摘要目的:基于网络药理学探讨川芎嗪治疗急性坏死性胰腺炎(ANP)大鼠的核心靶点及其潜在的分子机制。方法:通过中药系统药理学分析平台(TCMSP)及人类疾病信息相关数据库(CTD、DisGeNET、GeneCards、OMIM)筛选出川芎嗪作用靶点与ANP相关靶点，利用Uniprot数据库进行交集，导入STRING数据库构建蛋白质-蛋白质相互作用(PPI)网络，再导入Cytoscape软件进一步分析，利用cytoHubba插件得到关键靶点。对关键靶点进行基因本体(GO)功能富集分析和京都基因与基因组百科全书(KEGG)通路富集分析，最后通过PyMol和AutoDockTools软件进行分子对接。将30只雄性SD大鼠随机分为对照组、ANP组和川芎嗪治疗组(川芎嗪组)。采用胰胆管逆行注射4%牛磺胆酸钠建立ANP大鼠模型，川芎嗪组在制模后经腹腔注射10 ml/kg川芎嗪注射液。造模后12 h，取胰腺组织，常规行病理学检查，免疫组织化学染色观察关键靶点在胰腺组织中的蛋白表达；眼眶取血，采用酶联免疫吸附测定法(ELISA)测定血清IL-6和肿瘤坏死因子-α(TNF-α)水平。结果:药物平台筛选出137个川芎嗪作用靶点，疾病数据库筛选出513个ANP相关靶点，通过交集得到的25个靶点，最终获得白蛋白(ALB)、表皮生长因子受体(EGFR)、胱天蛋白酶3(CASP3)、丝裂原活化蛋白激酶1(MAPK1)和B细胞淋巴瘤样蛋白1(BCL2L1)共5个关键靶点。GO功能富集分析生物学过程主要涉及生殖结构、系统发育，对抗生素、化学应激和活性氧的反应等，细胞组成主要为囊泡腔、膜筏、膜微区和分泌颗粒腔等靶蛋白，分子功能主要包括SH2域、磷酸酪氨酸残基、蛋白酶结合及蛋白酪氨酸激酶和核受体活性等；KEGG通路富集分析主要为Ras信号通路、PI3K-Akt信号通路、铂类药物耐药、磷脂酶D信号通路和EGFR酪氨酸激酶抑制剂耐药等。5个关键靶点分子对接的平均结合能为-4.20 kcal/mol。胰腺组织病理学检查结果示ANP组大鼠腺体结构较紊乱，小叶间隙明显增大，腺泡、血管周围以及腺体间隙均可见中性粒细胞浸润；川芎嗪组大鼠胰腺小叶间隙略微增大，中性粒细胞轻度浸润。ANP组大鼠胰腺组织EGFR、CASP3和MAPK1蛋白表达量较对照组显著升高，川芎嗪组表达量较ANP组显著降低( P值均<0.01)；ANP组BCL2L1蛋白表达量较对照组显著升高，川芎嗪组又较ANP组显著升高( P值均<0.05)。ANP组大鼠血清IL-6和TNF-α水平较对照组显著增高，川芎嗪组则均较ANP组显著降低( P值均<0.01)。 结论:川芎嗪可以通过激活多种信号通路，减轻ANP后的炎症反应和氧化应激损伤，增强抗凋亡基因的表达，阻断细胞凋亡半胱天冬酶解级联反应，对ANP大鼠胰腺组织起保护性作用。

abstractsObjective: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.