目的 通過數(shù)據(jù)庫預測結合體外細胞實驗探討四妙丸調(diào)血脂的作用及機制。方法 通過中藥系統(tǒng)藥理學（TCMSP）數(shù)據(jù)庫與分析平臺、GeneCards、Disgenet、OMIM等數(shù)據(jù)庫篩選四妙丸關鍵成分及高脂血癥相關靶點；蛋白質(zhì)-蛋白質(zhì)相互作用（PPI）分析四妙丸治療高脂血癥的關鍵靶點；基因本體（GO）和京都基因與基因組百科全書（KEGG）分析此過程涉及的生物學過程及通路；進一步采用分子對接探討四妙丸成分與關鍵靶點間的相互作用。體外培養(yǎng)HepG2細胞，采用CCK-8實驗篩選游離脂肪酸造模和四妙丸、辛伐他汀給藥的最佳濃度，構建游離脂肪酸誘導的HepG2細胞高脂血癥模型，給予四妙丸（25、50、100 mg·L^-1）、辛伐他汀（25 μmol·L^-1）處理24 h，對照組不造模不加藥，模型組不加藥，通過細胞油紅O染色和細胞內(nèi)總膽固醇（TC）、三酰甘油（TG）含量測定研究細胞內(nèi)脂質(zhì)積累情況；實時熒光定量PCR（qRT-PCR）和Westernblotting檢測磷脂酰肌醇4,5-二磷酸3-激酶催化亞基α（PIK3CA）、絲氨酸/蘇氨酸激酶1（AKT1）、過氧化物酶體增殖物激活受體γ（PPARG）和細胞色素7A1（CYP7A1） mRNA和蛋白表達。結果 共篩選出56個四妙丸核心成分，對應蛋白靶點735個；高脂血癥疾病靶點共1 860個；有效成分靶點與疾病靶點的交集靶點為244個。PPI結果顯示核心靶點包括AKT1、IL6、EGFR、TNF、PIK3CA、PPARG等。GO和KEGG富集分析揭示四妙丸主要通過PI3K-AKT1信號通路、胰島素抵抗、炎癥反應信號等改善高脂血癥。細胞實驗結果表明，與模型組比較，四妙丸顯著降低游離脂肪酸誘導的HepG2高脂血癥細胞模型內(nèi)脂質(zhì)沉積（P＜0.05、0.01），顯著降低細胞內(nèi)TC、TG的含量（P＜0.05、0.01）；顯著降低細胞內(nèi)PIK3CA、AKT1mRNA和蛋白表達水平（P＜0.05、0.01），顯著升高PPARG、CYP7A1 mRNA和蛋白表達水平（P＜0.05、0.01）。結論 四妙丸改善游離脂肪酸誘導的HepG2細胞高脂血癥，作用機制與調(diào)節(jié)PIK3CA/AKT1/PPARG/CYP7A1信號通路有關。

Objective To explore the lipid-lowering effect and mechanism of Simiao Pill through database prediction and in vitro cell experiments. Methods The key components of Simiao Pill and the targets related to hyperlipidemia were screened through databases such as Traditional Chinese Medicine Systems Pharmacology (TCMSP) Database and Analysis Platform and GeneCards, the key targets of Simiao Pill in treating hyperlipidemia were analyzed by protein-protein interaction (PPI), the biological processes and pathways involved in this process were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), molecular docking was further used to explore the interaction between the components of Simiao Pill and the key targets. HepG2 cells were cultured in vitro, and the CCK-8 assay was used to screen the optimal concentrations of free fatty acid modeling and Simiao Pill and simvastatin administration. A free fatty acid-induced HepG2 cell hyperlipidemia model was constructed, and the cells were treated with Simiao Pill (25, 50, 100 mg·L^-1) and simvastatin (25 μmol·L^-1) for 24 h. The control group was not modeled and not treated with drugs, and the model group was not treated with drugs. The intracellular lipid accumulation was studied by oil red O staining and determination of total cholesterol (TC) and triglyceride (TG) content in cells. The mRNA and protein expressions of phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), serine/threonine kinase 1 (AKT1), peroxisome proliferator-activated receptor gamma (PPARG), and cytochrome P450 7A1 (CYP7A1) were detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blotting. Results A total of 56 core components of Simiao Pill were screened, corresponding to 735 protein targets; there were 1 860 disease targets for hyperlipidemia; the intersection targets of the effective component targets and disease targets were 244. The PPI results showed that the core targets included AKT1, IL6, EGFR, TNF, PIK3CA, PPARG, et al. GO and KEGG enrichment analysis revealed that Simiao Pill mainly improved hyperlipidemia through the PI3K-AKT signaling pathway, insulin resistance, and inflammatory response signaling. The cell experiment results showed that compared with the model group, Simiao Pill significantly reduced lipid deposition in the free fatty acid-induced HepG2 hyperlipidemia cell model (P <0.05, 0.01), significantly reduced the intracellular TC and TG content (P <0.05, 0.01); significantly reduced the mRNA and protein expression levels of PIK3CA and AKT1 in cells (P <0.05, 0.01), and significantly increased the mRNA and protein expression levels of PPARG and CYP7A1 (P <0.05, 0.01). Conclusion Simiao Pill improves free fatty acid-induced hyperlipidemia in HepG2 cells, and its mechanism of action is related to the regulation of the PIK3CA/AKT1/PPARG/CYP7A1 signaling pathway.

R285.5

廣東省中醫(yī)藥研究開發(fā)重點實驗室開放基金項目（ KFKT02-002）；廣東省中醫(yī)藥局開放項目（20233004）