| Objective:Based on the rat model of coronary heart disease with phlegm and blood stasis syndrome established earlier by the research group,the changes of differential small molecule metabolites in rats with coronary heart disease with phlegm and blood stasis syndrome before and after administration of Danlou tablets were observed through serum metabonomics technology,and their related metabolic pathways were analyzed.By detecting the protein expression in serum and myocardial tissue of model rats after the intervention of Danlou tablets,the pathway predicted by metabolomics is verified,aiming to explore the mechanism of Danlou tablets in treating coronary heart disease rats with phlegm and blood stasis syndrome.Methods:The chemical constituents in Danlou tablets were qualitatively analyzed by ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry(UPLC-Q-TOF/MS).Based on the model of Danlou tablet intervening coronary heart disease with phlegm and blood stasis syndrome established by the research group earlier,the metabolic profile of serum samples from coronary heart disease with phlegm and blood stasis syndrome rats was analyzed by using serum metabonomics technology.The changes of small molecule metabolites in coronary heart disease with phlegm and blood stasis syndrome rats before and after Danlou tablet administration were detected by principal component analysis(PCA)and partial least squares discriminant analysis(PLS-DA)and their related metabolic pathways were analyzed.Enzyme-linked immunosorbent assay(ELISA)was used to detect the content changes of oxidized low density lipoprotein(ox-LDL),malondialdehyde(MDA)and superoxide dismutase(SOD)in serum of rats in serum.Western-Blot was used to detect the expression level of epidermal growth factor receptor(EGFR)in myocardial tissue,and the pathway predicted by metabolomics was verified,so as to explore the action mechanism of danlou tablet in treating coronary heart disease rats with phlegm and blood stasis syndrome.Results:1.Based on UPLC-Q-TOF/MS technology,45 compounds were identified in this batch of Danlou tablets,including 25 flavonoids,7 tanshinones,5 phenolic acids,2lactones,4 triterpenes and 2 other compounds.2.Danlou tablet has a significant effect on the profile distribution characteristics of serum metabolites in rats with coronary heart disease and phlegm and blood stasis syndrome.Danlou tablet regulates 17 biomarkers with a significant change trend,mainly including betaine,γ-linolenic acid,linoleic acid,11,14,17-eicosatrienoic acid,docosahexaenoic acid,LPC(16:1),LPC(20:4),LPC(22:6),LPC(16:0),LPC(18:1),isobutyryl carnitine,19-hydroxydeoxycorticosterone,L-palmitoyl carnitine,arginine,5-hydroxyindoleacetaldehyde,L-octanoyl carnitine,and 5-hydroxytryptamine.These17 biomarkers play an important role in glycerol phospholipid metabolism,energy metabolism,steroid hormone metabolism and amino acid metabolism.3.Danlou Tablets have significant effects on the changes of serum ox-LDL,MDA,SOD and EGFR expression in myocardial tissue of rats with coronary heart disease and phlegm and blood stasis syndrome.Compared with blank group and sham operation group,ox-LDL and MDA contents in serum of rats in model group increased significantly(P<0.01),SOD contents decreased significantly(P<0.01),and EGFR expression in myocardial tissue increased significantly(P<0.01).Compared with model group,ox-LDL and MDA contents in serum of rats in low,medium and high dose groups and positive drug groups of danlou tablet were decreased(P<0.01),SOD contents were significantly increased(P<0.01),and EGFR expression in myocardial tissue of rats was significantly decreased(P<0.01).Conclusions:1.Based on UPLC-Q-TOF/MS technology,45 compounds were identified in Danlou tablets,mainly including flavonoids,tanshinones,phenolic acids,lactones,triterpenes and other types.The chemical composition of Danlou tablets was basically determined,which provided experimental basis for further study of its pharmacodynamic substance basis in the later stage.2.Danlou Tablet has a significant effect on the profile distribution characteristicsof serum metabolites in rats with coronary heart disease and phlegm and blood stasis syndrome.It regulates a total of 17 biomarkers with obvious change trends.These biomarkers mainly play an important role in glycerol phospholipid metabolism,energy metabolism,steroid hormone metabolism and amino acid metabolism.3.The mechanism of Danlou tablet in treating coronary heart disease rats with phlegm and blood stasis syndrome may be to inhibit the level of EGFR and MMP9 by regulating glycerophospholipid metabolism and energy metabolism such as lysophosphatidylcholine substances(LPCs),linoleic acid and gamma-linolenic acid,and affect the expression of inflammatory factors(TNF-α,IL-6)and oxidative stress(ox-LDL,SOD,MDA)related effect indexes,so as to play anti-inflammatory and antioxidant roles. |
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