Interleukin-17 alleviates erastin-induced alveolar bone loss by suppressing ferroptosis via interaction between NRF2 and p-STAT3.

AIM: To investigate the relationship between interleukin-17 (IL-17), ferroptosis and osteogenic differentiation. MATERIALS AND METHODS: We first analysed the changes in ferroptosis-related molecules in experimental periodontitis models. The effects of erastin, a small-molecule ferroptosis inducer, and IL-17 on alveolar bone loss and repair in animal models were then investigated. Primary mouse mandibular osteoblasts were exposed to erastin and IL-17 in vitro. Ferroptosis- and osteogenesis-related genes and proteins were detected. Further, siRNA, immunofluorescence co-localization and immunoprecipitation were used to confirm the roles of the nuclear factor erythroid-2-related factor 2 (NRF2) and phosphorylated signal transducer and activator of transcription 3 (p-STAT3), as well as their interaction. RESULTS: The levels of NRF2, glutathione peroxidase 4 and solute carrier family 7 member 11 were lower in the ligated tissues than in normal periodontal tissues. Alveolar bone loss in an in vivo experimental periodontitis model was aggravated by erastin and alleviated by IL-17. In vitro, IL-17 ameliorated erastin-inhibited osteogenic differentiation by reversing ferroptosis. Altered NRF2 expression correlated with changes in ferroptosis-related molecules and osteogenesis. Furthermore, the physical interaction between NRF2 and p-STAT3 was confirmed in the nucleus. In IL-17 + erastin-stimulated osteoblasts, the p-STAT3-NRF2 complex might actively participate in the downstream transcription of ferroptosis- and osteogenesis-related genes. CONCLUSIONS: IL-17 administration conferred resistance to erastin-induced osteoblast ferroptosis and osteogenesis. The possible mechanism may involve p-STAT3 directly interacting with NRF2.

Polysaccharide-Based Injectable Hydrogels with Fast Gelation and Self-Strengthening Mechanical Kinetics for Oral Tissue Regeneration.

Oral defects lead to a series of function disorders, severely threatening the patients' health. Although injectable hydrogels are widely studied in tissue regeneration, their mechanical performance is usually stationary after implant, without further self-adaption toward the microenvironment. Herein, an injectable hydrogel with programmed mechanical kinetics of instant gelation and gradual self-strengthening along with outstanding biodegradation ability is developed. The fast gelation is realized through rapid Schiff base reaction between biodegradable chitosan and aldehyde-modified sodium hyaluronate, while self-strengthening is achieved via slow reaction between redundant amino groups on chitosan and epoxy-modified hydroxyapatite. The resultant hydrogel also possesses multiple functions including (1) bio-adhesion, (2) self-healing, (3) bactericidal, (4) hemostasis, and (5) X-ray in situ imaging, which can be effectively used for oral jaw repair. We believe that the strategy illustrated here will provide new insights into dynamic mechanical regulation of injectable hydrogels and promote their application in tissue regeneration.

Systematic analysis of chemical profiles of Sophorae tonkinensis Radix et Rhizoma in vitro and in vivo using UPLC-Q-TOF-MSE.

Sophorae tonkinensis Radix et Rhizoma (S. tonkinensis) has been recorded as a 'poisonous' Chinese herbal medicine in Chinese Pharmacopoeia 2020. The clinical reaction reports of S. tonkinensis indicated its neurotoxicity; however, there still exists dispute about its toxic substances. At present, no report is available on the blood and brain prototype research of S. tonkinensis. Most studies focused on alkaloids and less on other compounds. Moreover, the constituents absorbed into the blood and brain have been rarely investigated so far. This study established a rapid and efficient qualitative analysis method using UPLC-Q-TOF-MSE to characterize the ingredients of S. tonkinensis and those entering into the rat's body after oral administration. A total of 91 compounds were identified in S. tonkinensis, of which 28 were confirmed by the standards. In addition, 30 and 19 prototypes were also first identified in the rat's blood and brain, respectively. It was found that most flavonoids, except alkaloids, were detected in the rat's body and distributed in the cerebrospinal fluid, suggesting that flavonoids may be one of the important toxic or effective substances of S. tonkinensis. This finding provides new clues and data for clarifying the toxicity or efficacy of this medicinal plant.

[Metabolites and metabolic pathways of maackiain in rats based on UPLC-Q-TOF-MS].

Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was used to investigate the metabolites of maackiain in rats based on the prediction function of UNIFI data processing system and liver microsomal incubation in vitro. Ten metabolites of maackiain after oral absorption were reasonably deduced and characterized. It was found that the biotransformation of maackiain mainly included phase Ⅰ oxidation, dehydrogenation, phase Ⅱ sulfate conjugation, glucosylation conjugation, and glucuronic acid conjugation. Among them, the product of glucosylation conjugation, trifolirhizin, was identified by comparison with the reference for the first time. Liver microsomal incubation in vitro further confirmed the metabolites and metabolic pathways of maackiain in rats. The metabolites in the blood, urine, and feces complemented each other, which revealed the migration, metabolism, and excretion modes of maackiain in rats. This study lays a foundation for the further investigation of the metabolic mechanism of maackiain in vivo and the in-depth research on the mechanism of pharmacodynamics and toxicity.

Diabetes aggravates myocardial ischaemia reperfusion injury via activating Nox2-related programmed cell death in an AMPK-dependent manner.

Cardiovascular diseases such as myocardial ischaemia have a high fatality rate in patients with diabetes. This study was designed to expose the crosstalk between oxidative stress and AMPK, a vital molecule that controls biological energy metabolism, in myocardial ischaemia reperfusion injury (I/RI) in diabetic rats. Diabetes was stimulated in rats using streptozotocin injection. Rats were separated on random into control, control + I/R, Diabetes, Diabetes + I/R, Diabetes + I/R + N-acetylcysteine and Diabetes + I/R + Vas2870 groups. Myocardial infarct size was determined, and the predominant Nox family isoforms were analysed. In vitro, the H9C2 cells were administered excess glucose and exposed to hypoxia/reoxygenation to mimic diabetes and I/R. The AMPK siRNA or AICAR was used to inhibit or activate AMPK expression in H9C2 cells, respectively. Then, myocardial oxidative stress and programmed cell death were measured. Diabetes or high glucose levels were found to aggravate myocardial I/RI or hypoxia/reoxygenation in H9C2 cells, as demonstrated by an increase in myocardial infarct size or lactate dehydrogenase levels, oxidative stress generation and induction of programmed cell death. In diabetic rat hearts, cardiac Nox1, Nox2 and Nox4 were all heightened. The suppression of Nox2 expression using Vas2870 or Nox2-siRNA treatment in vivo or in vitro, respectively, protected diabetic rats from myocardial I/RI. AMPK gene knockout increased Nox2 protein expression while AMPK agonist decreased Nox2 expression. Therefore, diabetes aggravates myocardial I/RI by generating of Nox2-associated oxidative stress in an AMPK-dependent manner, which led to the induction of programmed cell death such as apoptosis, pyroptosis and ferroptosis.

Dexmedetomidine alleviates H2O2-induced oxidative stress and cell necroptosis through activating of α2-adrenoceptor in H9C2 cells.

Oxidative stress induced necroptosis is important in myocardial ischemia/reperfusion injury. Dexmedetomidine (Dex), an α2-adrenoceptor (α2-AR) agonist, has protective effect on oxidative stress induced cell apoptosis, but effects of Dex and Dex-mediated α2-AR activation on oxidant induced necroptosis was unclear. H9C2 cardiomyocytes were pre-treated with or without Dex and α2-AR antagonist yohimbine hydrochloride (YOH) before being exposed to H2O2 to induce oxidative cellular damage. Cell viability and lactate dehydrogenase (LDH) were detected by ELISA kits, protein expressions of Heme Oxygenase 1(HO-1), receptor interacting protein kinase 1 (RIPK1) and receptor interacting protein kinase 3 (RIPK3) were observed by WB, and TUNEL was used to detected cell apoptosis. H2O2 significantly decreased cell viability and increased LDH release and necroptotic and apoptotic cell deaths (all p < 0.05, H2O2 vs. Control). Dex preconditioning alleviated these injuries induced by H2O2. Dex preconditioning significantly increased expression of protein HO-1 and decreased expressions of proteins RIPK1 and RIPK3 induced by H2O2, while all these protective effects of Dex were reversed by YOH (all p < 0.05, Dex + H2O2 vs. H2O2; and YOH + Dex + H2O2 vs. Dex + H2O2). However, YOH did not prevent this protective effect of Dex against H2O2 induced apoptosis (YOH + Dex + H2O2 vs. Dex + H2O2, p > 0.05). These findings indicated that Dex attenuates H2O2 induced cardiomyocyte necroptotic and apoptotic cell death respectively dependently and independently of α2-AR activation.

Chemical profiling of Honghua Xiaoyao tablet and simultaneous determination of its quality markers by liquid chromatography-tandem mass spectrometry combined with chemometrics methods.

Discovering marker components of traditional Chinese medicine formulas is challenging because of the hundreds of components they inherently contain. This study first proposed a reliable and validated method for the comprehensive profiling of chemical constituents in Honghua Xiaoyao tablet by using high-performance liquid chromatography coupled with mass spectrometry. After searching within the in-house library, a total of 55 constituents were unambiguously characterized or tentatively identified through reference standards and by comparing mass spectrometry data with literature values. Quantitative analysis of 14 compounds, which were selected as the quality marker components based on a serum pharmacochemistry study, has been performed by triple-quardrupole mass spectrometry technique. Multiple chemometric methods, including principal components analysis and hierarchical cluster analysis, were subsequently used to analyze the quantitative results, classify samples from three manufacturers, and distinguish the analytical markers. In method validation results, 14 quality marker compounds have shown good linearity (R2 ≥ 0.9965) with a relative wide concentration range and acceptable recovery at 98.39-102.46%. The proposed approach provides the chemical evidence for revealing the material basis of Honghua Xiaoyao tablet, and establishes a reliable statistical analysis-based strategy of quality marker investigation for controlling its quality.

S100a4 upregulation in Pik3caH1047R;Trp53R270H;MMTV-Cre-driven mammary tumors promotes metastasis.

BACKGROUND: PIK3CA mutations are frequent in human breast cancer. Pik3caH1047R mutant expression in mouse mammary gland promotes tumorigenesis. TP53 mutations co-occur with PIK3CA mutations in human breast cancers. We previously generated a conditionally activatable Pik3caH1047R;MMTV-Cre mouse model and found a few malignant sarcomatoid (spindle cell) carcinomas that had acquired spontaneous dominant-negative Trp53 mutations. METHODS: A Pik3caH1047R;Trp53R270H;MMTV-Cre double mutant mouse breast cancer model was generated. Tumors were characterized by histology, marker analysis, transcriptional profiling, single-cell RNA-seq, and bioinformatics. Cell lines were developed from mutant tumors and used to identify and confirm genes involved in metastasis. RESULTS: We found Pik3caH1047R and Trp53R270H cooperate in driving oncogenesis in mammary glands leading to a shorter latency than either alone. Double mutant mice develop multiple histologically distinct mammary tumors, including adenocarcinoma and sarcomatoid (spindle cell) carcinoma. We found some tumors to be invasive and a few metastasized to the lung and/or the lymph node. Single-cell RNA-seq analysis of the tumors identified epithelial, stromal, myeloid, and T cell groups. Expression analysis of the metastatic tumors identified S100a4 as a top candidate gene associated with metastasis. Metastatic tumors contained a much higher percentage of epithelial-mesenchymal transition (EMT)-signature positive and S100a4-expressing cells. CRISPR/CAS9-mediated knockout of S100a4 in a metastatic tumor-derived cell line disrupted its metastatic potential indicating a role for S100a4 in metastasis. CONCLUSIONS: Pik3caH1047R;Trp53R270H;MMTV-Cre mouse provides a preclinical model to mimic a subtype of human breast cancers that carry both PIK3CA and TP53 mutations. It also allows for understanding the cooperation between the two mutant genes in tumorigenesis. Our model also provides a system to study metastasis and develop therapeutic strategies for PIK3CA/TP53 double-positive cancers. S100a4 found involved in metastasis in this model can be a potential diagnostic and therapeutic target.

Low-Dose Arsenic Trioxide Modulates the Differentiation of Mouse Embryonic Stem Cells.

Arsenic (As) is a well-known environmental pollutant, while arsenic trioxide (ATO) has been proven to be an effective treatment for acute promyelocytic leukemia, however, the mechanism underlying its dual effects is not fully understood. Embryonic stem cells (ESCs) exhibit properties of stemness and serve as a popular model to investigate epigenetic modifiers including environmental pollutants. Herein, the effects of low-dose ATO on differentiation were evaluated in vitro using a mouse ESCs (mESCs) cell line, CGR8. Cells treated with 0.2-0.5 µM ATO for 3-4 days had slight inhibition of proliferation with elevation of apoptosis, but obvious alterations of differentiation by morphological checking and alkaline phosphatase (AP) staining. Moreover, ATO exposure significantly decreased the mRNA expression of the stemness maintenance genes including Oct4, Nanog, and Rex-1 ( P < 0.01), whereas obviously increased some tissue-specific differentiation marker genes such as Gata4, Gata-6, AFP, and IHH. These alterations were consistent with the differentiation phenotype induced by retinoic acid (RA) and the expression patterns of distinct pluripotency markers such as SSEA-1 and Oct4. Furthermore, low-dose ATO led to a quantitative increase in Caspase 3 (CASP3) activation and subsequent cleavage of Nanog around 27 kDa, which corresponded with the mouse Nanog cleaved by CASP3 in a tube cleavage assay. Taken together, we suggest that low-dose ATO exposure will induce differentiation, other than apoptosis, of ESCs, such effects might be tuned partially by ATO-induced CASP3 activation and Nanog cleavage coupling with other differentiation related genes involved. The present findings provide a preliminary action mechanism of arsenic on the cell fate determination.

Massively parallel nanowell-based single-cell gene expression profiling.

BACKGROUND: Technological advances have enabled transcriptome characterization of cell types at the single-cell level providing new biological insights. New methods that enable simple yet high-throughput single-cell expression profiling are highly desirable. RESULTS: Here we report a novel nanowell-based single-cell RNA sequencing system, ICELL8, which enables processing of thousands of cells per sample. The system employs a 5,184-nanowell-containing microchip to capture ~1,300 single cells and process them. Each nanowell contains preprinted oligonucleotides encoding poly-d(T), a unique well barcode, and a unique molecular identifier. The ICELL8 system uses imaging software to identify nanowells containing viable single cells and only wells with single cells are processed into sequencing libraries. Here, we report the performance and utility of ICELL8 using samples of increasing complexity from cultured cells to mouse solid tissue samples. Our assessment of the system to discriminate between mixed human and mouse cells showed that ICELL8 has a low cell multiplet rate (< 3%) and low cross-cell contamination. We characterized single-cell transcriptomes of more than a thousand cultured human and mouse cells as well as 468 mouse pancreatic islets cells. We were able to identify distinct cell types in pancreatic islets, including alpha, beta, delta and gamma cells. CONCLUSIONS: Overall, ICELL8 provides efficient and cost-effective single-cell expression profiling of thousands of cells, allowing researchers to decipher single-cell transcriptomes within complex biological samples.

Diverse somatic mutation patterns and pathway alterations in human cancers.

The systematic characterization of somatic mutations in cancer genomes is essential for understanding the disease and for developing targeted therapeutics. Here we report the identification of 2,576 somatic mutations across approximately 1,800 megabases of DNA representing 1,507 coding genes from 441 tumours comprising breast, lung, ovarian and prostate cancer types and subtypes. We found that mutation rates and the sets of mutated genes varied substantially across tumour types and subtypes. Statistical analysis identified 77 significantly mutated genes including protein kinases, G-protein-coupled receptors such as GRM8, BAI3, AGTRL1 (also called APLNR) and LPHN3, and other druggable targets. Integrated analysis of somatic mutations and copy number alterations identified another 35 significantly altered genes including GNAS, indicating an expanded role for galpha subunits in multiple cancer types. Furthermore, our experimental analyses demonstrate the functional roles of mutant GNAO1 (a Galpha subunit) and mutant MAP2K4 (a member of the JNK signalling pathway) in oncogenesis. Our study provides an overview of the mutational spectra across major human cancers and identifies several potential therapeutic targets.

Disruption of PH-kinase domain interactions leads to oncogenic activation of AKT in human cancers.

The protein kinase v-akt murine thymoma viral oncogene homolog (AKT), a key regulator of cell survival and proliferation, is frequently hyperactivated in human cancers. Intramolecular pleckstrin homology (PH) domain-kinase domain (KD) interactions are important in maintaining AKT in an inactive state. AKT activation proceeds after a conformational change that dislodges the PH from the KD. To understand these autoinhibitory interactions, we generated mutations at the PH-KD interface and found that most of them lead to constitutive activation of AKT. Such mutations are likely another mechanism by which activation may occur in human cancers and other diseases. In support of this likelihood, we found somatic mutations in AKT1 at the PH-KD interface that have not been previously described in human cancers. Furthermore, we show that the AKT1 somatic mutants are constitutively active, leading to oncogenic signaling. Additionally, our studies show that the AKT1 mutants are not effectively inhibited by allosteric AKT inhibitors, consistent with the requirement for an intact PH-KD interface for allosteric inhibition. These results have important implications for therapeutic intervention in patients with AKT mutations at the PH-KD interface.

Myocardin reverses insulin resistance and ameliorates cardiomyopathy by increasing IRS-1 expression in a murine model of lipodystrophy caused by adipose deficiency of vacuolar H+-ATPase V0d1 subunit.

Aim: Adipose tissue (AT) dysfunction that occurs in both obesity and lipodystrophy is associated with the development of cardiomyopathy. However, it is unclear how dysfunctional AT induces cardiomyopathy due to limited animal models available. We have identified vacuolar H+-ATPase subunit Vod1, encoded by Atp6v0d1, as a master regulator of adipogenesis, and adipose-specific deletion of Atp6v0d1 (Atp6v0d1AKO) in mice caused generalized lipodystrophy and spontaneous cardiomyopathy. Using this unique animal model, we explore the mechanism(s) underlying lipodystrophy-related cardiomyopathy. Methods and Results: Atp6v0d1AKO mice developed cardiac hypertrophy at 12 weeks, and progressed to heart failure at 28 weeks. The Atp6v0d1AKO mouse hearts exhibited excessive lipid accumulation and altered lipid and glucose metabolism, which are typical for obesity- and diabetes-related cardiomyopathy. The Atp6v0d1AKO mice developed cardiac insulin resistance evidenced by decreased IRS-1/2 expression in hearts. Meanwhile, the expression of forkhead box O1 (FoxO1), a transcription factor which plays critical roles in regulating cardiac lipid and glucose metabolism, was increased. RNA-seq data and molecular biological assays demonstrated reduced expression of myocardin, a transcription coactivator, in Atp6v0d1AKO mouse hearts. RNA interference (RNAi), luciferase reporter and ChIP-qPCR assays revealed the critical role of myocardin in regulating IRS-1 transcription through the CArG-like element in IRS-1 promoter. Reducing IRS-1 expression with RNAi increased FoxO1 expression, while increasing IRS-1 expression reversed myocardin downregulation-induced FoxO1 upregulation in cardiomyocytes. In vivo, restoring myocardin expression specifically in Atp6v0d1AKO cardiomyocytes increased IRS-1, but decreased FoxO1 expression. As a result, the abnormal expressions of metabolic genes in Atp6v0d1AKO hearts were reversed, and cardiac dysfunctions were ameliorated. Myocardin expression was also reduced in high fat diet-induced diabetic cardiomyopathy and palmitic acid-treated cardiomyocytes. Moreover, increasing systemic insulin resistance with rosiglitazone restored cardiac myocardin expression and improved cardiac functions in Atp6v0d1AKO mice. Conclusion: Atp6v0d1AKO mice are a novel animal model for studying lipodystrophy- or metabolic dysfunction-related cardiomyopathy. Moreover, myocardin serves as a key regulator of cardiac insulin sensitivity and metabolic homeostasis, highlighting myocardin as a potential therapeutic target for treating lipodystrophy- and diabetes-related cardiomyopathy.

Ferritin was involved in interleukin-17A enhanced osteogenesis through autophagy activation.

Periodontitis is a prevalent inflammatory immune disease that involves tissue inflammation and excessive bone loss. In murine periodontitis models and periodontitis patients, upregulated interleukin-17A (IL-17A) expression was observed, and its level seemed to correlate with the disease severity. In this study, we intended to investigate the specific role of ferritin, a critical iron storage protein, in IL-17A enhanced osteogenic differentiation as well as the underlying mechanism. Under osteogenic induction, IL-17A stimulation promoted differentiation and mineralization of murine calvarial osteoblasts. In addition, increased iron accumulation and ferritin expression were detected in osteoblasts treated with IL-17A, indicating an alteration in iron metabolism during osteogenesis. Administration of iron chelator deferoxamine (DFO) and transfection with small interfering RNA (siRNA) targeting ferritin heavy chain (FTH) further revealed that ferritin suppression consequently inhibited osteoblast differentiation. Autophagy activation was also found upon IL-17A stimulation, which played a positive role in osteogenic differentiation and was subsequently suppressed by DFO or siRNA targeting FTH. In conclusion, IL-17A induced ferritin expression in osteoblasts, which further enhanced osteogenic differentiation via autophagy activation. These findings may provide further insight into the role of IL-17A in osteoblast differentiation and demonstrate ferritin as a potential target in modulating alveolar bone homeostasis.

Traditional Chinese medicine Euodiae Fructus: botany, traditional use, phytochemistry, pharmacology, toxicity and quality control.

Euodiae Fructus, referred to as "Wuzhuyu" in Chinese, has been used as local and traditional herbal medicines in many regions, especially in China, Japan and Korea, for the treatment of gastrointestinal disorders, headache, emesis, aphtha, dermatophytosis, dysentery, etc. Substantial investigations into their chemical and pharmacological properties have been performed. Recently, interest in this plant has been focused on the different structural types of alkaloids like evodiamine, rutaecarpine, dehydroevodiamine and 1-methyl-2-undecyl-4(1H)-quinolone, which exhibit a wide range of pharmacological activities in preclinical models, such as anticancer, antibacterial, anti-inflammatory, anti-cardiovascular disease, etc. This review summarizes the up-to-date and comprehensive information concerning the botany, traditional uses, phytochemistry, pharmacology of Euodiae Fructus together with the toxicology and quality control, and discusses the possible direction and scope for future research on this plant.

Bayesian Design of Superiority Trials: Methods and Applications.

In this paper, we lay out the basic elements of Bayesian sample size determination (SSD) for the Bayesian design of a two-arm superiority clinical trial. We develop a flowchart of the Bayesian SSD that highlights the critical components of a Bayesian design and provides a practically useful roadmap for designing a Bayesian clinical trial in real world applications. We empirically examine the amount of borrowing, the choice of noninformative priors, and the impact of model misspecification on the Bayesian type I error and power. A formal and statistically rigorous formulation of conditional borrowing within the decision rule framework is developed. Moreover, by extending the partial borrowing power priors, a new borrowing-by-parts power prior for incorporating historical data is proposed. Computational algorithms are also developed to calculate the Bayesian type I error and power. Extensive simulation studies are carried out to explore the operating characteristics of the proposed Bayesian design of a superiority trial.

Interleukin-17 promotes osteoclastogenesis and periodontal damage via autophagy in vitro and in vivo.

OBJECTIVE: Because of its potent pro-inflammatory properties, interleukin-17 (IL-17) contributes to the pathogenesis of various inflammatory diseases. This study explored the effects of IL-17 on osteoclastogenesis in an osteoclast monoculture and osteoblast-osteoclast co-culture system, as tools to investigate the molecular mechanisms underlying the interactions between osteoclastogenesis and autophagy. METHODS: Various ratios of calvarial osteoblasts (OB) and osteoclast precursor cells (mouse macrophage cell line RAW264.7, hereinafter referred to as OC) were tested. Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the optimum osteoblasts:osteoclasts ratio. IL-17 was added to the co-culture system to test its effects on multinucleated osteoclast formation and osteoclast-related proteins. We assessed the effects of IL-17 on receptor activator of nuclear factor-kappa B ligand (RANKL) expression in osteoblasts, and determined if IL-17 alone could modulate osteoclast formation in an osteoclast monoculture. Administration of exogenous RANKL combined with IL-17 was employed to stimulate RAW264.7 cells osteoclastogenesis and to determine production of osteoclasts and autophagy-related proteins. We knocked down Beclin1 expression in RAW264.7 cells and examined the expression of autophagy-related and osteoclast-related proteins in RAW264.7 cells and the co-culture system, and the TAK1-binding protein 3 (TAB3)/ extracellular signal regulated kinase (ERK) pathway. RESULTS: A ratio of 20 OB : 1 OC yielded the highest rate of osteoclast formation. Low IL-17 concentrations increased osteoclastogenesis in co-cultures significantly, but high levels of IL-17 had the opposite effect. IL-17 alone could not induce formation of TRAP+ multinucleated cells in RAW264.7 cells. Low IL-17 concentrations promoted osteoclast differentiation and autophagy in RAW264.7 cells induced by exogenous RANKL, but high IL-17 concentrations inhibited this process. Knockdown of Beclin1 reversed the enhanced effects of 0.1 ng/mL IL-17 on osteoclastogenesis and autophagy in RAW264.7 cells. The TAB3/ERK pathway was also blocked after autophagy inhibition. CONCLUSION: In the co-culture model used in this study, a ratio of 20 OB:1 OC proved to be the optimal ratio to facilitate osteoclast formation. IL-17 regulated RANKL-induced osteoclastogenesis via autophagy. The Beclin1/TAB3/ERK pathway was involved in osteoclast autophagy.

Comprehensive Metabolic Profiling of Euphorbiasteroid in Rats by Integrating UPLC-Q/TOF-MS and NMR as Well as Microbial Biotransformation.

Euphorbiasteroid, a lathyrane-type diterpene from Euphorbiae semen (the seeds of Euphorbia lathyris L.), has been shown to have a variety of pharmacological effects such as anti-tumor and anti-obesity. This study aims to investigate the metabolic profiles of euphorbiasteroid in rats and rat liver microsomes (RLMs) and Cunninghamella elegans bio-110930 by integrating ultra-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry (UPLC-Q/TOF-MS), UNIFI software, and NMR techniques. A total of 31 metabolites were identified in rats. Twelve metabolites (M1-M5, M8, M12-M13, M16, M24-M25, and M29) were matched to the metabolites obtained by RLMs incubation and the microbial transformation of C. elegans bio-110930 and their structures were exactly determined through analysis of NMR spectroscopic data. In addition, the metabolic pathways of euphorbiasteroid were then clarified, mainly including hydroxylation, hydrolysis, oxygenation, sulfonation, and glycosylation. Finally, three metabolites, M3 (20-hydroxyl euphorbiasteroid), M24 (epoxylathyrol) and M25 (15-deacetyl euphorbiasteroid), showed significant cytotoxicity against four human cell lines with IC50 values from 3.60 µM to 40.74 µM. This is the first systematic investigation into the in vivo metabolic pathways of euphorbiasteroid and the cytotoxicity of its metabolites, which will be beneficial for better predicting the metabolism profile of euphorbiasteroid in humans and understanding its possible toxic material basis.

An integrated approach for structural characterization of Gui Ling Ji by traveling wave ion mobility mass spectrometry and molecular network.

Gui Ling Ji (GLJ), an ancient reputable traditional Chinese medicine (TCM) formula prescription, has been applied for the treatment of oligospermia and asthenospermia in clinical practice. However, its inherent compounds have not yet been systematically elucidated, which hampers developing standards or guidelines for quality evaluation and even the understanding of pharmacological effects. In this study, an integrated approach has been established for comprehensive structural characterization of GLJ. Mass spectrometry datasets of GLJ and each of the single herb medicines in this prescription have been developed by dynamic exclusion fast data-dependent acquisition and high-definition data-independent acquisition modes on ultra-high-performance liquid chromatography coupled with travelling wave ion mobility quadrupole time-of-flight mass spectrometry (UPLC-TWIMS-QTOF-MS). A global natural product social molecular networking (GNPS) platform was then applied for the visualization of chemical space of GLJ and further for the high throughput identification of the targeted or untargeted compounds due to the support of data-transmitting from each single herbal medicine to the formula GLJ. Moreover, drift time, predicted CCS, and diagnostic fragment ions were induced for annotating isomer compounds. Consequently, based on molecular network and library hits, a total of 257 compounds from GLJ, which were classified into 4 structural types, were positively or tentatively characterized. Among them, 20 potential new compounds were detected and 30 pairs of isomers were comprehensively distinguished. The established strategy was effective for attribution, classification, recognition of various constituents, and also was valuable for integrating large amounts of disordered MS/MS data and mining trace compounds in other complex chemical or biochemical systems.

3,4-Secocycloartane Triterpenoids from the Cones of Pseudolarix amabilis.

Four new 3,4-secocycloartane triterpenoids, pseudolactones A-D (1-4), were isolated from the ethanol extract of the cones of Pseudol arixamabilis. Their structures were established by extensive 1D- and 2D-NMR experiments. The cones of P. arixamabilis are enriched in the ring-expanded or cleaved cycloartane triterpenoids. This work provides new insight into cycloartane triterpenoids from the cones of P. arixamabilis.