| At present, TCM formulas are widely used in clinic in China, which are characteristic of excellent treatment and play an important role in preventing and treating diseases. However, TCM play the pharmacological actions through multi-components and multi-targets, which hinders the exploration of action mechanism in clinic. Hence, the development of TCM has been hampered due to the difficulty to evaluate TCM’s therapeutic effects by using the modern pharmaceutical technologies. It is hard to clarify the mechanism of TCM. The researchers should determine the active compounds in TCM and find the relative targets, then establish rational standards for quality control which could reflect the safety and therapeutic effects. Only doing so can the researchers overcome the limitations of the TCM development nowadays.Yang Xin Shi Tablet(YXST) is the major of Qingdao Guofeng Pharmaceutical Corporation, which is consisted of Astragalus membranaceus(called Huangqi in Chinese), Ganoderma lucidum(Lingzhi), Codonopsis(Dangshen), Radix salviae miltiorrhizae(Gegen), Radix puerariae(Huangqi), Radix Rehmanniae(Dihuang), Angelica sinensis(Danggui), Epimedium brevicornu Maxim.(Yinyanghuo), Rhizoma Corydalis(Yanhusuo), Crataegus pinnatifida Bunge(Shanzha), Liquorice(Gancao), Rhizoma Coptidis(Huanglian), Radix Ginseng(Renshen). At present, YXST has been used to treat coronary heart disease, angina, myocardial infarction and hyperlipidemia combined with hyperglycemia. The current studies show that YXST has a good therapeutic effect for cardiovascular diseases, especially heart failure. However, the exact mechanism of YXST is not clear, which hinders the clinical application.Hence, this article is aiming at those problems discussed above. The target of this study is to investigate the heart protective effect of YXST in a systematic strategy. The first step is to construct the chemical database of YXST. Then a UHPLC-Q-TOF/MS analysis system was used to characterize the constituents in YXST and active compounds in plasma. The pharmacological effect of YXST was evaluated on the animal models of heart failure. The potential mechanism of YXST on heart failure was deciphered through the strategy of metabolomics and proteomics. The content of this article could be divided into four parts:(1) Study of ingredients in Yangxinshi Tablet by using UHPLC-Q-TOF/MS Firstly, The compounds in YXST were collected from different jouranls and compound databases. A chemical database of YXST, which was consisted of 1500 compounds, was established by using the Agilent software ‘formula-database-generator’. The UPLC-Q-TOF/MS analysis system was adopted to separate and identify 34 mixed standards and chemical ingredients in YXST. Totally, 179 compounds were identified ambiguously according to their accurate formular weight by using UHPLC-Q-TOF/MS. These compounds could be classified into five categories, including saponins, phenolic acids, alkaloids, flavonoids, amino acids and their derivatives. Subsequently, the drug-contained rat plasma samples and blank plasma samples were obtained from the SD rats. In the rat plasma samples after the administration of YXST, 73 original compounds of YXST and 5 metabolites were identified by comparing with the chromatograms of blank rat plasma and YXST extracts. In this part, the major active compounds of YXST and their metabolites in rat plasma were identified by using UHPLC-Q-TOF/MS.(2) Evaluation of anti-heart failure action of YXST In this part, chronic ischemia-induced heart failure rat model was constructed. The ejection fraction was declined by 15% and the area of reduced blood flow reached 25%, which suggested that the myocardial infarction model was constructed successfully. There were obvious necroses in the pathological sections of myocardial tissue. Then the activity angiotensin converting enzyme and the level of aldosterone were evaluated. The aldosterone content of model group was significantly increased, and the activity of angiotensin converting enzyme was increased. In YXST high-dosage group, the activity of ACE was significantly decreased and the level of aldosterone was aslo decreased. The myocardium function of rats in YXST high dosage group and positive group was significantly improved. These experimental results suggested that YXST could ameliorate the symptom of chronic heart failure.(3) Study on the molecular mechanism of anti-heart failure of YXST In this research, omics was adopted to screen the biomarkers in rat heart tissue based on UHPLC-Q-TOF/MS. In the tissue samples of chronic ischemia-induced heart failure rats, 25 different metabolites were identified. The metabolites were mainly involved in TCA cycle, energy metabolism and mitochondrial transportation. According to the statistical analysis, YXST could perform an anti-heart failure action through the mechanism of energy metabolism and immunomodulation pathway. The iTRAQ quantitative proteomics strategy was adopted to evaluate the myocardium protein expression change after oral administration of YXST. Totally, 80 proteins were identified in comparison with model group and control group, in which 42 proteins were up-regulated and 38 proteins were down-regulated(p<0.05). And 67 proteins were identified in comparison with model group and control group, in which 44 proteins were up-regulated and 23 proteins were down-regulated(p<0.05). A series of proteins, such as myosin light chain, bone morphogenetic protein 10, ion channel receptor and paraoxonase-1, were screened as the potential target of YXST.(4) Study of ingredient-target interaction of YXST based on network pharmacology A database consisted of 179 compounds in YXST was constructed. And 24 compounds were selected as representative effective components. 1082 protein targets from immune system and cardiovascular system were collected. Affinities between 24 compounds and protein targets were calculated by Discovery Studio 3.0. Then an “ingredient-target network” was constructed by Cytoscape to interpret the mechanism of YXST. Several important protein targets of YXST, such as toll-like receptor 4, PPARγ, ACE, HMGCR and ADBR1 were found.In conclusion, this research investigated the cardiovascular protective effect in a systematic way. On the basis of determination of YXST’s chemical material basis and pharmacological effect, metabolomics and proteomics were adopted to investigate the underlying mechanism in a systematic way, which focus on the chronic ischemic heart failure model to evaluate the anti-heart failure effect of YXST. In addition, chemical database of YXST were docked in a high throughput way to find potential protein targets of YXST. ‘Ingredients-Targets’ interaction network was constructed by network pharmacological technology. According to the experimental results, 179 compounds were identified in YXST. Furthermore, 73 compounds and 5 metabolites were identified in rat serum. In the serum and heart tissue of heart failure rats, 25 metabolite biomarkers and 67 protein biomarkers were identified, which mainly involved in energy metabolism, immune regulation, ion exchange and cell proliferation. The result of network pharmacology indicated that the major targets of YXST were toll-like receptor 4, PPARγ, ACE, HMGCR, ADBR1 and so on. ‘Ingredient-Target’ interaction suggested that YXST could exert anti-heart failure therapeutic effect though improvement of cardiomyocyte validity, modulation of energy metabolism and regulation of immune system. In this article, different methods were integrated together to explore the new strategy of TCM formula analysis, which avoid the blindness of traditional method. Focused on the anti-heart failure effect of YXST, this research sets stone for precise quality control of YXST and systematic evaluation of safety and effectiveness. We hope our study could provide evidence for the clinical application of YXST and shed light on the investigation of TCM mechanism. |
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