Dan-Lou tablets reduces inflammatory response via suppression of the MyD88/p38 MAPK/NF-κB signaling pathway in RAW 264.7 macrophages induced by ox-LDL.

ABSTRACT

ETHNOPHARMACOLOGICAL EVIDENCE The anti-inflammatory effect of Dan-Lou tablets (DLT) have been reported; however, the signaling pathways involved and their role in foam cell formation remains unclear. AIM OF THE STUDY The purpose of this study was to determine the molecular target and mechanism of DLT in the treatment of coronary heart disease (CHD), and investigate the role of DLT in inhibiting foam cell formation and the anti-inflammatory effects of RAW 264.7 macrophages. MATERIALS AND METHODS: This study explored and elucidated the main active components, therapeutic targets, and pharmacological mechanisms of DLT treatment for CHD using network pharmacology. Secondly, the accuracy of the interaction of key active compounds with key proteins was verified by molecular docking analysis. Eight chemical compositions were determined from the ethanol extract of DLT (EEDL) by high-performance liquid chromatography. Finally, this study used EEDL intervention with oxidized low-density lipoprotein (ox-LDL) to induce RAW264.7 macrophages to verify the results of network pharmacology. RESULTS: According to network pharmacological analysis, 269 common targets of DLT and CHD were obtained from an online database, and 24 key targets were obtained from further analysis. GO enrichment and KEGG analyses were performed, mainly involving the cAMP, cGMP-PKG, MAPK, and NF-κB signaling pathways, and vascular smooth muscle contraction. Molecular docking showed that the active components in DLT docked well with MyD88, NF-κB, and p38 MAPK. The eight compounds from the EEDL have been identified as gallic acid, salvianolic acid, puerarin, daidzein, paeoniflorin, salvianolic acid B, cryptotanshinone, and tanshinone IIA with concentrations of 4.62, 4.76, 23.73, 34.24, 14.59, 21.69, 0.34, and 0.47 µg/mg, respectively. Further in vitro experiments showed that the levels of MyD88 and p-p38 MAPK in RAW 264.7 macrophages induced by ox-LDL increased noticeably. Stimulating the NF-κB signaling pathway increased the release of pro-flammatory factors (TNF-α and IL-1ß) and strengthened the inflammatory response (P < 0.05 or P < 0.01), while the levels of MyD88, p38 MAPK, NF-κB, TNF-α, and IL-1ß decreased after EEDL treatment (P < 0.05 or P < 0.01). CONCLUSION: The study demonstrated that the anti-inflammatory activity of the DLT intervention of ox-LDL-induced RAW 264.7 macrophages may involve the MyD88/p38 MAPK/NF-κB signaling pathway.