Exploring the molecular mechanism of Toddalia asiatica (L.) lam on the treatment of thrombosis based on zebrafish models, network pharmacology and experimental verification

Toddalia asiatica (L.) Lam. (TA) is a traditional folk medicine of ethnic minorities in the southwest of China. It is widely used in the treatment of dispersing blood stasis and activating blood. However, the effective substance and pharmacological mechanism have not been fully elucidated. The zebrafish larvae were treated with Phenylhydrazine (PHZ) to establish a thrombus model, and the staining intensity of zebrafish red blood cells was analyzed. The antithrombotic activity of TA was verified for the first time, and it was found that the inhibition rate of TA on thrombosis was up to 60.85 %. The chemical ingredients of TA were collected by combining UPLC-HRMS analysis and the literature research. Network pharmacology revealed that six key targets were obtained, which including TNF, AKT1, EGFR, PTGS2, PPARG, and IFNG. It showed that the PI3K-Akt pathway was a core signaling pathway. Coagulation factor III(TF), playing an important role in the process of hemostasis and thrombosis, which ranks high in the PPI network. Moreover, the results of molecular docking showed that the active components had a strong binding force with TF, which indicated that TF might be the key target of TA in treating thrombosis. In vitro experiments showed that TA could inhibit TNF-α-induced high expression of TF in EA.hy926 cells. In addition, TA could inhibit TNF-α-activated expression of Akt, IκBα and P65 protein phosphorylation in PI3K-Akt pathway. The results showed that TA had antithrombotic activity and exerted an antithrombotic effect by inhibiting the expression of TF through the PI3K-Akt-NF-κB signaling pathway.