Background
Peripheral arterial disease (PAD) is a typical disease of atherosclerosis, most commonly influencing the lower extremities. In patients with PAD, revascularization remains a preferred treatment strategy. Buyang Huanwu decoction (BHD) is a popular Chinese herbal prescription which has showed effects of cardiovascular protection through conducting antioxidant, antiapoptotic, and anti-inflammatory effects. Here, we intend to study the effect of BHD on promoting revascularization via the Akt/GSK3β/NRF2 pathway in diabetic hindlimb ischemia (HLI) model of mice. Materials and
Methods
All db/db mice (n = 60) were randomly divided into 6 groups by table of random number. (1) Sham group (N = 10): 7-0 suture thread passed through the underneath of the femoral artery and vein without occlusion. The remaining 5 groups were treated differently on the basis of the HLI (the femoral artery and vein from the inguinal ligament to the knee joint were transected and the vascular stump was ligated with 7-0 silk sutures) model: (2) HLI+NS group (N = 15): 0.2 ml NS was gavaged daily for 3 days before modeling and 14 days after occlusion; (3) HLI+BHD group (N = 15): 0.2 ml BHD (20 g/kg/day) was gavaged daily for 3 days before modeling and 14 days after occlusion; (4) HLI+BHD+sh-NC group (N = 8): local injection of adenovirus vector carrying the nonsense shRNA (Ad-GFP) in the hindlimbs of mice before treatment; (5) HLI+BHD+sh-NRF2 group (N = 8): knockdown of NRF2 in the hindlimbs of mice by local intramuscular injection of adenovirus vector carrying NRF2 shRNA (Ad-NRF2-shRNA) before treatment; and (6) HLI+BHD+LY294002 group (N = 4): intravenous injection of LY294002 (1.5 mg/kg) once a day for 14 days on the basis of the HLI+BHD group. Laser Doppler examination, vascular cast, and immunofluorescence staining were applied to detect the revascularization of lower limbs in mice. Western blot analysis was used to detect the expression of vascular endothelial growth factor (VEGF), interleukin-1beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor- (TNF-) α, heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase quinone-1 (NQO-1), catalase (CAT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphorylated protein kinase B (p-AKT), and phosphorylated glycogen synthase kinase-3 beta (p-GSK3β). HE staining was used to assess the level of muscle tissue damage and inflammation in the lower extremities. Local multipoint injection of Ad-NRF2-shRNA was used to knock down NRF2, and qPCR was applied to detect the mRNA level of NRF2. The blood glucose, triglyceride, cholesterol, MDA, and SOD levels of mice were tested using corresponding kits. The SPSS 20.0 software and GraphPad Prism 6.05 were used to do all statistics. Values of P < 0.05 were considered as statistically significant.