Therapeutic potential of ginseng leaf extract in inhibiting mast cell-mediated allergic inflammation and atopic dermatitis-like skin inflammation in DNCB-treated mice.

Ginseng leaves are known to contain high concentrations of bioactive compounds, such as ginsenosides, and have potential as a treatment for various conditions, including fungal infections, cancer, obesity, oxidative stress, and age-related diseases. This study assessed the impact of ginseng leaf extract (GLE) on mast cell-mediated allergic inflammation and atopic dermatitis (AD) in DNCB-treated mice. GLE reduced skin thickness and lymph node nodules and suppressed the expression and secretion of histamine and pro-inflammatory cytokines. It also significantly lowered the production of inflammatory response mediators including ROS, leukotriene C4 (LTC4), prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). GLE inhibited the phosphorylation of MAPKs (ERK, P38, JNK) and the activation of NF-κB, which are both linked to inflammatory cytokine expression. We demonstrated that GLE's inhibitory effect on mast cell-mediated allergic inflammation is due to the blockade of the NF-κB and inflammasome pathways. Our findings suggest that GLE can be an effective therapeutic agent for mast-cell mediated and allergic inflammatory conditions.

Strategically Designed Bifunctional Polydopamine Enwrapping Polycaprolactone-Hydroxyapatite-Doxorubicin Composite Nanofibers for Osteosarcoma Treatment and Bone Regeneration.

This study presents a new multifunctional nanofibrous drug delivery system to provide effective combination therapy with enormous potential for the treatment of osteosarcoma. We developed a composite nanofiber scaffold comprising poly(ε-caprolactone) (PCL) and hydroxyapatite (HAp)-loaded doxorubicin (DOX) coated with polydopamine (PDA) to combine cancer cell inhibition with bone tissue regeneration. DOX was conjugated with HAp and then mixed with a PCL solution to prepare a PCL/HAp-DOX (labeled PCLDH) nanofiber. Then, in situ polymerization of PDA on the PCLDH occurred to produce the PCLDH@PDA composite nanofiber. The morphology, XRD, FT-IR, wettability, photothermal characteristics, cumulative drug release, and in vitro bioactivities were evaluated. We found that the PDA coating not only enhanced the hydrophilic properties but also controlled drug release. The PCLDH@PDA composite scaffold significantly suppressed the proliferation of bone cancer cells initially and, consequently, improved the adhesion and proliferation of human mesenchymal stem cells (hMSCs). The PDA coating boosted the composite scaffold's bioactivity, as demonstrated through ALP activity, ARS assay, and biomineralization results. This strategy offers a promising dual-function scaffold to treat residual cancer and reconstruct defects after osteosarcoma surgery.

Effects of Social Defeat Stress on Microtubule Regulating Proteins and Tubulin Polymerization.

Objective: : Microtubule (MT) stability in neurons is vital for brain development; instability is associated with neuropsychiatric disorders. The present study examined the effects of social defeat stress (SDS) on MT-regulating proteins and tubulin polymerization. Methods: : After 10 days of SDS, defeated mice were separated into susceptible (Sus) and unsusceptible (Uns) groups based on their performance in a social avoidance test. Using extracted brain tissues, we measured the expression levels of α-tubulin, acetylated α-tubulin, tyrosinated α-tubulin, MT-associated protein-2 (MAP2), stathmin (STMN1), phospho stathmin serine 16 (p-STMN1 [Ser16]), phospho stathmin serine 25 (p-STMN1 [Ser25]), phospho stathmin serine 38 (p-STMN1 [Ser38]), stathmin2 (STMN2), phospho stathmin 2 serine 73 (p-STMN2 [Ser73]), 78-kDa glucose-regulated protein (GRP-78), and CCAAT/enhancer binding protein (C/EBP)-homologous protein (CHOP) using Western blot assay. The tubulin polymerization rate was also measured. Results: : We observed increased and decreased expression of acetylated and tyrosinated α-tubulin, respectively, decreased expression of p-STMN1 (Ser16) and increased expression of p-STMN1 (Ser25), p-STMN2 (Ser73) and GRP-78 and CHOP in the prefrontal cortex and/or hippocampus of defeated mice. A reduced tubulin polymerization rate was observed in the Sus group compared to the Uns and Con groups. Conclusion: : Our findings suggest that SDS has detrimental effects on MT stability, and a lower tubulin polymerization rate could be a molecular marker for susceptibility to SDS.

Selegiline Modulates Lipid Metabolism by Activating AMPK Pathways of Epididymal White Adipose Tissues in HFD-Fed Obese Mice.

Obesity, as a major cause of many chronic diseases such as diabetes, cardiovascular disease, and cancer, is among the most serious health problems. Increased monoamine oxidase (MAO) activity has been observed in the adipose tissue of obese humans and animals. Although previous studies have already demonstrated the potential of MAO-B inhibitors as a treatment for this condition, the mechanism of their effect has been insufficiently elucidated. In this study, we investigated the anti-obesity effect of selegiline, a selective MAO-B inhibitor, using in vivo animal models. The effect was evaluated through an assessment of body energy homeostasis, glucose tolerance tests, and biochemical analysis. Pharmacological inhibition of MAO-B by selegiline was observed to reduce body weight and fat accumulation, and improved glucose metabolism without a corresponding change in food intake, in HFD-fed obese mice. We also observed that both the expression of adipogenenic markers, including C/EBPα and FABP4, and lipogenic markers such as pACC were significantly reduced in epididymal white adipose tissues (eWATs). Conversely, increased expression of lipolytic markers such as ATGL and pHSL and AMPK phosphorylation were noted. Treating obese mice with selegiline significantly increased expression levels of UCP1 and promoted eWAT browning, indicating increased energy expenditure. These results suggest that selegiline, by inhibiting MAO-B activity, is a potential anti-obesity treatment.

Neural mechanism of acute stress regulation by trace aminergic signalling in the lateral habenula in male mice.

Stress management is necessary for vertebrate survival. Chronic stress drives depression by excitation of the lateral habenula (LHb), which silences dopaminergic neurons in the ventral tegmental area (VTA) via GABAergic neuronal projection from the rostromedial tegmental nucleus (RMTg). However, the effect of acute stress on this LHb-RMTg-VTA pathway is not clearly understood. Here, we used fluorescent in situ hybridisation and in vivo electrophysiology in mice to show that LHb aromatic L-amino acid decarboxylase-expressing neurons (D-neurons) are activated by acute stressors and suppress RMTg GABAergic neurons via trace aminergic signalling, thus activating VTA dopaminergic neurons. We show that the LHb regulates RMTg GABAergic neurons biphasically under acute stress. This study, carried out on male mice, has elucidated a molecular mechanism in the efferent LHb-RMTg-VTA pathway whereby trace aminergic signalling enables the brain to manage acute stress by preventing the hypoactivity of VTA dopaminergic neurons.

Ginsenoside CK Inhibits the Early Stage of Adipogenesis via the AMPK, MAPK, and AKT Signaling Pathways.

Obesity is considered a health hazard in part due to the associated multiple diseases. As rates of obesity continue to increase, a new strategy for its prevention and treatment is required. Compound-K, an active ingredient in ginseng, possesses antioxidant, anti-inflammatory, and anti-cancer properties. Although ginseng has used as various therapeutics, its potential ability to alleviate metabolic diseases by regulating adipocyte differentiation is still unknown. In this study, we found that CK treatment significantly inhibited lipid droplet and adipogenesis by downregulating the mRNA expression of C/ebpα, Ppar-γ, Fabp4, Srebp1, and adiponectin as well as protein levels of C/EBPα, PPAR-γ, and FABP4. CK also decreased the production of reactive oxygen species (ROS), while it increased endogeneous antioxidant enzymes such as catalase, glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD) 3 and SOD2. We observed that CK treatment suppressed the expression of cyclin-dependent kinase 1 (CDK1) and cyclin B1 during the mitotic clonal expansion (MCE) of adipocyte differentiation, and it arrested adipocytes at the G2/M stage due to the increased expression of p21 and p27. CK decreased the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 and protein kinase B (AKT) in early-stage adipogenesis. In addition, the inhibition of adipogenesis by CK significantly increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC). Interestingly, AMPK pharmacological inhibition with Dorsomorphin limited the effect of CK on suppressing PPAR-γ expression in differentiated 3T3-L1 cells. Our results suggest that CK exerts anti-adipogenic effects in 3T3-L1 cells through the activation of AMPK and inhibition of ERK/p38 and AKT signaling pathways.

Marine Plankton-Derived Whitlockite Powder-Based 3D-Printed Porous Scaffold for Bone Tissue Engineering.

Powder-based 3D printing is an excellent technique for the fabrication of complex structural shapes. The outstanding bone remodeling capacity of calcium phosphate bioceramics is a desirable characteristic for such fabrication. Whitlockite (WH) is a calcium phosphate-based ceramic that contains Mg ions and possesses good mechanical properties, rapid resorbability, and promotes osteogenesis. The aim of this study was to fabricate 3D-printed scaffolds using marine plankton-derived WH (MP-WH) powder. The surface morphology and composition of the fabricated scaffolds were characterized by scanning electron microscopy and X-ray diffraction. The biocompatibility and osteogenic effects were evaluated using human mesenchymal stem cells. We successfully obtained a 3D porous scaffold using MP-WH. The MP-WH 3D scaffold showed improved compressive strength compared to the tricalcium phosphate (TCP) 3D scaffold. The in vitro results showed that compared with TCP 3D scaffolds, MP-WH 3D scaffolds were biocompatible and enhanced cell proliferation and adhesion. In addition, alkaline phosphatase activity and real-time polymerase chain reaction assays demonstrated that osteoblast differentiation was improved on the MP-WH scaffold. These results suggest that marine plankton-derived WH is useful for fabricating 3D-printed scaffolds for bone tissue engineering applications.

The Peripheral Role of CCL2 in the Anti-Nociceptive Effect of Sigma-1 Receptor Antagonist BD1047 on Inflammatory Hyperalgesia in Rats.

Our recent study demonstrated that the CC-chemokine ligand 2 (CCL2) present in primary afferent fibers (PAFs) plays an important role in the microglia-dependent neuronal activation associated with zymosan-induced inflammatory pain. The present study was aimed to evaluate whether BD1047 (a prototypical sigma-1 receptor (Sig-1R) antagonist) is capable of modifying elevated levels of inflammation-evoked CCL2 as a peripheral antinociceptive mechanism. In DRG primary culture, zymosan dose-dependently increased CCL2 release from isolectin B4 (IB4)-positive DRG neurons, a process that was inhibited by co-culture with BD1047. Single treatment of BD1047 before intraplantar injection of zymosan in rats significantly reduced thermal hyperalgesia and mechanical hyperalgesia, as well as CCL2 expression in DRG neurons and microglia activation in the spinal dorsal horn. In the Complete Freund's adjuvant (CFA)-induced inflammation model, repeated administration of BD1047 dramatically attenuated thermal hyperalgesia and mechanical hyperalgesia, and significantly diminished CCL2 immunoreactivity and microglia activation. Notably, CFA-induced inflammation significantly increased Sig-1R immunoreactivity in DRG neurons, which was co-localized with CCL2 and IB4, respectively. Taken together, our results suggest that BD1047's anti-nociceptive property was substantially mediated by the inhibition of CCL2 release in unmyelinated PAFs and that this may, in turn, have attenuated the spinal microglia activation that is associated with inflammatory pain.

Synthesis and evaluation of multivalent nitroimidazole-based near-infrared fluorescent agents for neuroblastoma and colon cancer imaging.

Hypoxia is one of key characteristics of microenvironments of solid tumors, and evaluation of hypoxia status in solid tumors is important to determine cancer stage and appropriate treatment. In the present study, novel, multivalent, near-infrared (NIR) fluorescent imaging agents were developed to measure tumor hypoxia. These agents were synthesized using an amino acid as a backbone to connect mono-, bis-, or tris-2-nitroimidazole as a hypoxia-sensitive moiety to enhance uptake by the tumor and to attach sulfo-Cyanine 5.5 as an NIR fluorophore to visualize tumor accumulation. Studies of physical characteristics demonstrated that the novel NIR imaging agents showed suitable optical properties for in vitro and in vivo imaging and were stable in serum. In vitro cellular uptake studies in SK-N-BE(2) and SW620 cell lines demonstrated that NIR imaging agents bearing 2-nitroimidazole structures showed significantly higher tumor uptake in hypoxic cells than in normoxic cells. Moreover, in vivo optical imaging studies using SK-N-BE(2) and SW620 xenografted mice demonstrated that novel, multivalent, 2-nitroimadazole NIR imaging agents with two or three 2-nitroimidazole moieties showed higher uptake in tumor than the control agents with only one 2-nitroimidazole. These observations suggest that novel, multivalent, NIR agents could serve as potential optical imaging agents for evaluating tumor hypoxia.

Advances in multiplex PCR for Alzheimer's disease diagnostics targeting CDK genes.

Alzheimer's disease (AD) is a common neurodegenerative disease that lacks biomarkers for diagnosis. Biomarkers for accurate detection of AD are required for potential therapeutic approaches. Recent studies in mammalian cells have demonstrated an association between the expression of cell cycle proteins and AD occurrence. Therefore, we aimed to identify a potent biomarker among relevant cell cycle-regulating proteins such as cyclin-dependent kinases (CDKs) for the diagnosis of AD. We also developed a multiplex-PCR-based diagnostic method, which showed the rapid and accurate detection of AD biomarkers. Genome-wide association study (GWAS) results showed increased gene expression of CDKs in an AD mouse model. Based on genomic analysis, our multiplex-PCR method, which contained optimized primer sets and PCR conditions targeting genes of CDKs, accurately matched RT-PCR results in the AD mouse model. Interestingly, validation by in silico meta-analysis for the expression of each CDK gene showed significant expression in moderate and severe groups of AD patients. Accordingly, clinical applications relying on the diagnosis of AD using our results may shed light on AD therapeutics.

Discoveries for Long Non-Coding RNA Dynamics in Traumatic Brain Injury.

In recent years, our understanding of long non-coding RNAs (lncRNAs) has been challenged with advances in genome sequencing and the widespread use of high-throughput analysis for identifying novel lncRNAs. Since then, the characterization of lncRNAs has contributed to the establishment of their molecular roles and functions in transcriptional regulation. Although genetic studies have so far explored the sequence-based primary function of lncRNAs that guides the expression of target genes, recent insights have shed light on the potential of lncRNAs for widening the identification of biomarkers from non-degenerative to neurodegenerative diseases. Therefore, further advances in the genetic characteristics of lncRNAs are expected to lead to diagnostic accuracy during disease progression. In this review, we summarized the latest studies of lncRNAs in TBI as a non-degenerative disease and discussed their potential limitations for clinical treatment.

Ginsenoside Compound K Induces Adult Hippocampal Proliferation and Survival of Newly Generated Cells in Young and Elderly Mice.

Cognitive impairment can be associated with reduced adult hippocampal neurogenesis, and it may contribute to age-associated neurodegenerative diseases such as Alzheimer's (AD). Compound K (CK) is produced from the protopanaxadiol (PPD)-type ginsenosides Rb1, Rb2, and Rc by intestinal microbial conversion. Although CK has been reported as an inducing effector for neuroprotection and improved cognition in hippocampus, its effect on adult neurogenesis has not been explored yet. Here, we investigated the effect of CK on hippocampal neurogenesis in both young (2 months) and elderly (24 months) mice. CK treatment increased the number of cells co-labeled with 5-ethynyl-2'-deoxyuridine (EdU) and proliferating cell nuclear antigen (PCNA); also, Ki67, specific markers for progenitor cells, was more expressed, thus enhancing the generation of new cells and progenitor cells in the dentate gyrus of both young and elderly mice. Moreover, CK treatment increased the number of cells co-labeled with EdU and NeuN, a specific marker for mature neuron in the dentate gyrus, suggesting that newly generated cells survived and differentiated into mature neurons at both ages. These findings demonstrate that CK increases adult hippocampal neurogenesis, which may be beneficial against neurodegenerative disorders such as AD.

Enzymatic Biotransformation of Ginsenoside Rb2 into Rd by Recombinant α-L-Arabinopyranosidase from Blastococcus saxobsidens.

In this study, we used a novel α-L-arabinopyranosidase (AbpBs) obtained from ginsenoside-converting Blastococcus saxobsidens that was cloned and expressed in Escherichia coli BL21 (DE3), and then applied it in the biotransformation of ginsenoside Rb2 into Rd. The gene, termed AbpBs, consisting of 2,406 nucleotides (801 amino acid residues), and with a predicted translated protein molecular mass of 86.4 kDa, was cloned into a pGEX4T-1 vector. A BLAST search using the AbpBs amino acid sequence revealed significant homology with a family 2 glycoside hydrolase (GH2). The over-expressed recombinant AbpBs in Escherichia coli BL21 (DE3) catalyzed the hydrolysis of the arabinopyranose moiety attached to the C-20 position of ginsenoside Rb2 under optimal conditions (pH 7.0 and 40°;C). Kinetic parameters for α-Larabinopyranosidase showed apparent Km and Vmax values of 0.078 ± 0.0002 micrometer and 1.4 ± 0.1 µmol/min/mg of protein against p-nitrophenyl-α-L-arabinopyranoside. Using a purified AbpBs (1 µg/ml), 0.1% of ginsenoside Rb2 was completely converted to ginsenoside Rd within 1 h. The recombinant AbpBs could be useful for high-yield, rapid, and low-cost preparation of ginsenoside Rd from Rb2.

Tauroursodeoxycholic acid induces angiogenic activity in endothelial cells and accelerates bone regeneration.

Angiogenesis is a crucial process during bone tissue regeneration. The aim of this study was to investigate the angiogenic activity and the potentiation of bone regeneration via angiogenesis using tauroursodeoxycholic acid (TUDCA) in vitro and in vivo. We investigated the effect of TUDCA on proliferation and angiogenic differentiation in human umbilical vein endothelial cells (HUVECs) and the associated signaling pathway. Proliferation was determined using crystal violet assay. Angiogenic effects were evaluated based on cell migration and tube formation. In order to explore TUDCA-signaling pathways, phosphorylation of mitogen activated protein kinase, protein kinase B (AKT), and endothelial nitric oxide synthase (eNOS) was determined using western blot. Furthermore, in vivo bone formation and angiogenesis were determined using a New Zealand outbred albino rabbit calvarial defect model, while angiogenesis and bone formation were evaluated using micro-CT and histological analysis. Our results show that TUDCA significantly increased cell proliferation. Moreover, TUDCA enhanced cell migration and tube formation in HUVECs. TUDCA increased the phosphorylation of AKT, ERK1/2, c-Jun N-terminal kinase, and eNOS. Specific inhibitors of ERK1/2 (PD98059), JNK (SP600125), and AKT (AKT1/2) inhibited the TUDCA-induced migration and tube formation, while the p38 inhibitor (SB203580) did not. The in vivo study used TUDCA to accelerate new blood vessel formation and promoted bone formation in rabbit calvarial defect model. These results indicate that TUDCA plays a critical role in enhancing the angiogenesis of endothelial cells and in vivo new bone regeneration. The use of TUDCA may contribute to the regeneration of bone tissue by improving angiogenesis.

Synthesis of polypyrrole nanorods via sacrificial removal of aluminum oxide nanopore template: A study on cell viability, electrical stimulation and neuronal differentiation of PC12 cells.

Synthesis of nanomaterials having uniform shape and size is a challenging task. Properties exhibited by such substrates would be compatible and homogeneous compared to the average properties displayed by those substrates with heterogeneous size. Herein, we report the synthesis of polypyrrole nanorods (PPy-NRs) of almost uniform size via oxidative chemical polymerization of pyrrole within anodized aluminum oxide nanopores followed by sacrificial removal of the template. Field emission scanning electron microscopy (FE-SEM), fourier transformed infra-red (FT-IR) spectra, X-ray diffraction (XRD), and ultra-violet-visible-near infra-red (UV-Vis-NIR) spectra of the substrate were used to analyze the physicochemical properties of as-synthesized PPy-NRs. PPy-NRs treated MC3T3-E1 and PC12 cells exhibited good biocompatibility in CCK-8 and live/dead assays. The assay showed more cell viability on PC12 cell lines. Electrical stimulation through PPy-NRs treated PC12 cells accelerated neuronal differentiation compared to those without electrical stimulation during in vitro cell culture.

Ginsenoside Compound K Induces Ros-Mediated Apoptosis and Autophagic Inhibition in Human Neuroblastoma Cells In Vitro and In Vivo.

Autophagy can result in cellular adaptation, as well as cell survival or cell death. Modulation of autophagy is increasingly regarded as a promising cancer therapeutic approach. Ginsenoside compound K (CK), an active metabolite of ginsenosides isolated from Panax ginseng C.A. Meyer, has been identified to inhibit growth of cancer cell lines. However, the molecular mechanisms of CK effects on autophagy and neuroblastoma cell death have not yet been investigated. In the present study, CK inhibited neuroblastoma cell proliferation in vitro and in vivo. Treatment by CK also induced the accumulation of sub-G1 population, and caspase-dependent apoptosis in neuroblastoma cells. In addition, CK promotes autophagosome accumulation by inducing early-stage autophagy but inhibits autophagic flux by blocking of autophagosome and lysosome fusion, the step of late-stage autophagy. This effect of CK appears to be mediated through the induction of intracellular reactive oxygen species (ROS) and mitochondria membrane potential loss. Moreover, chloroquine, an autophagy flux inhibitor, further promoted CK-induced apoptosis, mitochondrial ROS induction, and mitochondria damage. Interestingly, those promoted phenomena were rescued by co-treatment with a ROS scavenging agent and an autophagy inducer. Taken together, our findings suggest that ginsenoside CK induced ROS-mediated apoptosis and autophagic flux inhibition, and the combination of CK with chloroquine, a pharmacological inhibitor of autophagy, may be a novel therapeutic potential for the treatment of neuroblastoma.

Rapid and simple colorimetric detection of multiple influenza viruses infecting humans using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform.

BACKGROUND: In addition to seasonal influenza viruses recently circulating in humans, avian influenza viruses (AIVs) of H5N1, H5N6 and H7N9 subtypes have also emerged and demonstrated human infection abilities with high mortality rates. Although influenza viral infections are usually diagnosed using viral isolation and serological/molecular analyses, the cost, accessibility, and availability of these methods may limit their utility in various settings. The objective of this study was to develop and optimized a multiplex detection system for most influenza viruses currently infecting humans. METHODS: We developed and optimized a multiplex detection system for most influenza viruses currently infecting humans including two type B (both Victoria lineages and Yamagata lineages), H1N1, H3N2, H5N1, H5N6, and H7N9 using Reverse Transcriptional Loop-mediated Isothermal Amplification (RT-LAMP) technology coupled with a one-pot colorimetric visualization system to facilitate direct determination of results without additional steps. We also evaluated this multiplex RT-LAMP for clinical use using a total of 135 clinical and spiked samples (91 influenza viruses and 44 other human infectious viruses). RESULTS: We achieved rapid detection of seasonal influenza viruses (H1N1, H3N2, and Type B) and avian influenza viruses (H5N1, H5N6, H5N8 and H7N9) within an hour. The assay could detect influenza viruses with high sensitivity (i.e., from 100 to 0.1 viral genome copies), comparable to conventional RT-PCR-based approaches which would typically take several hours and require expensive equipment. This assay was capable of specifically detecting each influenza virus (Type B, H1N1, H3N2, H5N1, H5N6, H5N8 and H7N9) without cross-reactivity with other subtypes of AIVs or other human infectious viruses. Furthermore, 91 clinical and spiked samples confirmed by qRT-PCR were also detected by this multiplex RT-LAMP with 98.9% agreement. It was more sensitive than one-step RT-PCR approach (92.3%). CONCLUSIONS: Results of this study suggest that our multiplex RT-LAMP assay may provide a rapid, sensitive, cost-effective, and reliable diagnostic method for identifying recent influenza viruses infecting humans, especially in locations without access to large platforms or sophisticated equipment.

Identification of novel glycoside hydrolases via whole genome sequencing of Niabella ginsenosidivorans for production of various minor ginsenosides.

In this study, many bacterial strains were screened for the production of minor ginsenosides, but based on conversion competence among the strains, the strain Niabella ginsenosidivorans BS26T has the good ginsenoside-transforming ability. Therefore, the strain BS26T was selected for complete genome sequence analysis to determine the target (glycoside hydrolase) functional genes. Whole genome analysis of strain BS26T showed 43 glycoside hydrolase genes in total. To determine the target functional gene, 12 sets of six different glycoside hydrolases (3 set of ß-glucosidase; 3 set of trehalase; 3 set of arabinofuranosidase; 2 set of xylosidase; and one set of each α-galactosidase and α-fucosidase, respectively) were selected and cloned in E. coli BL21 (DE3) using the pGEX4T-1 vector and were characterized. Among these 12 sets of clones, only one, ß-glucosidase (BglNg-767), showed ginsenoside conversion ability. The BglNg-767 comprised 767 amino acids and belonged to glycoside hydrolase family 3 (GH3). The recombinant GST-BglNg-767 was capable of altering the ginsenosides Rb1, Rd, and gypenoside XVII (Gyp-XVII) to F2; Rb2 to C-O; Rb3 to C-Mx1, and Rc to C-Mc1. Besides, complete genome sequence analysis of strain BS26T also indicates 30 endopeptidase genes, which may be responsible for self-hydrolysis of the proteins. Therefore, using SDS-PAGE analysis, we predict that the difference between the molecular weight of the expressed protein (around 90 kDa) and the predicted amino-acid sequence (102.7 kDa) is due to self-hydrolysis of the proteins.

The CCL2 elevation in primary afferent fibers produces zymosan-induced hyperalgesia through microglia-mediated neuronal activation in the spinal dorsal horn.

Although C-C motif chemokine ligand 2 (CCL2) plays a critical role in the pathogenesis of neuropathic pain through neuron-microglia interactions, its pronociceptive function underlying inflammatory pain remains to be fully understood. The present study aimed to elucidate the potential role of CCL2 in pain sensitization following zymosan-induced inflammation. Intraplantar injection of zymosan into the rat hind paw significantly increased the expression of CCL2 in both dorsal root ganglions and the superficial dorsal horn. The expression of CCL2 was exclusively present in the isolectin B4-positive unmyelinated primary afferent fibers, but no in other cells of the spinal cord. Intrathecal administration of RS504393 (a CCR2 antagonist) markedly reduced the zymosan-induced thermal and mechanical hyperalgesia accompanied with reduced expression in the spinal cord of c-Fos, CD11b, phosphorylated p38 mitogen activated protein kinases (p-p38), and interleukin-1ß. Whole cell patch-clamp recordings on spinal cord slices further revealed that the incubation of CCL2 evoked an evident inward current in substantia gelatinosa neurons and increased level of p-p38 in microglia. Moreover, co-incubation with minocycline (an inhibitor of microglial activation) prevented CCL2-mediated activation in the spinal cord slice. Taken together, we propose that the increased CCL2 expression from primary afferent fibers following zymosan-induced inflammation activates nociceptive neurons in the spinal dorsal horn via a microglia-dependent mechanism.