Exploring the Mechanism of Tetramethylpyrazine in the Treatment of Osteoarthritis Based on Network Pharmacology
Authors: Li Juncen , Song Daiying , Li Baihui , Wang Yajie , Sun Huilin , Li Qinglin , Lin Xiangming , Wang Di , Zhou Guangdong , Liu Yu 
Published: September 2023

Abstract
Background
Osteoarthritis(OA) is the most common joint disease, mainly damaging the articular cartilage and involving the whole joint tissue. It has the characteristics of a long course, repeated symptoms, and a high disability rate, and the incidence trend is gradually increasing. Tetramethylpyrazine(TMP) is the primary alkaloid active substance in Ligusticum wallichii, a traditional Chinese medicine, which has the effect of promoting blood circulation and dredging collaterals and has a good impact on the treatment of early OA. Still, its molecular mechanism has not been fully clarified so far. Based on network pharmacology, molecular docking simulation, and animal experiments, this study explored TMP's target and molecular mechanism in treating OA.
Methods
We used PubChem, SwissTargetPrediction, and PharmMapper databases to predict TMP's molecular structure and potential targets. GeneCards and DisGeNET databases were used to indicate the relevant targets of OA. Apply the UniProt database to convert targets into unified gene names and proofread and remove duplicates. The intersection targets of TMP and OA obtained on the venny2.1.0 website were submitted to the STRING database to construct a PPI network. CytoScape 3.8.2 software analyzed the PPI network and got the sub-network modules and ten key targets. The TMP and OA intersection targets were analyzed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment using the DAVID 6.8 database. The intersecting targets of TMP and OA, the biological process of GO enrichment, and the KEGG signaling pathway were imported into Cytoscape 3.8.2 software to construct the TMP-target-pathway network diagram. Use molecular docking technology to simulate the interaction between TMP molecules and critical targets and predict the binding mode and binding ability. Animal models of rabbit knee osteoarthritis were prepared, and a magnetic resonance imager(MRI) and fluorescence quantitative PCR (RT-qPCR) were used to observe the effect of TMP in treating OA and the expression of essential target genes.
Results
585 potential targets of TMP, 3857 potential targets of OA, and 49 intersecting targets of TMP and OA were obtained. The top 10 essential target genes were obtained in order of ranking: ALB, ESR1, IL10, CAT, F2, MPO, C3, CYP3A4, CYP2C9, and ANXA1. GO, and KEGG analysis implied that the key targets might act on OA by affecting endothelial cell permeability, peri-articular microcirculatory status, NETs production, activation of complement system and coagulation pathway, regulation of immune function of macrophages and T cells, and substance metabolism pathway in vivo, etc. The molecular mechanism might involve forming a neutrophil extracellular trap, regulating the actin cytoskeleton, complement and coagulation cascades, and T cell receptor signaling pathways. Molecular docking simulations showed that the binding energy of IL10 and ANXA1 to TMP was more significant than − 5Kcal/mol, but the other essential target proteins showed better binding to TMP, and the binding energy was less than − 5Kcal/mol. Animal experiments showed that TMP had a significant therapeutic effect on OA. The TMP group had significantly reduced knee joint effusion and bone marrow damage compared to the OA group (P < 0.05). The qRT-PCR results showed that compared with the OA group, the mRNA expression of ESR1, CAT, C3, CYP3A4, CYP2C9, and ANXA1 in the TMP group increased (P < 0.05), while there was no significant difference in mRNA expression of ALB, IL-10, F2, MPO, etc. (P > 0.05).
Conclusion
TMP effectively treats OA with multi-target and multi-pathway interactions. ESR1, CAT, C3, CYP3A4, CYP2C9, and ANXA1 may be potential targets for TMP treatment of OA. The molecular mechanism mainly involves the formation of neutrophil extracellular trap, regulation of the actin cytoskeleton, complement and coagulation cascades, and T cell receptor signaling pathways.