Study On The Antithrombotic Efficacy And Mechanism Of Leonurus And Its Specific Ingredients-Leonurine Hydrochloride

Object:Study the antithrombotic efficacy of Leonurus and its pharmacodynamic substance basis and investigate the potential antithrombotic effect and mechanism of Leonurine hydrochloride,the specific ingredients of Leonurus from multiple perspectives.Methods:1.Preparation and antithrombotic activity of Leonurus extract.The Leonurus extract was obtained according to the preparation method of Leonurus liquid extract in<Chinese Pharmacopoeia>.The content of two components of Leonurus extracts,leonurine hydrochloride and stachydrine hydrochloride,were determined by liquid chromatograpgy-mass spectrometry（LC-MS/MS）to provide scientific and reliable results for the subsequent study of Leonurus.A zebrafish thrombosis model was established using adrennaline hydrochloride（45μM）,and Leonurus extract（250,500,1000μg/m L）were given simultaneously for intervention treatment.After 16 hours,the antithrombotic effect of Leonurus extract was investigated by statistical analysis of the area of tail thrombus and the intensity of heart erythrocyte staining in zebrafish by using o-anisidine to stain the red blood cells in vivo.2.In vivo antithrombotic effect and mechanism of leonurine hydrochloride based on metabolomics.Adrenaline hydrochloride（45μM）was used to induce thrombosis in zebrafish,and leonurine hydrochloride（2.5,5,10μM）were used to intervene during the treatment.Erythrocyte staining area of tail vein,staining intensity of heart erythrocytes and histopathological observation of zebrafish were used to evaluate the effect of leonurine hydrochloride on erythrocyte aggregation in zebrafish.The content of ROS in zebrafish was detected by ROS fluorescence probe,the levels of vascular-related factors NO and ET-1 were measured by kits to investigate the antithrombotic pharmacological effects of leonurine hydrochloride.Also,metabolomics techniques were used to detect changes in the overall levels of endogenous small molecule metabolites in the tissues of thrombosed zebrafish induced by adrenaline hydrochloride after leonurine hydrochloride intervention.The differential metabolites of the effect of leonurine hydrochloride intervention on thrombosed zebrafish were searched by multivariate statistical analysis.We also searched for the metabolic pathways associated with the differential metabolites by KEGG pathway analysis to reveal the antithrombotic mechanism of leonurine hydrochloride from the level of organismal metabolism.Subsequently,the pharmacological efficacy of leonurine hydrochloride against thrombosis in zebrafish and its effect on metabolic pathways in thrombosed zebrafish were integrated to find the possible mechanism of leonurine hydrochloride against thrombosis,and the expression of related factors in zebrafish tissues was further detected by biochemical index assay kit,the expression of related genes was detected by RT-q PCR method to explore the mechanism of the antithrombotic effect of leonurine hydrochloride.3.Study on the protective effect and mechanism of endothelial oxidative damage by leonurine hydrochloride.H₂O₂（200μM）was used to stimulate HUVEC cells to establish a model of oxidative damage in HUVEC cells.The effects of leonurine hydrochloride（2.5,5,10μM）on the cell viability of the oxidative injury model were determined by MTT,and the effects of leonurine hydrochloride on endothelial cell migration and angiogenesis were explored by scratch assay and lumen formation assay.ROS fluorescence probe was used to detected the content of ROS in HUVEC cells,biochemical kits were used to detected the expression of LDH,MDA,SOD and NO in cells,and RT-q PCR and Western Blot molecular biology techniques were used to detect the expression levels of m RNA such as Bax,Bcl2,caspase3 and the expression of PI3K,Akt,e NOS,caspase3 and other proteins in the cells to study the effect and mechanism of LEO in protecting vascular endothelial cells.Results:1.After analysis,the contents of both leonurine hydrochloride and stachydrine hydrochloride in the Leonurus extract used by us are 0.132%and 1.219%respectively,which are higher than 0.05%and 0.5%specified in<Chinese Pharmacopoeia>,indicating that the quality of the Leonurus herb we used was in accordance with the Pharmacopoeia of the<Chinese Pharmacopoeia>.And the pharmacological study showed that Leonurus extract（250,500,1000μg/m L）has good antithrombotic effect,and it can inhibit thrombosis by inhibiting the expression of prothrombotic molecules v WF and TXB2.2.Leonurine hydrochloride（2.5,5,10μM）significantly inhibited the formation of thrombus in zebrafish,reduced the level of ROS in vivo,and regulated the secretion of vasodilatory factor NO and contractile factor ET-1.Also,leonurine hydrochloride significantly modulated several endogenous metabolites disturbed by AH,and inhibited thrombus formation by regulating tyrosine metabolism,alanine metabolism,aspartate metabolism,glutamate metabolism,inositol phosphate metabolism and citric acid cycle,which in turn interfered with oxidative stress response,coagulation cascade response and Ca²⁺signaling pathway.In further mechanistic studies,it was suggested that the mechanism of antithrombotic action of leonurine hydrochloride might be related to antioxidant stress response,and inhibition of the expression of genes related to platelet activation and coagulation cascade reaction.3.H₂O₂at 200μM significantly induced oxidative damage in human umbilical vein endothelial cells（HUVECs）and inhibited the proliferation and migration ability of HUVEC cells.Leonurine hydrochloride（2.5,5,10μM）significantly modulated H₂O₂-induced changes in the levels of oxidative stress indicators ROS,LDH,MDA,and SOD in HUVEC cells,increased the levels of vasodilatory factor NO,and maintained blood endoscopic homeostasis by activating PI3K/Akt-e NOS signaling pathway to repair H₂O₂-induced endothelial injury.Conclusion:1.The Leonurus extract（250,500,1000μg/m L）and Leonurine hydrochloride（2.5,5,10μM）reduced adrenaline hydrochloride-induced tail vein thrombosis in zebrafish,enhanced the intensity of cardiac red blood cell staining,and showed good antithrombotic activity.2.Leonurine hydrochloride（2.5,5,10μM）exhibited good anti-thrombotic effect against adrenaline hydrochloride induced thrombosis in zebrafish.This effect may be related to the intervention of metabolic pathways such as amino acid metabolism,lysine metabolism and tricarboxylic acid cycle,antioxidant,inhibition of platelet aggregation and anticoagulation cascade reaction.3.Leonurine hydrochloride（2.5,5,10μM）protects the normal function of vascular endothelial cells by activating PI3K/Akt signaling pathway and resisting H₂O₂-induced oxidative damage in HUVEC cells.