Li Zhu, Cheng Qi, Yu Lu, He Yuan-Yuan, Gao Li-Na, Wang Yue, Li Lin, Cui Yuan-Lu, Gao Shan, Yu Chun-Quan
Tianjin University of Traditional Chinese Medicine, No. 10, Poyang Lake Road, West Zone, Tuanbo New City, Jinghai District, Tianjin, China.
College of Pharmacy, Jining Medical University, Rizhao, China.
J Ethnopharmacol. 2022 Nov 15;298:115600. doi: 10.1016/j.jep.2022.115600. Epub 2022 Aug 12.
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.
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.
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.
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-1β) 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-1β decreased after EEDL treatment (P < 0.05 or P < 0.01).
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.
丹蒌片（DLT）的抗炎作用已有报道；然而，其涉及的信号通路及其在泡沫细胞形成中的作用仍不清楚。
本研究旨在确定丹蒌片治疗冠心病（CHD）的分子靶点和机制，并探讨丹蒌片在抑制RAW 264.7巨噬细胞泡沫细胞形成和抗炎作用中的作用。
本研究采用网络药理学方法探索并阐明丹蒌片治疗冠心病的主要活性成分、治疗靶点和药理机制。其次，通过分子对接分析验证关键活性化合物与关键蛋白相互作用的准确性。采用高效液相色谱法从丹蒌片乙醇提取物（EEDL）中测定了8种化学成分。最后，本研究采用EEDL干预氧化型低密度脂蛋白（ox-LDL）诱导RAW264.7巨噬细胞，以验证网络药理学的结果。
通过网络药理学分析，从在线数据库中获得了丹蒌片和冠心病的269个共同靶点，并通过进一步分析获得了24个关键靶点。进行了基因本体（GO）富集分析和京都基因与基因组百科全书（KEGG）分析，主要涉及环磷酸腺苷（cAMP）、环磷酸鸟苷-蛋白激酶G（cGMP-PKG）、丝裂原活化蛋白激酶（MAPK）和核因子κB（NF-κB）信号通路以及血管平滑肌收缩。分子对接显示，丹蒌片中的活性成分与髓样分化因子88（MyD88）、NF-κB和p38丝裂原活化蛋白激酶（p38 MAPK）对接良好。已鉴定出EEDL中的8种化合物为没食子酸、丹酚酸、葛根素、大豆苷元、芍药苷、丹酚酸B、隐丹参酮和丹参酮IIA，浓度分别为4.62、4.76、23.73、34.24、14.59、21.69、0.34和0.47μg/mg。进一步的体外实验表明，ox-LDL诱导的RAW 264.7巨噬细胞中MyD88和磷酸化p38 MAPK（p-p38 MAPK）的水平显著升高。刺激NF-κB信号通路会增加促炎因子（肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β））的释放并增强炎症反应（P<0.05或P<0.01），而EEDL处理后MyD88、p38 MAPK、NF-κB、TNF-α和IL-1β的水平降低（P<0.05或P<0.01）。
该研究表明，丹蒌片干预ox-LDL诱导的RAW 264.7巨噬细胞的抗炎活性可能涉及MyD88/p38 MAPK/NF-κB信号通路。
