Lu Chen1, Lusha Zhang2, Zhirui Fang2, Chunxiao Li2, Yue Yang3, Xingyu You4, Min Song5, Joel Coffie2, Liyuan Zhang2, Xiumei Gao6, Hong Wang7. 1. Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin 300193, China; Key Laboratory of Pharmacology of Traditional Chinese Medical Formulae, Ministry of Education, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China; Tianjin Key Laboratory of Chinese Medicine Pharmacology, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China; Institute of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China. Electronic address: chenlutjutcm@tjutcm.edu.cn. 2. Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin 300193, China; Key Laboratory of Pharmacology of Traditional Chinese Medical Formulae, Ministry of Education, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China. 3. Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin 300193, China. Electronic address: yangyue19901007@sina.com. 4. Key Laboratory of Pharmacology of Traditional Chinese Medical Formulae, Ministry of Education, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China. 5. Tianjin Key Laboratory of Chinese Medicine Pharmacology, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China. 6. Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin 300193, China; Key Laboratory of Pharmacology of Traditional Chinese Medical Formulae, Ministry of Education, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China; Tianjin Key Laboratory of Chinese Medicine Pharmacology, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China. Electronic address: gaoxiumei@tjutcm.edu.cn. 7. Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin 300193, China; Key Laboratory of Pharmacology of Traditional Chinese Medical Formulae, Ministry of Education, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China; School of Integrative Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China. Electronic address: wanghongsys@tjutcm.edu.cn.
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.
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.
Authors: Fatima Noor; Muhammad Tahir Ul Qamar; Usman Ali Ashfaq; Aqel Albutti; Ameen S S Alwashmi; Mohammad Abdullah Aljasir Journal: Pharmaceuticals (Basel) Date: 2022-05-04