4'-O-MethylbavachalconeB Targeted 14-3-3ζ Blocking the Integrin ß3 Early Outside-In Signal to Inhibit Platelet Aggregation and Thrombosis.

14-3-3ζ protein, the key target in the regulation and control of integrin ß3 outside-in signaling, is an attractive new strategy to inhibit thrombosis without affecting hemostasis. In this study, 4'-O-methylbavachalconeB (4-O-MB) in Psoraleae Fructus was identified as a 14-3-3ζ ligand with antithrombosis activity by target fishing combined with ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) analysis. The competitive inhibition analysis showed that 4-O-MB targeted 14-3-3ζ and blocked the 14-3-3ζ/integrin ß3 interaction with inhibition constant (Ki) values of 9.98 ± 0.22 µM. Molecular docking and amino acid mutation experiments confirmed that 4-O-MB specifically bound to 14-3-3ζ through LSY9 and SER28 to regulate the 14-3-3ζ/integrin ß3 interaction. Besides, 4-O-MB affected the integrin ß3 early outside-in signal by inhibiting AKT and c-Src phosphorylation. Meanwhile, 4-O-MB could inhibit ADP-, collagen-, or thrombin-induced platelet aggregation function but had no effect on platelet adhesion to collagen-coated surfaces in vivo. Administration of 4-O-MB could significantly inhibit thrombosis formation without disturbing hemostasis in mice. These findings provide new prospects for the antithrombotic effects of Psoraleae Fructus and the potential application of 4-O-MB as lead compounds in the therapy of thrombosis by targeting 14-3-3ζ.

Blockade of spinal dopamine D1/D2 receptor heteromers by levo-Corydalmine suppressed calcium signaling cascade in spinal neurons to alleviate bone cancer pain in rats.

Background: Dopamine receptors have been reported to be involved in pain, while the exact effects and mechanism in bone cancer pain have not been fully explored. Methods: Bone cancer pain model was created by implanting walker 256 mammary gland carcinoma into right tibia bone cavity. Primary cultured spinal neurons were used for in vitro evaluation. FLIPR, western-blot, immunofluorescence, and Co-IP were used to detect cell signaling pathway. Results: Our results indicated that spinal dopamine D1 receptor (D1DR) and spinal dopamine D2 receptor (D2DR) could form heteromers in TCI rats, and antagonizing spinal D1DR and D2DR reduced heteromers formation and alleviated TCI-induced bone cancer pain. Further results indicated that D1DR or D2DR antagonist induced antinociception in TCI rats could be reversed by D1DR, D2DR, and D1/D2DR heteromer agonists. And Gq, IP3, and PLC inhibitors also attenuated TCI-induced bone cancer pain. In vitro results indicated that D1DR or D2DR antagonist decreased the Ca2+ oscillations upregulated by D1DR, D2DR, and D1/D2DR heteromer agonists in activated primary cultured spinal neurons. Moreover, inhibition of D1/D2DR heteromers induced antinociception in TCI rats was partially mediated by the CaMKII and MAPKs pathway. In addition, a natural compound levo-Corydalmine (l-CDL), could inhibit D1/D2DR heteromers and attenuate bone cancer pain. Results: Inhibition of spinal D1/D2DR heteromers via l-CDL decreases excitability in spinal neurons, which might present new therapeutic strategy for bone cancer pain.

Hybrid Cell Membrane-Functionalized Biomimetic Nanoparticles for Targeted Therapy of Osteosarcoma.

PURPOSE: In order to prepare a biomimetic nano-carrier which has inflammatory chemotaxis, homologous targeting and reduce immune clearance, for targeted chemotherapy of osteosarcoma, we fabricated the paclitaxel-loaded poly(lactic-co-glycolic) acid (PLGA) nanoparticles coated with 143B-RAW hybrid membrane (PTX-PLGA@[143B-RAW] NPs) and evaluate its anti-cancer efficacy in vitro and vivo. METHODS: PTX-PLGA@[143B-RAW] NPs were prepared by the ultrasonic method and were characterized by size, zeta potential, polymer dispersion index (PDI), Coomassie bright blue staining, transmission electron microscopy (TEM) and high performance liquid chromatography (HPLC). Cellular uptake, cell viability assay, flow cytometry and chemotactic effect of PTX-PLGA@[143B-RAW] NPs were evaluated in vitro. Biodistribution, anti-cancer therapeutic efficacy and safety of PTX-PLGA@[143B-RAW] NPs were evaluated in 143B osteosarcoma xenograft mice. RESULTS: The hybrid membrane successfully coated onto the surface of PLGA nanoparticles. PTX-PLGA@[143B-RAW] NPs had a drug loading capacity of 4.24 ± 0.02% and showed targeting ability to osteosarcoma. PTX-PLGA@[143B-RAW] NPs showed high cellular uptake and improved anti-cancer efficacy against 143B cells. More importantly, PTX-PLGA@[143B-RAW] NPs treatment suppressed tumor growth in tumor-bearing mice with minimal damage to normal tissues. CONCLUSION: PTX-PLGA@[143B-RAW] NPs could be used for targeted drug delivery and osteosarcoma therapy.

The dopamine D1-D2DR complex in the rat spinal cord promotes neuropathic pain by increasing neuronal excitability after chronic constriction injury.

Dopamine D1 receptor (D1DR) and D2 receptor (D2DR) are closely associated with pain modulation, but their exact effects on neuropathic pain and the underlying mechanisms remain to be identified. Our research revealed that intrathecal administration of D1DR and D2DR antagonists inhibited D1-D2DR complex formation and ameliorated mechanical and thermal hypersensitivity in chronic constriction injury (CCI) rats. The D1-D2DR complex was formed in the rat spinal cord, and the antinociceptive effects of D1DR and D2DR antagonists could be reversed by D1DR, D2DR, and D1-D2DR agonists. Gαq, PLC, and IP3 inhibitors also alleviated CCI-induced neuropathic pain. D1DR, D2DR, and D1-D2DR complex agonists all increased the intracellular calcium concentration in primary cultured spinal neurons, and this increase could be reversed by D1DR, D2DR antagonists and Gαq, IP3, PLC inhibitors. D1DR and D2DR antagonists significantly reduced the expression of p-PKC γ, p-CaMKII, p-CREB, and p-MAPKs. Levo-corydalmine (l-CDL), a monomeric compound in Corydalis yanhusuo W.T. Wang, was found to obviously suppress the formation of the spinal D1-D2DR complex to alleviate neuropathic pain in CCI rats and to decrease the intracellular calcium concentration in spinal neurons. l-CDL-induced inhibition of p-PKC γ, p-MAPKs, p-CREB, and p-CaMKII was also reversed by D1DR, D2DR, and D1-D2DR complex agonists. In conclusion, these results indicate that D1DR and D2DR form a complex and in turn couple with the Gαq protein to increase neuronal excitability via PKC γ, CaMKII, MAPK, and CREB signaling in the spinal cords of CCI rats; thus, they may serve as potential drug targets for neuropathic pain therapy.

Blockade of spinal dopamine D1/D2 receptor suppresses activation of NMDA receptor through Gαq and Src kinase to attenuate chronic bone cancer pain.

INTRODUCTION: Spinal N-methyl-D-aspartate receptor (NMDAR) is vital in chronic pain, while NMDAR antagonists have severe side effects. NMDAR has been reported to be controlled by G protein coupled receptors (GPCRs), which might present new therapeutic targets to attenuate chronic pain. Dopamine receptors which belong to GPCRs have been reported could modulate the NMDA-mediated currents, while their exact effects on NMDAR in chronic bone cancer pain have not been elucidated. OBJECTIVES: This study was aim to explore the effects and mechanisms of dopamine D1 receptor (D1DR) and D2 receptor (D2DR) on NMDAR in chronic bone cancer pain. METHODS: A model for bone cancer pain was established using intra-tibia bone cavity tumor cell implantation (TCI) of Walker 256 in rats. The nociception was assessed by Von Frey assay. A range of techniques including the fluorescent imaging plate reader, western blotting, and immunofluorescence were used to detect cell signaling pathways. Primary cultures of spinal neurons were used for in vitro evaluation. RESULTS: Both D1DR and D2DR antagonists decreased NMDA-induced upregulation of Ca2+ oscillations in primary culture spinal neurons. Additionally, D1DR/D2DR antagonists inhibited spinal Calcitonin Gene-Related Peptide (CGRP) and c-Fos expression and alleviated bone cancer pain induced by TCI which could both be reversed by NMDA. And D1DR/D2DR antagonists decreased p-NR1, p-NR2B, and Gαq protein, p-Src expression. Both Gαq protein and Src inhibitors attenuated TCI-induced bone cancer pain, which also be reversed by NMDA. The Gαq protein inhibitor decreased p-Src expression. In addition, D1DR/D2DR antagonists, Src, and Gαq inhibitors inhibited spinal mitogen-activated protein kinase (MAPK) expression in TCI rats, which could be reversed by NMDA. CONCLUSIONS: Spinal D1DR/D2DR inhibition eliminated NMDAR-mediated spinal neuron activation through Src kinase in a Gαq-protein-dependent manner to attenuate TCI-induced bone cancer pain, which might present a new therapeutic strategy for bone cancer pain.

The Value of Enhanced MR Radiomics in Estimating the IDH1 Genotype in High-Grade Gliomas.

BACKGROUND: The prognosis of IDH1-mutant glioma is significantly better than that of wild-type glioma, and the preoperative identification of IDH mutations in glioma is essential for the formulation of surgical procedures and prognostic assessment. PURPOSE: To explore the value of a radiomic model based on preoperative-enhanced MR images in the assessment of the IDH1 genotype in high-grade glioma. MATERIALS AND METHODS: A retrospective analysis was performed on 182 patients with high-grade glioma confirmed by surgical pathology between December 2012 and January 2019 in our hospital with complete preoperative brain-enhanced MR images, including 79 patients with an IDH1 mutation (45 patients with WHO grade III and 34 patients with WHO grade IV) and 103 patients with wild-type IDH1 (33 patients with WHO grade III and 70 patients with WHO grade IV). Patients were divided into a primary dataset and a validation dataset at a ratio of 7 : 3 using a stratified random sampling; radiomic features were extracted using A.K. (Analysis Kit, GE Healthcare) software and were initially reduced using the Kruskal-Wallis and Spearman analyses. Lasso was finally conducted to obtain the optimized subset of the feature to build the radiomic model, and the model was then tested with cross-validation. ROC (receiver operating characteristic curve) analysis was performed to evaluate the performance of the model. RESULTS: The radiomic model showed good discrimination in both the primary dataset (AUC = 0.87, 95% CI: 0.754 to 0.855, ACC = 0.798, sensitivity = 85.5%, specificity = 75.4%, positive predictive value = 0.734, and negative predictive value = 0.867) and the validation dataset (AUC = 0.86, 95% CI: 0.690 to 0.913, ACC = 0.789, sensitivity = 91.3%, specificity = 69.0%, positive predictive value = 0.700, and negative predictive value = 0.909). CONCLUSION: The radiomic model, based on the preoperative-enhanced MR, can effectively predict the IDH1 genotype in high-grade glioma.

Regulation of the KATP-JNK gap junction signaling pathway by immunomodulator astragaloside IV attenuates neuropathic pain.

BACKGROUND AND OBJECTIVES: Gap junctions play a pivotal role in contributing to the formation of astroglial networks and in chronic pain. However, the mechanisms underlying the dysfunction of astroglial gap junctions in chronic pain have not been fully elucidated. METHODS: Chronic constriction injury (CCI) of the sciatic nerve was used to establish rat neuropathic pain model. C6 cells were used to perform experiments in vitro. Von Frey hairs and Hargreave's method were used to determine the withdrawal threshold of rats. Protein expression was detected by immunofluorescence and western blotting. RESULTS: Astragaloside IV (AST IV) significantly attenuated neuropathic pain and suppressed the excitation of spinal astrocytes in rats with CCI. The antinociceptive effect of AST IV was reversed by the gap junction decoupler carbenoxolone (CBX). AST IV inhibited the high expression of phosphorylated connexin 43 (p-Cx43) and p-c-Jun N-terminal kinase (p-JNK) in spinal cord of rats with CCI. JNK inhibitor alleviated neuropathic pain, which was reversed by CBX. JNK inhibitor decreased the high expression of p-Cx43 in both rats with CCI and tumor necrosis factor-alpha (TNF-α)-treated C6 cells. Additionally, the analgesic effect of AST IV was reversed by the adenosine triphosphate-sensitive potassium (KATP) channel blocker, glibenclamide (Glib). Glib abolished the inhibitory effects of AST IV on p-JNK and p-Cx43 both in vivo and in vitro. KATP channel opener (KCO) mimicked the inhibitory effects of AST IV on p-JNK and p-Cx43 in TNF-α-treated C6 cells. CONCLUSION: Our results indicate that the sciatic nerve CCI induces the dysfunction of gap junctions in the spinal cord by activating KATP/JNK signaling to contribute to neuropathic pain. AST IV attenuates neuropathic pain via regulating the KATP-JNK gap junction axis.

Suppression of peripheral NGF attenuates neuropathic pain induced by chronic constriction injury through the TAK1-MAPK/NF-κB signaling pathways.

BACKGROUND: Anti-nerve growth factor (NGF) monoclonal antibodies (anti-NGF mAbs) have been reported to significantly attenuate pain, but the mechanism involved has not been fully elucidated, and the serious adverse events associated with mAbs seriously limit their clinical use. This study further investigated the mechanism by which peripheral NGF is involved in neuropathic pain and found safe, natural compounds that target NGF to attenuate neuropathic pain. METHODS: Nociception was assessed by the Von Frey hair and Hargreaves' methods. Western-blotting, qPCR and immunofluorescence were used to detect the cell signaling pathway. RAW264.7 macrophages and RSC96 Schwann cells were cultured for in vitro evaluation. RESULTS: Intraplantar administration of anti-NGF mAbs suppressed the expression of phosphorylated transforming growth factor-ß-activated kinase 1 (TAK1) in the dorsal root ganglion (DRG) and sciatic nerve. Intraplantar administration of a TAK1 inhibitor attenuated CCI-induced neuropathic pain and suppressed the expression of phosphorylated mitogen-activated protein kinases (MAPKs) in the DRG and sciatic nerve. Perisciatic nerve administration of levo-corydalmine (l-CDL) on the operated side obviously attenuated CCI-induced neuropathic pain and suppressed the expression of mNGF and proNGF. In addition, l-CDL-induced antinociception was reversed by intraplantar administration of NGF. Further results indicated that l-CDL-induced suppression of phosphorylated TAK1, MAPKs, and p65 and expression of the proinflammatory cytokines TNF-α and IL-1ß in the DRG and sciatic nerve were all abolished by NGF. In addition, in vitro experiments indicated that l-CDL suppressed the secretion of NGF and proNGF in RAW264.7 macrophages and RSC96 Schwann cells, which was abolished by AP-1 and CREB agonists, respectively. CONCLUSIONS: This study showed NGF inhibition suppressed TAK1 in the periphery to attenuate CCI-induced neuropathic pain through inhibition of downstream MAPK and p65 signaling. The natural compound l-CDL inhibited NGF secretion by macrophages and Schwann cells and downstream TAK1-MAPK/NF-κB signaling in the periphery to attenuate CCI-induced neuropathic pain. Video abstract Proposed mechanisms underlying the effect of l-CDL in periphery of CCI rats. In CCI rats, macropahages and Schwann cells could secret NGF to act on the receptors in the periphery to activate TAK1-MAPK/NF-κB axis and promote the release of proinflammatory cytokines, including TNF-α and IL-1ß to promote neuropathic pain. l-CDL decreased the secretion of NGF through inhibiting AP-1 and CREB respectively in RAW264.7 and RSC96 Schwann cells to attenuate CCI-induced neuropathic pain by inhibiting the TAK1-p38 MAPK/NF-κB signaling pathway.

Rapid and sensitive detection of NGAL for the prediction of acute kidney injury via a polydopamine nanosphere/aptamer nanocomplex coupled with DNase I-assisted recycling amplification.

Early detection of acute kidney injury (AKI) is important, as early intervention and treatment can prevent further kidney injury and improve kidney health. Neutrophil gelatinase-associated lipocalin (NGAL) has emerged as the earliest and promising non-invasive biomarker of AKI in urine, and has been used as a new predictive biomarker of AKI in the bench-to-bedside journey. In this work, a nanocomplex composed of a polydopamine nanosphere (PDANS) and a fluorophore-labelled aptamer has been constructed for the detection of NGAL using a DNase I-assisted recycling amplification strategy. After the addition of NGAL, the fluorescence intensity increases linearly over the NGAL concentration range from 12.5 to 400 pg mL-1. The limit of detection of this strategy is found to be 6.25 pg mL-1, which is almost 5 times lower than that of the method that does not involve DNase I. The process can be completed within 1 h, indicating a fast fluorescence response. Furthermore, the method using the nanocomplex coupled with DNase I has been successfully utilized for the detection of NGAL in the urine from cisplatin-induced AKI and five-sixths nephrectomized mice, demonstrating its promising ability for the early prediction of AKI. This method also demonstrates the protective effect of the Huangkui capsule on AKI, and provides an effective way to screen potentially protective drugs for renal disease.

Levo-corydalmine attenuates microglia activation and neuropathic pain by suppressing ASK1-p38 MAPK/NF-κB signaling pathways in rat spinal cord.

BACKGROUND AND OBJECTIVES: Neuropathic pain is partially refractory to currently available treatments. Although some studies have reported that apoptosis signal-regulating kinase 1 (ASK1) may inhibit chronic pain, the mechanisms underlying this process have not been fully elucidated. METHODS: Chronic constriction injury (CCI) of the rat sciatic nerve was used to establish a neuropathic pain model. Nociception was assessed using von Frey hair and Hargreaves' methods. Western blot and immunofluorescence were used to detect the cell signaling pathway. BV2 cell line was cultured for in vitro evaluation. RESULTS: Our results indicated that spinal ASK1 was co-expressed with the microglia marker ionized calcium binding adaptor 1. Additionally, intrathecal administration of ASK1 inhibitor suppressed the activation of spinal microglia and attenuated CCI-induced neuropathic pain. The ASK1 inhibitor also decreased the levels of phosphorylated ASK1 (p-ASK1), p65, p38 mitogen-activated protein kinase (MAPK) and tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) messenger RNA in lipopolysaccharide-stimulated BV2 microglia cells. Intragastric administration of levo-corydalmine (l-CDL) significantly attenuated CCI-induced neuropathic pain and inhibited the expression of p-ASK1 in the spinal cord. l-CDL conspicuously suppressed the activation of spinal microglia in vitro and in vivo. Translocation of nuclearfactor-kappa B (NF-κB) and upregulation of p-p65, TNF-α, IL-1ß were inhibited by l-CDL. Further, the analgesic effects of l-CDL were associated with reduced levels of phosphorylated protein kinase C (PKC γ), c-JunNH2-terminal kinase, and extracellular signal-regulated kinase. CONCLUSIONS: This study showed that the expression of ASK1 in spinal microglia and ASK1 inhibitor suppressed microglia activation via suppression of p38 MAPK/NF-κB, which ultimately attenuated CCI-induced neuropathic pain. l-CDL also inhibited the ASK1-P38 MAPK/NF-κB axis to attenuate CCI-induced neuropathic pain.

Dual signal amplification for microRNA-21 detection based on duplex-specific nuclease and invertase.

MicroRNA-21 (miRNA-21) is a significant biomarker which is closely related to some kinds of diseases, such as cancer, cardiovascular disease and kidney disease. Therefore, the detection of miRNA-21 is of great importance and can provide essential information for disease diagnosis. In this study, we report a facile, sensitive assay for miRNA-21 detection using personal glucose meters (PGM). Biotinylated DNA strand linked invertase (Inv) is conjugated on the surface of streptavidin-coated magnetic beads (MBs) to form a MBs-DNA-Inv complex. Target miRNA-21 in the detection system is captured by the MBs-DNA-Inv probe through DNA/RNA hybridization. The duplex-specific nuclease (DSN) enzyme specifically cleaves the DNA to recycle the target miRNA and release invertase, thereby triggering the dual signal amplification and ensuring high sensitivity. Besides, we establish a linear relationship between PGM and different concentrations of miRNA-21 in the range of 10 to 200 pM. The limit of detection is 1.8 pM, which is more sensitive than some of the previous reports. In addition, the biosensor exhibits excellent sequence selectivity and single-base mutation can be discriminated. Moreover, the expression of miRNA-21 is confirmed in urine from mice by our method, which is in good accordance with the qRT-PCR result. Therefore, a dependable, low-cost strategy for the detection of miRNA has been established and it meets the latest analytical demands for miRNA determination that is suitable for the public.

A rapid strategy for screening high-efficiency PCSK9 inhibitors from Ginkgo biloba leaves by ligand fishing, HPLC-Q-TOF-MS and interdisciplinary assay.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an attractive target for new cholesterol-lowering drug development. Here, we developed a method integrating ligand fishing, HPLC-Q-TOF-MS and interdisciplinary assay, aiming to explore potential PCSK9 inhibitors from mixtures rapidly and accurately. PCSK9 was expressed and purified firstly, and then the recombined PCSK9 was coated on the surface of magnetic beads (MBs). The PCSK9-immobilized MBs (PCSK9-MBs) were used for ligand fishing combined with HPLC and Q-TOF-MS/MS. Ginkgo biloba leaves (GBL), an herbal medicine widely used in Asia and Europe with good efficacy in treatment of hypercholesterolemia, were chosen as an illustration for ligand fishing. Two PCSK9 ligands were discovered from GBL and identified as kaempferol-3-O-rutinoside (1) and kaempferol 3-O-26″-(6‴-p-coumaroyl) glucosylrhamnoside (KCGR) (2). In order to verify fishing results and pick out more powerful PCSK9 inhibitors, molecular docking assay was further performed and KCGR was optimized to be an excellent PCSK9 inhibitor by the confirmation of affinity and activity bioassay. These results suggested that the developed approach could be applied to screen and analysis potential bioactive constituents from mixtures, which may improve the efficiency of drug discovery. Moreover, KCGR separated from GBL was expected to be a potential candidate of PCSK9 inhibitors.

Dihydroartemisinin derivative DC32 inhibits inflammatory response in osteoarthritic synovium through regulating Nrf2/NF-κB pathway.

Synovitis is an aseptic inflammation that leads to joint effusion, pain and swelling. As one of the main drivers of pathogenesis in osteoarthritis (OA), the presence of synovitis contributes to pain, incidence and progression of OA. In our previous study, DC32 [(9α,12α-dihydroartemisinyl) bis(2'-chlorocinnmate)], a dihydroartemisinin derivative, was found to have an antirheumatic ability via immunosuppression, but the effect of DC32 on synovitis has not been fully illuminated. In this study, we chose to evaluate the effect and mechanism of DC32 on attenuating synovial inflammation. Fibroblast-like synoviocytes (FLSs) of papain-induced OA rats were isolated and cultured. And DC32 significantly inhibited the invasion and migration of cultured OA-FLSs, as well as the transcription of IL-6, IL-1ß, CXCL12 and CX3CL1 in cultured OA-FLSs measured by qPCR. DC32 remarkably inhibited the activation of ERK and NF-κB pathway, increased the expression of Nrf2 and HO-1 in cultured OA-FLSs detected by western blot. DC32 inhibited the degradation and phosphorylation of IκBα which further prevented the phosphorylation of NF-κB p65 and the effect of DC32 could be relieved by siRNA for Nrf2. In papain-induced OA mice, DC32 significantly alleviated papain-induced mechanical allodynia, knee joint swelling and infiltration of inflammatory cell in synovium. DC32 upregulated the mRNA expression of Type II collagen and aggrecan, and downregulated the mRNA expression of MMP2, MMP3, MMP13 and ADAMTS-5 in the knee joints of papain-induced OA mice measured by qPCR. The level of TNF-α in the serum and secretion of TNF-α in the knee joints were also reduced by DC32 in papain-induced OA mice. In conclusion, DC32 inhibited the inflammatory response in osteoarthritic synovium through regulating Nrf2/NF-κB pathway and attenuated OA. In this way, DC32 may be a potential agent in the treatment of OA.

Determination of the chemical components and phospholipids of velvet antler using UPLC/QTOF-MS coupled with UNIFI software.

Velvet antler, which exhibits immune and growth enhancing effects, is commonly used in a variety of Asian health care products, but its complex components remain unknown. The current study analyzed extracts using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry in the MSE mode. Automated detection and data filtering were performed using UNIFI software and peaks were compared with a proprietary scientific library (Traditional Medicine Library; TML). The results obtained using different data processing parameters (including 3D peak detection, target by mass and fragment identification) were evaluated against 87 compounds comprising 1 lignan, 30 terpenoids (including 20 triterpenes), 39 steroids, 8 alkaloids, 4 organic acids and 5 esters in the TML. Using a screening method with a mass accuracy cutoff of ±2 mDa, a retention time cutoff of ±0.2 min, a minimum response threshold of 1,000 counts and an average of 10 false detects per sample analysis, 16 phospholipids were identified in the extracts of velvet antler, three of which were quantified. The results demonstrated that there was 1.07±0.02 µg/g of 1-myristoyl-sn-glycero-3-phosphocholine, 7.05±0.52 ng/g of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 18.81±0.55 ng/g of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in velvet antler. The current study successfully identified certain components of velvet antler. Furthermore, the results may provide an experimental basis for further pharmacological and clinical study.

Rapid identification of urokinase plasminogen activator inhibitors from Traditional Chinese Medicines based on ultrafiltration, LC-MS and in silico docking.

The urokinase plasminogen activator (uPA) is regarded as the crucial trigger for plasmin generation, which is involved in several diseases especially for neoplasm metastasis. In this study, an efficient approach integrating ultrafiltration, LC/MS, bioassay and in silico docking, was proposed for rapidly detecting uPA ligands from Traditional Chinese Medicines (TCMs). Forty-two TCMs were initially assessed, and as illustrative case studies, Galla Chinensis and Sanguisorbae Radix, which appeared significant inhibitory activities on uPA, were chosen to develpe and verify the strategy. A total of seven uPA ligands were successfully detected and identified. Two of them, pentagalloylglucose and 28-O-ß-d-glucopyranosyl pomolic acid, were demonstrated to be potential inhibitors, with IC50 at 1.639 µM and 37.82 µM repectively. Furthermore, a combinatorial compound library screening combined with in silico docking assay, was revealed that ursolic acid (IC50 = 2.623 µM) was also speculated to be a potent parent structure for inhibition of uPA. This approach offers a multidimensional perspective to discover uPA-binding leading compounds from TCMs or other complex mixtures, which would provide an efficient route for drug discovery.

Selective blockade of spinal D2DR by levo-corydalmine attenuates morphine tolerance via suppressing PI3K/Akt-MAPK signaling in a MOR-dependent manner.

Morphine tolerance remains a challenge in the management of chronic pain in the clinic. As shown in our previous study, the dopamine D2 receptor (D2DR) expressed in spinal cord neurons might be involved in morphine tolerance, but the underlying mechanisms remain to be elucidated. In the present study, selective spinal D2DR blockade attenuated morphine tolerance in mice by inhibiting phosphatidylinositol 3 kinase (PI3K)/serine-threonine kinase (Akt)-mitogen activated protein kinase (MAPK) signaling in a µ opioid receptor (MOR)-dependent manner. Levo-corydalmine (l-CDL), which exhibited micromolar affinity for D2DR in D2/CHO-K1 cell lines in this report and effectively alleviated bone cancer pain in our previous study, attenuated morphine tolerance in rats with chronic bone cancer pain at nonanalgesic doses. Furthermore, the intrathecal administration of l-CDL obviously attenuated morphine tolerance, and the effect was reversed by a D2DR agonist in mice. Spinal D2DR inhibition and l-CDL also inhibited tolerance induced by the MOR agonist DAMGO. l-CDL and a D2DR small interfering RNA (siRNA) decreased the increase in levels of phosphorylated Akt and MAPK in the spinal cord; these changes were abolished by a PI3K inhibitor. In addition, the activated Akt and MAPK proteins in mice exhibiting morphine tolerance were inhibited by a MOR antagonist. Intrathecal administration of a PI3K inhibitor also attenuated DAMGO-induced tolerance. Based on these results, l-CDL antagonized spinal D2DR to attenuate morphine tolerance by inhibiting PI3K/Akt-dependent MAPK phosphorylation through MOR. These findings provide insights into a more versatile treatment for morphine tolerance.

Novel dihydroartemisinin derivative DHA-37 induces autophagic cell death through upregulation of HMGB1 in A549 cells.

Dihydroartemisinin (DHA) and its analogs are reported to possess selective anticancer activity. Here, we reported a novel DHA derivative DHA-37 that exhibited more potent anticancer activity on the cells tested. Distinct from DHA-induced apoptosis, DHA-37 triggered excessive autophagic cell death, and became the main contributor to DHA-37-induced A549 cell death. Incubation of the cells with DHA-37 but not DHA produced increased dots distribution of GFP-LC3 and expression ratio of LC3-II/LC3-I, and enhanced the formation of autophagic vacuoles as revealed by TEM. Treatment with the autophagy inhibitor 3-MA, LY294002, or chloroquine could reverse DHA-37-induced cell death. In addition, DHA-37-induced cell death was associated significantly with the increased expression of HMGB1, and knockdown of HMGB1 could reverse DHA-37-induced cell death. More importantly, the elevated HMGB1 expression induced autophagy through the activation of the MAPK signal but not PI3K-AKT-mTOR pathway. In addition, DHA-37 also showed a wonderful performance in A549 xenograft mice model. These findings suggest that HMGB1 as a target candidate for apoptosis-resistant cancer treatment and artemisinin-based drugs could be used in inducing autophagic cell death.

A New Strategy for Rapidly Screening Natural Inhibitors Targeting the PCSK9/LDLR Interaction In Vitro.

The interaction between proprotein convertase subtilisin/kexin type 9 (PCSK9) and the low-density lipoprotein receptor (LDLR) is a promising target for the treatment of hyperc-holesterolemia. In this study, a new method based on competitive affinity and tag detection was developed, which aimed to evaluate potent natural inhibitors preventing the interaction of PCSK9/LDLR directly. Herein, natural compounds with efficacy in the treatment of hypercholesterolemia were chosen to investigate their inhibitory activities on the PCSK9/LDLR interaction. Two of them, polydatin (1) and tetrahydroxydiphenylethylene-2-O-glucoside (2), were identified as potential inhibitors for the PCSK9/LDLR interaction and were proven to prevent PCSK9-mediated LDLR degradation in HepG2 cells. The results suggested that this strategy could be applied for evaluating potential bioactive compounds inhibiting the interaction of PCSK9/LDLR and this strategy could accelerate the discovery of new drug candidates for the treatment of PCSK9-mediated hypercholesterolemia.

Identification of active compound combination contributing to anti-inflammatory activity of Xiao-Cheng-Qi Decoction via human intestinal bacterial metabolism.

Human intestinal bacteria play an important role in the metabolism of herbal medicines, leading to the variations in their pharmacological profile. The present study aimed to investigate the metabolism of Xiao-Cheng-Qi decoction (XCQD) by human intestinal bacteria and to discover active component combination (ACC) contributing to the anti-inflammatory activity of XCQD. The water extract of XCQD was anaerobically incubated with human intestinal bacteria suspensions for 48 h at 37 °C. A liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS) method was performed for identification of the metabolites. In addition, the anti-inflammatory effects of XCQD and biotransformed XCQD (XCQD-BT) were evaluated in vitro with cytokines in RAW264.7 cells induced by lipopolysaccharide (LPS). A total of 51 compounds were identified in XCQD and XCQD-BT. Among them, 20 metabolites were proven to be transformed by human intestinal bacteria. Significantly, a combination of 14 compounds was identified as ACC from XCQD-BT, which was as effective as XCQD in cell models of inflammation. In conclusion, this study provided an applicable method, based on intestinal bacterial metabolism, for identifying combinatory compounds responsible for a certain pharmacological activity of herbal medicines.

Computed tomography and magnetic resonance imaging features of cervical chordoma.

Computed tomography (CT) and magnetic resonance imaging (MRI) scans of 11 patients with histologically proven cervical chordoma were retrospectively evaluated. Imaging features assessed included location, morphology, association with adjacent structures, vertebral destruction, status of cortical bone, periosteal reaction, attenuation and calcification by CT, and signal intensity and enhancement pattern by MRI. Of 7 cases with CT, 6 exhibited lytic-sclerotic bone destruction. A total of 5 cases exhibited pressure erosion of outer cortex, 3 of which had spiculated periosteal reaction. Calcification was observed in 3 cases. All cases were heterogeneous and hypodense. MRI T2-weighted images (n=10) revealed heterogeneous hyperintense (n=5), intermediate (n=2) and intermediate-hyperintense signal intensity (n=3). Hypointense septa between lobules (n=5) and stripes (n=3) were observed on T2-weighted images. Post-contrast magnetic resonance images (n=6) demonstrated marked heterogeneous (n=3) and ring-like (n=3) enhancement. CT scanning is valuable in revealing the lytic-sclerotic bone destruction, pressure erosion of outer cortex and calcification. MRI is useful in demonstrating the results of soft tissue mass. The two examinations are necessary for differential diagnosis of patients with suspected cervical chordoma.