丹参山楂组分配伍抗动脉粥样硬化及作用机制研究
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摘要
心血管疾病(cardiovascular disease, CVD)是世界上发病率和病死率较高的疾病之一。动脉粥样硬化(atherosclerosis, AS)是导致CVD的重要病理基础。一些中药在临床防治AS上有较好的疗效,但是中药研究存在的一些问题阻碍了对其的深入研究：1)中药在临床多以饮片形式入药,众多的影响因素使饮片的质量不易控制；2)现代药效评价模型不能很好地与中医的“证”相对应；3)中药多成分、多靶点的特点较难体现等。随着分析仪器的不断更新,其他学科理论和技术的引入,目前对上述研究中存在的问题,已经有了一些解决对策和实践：1)制备成分相对稳定的中药有效组分进行配伍,探讨组分配伍替代饮片配伍的可行性；2)生物体内源性成分(小分子代谢物)在生理、病理及中药治疗状态下,表达谱的改变可能反映了中医证候规律的物质基础在代谢物水平上的变化,因此以体内代谢物为研究对象的代谢组学,为从系统生物学的角度诠释中药的整体作用提供了研究思路；3)基于生物调控网络角度的网络药理学方法,可用于分析中药多种活性成分所发挥的综合或整合调节作用,为解析中药多靶点、多途径的作用机制及配伍规律提供了新的方法。
丹参山楂是中医治疗CVD的常用药对之一,临床和动物实验研究表明二者配伍在防治AS上具有较好的疗效。为了更好的阐释丹参山楂配伍的内涵和作用机制,本研究拟从丹参山楂组分配伍入手,建立AS大鼠模型,在此基础上,采用多药效指标综合评价和代谢组学相结合的方法,筛选丹参山楂抗AS的有效组分；进而采用正交设计优化抗AS组分的配伍；另一方面,对有效组分进行化学成分定性定量分析,以网络药理学预测作用机制；进而考察较优配伍抗AS的作用机制。
1.组分的制备及其化学成分分析
制备丹参山楂单煎及合煎的不同组分：丹参总糖组分(DST)、丹参总酚酸组分(DSF)、丹参脂溶性组分(DSZ)；山楂糖组分(SZT)、山楂黄酮组分(SZH)、山楂脂溶性组分(SZZ)；合煎糖组分(HJT)、合煎水溶性组分(HJS)、合煎脂溶性组分(HJZ)。采用HPLC法对各组分中的化学成分进行定性定量分析,检出丹参素、迷迭香酸、紫草酸、丹酚酸B、丹酚酸A、绿原酸、原花青素B2、表儿茶素、芦丁/金丝桃苷、异槲皮素、隐丹参酮、丹参酮Ⅰ、丹参酮ⅡA、齐墩果酸/熊果酸等成分。定量分析结果显示SZH中原花青素B2、表儿茶素、绿原酸、金丝桃苷、异槲皮素含量分别为4.84%、4.00%、0.23%、0.28%、0.14%；DSF中迷迭香酸、紫草酸、丹酚酸B、丹酚酸A含量分别为3.50%、1.67%、64.78%、0.96%；HJS中迷迭香酸、紫草酸、丹酚酸B、丹酚酸A、原花青素B2、表儿茶素的含量分别为1.93%、2.35%、0.47%、39.52%、1.74%、1.05%；DSZ中隐丹参酮、丹参酮ⅡA的含量分别为3.40%、5.31%；HJZ中隐丹参酮、丹参酮ⅡA的含量分别为3.01%、4.54%。
2.抗AS有效组分的筛选
2.1各组分对AS大鼠影响的考察
采用高脂饲养、腹腔注射维生素D3及卵清白蛋白诱发炎症的复合方法建立AS大鼠模型。以辛伐他汀为阳性对照,从降脂活性(total cholesterol, TC; triglyceride, TG; high-density lipoprotein cholesterol, HDL-C; low-density lipoprotein cholesterol, LDL-C)、抗氧化活性(malondialdehyde, MDA; superoxide dismutase, SOD)、内皮保护活性(nitric oxide, NO; endothelin, ET;6-keto-prostaglandin F1α,6-keto-PGF1α; thromboxane B2, TXB2)、抗炎活性(C-reactive protein, CRP; interleukin8, IL-8; interleukin18, IL-18; interleukin1β,IL-1β)上考察各组分的抗AS作用(口服灌胃给药4周),同时进行病理形态学观察；采用主成分分析(principal component analysis, PCA)和聚类分析进行综合评价,筛选有效组分；析因分析观察丹参山楂的交互作用。结果显示,不同组分干预后的抗AS作用不同,其中DSF降低了AS大鼠血中TC、TG、LDL-C、ET、TXB2、CRP、IL-18、IL-1β水平(P<0.05),升高了SOD、6-keto-PGF1α水平(P<0.05)；SZH降低了TC、TG、LDL-C、ET、TXB2、CRP、 IL-8、IL-18、IL-1β水平(P<0.05),升高了HDL-C、SOD、6-keto-PGF1α水平(P<0.05)；DSZ降低了TC、TG、LDL-C、ET、IL-1β水平,升高了6-keto-PGF1α、 HDL-C、NO水平(P<0.05)；SZZ降低了TC、LDL-C、ET水平(P<0.05)；SZT降低了IL-18水平(P<0.05)。PCA结果表明DSZ-1、DSF-4、SZH-1的综合效应较好,聚类分析与PCA的趋势一致。病理结果显示DSZ-1、SZH-1、HJS-1、SZT-4的主动脉病变较模型组明显减轻,DSZ-1、HJS-4、SZT-4组显著抑制了主动脉内膜变厚(P<0.05)；析因分析结果显示DSF与SZH在降脂、内皮保护、抗炎活性上存在交互作用。综上,DSF、SZH、DSZ为丹参山楂抗AS的有效组分。
2.2有效组分的代谢组学评价
采用三甲基硅烷化衍生的方法,建立AS大鼠血清代谢物的GC/MS分析方法,采用PCA和偏最小二乘法-判别分析(partial least-squares discriminant ana-lysis, PLS-DA)观察代谢物轮廓变化,筛选生物标志物,观察有效组分的干预作用。PLS-DA结果表明,模型组与正常组可明显地聚为两类,与正常组比较,模型组大鼠血清乳酸、缬氨酸、甘油、异亮氨酸、谷氨酰胺、苯丙氨酸、核糖、酪氨酸、十七酸、肌醇、亚油酸、花生四烯酸和二十二碳六烯酸水平显著降低(P<0.05),赖氨酸和油酸水平显著升高(P<0.05),表明这15个成分是差异代谢物。进一步的分析表明,AS大鼠在脂肪酸代谢、氨基酸代谢和糖代谢通路上发生紊乱。不同有效组分在一定程度上可逆转这种紊乱,其中SZH显著升高了乳酸、甘油、异亮氨酸、谷氨酰胺、苯丙氨酸、核糖、酪氨酸、花生四烯酸、肌醇和亚油酸水平(P<0.05或P<0.01)；DSF显著升高了乳酸、甘油、苯丙氨酸、异亮氨酸水平(P<0.05或P<0.01),降低赖氨酸水平(P<0.01),且呈量效关系；HJS可显著升高乳酸、甘油、异亮氨酸水平(P<0.01),降低赖氨酸水平(P<0.01)；DSZ可升高乳酸、甘油、异亮氨酸、酪氨酸水平(P<0.05或P<0.01)。与合煎相比,单味药对代谢轮廓的回调作用较优,提示丹参山楂合用比例和剂量需要优化。
3.抗AS有效组分配伍的研究
在AS大鼠模型上,采用正交设计(L934)的方法,对DSF、SZH、DSZ高(1)、中(2)、低(3)3个剂量水平的不同配比进行实验,采用多药效指标观察其抗AS作用；以多因素方差分析、PCA和聚类分析对实验数据进行综合评价。结果表明,各配伍组具一定的降脂、抗氧化、内皮保护和抗炎活性；多因素方差分析结果显示DSF(2)-SZH(2)-DSZ(1)为侧重降脂的较优配比,DSF(3)-SZH(2)-DSZ(1)为侧重抗氧化的较优配比,DSF(3)-SZH(3)-DSZ(3)为侧重内皮保护的较优配比,DSF(3)-SZH(1)-DSZ(1)为侧重抗炎较优配比；PCA和聚类分析综合评价结果显示DSF(2)-SZH(1)-DSZ(2)(SC121)为抗AS的较优配伍；病理结果显示SC121可显著抑制主动脉斑块进展,同时减轻肝脏的脂肪变性和炎性浸润。
4.网络药理学预测有效组分的作用机制
采用系统药理学软件TCMSP (http://tcmspnw.com/default),对SZH中5个主要成分进行口服利用度和类药性预测,提取主要成分的作用靶标,通过搜索PharmGKb、TTD和DrugBank数据库,确定了与CVD密切相关的靶标20个,这些靶标涉及花生四烯酸代谢、精氨酸/一氧化氮、过氧化物增殖物受体和血小板聚集等通路。结合文献报道的有关丹参网络药理学的分析,推测上述有效组分配伍的主要作用靶位为内皮细胞、炎性细胞及血小板等,介质为脂质,此预测结果与整体动物实验结果相似。
5.较优配伍方的作用机制研究
采用体外Ox-LDL和H2O2诱导人脐静脉血管内皮细胞(human umbilical vein endothelial cells, HUVEC)损伤模型评价SC121的保护作用,实验结果表明,SC121可提高HUVEC细胞存活率(P<0.05或P<0.01),并呈一定的剂量依赖性。
采用Ox-LDL和H2O2诱导巨噬细胞RAW264.7损伤模型评价SC121的保护作用；采用荧光素法测定SC121对RAW264.7细胞内活性氧的影响；采用油红O染色法评价SC121对RAW264.7泡沫化形成的影响。结果显示,SC121可提高RAW264.7细胞存活率(P<0.05或P<0.01),及抑制细胞内活性氧的产生和泡沫化的形成,并呈一定的剂量依赖性。
综上所述,本论文以AS大鼠模型为基础,从丹参山楂组分配伍入手,通过多药效指标综合评价和代谢组学相结合的方法,筛选出了丹参山楂抗AS的有效组分(DSF、SZH、DSZ),对其化学成分进行了定性定量分析；通过正交设计实验确定了较优配伍SC121；以网络药理学预测了有效组分的作用靶位为内皮细胞和炎性细胞等。体外结果表明,SC121可干预Ox-LDL、H2O2诱导的内皮细胞和巨噬细胞损伤,抑制巨噬细胞活性氧的产生,抑制巨噬细胞泡沫细胞的形成等多环节。以上结果在一定程度上揭示了丹参山楂配伍的内涵,为二者在临床的合理应用奠定了基础。
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 constituents
In 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 components
2.1Establishment of AS rat model and assessment of anti-atherosclerotic effect of different components
Atherosclerotic 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 metabolomics
A 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 combination
A 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 DS
TCMSP (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 SC121
Ox-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.
丹参山楂是中医治疗CVD的常用药对之一,临床和动物实验研究表明二者配伍在防治AS上具有较好的疗效。为了更好的阐释丹参山楂配伍的内涵和作用机制,本研究拟从丹参山楂组分配伍入手,建立AS大鼠模型,在此基础上,采用多药效指标综合评价和代谢组学相结合的方法,筛选丹参山楂抗AS的有效组分；进而采用正交设计优化抗AS组分的配伍；另一方面,对有效组分进行化学成分定性定量分析,以网络药理学预测作用机制；进而考察较优配伍抗AS的作用机制。
1.组分的制备及其化学成分分析
制备丹参山楂单煎及合煎的不同组分：丹参总糖组分(DST)、丹参总酚酸组分(DSF)、丹参脂溶性组分(DSZ)；山楂糖组分(SZT)、山楂黄酮组分(SZH)、山楂脂溶性组分(SZZ)；合煎糖组分(HJT)、合煎水溶性组分(HJS)、合煎脂溶性组分(HJZ)。采用HPLC法对各组分中的化学成分进行定性定量分析,检出丹参素、迷迭香酸、紫草酸、丹酚酸B、丹酚酸A、绿原酸、原花青素B2、表儿茶素、芦丁/金丝桃苷、异槲皮素、隐丹参酮、丹参酮Ⅰ、丹参酮ⅡA、齐墩果酸/熊果酸等成分。定量分析结果显示SZH中原花青素B2、表儿茶素、绿原酸、金丝桃苷、异槲皮素含量分别为4.84%、4.00%、0.23%、0.28%、0.14%；DSF中迷迭香酸、紫草酸、丹酚酸B、丹酚酸A含量分别为3.50%、1.67%、64.78%、0.96%；HJS中迷迭香酸、紫草酸、丹酚酸B、丹酚酸A、原花青素B2、表儿茶素的含量分别为1.93%、2.35%、0.47%、39.52%、1.74%、1.05%；DSZ中隐丹参酮、丹参酮ⅡA的含量分别为3.40%、5.31%；HJZ中隐丹参酮、丹参酮ⅡA的含量分别为3.01%、4.54%。
2.抗AS有效组分的筛选
2.1各组分对AS大鼠影响的考察
采用高脂饲养、腹腔注射维生素D3及卵清白蛋白诱发炎症的复合方法建立AS大鼠模型。以辛伐他汀为阳性对照,从降脂活性(total cholesterol, TC; triglyceride, TG; high-density lipoprotein cholesterol, HDL-C; low-density lipoprotein cholesterol, LDL-C)、抗氧化活性(malondialdehyde, MDA; superoxide dismutase, SOD)、内皮保护活性(nitric oxide, NO; endothelin, ET;6-keto-prostaglandin F1α,6-keto-PGF1α; thromboxane B2, TXB2)、抗炎活性(C-reactive protein, CRP; interleukin8, IL-8; interleukin18, IL-18; interleukin1β,IL-1β)上考察各组分的抗AS作用(口服灌胃给药4周),同时进行病理形态学观察；采用主成分分析(principal component analysis, PCA)和聚类分析进行综合评价,筛选有效组分；析因分析观察丹参山楂的交互作用。结果显示,不同组分干预后的抗AS作用不同,其中DSF降低了AS大鼠血中TC、TG、LDL-C、ET、TXB2、CRP、IL-18、IL-1β水平(P<0.05),升高了SOD、6-keto-PGF1α水平(P<0.05)；SZH降低了TC、TG、LDL-C、ET、TXB2、CRP、 IL-8、IL-18、IL-1β水平(P<0.05),升高了HDL-C、SOD、6-keto-PGF1α水平(P<0.05)；DSZ降低了TC、TG、LDL-C、ET、IL-1β水平,升高了6-keto-PGF1α、 HDL-C、NO水平(P<0.05)；SZZ降低了TC、LDL-C、ET水平(P<0.05)；SZT降低了IL-18水平(P<0.05)。PCA结果表明DSZ-1、DSF-4、SZH-1的综合效应较好,聚类分析与PCA的趋势一致。病理结果显示DSZ-1、SZH-1、HJS-1、SZT-4的主动脉病变较模型组明显减轻,DSZ-1、HJS-4、SZT-4组显著抑制了主动脉内膜变厚(P<0.05)；析因分析结果显示DSF与SZH在降脂、内皮保护、抗炎活性上存在交互作用。综上,DSF、SZH、DSZ为丹参山楂抗AS的有效组分。
2.2有效组分的代谢组学评价
采用三甲基硅烷化衍生的方法,建立AS大鼠血清代谢物的GC/MS分析方法,采用PCA和偏最小二乘法-判别分析(partial least-squares discriminant ana-lysis, PLS-DA)观察代谢物轮廓变化,筛选生物标志物,观察有效组分的干预作用。PLS-DA结果表明,模型组与正常组可明显地聚为两类,与正常组比较,模型组大鼠血清乳酸、缬氨酸、甘油、异亮氨酸、谷氨酰胺、苯丙氨酸、核糖、酪氨酸、十七酸、肌醇、亚油酸、花生四烯酸和二十二碳六烯酸水平显著降低(P<0.05),赖氨酸和油酸水平显著升高(P<0.05),表明这15个成分是差异代谢物。进一步的分析表明,AS大鼠在脂肪酸代谢、氨基酸代谢和糖代谢通路上发生紊乱。不同有效组分在一定程度上可逆转这种紊乱,其中SZH显著升高了乳酸、甘油、异亮氨酸、谷氨酰胺、苯丙氨酸、核糖、酪氨酸、花生四烯酸、肌醇和亚油酸水平(P<0.05或P<0.01)；DSF显著升高了乳酸、甘油、苯丙氨酸、异亮氨酸水平(P<0.05或P<0.01),降低赖氨酸水平(P<0.01),且呈量效关系；HJS可显著升高乳酸、甘油、异亮氨酸水平(P<0.01),降低赖氨酸水平(P<0.01)；DSZ可升高乳酸、甘油、异亮氨酸、酪氨酸水平(P<0.05或P<0.01)。与合煎相比,单味药对代谢轮廓的回调作用较优,提示丹参山楂合用比例和剂量需要优化。
3.抗AS有效组分配伍的研究
在AS大鼠模型上,采用正交设计(L934)的方法,对DSF、SZH、DSZ高(1)、中(2)、低(3)3个剂量水平的不同配比进行实验,采用多药效指标观察其抗AS作用；以多因素方差分析、PCA和聚类分析对实验数据进行综合评价。结果表明,各配伍组具一定的降脂、抗氧化、内皮保护和抗炎活性；多因素方差分析结果显示DSF(2)-SZH(2)-DSZ(1)为侧重降脂的较优配比,DSF(3)-SZH(2)-DSZ(1)为侧重抗氧化的较优配比,DSF(3)-SZH(3)-DSZ(3)为侧重内皮保护的较优配比,DSF(3)-SZH(1)-DSZ(1)为侧重抗炎较优配比；PCA和聚类分析综合评价结果显示DSF(2)-SZH(1)-DSZ(2)(SC121)为抗AS的较优配伍；病理结果显示SC121可显著抑制主动脉斑块进展,同时减轻肝脏的脂肪变性和炎性浸润。
4.网络药理学预测有效组分的作用机制
采用系统药理学软件TCMSP (http://tcmspnw.com/default),对SZH中5个主要成分进行口服利用度和类药性预测,提取主要成分的作用靶标,通过搜索PharmGKb、TTD和DrugBank数据库,确定了与CVD密切相关的靶标20个,这些靶标涉及花生四烯酸代谢、精氨酸/一氧化氮、过氧化物增殖物受体和血小板聚集等通路。结合文献报道的有关丹参网络药理学的分析,推测上述有效组分配伍的主要作用靶位为内皮细胞、炎性细胞及血小板等,介质为脂质,此预测结果与整体动物实验结果相似。
5.较优配伍方的作用机制研究
采用体外Ox-LDL和H2O2诱导人脐静脉血管内皮细胞(human umbilical vein endothelial cells, HUVEC)损伤模型评价SC121的保护作用,实验结果表明,SC121可提高HUVEC细胞存活率(P<0.05或P<0.01),并呈一定的剂量依赖性。
采用Ox-LDL和H2O2诱导巨噬细胞RAW264.7损伤模型评价SC121的保护作用；采用荧光素法测定SC121对RAW264.7细胞内活性氧的影响；采用油红O染色法评价SC121对RAW264.7泡沫化形成的影响。结果显示,SC121可提高RAW264.7细胞存活率(P<0.05或P<0.01),及抑制细胞内活性氧的产生和泡沫化的形成,并呈一定的剂量依赖性。
综上所述,本论文以AS大鼠模型为基础,从丹参山楂组分配伍入手,通过多药效指标综合评价和代谢组学相结合的方法,筛选出了丹参山楂抗AS的有效组分(DSF、SZH、DSZ),对其化学成分进行了定性定量分析；通过正交设计实验确定了较优配伍SC121；以网络药理学预测了有效组分的作用靶位为内皮细胞和炎性细胞等。体外结果表明,SC121可干预Ox-LDL、H2O2诱导的内皮细胞和巨噬细胞损伤,抑制巨噬细胞活性氧的产生,抑制巨噬细胞泡沫细胞的形成等多环节。以上结果在一定程度上揭示了丹参山楂配伍的内涵,为二者在临床的合理应用奠定了基础。
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 constituents
In 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 components
2.1Establishment of AS rat model and assessment of anti-atherosclerotic effect of different components
Atherosclerotic 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 metabolomics
A 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 combination
A 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 DS
TCMSP (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 SC121
Ox-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.
引文
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