Metabolic characteristics of evodiamine were associated with its hepatotoxicity via PPAR/PI3K/AKT/NF-кB/tight junction pathway-mediated apoptosis in zebrafish.

Evodiae Fructus (EF), an herbal medicine, possesses remarkable anti-inflammatory and analgesic properties. It exhibits insecticidal activity as a potent insecticide candidate. However, the toxic characteristics of EF and the underlying mechanisms have not been comprehensively elucidated comprehensively. Thus, we comprehensively explored the toxic components of EF and established the relationship between the therapeutic and toxic effects of EF, encouraging its therapeutic use. We found that evodiamine (EVO), one of the main ingredients of EF, can truly reflect its analgesic properties. In phenotype observation trials, low doses of EVO (< 35 ng/mL) exhibited distinct analgesic activity without any adverse effects in zebrafish. However, EVO dose-dependently led to gross morphological abnormalities in the liver, followed by pericardial edema, and increased myocardial concentrations. Furthermore, the toxic effects of EVO decreased after processing in liver microsomes but increased after administering CYP450 inhibitors in zebrafish, highlighting the prominent effect of CYP450s in EVO-mediated hepatotoxicity. EVO significantly changed the expression of genes enriched in multiple pathways and biological processes, including lipid metabolism, inflammatory response, tight junction damage, and cell apoptosis. Importantly, the PPAR/PI3K/AKT/NF-кB/tight junction-mediated apoptosis pathway was confirmed as a critical functional signaling pathway inducing EVO-mediated hepatotoxicity. This study provided a typical example of the overall systematic evaluation of traditional Chinese medicine (TCM) and its active ingredients with significant therapeutic effects and simultaneous toxicities, especially metabolic toxicities.

Investigation of the applicability of the zebrafish model for the evaluation of aristolochic acid-related nephrotoxicity.

BACKGROUND: The risk of compounds/drugs, including aristolochic acid-induced nephrotoxicity remains high and is a significant public health concern. Therefore, it is particularly important to select reasonable animal models for rapid screening and evaluation of different samples with complex chemical systems. The zebrafish (Danio rerio) has been used to study chemical-induced renal toxicity. However, most of the published literature was performed on individual components or drugs, and the key evidence confirming the applicability of zebrafish larvae for the evaluation of aristolochic acid-related nephrotoxicity in complex chemical systems, such as in traditional Chinese medicine (TCM), was insufficient. METHODS: High-performance liquid chromatography (HPLC) was used to determine the content of aristolochic acid (AA) in herbs and Chinese patent medicines. The zebrafish larvae at 4 days post-fertilization (dpf) were used to evaluate the nephrotoxicity of various samples, respectively, based on the phenotype of the kidney and histological, and biochemical. Transcriptome technology was used to investigate the related signaling pathways and potential mechanisms after treatment with AA, which was verified by RT-PCR technology. RESULTS: The results showed that the total amounts of AAI, AAII, and ALI ranged from 0.0004 to 0.1858 g·g-1( %) from different samples, including Aristolochia debilis, Fibraurea recisa, Asarum, Wantongjingu tablets, Jiuweiqianghuo granules, and Xiaoqinglong granules in descending order. Moreover, compared with the negative/blank control, substantial changes in phenotype, histomorphology and biochemical parameters of renal function were observed in the groups challenged with the sublethal concentration of drugs. The transcriptomics results showed the upregulation of most genes in PERK/ATF4/CHOP, ATM/Chk2/p53, Caspase/Bax/Bcl-2a, TGF/Smad/ERK, PI3K/Akt, induced by aristolochic acid analogues, which were essentially consistent with those of the q-RT-PCR experiments, highlighting the similar toxicity response to the previously published article with the other traditional evaluation model. CONCLUSION: The stability, accuracy and feasibility of the zebrafish larval model in screening and evaluating the nephrotoxicity of TCM were validated for the first time on the AAs-related drugs in a unified manner, confirming and promoting the applicability of zebrafish in assessing nephrotoxicity of samples with complex chemical character.

[Hepatotoxicity and mechanism of Rhododendri Mollis Flos based on zebrafish model].

This study used the zebrafish model to explore the hepatotoxicity of Rhododendri Mollis Flos(RMF). The mortality was calculated according to the number of the survival of zebrafish larvae 4 days after fertilization under different concentration of RMF, and the dose-toxicity curve was fitted to preliminarily evaluate the toxicity of RMF. The liver phenotypes under the sublethal concentration of RMF in the treatment group and the blank control group were observed by hematoxylin-eosin(HE) staining and acridine orange(AO) staining. Meanwhile, the activities of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were determined to confirm the hepatotoxicity of RMF. Real-time quantitative polymerase chain reaction(real-time PCR) and Western blot were used to determine the expressions of genes and proteins in zebrafish larvae. Gas chromatography time-of-flight mass spectrometry(GC-TOF-MS) was used to conduct untargeted metabolomics testing to explore the mechanism. The results showed that the toxicity of RMF to zebrafish larvae was dose-dependent, with 1 100 µg·mL~(-1) of the absolute lethal concentration and 448 µg·mL~(-1) of sublethal concentration. The hepatocyte apoptosis and degeneration appeared in the zebrafish larvae under the sublethal concentration of RMF. The content of ALT and AST in zebrafish larvae at the end of the experiment was significantly increased in a dose-dependent manner. Under the sublethal concentration, the expressions of genes and proteins related to apoptosis in zebrafish larvae were significantly increased as compared with the blank control group. The results of untargeted metabolomics showed that the important metabolites related to the he-patotoxicity of RMF were mainly enriched in alanine, aspartic acid, glutamic acid, and other pathways. In conclusion, it is inferred that RMF has certain hepatotoxicity to zebrafish larvae, and its mechanism may be related to apoptosis.

The interaction between polyphyllin I and SQLE protein induces hepatotoxicity through SREBP-2/HMGCR/SQLE/LSS pathway.

Polyphyllin I (PPI) and polyphyllin II (PII) are the main active substances in the Paris polyphylla. However, liver toxicity of these compounds has impeded their clinical application and the potential hepatotoxicity mechanisms remain to be elucidated. In this work, we found that PPI and PII exposure could induce significant hepatotoxicity in human liver cell line L-02 and zebrafish in a dose-dependent manner. The results of the proteomic analysis in L-02 cells and transcriptome in zebrafish indicated that the hepatotoxicity of PPI and PII was associated with the cholesterol biosynthetic pathway disorders, which were alleviated by the cholesterol biosynthesis inhibitor lovastatin. Additionally, 3-hydroxy-3-methy-lglutaryl CoA reductase (HMGCR) and squalene epoxidase (SQLE), the two rate-limiting enzymes in the cholesterol synthesis, selected as the potential targets, were confirmed by the molecular docking, the overexpression, and knockdown of HMGCR or SQLE with siRNA. Finally, the pull-down and surface plasmon resonance technology revealed that PPI could directly bind with SQLE but not with HMGCR. Collectively, these data demonstrated that PPI-induced hepatotoxicity resulted from the direct binding with SQLE protein and impaired the sterol-regulatory element binding protein 2/HMGCR/SQLE/lanosterol synthase pathways, thus disturbing the cholesterol biosynthesis pathway. The findings of this research can contribute to a better understanding of the key role of SQLE as a potential target in drug-induced hepatotoxicity and provide a therapeutic strategy for the prevention of drug toxic effects with similar structures in the future.

Berberine inhibits RA-FLS cell proliferation and adhesion by regulating RAS/MAPK/FOXO/HIF-1 signal pathway in the treatment of rheumatoid arthritis.

AIMS: Rheumatoid arthritis (RA) is a common chronic immune disease. Berberine, as its main active ingredient, was also contained in a variety of medicinal plants such as Berberaceae, Buttercup, and Rutaceae, which are widely used in digestive system diseases in traditional Chinese medicine with anti-inflammatory and antibacterial effects. The aims of this article were to explore the therapeutic effect and mechanism of berberine on rheumatoid arthritis. METHODS: Cell Counting Kit-8 was used to evaluate the effect of berberine on the proliferation of RA fibroblast-like synoviocyte (RA-FLS) cells. The effect of berberine on matrix metalloproteinase (MMP)-1, MMP-3, receptor activator of nuclear factor kappa-Β ligand (RANKL), tumour necrosis factor alpha (TNF-α), and other factors was determined by enzyme-linked immunoassay (ELISA) kit. Transcriptome technology was used to screen related pathways and the potential targets after berberine treatment, which were verified by reverse transcription-polymerase chain reaction (RT-qPCR) and Western blot (WB) technology. RESULTS: Berberine inhibited proliferation and adhesion of RA-FLS cells, and significantly reduced the expression of MMP-1, MMP-3, RANKL, and TNF-α. Transcriptional results suggested that berberine intervention mainly regulated forkhead box O (FOXO) signal pathway, prolactin signal pathway, neurotrophic factor signal pathway, and hypoxia-inducible factor 1 (HIF-1) signal pathway. CONCLUSION: The effect of berberine on RA was related to the regulation of RAS/mitogen-activated protein kinase/FOXO/HIF-1 signal pathway in RA-FLS cells.Cite this article: Bone Joint Res 2023;12(2):91-102.

Pharmacokinetic/Pharmacodynamic Study of Salt-Processed product of Cuscutae Semen with Hepatoprotective Effects.

BACKGROUND: Salt-processed product of cuscutae semen (SCS), which is documented in Chinese pharmacopoeia (2020 edition), is one of the processed products of cuscustae semen. SCS possesses hepatoprotective effects. However, Pharmacokinetic / Pharmacodynamic (PK-PD) study of SCS with intervening acute liver injury (ALI) has not been reported yet. Effective constituents are still not well addressed. OBJECTIVE: This study was performed to study PK-PD properties with the purpose of linking SCS hepatoprotective effects to key therapeutic outlines to guide therapeutic use in clinical settings. METHODS: Rats were orally administered SCS after the acute liver injury model was established. Plasma biochemical analysis, antioxidative analysis, and liver histopathology were measured to evaluate the hepatoprotective effects of SCS. Blood samples were collected at different time points (0 h, 0.083 h, 0.25 h, 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 8 h, 12 h, 24 h) for PK/PD study after SCS administration. Contents of chlorogenic acid, hyperoside and astragalin were estimated by UHPLC-ESI-MS. The relationship between concentrations of chlorogenic acid, hyperoside, and astragalin and hepatoprotective effects was assessed by PK-PD modeling. RESULTS: The results showed that SCS ameliorated liver repair and decreased the serum levels of alanine aminotransferase (ALT), aspartate transaminase (AST) markedly. Hepatic oxidative stress was inhibited by SCS, as evidenced by a decrease in malondialdehyde (MDA) and an increase in glutathione (GSH) and superoxide dismutase (SOD) in the liver. PK-PD correlation analysis indicated that concentrations of chlorogenic acid, hyperoside, and astragalin were negatively correlated with level of AST and ALT. CONCLUSION: The encouraging finding indicates that SCS has beneficial effects on CCl4-induced liver damage. Chlorogenic acid, hyperoside, and astragalin are three effective constituents to exert hepatoprotective effects while astragalin may have maximum pharmacological activity. PK-PD study reveals the positive relationship between drug concentration and action intensity of SCS against liver injury. This research provides a robust foundation for future studies.

Transcriptomic profiling revealed the role of apigenin-4'-O-α-L-rhamnoside in inhibiting the activation of rheumatoid arthritis fibroblast-like synoviocytes via MAPK signaling pathway.

BACKGROUND: Activated fibroblast-like synoviocyte (FLS) played a significant role in the pathogenesis and progression of rheumatoid arthritis (RA). Apigenin-4'-O-α-L-rhamnoside showed remarkable effects against RA, however, no relevant studies on pharmacology of apigenin-4'-O-α-L-rhamnoside yet, the effects and underlying molecular mechanism of apigenin-4'-O-α-L-rhamnoside on RA are still unclear. PURPOSE: This study aimed to investigate the therapeutic effects and mechanisms of apigenin-4'-O-α-L-rhamnoside on RA-FLS cells by transcriptomic analysis. METHODS: In vitro, RA-FLS cell viability and migration were measured by CCK-8 and scratch assays, respectively. The effects of apigenin-4'-O-α-L-rhamnoside on inflammatory levels of MMP-1, MMP-3, RANKL and TNF-α in RA-FLS cells were detected using ELISA kits. High-throughput transcriptome analysis was performed to screen the key genes and related pathways of apigenin-4'-O-α-L-rhamnoside inhibit RA-FLSs, and the result of which were validated by RT-qPCR and western blot. Furthermore, in vivo, we also evaluated the effects of apigenin-4'-O-α-L-rhamnoside in rat with CIA. RESULTS: Apigenin-4'-O-α-L-rhamnoside significantly suppressed RA-FLS migration, exerted remarkable inhibiting effects on the expression levels on MMP-1, MMP3, RANKL and TNF-α in RA-FLS cells. It seemed that MAPK signaling pathway might be closely related to the pathogenesis of RA by down-regulated relevant core targets (MAPK1, HRAS, ATF-2, p38 and JNK). Moreover, apigenin-4'-O-α-L-rhamnoside attenuated the severity of arthritis in CIA rat. CONCLUSION: Apigenin-4'-O-α-L-rhamnoside inhibited pro-inflammatory cytokine, chemokine and MMPs factors production of RA-FLS by targeting the MAPK signaling pathway, which provided a scientific basis for potential application in the treatment of RA.

[Anti-tumor and analgesic activity evaluation and mechanism of Compound Kushen Injection].

This study aims to evaluate the anti-tumor and analgesic activities of Compound Kushen Injection(CKI) based on zebrafish model in vivo and investigate the anti-tumor mechanism. To be specific, zebrafish tumor xenotransplantation model was established by microinjection of murine LPC H12 cells into yolk sac. Then the high-dose CKI(H-CKI), medium-dose CKI(M-CKI), low-dose CKI(L-CKI) groups, and the model group were set. The anti-tumor activity of CKI was evaluated with the tumor area growth fold and integral absorbance(IA) growth fold 72 h after administration. The peripheral pain and central pain in zebrafish were respectively induced with acetic acid(AA) and phorbol myristate acetate(PMA). Zebralab ViewPoint system was employed to monitor behavioral trajectory of zebrafish, and movement times, movement time, movement distance, and movement velocity were used to evaluate the analgesic activity of CKI. Finally, real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) was performed to detect the expression levels of apoptosis-related B lymphocyte tumor-2(Bcl-2) and phosphatidylinositol-3-kinase(PI3 K)/protein kinase B(Akt or PKB) pathway-related genes, for the verification of the anti-tumor mechanism. Compared with the model group, M-CKI and H-CKI significantly reduced the growth folds of tumor area and IA, relief the peripheral pain and central pain. The mechanism was that CKI can up-regulate the expression of cysteine aspartic acid specific protease-3(caspase-3, Casp3) and caspase-9(Casp9), down-regulate the expression of phosphoinositide 3-kinase(PI3 K) and Akt, and significantly reduce the expression of Bcl-2, hypoxia-inducible factor-1α(HIF-1α), and vascular endothelial growth factor(VEGF). In conclusion, CKI has significant inhibitory effect on tumor growth and pain, which is related to the PI3 K/Akt signaling pathway. The pathway mediates cell apoptosis, suppresses tumor growth, and alleviates tumor pain.

Lipidomics Indicates the Hepatotoxicity Effects of EtOAc Extract of Rhizoma Paridis.

Rhizoma Paridis is a traditional Chinese medicine commonly used in the clinical treatment of gynecological diseases. Previous studies have shown that aqueous extracts of Rhizoma Paridis exhibit some hepatotoxicity to hepatocytes. Here, using lipidomics analysis, we investigated the potential hepatotoxicity of Rhizoma Paridis and its possible mechanism. The hepatic damaging of different solvent extracts of Rhizoma Paridis on zebrafish larvae were determined by a combination of mortality dose, biochemical, morphological, and functional tests. We found that ethyl acetate extracts (AcOEtE) were the most toxic fraction. Notably, lipidomic responsible for the pharmacological effects of AcOEtE were investigated by Q-Exactive HF-X mass spectrometer (Thermo Scientific high-resolution) coupled in tandem with a UHPLC system. Approximately 1958 unique spectral features were detected, of which 325 were identified as unique lipid species. Among these lipid species, phosphatidylethanolamine cardiolipin Ceramide (Cer), lysophosphatidylinositol sphingosine (Sph), etc., were significantly upregulated in the treated group. Pathway analysis indicates that Rhizoma Paridis may cause liver damage via interfering with the glycerophospholipid metabolism. Collectively, this study has revealed previously uncharacterized lipid metabolic disorder involving lipid synthesis, metabolism, and transport that functionally determines hepatic fibrosis procession.

Screening and identification of Euphorbiae pekinensis Rupr. anti-angiogenic multi-components with UPLC-QTOF-MS in zebrafish.

Euphorbia pekinensis Rupr. (EP) (Euphorbiaceae), as Traditional Chinese Medicine (TCM), exhibits therapeutic effects on tumors in clinical practice. Anti-angiogenesis may be an underlying molecular mechanism of EP's actions. However, the anti-angiogenic active ingredients of EP remain unclear. The screening and analysis of anti-angiogenic agents were essential for the sufficient utilization and development of EP. Thus, we established a UPLC-QTOF-MS method based on a transgenic zebrafish model to screen anti-angiogenesis activity components in EP. UPLC-QTOF-MS was used to characterize compounds from EP and in vivo compounds in Tg (flk1: mCherry) zebrafish larvae treated with EP. Based on the identification results, five components were selected, and their anti-angiogenesis activity were investigated via assessment of intersegmental blood vessels during the development of the transgenic zebrafish. Three of these components (3,3'-O-dimethoxy ellagic acid, quercetin, and ingenol) are active components of EP with anti-angiogenic effects. Among them, 3, 3'-O-dimethoxy ellagic acid and ingenol were first demonstrated with anti-angiogenesis effects. UPLC-PDA analysis was performed on EP water extracts to determine anti-angiogenesis active ingredients quantitatively. In the concentration range of 100-200 µg/mL, EP and the active ingredient compositions, mixed according to the content of EP, had equivalent anti-angiogenesis activities. These experimental results indicate that the UPLC-QTOF-MS method, combined with a transgenic zebrafish model, is rapid, sensitive and reliable. The combination in TCM offers the potential to achieve certain effect levels with lower concentrations of the individual compound.

Hepatotoxicity induced by PPâ ¥ and PPâ ¦ in zebrafish were related to the Cholesterol disorder.

BACKGROUND: PPⅥ2 and PPⅦ3 were a group of Pennogenin compounds extracted from the Paris polyphylla and caused hepatotoxicity in human, while the potential underlying mechanism was unclear. PURPOSE: To evaluated the adverse effects of PPⅥ and PPⅦ on the liver in the zebrafish. METHOD: In this study, 4dpf zebrafish were used for acute toxicity test, LC0 was calculated, and 1/2LC0 and 3/5LC0 were selected for pathological section and liver area measurement to verify the hepatotoxicity of PPⅥ and PPⅦ. Etabonomics study was then conducted to further explore the mechanism of hepatotoxicity of PPⅥ and PPⅦ. Lovastatin was used as an inhibitor, and PCR was used to verify the results. RESULT: The result showed that under the condition of sub-lethal concentration exposure, hepatotoxicity-included changes in liver phenotype (liver area), hepatocyte swelling and degeneration, liver cell apoptosis and disturbed biochemical index were observed in zebrafish treated with PPⅥ and PPⅦ. Furthermore, the transcriptome was conducted to confirm the toxicity mechanism shared with PPⅥ and PPⅦ, and we found that steroid biosynthesis process and the related target genes were mainly affected. While, lovastatin treatment effectively ameliorated PPⅦ-induced zebrafish liver injury by improving the liver tissue structure and regulate the expression of associated genes including HMGCRA, SREBP, LSS, CYP2R1, PIK3R3A, GDPD1 and PFKFB-2. CONCLUSION: This study was the first investigation to provide the direct evidence of hepatotoxicity of PPⅥ and PPⅦ in vivo zebrafish model, which were related to the steroid biosynthesis. furthermore, in lovastatin played an important role in protection against hepatotoxicity induced by PPVI and PPⅦ by regulating the cholesterol metabolism.

Combined Metabolomics and Network Toxicology to Explore the Molecular Mechanism of Phytolacca acinose Roxb-Induced Hepatotoxicity in Zebrafish Larvae in Vivo.

Phytolacca acinosa Roxb (PAR), a traditional Chinese medicine, has been widely used as a diuretic drug for a long period of time for the treatment edema, swelling, and sores. However, it has been reported that PAR might induce hepatotoxicity, while the mechanisms of its toxic effect are still unclear. In this study, network toxicology and metabolomic technique were applied to explore PAR-induced hepatotoxicity on zebrafish larvae. We evaluated the effect of PAR on the ultrastructure and the function of the liver, predictive targets, and pathways in network toxicology, apoptosis of liver cells by PCR and western blot, and metabolic profile by GC-MS. PAR causes liver injury, abnormal liver function, and apoptosis in zebrafish. The level of arachidonic acid in endogenous metabolites treated with PAR was significantly increased, leading to oxidative stress in vivo. Excessive ROS further activated the p53 signal pathway and caspase family, which were obtained from KEGG enrichment analysis of network toxicology. The gene levels of caspase-3, caspase-8, and caspase-9 were significantly increased by RT-PCR, and the level of Caps3 protein was also significantly up-regulated through western blot. PAR exposure results in the liver function abnormal amino acid metabolism disturbance and motivates hepatocyte apoptosis, furthermore leading to liver injury.

[Identification of geographical origins of Cordyceps based on data of amino acids with self-organizing map neural network].

In this study, data of amino acids of Cordyceps samples from Qinghai and Tibet was analyzed with self-organizing map neural network. A model of XY-Fused network was established with the content of 8 major amino acids and total amino acids for the identification of geographical origins of Cordyceps from Qinghai and Tibet. It had the prediction accuracy of 83.3% for the test set. In addition, data mining indicated that methionine was a special kind of amino acid in Cordyceps which could serve as a marker to identify its geographical origins. On this basis, the content ratio of methionine to total amino acids was proposed to be a quantifiable indicator to distinguish Cordyceps from Qinghai and Tibet.

[Overview of metalomics research and its application prospect in mineral Chinese medicine research].

Metallomics is a frontier interdisciplinary subject at its vigorous development stage. Its goal is to systematically study the content, distribution, chemical species, structural characteristics and functions of metal elements in biological system. It is also a comprehensive discipline to study the existing state and function of free or complex metal elements in life. Metallomics is an ideal tool to study the biological behavior of inorganic elements, which can be used to solve many problems in the research of mineral Chinese medicine(MCM). It provides a strong theoretical basis and technical support for the research of MCM. Its theory and methods provide re-ference and enlightenment for the in-depth study of MCM, and also provide new ideas and open up new ways for the research of MCM. The application of metallomics theory and methods in the research of MCM is of great significance to reveal the material basis and mec-hanism of MCM, promote the process of basic research on MCM, fully exploit and utilize medicinal mineral resources and carry forward the traditional MCM treasure in China. In this paper, we introduced the concept, academic development, research content and research methods of metallomics, and discussed the application prospects of metallomics in the analysis of inorganic element composition characteristics and quality control, material basis and mechanism of MCM, so as to provide reference for further researches on MCM.

[Study on liver diseases with zebrafish as an important tool].

With the increasing incidence of hepatobiliary diseases, it is particularly important to understand the role of molecular, cellular and physiological factors in the clinical diagnosis and treatment with traditional Chinese medicine(TCM) in the development of liver disease. Appropriate animal models can help us identify the possible mechanisms of relevant diseases. Danio rerio(zebrafish) model was traditionally used to study embryonic development, and has been gradually used in screening and evaluation of liver diseases and relevant drug in recent years. Zebrafish embryos develop rapidly and the digestive organs of 5-day-old juvenile fish are all mature. At this stage, they may develop hepatobiliary diseases induced by developmental defects or compounds. Zebrafish liver is similar to human liver in cell composition, function, signal transduction, response to injury and cell process mediating liver disease. Furthermore, due to the high conservation of genes and proteins between humans and zebrafish, zebrafish becomes an alternative system for studying basic mechanisms of liver disease. Therefore, genetic screening could be performed to identify new genes involving specific disease processes, and chemical screening could be made for drugs in specific processes. This paper briefly introduced the experimental properties of zebrafish as model system, emphasized the study progress of zebrafish models for pathological mechanism of liver diseases, especially fatty liver, and drug screening and evaluation, so as to provide ideas and techniques for the future liver toxicity assessment of TCM.

Effect of cultivation ages on anti-inflammatory activity of a new type of red ginseng.

Ginseng has been widely applied in clinical practice, but the cultivation age cannot be ignored as it influences the quality of ginseng and its products. In this work, different cultivation ages of fresh ginseng (FG) from four to seven years were analysed by UPLC-Q-TOF-MS/MS. Principal component analysis and supervised orthogonal partial least squared discrimination analysis, which belong to the normal method of multivariate statistical analysis, were applied to discover the characteristic components of FG at different cultivation ages. The components of new type of red ginseng (NRG) derived from FG at different cultivation ages were compared by HPLC analysis. The pharmacological anti-inflammatory activity was evaluated by ELISA and qPCR. The result showed that the characteristic components of both 6- and 7-year-old ginseng were ginsenoside Rb1, mal-ginsenoside Rb1, ginsenoside Rc, mal-ginsenoside Rc, mal-ginsenoside Rb1 isomer, and mal-ginsenoside Rb2. Moreover, the characteristic components of both 4- and 5-year-old ginseng were ADP-glucose and 3-hydroxyhexanoyl CoA. In addition, 6-year-old NRG has higher rare ginsenosides than 4-year-old NRG, which possesses great anti-inflammatory activity in vitro. The results reveal the ginsenoside transformation law of NRG processing and suggest that the cultivation age of FG influences the content of ginsenosides in NRG. Therefore, 6-year-old ginseng is more suitable for red ginseng processing and clinical use.

Integrated strategy of LC-MS and network pharmacology for predicting active constituents and pharmacological mechanisms of Ranunculus japonicus Thunb. for treating rheumatoid arthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Ranunculus japonicus Thunb. (short for R. japonicus) is a topically applied herb with the activities of removing jaundice, nebula and edema, preventing malaria, stopping asthma, promoting diuresis and relieving pain. It was firstly recorded in Zhouhou Beiji Fang and has been used for the treatment of malaria, ulcers, carbuncle, jaundice, migraine, stomachache, toothache and arthritis for over 1800 years. AIM OF THE STUDY: This study aimed to uncover the potentially effective components of R. japonicus and the pharmacological mechanisms against rheumatoid arthritis (RA) by combing LC-MS and network pharmacology. MATERIALS AND METHODS: Firstly, the chemical constituents of R. japonicus were qualitatively identified by UPLC-ESI-LTQ-Orbitrap MS. Then we performed target prediction by PharmMapper, protein-protein interaction (PPI) analysis via String, GO and KEGG pathway enrichment analysis by DAVID and constructed the compound-target-pathway network using Cytoscape. Thirdly, crucial compounds in the network were quantitatively analyzed to achieve quality control of R. japonicus. Finally, the pharmacological activities of R. japonicus and two potentially bioactive ingredients were validated in RA-FLSs (Rheumatoid Arthritis Fibroblast-like Synoviocytes) in vitro. RESULTS: Overall fifty-four ingredients of R. japonicus were identified and forty-five components were firstly discovered in R. japonicus. Among them, twenty-seven validated compounds were predicted to act on twenty-five RA-related targets and they might exhibit therapeutic effects against RA via positive regulation of cell migration, etc. Nine potentially bioactive components of R. japonicus which played important roles in the compound-target-pathway network were simultaneously quantified by an optimized UPLC-ESI-Triple Quad method. In vitro, compared to control group, R. japonicus extract, berberine and yangonin significantly inhibited the migration capacity of RA-FLSs after 24 h treatment. CONCLUSION: This study clarified that R. japonicus and the bioactive ingredients berberine and yangonin might exert therapeutic actions for RA via suppressing the aggressive phenotypes of RA-FLSs through combined LC-MS technology and network pharmacology tools for the first time. The present research provided deeper understanding into the chemical profiling, pharmacological activities and quality control of R. japonicus and offered reference for further scientific research and clinical use of R. japonicus in treating RA.

Integrated UPLC-MS and Network Pharmacology Approach to Explore the Active Components and the Potential Mechanism of Yiqi Huoxue Decoction for Treating Nephrotic Syndrome.

Background: Yiqi Huoxue Decoction (YQHXD) is a traditional Chinese medicine that promotes blood circulation, removes blood stasis, facilitates diuresis, and alleviates edema. It is composed of 10 herbal medicines and has extensive application in treating nephrotic syndrome (NS). However, the active components and the potential mechanism of YQHXD for treating NS remain unclear. Methods: We set up a sensitive and rapid method based on Ultra-High Performance Liquid Chromatograph-Mass (UPLC-MS) to identify the compounds in YQHXD and constituents absorbed into the blood. Disease genes were collected through GeneCards, DisGeNET, and OMIM database. Genes of compounds absorbed into blood were predicted by the TCMSP database. We constructed Disease-Drug-Ingredient-Gene (DDIG) network using Cytoscape, established a Protein-protein interaction (PPI) network using String, Gene biological process (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed using DAVID. Cellular experiments were performed to validate the results of network pharmacology. Result: A total of 233 compounds in YQHXD and 50 constituents absorbed into the blood of rats were identified. The 36 core targets in the PPI network were clustered in the phosphatidylinositol 3 kinase-RAC serine/threonine-protein kinase (PI3K-AKT) and nuclear factor kappa-B (NF-κB) signaling pathways. Luteolin, Wogonin, Formononetin, and Calycosin were top-ranking components as potentially active compounds. Conclusion: The results of our studies show that YQHXD is able to enhance renal function, alleviate podocyte injury, and improve adriamycin nephrotic syndrome.

Integrative analysis of proteomic and metabonomics data for identification of pathways related to Rhizoma Paridis-induced hepatotoxicity.

Clinical reports on hepatotoxicity that arise from Rhizoma Paridis have recently received widespread attention. Because the hepatotoxicity mechanism is little understood, this research strived to investigate the hepatotoxicity mechanism of Rhizoma Paridis extracts based on iTRAQ quantitative proteomics and metabonomics. The extraction solutions were administrated to rats for 7 days by gavage, and the hepatotoxicity was assessed through quantification of biochemical indexes and Oil red O staining. Additionally, the mechanism of hepatotoxicity was investigated by metabonomics based upon GC-MS and iTRAQ quantitative proteomics. The biochemical and histopathological analysis stood out that Rhizoma Paridis extract could induce liver injury, which was proved by the formation of fat droplets, the changes of mitochondrial structure, and biochemical parameters. The iTRAQ proteomics and metabonomics revealed that Rhizoma Paridis-induced hepatotoxicity was chiefly connected with the abnormal activity of mitochondrion function, which brought about oxidative stress injuries and inflammation, finally causing cell apoptosis. Collectively, we have provided previously uncharacterized hepatotoxic mechanism induced by Rhizoma Paridis and a reference to ensure its safe use in the future.