Anti-Inflammatory Action of Dexmedetomidine on Human Microglial Cells

Neuroinflammation, where inflammatory cytokines are produced in excess, contributes to the pathogenesis of delirium. Microglial cells play a central role in neuroinflammation by producing and releasing inflammatory cytokines in response to infection, tissue damage and neurodegeneration. Dexmedetomidine (DEX) is a sedative, which reduces the incidence of delirium. Thus, we hypothesized that DEX may alleviate delirium by exhibiting anti-inflammatory action on microglia. In the present study, we investigated the anti-inflammatory action of DEX on human microglial HMC3 cells. The results indicated that DEX partially suppressed the IL-6 and IL-8 production by lipopolysaccharide (LPS)-stimulated HMC3 cells as well as the phosphorylation of p38 MAPK and IκB and the translocation of NF-κB. Furthermore, DEX substantially suppressed IL-6 and IL-8 production by unstimulated HMC3 cells as wells as the phosphorylation of p38 MAPK and IκB and the translocation of NF-κB. These observations suggest that DEX exhibits anti-inflammatory action on not only LPS-stimulated but also unstimulated microglial cells via the suppression of inflammatory signaling and cytokine production.     

HPLC法测定红参中人参皂苷Rg1的含量

建立红参药材中人参皂苷Rg1的含量测定方法。利用高效液相色谱(HPLC)法测定红参中人参皂苷Rg1的含量,采用Ulitimate XB-C18(2)柱(250 mm×4.6 mm,5μm);流动相∶乙腈-水(23∶77);柱温:室温;流速:0.7 m L·min-1;检测波长:203 nm;进样量为10μL。人参皂苷Rg1的线性回归方程为A=438355.8C-1328.9(r=0.9999)。人参皂苷Rg1在0.1~5μg范围内呈良好的线性关系。人参皂苷Rg1的平均回收率为103.6%,RSD为1.581%。采用此法测定红参中人参皂苷Rg1的含量,准确可靠,可用于该药材的质量控制。     

Prenylated Flavonoids from Cudrania tricuspidata Suppress Lipopolysaccharide-Induced Neuroinflammatory Activities in BV2 Microglial Cells

In Korea and China, Cudrania tricuspidata Bureau (Moraceae) is an important traditional medicinal plant used to treat lumbago, hemoptysis, and contusions. The C. tricuspidata methanol extract suppressed both production of NO and PGE₂ in BV2 microglial cells. Cudraflavanone D (1), isolated from this extract, remarkably suppressed the protein expression of inducible NO synthase and cyclooxygenase-2, and decreased the levels of NO and PGE₂ in BV2 microglial cells exposed to lipopolysaccharide. Cudraflavanone D (1) also decreased IL-6, TNF-α, IL-12, and IL-1β production, blocked nuclear translocation of NF-κB heterodimers (p50 and p65) by interrupting the degradation and phosphorylation of inhibitor of IκB-α, and inhibited NF-κB binding. In addition, cudraflavanone D (1) suppressed the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK pathways. This study indicated that cudraflavanone D (1) can be a potential drug candidate for the cure of neuroinflammation.     

HPLC法同步测定三七粉中人参皂苷Rg1、Rb1及三七皂苷R1的含量

目的:采用HPLC法同步测定三七粉中人参皂苷Rg1、Rb1及三七皂苷R1含量。方法:采用SGE protecol C18(5μm,4.6×250mm)色谱柱,流动相为乙腈—水梯度洗脱(0~12min,19∶81;12~60min,19~36∶81~64),检测波长为203nm,流速1.0mL/min,进样量20μL。结果:人参皂苷Rg1、人参皂苷Rb1、三七皂苷R1分别在25.625~430mg/L、26.875~410mg/L、10.625~170mg/L范围呈线性,相关系数r分别为0.9999、0.9998、0.9996;该方法重复性及回收率均符合要求。结论:本法用于同步测定三七粉中人参皂苷Rg1、Rb1及三七皂苷R1的含量,具有简便、准确、高效等特点。     

Ginsenoside Rg3 Reduces Lipid Accumulation with AMP-Activated Protein Kinase (AMPK) Activation in HepG2 Cells

Cardiovascular disease (CVD) is one of the main causes of mortality worldwide, and dyslipidemia is a major risk factor for CVD. Ginseng has been widely used in the clinic to treat CVD. Ginsenoside Rg3, one of the major active components of ginseng, has been reported to exhibit antiobesity, antidiabetic, and cardioprotective effects. However, the effect of ginsenoside Rg3 on hepatic lipid metabolism remains unclear. Therefore, we investigated whether ginsenoside Rg3 would regulate hepatic lipid metabolism with AMP-activated protein kinase (AMPK) activation in HepG2 cells. Ginsenoside Rg3 significantly reduced hepatic cholesterol and triglyceride levels. Furthermore, ginsenoside Rg3 inhibited expression of sterol regulatory element binding protein-2 (SREBP-2) and 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGCR). Ginsenoside Rg3 increased activity of AMPK, a major regulator of energy metabolism. These results suggest that ginsenoside Rg3 reduces hepatic lipid accumulation with inhibition of SREBP-2 and HMGCR expression and stimulation of AMPK activity in HepG2 cells. Therefore, ginsenoside Rg3 may be beneficial as a food ingredient to lower the risk of CVD by regulating dyslipidemia.     

Regulatory Effects of Caffeic Acid Phenethyl Ester on Neuroinflammation in Microglial Cells

Microglial activation has been widely demonstrated to mediate inflammatory processes that are crucial in several neurodegenerative disorders. Pharmaceuticals that can deliver direct inhibitory effects on microglia are therefore considered as a potential strategy to counter balance neurodegenerative progression. Caffeic acid phenethyl ester (CAPE), a natural phenol in honeybee propolis, is known to possess antioxidant, anti-inflammatory and anti-microbial properties. Accordingly, the current study intended to probe the effects of CAPE on microglia activation by using in vitro and in vivo models. Western blot and Griess reaction assay revealed CAPE significantly inhibited the expressions of inducible nitric oxide synthase (NOS), cyclooxygenase (COX)-2 and the production of nitric oxide (NO). Administration of CAPE resulted in increased expressions of hemeoxygenase (HO)-1and erythropoietin (EPO) in microglia. The phosphorylated adenosine monophosphate-activated protein kinase (AMPK)-α was further found to regulate the anti-inflammatory effects of caffeic acid. In vivo results from immunohistochemistry along with rotarod test also revealed the anti-neuroinflammatory effects of CAPE in microglia activation. The current study has evidenced several possible molecular determinants, AMPKα, EPO, and HO-1, in mediating anti-neuroinflammatory responses in microglial cells.     

Inhibitory Activities of Rare Ginsenoside Rg4 on Cecal Ligation and Puncture-Induced Sepsis

Sepsis is an uncontrolled response to inflammatory infection and is associated with high levels of mortality and morbidity. Rg4 is a rare ginsenoside mainly found in the leaves of Panax ginseng C. A. Meyer and the major protopanaxatriol-type ginsenoside of black ginseng. In this study, we determined whether Rg4 affects cecal ligation and puncture (CLP)-induced sepsis. Animals were separated into the following six groups: control group, CLP-operated group, CLP plus maslinic acid (MA), and CLP plus Rg4 (5, 10, or 15 mg/kg). Survival rate, body weight changes, inflammatory cytokines, and histological analyses were assessed. Human endothelial cells were activated with the high-mobility group box 1 (HMGB1) protein and Rg4. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis were used to assess inflammation and gene expression, respectively. After CLP surgery, the Rg4-administered group exhibited a higher survival rate and body weight compared with the untreated control group. Rg4 treatment reduced cytokine levels, including tumor necrosis factor (TNF)-α and interleukin (IL)-1β, as well as nitric oxide (NO) levels and renal inflammation. After Rg4 treatment of HMGB1-activated cells, the expressions of toll-like receptor (TLR) 4 and TNF-α were decreased, and the activation of phosphoinositide 3-kinase (PI3K)/AKT signaling increased cell viability. In summary, Rg4 inhibited inflammation and exhibited a protective effect against CLP-induced sepsis, thereby reinforcing cell survival against septic responses.     

MAO-A Inhibition by Metaxalone Reverts IL-1β-Induced Inflammatory Phenotype in Microglial Cells

Experimental and clinical studies have suggested that several neurological disorders are associated with the occurrence of central nervous system neuroinflammation. Metaxalone is an FDA-approved muscle relaxant that has been reported to inhibit monoamine oxidase A (MAO-A). The aim of this study was to investigate whether metaxalone might exert antioxidant and anti-inflammatory effects in HMC3 microglial cells. An inflammatory phenotype was induced in HMC3 microglial cells through stimulation with interleukin-1β (IL-1β). Control cells and IL-1β-stimulated cells were subsequently treated with metaxalone (10, 20, and 40 µM) for six hours. IL-1β stimulated the release of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), but reduced the anti-inflammatory cytokine interleukin-13 (IL-13). The upstream signal consisted of an increased priming of nuclear factor-kB (NF-kB), blunted peroxisome proliferator-activated receptor gamma (PPARγ), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression. IL-1β also augmented MAO-A expression/activity and malondialdehyde levels and decreased Nrf2 mRNA expression and protein levels. Metaxalone decreased MAO-A activity and expression, reduced NF-kB, TNF-α, and IL-6, enhanced IL-13, and also increased PPARγ, PGC-1α, and Nrf2 expression. The present experimental study suggests that metaxalone has potential for the treatment of several neurological disorders associated with neuroinflammation.     

The Safety Evaluation of Salvianolic Acid B and Ginsenoside Rg1 Combination on Mice

Our previous study indicated that the combination of salvianolic acid B (SalB) and ginsenoside Rg1 (Rg1), the main components of Salvia miltiorrhizae and Panax notoginseng, improves myocardium structure and ventricular function in rats with ischemia/reperfusion injury. The present study aimed to determine the safety of the combined SalB and Rg1 (SalB-Rg1) in mice. The safety of SalB-Rg1 was evaluated through acute toxicity and repeated-dose toxicity. In the acute toxicity study, the up and down procedure was carried out firstly, and then, the Bliss method was applied. In the toxicity study for seven-day repeated treatment of SalB-Rg1, forty Kunming mice were randomly divided into four groups. The intravenous median lethal dose (LD₅₀) of the SalB-Rg1 combination was 1747 mg/kg using the Bliss method. For both the acute toxicity study and the seven-day repeated toxicity study, SalB-Rg1 did not induce significant abnormality on brain, heart, kidney, liver and lung structure at any dose based on H&E stain. There were no significant changes related to the SalB-Rg1 toxicity detected on biochemical parameters for two kinds of toxicity studies. The LD₅₀ in mice was 1747 mg/kg, which was more than one hundred times higher than the effective dose. Both studies of acute toxicity and seven-day repeated dose toxicity indicated the safety of the SalB-Rg1 combination.     

Novel Antidepressant Mechanism of Ginsenoside Rg1 in Regulating the Dysfunction of the Glutamatergic System in Astrocytes

Ginsenoside Rg1, a traditional Chinese medicine monomer, has been shown to have antidepressant effects. We previously found that Rg1 exerts antidepressant effects by improving the gap junction channels (GJCs) dysfunction; however, the downstream mechanisms through which Rg1 ameliorates GJC dysfunction remain unclear. Since hemichannels directly release glutamate, GJC dysfunction decreases the expression levels of glutamate transporters in astrocytes, and glutamatergic system dysfunction plays an essential role in the pathogenesis of depression. The glutamatergic system may be a potential downstream target of Rg1 that exerts antidepressant effects. Therefore, in this study, we aimed to determine the downstream mechanisms by which Rg1 ameliorated GJC dysfunction and exerted its antidepressant effects. Corticosterone (CORT) is used to mimic high glucocorticoid levels in patients with depression in vitro. Primary cortical astrocytes were isolated and phosphorylation of connexin43 (Cx43) as well as the functions of hemichannels, GJCs, and the glutamatergic system were evaluated after drug treatment. Rg1 pretreatment reversed the anomalous activation of Cx43 phosphorylation as well as the dysfunction of hemichannels, GJCs, and the glutamatergic system induced by CORT. These results suggest that Rg1 can ameliorate CORT-induced dysfunction of the glutamatergic system in astrocytes by potentially reducing Cx43 phosphorylation and inhibiting opening of hemichannels, thereby improving GJC dysfunction.     

双波长薄层扫描法测定乳癖消片中人参皂苷Rg1含量

应用双波长薄层扫描法测定乳癖消片中人参皂苷Rg1含量 ,平均回收率 97.0 % ,RSD为1 .98%。结果 :方法重现性良好 ,结果准确可靠     

Effects of Paeonol on Anti-Neuroinflammatory Responses in Microglial Cells

Increasing studies suggest that inflammatory processes in the central nervous system mediated by microglial activation plays an important role in numerous neurodegenerative diseases. Development of planning for microglial suppression is considered a key strategy in the search for neuroprotection. Paeonol is a major phenolic component of Moutan Cortex, widely used as a nutrient supplement in Chinese medicine. In this study, we investigated the effects of paeonol on microglial cells stimulated by inflammagens. Paeonol significantly inhibited the release of nitric oxide (NO) and the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Treatment with paeonol also reduced reactive oxygen species (ROS) production and inhibited an ATP-induced increased cell migratory activity. Furthermore, the inhibitory effects of neuroinflammation by paeonol were found to be regulated by phosphorylated adenosine monophosphate-activated protein kinase-α (AMPK-α) and glycogen synthase kinase 3 α/β (GSK 3α/β). Treatment with AMPK or GSK3 inhibitors reverse the inhibitory effect of neuroinflammation by paeonol in microglial cells. Furthermore, paeonol treatment also showed significant improvement in the rotarod performance and microglial activation in the mouse model as well. The present study is the first to report a novel inhibitory role of paeonol on neuroinflammation, and presents a new candidate agent for the development of therapies for inflammation-related neurodegenerative diseases.     

Regulation of the Fructose Transporter Gene Slc2a5 Expression by Glucose in Cultured Microglial Cells

Microglia play a role in the regulation of metabolism and pathogenesis of obesity. Microglial activity is altered in response to changes in diet and the body’s metabolic state. Solute carrier family 2 member 5 (Slc2a5) that encodes glucose transporter 5 (GLUT5) is a fructose transporter primarily expressed in microglia within the central nervous system. However, little is known about the nutritional regulation of Slc2a5 expression in microglia and its role in the regulation of metabolism. The present study aimed to address the hypothesis that nutrients affect microglial activity by altering the expression of glucose transporter genes. Murine microglial cell line SIM-A9 cells and primary microglia from mouse brain were exposed to different concentrations of glucose and levels of microglial activation markers and glucose transporter genes were measured. High concentration of glucose increased levels of the immediate-early gene product c-Fos, a marker of cell activation, Slc2a5 mRNA, and pro-inflammatory cytokine genes in microglial cells in a time-dependent manner, while fructose failed to cause these changes. Glucose-induced changes in pro-inflammatory gene expression were partially attenuated in SIM-A9 cells treated with the GLUT5 inhibitor. These findings suggest that an increase in local glucose availability leads to the activation of microglia by controlling their carbohydrate sensing mechanism through both GLUT5-dependent and –independent mechanisms.     

Microglial FABP4-UCP2 Axis Modulates Neuroinflammation and Cognitive Decline in Obese Mice

The microglial fatty-acid-binding protein 4-uncoupling protein 2 (FABP4-UCP2) axis is a key regulator of neuroinflammation in high-fat-diet (HFD)-fed animals, indicating a role for FABP4 in brain immune response. We hypothesized that the FABP4-UCP2 axis is involved in regulating diet-induced cognitive decline. We tested cognitive function in mice lacking microglial FABP4 (AKO mice). Fifteen-week-old male AKO and wild-type (WT) mice were maintained on 60% HFD or normal chow (NC) for 12 weeks. Body composition was measured using EchoMRI. Locomotor activity, working memory, and spatial memory were assessed using behavioral tests (open field, T-maze, and Barnes maze, respectively). Hippocampal microgliosis was assessed via immunohistochemical staining. An inflammatory cytokine panel was assayed using hippocampal tissue. Real-time RT-PCR was performed to measure microglial UCP2 mRNA expression. Our data support that loss of FABP4 prevents cognitive decline in vivo. HFD-fed WT mice exhibited impaired long- and short-term memory, in contrast with HFD-fed AKO mice. HFD-fed WT mice had an increase in hippocampal inflammatory cytokine expression (IFNγ, IL-1β, IL-5, IL-6, KC/GRO(CXCL1), IL-10, and TNFα) and microgliosis, and decreased microglial UCP2 expression. HFD-fed AKO mice had decreased hippocampal inflammatory cytokine expression and microgliosis and increased microglial UCP2 expression compared to HFD-fed WT mice. Collectively, our work supports the idea that the FABP4-UCP2 axis represents a potential therapeutic target in preventing diet-induced cognitive decline.     

Biochemical Targets and Molecular Mechanism of Ginsenoside Compound K in Treating Osteoporosis Based on Network Pharmacology

To investigate the potential of ginsenosides in treating osteoporosis, ginsenoside compound K (GCK) was selected to explore the potential targets and mechanism based on network pharmacology (NP). Based on text mining from public databases, 206 and 6590 targets were obtained for GCK and osteoporosis, respectively, in which 138 targets were identified as co-targets of GCK and osteoporosis using intersection analysis. Five central gene clusters and key genes (STAT3, PIK3R1, VEGFA, JAK2 and MAP2K1) were identified based on Molecular Complex Detection (MCODE) analysis through constructing a protein–protein interaction network using the STRING database. Gene Ontology (GO) analysis implied that phosphatidylinositol-related biological process, molecular modification and function may play an important role for GCK in the treatment of osteoporosis. Function and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that the c-Fms-mediated osteoclast differentiation pathway was one of the most important mechanisms for GCK in treating osteoporosis. Meanwhile, except for being identified as key targets based on cytoHubba analysis using Cytoscape software, MAPK and PI3K-related proteins were enriched in the downstream of the c-Fms-mediated osteoclast differentiation pathway. Molecular docking further confirmed that GCK could interact with the cavity on the surface of a c-Fms protein with the lowest binding energy (−8.27 Kcal/moL), and their complex was stabilized by hydrogen bonds (Thr578 (1.97 Å), Leu588 (2.02 Å, 2.18 Å), Ala590 (2.16 Å, 2.84 Å) and Cys 666 (1.93 Å)), van der Waals and alkyl hydrophobic interactions. Summarily, GCK could interfere with the occurrence and progress of osteoporosis through the c-Fms-mediated MAPK and PI3K signaling axis regulating osteoclast differentiation.     

Effects of a Novel GPR55 Antagonist on the Arachidonic Acid Cascade in LPS-Activated Primary Microglial Cells

Neuroinflammation is a crucial process to maintain homeostasis in the central nervous system (CNS). However, chronic neuroinflammation is detrimental, and it is described in the pathogenesis of CNS disorders, including Alzheimer’s disease (AD) and depression. This process is characterized by the activation of immune cells, mainly microglia. The role of the orphan G-protein-coupled receptor 55 (GPR55) in inflammation has been reported in different models. However, its role in neuroinflammation in respect to the arachidonic acid (AA) cascade in activated microglia is still lacking of comprehension. Therefore, we synthesized a novel GPR55 antagonist (KIT 10, 0.1–25 µM) and tested its effects on the AA cascade in lipopolysaccharide (LPS, 10 ng / mL)-treated primary rat microglia using Western blot and EIAs. We show here that KIT 10 potently prevented the release of prostaglandin E₂ (PGE₂), reduced microsomal PGE₂ synthase (mPGES-1) and cyclooxygenase-2 (COX-2) synthesis, and inhibited the phosphorylation of Ikappa B-alpha (IκB-α), a crucial upstream step of the inflammation-related nuclear factor-kappaB (NF-κB) signaling pathway. However, no effects were observed on COX-1 and -2 activities and mitogen-activated kinases (MAPK). In summary, the novel GPR55 receptor antagonist KIT 10 reduces neuroinflammatory parameters in microglia by inhibiting the COX-2/PGE₂ pathway. Further experiments are necessary to better elucidate its effects and mechanisms. Nevertheless, the modulation of inflammation by GPR55 might be a new therapeutic option to treat CNS disorders with a neuroinflammatory background such as AD or depression.