[Efficacy and mechanism of Xuefu Zhuyu Decoction on model rats of coronary heart disease with heart blood stasis syndrome based on metabolomics].

This study investigated the effects of Xuefu Zhuyu Decoction on myocardial metabolites in a rat model of coronary heart disease with heart blood stasis syndrome and explored the therapeutic mechanism of blood circulation-promoting and blood stasis-removing therapy. SD rats were randomly divided into a sham operation group, a model group, a Xuefu Zhuyu Decoction group(14.04 g·kg~(-1)), and a trimetazidine group(5.4 mg·kg~(-1)). The sham operation group underwent thread insertion without ligation, while the other groups underwent coronary artery left anterior descending branch ligation to induce a model of coronary heart disease with heart blood stasis syndrome. Three days after modeling, drug intervention was performed, and samples were taken after 14 days of intervention. General conditions were observed, and electrocardiogram and cardiac ultrasound indices were measured. Hematoxylin-eosin(HE) staining and Masson staining were used to observe tissue pathological morphology. The enzyme linked immunosorbent assay(ELISA) was used to measure the levels of triglyceride(TG) and total cholesterol(TC) in the serum. Ultra high performance liquid chromatography-quantitative exactive-mass spectrometry(UHPLC-QE-MS) technology was used to screen differential metabolites in myocardial tissue and conduct metabolic pathway enrichment analysis. The results showed that Xuefu Zhuyu Decoction significantly improved the general condition of the model rats, reduced heart rate and ST segment elevation in the electrocardiogram, increased left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS), and decreased left ventricular internal diameter in diastole(LVIDd) and left ventricular internal diameter in systole(LVIDs). HE staining and Masson staining showed that Xuefu Zhuyu Decoction effectively alleviated myocardial tissue structural disorders, inflammatory cell infiltration, and collagen fiber deposition in the model rats. ELISA results showed that Xuefu Zhuyu Decoction effectively regulated serum TG and TC levels in the model rats. There were significant differences in the metabolic phenotypes of myocardial samples in each group. Fourteen differential metabolites were identified in the Xuefu Zhuyu Decoction group, involving five metabolic pathways, including arginine and proline metabolism, glycerophospholipid metabolism, aminoacyl-tRNA biosynthesis, ether lipid metabolism, and alanine, aspartate, and glutamate metabolism. Xuefu Zhuyu Decoction improved cardiac function and myocardial structural damage in the rat model of coronary heart disease with heart blood stasis syndrome, and its biological mechanism involved the regulation of lipid metabolism, choline metabolism, amino acid metabolism, energy metabolism, and protein synthesis pathways.

Mechanisms of Xuefu Zhuyu Decoction in the treatment of coronary heart disease based on integrated metabolomics and network pharmacology approach.

Coronary heart disease (CHD) has become the leading cause of mortality, morbidity, and disability worldwide. Though the therapeutic effect of Xuefu Zhuyu Decoction (XFZY) on CHD has been demonstrated in China, the active ingredients and molecular mechanisms of XFZY have not been elucidated. The purpose of the current study is to explore the molecular mechanisms of XFZY in the treatment of CHD via network pharmacology, metabolomics, and experimental validation. First, we established a CHD rat model by permanently ligating the left anterior descending coronary artery (LAD), and evaluated the therapeutic effect of XFZY by hemorheology and histopathology. Second, network pharmacology was employed to screen the active ingredients and potential targets of XFZY for the treatment of CHD. Metabolomic was applied to identify the molecules present in the serum after XFZY treatment. Third, the results of network pharmacology and metabolomics were further analyzed by Cytoscape to elucidate the core ingredients and pathways. Finally, the obtained key pathways were verified by transmission electron microscopy and immunofluorescence assay. The results showed that XFZY was effective in the treatment of CHD in the rat model, and the highest dose exerted the best effect. Network pharmacology analysis revealed 215 active ingredients and 129 key targets associated with XFZY treatment of CHD. These targets were enriched in pathways of cancer, lipid and atherosclerosis, fluid shear stress and atherosclerosis, proteoglycans in cancer, chemical carcinogenesis - receptor activation, HIF-1 signaling, et al. Serum metabolomic identified 1081 metabolites involved in the therapeutic effect of XFZY on CHD. These metabolites were enriched in taurine and hypotaurine metabolism, histidine metabolism, retrograde endocannabinoid signaling pathways, et al. Cytoscape analysis combining the data from serum metabolomic and network pharmacology revealed that energy metabolism as the core pathway for XFZY treatment of CHD. Electron microscope observation identified changes in the level of autophagy in the mitochondrial structure of cardiomyocytes. Immunofluorescence assay showed that the expression levels of autophagy-related proteins LC3-B and P62/SQSTM1 were consistent with the levels of autophagy observed in mitochondria. In conclusion, our findings suggest that the possible mechanisms of XFZY in the treatment of CHD are reducing the level of autophagy, improving energy metabolism, and maintaining mitochondrial homeostasis in cardiomyocytes. Our study also shows that the combined strategies of network pharmacology, metabolomics, and experimental validation may provide a powerful approach for TCM pharmacology study.

Chemical profiling and quality evaluation of Pogostemon cablin Benth by liquid chromatography tandem mass spectrometry combined with multivariate statistical analysis.

Pogostemon cablin Benth (PCB) is a well-known traditional Chinese medicine that has been used for treatment of many ailments for several centuries. In presently, the chemical profiling and quality control study of PCB has mainly concentrated on the volatile fractions. However, the non-volatile chemical profile of PCB was still unclear. In this study, 73 non-volatile constituents (i.e., 33 flavonoids, 21 organic acids, 9 phenylpropanoids, 4 sesquiterpenes, 3 alkaloids, and 3 other types of compounds) were identified and characterized in PCB using high performance liquid chromatography coupled with quadruple time-of-flight tandem mass spectrometry (HPLC-Q-TOF-MS). Meanwhile, to assess PCB samples, an established HPLC-Q-TOF-MS fingerprint was combined with multivariate statistical analysis that included similarity analysis (SA), hierarchical cluster analysis (HCA), principal component analysis (PCA), and orthogonal partial least squares-discriminant analysis (OPLS-DA). The PCB samples could be classified into two groups (herbal decoction pieces and processed medicinal materials), and acteoside, isoacteoside, 4',6-Dihydroxy-5,7-dimethoxyflavone, pachypodol and pogostone were screened as the potential chemical markers that attributed classification. In addition, nine representative components (pachypodol, vicenin-2, apigenin, rhamnocitrin, acteoside, isoacteoside, chlorogenic acid, azelaic acid and pogostone) in PCB were simultaneously determined by using an ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UPLC-QQQ-MS/MS). This study is the first to describe the chemical profile of PCB using liquid chromatography tandem mass spectrometry, which would improve our understanding of the substance basis of PCB and is helpful to the PCB further quality evaluation.

SARS-CoV-2 replicon for high-throughput antiviral screening.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus, which is highly pathogenic and classified as a biosafety level 3 (BSL-3) agent, has greatly threatened global health and efficacious antivirals are urgently needed. The high requirement of facilities to manipulate the live virus has limited the development of antiviral study. Here, we constructed a reporter replicon of SARS-CoV-2, which can be handled in a BSL-2 laboratory. The Renilla luciferase activity effectively reflected the transcription and replication levels of the replicon genome. We identified the suitability of the replicon in antiviral screening using the known inhibitors, and thus established the replicon-based high-throughput screening (HTS) assay for SARS-CoV-2. The application of the HTS assay was further validated using a few hit natural compounds, which were screened out in a SARS-CoV-2 induced cytopathic-effect-based HTS assay in our previous study. This replicon-based HTS assay will be a safe platform for SARS-CoV-2 antiviral screening in a BSL-2 laboratory without the live virus.

Long read sequencing of Toona sinensis (A. Juss) Roem: A chromosome-level reference genome for the family Meliaceae.

Chinese mahogany (Toona sinensis) is a woody plant that is widely cultivated in China and Malaysia. Toona sinensis is important economically, including as a nutritious food source, as material for traditional Chinese medicine and as a high-quality hardwood. However, the absence of a reference genome has hindered in-depth molecular and evolutionary studies of this plant. In this study, we report a high-quality T. sinensis genome assembly, with scaffolds anchored to 28 chromosomes and a total assembled length of 596 Mb (contig N50 = 1.5 Mb and scaffold N50 = 21.5 Mb). A total of 34,345 genes were predicted in the genome after homology-based and de novo annotation analyses. Evolutionary analysis showed that the genomes of T. sinensis and Populus trichocarpa diverged ~99.1-103.1 million years ago, and the T. sinensis genome underwent a recent genome-wide duplication event at ~7.8 million years and one more ancient whole genome duplication event at ~71.5 million years. These results provide a high-quality chromosome-level reference genome for T. sinensis and confirm its evolutionary position at the genomic level. Such information will offer genomic resources to study the molecular mechanism of terpenoid biosynthesis and the formation of flavour compounds, which will further facilitate its molecular breeding. As the first chromosome-level genome assembled in the family Meliaceae, it will provide unique insights into the evolution of members of the Meliaceae.

Generation and characterization of Japanese encephalitis virus expressing GFP reporter gene for high throughput drug screening.

Japanese encephalitis virus (JEV), a major cause of Japanese encephalitisis, is an arbovirus that belongs to the genus Flavivirus of the family Flaviviridae. Currently, there is no effective drugs available for the treatment of JEV infection. Therefore, it is important to establish efficient antiviral screening system for the development of antiviral drugs. In this study, we constructed a full-length infectious clone of eGFP-JEV reporter virus by inserting the eGFP gene into the capsid-coding region of the viral genome. The reporter virus RNA transfected-BHK-21 cells generated robust eGFP fluorescence signals that were correlated well with viral replication. The reporter virus displayed growth kinetics similar to wild type (WT) virus although replicated a little slower. Using a known JEV inhibitor, NITD008, we demonstrated that the reporter virus could be used to identify inhibitors against JEV. Furthermore, an eGFP-JEV-based high throughput screening (HTS) assay was established in a 96-well format and used for screening of 1443 FDA-approved drugs. Sixteen hit drugs were identified to be active against JEV. Among them, five compounds which are lonafarnib, cetylpyridinium chlorid, cetrimonium bromide, nitroxoline and hexachlorophene, are newly discovered inhibitors of JEV, providing potential new therapies for treatment of JEV infection.

[Comparison between Ye Tianshi and Wu Jutong in treatment of summer-heat disease].

Through the traditional Chinese medicine inheritance platform system, with the help of medical records, Ye Tianshi and Wu Jutong's medication characteristics for summer heat sickness were analyzed, the laws of the two people's medication were summarized, and the similarities and differences between the two were explored to explore the relationship. As a result, it was found that both of them recognized the relationship between summer heat and wetness, and Wu Jutong believed that "wind" was also an important pathogenic factor. Both of the patients were treated with cold medicine and warm medicine. They used mostly bitter, sweet, pungent taste and lungs, spleen, stomach, and heart meridian are the main components; two are commonly used Armeniacae Semen Amarum, Talcum, Rehmanniae Radix, Ophiopogonis Radix, Pinelliae Rhizoma and other drugs, Ye Tianshi use Scrophulariae Radix, Tetrapanacis Medulla, Coicis Semen and other drugs more, Wu Jutong use Gypsum Fibrosum, Sojae Semen Praeparatum, Menthae Haplocalycis Herba and other drugs more; at the same time, a combination of two high-frequency medicines used by two people has been excavated, and a new prescription has been deduced to provide a reference for further understanding and treatment of summer diseases.

Protective role of berberine and Coptischinensis extract on T2MD rats and associated islet Rin­5f cells.

The aim of the present study was to compare the different effects of berberine (Ber) and Coptischinensis extract (CCE) on a rat model of type 2 diabetes mellitus (T2DM), and the islet Rin­5f cell line was used to examine the differences between Ber and CCE and the underlying mechanisms. CCE was extracted and purified prior to analysis. Male Sprague­Dawley rats were provided with a high­fat diet to induce insulin resistance prior to injecting with streptozotocinto establish the T2DM model, the T2DM rats were treated with Ber and CCE, and blood samples and pancreatic tissues were obtained and compared to examine T2DM metabolic syndromes among the groups of rats, which included healthy rats, model rats, and model rats treated with Ber and CCE at different doses between 0 and 8 weeks. The protective effects of Ber and CCE on the Rin­5f islet cell line were also evaluated. The effects on Rin­5f cell proliferation and cell cycle, glucose­stimulated insulin release test (GSIS), the anti­apoptotic effects caused by fat induction, and protein expression levels of poly ADP­ribose polymerase (PARP­1) were evaluated. The results showed that the content of the prepared CCE was 96.07% for five alkaloids. When it was used for treatment of the T2DM rats, compared with Ber, metformin and rosiglitazone, the fasting blood glucose, glucosylated serum protein (GSP) and glucose infusion rate indicesin the fasting rats were ameliorated, compared with those in the T2MD rats, with no significant differences between treatment with Ber or CCE and metformin or rosiglitazone. The indices of mean optical density and fasting ß­cell function index (FBCI) were different following treatment with Ber and CCE, compared with those in the model rats, which may have stimulated the pancreatic secretion of insulin. When Ber and CCE were used to examine the protective effects on Rin­5F cells, it was found that the Rin­5f cell GSIS, cell cycle, lipotoxic islet cell proliferation and protein expression of PARP­1 were altered and improved, which may have protected pancreatic islet ß­cells by improving islet ß­cell proliferation and the protein expression of PARP­1. CCE and Ber exerted similar effects when used for the treatment of T2MD rats, and may have stimulated the pancreatic secretion of insulin through the protective effect on islet ß­cells via improving islet ß­cell proliferation and the protein expression of PARP­1.

S-propargyl-cysteine attenuates inflammatory response in rheumatoid arthritis by modulating the Nrf2-ARE signaling pathway.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder. Hydrogen sulfide (H2S), the third physiological gasotransmitter, is well recognized as an anti-inflammatory mediator in various inflammatory conditions. Herein, we explored the protective effects of S-propargyl-cysteine (SPRC, also known as ZYZ-802), an endogenous H2S modulator, on RA and determined the underlying mechanisms. In the present study, SPRC concentration-dependently attenuated inflammatory mediator expression, reactive oxidase species generation, and the expression and activity of matrix metalloproteinases (MMP)-9 in interleukin (IL)-1ß-induced human rheumatoid fibroblast-like synoviocytes MH7A. In addition, SPRC blocked IL-1ß-mediated migration and invasion of MH7A cells. As expected, the protective effects of SPRC were partially abrogated by DL-propargylglycine (PAG, a H2S biosynthesis inhibitor). In vivo study also demonstrated that SPRC treatment markedly ameliorated the severity of RA in adjuvant-induced arthritis rats, and this effect was associated with the inhibition of inflammatory response. We further identified that SPRC remarkably induced heme oxygenase-1 expression associated with the degradation of Kelch-like ECH-associated protein 1 (Keap1) and nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2); this effect was attributed to the sulfhydrylation of the cysteine residue of Keap1. Our data demonstrated for the first time that SPRC, an endogenous H2S modulator, exerted anti-inflammatory properties in RA by upregulating the Nrf2-antioxidant response element (ARE) signaling pathway.

Poor permeability and absorption affect the activity of four alkaloids from Coptis.

Coptidis rhizoma (Coptis) and its alkaloids exert various pharmacological functions in cells and tissues; however, the oral absorption of these alkaloids requires further elucidation. The present study aimed to examine the mechanism underlying the poor absorption of alkaloids, including berberine (BER), coptisine (COP), palmatine (PAL) and jatrorrhizine (JAT). An ultra­performance liquid chromatography (UPLC) method was validated for the determination of BER, COP, PAL and JAT in the above experimental medium. In addition, the apparent oil­water partition coefficient (Po/w); apparent permeability coefficient (Papp), determined using a parallel artificial membrane permeability assay (PAMPA) plate; membrane retention coefficient (R %); and effect of P­glycoprotein (P­gp) inhibitor on the Papp of the four alkaloids were investigated. The intestinal absorption rate constant (Ka) and absorption percentage (A %) of the four alkaloids were also determined. The results of the present study demonstrated that the Po/w of the four alkaloids in 0.1 mol·l­1 HCl medium was significantly higher (P<0.01), compared with those of the alkaloids in phosphate buffer (pH 7.4). The Papp of BER was 1.0­1.2x10­6 cm·s­1, determined using a PAMPA plate, and the Papp of BER, COP, PAL and JAT decreased sequentially. The concentrations of the four alkaloids on the apical­to­basolateral (AP­BL) surface and the basolateral­to­apical (BL­AP) surface increased in a linear manner, with increasing concentrations between 10 and 100 µmol. In addition, the transportation of BER on the BL­AP surface was significantly faster (P<0.01), compared with that on the AP­BL surface and, following the addition of verpamil (a P­gp inhibitor), the Papp (AP­BL) of the four alkaloids increased, whereas the Papp (BL­AP) was significantly decreased (P<0.01). The rat intestinal perfusion experiment demonstrated that the four alkaloids were poorly absorbed; however, the Ka of BER was significantly higher, compared with the three other alkaloids. Furthermore, the A % and Ka provided evidence that the absorption of BER was increased in the jejunum, compared with in the ileum. In conclusion, the four alkaloids from Coptis appeared to be poorly absorbed, determined using a shake flask, pre­coated PAMPA plates, a Caco­2 cell monolayer model and intestinal perfusion; however, absorption was higher in the jejunum than in the ileum. Among the four alkaloids, the permeability of BER was markedly higher than the others, and P­gp efflux had a significant effect on the absorption of those alkaloids.

Correlations between Phlegm Syndrome of Chinese Medicine and Coronary Angiography: A Systematic Review and Meta-Analysis.

Phlegm is one of the most common patterns of coronary artery disease (CAD) in Chinese medicine. Our research was aimed at investigating the association between phlegm syndrome of CAD and coronary angiography (CAG) by meta-analysis. According to inclusion criteria, a total of 30 studies involving 5,055 CAD patients were included. The meta-analysis showed that phlegm syndrome patients were prone to multivessel disease (28 studies, OR = 1.53, 95% CI, 1.24 to 1.88, P < 0.01) and higher Gensini score (2 studies, OR = 5.90, 95% CI, 1.86 to 9.94, P = 0.004), but not obviously relevant to severe stenosis (≥75%) of coronary arteries (13 studies, OR = 1.20, 95% CI, 0.63 to 2.27, P = 0.57). We concluded that the coronary arteries lesions of CAD patients with phlegm syndrome were more severe than those with nonphlegm syndromes. Phlegm syndrome should, therefore, be regarded as a dangerous pattern of CAD with worse prognosis.

In vitro studies of berberine metabolism and its effect of enzyme induction on HepG2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Berberine (BER) and BER-original herbal medicines have a variety of pharmacological functions and have been widely used in clinical. However, its effect of enzyme induction on cytochrome P450 (CYP) in human hepatocytes is unknown. MATERIAL AND METHOD: Metabolism of berberine and its effect on main metabolic enzymes in HepG2 cell in vitro was investigated. Cocktail probe drugs, mRNA expression and protein expression were used to evaluate the metabolism potency. Meanwhile, an UPLC-MS/MS method was validated for the analysis of BER and four probe drugs in HepG2 cell. RESULT: BER significantly increased the metabolism of midazolam, phenacetin and tolbutamide by inducing the CYP1A2 and 3A4 enzyme in a dose-dependent manner, the mRNA and protein expression of CYP1A2 and 3A4 were increased by berberine at 1000ng·mL(-1). The activity of CYP1A2 and 3A4 could be induced by BER more than 500ng·mL(-1) in HepG2 cell, which was confirmed by the increase of its mRNA and protein expression. CONCLUSION: BER increases the metabolism of cocktail drugs such as midazolam, phenacetin and tolbutamide by increasing the mRNA and protein expression of CYP1A2 and 3A4.

[Study on HPLC-fingerprint-based identification of dao-di herb and non dao-di herb of scutellariae radix].

OBJECTIVE: To compare the discrepancies between chemical constituents in Dao-di herb and non Dao-di herb of Huangqin (the root of Scutellaria baicalensis), study the impact of habitat and growth pattern (including cultivated and wild Huangqin) on chemical substances of Huangqin, and then provide evidence for the identification of Dao-di herb and quality evaluation of Huangqin. METHOD: The chemical constituents in Huangqin collected from different habitats and under different growth patterns, were analyzed using HPLC fingerprint. The fingerprints obtained were then evaluated by hierarchical clustering analysis, principal component analysis and components peak area pattern. RESULT: The fingerprints' chemical profiles of Dao-di herb and non Dao-di Huangqin had significant disparity. The fingerprints of modem Dao-di herb Huangqin samples originated from Chengde (Hebei Province) were significantly different from those from other habitats, though the fingerprints of the non Dao-di Huangqin collected from Chifeng (Inner Mongolia) and Chengde had high similarity to each other. The chemical characteristics of Huangqin samples collected from the habitats recorded in ancient herbals, such as Qingyang (Gansu Province), Yan'an (Shaanxi Province), Linyi (Shangdong Province), Changzhi and Jinzhong (Shanxi Province) were similar. The fingerprints of modern non Dao-di samples collected from Dingxi and Longnan (Gansu Province) and Shangluo (Shaanxi Province) had high similarity. In addition, the content of acteoside in wild Huangqin was higher than that in cultivated Huangqin. CONCLUSION: Dao-di herb and non Dao-di herb of Huangqin could be distinguished using the developed HPLC fingerprints. The results obtained may provide evidence for the quality control and pharmcodynamical research of Dao-di herb and non Dao-di Huangqin.

[The endothelium injuries caused by homocysteine and treatmental effects of Tongxinluo powder].

AIM: To observe the effect of homocysteine (HCY) on the function of endothelium cell, and to discuss the possible mechanisms that Tongxinluo super powder affected. METHODS: Healthy male Wistar rats were divided into randomly the control group, the model group, the Tongxinluo group. The effect of Ach on isolated rat thoracic aorta in vitro was examined, the microcirculation was observed by microcirculation meter, the activity of SOD and GSH-PX and content of NO, MDA, ET, Ang II, TXA2, PGI2 was detected. RESULTS: Compared with control group, the effect of Ach on isolated rat thoracic aorta in vitro weakened markablely (P < 0.01), the format and percentage that capillary dilated declined significantly (P < 0.05), after treatment with Tongxinluo powder, the effect of Ach on isolated rat thoracic aorta in vitro was improved obviously (p < 0.01), and the format and percentage that capillary dilated were increased compared with model group; comparing with the control group, the level of Ang II and ET, TXA2 in plasm increased obviously (P < 0.05, P < 0.01), while the content of PGI2 depressed manifestly (P < 0.05), at the same time, both content of NO and activity of SOD, (GSH-PX declined obviously (P < 0.001, P < 0.05). After treatment with Tongxinluo powder, the level of ET, AngII and TXA2 reduced significantly in different degree (P < 0.01), while the content of PGI2 appeared stepping up notably (P < 0.01), and both activity of SOD and NO level increased obviously (P < 0.01, P < 0.05). CONCLUSION: (1) The high homocystein might cause the contracted and dilated function decreased, it might get involved in endothelium disfunction as a result of the massive free radicals production and diastolic-contract factors balance disorder induced by high homocystein. (2) Tongxinluo powder could improve the function of endothelium-dependment dilation induced by high homocystein, that associated with inhibitting the excessive production of free radicals, and improved function of endothelium.