ABSTRACT
Objective:To explore the core targets and potential molecular mechanisms of tetramethylpyrazine in the treatment of rats with acute necrotizing pancreatitis (ANP) based on network pharmacology.Methods:The related co-targets of tetramethylpyrazine and ANP were screened out by traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP) and human disease information-related databases (CTD, DisGeNET, GeneCards, OMIM); Uniprot data were used to co-link and put into the STRING database to build protein-protein interaction (PPI) networks; the Cytoscape software was used for further analysis and the key targets were obtained by using the cytoHubba plug-in. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on these key targets, and finally the molecular docking models were constructed by using PyMol and AutoDockTools software. 30 male SD rats were randomly divided into the control group, ANP group, and tetramethylpyrazine treatment group (tetramethylpyrazine group). ANP rats were induced by retrograde infusion of 4% sodium taurocholate into the biliary-pancreatic duct, and the tetramethylpyrazine group rats were injected with 10 ml/kg tetramethylpyrazine through the abdominal cavity after ANP was induced. After 12 h, pancreatic tissue was taken, a pathological examination was performed routinely, and immunohistochemical staining was performed to observe the protein expression of key targets in pancreatic tissue. Blood was taken from orbits, and then the serum IL-6 and tumor necrosis factor-α (TNF-α) levels were measured by enzyme-linked immunosorbent assay (ELISA).Results:The drug platform screened 137 tetramethylpyrazine action targets, and the disease database screened out 513 ANP-related targets; then 25 targets were obtained through intersection, finally resulting in a total of 5 key targets: albumin (ALB), epidermal growth factor receptor (EGFR), caspase 3 (CASP3), mitogen-activated protein kinase 1 (MAPK1) and B-cell lymphoma-2-like protein 1 (BCL2L1). GO functional enrichment analysis of biological processes mainly involved reproductive structure or system development, response to antibiotics, chemical stress and reactive oxygen species, and the cellular components were mainly vesicle lumen, membrane raft, membrane microdomain, and secretory granule lumen; molecular functions mainly included SH2 domain, phosphotyrosine residue, protease binding, protein tyrosine kinase and nuclear receptor activity; KEGG pathway enrichment analysis were mainly enriched in Ras signaling pathway, PI3K-Akt signaling pathway, platinum drug resistance, phospholipase D signaling pathway, and EGFR tyrosine kinase inhibitor resistance. The average binding energy of the 5 key targets molecule docking was -4.20 kcal/mol. After hematoxylin-eosin staining, it could be seen that the gland structure of rats in the ANP group was disordered, the interlobular space was significantly increased, and neutrophil infiltration was observed in the acinar, perivascular and gland space. The pancreatic lobule space of tetramethylpyrazine group rats was slightly increased, with mild neutrophil infiltration. The protein expressions of EGFR, CASP3 and MAPK1 in the ANP group were significantly higher compared with those in the control group, and EGFR, CASP3 and MAPK1 expression in tetramethylpyrazine group was significantly lower than those in ANP group ( P<0.01); the protein expression of BCL2L1 in the ANP group were significantly higher than that in control group, and the protein expression of BCL2L1 in tetramethylpyrazine group were significantly higher than that in ANP group (all P value <0.05). The serum levels of IL-6 and TNF-α in the ANP group were significantly higher than those in the control group, and IL-6 and TNF-α in tetramethylpyrazine group were significantly lower than those in the ANP group (all P value <0.01). Conclusions:Tetramethylpyrazine could reduce the inflammatory response and oxidative stress injury after ANP by activating a variety of signaling pathways, enhancing the expression of anti-apoptotic genes, and blocking the enzymatic cascade reaction of apoptotic caspase, thus playing a protective role in pancreatic tissues of rats with ANP.