Cell Line BV-2 Research Articles

CXCL12 is involved in \u03b1-synuclein-triggered neuroinflammation of Parkinson\u2019s disease

BackgroundThe mechanisms underlying the pathogenesis and progression of Parkinson’s disease (PD) remain elusive, but recent opinions and perspectives have focused on whether the inflammation process induced by microglia contributes to α-synuclein-mediated toxicity. Migration of microglia to the substantia nigra (SN) could precede neurodegeneration in A53T mice. We hypothesized that CXCL12 could be a mediator in the α-synuclein-induced migration of microglia.MethodsAfter establishing appropriate animal and cell culture models, we explored the relationship between α-synuclein and CXCL12 in A53T mice, primary microglia, and BV-2 cell lines. We also explored the mechanisms of these interactions and the signaling processes involved in neuroinflammation.ResultsWe confirmed the positive correlation between α-synuclein and CXCL12 in the postmortem brain tissue of PD patients and the upregulated CXCR4 expression in SN microglia of A53T mice. In addition, as expected, α-synuclein increased the production of CXCL12 in microglia via TLR4/IκB-α/NF-κB signaling. Importantly, CXCL12/CXCR4/FAK/Src/Rac1 signaling was shown to be involved in α-synuclein-induced microglial accumulation.ConclusionsOur study suggests that CXCL12 could be a novel target for the prevention of α-synuclein-triggered ongoing microglial responses. Blocking CXCL12/CXCR4 may be a potential therapeutic approach for PD progression.

BAP31 regulates IRAK1-dependent neuroinflammation in microglia

BackgroundMicroglia, the mononuclear immune cells of the central nervous system (CNS), are essential for the maintenance of CNS homeostasis. BAP31, a resident and ubiquitously expressed protein of the endoplasmic reticulum, serves as a sorting factor for its client proteins, mediating the subsequent export, retention, and degradation or survival. Recently, BAP31 has been defined as a regulatory molecule in the CNS, but the function of BAP31 in microglia has yet to be determined. In the present study, we investigated whether BAP31 is involved in the inflammatory response of microglia.MethodsThis study used the BV2 cell line and BAP31 conditional knockdown mice generated via the Cre/LoxP system. A BAP31 knockdown experiment was performed to elucidate the role of BAP31 in the endogenous inflammatory cytokine production by microglial BV2 cells. A mouse model of lipopolysaccharide (LPS)-induced cognitive impairment was established to evaluate the neuroprotective effect of BAP31 against neuroinflammation-induced memory deficits. Behavioral alterations were assessed with the open field test (OFT), Y maze, and Morris water maze. The activation of microglia in the hippocampus of mice was observed by immunohistochemistry. Western blot, enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining, and reverse transcription quantitative real-time polymerase chain reaction (RT-PCR) were used to clarify the mechanisms.ResultsBAP31 deficiency upregulates LPS-induced proinflammatory cytokines in BV2 cells and mice by upregulating the protein level of IRAK1, which in turn increases the translocation and transcriptional activity of NF-κB p65 and c-Jun, and moreover, knockdown of IRAK1 or use of an IRAK1 inhibitor reverses these functions. In the cognitive impairment animal model, the BAP31 knockdown mice displayed increased severity in memory deficiency accompanied by an increased expression of proinflammatory factors in the hippocampus.ConclusionsThese findings indicate that BAP31 may modulate inflammatory cytokines and cognitive impairment induced by neuroinflammation through IRAK1, which demonstrates that BAP31 plays an essential role in microglial inflammation and prevention of memory deficits caused by neuroinflammation.

Dopamine attenuates lipopolysaccharide-induced expression of proinflammatory cytokines by inhibiting the nuclear translocation of NF-κB p65 through the formation of dopamine quinone in microglia.

Many reports have indicated that dopamine has immunomodulatory effects on peripheral immune cells. The purpose of this study was to reveal the immunomodulatory effect of dopamine on the expression of proinflammatory cytokines in microglial cells, which are the immune cells of the central nervous system. In murine microglial cell line BV-2 cells, pretreatment with dopamine for 24 h attenuated the lipopolysaccharide (LPS)-induced expression of proinflammatory cytokines such as tumor-necrosis factor-α, interleukin-1β, and interleukin-6. Neither (5R)-8-chloro-3-methyl-5-phenyl-1,2,4,5-tetrahydro-3-benzazepin-7-ol; hydrochloride (SCH-23390) nor sulpiride, which are dopamine D1-like and D2-like receptor antagonists, respectively, affected the attenuation of LPS-induced expression of cytokines by dopamine. In addition, pretreatment with neither (−)-(6aR,12bR)-4,6,6a,7,8,12b-Hexahydro-7-methylindolo[4,3-a]phenanthridin (CY208-243) nor bromocriptine, dopamine D1-like and D2-like receptor agonists, respectively, was effective in doing so. However, N-acetylcysteine (NAC), which inhibits dopamine oxidation to dopamine quinone, did inhibit this attenuated expression. Dopamine increased the level of quinoproteins, and this increase was inhibited by NAC. Western blot and immunocytochemical analyses revealed that dopamine inhibited LPS-induced nuclear translocation of nuclear factor-kappa B (NF-κB) p65. Dopamine also attenuated the expression of cytokines and the nuclear translocation of NF-κB p65 induced by LPS in mouse microglial cells in primary culture. These results suggest that dopamine attenuated LPS-induced expression of cytokines by inhibiting the nuclear translocation of NF-κB p65 through the formation of dopamine quinone in microglial cells.

Simultaneous characterisation of multiple Mahonia fortunei bioactive compounds in rat plasma by UPLC–MS/MS for application in pharmacokinetic studies and anti-inflammatory activity in vitro

The stems of Mahonia fortunei (MF) are commonly used in Chinese Traditional Medicine and contain multiple bioactive compounds, including 3,4,5-trimethoxyphenol-1-O-β-d-glucopyranoside (1), 5-hydroxypicolinic acid methyl ester (2), acortatarin A (3), syringic acid (4), 9-epi-acortatarin A (5), vomifoliol (6), corydaldine (7), noroxyhydrastinine (8), columbamine (9), jatrorrhizine (10), palmatine (11), berberine (12) and schisandrin (13). The pharmacokinetics of these 13 compounds in the rat plasma were assessed using a novel sensitive, rapid, and specific UPLC-ESI-MS/MS method after oral administration of an aqueous extract of MF stems. Carbamazepine was employed as the internal standard (IS) and all samples were precipitated with acetonitrile. Chromatographic separation was performed on a C18 column using a gradient elution at 0.3 mL/min, with the mobile phase consisting of acetonitrile and 0.06 % formic acid and 5 mM ammonium acetate aqueous solution. The calibration curves showed satisfactory linearity in the examination area (r2 ≥ 0.99). The accuracy, precision, extraction recovery, matrix effect, and stability were within acceptable ranges. The method successfully assessed the pharmacokinetics of these 13 compounds. In vitro, compound 12 exhibited potent inhibitory activity against production of nitric oxide (NO) in the RAW264.7 cell line when stimulated by lipopolysaccharide (LPS), while compounds 7, 12, and 13 were the most potent inhibitors of NO production in the BV2 cell line when stimulated by LPS. The IC50 values of compounds 7, 12 and 13 were 42.81, 20.55 and 22.74 μM. We conclude that these compounds have promise for clinical application, although their synergistic action may be more effective than that by any single compound alone.

Suppressors of cytokine signalling (SOCS)-1 inhibits neuroinflammation by regulating ROS and TLR4 in BV2 cells.

The suppressors of cytokine signaling (SOCS) proteins are physiological suppressors of cytokine signaling which have been identified as a negative feedback loop to weaken cytokine signaling. However, the underlying molecular mechanisms is unknown. This study was to investigate the role of SOCS1 in the oxygen-glucose deprivation and reoxygenation (OGDR) or LPS-induced inflammation in microglia cell line BV-2 cells. BV-2 microglial cells were used to construct inflammation model. A SOCS1 over-expression plasmid was constructed, and the SOCS1-deficient cells were generated by utilizing the CRISPR/CAS9 system. BV-2 microglial cells were pretreated with over-expression plasmid or SOCS1 CRISPR plasmid before OGDR and LPS stimulation. The effect of SOCS1 on proinflammatory cytokines, toll-like receptor 4 (TLR4), and reactive oxygen species (ROS) were evaluated. We found that SOCS1 increased in OGDR or LPS-treated BV-2 microglial cells in vitro. SOCS1 over-expression significantly reduced the production of proinflammatory cytokines including tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6, and CRISPR/CAS9-mediated SOCS1 knockout reversed this effect. Also we determined that SOCS1 over-expression reduced the level of reactive oxygen species (ROS) while the absence of SOCS1 increased the production of ROS after OGDR or LPS-stimulated inflammation. Furthermore, we found that OGDR and LPS induced the expression of toll-like receptor 4 (TLR4) in BV2 cells. Nevertheless, SOCS1 over-expression attenuated the expression of TLR4, while knockdown of SOCS1 upregulated TLR4. Our study indicated that SOCS1 played a protective role under inflammatory conditions in OGDR or LPS treated BV-2 cells through regulating ROS and TLR4. These data demonstrated that SOCS1 served as a potential therapeutic target to alleviate inflammation after ischemic stroke.

Dietary Supplementation of the Antioxidant Curcumin Halts Systemic LPS-Induced Neuroinflammation-Associated Neurodegeneration and Memory/Synaptic Impairment via the JNK/NF-κB/Akt Signaling Pathway in Adult Rats.

Curcumin is a natural polyphenolic compound widely known to have antioxidant, anti-inflammatory, and antiapoptotic properties. In the present study, we explored the neuroprotective effect of curcumin against lipopolysaccharide- (LPS-) induced reactive oxygen species- (ROS-) mediated neuroinflammation, neurodegeneration, and memory deficits in the adult rat hippocampus via regulation of the JNK/NF-κB/Akt signaling pathway. Adult rats were treated intraperitoneally with LPS at a dose of 250 μg/kg for 7 days and curcumin at a dose of 300 mg/kg for 14 days. After 14 days, the rats were sacrificed, and western blotting and ROS and lipid peroxidation assays were performed. For immunohistochemistry and confocal microscopy, the rats were perfused transcardially with 4% paraformaldehyde. In order to verify the JNK-dependent neuroprotective effect of curcumin and to confirm the in vivo results, HT-22 neuronal and BV2 microglial cells were exposed to LPS at a dose of 1 μg/ml, curcumin 100 μg/ml, and SP600125 (a specific JNK inhibitor) 20 μM. Our immunohistochemical, immunofluorescence, and biochemical results revealed that curcumin inhibited LPS-induced oxidative stress by reducing malondialdehyde and 2,7-dichlorofluorescein levels and ameliorating neuroinflammation and neuronal cell death via regulation of the JNK/NF-κB/Akt signaling pathway both in vivo (adult rat hippocampus) and in vitro (HT-22/BV2 cell lines). Moreover, curcumin markedly improved LPS-induced memory impairment in the Morris water maze and Y-maze tasks. Taken together, our results suggest that curcumin may be a potential preventive and therapeutic candidate for LPS-induced ROS-mediated neurotoxicity and memory deficits in an adult rat model.

Bone marrow mesenchymal stem cell-derived exosome uptake and retrograde transport can occur at peripheral nerve endings

We investigated the occurrence of mesenchymal stem cell (MSC)-derived exosome uptake and retrograde transport at peripheral nerve endings using bone marrow MSCs (bMSCs) transduced with recombinant CD63-green fluorescent protein (GFP) lentiviral plasmid. GFP was used to track the release of bMSC-derived exosomes and the uptake and transport at peripheral nerve terminals, the dorsal root ganglion (DRG), and the spinal cord. In vitro cell culture and injection of a CD63-GFP exosome suspension into the right gastrocnemius muscle of an in vivo rat model were also performed. Fluorescence microscopy of co-cultured CD63-GFP exosomes and SH-SY5Y or BV2 cell lines and primary cultured DRG cells in a separate experiment demonstrated exosome uptake into DRG neurons and glia. Moreover, we observed both retrograde axoplasmic transport and hematogenous transport of exosomes injected into rat models at the DRG and the ipsilateral side of the anterior horn of the spinal cord using fluorescence microscopy, immunohistochemistry, and Western blot analyses. In conclusion, we showed that exosome uptake at peripheral nerve endings and retrograde transport of exosomes to DRG neurons and spinal cord motor neurons in the anterior horn can occur. In addition, our findings propose a novel drug delivery approach for treating neuronal diseases.

Tripartite motif containing protein 37 involves in thrombin stimulated BV-2 microglial cell apoptosis and interleukin 1β release

Intracerebral hemorrhage (ICH) is the most common of stroke with high mortality and severe morbidity. Peroxisome proliferator-activated receptor gamma (PPARγ) plays a neuronprotective role in ICH. In the current study, TRIM37 mRNA expression in peripheral blood mononuclear cells (PBMCs) was found to be increased in ICH patients compared to that in healthy controls (n = 15). TRIM37 bound to PPARγ and enhanced its ubiquitination in mouse microglial BV-2 cell line. According to previous studies, thrombin is produced in the brain instantaneously after ICH and triggers the activation of microglia. Here, thrombin induced TRIM37 expression, cell apoptosis and interleukin-1β (IL-1β) release in BV-2 cells, while TRIM37 knockdown partially reversed the effects of thrombin on BV-2 cells. TRIM37 overexpression showed similar effects as thrombin on BV-2 cells, and PPARγ agonist rosiglitazone abolished the effects of TRIM37. In summary, TRIM37 involved in apoptosis and IL-1β release in BV-2 microglia by regulating PPARγ ubiquitination. The present data established a potential biological role of TRIM37 in ICH-induced brain damage and may provide insight into the development of therapy strategies for ICH.

Oligostilbenes from the leaves of Gnetum latifolium and their biological potential to inhibit neuroinflammation

Oligostilbenes are polyphenol oligomers derived from resveratrol and are commonly produced by members of the Gnetaceae family, and many researchers have focused on their anti-inflammatory activities. The EtOAc fraction of a Gnetum latifolium extract showed inhibitory activity against neuroinflammation induced by the transfection of Aβ1-42 into microglial BV-2 cells. The bioassay-guided isolation of the 70% EtOH extract of this plant resulted in three previously undescribed resveratrol oligostilbenes and ten known stilbene derivatives. The structures of the isolated compounds were established based on extensive NMR spectroscopic analysis. The absolute configurations of the three undescribed compounds were confirmed by comparison with available compounds with known stereochemistry and by ECD calculations and molecular modelling. Latifoliols A and B are the first reported oligostilbenes with a bridged 3-oxabicyclo[3.3.0]octane moiety, and latifoliol C was formed by the condensation of gnemontanin G with oxyresveratrol. Moreover, the hypothetical biogenetic pathway of latifoliols A, B and C was proposed. The potential anti-inflammatory activities of the thirteen isolated compounds were tested by measuring their effect on the secreted NO concentrations induced by transfection with plasmids expressing the Aβ1-42 gene in the BV-2 cell line. Interestingly, cis- and trans-shegansu B and latifolol, whose structures contained double bonds, strongly inhibited NO secretion in BV-2 cells, supporting the double binding effect of the stilbene derivative on inhibitory activity.

Stereochemistry and innate immune recognition: (+)-norbinaltorphimine targets myeloid differentiation protein 2 and inhibits toll-like receptor 4 signaling.

Deregulation of innate immune TLR4 signaling contributes to various diseases including neuropathic pain and drug addiction. Naltrexone is one of the rare TLR4 antagonists with good blood-brain barrier permeability and showing no stereoselectivity for TLR4. By linking 2 naltrexone units through a rigid pyrrole spacer, the bivalent ligand norbinaltorphimine was formed. Interestingly, (+)-norbinaltorphimine [(+)-1] showed ∼25 times better TLR4 antagonist activity than naltrexone in microglial BV-2 cell line, whereas (-)-norbinaltorphimine [(-)-1] lost TLR4 activity. The enantioselectivity of norbinaltorphimine was further confirmed in primary microglia, astrocytes, and macrophages. The activities of meso isomer of norbinaltorphimine and the molecular dynamic simulation results demonstrate that the stereochemistry of (+)-1 is derived from the (+)-naltrexone pharmacophore. Moreover, (+)-1 significantly increased and prolonged morphine analgesia in vivo. The efficacy of (+)-1 is long lasting. This is the first report showing enantioselective modulation of the innate immune TLR signaling.-Zhang, X., Peng, Y., Grace, P. M., Metcalf, M. D., Kwilasz, A. J., Wang, Y., Zhang, T., Wu, S., Selfridge, B. R., Portoghese, P. S., Rice, K. C., Watkins, L. R., Hutchinson, M. R., Wang, X. Stereochemistry and innate immune recognition: (+)-norbinaltorphimine targets myeloid differentiation protein 2 and inhibits toll-like receptor 4 signaling.

Inhibitory Effects of the Two Novel TSPO Ligands 2-Cl-MGV-1 and MGV-1 on LPS-induced Microglial Activation.

The 18 kDa translocator protein (TSPO) ligands 2-Cl-MGV-1 and MGV-1 can attenuate cell death of astrocyte-like cells (U118MG) and induce differentiation of neuronal progenitor cells (PC-12). Lipopolysaccharide (LPS) is a bacterial membrane endotoxin that activates cellular inflammatory pathways by releasing pro-inflammatory molecules, including cytokines and chemokines. The aim of the present study was to assess the immuno-modulatory effect of TSPO ligands in activated microglial cells. We demonstrated that the TSPO ligands 2-Cl-MGV-1 and MGV-1 can prevent LPS-induced activation of microglia (BV-2 cell line). Co-treatment of LPS (100 ng/mL) with these TSPO ligands (final concentration- 25 µM) reduces significantly the LPS-induced release of interleukin-6 (IL-6) from 16.9-fold to 2.5-fold, IL-β from 8.3-fold to 1.6-fold, interferon-γ from 16.0-fold to 2.2-fold, and tumor necrosis factor-α from 16.4-fold to 1.8-fold. This anti-inflammatory activity seems to be achieved by inhibition of NF-κB p65 activation. Assessment of initiation of ROS generation and cell metabolism shows significant protective effects of these two novel TSPO ligands. The IL-10 and IL-13 levels were not affected by any of the TSPO ligands. Thus, it appears that the ligands suppress the LPS-induced activation of some inflammatory responses of microglia. Such immunomodulatory effects may be relevant to the pharmacotherapy of neuro-inflammatory diseases.

Icariin targets Nrf2 signaling to inhibit microglia-mediated neuroinflammation

Microglia-mediated neuroinflammation is an important contributor to the pathogenesis of neurodegenerative diseases. Inhibition of neuroinflammation has been proved to be effective in neurodegenerative diseases treatment. Nuclear factor erythroid 2 related factor 2 (Nrf2) is a key mediator of endogenous inducible defense systems in the body. In response to oxidative stress, Nrf2 translocates to the nucleus and binds to specific DNA sites termed as anti-oxidant response elements to initiate transcription of cytoprotective genes, such as hemeoxygenase-1 (HO-1) and nicotinamide adenine dinucleotide phosphate: quinine oxidoreductase-1 (NQO1). However, insufficient Nrf2 activation has been closely associated with the progress of neurodegenerative diseases. New findings have linked activation of Nrf2 signaling to anti-inflammatory effects. Icariin (ICA), a natural compound derived from Herba Epimedii, possesses amounts of pharmacological activities, such as anti-aging, anti-oxidation and anti-inflammatory effects. Recent studies have confirmed that ICA exerted neuroprotection against neurodegenerative diseases. However, the mechanisms underlying ICA-mediated neuroprotection were not fully understood. In the present study, microglia BV-2 cell lines were performed to investigate the anti-neuroinflammatory effects of ICA and the mechanisms of actions. Results showed that ICA suppressed lipopolysaccharide (LPS)-induced microglial pro-inflammatory factors production. In addition, activation of Nrf2 signaling pathway participated in ICA-mediated anti-neuroinflammation, as evidenced by the following observations. First, Nrf2 siRNA reversed ICA-reduced microglial activation and pro-inflammatory factors release. Second, a selective inhibitor of HO-1 abolished ICA-mediated anti-neuroinflammatory actions. This study will give us an insight into the potential of Nrf2 and neuroinflammation in terms of opening up an alternative therapeutic strategy for neurodegenerative diseases.

Caffeine Modulates Cadmium-Induced Oxidative Stress, Neuroinflammation, and Cognitive Impairments by Regulating Nrf-2/HO-1 In Vivo and In Vitro.

Cadmium (Cd), a nonbiodegradable heavy metal and one of the most neurotoxic environmental and industrial pollutants, promotes disturbances in major organs and tissues following both acute and chronic exposure. In this study, we assessed the neuroprotective potential of caffeine (30 mg/kg) against Cd (5 mg/kg)-induced oxidative stress-mediated neuroinflammation, neuronal apoptosis, and cognitive deficits in male C57BL/6N mice in vivo and in HT-22 and BV-2 cell lines in vitro. Interestingly, our findings indicate that caffeine markedly reduced reactive oxygen species (ROS) and lipid peroxidation (LPO) levels and enhanced the expression of nuclear factor-2 erythroid-2 (Nrf-2) and hemeoxygenase-1 (HO-1), which act as endogenous antioxidant regulators. Also, 8-dihydro-8-oxoguanine (8-OXO-G) expression was considerably reduced in the caffeine-treated group as compared to the Cd-treated group. Similarly, caffeine ameliorated Cd-mediated glial activation by reducing the expression of glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (Iba-1), and other inflammatory mediators in the cortical and hippocampal regions of the mouse brain. Moreover, caffeine markedly attenuated Cd-induced neuronal loss, synaptic dysfunction, and learning and cognitive deficits. Of note, nuclear factor-2 erythroid-2 (Nrf-2) gene silencing and nuclear factor-κB (NF-κB) inhibition studies revealed that caffeine exerted neuroprotection via regulation of Nrf-2- and NF-κB-dependent mechanisms in the HT-22 and BV-2 cell lines, respectively. On the whole, these findings reveal that caffeine rescues Cd-induced oxidative stress-mediated neuroinflammation, neurodegeneration, and memory impairment. The present study suggests that caffeine might be a potential antioxidant and neuroprotective agent against Cd-induced neurodegeneration.

Anti-inflammatory and antiproliferative compounds from Sphaeranthus africanus

BackgroundSphaeranthus africanus has been used in traditional Vietnamese medicine to treat sore throat, and to relieve pain and swelling. However, the anti-inflammatory activity of this plant had not yet been investigated. Previously, we isolated five carvotacetones (1–5) from this plant that displayed cytotoxicity against several cancer cell lines. PurposeThe objective of this study was to isolate further constituents from S. africanus and to investigate the anti-inflammatory activity of all constituents. Furthermore, the anti-proliferative activity of the newly isolated compounds was evaluated. Study design and methodsCompounds were isolated from the upper parts of S. africanus by chromatographic methods. Structures were determined using spectroscopic techniques, like NMR and MS. All nine compounds isolated from S. africanus were evaluated for inhibitory activity against COX-1 and COX-2 isoenzymes in-vitro, COX-2 mRNA expression and influence on NO production. The anti-proliferative activities of newly isolated compounds (6–9) were evaluated by XTT viability assay with four cancer cell lines, namely CCRF-CEM, MDA-MB-231, HCT-116, and U-251 cells. ResultsTwo diastereomeric carvotacetones (3-angeloyloxy-5-[2″S,3″R-dihydroxy-2″-methyl-butanoyloxy]-7-hydroxycarvotacetone (6) and 3-angeloyloxy-5-[2″R,3″R-dihydroxy-2″-methyl-butanoyloxy]-7-hydroxycarvotacetone (7), asperglaucide (8) and chrysoplenol D (9) were isolated from S. africanus. COX-1 and COX-2 assays of compounds 1–9 revealed that compounds 1 and 2 possess potent and selective COX-2 inhibitory activity with IC50 values of 3.6 and 0.5 μM, respectively. COX-2 gene expression assay showed that some carvotacetones exhibited inhibitory effects on COX-2 gene expression in THP-1 macrophages. Compound 4 is the most active compound inhibiting the synthesis of COX-2 by 55% at 2.06 μM. In the iNOS assay, all seven carvotacetones inhibited NO production in BV2 and RAW cell lines with IC50 values ranging from 0.2 to 2.9 μM. Compound 4 showed potent inhibitory activity with IC50 values of 0.2 μM in both BV2 and RAW cell lines. Molecular docking studies revealed the binding orientations of 1 and 2 in the active sites of COX-2. XTT assay of the newly isolated compounds revealed that the two isomeric carvotacetones (6–7) exhibited considerable anti-proliferative activity against four cancer cell lines (CCRF-CEM, MDA-MB-231, HCT-116, U-251) with IC50 values ranging from 1.23 to 8 μM. ConclusionFor the first-time, the diastereomeric carvotacetones (6–7) were isolated as separate compounds, and their anti-proliferative activity was determined. Selective COX-2 inhibitory, COX-2 mRNA expression and NO production inhibitory activities by some of the major constituents of S. africanus supports the traditional medical application of this plant for the treatment of inflammation-related disorders.

Involvement of the microglial NLRP3 inflammasome in the anti-inflammatory effect of the antidepressant clomipramine

Background: Depression has recently been referred to as a neuroimmune disease because it is characterized by inflammatory changes in the cerebral cortex and hippocampus. Studies have demonstrated that microglial activation plays a crucial role in releasing inflammatory cytokines in the central nervous system (CNS), thereby contributing to depression, the mechanism underlying which remains unclear. Methods: First, we examined microglial activation and inflammatory changes in C57BL/6 male mice injected with lipopolysaccharide (LPS; 1 mg/kg), which leads to depressive behaviors in mice that were attenuated by the antidepressant clomipramine. Second, we utilized a BV2 cell line and primary microglial cultures to determine the inflammatory response in vitro, and the effects of clomipramine exerted on the inflammatory response using real-time polymerase chain reaction and ELISA. Third, we utilized NLRP3 (NOD-like receptor protein 3) knock-out (KO) mice to prove that NLRP3 is involved in the effects of clomipramine. Results: The results showed that LPS injection induced depressive-like behaviors in mice, as assessed using several behavioral tests including body weight, and forced swimming and tail suspension tests. The LPS-induced expression of interleukin-1beta (IL-1β), IL-6, and tumor necrosis factor alpha could be downregulated by clomipramine pre-treatment both in vivo and in vitro. The inhibitory effect of clomipramine on the LPS-induced increase in cytokines was found at both the protein and gene levels. Clomipramine significantly reduced the LPS-induced increase in NLRP3 gene expression in BV2 cells. Furthermore, we utilized NLRP3 KO mice to explore whether NLPP3 was involved in this process and found that clomipramine treatment inhibits the LPS-induced increased expression of IL-1β. Conclusion: These results imply that clomipramine could attenuate depressive behaviors and neuroinflammation induced by LPS via partial regulation of NLRP3.

Naringenin Produces Neuroprotection Against LPS-Induced Dopamine Neurotoxicity via the Inhibition of Microglial NLRP3 Inflammasome Activation.

Background: Parkinson's disease (PD) is the second most prevalent central nervous system (CNS) degenerative disease and characterized by slow and progressive loss of dopamine (DA) neurons in the midbrain substantia nigra. Microglia-mediated neuroinflammation has been considered as the major central event in the process of DA neuronal loss. Thus, inhibition of neuroinflammation could possess a more viable strategy for PD treatment. Naringenin (NAR), a natural flavanoid contained in citrus fruit and grapefruits, possesses amounts of pharmacological activities. Recent studies indicated that NAR produced neuroprotection against several neurological disorders. However, the mechanisms underlying NAR-generated neuroprotection are not fully illuminated.Methods: In the present study, rat nigral stereotaxic injection of lipopolysaccharide (LPS)-induced DA neuronal loss was performed to investigate NAR-mediated neuroprotection. In addition, BV-2 and MN9D cell lines were applied to explore the underlying mechanisms.Results: NAR protected DA neurons against LPS-induced neurotoxicity. Also, NAR suppressed microglial nod-like receptor protein 3 (NLRP3) inflammasome signaling activation and the subsequent pro-inflammatory factors release. In addition, NAR-mediated DA neuroprotection was dependent on the inhibition of microglial NLRP3 inflammasome activation, as evidenced by the observations that NAR-reduced pro-inflammatory factors production and further NAR-exerted DA neuroprotection against LPS-induced neuronal damage was not discerned after microglial NLRP3 siRNA treatment.Conclusions: This study demonstrated that NAR targeted microglial NLRP3 inflammasome to protect DA neurons against LPS-induced neurotoxicity. These findings suggest NAR might hold a promising therapeutic potential for PD.

MicroRNA-124 regulates the expression of p62/p38 and promotes autophagy in the inflammatory pathogenesis of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor and nonmotor symptoms due to the selective loss of midbrain dopaminergic neurons. The evidence for a chronic inflammatory reaction mediated by microglial cells in the brain is particularly strong in PD. In our previous study, we have shown that brain-specific microRNA-124 (miR-124) is significantly down-regulated in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD and that it can also inhibit neuroinflammation during the development of PD. However, further investigation is required to understand whether the abnormal expression of miR-124 regulates microglial activation. In this study, we found that the expression of sequestosome 1 (p62) and phospho-p38 mitogen-activated protein kinases (p-p38) showed a significant increase in LPS-treated immortalized murine microglial cell line BV2 cells in an MPTP-induced mouse model of PD. Knockdown of p62 could suppress the secretion of proinflammatory cytokines and p-p38 of microglia. Besides, inhibition of p38 suppressed the secretion of proinflammatory cytokines and promoted autophagy in BV2 cells. Moreover, our study is the first to identify a unique role of miR-124 in mediating the microglial inflammatory response by targeting p62 and p38 in PD. In the microglial culture supernatant transfer model, the knockdown of p62 in BV2 cells prevented apoptosis and death of human neuroblastoma cell lines SH-SY5Y (SH-SY5Y) cells following microglia activation. In addition, the exogenous delivery of miR-124 could suppress p62 and p-p38 expression and could also attenuate the activation of microglia in the substantia nigra par compacta of MPTP-treated mice. Taken together, our data suggest that miR-124 could inhibit neuroinflammation during the development of PD by targeting p62, p38, and autophagy, indicating that miR-124 could be a potential therapeutic target for regulating the inflammatory response in PD.-Yao, L., Zhu, Z., Wu, J., Zhang, Y., Zhang, H., Sun, X., Qian, C., Wang, B., Xie, L., Zhang, S., Lu, G. MicroRNA-124 regulates the expression of p62/p38 and promotes autophagy in the inflammatory pathogenesis of Parkinson's disease.

MicroRNA-181b-5p attenuates early postoperative cognitive dysfunction by suppressing hippocampal neuroinflammation in mice

BackgroundPostoperative cognitive dysfunction (POCD) is a common complication after surgery and its occurrence is associated with increased morbidity and mortality. However, the pathophysiology of this complication remains largely unknown. Efforts to identify causes of POCD have focused on the hippocampal neuroinflammation. Recently, accumulated evidence indicates that NeurimmiRs, a subset of microRNAs (miRNAs), which modulate both neuronal and immune processes, play an important role in neuroinflammation. However, the impact of NeurimmiRs on POCD has not been investigated. We hypothesized that NeurimmiRs is involved in surgery-induced cognitive impairment in adult mice via mediating hippocampal neuroinflammation. MethodsMicroRNA(miR)-181b-5p was found to be downregulated in the hippocampi of mice with POCD using microRNA array, which was also verified in vivo in the mouse model of POCD by Quantitative real-time polymerase chain reaction (qPCR). Subsequently, the expression of miR-181b-5p was measured in lipopolysaccharide (LPS) stimulated BV-2 microglial cells and hippocampal tissues of the mice with POCD. Then, loss of function and overexpression studies were performed by transfection with miR-181b-5p mimic/ inhibitor in cultured BV-2 cell lines and intrahippocampal injection of miR-181b-5p agomir before Surgery/Anesthesia, to identify the role of miR-181b-5p in neuroinflammation and cognitive impairments. QPCR, western blot and ELISA were used to determine the expression of proinflammatory mediators. Immunofluorescence staining was applied to evaluate the activation of microglia. Furthermore, we used bioinformatics analysis and dual-luciferase assay to predict and verify the potential target of miR-181b-5p. ResultsThe results indicated that miR-181b-5p mimic could repress the mRNA and protein expression of proinflammatory mediators, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and monocyte chemoattractant protein (MCP)-1 in LPS-stimulated BV-2 microglial cells, while the miR-181b-5p inhibitor induced upregulation of the above-mentioned proinflammatory factors. Further bioinformatics analysis showed that miR-181b-5p was predicted to potentially target the 3′-untranslated region (UTR) of TNF-α, and binding sites of miR-181b-5p in the 3′-UTR of TNF-α were identified by dual-luciferase assay. Importantly, injecting miR-181b-5p agomir into the hippocampus of mice before surgery, ameliorated the hippocampus-dependent memory, and was accompanied by downregulation of proinflammatory factors expression and reduced microglial activation in the hippocampus of POCD mice. ConclusionsCollectively, these findings suggest that miR-181b-5p attenuates early POCD by suppressing hippocampal neuroinflammation in mice. They also highlight the importance of studying miRNAs in the context of POCD and identify miR-181b-5p as a novel potential therapeutic target for improving POCD.

Identification of phenolic compounds from a unique citrus species, finger lime (Citrus australasica) and their inhibition of LPS-induced NO-releasing in BV-2 cell line

In this study, a unique citrus species (Citrus australasica) was selected, and its fruit characteristics, phenolic compounds and ability to inhibit inflammation were preliminarily studied. Finger lime fruits showed distinctive features in shape, size, weight, colour, total soluble solids, water-soluble pectin, sugar and acids contents. Combining UPLC-HRMS and UPLC-DAD analysis, 31 phenolics, 1 secoiridoid derivative and 1 neolignan glycoside were preliminarily identified and quantified. The phenolics composition of finger limes showed cultivar and tissue specificity. Antioxidant evaluation showed that extracts from finger lime cultivar of ‘XiangBin’ exhibited better antioxidant capacities than cultivar of ‘LiSiKe’, especially in peel. LPS-induced NO-releasing model was performed in the mouse microglia BV-2 cell line. Results illustrated that finger limes inhibited the NO-releasing and the inflammation-related cytokines including IL-1β, IL-6 and TNFα elevation. QRT-PCR revealed that finger lime extracts alleviated LPS-induced upregulation of iNOS, IL-6, JAK2, TNFα, TLR2, TLR4, IL-1β, NF-κB and LPS-induced downregulation of IκBα. This study may expand our knowledge on the physiochemical characteristics and bioactive properties of citrus fruits.

Three Polymethoxyflavones Purified from Ougan (Citrus reticulata Cv. Suavissima) Inhibited LPS-Induced NO Elevation in the Neuroglia BV-2 Cell Line via the JAK2/STAT3 Pathway.

In order to establish an efficient method for separation of polymethoxyflavones (PMFs) and explore the anti-inflammatory mechanism of PMF monomers, a citrus variety rich in PMFs, Ougan (Citrus reticulata cv. Suavissima), was selected, and three monomers, including nobiletin, tangeretin, and 5-demethylnobiletin, were purified by ultrasonic-assisted extraction, solid phase extraction, and high-speed countercurrent chromatography separation. UPLC-MS was used to identify the three monomers. UPLC determined purities of 99.87% to nobiletin, 99.76% to tangeretin, and 98.75% to 5-demethylnobiletin with the standard curve method. A lipopolysaccharide (LPS)-induced NO releasing model was performed in the mouse microglia BV-2 cell line. Results illustrated that PMF monomers inhibited the NO release and the inflammation-related cytokines, including IL-1β, IL-6, and TNFα elevation. QRT-PCR revealed that PMFs alleviated LPS-induced upregulation of iNOS, IL-6, JAK2, TNFα, IL-1β, and NF-κB and LPS-induced downregulation of IκBα, while they did not affect TLR1, TLR2, TLR4, and TLR6. STAT3 expression was repressed by tangeretin and 5-demethylnobiletin, but not by nobiletin. Western blot assay also showed a suppression of expression and phosphorylation of JAK2 by all three PMF monomers, while STAT3 phosphorylation was restrained by tangeretin and 5-demethylnobiletin. The mechanism was primarily verified by the JAK2 inhibitor Ruxolitinib and the STAT3 inhibitor Stattic.