Study Of Component Combination With Danshen-shanzha Herb Couple On Anti-atherosclerotic Effects And Its Mechanisms

Cardiovascular disease (CVD) is a leading cause of morbidity and mortality worldwide. The underlying pathogenesis of CVD is atherosclerosis (AS). Some traditional Chinese medicines (TCMs) have significant effects on clinical prevention and treatment of AS, however several problems prevent the further investigation of these TCMs. Firstly, TCM is often clinical applied with decoction pieces, whose quality was heavily affected by many parameters, such as the varieties, ecology and processing, et al. It is difficult to control the quality of decoction pieces. Secondly, pharmacological models we usually used to evaluate the effects of TCM are not always equal to syndrome model. Thirdly, it’s difficult to clarify the synergistic interaction among multi-components in TCM. With the development of analytical instrument and the introduction of other disciplines theory and technology, some strategies and practices have been used in TCM study:1) Component combination is used instead of the decoction pieces, in that case, the quality of the tested sample can be controlled easily.2) Metabonomics is the comparative analysis of metabolites and their dynamic flux associated with the response of living systems to pathophysiological and chemical constituents stimuli. The global changes of endogenous metabolites may character the states of TCM syndromes. Since TCM is based on "holism" philosophy, it is sensible conceivable that metabonomics may play some roles in evaluation of therapeutic effect of TCM and provide important information.3) Network pharmacology is a novel subject based on the construction of multi-layer networks of disease-phenotype-gene-drug to predict the drug targets in a holistic view, and then study the function and mechanism of drug. Network pharmacology integrated the notions of comprehensive research and systematic assessment, and these characteristics agree with the combined effects and mechanism of TCM treatment. So Network pharmacology shows potential in analysis of multi-target and multi-pathway mechanism of TCM.The root of Salvia miltiorrhiza Bge.(danshen) and the fruit of Crataegus pinnatifida Bge. var. major N. E. Br.(shanzha) are common TCM in China. And danshen-shanzha herb couple (DS) is the main herb couple in many TCM formulas for treatment of CVD. Clinical and animal experiments showed that DS have remark effects in prevention and treatment of AS. To understand the effects of DS, the components of DS were prepared and an AS rat model was established to evaluate the anti-atherosclerotic EC. Further, an orthogonal test was applied for screening the optimum anti-atherosclerotic combination. On the other hand, an HPLC method was used for qualitative and quantitative analysis of EC. Network pharmacology was used for prediction of mechanism, finally the anti-atherosclerotic mechanisms were explored.1. Preparation of different components and analysis of chemical constituentsIn present research, the different components of danshen, shanzha and DS were extracted and purified, including danshen sugar components (DST), danshen tanshinone components (DSZ), danshen phenolic acid components (DSF); shanzha sugar components (SZT), shanzha flavonoid components (SZH), shanzha liposoluble components (SZZ); DS sugar components (HJT), DS aqueous components (HJS), DS liposoluble components (HJZ). An HPLC method was used for qualitative and quantitative analysis of the chemical constituents of components, danshensu, rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A, chlorogenic acid, procyanidin B2,(-)-epicatechin, rutin/hyperoside, isoquercetin, cryptotanshinone, tanshinone I, tanshinone ⅡA, oleanolic acid/ursolic acid were tested. The quantitative analysis results showed that the contents of procyanidin B2,(-)-epicatechin, chlorogenic acid, hyperoside and isoquercetin in SZH were4.84%,4.00%,0.23%,0.28%,0.14%, respectively; rosmarinic acid, lithospermic acid, salvianolic acid B and salvianolic acid A in DSF were3.50%,1.67%,64.78%,0.96%; rosmarinic acid, lithospermic acid, salvianolic acid B, salvianolic acid A, procyanidin B2and (-)-epicatechin in HJS were1.93%,2.35%,0.47%,39.52%,1.74%,1.05%, respectively; cryptotanshinone and tanshinone ⅡA in DSZ and HJZ were3.40%,5.31%,3.01%,4.54%, respectively.2. Screening of anti-atherosclerotic components2.1Establishment of AS rat model and assessment of anti-atherosclerotic effect of different componentsAtherosclerotic rats were fed a high-fat diet and injected with vitamin D3and ovalbumin. The anti-atherosclerotic effects were tested from four aspects including lipid-lowering activity (total cholesterol, TC; triacylglycerol, TG; high-density lipoprotein cholesterol, HDL-C; low-density lipoprotein cholesterol, LDL-C), antioxidant activity (malondialdehyde, MDA; superoxide dismutase, SOD), endothelial proctection activity (nitric oxide, NO; endothelin, ET;6-keto-prostaglandin F1α,6-keto-PGF1α; thromboxane B2, TXB2), anti-inflammatory activity (C-reactive protein, CRP; interleukin8, IL-8; interleukin18, IL-18; interleukin1β, IL-1β). On the other hand, the histopathological changes of aorta were observed by HE staining. Principal component analysis (PCA) and cluster analysis were used for screening the anti-atherosclerotic components. Factorial analysis was used for observing the interactions. Experimental results showed DSF reduced the levels of TG, LDL-C, ET, TXB2, CRP, IL-18, IL-1β (P<0.05) and increased the levels of SOD,6-keto-PGF1α (P<0.05); SZH reduced the levels of TC, TG, LDL-C, ET, TXB2, CRP, IL-8, IL-18, IL-1β (P<0.05) and increased the levels of HDL-C, SOD,6-keto-PGF1α (P<0.05); DSZ reduced the levels of TC, TG, LDL-C, ET, IL-1β and increased the levels of6-keto-PGF1α, HDL-C, NO (P<0.05); SZZ reduced the levels of TC, LDL-C, ET (P<0.05); SZT reduced the level of IL-18(P<0.05). PCA and cluster analysis showed that the preferably comprehensive effect components were DSZ-1, DSF-4and SZH-1. Above mentional components significant reduced the pathological changes of rat aorta compared with the model rats, DSZ-1, HJS-4, SZT-4significantly inhibited the thickening of rat intima (P<0.05). DSF and SZH had the interactive impacts on lipid-lowering, endothelial protection, anti-inflammation activities. Conclusively, above results demonstrated that DSF, SZH and DSZ were anti-atherosclerotic components.2.2Assessment of effective components by metabolomicsA GC/MS method was established for characterizing the metabolite profile changes of the rat serum with trimethylsilylation derivatization. PCA and partial least-squares discriminant analysis (PLS-DA) were used for recognizing the different metabolic pattern in samples. The biomarkers of AS rat were identified, and then the interventions of EC were tested. The metabonomic differences between model and control rats were clearly visualized in PLS-DA score plots.15endogenous metabolites were identified; the contents of lactic acid, valine, glycerol, isoleucine, glutamine, phenylalanine, ribose, tyrosine, daturic acid, inositol, arachidonic acid, linoleic acid, and docosahexaenoic acid in the model group were significantly reduced compared with those in the control group (P<0.05). The contents of lysine and oleic acid in the model group were significantly increased (P<0.05). The changes of metabolites predicted the abnormality of fatty acid metabolism, amino acid metabolism and energy metabolism in atherosclerotic rats. Atherosclerotic rats with different EC intervention showed a tendency of bringing the levels of biomarkers to normal, SZH significantly increased the levels of lactic acid, glycerol, isoleucine, glutamine, phenylalanine, ribose, tyrosine, arachidonic acid, inositol and linoleic acid compared with those in model group (P<0.05or P<0.01); DSF significantly increased the levels of lactic acid, glycerol, phenylalanine, isoleucine and reduced the level of tyrosine (P<0.01) in dose-dependent manner; HJS significantly increased the levels of lactic acid, glycerol, isoleucine (P<0.01) and reduced the level of tyrosine (P<0.01); DSZ significantly increased the levels of lactic acid, glycerol and tyrosine (P<0.05or P<0.01). Combined with EC and HJS results, the evidence indicated the EC alone showed obvious anti-atherosclerotic effects, which was stronger than HJS. At the same time, metabolomics results showed the dose and proportion of EC from DS needed to be optimized.3. Optimization of anti-atherosclerotic component combinationA L934orthogonal design was used in experiment for the different proportion of DSF, SZH and DSZ. Multiple indexes were used to evaluate the anti-atherosclerotic effects of samples. The experimental data were analyzed by multi-factor variance analysis, PCA and cluster analysis. The results indicated that DSF (2)-SZH (2)-DSZ (1) was the optimal proportion on lipid-lowering activity, DSF (3)-SZH (2)-DSZ (1) was the optimal proportion on antioxidant activity, DSF (3)-SZH (3)-DSZ (3) was the optimal proportion on endothelial protection activity, DSF (3)-SZH (1)-DSZ (1) was the optimal proportion on anti-inflammatory activity; DSF (2)-SZH (1)-DSZ (2) was the optimal proportion (SC121) on anti-atherosclerotic effects. Furtherly, SC121could inhibit the progress of aorta plaques and alleviated the steatosis and inflammation infiltration of liver in atherosclerotic rats.4. Network pharmacology study on the major compounds of DSTCMSP (http://tcmspnw.com/default) was used for network pharmacology ana-lysis.5chemical ingredients of SZH were analyzed with oral bioavailability and drug likeness predication; the targets were extracted and confirmed in PharmGKb, TTD, DrugBank;20targets associated with CVD were ascertained. These targets are thought to be related with arachidonic acid metabolism, L-argine/NO, peroxisome proliferator activated receptor and platelet aggregation pathway. Combined with recent danshen results, the target sites of DS were predicted as lipids, endothelial cells, inflammatory cells and platelet.5. Mechanism of SC121Ox-LDL and H2O2-induced damage in human umbilical vein endothelial cells (HUVEC) were used for investigating the protective effects of SC121. The results showed that SC121could increase the survival rate of HUVEC in dose-dependent manner.Ox-LDL and H2O2-induced damage in murine macrophage RAW264.7cells were used for evaluating the protective effects of SC121; fluorescent method was used for measuring reactive oxygen species (ROS) in RAW264.7cells; oil red O staining was used for assessing the formation of macrophage-derived foam cells. The results showed that SC121could increase the survival rate of RAW264.7cells, inhibit the production of ROS and decrease the formation of foam cells in dose-dependent manner.To sum up, the AS rat model was established and the components of danshen, shanzha were prepared, at the same time, multiple indexes and metabonomics were integrated to study the anti-atherosclerotic effects of different components. The anti-atherosclerotic EC (DSF, SZH and DSZ) in DS were screened and the chemical ingredients were qualitative and quantitative analyzed; the optimal proportion was screened by an orthogonal design; the predicted target sites of DS were lipids, endothelial cells and inflammtory cells; in vitro study showed that SC121could attenuate Ox-LDL, H2O2-induced HUVEC and RAW264.7damage, inhibit ROS generation and decrease the formation of foam cells. Above results revealed the connotation of DS combination and then provided basement for clinical application.