Anti-Neuroinflammatory Effects of Arecae pericarpium on LPS-Stimulated BV2 Cells.

Arecae pericarpium (AP), the fruit peel of the betel palm, is a traditional Oriental herbal medicine. AP is used to treat various diseases and conditions, such as ascites, edema, and urinary retention, in traditional Korean medicine. Recent studies have demonstrated its anti-obesity and antibacterial effects; however, its anti-neuroinflammatory effects have not yet been reported. Therefore, we investigated the anti-neuroinflammatory effects of AP on lipopolysaccharide (LPS)-stimulated mouse microglia in this study. To determine the anti-neuroinflammatory effects of AP on BV2 microglial cells, we examined the production of nitric oxide (NO) using Griess assay and assessed the mRNA expression levels of inflammatory mediators, such as inducible NO synthase (iNOS) and cyclooxygenase (COX)-2, and pro-inflammatory cytokines, such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α, using a real-time reverse transcription-polymerase chain reaction. Furthermore, we determined the levels of mitogen-activated protein kinases and IκBα via Western blotting to understand the regulating mechanisms of AP. AP treatment decreased NO production in LPS-stimulated BV2 cells. Additionally, AP suppressed the expression of iNOS and COX-2 and the production of pro-inflammatory cytokines. AP also inhibited the activation of p38 and nuclear factor-kappa B (NF-κB) in LPS-stimulated BV2 cells. Therefore, AP exerts anti-neuroinflammatory effects via inactivation of the p38 and NF-κB pathways.

Clerodane diterpenoids with in-vitro anti-neuroinflammatory activity from the tuberous root of Tinospora sagittata (Menispermaceae).

Twenty-six clerodane diterpenoids have been isolated from T. sagittata, a plant species of traditional Chinese medicine Radix Tinosporae, also named as "Jin Guo Lan". Among them, there are eight previously undescribed clerodane diterpenoids (tinotanoids A-H: 1-8), and 18 known diterpenoids (9-26). The absolute configurations of compounds 1, 2, 5, 8, 13, 17 and 20 were determined by single-crystal X-ray diffraction. Compound 1 is the first example of rotameric clerodane diterpenoid with a γ-lactone ring which is constructed between C-11 and C-17; meanwhile, compounds 3 and 4 are two pairs of inseparable epimers. Compounds 2, 12 and 17 demonstrated excellent inhibitory activity on NO production against LPS-stimulated BV-2 cells with IC50 values of 9.56 ± 0.69, 9.11 ± 0.53 and 11.12 ± 0.70 µM, respectively. These activities were significantly higher than that of the positive control minocycline (IC50 = 23.57 ± 0.92 µM). Moreover, compounds 2, 12 and 17 dramatically reduced the LPS-induced upregulation of iNOS and COX-2 expression. Compounds 2 and 12 significantly inhibited the levels of pro-inflammatory cytokines TNF-α, IL-1ß and IL-6 that were increased by LPS stimulation.

Essential oil from Ligusticum chuanxiong Hort. Alleviates lipopolysaccharide-induced neuroinflammation: Integrating network pharmacology and molecular mechanism evaluation.

ETHNOPHARMACOLOGICAL RELEVANCE: Chuanxiong, the rhizome of Ligusticum chuanxiong Hort., is an ancient herbal medicine that has gained extensive popularity in alleviating migraines with satisfying therapeutic effects in China. As the major bioactive component of Chuanxiong, the essential oil also exerts a marked impact on the treatment of migraine. It is widely recognized that neuroinflammation contributes to migraine. However, it remains unknown whether Chuanxiong essential oil has anti-neuroinflammatory activity. AIM OF THE STUDY: To explore the anti-neuroinflammatory properties of Chuanxiong essential oil and its molecular mechanisms by network pharmacology analysis and in vitro experiments. MATERIALS AND METHODS: Gas chromatography-mass spectrometry (GC-MS) was used to identify the chemical components of Chuanxiong essential oil. Public databases were used to predict possible targets, build the protein-protein interaction network (PPI), and perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Moreover, cytological experiments, nitric oxide assay, enzyme-link immunosorbent assay, western blotting, and immunofluorescence assay were adopted to prove the critical signaling pathway in lipopolysaccharide (LPS)-induced BV2 cells. RESULTS: Thirty-six compounds were identified from Chuanxiong essential oil by GC-MS, and their corresponding putative targets were predicted. The network pharmacology study identified 232 candidate targets of Chuanxiong essential oil in anti-neuroinflammation. Furthermore, Chuanxiong essential oil was found to potentially affect the C-type lectin receptor, FoxO, and NF-κB signaling pathways according to the KEGG analysis. Experimentally, we verified that Chuanxiong essential oil could significantly reduce the overproduction of inflammatory mediators and pro-inflammatory factors via the NF-κB signaling pathway. CONCLUSION: Chuanxiong essential oil alleviates neuroinflammation through the NF-κB signaling pathway, which provides a theoretical foundation for a better understanding of the clinical application of Chuanxiong essential oil in migraine treatment.

Huang-Lian-Jie-Du decoction drug-containing serum inhibits IL-1ß secretion from D-glucose and PA induced BV2 cells via autophagy/NLRP3 signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Huang-Lian-Jie-Du decoction (HLJDD), a famous traditional Chinese medicine prescription with heat-clearing and detoxifying effects, has been widely used to treat diabetes, dementia, stroke, and other diseases. However, the detailed mechanisms of HLJDD against type 2 diabetes associated cognitive dysfunction (DACD) through inhibiting interleukin-1ß (IL-1ß) mediated neuroinflammation remain to be further elucidated. AIM OF THE STUDY: The aim of this study was to investigate the effect and potential mechanism of HLJDD on IL-1ß secretion in a DACD model of BV2 cells induced by D-glucose and palmitic acid (PA). MATERIALS AND METHOD: sUltra-performance liquid chromatography-quadrupole/electrostatic field orbital well high-resolution mass spectrometry technology was used to analyze the compounds in HLJDD drug-containing serum. The cytotoxicity was detected by cell counting kit-8. Enzyme-linked immunosorbent assay was used to measure the secretion of IL-1ß in BV2 cells. Reactive oxygen species, glutathione, superoxide dismutase, and malondialdehyde kits were used to detect the intracellular oxidative stress levels. The autophagy level was determined by autophagy staining kit and transmission electron microscope. The expression levels of autophagy-related 7 (Atg7), P62, LC3, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3(NLRP3), Caspase1, and IL-1ß were detected by real-time PCR, immunofluorescence, and western blotting. The Atg7siRNA was transfected into BV2 cells to produce autophagy inhibitory effect. Then the effect of HLJDD drug-containing serum on IL-1ß secretion in D-glucose and PA induced BV2 cells and the potential mechanism of autophagy-NLRP3 inflammasome activation were further observed. RESULTS: Eighty-eight compounds were preliminarily identified in HLJDD drug-containing serum, among which geniposide, baicalin, palmatine, berberine, wogonoside, wogonin, and geniposidic acid were identified as the main prototype components of HLJDD into the blood. In this study, the DACD model of BV2 cells induced by high concentrations of glucose and PA was successfully constructed. HLJDD drug-containing serum significantly reduced the secretion of IL-1ß and the activity of NLRP3 inflammasome with improving the oxidative stress level. Interestingly, the enhanced autophagy level was also found. After transfection of Atg7siRNA into BV2 cells, the effect of HLJDD drug-containing serum on autophagy promotion was reversed, but the inhibitory effects on IL-1ß secretion, NLRP3 inflammasome activation, and oxidative stress were reduced. CONCLUSIONS: These results indicated that the inhibition of HLJDD drug-containing serum on the IL-1ß secretion in D-glucose and PA induced BV2 cells was related to autophagy promotion, the decreased NLRP3 inflammasome activation, and the improved oxidative stress. Moreover, the improvement of HLJDD drug-containing serum on IL-1ß secretion, NLRP3 inflammasome activation, and oxidative stress were all closely associated with Atg7 mediated autophagy promotion. Geniposide, baicalin, palmatine, berberine, wogonoside, wogonin, and geniposidic acid may be the potential active ingredients of HLJDD drug-containing serum.

Cocktail of three isoquinoline alkaloids derived from Glaucium grandiflorum Boiss. & A. Huet subsp. refractum (Nábelek) Mory inhibits the production of LPS-induced ROS, pro-inflammatory cytokines, and mediators through the down-regulation of p38 MAPK in BV-2 cells.

Glaucium grandiflorum extracts have traditionally been used to treat brain-related disorders. G. grandiflorum extracts also exhibited inhibitory effects on cholinesterase enzymes, as well as antigenotoxic activity. However, no research has been done on the effect of G. grandiflorum alkaloid extracts on the anti-oxidative and anti-inflammatory mechanisms. In this study we aimed to evaluate the anti-oxidative and anti-inflammatory activities of the alkaloid extract obtained from G. grandiflorum as well as the mechanisms responsible for their neuroprotective effects in neuronal damage caused by LPS in BV2 cells. We used LC-MS/MS and 1H, 13C NMR analysis to determine the presence of major alkaloids (allocryptopine, tetrahydropalmatine, and tetrahydroberberine N-oxide (trans-cannadine-N-oxide) in the alkaloid extracts. We used flow cytometry to study the alkaloid extracts' effects on ROS production; we also employed qRT-PCR and Western Blot to analyze the effects of oxidative stress and inflammation-related genes and proteins. ROS production within the cell was inhibited by chloroform alkaloid extract (CAE). There occurred marked CAE-induced reductions in IL-1ß, Cox-2, and iNOS mRNA expressions. We also observed marked reductions in IL-6 and TNF-α mRNA expressions with methanol alkaloid extract (MAE). CAE effectively suppressed IL-1ß and iNOS protein levels, especially as in qRT-PCR studies, while MAE effectively reduced IL-6 and TNF-α protein levels. Additionally, MAE was found to be prominent in suppressing the levels of Cox-2 protein, unlike qRT-PCR studies. According to our study findings, oxidative stress brought about by inflammation was suppressed by alkaloid extracts from G. grandiflorum which can be attributed to their suppressor effects on the pro-inflammatory cytokines-mediators, and p38 MAPK. As a result, a drug active substance that suppresses oxidative stress and inflammation has been brought to the neuropharmacological field.

Anti-Neuroinflammatory Components from Clausena lenis Drake.

Clausena lenis Drake (C. lenis) is a folk medicinal herb to treat influenza, colds, bronchitis, and malaria. The 95% and 50% ethanol extract of C. lenis showed significant nitric oxide (NO) inhibition activity in BV-2 microglial cells stimulated by lipopolysaccharide (LPS). Bio-guided isolation of the active extract afforded five new compounds, including a chlorine-containing furoquinoline racemate, (±)-claulenine A (1), an amide alkaloid, claulenine B (2), a prenylated coumarin, claulenin A (3), a furocoumarin glucoside, clauleside A (4), and a multi-prenylated p-hydroxybenzaldehyde, claulenin B (5), along with 33 known ones. Their structures were determined via spectroscopic methods, and the absolute configurations of new compounds were assigned via the electronic circular dichroism (ECD) calculations and single-crystal X-ray diffraction analysis. Compounds 2, 23, 27, 28, 33, and 34 showed potent anti-neuroinflammatory effects on LPS-induced NO production in BV-2 microglial cells, with IC50 values in the range of 17.6-40.9 µM. The possible mechanism was deduced to interact with iNOS through molecular docking.

Effects of Gynostemma pentaphyllum on spinal cord motor neurons and microglial cells in vitro.

The regenerative capability of spinal cord neurons is limited to impossible. Thus, experimental approaches supporting reconstruction/regeneration are in process. This study focused on the evaluation of the protective potency of an extract from Gynostemma pentaphyllum (GP), a plant used in traditional medicine with anti-oxidative and neuroprotective activities, in vitro on organotypic spinal cord cultures, the motor-neuron-like NSC-34 cell line and the microglial cell line BV-2. Organotypic cultures were mechanically stressed by the slicing procedure and the effect of GP on motor neuron survival and neurite sprouting was tested by immunohistochemistry. NSC-34 cells were neuronal differentiated by using special medium. Afterwards, cell survival (propidium iodide/fluorescein diacetate labeling), proliferation (BrdU-incorporation), and neurite sprouting were evaluated. BV-2 cells were stimulated with LPS/interferon γ and subjected to migration assay and nanoparticle uptake. Cell survival, proliferation and the expression pattern of different microglial activation markers (cFOS, iNOS) as well as transcription factors (PPARγ, YB1) were analyzed. In organotypic cultures, high-dose GP supported survival of motor neurons and especially of the neuronal fiber network. Despite reduced neurodegeneration, however, there was a GP-mediated activation of astro- and microglia. In NSC-34 cells, high-dosed GP had degenerative and anti-proliferative effects, but only in normal medium. Moreover, GP supported the neuro-differentiation ability. In BV-2 cells, high-dosed GP was toxic. In lower dosages, GP affected cell survival and proliferation when combined with LPS/interferon γ. Nanoparticle uptake, migration ability, and the transcription factor PPARγ, however, GP affected directly. The data suggest positive effects of GP on injured spinal motor neurons. Moreover, GP activated microglial cells. The dual role of microglia (protective/detrimental) in neurodegenerative processes required further experiments to enhance the knowledge about GP effects. Therefore, a possible clinical use of GP in spinal cord injuries is still a long way off.

Comparison of anti-inflammatory effects of Mecasin and its constituents on lipopolysaccharide-stimulated BV2 cells.

Mecasin, a traditional medicine, contains nine herbal constituents: Curcuma longa, Salvia miltio rhiza, Gastrodia elata, Chaenomeles sinensis, Polygala tenuifolia, Paeonia japonica, Glycyrrhiza uralensis, Atractylodes japonica and processed Aconitum carmichaeli. Several biological effects of mecasin have been described both in vivo and in vitro. Previous studies have demonstrated that mecasin has anti-inflammatory effects. The purpose of the present study was to determine anti-inflammatory effects of mecasin and its natural product constituents on lipopolysaccharide (LPS)-stimulated BV2 cells by measuring nitrite and nitric oxide contents. Nitrite production levels in LPS-stimulated BV2 cells incubated with mecasin and each individual constituent of mecasin were measured. The results suggested that C. longa, P. tenuifolia and P. japonica inhibited nitrite production in a pattern similar to that of mecasin. The effect of mecasin was likely a result of synergistic effects of its natural herb constituents.

Blueberry Counteracts BV-2 Microglia Morphological and Functional Switch after LPS Challenge.

Microglia, the innate immune cells of the CNS, respond to brain injury by activating and modifying their morphology. Our study arises from the great interest that has been focused on blueberry (BB) for the antioxidant and pharmacological properties displayed by its components. We analyzed the influence of hydroalcoholic BB extract in resting or lipopolysaccharide (LPS)-stimulated microglia BV-2 cells. BB exerted a protective effect against LPS-induced cytotoxicity, as indicated by cell viability. BB was also able to influence the actin cytoskeleton organization, to recover the control phenotype after LPS insult, and also to reduce LPS-driven migration. We evaluated the activity of Rho and Rac1 GTPases, which regulate both actin cytoskeletal organization and migratory capacity. LPS caused an increase in Rac1 activity, which was counteracted by BB extract. Furthermore, we demonstrated that, in the presence of BB, mRNA expression of pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α decreased, as did the immunofluorescence signal of iNOS, whereas that of Arg-1 was increased. Taken together, our results show that, during the inflammatory response, BB extract shifts the M1 polarization towards the M2 phenotype through an actin cytoskeletal rearrangement. Based on that, we might consider BB as a nutraceutical with anti-inflammatory activities.

Effects of Selected Resveratrol Analogues on Activation and Polarization of Lipopolysaccharide-Stimulated BV-2 Microglial Cells.

Increasing health-promoting effects of resveratrol and its molecular structural analogues have been discovered, and the acting mechanism has been explored. However, the activity comparison of such compounds in targeting macrophage-related inflammation associated with neurodegenerative diseases remains untouched. In this study, we evaluated the activation and polarization transition of lipopolysaccharide (LPS)-stimulated BV-2 mouse microglial macrophages exposed to resveratrol (RES) and its analogues pterostilbene (PTE), oxyresveratrol (ORES), acetyl-trans-resveratrol (ARES), and trans-2,3,5,4'-tetrahydroxystilbene-2-O-glucopyranoside (TSG). At 10 µM, all of the five stilbene compounds have effectively suppressed the LPS-stimulated BV-2 cell release of proinflammatory mediators such as NO, TNF-α, iNOS, IL-1ß, and IL-6. Mechanism study elucidated that they exert anti-inflammatory effects through MAPKs (ERK1/2, JNK, and p38) and NF-κB signaling pathways. Further investigation in treating BV-2 cells with resveratrol and its analogues revealed the reversal of LPS-induced phenotype molecules from M1 (iNOS, IL-1ß, IL-6, and CD86) to M2 (Arg1, CD163, and IL-10) subtypes, manifesting that these five stilbenes suppressed inflammation through modulating the polarized phenotypes of BV-2 microglia. Most importantly, PTE demonstrated the most potent inhibitory activity among these five stilbene compounds. Therefore, this study not only highlights microglia-induced inflammatory responses as a potential therapeutic target but also suggests future insights in considering the options of nutraceutical development for resveratrol and its analogues.

Protective Effect and Mechanism of Boswellic Acid and Myrrha Sesquiterpenes with Different Proportions of Compatibility on Neuroinflammation by LPS-Induced BV2 Cells Combined with Network Pharmacology.

Frankincense and myrrha (FM), commonly used as a classical herbal pair, have a wide range of clinical applications and definite anti-inflammatory activity. However, anti-neuroinflammation effects and mechanisms are not clear. In this study, we adopted a lipopolysaccharide (LPS)-induced microglial (BV2) cell model and a network pharmacology method to reveal the anti-neuroinflammatory effects and mechanisms of boswellic acid (BA) and myrrha sesquiterpenes (MS) with different proportions of compatibility. The data showed that the different ratios of BA and MS had different degrees of inhibition of interleukin-1ß (IL-1ß), IL-6, and inducible nitric oxide synthase (iNOS) mRNA expression, down-regulated the phosphor-nuclear factor kappa B/nuclear factor kappa B (p-NF-Ò¡B)/(NF-Ò¡B), phosphorylated protein kinase b/protein kinase b (p-AKT/AKT), and Toll-like receptor 4 (TLR4) protein expression levels, and increased phospho-PI3 kinase (p-PI3K) protein expression levels. When the ratios of BA and MS were 10:1, 5:1, and 20:1, better effective efficacy was exhibited. According to the correlation analysis between the effect index and bioactive substances, it was suggested that 2-methoxy-5-acetoxy -fruranogermacr-1(10)-en-6-one (Compound 1), 3α-acetyloxylanosta-8,24-dien-21-oic acid (Compound 2), 11-keto-boswellic acid (Compound 3), and 3-acetyl-11-keto-ß -boswellic acid (Compound 4) made important contributions to the treatment of neuroinflammation. Furthermore, based on the network pharmacological analysis, it was found that these four active compounds acted on 31 targets related to neuroinflammation and were involved in 32 signaling pathways which mainly related to the immune system, cardiovascular system, and nervous system, suggesting that BA and MS could be used to treat neuroinflammation.

Neuroprotective effect of biosynthesised gold nanoparticles synthesised from root extract of Paeonia moutan against Parkinson disease - In vitro &In vivo model.

Parkinson disease is one of the most common neurological movement disorders affecting geriatric population. Biosynthesized gold nanoparticles are the ideal alternatives spotlighted by many researchers to treat various diseases. In the present study we synthesized gold nanoparticles using the root extract of Paeonia mountan, woody trees which are used in traditional Chinese medicine to be prescribed for diverse diseases. The synthesis of gold nanoparticles was confirmed with UV-Vis spectroscopic analysis and characterized using FTIR, HR-TEM, EDAX and XRD analysis. The cytotoxicity property of synthesized gold nanoparticles was assessed using MTT assay in the murine microglial BV2 cells. The neuroprotective effect of synthesized gold nanoparticles in inflammatory agent lipopolysaccharides triggered murine microglial BV2 cells was evaluated using nitric oxide, prostaglandin E2 and inflammatory cytokines assays such as IL-6&IL-1ß. Further to confirm in vivo effect of synthesized nanoparticles, the nanoparticles were treated to Parkinson induced C57BL/6 mice. Behavioral, biochemical and molecular analysis were performed to estimate the potency of synthesized gold nanoparticles against the Parkinson induction in mice model. Our characterization results prove the gold nanoparticles synthesized using Paeonia mountan fulfills the requirement of ideal nanodrug and it potentially inhibited the inflammation in in vitro murine microglial BV2. The results of in vivo experiments authentically confirm gold nanoparticles synthesized using Paeonia mountan alleviates the neuroinflammation and improves the motor coordination in Parkinson induced mice.

Anti-neuroinflammatory activities of extract and polymethoxyflavonoids from immature fruit peels of Citrus 'Hebesu'.

Chronic neuroinflammation is reported to be associated in the progression of many neurodegenerative diseases and there is an increasing interest for the natural products as neuroprotective and anti-neuroinflammatory agents. In present research, we evaluated the potential inhibitory effects of extract, fractions, and isolated compounds from Citrus 'Hebesu' on lipopolysaccharide (LPS)-induced inflammatory responses using BV-2 murine microglial cells. The dried methanol extract (CH) was suspended in water and partitioned with hexane and ethyl acetate to give hexane soluble (CHH), ethyl acetate soluble (CHE), and water soluble (CHW) fractions. The extract (CH) and fractions CHH and CHE inhibited the expression of mRNA encoding pro-inflammatory cytokine interleukin (IL)-1ß. CHE and CHH were further purified by various column chromatographic methods to obtain hesperidin (1), tangeretin (2), 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone (3), 3,5,6,7,8,3',4'-heptamethoxyflavone (4), nobiletin (5), 3,4,5-trimethoxy-trans-cinnamic alcohol (6), and meranzin hydrate (7). Among them, three polymethoxyflavonoids, 3, 4, and 5 significantly inhibited the expression of IL-1ß mRNA. PRACTICAL APPLICATIONS: Citrus 'Hebesu' is a local cultivar in Hyuga City, Miyazaki prefecture, Japan and its immature fruits are consumed with different food recipes. Till now, there is no detailed study on the chemical constituents and anti-neuroinflammatory activity of this cultivar. In this study, seven compounds were isolated from the peels of immature fruits. Methanol extract, hexane, and ethyl acetate fractions and three polymethoxyflavonoids showed a significant inhibitory activity against expression of IL-1ß mRNA. Consumption of peels of Citrus 'Hebesu' might play important role in the prevention and treatment of neurodegenerative diseases, however, detailed mechanism based in vivo studies are necessary in future for providing more scientific evidences.

Anti-Inflammatory Activity of A Polyphenolic Extract from Arabidopsis thaliana in In Vitro and In Vivo Models of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder and the primary form of dementia in the elderly. One of the main features of AD is the increase in amyloid-beta (Aß) peptide production and aggregation, leading to oxidative stress, neuroinflammation and neurodegeneration. Polyphenols are well known for their antioxidant, anti-inflammatory and neuroprotective effects and have been proposed as possible therapeutic agents against AD. Here, we investigated the effects of a polyphenolic extract of Arabidopsis thaliana (a plant belonging to the Brassicaceae family) on inflammatory response induced by Aß. BV2 murine microglia cells treated with both Aß25⁻35 peptide and extract showed a lower pro-inflammatory (IL-6, IL-1ß, TNF-α) and a higher anti-inflammatory (IL-4, IL-10, IL-13) cytokine production compared to cells treated with Aß only. The activation of the Nrf2-antioxidant response element signaling pathway in treated cells resulted in the upregulation of heme oxygenase-1 mRNA and in an increase of NAD(P)H:quinone oxidoreductase 1 activity. To establish whether the extract is also effective against Aß-induced neurotoxicity in vivo, we evaluated its effect on the impaired climbing ability of AD Drosophila flies expressing human Aß1⁻42. Arabidopsis extract significantly restored the locomotor activity of these flies, thus confirming its neuroprotective effects also in vivo. These results point to a protective effect of the Arabidopsis extract in AD, and prompt its use as a model in studying the impact of complex mixtures derived from plant-based food on neurodegenerative diseases.

Neuroprotective effect of tempeh against lipopolysaccharide-induced damage in BV-2 microglial cells.

Objectives: This study evaluated the bioactive composition of tempeh products and examined the effects of tempeh on BV-2 microglial cell cytotoxicity, neurotrophic effects, and expression of inflammatory genes.Methods: Tempeh products included soybean fermented by Rhizopus, soybean fermented through cocultivation with Rhizopus and Lactobacillus, and red bean fermented through cocultivation with Rhizopus and Lactobacillus (RT-C). We analyzed the bioactive contents of tempeh extracts and evaluated the effects of tempeh water extract on lipopolysaccharide (LPS)-treated BV-2 cells.Results: The results showed that RT-C water extract had the highest concentrations of γ-aminobutyric acid (GABA) and anthocyanin. The tempeh water extracts, especially RT-C, reduced the formation of LPS-induced reactive oxygen species, downregulated the levels of nitric oxide synthase and phospho-cyclic-AMP response element-binding protein, and upregulated the expression of brain-derived neurotrophic factor (BDNF).Discussion: Our data demonstrate that RT-C has the highest concentrations of GABA and anthocyanin, more effectively reduces oxidative stress and inflammation, and increases the expression of BDNF in LPS-induced BV-2 cells.

Yokukansan Reduces Cuprizone-Induced Demyelination in the Corpus Callosum Through Anti-inflammatory Effects on Microglia.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). The release of inflammatory cytokines and pro-oxidant molecules from microglia has been shown to play a key role in the pathology of MS. Thus, suppression of microglial cell activation is an attractive therapeutic option. Yokukansan, a traditional Japanese herbal medicine, has been shown to suppress microglial activity in the CNS. However, whether or not yokukansan reduces demyelination observed in the CNS during MS remains unknown. In this study, female C57BL/6 mice were fed a diet containing 0.2% cuprizone (bis-cyclohexanone oxaldihydrazone) to induce demyelination in the corpus callosum. We investigated whether or not yokukansan reduces cuprizone-induced demyelination using immunohistochemical analyses. Furthermore, we examined the in vitro anti-inflammatory effects of yokukansan on LPS-stimulated BV2 cells, a murine microglial cell line. Luxol fast blue staining and immunostaining for myelin basic protein demonstrated that yokukansan reduces demyelination of the corpora callosa of cuprizone-fed mice. In addition, yokukansan significantly decreased the number of activated microglial cells in the corpora callosa of cuprizone-fed mice. Furthermore, treatment with 500 µg/ml yokukansan suppressed the expression of interleukin-1ß and inducible nitric-oxide synthase mRNA and protein in LPS-stimulated BV2 cells. These findings suggest that yokukansan reduces demyelination owing to anti-inflammatory effects on microglia. As yokukansan has few adverse effects, yokukansan has the potential to be a novel option to treat MS.

Protective Effects of α-Tocopherol, γ-Tocopherol and Oleic Acid, Three Compounds of Olive Oils, and No Effect of Trolox, on 7-Ketocholesterol-Induced Mitochondrial and Peroxisomal Dysfunction in Microglial BV-2 Cells.

Lipid peroxidation products, such as 7-ketocholesterol (7KC), may be increased in the body fluids and tissues of patients with neurodegenerative diseases and trigger microglial dysfunction involved in neurodegeneration. It is therefore important to identify synthetic and natural molecules able to impair the toxic effects of 7KC. We determined the impact of 7KC on murine microglial BV-2 cells, especially its ability to trigger mitochondrial and peroxisomal dysfunction, and evaluated the protective effects of α- and γ-tocopherol, Trolox, and oleic acid (OA). Multiple complementary chemical assays, flow cytometric and biochemical methods were used to evaluate the antioxidant and cytoprotective properties of these molecules. According to various complementary assays to estimate antioxidant activity, only α-, and γ-tocopherol, and Trolox had antioxidant properties. However, only α-tocopherol, γ-tocopherol and OA were able to impair 7KC-induced loss of mitochondrial transmembrane potential, which is associated with increased permeability to propidium iodide, an indicator of cell death. In addition, α-and γ-tocopherol, and OA were able to prevent the decrease in Abcd3 protein levels, which allows the measurement of peroxisomal mass, and in mRNA levels of Abcd1 and Abcd2, which encode for two transporters involved in peroxisomal ß-oxidation. Thus, 7KC-induced side effects are associated with mitochondrial and peroxisomal dysfunction which can be inversed by natural compounds, thus supporting the hypothesis that the composition of the diet can act on the function of organelles involved in neurodegenerative diseases.

The ethyl acetate fraction from Physalis alkekengi inhibits LPS-induced pro-inflammatory mediators in BV2 cells and inflammatory pain in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Physalis alkekengi is an edible herb whose fruit and calyx are traditionally used to treat a wide range of diseases including inflammation, toothache, and rheumatism. However, the effects of Physalis alkekengi fruit along with its calyx (PAF) on neuroinflammation and inflammatory pain behavior have not been reported yet. AIM OF THE STUDY: This study evaluated the anti-inflammatory effect of PAF on lipopolysaccharide (LPS)-induced neuroinflammation and several in vivo model of inflammatory pain in mice. MATERIALS AND METHODS: Here, first we studied the effects of PAF fractions on the production of pro-inflammatory mediators in LPS-treated BV2 microglial cells using enzyme-linked immunosorbent assay. The translocation of nuclear factor-kappa B (NF-κB) and the involvements of Akt and mitogen-activated protein (MAP) kinases in ethyl acetate fraction of PAF (PAF-EA)-mediated anti-inflammatory effect were measured using Western blotting. In in vivo experiments, the efficacy of PAF-EA was evaluated at the doses of 100 and 200mg/kg using several chemical-induced models of inflammatory pain such as acetic acid-induced writhing, formalin-induced paw licking and edema. RESULTS: We found that compared to other fractions, the PAF-EA more potently inhibited the LPS-induced generation of nitric oxide, tumor necrosis factor-α, interleukin-6 and reactive oxygen species. It also inhibited LPS-induced nuclear translocation of NF-κB. These actions of EA fraction were found to be associated with a disruption of Akt and MAP kinases signaling pathways. The EA fraction also significantly inhibited acetic acid-induced writhing, formalin-induced licking time and edema in mice. CONCLUSIONS: Our findings support the ethnopharmacological use of P. alkekengi fruit along with its calyx as an anti-inflammatory agent and suggest that the EA fraction of PAF may serve as a potential candidate to treat different neurological disorders and pain associated with inflammation.

Safflower Yellow regulates microglial polarization and inhibits inflammatory response in LPS-stimulated Bv2 cells.

Activated microglia, especially polarized M1 cells, produce pro-inflammatory cytokines and free radicals, thereby contributing directly to neuroinflammation and various brain disorders. Given that excessive or chronic neuroinflammation within the central nervous system (CNS) exacerbates neuronal damage, molecules that modulate neuroinflammation are candidates as neuroprotective agents. In this study, we provide evidence that Safflor yellow (SY), the main active component in the traditional Chinese medicine safflower, modulates inflammatory responses by acting directly on BV2 microglia. LPS stimulated BV2 cells to upregulate expression of TLR4-Myd88 and MAPK-NF-κB signaling pathways and to release IL-1ß, IL-6, TNF-α, and COX-2. However, SY treatment inhibited expression of TLR4-Myd88 and p-38/p-JNK-NF-κB, downregulated expression of iNOS, CD16/32, and IL-12, and upregulated CD206 and IL-10. In conclusion, our results demonstrate that SY exerts an anti-inflammatory effect on BV2 microglia, possibly through TLR-4/p-38/p-JNK/NF-κB signaling pathways and the conversion of microglia from inflammatory M1 to an anti-inflammatory M2 phenotype.

Dimeric guaianolides from Artemisia absinthium.

Five dimeric guaianolides, absinthins A-E, and seven known dimeric guaianolides were isolated from Artemisia absinthium. Their structures were elucidated based on 1D- and 2D-NMR experiments, including (1)H NMR, (13)C NMR, DEPT, (1)H-(1)H COSY, HSQC, HMBC, and NOESY, and through HRESIMS data analysis. The absolute configuration of the known compound, anabsinthin, was determined by X-ray crystallographic analysis. The isolated compounds were tested to assess their inhibitory activities on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV-2 cells; absinthin C and isoanabsinthin exhibited significant inhibitory effects with IC50 values of 1.52 and 1.98µM, respectively.