Study On Antithrombotic Molecular Mechanism Of Ligusticum Chuanxiong Based On Network Pharmacology

With the aging of the population and the change of dietary structure and habits,the incidence of cardiovascular disease is increasing,in which thrombotic disease plays an important role in cardio-cerebrovascular events.At present,there are many kinds of antithrombotic drugs in clinic,but most of them have certain side effects,and their therapeutic effects are different.Ligusticum chuanxiong removing blood stasis and promoting blood circulation is a traditional Chinese medicine,it has a good effect in the prevention and treatment of thrombosis.however,its related molecular mechanism is not clear and the material basis of its efficacy has not been fully elucidated.In this study,the research method of network pharmacology was used to analyze the interaction between the active components of Ligusticum chuanxiong and to predict the potential mechanism of antithrombotic effect of Ligusticum chuanxiong.Then computer virtual screening and pharmacological experiments were carried out to further explore the molecular mechanism of Ligusticum chuanxiong antithrombotic.The achievements of this paper are mainly reflected in the following aspects:1.In this study,using the research method of network pharmacology,8 key components and 6key targets of Ligusticum chuanxiong antithrombotic were obtained by analyzing the network topology parameters through network analysis.Kyoto Encyclopedia of Genes and Genomes signal pathway analysis shows that the potential mechanism of Ligusticum chuanxiong anti-thrombus may be through PI3K-Akt signal pathway to play a role in the treatment of thrombus.2.Using network analysis to analyze tissue factor and its related target network,it is found that tissue factor is closely related to thrombus targets such as inflammation,angiogenesis and fibrin aggregation,and is the key target of thrombosis.Then the computer simulation method is used to explore whether the active components of Ligusticum chuanxiong can act on the target of TF.The results show that most of the active components of Ligusticum chuanxiong can strongly bind to TF protein,and the representative compound is levistilide A.The inhibitory activity of representative compounds against TF was evaluated by 3D-QSAR.The results showed that all the18 active components of Ligusticum chuanxiong had good inhibitory activity against TF,and 13of them had strong inhibitory activity against TF(their IC₅₀ value was less than 1μM).It is further proved that the main active components of Ligusticum chuanxiong may treat related cardiovascular diseases by inhibiting the activity of TF and promoting blood circulation and removing blood stasis.3.MTT was used to measuring the cytotoxicity of Ligusticum chuanxiong including main active components.The result showed that these components of Ligusticum chuanxiong had no cytotoxicity in the experimental dose range,which proved the safety of natural products.Then,RT-PCR method was used to study the effect of active components of Ligusticum chuanxiong on the expression of TF and ICAM-1 m RNA in EA.hy926 induced by lipopolysaccharide（LPS）or tumor necrosis factorα（TNF-α）.The results showed that ferulic acid,levistilide A and ligustilide were in a dose-dependent manner to inhibit the expression of TF and ICAM-1 m RNA EA.hy926cells induced by LPS or TNF-α,which may be one of the molecular mechanisms of antithrombotic effect of Ligusticum chuanxiong.4.The results of network pharmacology showed that ligustilide was the key component of ligustilide chuanxiong to exert its antithrombotic effect.Western blot and flow cytometry showed ligustilide could inhibit the expression of TF protein at the protein level.Therefore,ligustilide was selected as the representative component of Ligusticum chuanxiong to study the pharmacological mechanism,and western blot technique was used to detect the protein expression in PI3K-Akt pathways.It was found that ligustilide inhibited the phosphorylation of Akt and p65 in endothelial cells induced by TNF-αin a dose-dependent manner.It is speculated that ligustilide can inhibit the expression of TF by regulating PI3K-Akt signal pathway,which further validates the prediction results of network pharmacology.In this paper,by using the research idea of network pharmacology,the relationship network among pharmacological molecules,targets and signal pathways of Ligusticum chuanxiong was constructed systematically,and the core components,targets and signal pathways were scr eened,and then pharmacological experiments were used to verify that the active components of Ligusticum chuanxiong could prevent and treat related cardiovascular diseases by regulating/inhibiting TF and related core signal pathways.This study partially clarified the modern pharmacological mechanism of Ligusticum chuanxiong,a traditional Chinese medicine for promoting blood circulation and removing blood stasis,in the treatment of related cardiovascular diseases,and found some active components with strong inhibitory effect on TF,which may be the material basis of antithrombotic effect of Ligusticum chuanxiong.