Jianxiu Zhai1, Zhaohui Ren2, Yuwei Wang3, Mingshu Han4, Na Han5, Zhihui Liu6, Sikai Li7, Jun Yin8. 1. School of Traditional Chinese Material, Shenyang Pharmaceutical University, Shenyang, 110016, China. Electronic address: lookingformilk@163.com. 2. School of Traditional Chinese Material, Shenyang Pharmaceutical University, Shenyang, 110016, China. Electronic address: 583194513@qq.com. 3. School of Traditional Chinese Material, Shenyang Pharmaceutical University, Shenyang, 110016, China. Electronic address: 1372689787@qq.com. 4. School of Traditional Chinese Material, Shenyang Pharmaceutical University, Shenyang, 110016, China. Electronic address: 675588190@qq.com. 5. School of Traditional Chinese Material, Shenyang Pharmaceutical University, Shenyang, 110016, China. Electronic address: hanna82@163.com. 6. School of Traditional Chinese Material, Shenyang Pharmaceutical University, Shenyang, 110016, China. Electronic address: 48867414@qq.com. 7. School of Traditional Chinese Material, Shenyang Pharmaceutical University, Shenyang, 110016, China. Electronic address: 672406281@qq.com. 8. School of Traditional Chinese Material, Shenyang Pharmaceutical University, Shenyang, 110016, China. Electronic address: yinjun826@sina.com.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: Chinese patent medicine Zhixiong Capsule (ZXC) is the equal mixture of the extract of leech, Ligusticum chuanxiong Hort., Salvia miltiorrhiza Bunge, Leonurus japonicus Houtt., and Pueraria lobate (Willd.) Ohwi, which have been long used against inflammation, hyperlipidemia or blood stasis. In our previous study, ZXC showed good efficacy in preventing atherosclerosis (AS) plaque formation in rabbits. AIM OF THE STUDY: In actual clinic practice, patients are more likely to receive treatments after AS plaque formation. Therefore, the efficacy of ZXC on formed AS plaques and the underlying mechanisms were further investigated in this study. MATERIALS AND METHODS: Simvastatin (positive control) and ZXC (420 mg/kg and 840 mg/kg) were administrated to rats which first received long-term high fat diet administration (12 weeks). The blood lipid profiles of rats were monitored during the whole experiment, and the thoracic arteries were collected at the end of experiment for AS assessment (18th week). The blood-dissolved ZXC components were determined using an UPLC-QTOF-MS method, and the attained components were then used for network pharmacology analysis to predict the key ZXC components and targets. At last, the predicted targets were validated by ELISA and western blot methods. RESULTS: ZXC administration showed good blood lipid-lowering effect by significantly reduced LDL-C and TC levels in rats while significantly increased HDL-C level. Compared with model group, simvastatin, low- and high-dose of ZXC administration decreased the ratio of intimal area and medial area by 81.1%, 71.1% and 71.4%, respectively (p < 0.01), and significantly alleviated collagen deposition and mineralization in rat arteries. It was found by network pharmacology analysis that leech and four components (namely daidzein, 4-methylenemiltirone, isorhamnetin and 2-isopropyl-8-methylphenanthrene-3,4-dione) are vital components for the anti-AS efficacy of ZXC. Combing the results from biochemical validation, IL-4, IL-13, MAPK1, MAPK14, JUN and P53 were confirmed as key targets of ZXC. CONCLUSION: It could be concluded that ZXC has value as an anti-AS agent in clinical treatment against formed AS plaque at the current application dosage.
ETHNOPHARMACOLOGICAL RELEVANCE: Chinese patent medicine Zhixiong Capsule (ZXC) is the equal mixture of the extract of leech, Ligusticum chuanxiong Hort., Salvia miltiorrhiza Bunge, Leonurus japonicus Houtt., and Pueraria lobate (Willd.) Ohwi, which have been long used against inflammation, hyperlipidemia or blood stasis. In our previous study, ZXC showed good efficacy in preventing atherosclerosis (AS) plaque formation in rabbits. AIM OF THE STUDY: In actual clinic practice, patients are more likely to receive treatments after AS plaque formation. Therefore, the efficacy of ZXC on formed AS plaques and the underlying mechanisms were further investigated in this study. MATERIALS AND METHODS:Simvastatin (positive control) and ZXC (420 mg/kg and 840 mg/kg) were administrated to rats which first received long-term high fat diet administration (12 weeks). The blood lipid profiles of rats were monitored during the whole experiment, and the thoracic arteries were collected at the end of experiment for AS assessment (18th week). The blood-dissolved ZXC components were determined using an UPLC-QTOF-MS method, and the attained components were then used for network pharmacology analysis to predict the key ZXC components and targets. At last, the predicted targets were validated by ELISA and western blot methods. RESULTS:ZXC administration showed good blood lipid-lowering effect by significantly reduced LDL-C and TC levels in rats while significantly increased HDL-C level. Compared with model group, simvastatin, low- and high-dose of ZXC administration decreased the ratio of intimal area and medial area by 81.1%, 71.1% and 71.4%, respectively (p < 0.01), and significantly alleviated collagen deposition and mineralization in rat arteries. It was found by network pharmacology analysis that leech and four components (namely daidzein, 4-methylenemiltirone, isorhamnetin and 2-isopropyl-8-methylphenanthrene-3,4-dione) are vital components for the anti-AS efficacy of ZXC. Combing the results from biochemical validation, IL-4, IL-13, MAPK1, MAPK14, JUN and P53 were confirmed as key targets of ZXC. CONCLUSION: It could be concluded that ZXC has value as an anti-AS agent in clinical treatment against formed AS plaque at the current application dosage.