Ratiometric SERS quantification of SO2 vapor based on Au@Ag-Au with Raman reporter as internal standard.

Practical gas sensing application requires sensors to quantify target analytes with high sensitivity and reproducibility. However, conventional surface enhanced Raman scattering (SERS) sensor lacks reproducibility and quantification arising from variations of "hot spot" distribution and measurement conditions. Here, a ratio-dependent SERS sensor was developed for quantitative label-free gas sensing. Au@Ag-Au nanoparticles (NPs) were filtered onto anodic aluminum oxide (AAO) forming Au@Ag-Au@AAO SERS substrate. 4-MBA was encapsulated in the gap of Au@Ag-Au and served as the internal standard (IS) to calibrate SERS signal fluctuation for improved quantification ability. Combined with headspace sampling method, SO2 residue in traditional Chinese medicine (TCM) can be extracted and captured on the immediate vicinity of Au@Ag-Au surface. The intensity ratio I613 cm-1/I1078 cm-1 showed excellent linearity within the range of 0.5 mg/kg-500 mg/kg, demonstrating superior quantification performance for SO2 detection. Signals for concentration as low as 0.05 mg/kg of SO2 could be effectively collected, much lower than the strictest limit 10 mg/kg in Chinese Pharmacopoeia. Combined with a handheld Raman spectrometer, handy and quantitative TCM quality evaluation in aspect of SO2 residue was realized. This ratiometric SERS sensor functioned well in rapid on-site SO2 quantification, exhibiting excellent sensitivity and simple operability.

Tripterygium wilfordii Hook. F. and Its Extracts for Psoriasis: Efficacy and Mechanism.

Psoriasis is an inflammatory autoimmune skin condition that is clinically marked by chronic erythema and scaling. The traditional Chinese herb Tripterygium wilfordii Hook. F. (TwHF) is commonly used in the treatment of immune-related skin illnesses, such as psoriasis. In clinical studies, PASI (Psoriasis Area and Severity Index) were dramatically decreased by TwHF and its extracts. Their benefits for psoriasis also include relief from psoriasis symptoms such as itching, dryness, overall lesion scores and quality of life. And the pathological mechanisms include anti-inflammation, immunomodulation and potentially signaling pathway modulations, which are achieved by modulating type-3 inflammatory cytokines including IL-22, IL-23, and IL-17 as well as immune cells like Th17 lymphocytes, γδT cells, and interfering with IFN-SOCS1, NF-κB and IL- 36α signaling pathways. TwHF and its extracts may cause various adverse drug reactions, such as gastrointestinal responses, aberrant hepatocytes, reproductive issues, and liver function impairment, but at adequate doses, they are regarded as an alternative therapy for the treatment of psoriasis. In this review, the effectiveness and mechanisms of TwHF and its extracts in psoriasis treatment are elucidated.

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte modelling of cardiovascular diseases for natural compound discovery.

Cardiovascular disease (CVD) remains the leading cause of death worldwide. Natural compounds extracted from medicinal plants characterized by diverse biological activities and low toxicity or side effects, are increasingly taking center stage in the search for new drugs. Currently, preclinical evaluation of natural products relies mainly on the use of immortalized cell lines of human origin or animal models. Increasing evidence indicates that cardiomyopathy models based on immortalized cell lines do not recapitulate pathogenic phenotypes accurately and a substantial physiological discrepancy between animals and humans casts doubt on the clinical relevance of animal models for these studies. The newly developed human induced pluripotent stem cell (hiPSC) technology in combination with highly-efficient cardiomyocyte differentiation methods provides an ideal tool for modeling human cardiomyopathies in vitro. Screening of drugs, especially screening of natural products, based on these models has been widely used and has shown that evaluation in such models can recapitulate important aspects of the physiological properties of drugs. The purpose of this review is to provide information on the latest developments in this area of research and to help researchers perform screening of natural products using the hiPSC-CM platform.

Discovery of anti-stroke active substances in Guhong injection based on multi-phenotypic screening of zebrafish.

Ischemic stroke is a leading cause of death worldwide, and it remains an urgent task to develop novel and alternative therapeutic strategies for the disease. We previously reported the positive effects of Guhong injection (GHI), composed of safflower extract and aceglutamide, in promoting functional recovery in ischemic stroke mice. However, the active substances and pharmacological mechanism of GHI is still elusive. Aiming to identify the active anti-stroke components in GHI, here we conducted a multi-phenotypic screening in zebrafish models of phenylhydrazine-induced thrombosis and ponatinib-induced cerebral ischemia. Peripheral and cerebral blood flow was quantified endogenously in erythrocytes fluorescence-labeled thrombosis fish, and baicalein and rutin were identified as major anti-thrombotic substances in GHI. Moreover, using a high-throughput video-tracking system, the effects of locomotion promotion of GHI and its main compounds were analyzed in cerebral ischemia model. Chlorogenic acid and gallic acid showed significant effects in preventing locomotor dyfunctions. Finally, GHI treatment greatly decreased the expression levels of coagulation factors F7 and F2, NF-κB and its mediated proinflammatory cytokines in the fish models. Molecular docking suggested strong affinities between baicalein and F7, and between active substances (baicalein, chlorogenic acid, gallic acid, and rutin) and NF-κB p65. In summary, our findings established a novel drug discovery method based on multi-phenotypic screening of zebrafish, provided endogenous evidences of GHI in preventing thrombus formation and promoting behavioral recovery after cerebral ischemia, and identified baicalein, rutin, chlorogenic acid, and gallic acid as active compounds in the management of ischemic stroke.

Pien Tze Huang Inhibits Migration and Invasion of Hepatocellular Carcinoma Cells by Repressing PDGFRB/YAP/CCN2 Axis Activity.

OBJECTIVE: To investigate the effects of Pien Tze Huang (PZH) on the migration and invasion of HCC cells and underlying molecular mechanism. METHODS: Cell counting kit-8 (CCK-8) was applied to evaluate the cell viabilities of SMMC-7721, SK-Hep-1, C3A and HL-7702 (6 × 103 cells/well) co-incubated with different concentrations of PZH (0, 0.2, 0.4, 0.6, 0.8 mg/mL) for 24 h. Transwell, wound healing assay, CCK-8 and Annexin V-FITC/PI staining were conducted to investigate the effects of PZH on the migration, invasion, proliferation and apoptosis of SK-Hep-1 and SMMC-7721 cells (650 µ g/mL for SK-Hep-1 cells and 330 µ g/mL for SMMC-7721 cells), respectively. In vivo, lung metastasis mouse model constructed by tail vein injection of HCC cells was used for evaluating the anti-metastasis function of PZH. SK-Hep-1 cells (106 cells/200 µ L per mice) were injected into B-NDG mice via tail vein. Totally 8 mice were randomly divided into PZH and control groups, 4 mice in each group. After 2-d inoculation, mice in the PZH group were administered with PZH (250 mg/kg, daily) and mice in the control group received only vehicle (PBS) from the 2nd day after xenograft to day 17. Transcriptome analysis based on RNA-seq was subsequently used for deciphering anti-tumor mechanism of PZH. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were applied to verify RNA-seq results. Luciferase reporter assay was performed to examine the transcriptional activity of yes-associated protein (YAP). RESULTS: PZH treatment significantly inhibited the migration, invasion, proliferation and promoted the apoptosis of HCC cells in vitro and in vivo (P<0.01). Transcriptome analysis indicated that Hippo signaling pathway was associated with anti-metastasis function of PZH. Mechanical study showed PZH significantly inhibited the expressions of platelet derived growth factor receptor beta (PDGFRB), YAP, connective tissue growth factor (CCN2), N-cadherin, vimentin and matrix metallopeptidase 2 (MMP2, P<0.01). Meanwhile, the phosphorylation of YAP was also enhanced by PZH treatment in vitro and in vivo. Furthermore, PZH played roles in inhibiting the transcriptional activity of YAP. CONCLUSION: PZH restrained migration, invasion and epithelial-mesenchymal transition of HCC cells through repressing PDGFRB/YAP/CCN2 axis.

Identification of bioactive ingredients from Babaodan using UPLC-QTOF-MS analysis combined with network pharmacology guided bioassays.

Babaodan (BBD) is a traditional Chinese medicine (TCM) prescribed for various inflammatory diseases, including viral hepatitis and acute genitourinary tract infection. Like other TCMs, BBD is a multi-component formula whose chemical composition and mode of action are largely unknown. The current study identified the bioactive ingredients of BBD using ultrahigh-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) followed by mass spectrometry molecular networking analysis. Subsequently, network pharmacology analysis was performed to predict the potential targets and pathways regulated by BBD. Eventually, a panel of compounds was selected and examined for their anti-inflammatory effects using lipopolysaccharide-stimulated RAW264.7 cells. Eighty-six compounds, including saponins, bile acids, and fatty acids, were identified. Tumor necrosis factor-alpha was identified as a key molecule. Pathways in cancer, inflammatory bowel disease, and hepatitis were predicted to be the major regulatory pathways. The results from bioassays validated ginsenoside Rb1, ginsenoside Rd, deoxycholic acid, chenodeoxycholic acid, and taurochenodeoxycholic acid as novel bioactive ingredients in BBD with anti-inflammatory effects. In conclusion, our study explains the anti-inflammatory efficacy of BBD from both chemical and biological aspects, which provides a scientific basis for the clinical application of BBD in inflammation-related diseases.

Guhong injection promotes post-stroke functional recovery via attenuating cortical inflammation and apoptosis in subacute stage of ischemic stroke.

BACKGROUND: As a leading cause of death and disability, alternative therapies for stroke are still limited by its complicated pathophysiological manifestations. Guhong injection (GHI), consisting of safflower aqueous extract and aceglutamide, has been widely applied for the clinical treatment of cerebrovascular diseases, especially ischemic stroke and post-stroke recovery, in China. Recently, a series of studies have reported the positive effect of GHI against cerebral ischemia/reperfusion injury via targeting various molecular mechanisms. However, questions remain on whether treatment with GHI contributes to better functional recovery after stroke and if so, the potential mechanisms and active substances. PURPOSE: The aim of this work was to explore the potential therapeutic possibilities of GHI for the neurological and behavioral recovery after stroke and to investigate the underlying molecular mechanisms as well as active substances. METHODS: The neural and motor deficits as well as cortical lesions after GHI treatment were investigated in a mouse model of transient ischemic stroke. Based on the substance identification of GHI, network pharmacology combined with an experimental verification method was used to systematically decipher the biological processes and signaling pathways closely related to GHI intervention in response to post-stroke functional outcomes. Subsequently, ingenuity pathway analysis (IPA) analysis was performed to determine the anti-stroke active substances targeting to the hub targets involved in the significant molecular pathways regulated by GHI treatment. RESULTS: Therapeutically, administration of GHI observably ameliorated the post-stroke recovery of neural and locomotor function as well as reduced infarct volume and histopathological damage to the cerebral cortex in subacute stroke mice. According to 26 identified or tentatively characterized substances in GHI, the compound-target-pathway network was built. Bioinformatics analysis suggested that inflammatory and apoptotic pathways were tightly associated with the anti-stroke effect of GHI. Based on protein-protein interaction network analysis, the hub targets (such as NF-κB p65, TNF-α, IL-6, IL-1ß, Bax, Bcl-2, and Caspase-3) involved in inflammation and apoptosis were selected. On the one hand, immunofluorescence and ELISA results showed that GHI (10 ml/kg) treatment obviously reduced NF-κB p65 nuclear translocation as well as decreased the abnormally elevated concentrations of proinflammatory cytokines (TNF-α, IL-6, and IL-1ß) in damaged cortex tissues. On the other hand, GHI (10 ml/kg) treatment significantly downregulated the number of TUNEL-positive apoptotic cells in ischemic cortex and effectively restored the abnormal expression of Bax, Bcl-2, and Caspase-3. Based on the results of IPA, hydroxysafflor yellow A, baicalin, scutellarin, gallic acid, syringin, chlorogenic acid, kaempferol, kaempferol-3-O-ß-rutinoside, and rutin acted synergistically on core targets, which could be considered as the active substances of GHI. CONCLUSION: Overall, the current findings showed that the beneficial action of GHI on improving post-stroke functional recovery of subacute stroke mice partly via the modulation of cortical inflammation and apoptosis. These findings not only provide a reliable reference for the clinical application of GHI, but also shed light on a promising alternative therapeutic strategy for ischemic stroke patients.

Salvianolic acid B and ferulic acid synergistically promote angiogenesis in HUVECs and zebrafish via regulating VEGF signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Induced vascular growth in the myocardium has been widely acknowledged as a promising intervention strategy for patients with ischemic coronary artery disease. Yet despite long-term efforts on gene, protein or cell-based pro-angiogenic therapies, the clinical translation remains challenging. Noticeably, multiple medicinal herbs have long-term documented effects in promoting blood circulation. Salvia miltiorrhiza and Ligusticum stratum are two representative traditional Chinese medicine herbs with suggested roles in enhancing organ blood supply, and Guanxinning Tablet (GXNT), a botanical drug which is formulated with these two herbs, exhibited significant efficacy against angina pectoris in clinical practices. AIM OF THE STUDY: This study aimed to examine the pro-angiogenic activity of GXNT and its major components, as well as to explore their pharmacological mechanism in promoting angiogenesis. MATERIALS AND METHODS: In vitro, the pro-angiogenic effects of GXNT and its major components were examined on human umbilical vein endothelial cells by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), scratch assay, and endothelial cell tube formation assay. In vivo, the pro-angiogenic effects were examined on the ponatinib-induced angiogenesis defective zebrafish model. The active compounds were identified through phenotype-based screening in zebrafish, and their pharmacological mechanism was explored in both in vitro and in vivo models by immunofluorescent staining, cell cycle analysis, quantitative PCR and whole embryo in-situ hybridization. RESULTS: We demonstrated strong pro-angiogenic effects of GXNT in both human umbilical vein endothelial cells and zebrafish model. Moreover, through phenotype-based screening in zebrafish for active compounds, pro-angiogenic effects was discovered for salvianolic acid B (Sal B), a major component of Salvia miltiorrhiza, and its activity was further enhanced when co-administered with ferulic acid (FA), which is contained in Ligusticum stratum. On the cellular level, Sal B and FA cotreatment increased endothelial cell proliferation of sprouting arterial intersomitic vessels in zebrafish, as well as largely restored G1-S cell cycle progression and cyclin D1 expression in angiogenic defective HUVECs. Through quantitative transcriptional analysis, increased expression of vegfr2 (kdr, kdrl) and vegfr1 was detected after GXNT or SalB/FA treatment, together with upregulated transcription of their ligands including vegf-a, vegf-b, and pgfb. Bevacizumab, an anti-human VEGF-A monoclonal antibody, was able to significantly, but not completely, block the pro-angiogenic effects of GXNT or SalB/FA, suggesting their multi-targeting properties. CONCLUSIONS: In conclusion, from a traditional Chinese medicine with effects in enhancing blood circulation, we demonstrated the synergistic pro-angiogenic effects of Sal B and FA via both in vitro and in vivo models, which function at least partially through regulating the expression of VEGF receptors and ligands. Future studies are warranted to further elaborate the molecular interaction between these two compounds and the key regulators in the process of neovascularization.

Discovery of Herbacetin as a Novel SGK1 Inhibitor to Alleviate Myocardial Hypertrophy.

Cardiac hypertrophy is a pivotal pathophysiological step of various cardiovascular diseases, which eventually leads to heart failure and death. Extracts of Rhodiola species (Ext.R), a class of commonly used medicinal herbs in Europe and East Asia, can attenuate cardiac hypertrophy both in vitro and in vivo. Serum/glucocorticoid regulated kinase 1 (SGK1) is identified as a potential target of Ext. R. By mass spectrometry-based kinase inhibitory assay, herbacetin (HBT) from Ext.R is identified as a novel SGK1 inhibitor with IC50 of 752 nmol. Thermal shift assay, KINOMEscan in vitro assay combined with molecular docking proves a direct binding between HBT and SGK1. Site-specific mutation of Asp177 in SGK1 completely ablates the inhibitory activity of HBT. The presence of OH groups at the C-3, C-8, C-4' positions of flavonoids is suggested to be favorable for the inhibition of SGK1 activity. Finally, HBT significantly suppresses cardiomyocyte hypertrophy in vitro and in vivo, reduces reactive oxygen species (ROS) synthesis and calcium accumulation. HBT decreases phosphorylation of SGK1 and regulates its downstream forkhead box protein O1 (FoxO1) signaling pathway. Taken together, the findings suggest that a panel of flavonoids structurally related to HBT may be novel leads for developing new therapeutics against cardiac hypertrophy.

Si@Ag@PEI substrate-based SERS sensor for rapid detection of illegally adulterated sulfur dioxide in traditional Chinese medicine.

The illegal adulteration of sulfur dioxide in natural healthcare products may lead to serious health problems, which raise an urgent demand of straightforward approach for detecting sulfur dioxide. In this paper, surface-enhanced Raman scattering (SERS) sensor with sample preparation apparatus for headspace adsorption of SO2 has been developed, which was successfully applied to detect illegal adulteration of sulfur dioxide in traditional Chinese medicine (TCM). Functional membrane substrate of Si@Ag@PEI composite was synthesized to enhance the adsorption and Raman signal of SO2. A 3D-printed headspace extraction device was designed to adsorbed SO2 by Si@Ag@PEI membrane after micro-extraction of TCM samples in 15 min. The content of sulfur dioxide was subsequently quantitatively measured by SERS sensor. The linear range of sensor is between 2.5 and 250 mg/kg with limit of detection of 0.25 mg/kg, which is lower than the strictest standard of Chinese Pharmacopoeia (10 mg/kg). The proposed approach was used to detect the SO2 residue in TCMs including ginseng, Salvia miltiorrhiza, and bitter almonds. The fabricated sensor exhibited satisfied sensitivity and stability, which provide a simple approach for on-site detection of illegal adulteration of sulfur dioxide.

Discovery of tetrahydropalmatine and protopine regulate the expression of dopamine receptor D2 to alleviate migraine from Yuanhu Zhitong formula.

BACKGROUND: Migraine is the third most common disease worldwide, leading to severely decreased quality of life for the patients. In spite of great efforts endeavored in pharmacological and nonpharmacological therapeutic strategies for treating migraine, the outcome is rather disappointing in terms of efficacy. Compelling evidence shows that the expression level of dopamine receptor D2 (DRD2) plays an essential role in progression of migraine. PURPOSE: To explore potential therapeutical possibilities, the attention was paid to Yuanhu Zhitong formula (YHZTF), which is a classical traditional Chinese medicine prescription frequently applied to relieve pain. The aim of this study was to identify the promising compounds derived from YHZTF with anti-migraine effects and investigate the underlying molecular mechanism. METHODS: The high-resolution mass spectrometry and molecular networking were performed for comprehensive chemical profiling of YHZTF. Network pharmacology was used to generate herbal-component-target-pathway network. Based on the pathway enrichment analysis, the active substances of anti-migraine and the potential molecular mechanism were further determined by performing animal experiments combined with molecular docking strategy. RESULTS: In total, 31 substances were identified in YHZTF, including alkaloids such as tetrahydropalmatine and protopine. The analysis of herbal-component-target-pathway network suggests that the alkaloid substances (e.g. tetrahydropalmatine and protopine) from YHZTF target dopamine receptors, thus can be linked to neuroactive ligand-receptor interaction pathways. In a nitroglycerin-induced migraine animal model, pretreatment with tetrahydropalmatine or protopine substantially lessened the aberrant migraine-like symptoms. The results of molecular docking analysis showed that tetrahydropalmatine and protopine had strong affinities to dopamine receptor D2 (DRD2). Using RT-qPCR, the investigators found that DRD2 was significantly down-regulated at the mRNA level in brain tissues of tetrahydropalmatine and protopine-treated group compared to the control group. CONCLUSION: Collectively, the results provide reliable evidence showing that the active substances tetrahydropalmatine and protopine from YHZTF lessens migraine symptoms in an in vivo mouse model suggestively via regulating expression of DRD2. These findings shed light on novel therapeutic strategies and targets to treat migraine using natural products.

Identification of anti-inflammatory compounds from Zhongjing formulae by knowledge mining and high-content screening in a zebrafish model of inflammatory bowel diseases.

BACKGROUND: Inflammatory bowel diseases (IBD) are chronic relapsing intestinal inflammations with increasing global incidence, and new drug development remains in urgent demand for IBD management. To identify effective traditional Chinese medicine (TCM) formulae and compounds in IBD treatment, we innovatively combined the techniques of knowledge mining, high-content screening and high-resolution mass spectrometry, to conduct a systematic screening in Zhongjing formulae, which is a large collection of TCM prescriptions with most abundant clinical evidences. METHODS: Using Word2vec-based text learning, the correlations between 248 Zhongjing formulae and IBD typical symptoms were analyzed. Next, from the top three formulae with predicted relationship with IBD, TCM fractions were prepared and screened on a transgenic zebrafish IBD model for their therapeutic effects. Subsequently, the chemical compositions of the fraction hits were analyzed by mass spectrometry, and the major compounds were further studied for their anti-IBD effects and potential mechanisms. RESULTS: Through knowledge mining, Peach Blossom Decoction, Pulsatilla Decoction, and Gegen Qinlian Decoction were predicted to be the three Zhongjing formulae mostly related to symptoms typical of IBD. Seventy-four fractions were prepared from the three formulae and screened in TNBS-induced zebrafish IBD model by high-content analysis, with the inhibition on the intestinal neutrophil accumulation and ROS level quantified as the screening criteria. Six herbal fractions showed significant effects on both pathological processes, which were subsequently analyzed by mass spectrometry to determine their chemical composition. Based on the major compounds identified by mass spectrometry, a second-round screen was conducted and six compounds (palmatine, daidzin, oroxyloside, chlorogenic acid, baicalin, aesculin) showed strong inhibitory effects on the intestinal inflammation phenotypes. The expression of multiple inflammatory factors, including il1ß, clcx8a, mmp and tnfα, were increased in TNBS-treated fish, which were variously inhibited by the compounds, with aesculin showing the most potent effects. Moreover, aesculin and daidzin also upregulated e-cadherin's expression. CONCLUSION: Taken together, we demonstrated the regulatory effects of several TCM formulae and their active compounds in the treatment of IBD, through a highly efficient research strategy, which can be applied in the discovery of effective TCM formulae and components in other diseases.

Hongjingtian injection protects against myocardial ischemia reperfusion-induced apoptosis by blocking ROS induced autophagic- flux.

BACKGROUND: Hongjingtian injection (HJT) has been widely used in the clinic to treat coronary heart disease in China. However, the underlying mechanisms of therapies still need to be illustrated. The present study aims to determine whether HJT protects against myocardial ischemia reperfusion injury via Reactive Oxygen Species (ROS)-induced autophagic flux and apoptosis and, if so, to explore the underlying mechanisms. METHODS: In vivo myocardial protection and autophagy regulation of HJT in myocardial ischemia reperfusion injury in C57BL/6 J and CAG-RFP-EGFP-LC3 transgenic C57BL/6 J mice were investigated. In vitro, the effects of HJT on apoptosis, autophagic flux, oxidative stress and mitochondrial function were observed in H2O2-induced H9c2 cells. In addition, apoptosis-related proteins and autophagy-related proteins were assessed to explore the underlying mechanisms. RESULTS: HJT significantly decreased the infarct area and cell apoptosis after myocardial ischemia reperfusion injury in C57BL/6 J mice. Autophagic flux was reduced by HJT treatment after myocardial ischemia reperfusion injury in CAG-RFP-EGFP-LC3 transgenic C57BL/6 J mice. HJT inhibited H2O2-induced cell apoptosis by significantly decreasing the levels of cleaved caspase 3 and increasing the Bcl-2/Bax ratio. HJT inhibited autophagic flux after H2O2 stimulation by significantly decreasing LC3-Ⅱ and p-AMPK expression and increasing p-mTOR. HJT inhibited ROS production and improved mitochondrial function in H2O2-induced cells by significantly increasing the mitochondrial membrane potential, intracellular ATP contents and oxygen consumption. CONCLUSION: The beneficial effects of HJT in treating myocardial ischemia reperfusion are partially due to improved mitochondrial function and regulated autophagy to inhibit cell apoptosis through the AMPK/mTOR pathway.

Untargeted metabolomics reveals the synergistic mechanisms of Yuanhu Zhitong oral liquid in the treatment of primary dysmenorrhea.

Primary dysmenorrhea is a prevalent gynecological disorder that severely affects the quality of life in women. Yuanhu Zhitong oral liquid (YZOL) is a standardized herbal preparation frequently used in clinical practice and is a promising alternative therapy for primary dysmenorrhea. The findings of previous studies show that YZOL exhibits significant analgesic and spasmolytic effects, however, the involved mechanism remains unclear. Herein, we performed an untargeted plasma metabolomic analysis on a mouse model of oxytocin-induced primary dysmenorrhea to investigate the underlying mechanism of YZOL. We used multivariate and pathway-driven analyses to uncover the treatment targets linked with YZOL therapy and verified the possible mechanisms through biochemical assays. Therefore, we identified 47 plasma biomarkers primarily associated with sphingolipid metabolism, amino acid metabolism, arachidonic acid metabolism, and biosynthesis of steroid hormone as well as primary bile acid. We established that the analgesic effect of YZOL on primary dysmenorrhea relies on multiple constituents that act on multiple targets in multiple pathways. Our correlation analysis showed significant correlations between the biomarkers and biochemical indicators, which is of considerable significance in elucidating the YZOL mechanisms. Moreover, we identified some novel prospective biomarkers linked to primary dysmenorrhea, including bile acids. Collectively, these data provide new insights into the mechanism of YZOL and provide evidence for the analgesic effect of YZOL in the treatment of primary dysmenorrhea.

Identification of cryptotanshinone from Tongmai to inhibit thrombosis in zebrafish via regulating oxidative stress and coagulation cascade.

BACKGROUND: Thromboembolic events are leading causes of mortality and morbidity all over the world. Tongmai (TM) is a botanical drug with valid clinical efficacy and safety in the management of thrombosis and ischemic cardiovascular diseases, however, its active compounds and underlying mechanism are largely unclear. PURPOSE: To investigate the endogenous effects, therapeutic mechanism and active compounds of TM in thrombus formation. STUDY DESIGN: Combined with transgenic zebrafish models and high-content imaging system, this study evaluated the endogenous antithrombotic effects of TM and screened for the active compounds. METHODS: The PHZ-induced thrombotic model in erythrocytes or platelets labeled transgenic zebrafish were established, to dynamically evaluate the antithrombotic effects of TM. The oxidative damage levels were analyzed by specific fluorescent probes, and the expression levels of key factors in coagulation cascades and platelet activation were examined by QPCR. TM were dissected into fractions by reverse phase chromatography and subsequently screened for their antithrombotic effects in the transgenic fish models. The compounds of the active TM fraction were then analyzed by UPLC-Q-TOF analysis and further verified for their antithrombotic effects and mechanisms. RESULTS: In PHZ-induced zebrafish thrombotic model, TM incubation markedly increased cardiac blood flow, decreased peripheral erythrocytes aggregation, and recovered peripheral platelet circulation. Besides, the levels of oxidative stress and lipid peroxidation were increased in the PHZ-induced thrombotic fish, which were greatly decreased by TM treatment. Moreover, TM significantly reduced the expression of coagulation factor II (thrombin) and the downstream fibrinogen. In order to identify the active compounds of TM, four fractions were separated from the extract by reverse phase chromatography, which were subsequently screened for their antithrombotic effects in the fish model. As a result, fraction 4 showed the strongest effect in inhibiting thrombosis. Finally, through UPLC-Q-TOF analysis and endogenous screening, cryptotanshione was identified as the main active compound with antithrombotic effects. CONCLUSION: Our study demonstrated the endogenous antithrombotic effects of TM, which is possibly mediated by inhibiting oxidative stress and coagulation cascade. Cryptotanshione was identified as a major compound with antithrombotic activity and is a promising candidate for novel antithrombotic therapy.

Chemical composition and pharmacological mechanism of Qingfei Paidu Decoction and Ma Xing Shi Gan Decoction against Coronavirus Disease 2019 (COVID-19): In silico and experimental study.

The Coronavirus Disease 2019 (COVID-19) pandemic has become a huge threaten to global health, which raise urgent demand of developing efficient therapeutic strategy. The aim of the present study is to dissect the chemical composition and the pharmacological mechanism of Qingfei Paidu Decoction (QFPD), a clinically used Chinese medicine for treating COVID-19 patients in China. Through comprehensive analysis by liquid chromatography coupled with high resolution mass spectrometry (MS), a total of 129 compounds of QFPD were putatively identified. We also constructed molecular networking of mass spectrometry data to classify these compounds into 14 main clusters, in which exhibited specific patterns of flavonoids (45 %), glycosides (15 %), carboxylic acids (10 %), and saponins (5 %). The target network model of QFPD, established by predicting and collecting the targets of identified compounds, indicated a pivotal role of Ma Xing Shi Gan Decoction (MXSG) in the therapeutic efficacy of QFPD. Supportively, through transcriptomic analysis of gene expression after MXSG administration in rat model of LPS-induced pneumonia, the thrombin and Toll-like receptor (TLR) signaling pathway were suggested to be essential pathways for MXSG mediated anti-inflammatory effects. Besides, changes in content of major compounds in MXSG during decoction were found by the chemical analysis. We also validate that one major compound in MXSG, i.e. glycyrrhizic acid, inhibited TLR agonists induced IL-6 production in macrophage. In conclusion, the integration of in silico and experimental results indicated that the therapeutic effects of QFPD against COVID-19 may be attributed to the anti-inflammatory effects of MXSG, which supports the rationality of the compatibility of TCM.

High content screening identifies licoisoflavone A as a bioactive compound of Tongmaiyangxin Pills to restrain cardiomyocyte hypertrophy via activating Sirt3.

BACKGROUND: Cardiac hypertrophy is a prominent feature of heart remodeling, which may eventually lead to heart failure. Tongmaiyangxin (TMYX) pills are a clinically used botanical drug for treating multiple cardiovascular diseases including chronic heart failure. The aim of the current study was to identify the bioactive compounds in Tongmaiyangxin pills that attenuate cardiomyocytes hypertrophy, and to investigate the underlying mechanism of action. METHODS AND RESULTS: The anti-hypertrophy effect of TMYX was validated in isoproterenol-induced cardiac hypertrophy model in C57BL/6 mice. After TMYX treatment for 2 weeks, the heart ejection fraction and fractional shortening of the mice model was increased by approximately 20% and 15%, respectively, (p < 0.05). Besides, TMYX dose-dependently reduced the cross section area of cardiomyocytes in the angiotensin-II induced hypertrophy H9c2 model (p < 0.01). Combining high content screening and liquid chromatography mass spectrometry, four compounds with anti-cardiac hypertrophy effects were identified from TMYX, which includes emodin, licoisoflavone A, licoricone and glyasperin A. Licoisoflavone A is one of the compounds with most significant protective effect and we continued to investigate the mechanism. Primary cultures of neonatal rat cardiomyocytes were treated with a hypertrophic agonist phenylephrine (PE) in the presence or absence of licoisoflavone A. After 48 h of treatment, cells were harvested and mitochondrial acetylation was analyzed by western blotting and Image analysis. Interestingly, the results suggested that the anti-hypertrophic effects of licoisoflavone A depend on the activation of the deacetylase Sirt3 (p < 0.01). Finally, we showed that licoisoflavone A-treatment was able to decrease relative ANF and BNP levels in the hypertrophic cardiac cells (p < 0.01), but not in cells co-treated with Sirt3 inhibitors (3-TYP) (p > 0.05). CONCLUSION: TMYX exerts its anti-hypertrophy effect possibly through upregulating Sirt3 expression. Four compounds were identified from TMYX which may be responsible for the anti-hypertrophy effect. Among these compounds, licoisoflavone A was demonstrated to block the hypertrophic response of cardiomyocytes, which required its positive regulation on the expression of Sirt3. These results suggested that licoisoflavone A is a potential Sirt3 activator with therapeutic effect on cardiac hypertrophy.

MiR-30c-5p mediates the effects of panax notoginseng saponins in myocardial ischemia reperfusion injury by inhibiting oxidative stress-induced cell damage.

Myocardial ischemia reperfusion (MI/R) injury is a severe pathological process that threatens human health all over the world. The role of microRNAs (miRNAs) in the pathogenesis of MI/R injury has been increasingly recognized in recent years. Here, we conducted a miRNA profiling of the hearts of MI/R injured rat model, and identified 46 miRNAs which were differentially expressed between the MI/R injury and the control groups. With a special focus on one of the most significantly changed miRNA, miR-30c-5p, we demonstrated its protective role against cardiomyocyte injury in tBHP-treated H9c2 cells. Overexpression of miR-30c-5p increased cell viability, decreased LDH release, and reduced cell apoptosis of cardiomyocytes after tBHP stimulation, accompanied with downregulated p53 expression. Noticeably, the level of miR-30c-5p was markedly upregulated in MI/R injury cells treated with panax notoginseng saponins (PNS), a traditional Chinese Medicine with significant clinical effects in the treatment of human MI/R injury. Moreover, miR-30c-5p inhibitor is sufficient to block the protection of PNS, as well as its active ingredient ginsenoside Re, against tBHP induced cardiomyocyte injury. The expression of p53 protein was also reduced in PNS treated cells. In summary, our study identified novel miRNA hits of MI/R injury, revealed a pivotal role of miR-30c-5p in cardiomyocyte damage and apoptosis after MI/R, and illustrated a miR-30c-5p-dependent therapeutic mechanism of PNS of this pathologic process. Future studies are warranted to examine the endogenous significance of miR-30c-5p, along with multiple other miRNA hits, in the pathogenesis and treatment of MI/R injury.

Low-Frequency Electroacupuncture Alleviates Chronic Constrictive Injury-Induced Mechanical Allodynia by Inhibiting NR2B Upregulation in Ipsilateral Spinal Dorsal Horn in Rats.

OBJECTIVE: To study the effects of electroacupuncture (EA) in chronic constrictive injury (CCI) rat model and the expression of N-methyl-D-aspartate receptor type 2B (NR2B) in ipsilateral spinal dorsal horn in rats to explore the analgesic mechanisms of EA. METHODS: According to the random number table, totally 180 rats were evenly divided into a sham group, a CCI group, and an EA group. CCI model was conducted with four 4-0 chromic gut ligatures loosely ligated around the left sciatic nerve 1 cm above the trifurcation. Rats in the EA group received 2 Hz EA therapy bilaterally at acupoints of Zusanli (ST 36) and Sanyinjiao (SP 6) once daily (30 min/d) for 30 days after surgery. Paw withdrawal thresholds (PWTs) were measured on 0 (baseline), 1, 3, 7, 15, 30 days after surgery. Rats were sacrificed on 0, 1, 3, 7, 15 and 30 days after surgery, and the L4-5 segments of spinal cord were removed to detect the expression of NR2B by immunohistochemistry and quantitative polymerase chain reaction. RESULTS: PWTs in the CCI group were significantly lower than the sham group at Day 1-30 after surgery, and reached its lowest at Day 1 (P<0.01). After EA treatment, the PWTs recovered rapidly and were significantly higher than those in the CCI group on 3, 7, 15 and 30 days after surgery (P<0.01). The numbers of NR2B-immunoreactive cells of the CCI group significantly increased after CCI surgery compared with the sham group (P<0.01). Compared with the CCI group, stimulation of EA markedly decreased the numbers of NR2B-immunoreactive cells at Day 3, 7, 15 and 30 (P<0.05). In the sham group, NR2B mRNA was expressed at a low level. It increased after CCI surgery, which increased rapidly at Day 7 (P<0.01) and reached its peak value at Day 15 (P<0.01). After EA stimulation, relative quantity of NR2B mRNA expression was less than that in the CCI group at Day 15 and 30 (P<0.05). CONCLUSIONS: Low frequency of EA had antinociceptive effect in CCI rat model. The analgesic effects of EA might be through the inhibition of NR2B.

[Vasodilation effect and mechanism of extraction of Tongmai Yangxin Pills (TMYX) on isolated rat mesenteric artery].

The aim of the present study is to evaluate the vasodilation effects of Tongmai Yangxin Pills (TMYX) on rat mesenteric artery as well as its mechanism of action. The relaxation effects of TMYX extracts with different concentrations were determined on isolated rat mesenteric artery in normal condition as well as pretreating by phenylephrine and KCl. Vascular relaxation effects of TMTX were also determined in mesenteric artery preincubated with L-ANME and indomethacin or in endothelium denuded mesenteric artery. Moreover, effects of TMYX by 50 mg·L⁻¹ on NO secretion and the phosphorylation of eNOS in a cellular model of human umbilical vein endothelial cell (HUVEC) pretreated with or without L-NAME were also observed. The experimental results showed that TMYX has no obvious effect on vasodilation of arteries in normal or KCl pretreated condition, while it can dose-dependently relax the rat mesenteric artery with intact endothelium stimulated with phenylephrine at a maximal diastolic rate of (64.71±10.03)%. After preincubating with L-NAME for 15 min or removal of mesenteric artery endothelium, the maximal diastolic rate was decreased to (35.77±8.93)% and (25.85±10.84)% respectively. However, preincubating with indomethacin had no inhibitory effect on TMYX induced vascular relaxation. Meanwhile, TMYX at 50 mg·L⁻¹ could increase the expression of P-eNOS and the secretion of NO in HUVEC. L-NAME significantly inhibited NO release and phosphorylation of eNOS induced by TMYX. The results suggested TMYX exerted endothelium-dependent relaxation effects against PE-induced contractions of isolated rat mesenteric artery through NO-cGMP signaling pathway.