group （9.1 ± 2. 0）, model therapeutic group （ 16.4±2.9）, P 〈 0.05 ]. The expression of p- Akt was obviously increased[model therapeutic group（0.28 ±0.56） vs model group（0.20 ±0.07）, P 〈0.05], and P38 was decreased [ model therapeutic group （ 0.99 ± 0.04 ） vs model group （ 1. 18 ± 0.23 ）, P 〈 0. 05 ]. Conclusion Simiaoyongan recipe can significantly improve and accelerate angiogenesis process on diabetic rats of limb ischemia, which is related to up-reguation of Akt signal pathway and down-regulation of P38 signal pathway.