Naoxintong restores collateral blood flow in a murine model of hindlimb ischemia through PPARδ-dependent mechanism

Abstract Ethnopharmacological relevance Naoxintong (NXT) is a compound preparation that is widely used in patients with cardiovascular and cerebrovascular diseases. Aim of study The aim of this study is to investigate the protective mechanism of NXT on the mice model of peripheral vascular disease (PAD). Materials and methods In the study, hindlimb ischemia was induced by ligation of femoral artery on the right leg of mice. After surgery, the mice were administrated with saline solution, 10 mg/kg/d simvastatin and 700 mg/kg/d NXT for 4 weeks. The blood flow perfusion was measured by laser Doppler perfusion imaging system. Histological and immunofluorescent staining was used to determine muscle recovery, capillary density, tissue vascular endothelial growth factor (VEGF), phosphorylated-Akt (p-Akt) and phosphorylated-endothelial nitric oxide synthase (p-eNOS) expression. Terminal deoxynucleotidyl transferased UTP nick end labeling (TUNEL) was performed to detect the apoptosis of myocytes in hindlimb. The autophagy-associated gene expression and peroxisome proliferator-activated receptors (PPARs) expression were measured by Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR). Western blotting was performed to detect the expressions of light-chain 3 (LC3), VEGF, p-Akt, p-eNOS and PPARs. The EMSA experiment was performed to figure out whether PPARδ could directly bind to the predicted PPRE motif of VEGF. Results NXT treatment significantly accelerated perfusion recovery and reduced tissue injury in mice muscle. Apoptosis and autophagy were decreased within the ischemic muscle of NXT-treated mice. Quantification of vessels in hindlimb muscles provided evidences that NXT promoted angiogenesis in peripheral ischemia. In addition, results from western blotting and immunofluorescent staining suggested NXT induced angiogenesis via VEGF/Akt/eNOS signaling pathway. More interestingly, NXT specifically increased the expression of PPARδ in both mRNA and protein levels. EMSA results showed that PPARδ associated with PPRE site of VEGF promoter, suggesting that NXT-induced VEGF expression is mediated, at least in part, by PPARδ. Conclusion In conclusion, the present study implicated that the restoration of hindlimb blood perfusion and recovery of limb functions were improved in NXT-treated mice with significant improvement of angiogenesis mediated by PPARδ-VEGF-Akt-eNOS axis as well as attenuation of autophagy and apoptosis. These results expand knowledge about the beneficial effects of NXT in angiogenesis and blood flow recovery. It might provide insight into the PPARδ regulating neovascularization in hindlimb ischemia and identify NXT as a potent new compound used for the treatment of peripheral vascular disease.