Han Liu1, Peng Li1, Juan Lin2, Wenpei Chen1, Haibiao Guo2, Jianyun Lin2, Jin Liu1, Ziqi Lu1, Xiaolan Yao1, Yang Chen1, Baoqin Lin3. 1. School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, 510006, China. 2. Guangzhou Baiyun Mountain and Hutchison Whampoa Ltd., Guangzhou, Guangdong, 510515, China. 3. School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, 510006, China. Electronic address: linbaoqin@gzucm.edu.cn.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: Danhong Huayu Koufuye (DHK), a compound traditional Chinese medicine, is composed of Salvia miltiorrhiza radix (Salvia miltiorrhiza Bge.), Angelicae Sinensis radix (Angelicae Sinensis (Oliv.) Diels.), Chuanxiong rhizoma (Ligusticum chuanxiong Hort.), Persicae semen (Prunus persica (L.) Batsch), Carthami flos (Carthamus tinctorius L.), Bupleuri radix (Bupleurum chinense DC.) and Aurantii fructus (Citrus aurantium L.). DHK prevents deep vein thrombosis (DVT) through antiinflammation. However, the antiinflammatory mechanism of DHK is still unknown. OBJECTIVE: The aim of this study was to evaluate whether DHK prevented venous thrombosis through antiinflammation via Sirtuin 1 (SIRT1)/NF-κB signaling pathway. METHODS: Inferior vena cava (IVC) stenosis-induced DVT rat model was established. Rats were administered with DHK (1.6, 3.2 or 6.4 mL/kg/d, p.o.), heparin (200 U/kg/d, i.v.), clopidogrel (25 mg/kg/d, p.o.), resveratrol (50 mg/kg/d, p.o.) or vehicle (p.o.) once daily for two days. Blood coagulation, blood fibrinolysis, blood viscosity, blood cell counts and platelet activity were evaluated. Serum levels of inflammatory cytokines were analyzed by enzyme-linked immunosorbent assay. Pathological changes were observed by hematoxylin-eosin (HE) staining. Protein expressions in thrombosed IVCs were evaluated by Western blot and/or immunofluorescence analyses. SIRT1 mRNA expression was analyzed by real-time quantitative polymerase chain reaction. Besides, SIRT1-specific inhibitor EX527 was pretreated to confirm the role of SIRT1/NF-κB signaling pathway in the antithrombotic effect of DHK. RESULTS: DHK remarkably prevented DVT. DHK had no effects on blood coagulation, blood fibrinolysis, blood viscosity, blood cell counts or platelet activity. But DHK significantly up-regulated protein and mRNA expressions of SIRT1, and reduced leukocytes infiltration into thrombus and vein wall, serum levels of inflammatory cytokines, and protein expressions of acetylated p65 (Ace-p65), phosphorylated p65 (p-p65) and tissue factor (TF). Moreover, the antithrombotic effect of DHK was significantly abolished by EX527. CONCLUSION: DHK may prevent DVT by inhibiting inflammation via SIRT1/NF-κB signaling pathway.
ETHNOPHARMACOLOGICAL RELEVANCE: Danhong Huayu Koufuye (DHK), a compound traditional Chinese medicine, is composed of Salvia miltiorrhiza radix (Salvia miltiorrhiza Bge.), Angelicae Sinensis radix (Angelicae Sinensis (Oliv.) Diels.), Chuanxiong rhizoma (Ligusticum chuanxiong Hort.), Persicae semen (Prunus persica (L.) Batsch), Carthami flos (Carthamus tinctorius L.), Bupleuri radix (Bupleurum chinense DC.) and Aurantii fructus (Citrus aurantium L.). DHK prevents deep vein thrombosis (DVT) through antiinflammation. However, the antiinflammatory mechanism of DHK is still unknown. OBJECTIVE: The aim of this study was to evaluate whether DHK prevented venous thrombosis through antiinflammation via Sirtuin 1 (SIRT1)/NF-κB signaling pathway. METHODS: Inferior vena cava (IVC) stenosis-induced DVT rat model was established. Rats were administered with DHK (1.6, 3.2 or 6.4 mL/kg/d, p.o.), heparin (200 U/kg/d, i.v.), clopidogrel (25 mg/kg/d, p.o.), resveratrol (50 mg/kg/d, p.o.) or vehicle (p.o.) once daily for two days. Blood coagulation, blood fibrinolysis, blood viscosity, blood cell counts and platelet activity were evaluated. Serum levels of inflammatory cytokines were analyzed by enzyme-linked immunosorbent assay. Pathological changes were observed by hematoxylin-eosin (HE) staining. Protein expressions in thrombosed IVCs were evaluated by Western blot and/or immunofluorescence analyses. SIRT1 mRNA expression was analyzed by real-time quantitative polymerase chain reaction. Besides, SIRT1-specific inhibitor EX527 was pretreated to confirm the role of SIRT1/NF-κB signaling pathway in the antithrombotic effect of DHK. RESULTS: DHK remarkably prevented DVT. DHK had no effects on blood coagulation, blood fibrinolysis, blood viscosity, blood cell counts or platelet activity. But DHK significantly up-regulated protein and mRNA expressions of SIRT1, and reduced leukocytes infiltration into thrombus and vein wall, serum levels of inflammatory cytokines, and protein expressions of acetylated p65 (Ace-p65), phosphorylated p65 (p-p65) and tissue factor (TF). Moreover, the antithrombotic effect of DHK was significantly abolished by EX527. CONCLUSION: DHK may prevent DVT by inhibiting inflammation via SIRT1/NF-κB signaling pathway.