基于UPLC-Q-Exactive Orbitrap MS探究朱日亨滴丸抑制巨噬细胞泡沫化的谱效关系

Exploration of Spectrum-effect Relationship of Zhuriheng Dropping Pills Against Macrophage Foaming Based on UPLC-Q-Exactive Orbitrap MS

目的:通过对朱日亨滴丸(ZRH)的肠吸收液物质组成与抑制巨噬细胞泡沫化进行相关性分析,探究ZRH的药效成分群,为建立其质量标准提供依据。方法:采用大鼠外翻肠囊法制备ZRH的0、5、10、15、20倍临床等效剂量肠吸收液,并使用氧化型低密度脂蛋白(ox-LDL)诱导的RAW264.7巨噬细胞泡沫化模型,通过油红O染色及胆固醇含量测定考察不同剂量ZRH肠吸收液对RAW264.7细胞脂质积累的影响,筛选最佳剂量;采用超高效液相色谱-四级杆-静电场轨道阱高分辨质谱法(UPLC-Q-Exactive Orbitrap MS)对ZRH肠吸收液中的肠吸收组分进行分析鉴定,以峰面积为自变量,药效指标为因变量,采用SIMCA 13.0软件对不同剂量ZRH肠吸收液进行偏最小二法-乘判别分析(PLS-DA)和正交偏最小二乘法-判别分析(OPLSDA),筛选变量重要性投影(VIP)值>1.0的化合物作为贡献性成分,并利用Pearson相关性分析确定谱效相关性;使用分子对接验证过氧化物酶体增殖物激活受体α(PPARα)、PPARγ、PPARβ、人视黄醇X受体α(RXRA)和核转录因子-κB(NF-κB)与ZRH肠吸收液中活性成分的结合能;实时荧光定量聚合酶链式反应(Real-time PCR)检测RAW264.7细胞中PPARγ、A类Ⅰ型清道夫受体(SRA1)和三磷酸腺苷结合盒转运体A1(ABCA1)的mRNA表达水平;蛋白免疫印迹法(Westen blot)检测RAW264.7细胞中PPARγ蛋白表达水平;酶联免疫吸附测定法(ELISA)检测RAW264.7细胞中白细胞介素(IL)-1β和NF-κB水平。结果:油红O染色、胆固醇含量测定结果显示,ZRH肠吸收液均能够降低巨噬细胞泡沫化程度,以15倍和20倍ZRH肠吸收液效果最佳,最终确定以15倍ZRH肠吸液作为研究对象。谱效关联分析显示,ZRH含药肠液中有52个对应峰与药效呈正相关,包括有机酸类、苯丙素类、环烯醚萜类、黄酮类、胆汁酸类、香豆素类和色酮类。分子对接靶标验证结果显示,86.9%~96.2%的成分与关键靶点PPARα、PPARγ、PPARβ、RXRA和NF-κB具有良好的结合活性,对接能量值均<-6.0 kcal·mol^(-1)(1 cal≈4.19 J);验证结果显示,与正常组比较,模型组SRA1、PPARγmRNA表达水平显著上升,ABCA1 mRNA表达水平显著下降,PPARγ蛋白表达水平显著上升,IL-1β、NF-κB水平显著上升(P<0.01);与模型组比较,15倍肠吸收液组SRA1、PPARγmRNA表达水平明显降低(P<0.05,P<0.01),ABCA1 mRNA表达水平显著上调,IL-1β、NF-κB水平显著降低(P<0.01),PPARγ蛋白表达水平明显降低(P<0.05)。结论:该研究确定了ZRH肠吸收液中52个与抑制巨噬细胞泡沫化呈正相关的药物成分及其代谢产物,可能与调节细胞中PPARs通路并降低炎症因子水平有关,为ZRH的质量控制和临床应用提供借鉴。

Objective:Through the correlation analysis between intestinal absorption profile and inhibition of macrophage foaming,the pharmacodynamic components of Zhuriheng dripping pills(ZRH)were explored to provide a basis for establishing its quality standard.Method:Intestinal absorption fluids with 0,5,10,15,20 times clinical equivalent doses were prepared by a rat everted gut sac(EGS),and the oxidized low density lipoprotein(ox-LDL)-induced RAW264.7 macrophage foaming model was used to investigate the effect of intestinal absorption fluid with different doses on the accumulation of lipids in RAW264.7 cells by oil red O staining and cholesterol content determination,and to screen for the optimal dose.Ultra performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry(UPLC-Q-Exactive Orbitrap MS)was used to analyze and identify intestinal absorption fractions of ZRH intestinal absorption fluids,and partial least squares-discriminant analysis(PLS-DA)and orthogonal partial least squares-discriminant analysis(OPLS-DA)were performed on different doses of ZRH intestinal absorption fluids using SIMCA 13.0 with peak area as the independent variable and the pharmacodynamic indicators as the dependent variables to screen the compounds with variable importance in the projection(VIP)value>1.0 as contributing components,and Pearson correlation analysis was used to determine the spectral effect relationship,determined the compounds and positive correlation with pharmacodynamic were as active ingredients.Molecular docking was used to verify the binding energy of peroxisome proliferator-activated receptorα(PPARα),PPARγ,PPARβ,human retinoid X receptorα(RXRA)and nuclear transcription factor-κB(NF-κB)with the active ingredients in ZRH intestinal absorption fluids.Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was performed to detect the mRNA levels of PPARγ,scavenger receptor A1(SRA1)and adenosine triphosphatebinding cassette transporter A1(ABCA1)in RAW264.7 cells,Westen blot was used to detect the expression level of PPARγprotein in RAW264.7 cells,and enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of interleukin(IL)-1βand NF-κB in RAW264.7 cells.Result:According to the results of oil red O staining and cholesterol content determination,the ZRH intestinal absorption fluids could significantly reduce macrophage foaming,and intestinal absorption fluids with 15,20 times clinical equivalent doses had the best effect,the 15-fold ZRH intestinal absorption fluid was finally determined as the study subject.Spectral effect relationship showed that 52 corresponding peaks in the ZRH-containing intestinal fluid were positively correlated with the efficacy,including organic acids,phenylpropanoids,iridoids,flavonoids,bile acids,coumarins and chromones.Target validation results showed that 86.9%-96.2%of the total components processed good binding activities with the key targets of PPARα,PPARγ,PPARβ,RXRA and NF-κB,and the docking energy values were all less than-6.0 kcal·mol^(-1)(1 cal≈4.19 J).The results of validation showed that,compared with the normal group,the model group showed a significant increase in the levels of SRA1 and PPARγmRNA expression,a significant decrease in ABCA1 mRNA expression,a significant increase in the level of PPARγprotein expression,and a significant increase in the levels of IL-1βand NF-κB(P<0.01),compared with the model group,the 15-fold intestinal absorption fluid group showed a significant decrease in the levels of SRA1 and PPARγmRNA expression(P<0.05,P<0.01),ABCA1 mRNA expression level was significantly up-regulated,the levels of IL-1βand NF-κB were significantly reduced(P<0.01),and PPARγprotein expression level was significantly reduced(P<0.05).Conclusion:This study identifies 52 components and their metabolites in ZRH intestinal absorption fluid that are positively correlated with the inhibition of macrophage foaming,which may be related to the regulation of the PPARs pathway in cells and the reduction of the levels of inflammatory factors,and can provide a reference for the quality control and clinical application of ZRH.