LPS-stimulated BV2 Cells Research Articles

Cirrhosinones A-H, 24-hydroxy cevanine-type alkaloids from Fritillariacirrhosa

Eight undescribed isosteroidal alkaloids cirrhosinones A-H (1–8), along with six known isosteroidal alkaloids (9–14), were isolated from the bulbs of Fritillaria cirrhosa D. Don. Their structures were determined by HRESIMS and 2D NMR analysis, and their absolute configurations were established by X-ray analysis. Compounds 1–8 possessed a typical cevanine-type alkaloid skeleton with a hydroxyl group rarely substituted at C-24 and compounds 4–8 possessed rare 7α or 7β-hydroxyl groups. This was the first report of both C-7 and C-24 hydroxyl groups substituted cevanine-type alkaloids. In addition, an approach for distinguishing D/E cis and trans conformations of cevanine-type alkaloids by 1H NMR data was developed. Moreover, the correlations between the relative configurations of 3-OH, 7-OH, 22-C, 24-OH, and 25-Me and the 1H NMR and 13C NMR data were also summarized. Compounds 1–9 exhibited moderate NO inhibitory activities in LPS-stimulated BV-2 cells at the concentration of 40 μM. The acetylcholinesterase inhibitory activities of compounds 1–7 and 9–10 were also evaluated and none of them showed acetylcholinesterase inhibitory activities at the concentrations of 20–80 μM.

The Novel KLF4/BIG1 Regulates LPS-mediated Neuro-inflammation and Migration in BV2 Cells via PI3K/Akt/NF-kB Signaling Pathway

Excessive microglia activation occurred in many neurodegenerative diseases. Brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1, ARFGEF1) is involved in cell migration and neurite growth. In the present study, we aimed to explore the effects and potential mechanisms of BIG1 in LPS-mediated neuro-inflammation and migration in BV2 cells. Loss-of-function and gain-of-function experiments were performed. Inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-10) mRNA levels and concentrations were analyzed by Quantitative Real-time PCR (RT-qPCR) and ELISA kits. The NO concentration was tested by ELISA kit. iNOS and COX-2 mRNA and protein levels were measured by RT-qPCR and western blot. Cell migration was determined by transwell assay. The results demonstrated that BIG1 silencing reduced TNF-α, IL-1β, and IL-6 expression, while increased IL-10 expression. The NO production, iNOS and COX-2 expression were clearly inhibited by BIG1 knockdown in the presence of LPS. Furthermore, ablation of BIG1 attenuated the migration capacity of BV2 cells. Overexpression of BIG1 displayed the opposite trends. Moreover, we found BIG1 suppression inhibited PI3K/Akt/NF-κB pathway activation. 740Y-P, an agonist of PI3K, abolished the roles of BIG1 silencing in neuro-inflammation and migration. Additionally, ChIP-qPCR and Dual-luciferase reporter assay determined that KLF4 binds to the promoter of BIG1, western blot analysis demonstrated that KLF4 could regulate BIG1 positively. In addition, we observed that BIG1 overexpression partly rescued the biological activities of KLF4 silencing in neuro-inflammation and migration in LPS-stimulated BV2 cells. Taken together, BIG1 was mediated by KLF4 regulated LPS-mediated neuro-inflammation and migration in BV2 cells via PI3K/Akt/NF-kB signaling pathway.

Antioxidant and anti-inflammatory activities of Oroxylum indicum Kurz (L.) fruit extract in lipopolysaccharide-stimulated BV2 microglial cells


 
 
 
 Purpose: To investigate the anti-inflammatory effects and antioxidant activity of Oroxylum indicum (L.) Kurz fruit extract in lipopolysaccharide (LPS)-stimulated BV2 microglia.
 Methods: BV2 cells were treated with LPS for 24 h in the presence or absence of O. indicum fruit extract. Then, nitric oxide (NO), reactive oxygen species (ROS) and interleukin 6 (IL-16) levels were measured using Griess reagent assay, CM-H2DCFDA and enzyme-linked immunosorbent (ELISA) assays, respectively. The in vitro antioxidant property of the extract was also investigated by 1,1- diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) assays.
 Results: Levels of IL-6, NO, and ROS in LPS-treated BV2 cells were significantly higher than those in control (p < 0.01). However, exposure of LPS-treated BV2 cells to O. indicum extract led to a marked decrease in the levels of these parameters, when compared to the untreated cells (p < 0.01). Results from DPPH and ABTS assays showed that the O. indicum extract exhibited good antioxidant properties, with total flavonoid and total phenolic contents of 115.58 ± 1.09 and 131.04 ± 2.37 mg/g of dried extract, respectively.
 Conclusion: The results demonstrate that O. indicum fruit exerts anti-oxidant and anti-inflammatory effects in LPS-stimulated BV2 cells. Thus O. indicum fruit might be beneficial in the development of novel anti-oxidative and anti-neuroinflammatory herbal medicines. However, the mechanisms by which O. indium fruits reduces NO and IL-6 needs to be further investigated.
 
 
 

6-Benzyloxyphthalides as selective and reversible monoamine oxidase B inhibitors with antioxidant and anti-neuroinflammatory activities for Parkinson’s disease treatment

A series of 6-benzyloxyphthalides were designed and synthesized as potent monoamine oxidase B inhibitors with antioxidant and anti-neuroinflammatory activities. The representative compounds 8f and 14a exhibited excellent selective MAO-B inhibition activity (IC50 = 1.33 nM, SI = 865; IC50 = 0.02 nM, SI = 40250, respectively) and moderate antioxidant activity (0.34 and 0.36 Trolox equivalent, respectively). Further studies showed that they were competitive and quasi-reversible MAO-B inhibitors. In cellular experiments, they could significantly decrease the production of NO and TNF-α in LPS-stimulated BV-2 cells to perform their in vitro anti-neuroinflammatory activities. Moreover, BBB permeability study and the predicted physicochemical properties indicated they were suitable for the CNS. Finally, in in vivo acute and subacute MPTP-induced mice model of PD, 8f and 14a could significantly improve most behavioral disorders, restore the DA content and decrease the MDA content in the mice striatum, exhibiting better anti-PD effects than clinically used safinamide. Hence, compounds 8f and 14a are identified in our studies as prospective prototype in the research of innovative multifunctional drugs for Parkinson’s disease treatment.

Forsythoside A Mitigates Alzheimer's-like Pathology by Inhibiting Ferroptosis-mediated Neuroinflammation via Nrf2/GPX4 Axis Activation.

Ferroptosis and neuroinflammation play crucial roles in Alzheimer's disease (AD) pathophysiology. Forsythoside A (FA), the main constituent of Forsythia suspensa (Thunb.) Vahl., possesses anti-inflammatory, antibacterial, antioxidant, and neuroprotective properties. The present study aimed to investigate the potential role of FA in AD neuropathology using male APP/PS1 double transgenic AD mice, Aβ1-42-exposed N2a cells, erastin-stimulated HT22 cells, and LPS-induced BV2 cells. FA treatment significantly improved mitochondrial function and inhibited lipid peroxidation in Aβ1-42-exposed N2a cells. In LPS-stimulated BV2 cells, FA treatment decreased the formation of the pro-inflammatory factors IL-6, IL-1β, and NO. In male APP/PS1 mice, FA treatment ameliorated memory and cognitive impairments and suppressed Aβ deposition and p-tau levels in the brain. Analyses using proteomics, immunohistochemistry, ELISA, and western blot revealed that FA treatment significantly augmented dopaminergic signaling, inhibited iron deposition and lipid peroxidation, prevented the activation of IKK/IκB/NF-κB signaling, reduced the secretion of pro-inflammatory factors, and promoted the production of anti-inflammatory factors in the brain. FA treatment exerted anti-ferroptosis and anti-neuroinflammatory effects in erastin-stimulated HT22 cells, and the Nrf2/GPX4 axis played a key role in these effects. Collectively, these results demonstrate the protective effects of FA and highlight its therapeutic potential as a drug component for AD treatment.

Ethanol Extract of Elaeagnus glabra f. oxyphylla Branches Alleviates the Inflammatory Response Through Suppression of Cyclin D3/Cyclin-Dependent Kinase 11p58 Coupled to Lipopolysaccharide-Activated BV-2 Microglia

Neuroinflammation plays a pivotal role in the pathogenesis of neurodegenerative diseases and is characterized by microglial dysregulation. Here, we explored the beneficial effects of a leaf extract of Elaeagnus glabra f. oxyphylla (EGFO), a native medicinal plant to Korea, South China, Japan, and Taiwan, on neuroinflammation using lipopolysaccharide (LPS)-stimulated BV-2 microglia. Levels of the inflammatory mediators were determined by enzyme-linked immunosorbent assays and reverse transcription–polymerase chain reaction. The phospho levels of mitogen-activated protein kinases, which are key kinase molecules in the inflammatory signaling pathway in microglia, were analyzed by Western blotting. Treatment with EGFO significantly suppressed the LPS-mediated induction of nitric oxide and prostaglandin E2. Consistently, EGFO treatment in LPS-stimulated BV-2 cells markedly reduced the inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) levels. The best concentration of EGFO that could reduce TNF-α and IL-6 was 100 μg/mL. EGFO relatively reduced the messenger RNA expression of TNF-α and IL-6 by 0.36 and 0.32-fold ratio, respectively, compared to LPS treatment. Moreover, EGFO markedly reduced the phospho levels of p38 and the c-jun N-terminal kinase. Furthermore, antibody microarray and immunoblotting data revealed that the pharmacological mechanisms driving the antineuroinflammatory action of EGFO involve prevention of the cyclin D3/cyclin-dependent kinase 11p58 (CDK11p58) interaction. In conclusion, our results demonstrate that EGFO alleviates the inflammatory response through the suppression of cyclin D3/CDK11p58 coupling in LPS-activated BV-2 microglia. We propose the potential of EGFO as a novel drug candidate for neurodegenerative diseases by targeting neuroinflammation.

Cytotoxic and nitric oxide inhibitory activities of triterpenoids from Lycopodium clavatum L.

Using combined chromatographic separation techniques, three new triterpenoids named lycomclavatols A–C (1–3), a new natural product, methyl lycernuate-A (4), as well as seven known compounds (5–11), were isolated from the methanol extract of the whole plants of Lycopodium clavatum. Their chemical structures were established based on 1 D/2D NMR and HR-ESI-MS spectroscopic analyses. Among the isolates, compound 1 exhibited inhibitory activity on NO production in LPS-stimulated BV2 cells (IC50 = 36.0 μM). In addition, 1 was cytotoxic against both HepG2 and A549 cancer cell lines, with IC50 values of 40.7 and 87.0 μM, respectively. Compounds 10 and 11 showed cytotoxicity on only HepG2 and A549 cells, with IC50 values of 91.2 and 57.6 μM, respectively. Our results contribute to understanding more the secondary metabolites produced by L. clavatum and provide a scientific rationale for further investigations of anti-inflammatory and anticancer effects for this valuable medicinal plant.

TLR4-IN-C34 Inhibits Lipopolysaccharide-Stimulated Inflammatory Responses via Downregulating TLR4/MyD88/NF-κB/NLRP3 Signaling Pathway and Reducing ROS Generation in BV2 Cells.

TLR4 signal activated by lipopolysaccharide (LPS) is involved in the pathological process of the central nervous system (CNS) diseases and the suppression of TLR4 signal may become an effective treatment. TLR4-IN-C34, a TLR4 inhibitor, is expected to become a candidate compound with anti-neuroinflammatory response. In the present study, the anti-neuroinflammatory effects and possible mechanism of TLR4-IN-C34 were investigated in BV2 microglia cells stimulated by LPS. The results showed that TLR4-IN-C34 decreased the levels of pro-inflammatory factors and chemokines including NO, TNF-α, IL-1β, IL-6, and MCP-1 in the supernatant of LPS-stimulated BV2 cells. Further research indicated that TLR4-IN-C34 suppressed the expression or phosphorylation levels of inflammatory proteins regarding TLR4/MyD88/NF-κB/NLRP3 signaling pathway. In addition, TLR4-IN-C34 reduced ROS production in BV2 cells after LPS treatment. In conclusion, our findings suggest that anti-neuroinflammatory activity of TLR4-IN-C34 may be interrelated to the inhibition of TLR4/MyD88/NF-κB/NLRP3 signaling pathway and reduction of ROS generation.

Clock Gene Nr1d1 Alleviates Retinal Inflammation Through Repression of Hmga2 in Microglia.

PurposeRetinal inflammation is involved in the pathogenesis of several retinal diseases. As one of the core clock genes, Nr1d1 has been reported to suppress inflammation in many diseases. We investigated whether pharmacological activation of Nr1d1 can inhibit retinal inflammation and delineated the mechanisms of Nr1d1 in alleviating microglia activation.MethodsLipopolysaccharide (LPS) induced mice models were used to examine the effects of SR9009 (agonist of NR1D1) treatment on inflammatory phenotypes in vivo. Anti-inflammatory effects of Nr1d1 and associated mechanisms were investigated in the BV2 microglia cell line, and in primary retinal microglia in vitro.ResultsSR9009 treatment alleviated LPS-induced inflammatory cell infiltration, elevated cytokine levels and morphological changes of the microglia in mice models. In LPS-stimulated BV2 cells and primary retinal microglia, SR9009 suppressed cytokine expressions by inhibiting the NF-κB signaling pathway. Moreover, SR9009 treatment increased the levels of the M2 phenotype marker (CD206) and the proportions of ramified microglia. Suppression of Nr1d1 with siRNA reversed the inhibitory effects of SR9009 on cytokine production in BV2 cells. RNA-seq analysis showed that genes that were upregulated following Nr1d1 knockdown were enriched in inflammatory-associated biological processes. Subsequently, ChIP-seq of NR1D1 in BV2 was performed, and the results were integrated with RNA-seq results using the Binding and Expression Target Analysis (BETA) tool. Luciferase assays, electrophoretic mobility shift assay (EMSA), qPCR and Western blotting assays revealed that NR1D1 binds the promoter of Hmga2 to suppress its transcription. Notably, overexpressed Hmga2 in activated microglia could partly abolish the anti-inflammatory effects of Nr1d1.ConclusionThe clock gene Nr1d1 protects against retinal inflammation and microglia activation in part by suppressing Hmga2 transcription.

LINCS Dataset-Based Repositioning of Dutasteride as an Anti-Neuroinflammation Agent.

Neuroinflammation is often accompanied by central nervous system (CNS) injury seen in various CNS diseases, with no specific treatment. Drug repurposing is a strategy of finding new uses for approved or investigational drugs, and can be enabled by the Library of Integrated Network-based Cellular Signatures (LINCS), a large drug perturbation database. In this study, the signatures of Lipopolysaccharide (LPS) were compared with the signatures of compounds contained in the LINCS dataset. To the top 100 compounds obtained, the Quantitative Structure-Activity Relationship (QSAR)-based tool admetSAR was used to identify the top 10 candidate compounds with relatively high blood–brain barrier (BBB) penetration. Furthermore, the seventh-ranked compound, dutasteride, a 5-α-reductase inhibitor, was selected for in vitro and in vivo validation of its anti-neuroinflammation activity. The results showed that dutasteride significantly reduced the levels of IL-6 and TNF-α in the supernatants of LPS-stimulated BV2 cells, and decreased the levels of IL-6 in the hippocampus and plasma, and the number of activated microglia in the brain of LPS administration mice. Furthermore, dutasteride also attenuated the cognitive impairment caused by LPS stimulation in mice. Taken together, this study demonstrates that the LINCS dataset-based drug repurposing strategy is an effective approach, and the predicted candidate, dutasteride, has the potential to ameliorate LPS-induced neuroinflammation and cognitive impairment.

Bioactive secondary metabolites from a soybean-derived fungus Aspergillus versicolor IMBC-NMTP02

• Isolation of 10 compounds from Aspergillus versicolor IMBC-NMTP02. • Structural identification of 3 new, 2 new natural, and 5 known compounds. • Cytotoxicity toward HepG2, KB, HL-60, MCF-7, SK-Mel-2, and LNCaP cancer cells. • Nitric oxide inhibitory effects of the isolated compounds. • Antimicrobial effects of the isolated compounds. In the present study, 10 secondary metabolites, including three new asperversilactones A–C ( 2 , 4 , and 5 ) and two new natural metabolites (4 Z ,6 S ,7 R )-6,7-dihydroxyocta-2,4-dien-4-lactone ( 1 ) and (4 E ,6 S ,7 R )-6,7-dihydroxyocta-2,4-dien-4-lactone ( 3 ) were isolated from a culture fermentation of the soybean-derived fungus Aspergillus versicolor IMBC-NMTP02. Their structures were elucidated by comprehensive analyses of spectroscopic methods, including NMR, mass, and ECD spectra. Compounds 5 , 6 , and 9 were shown to have cytotoxic effects toward the HepG2, KB, HL-60, MCF-7, SK-Mel-2, and LNCaP human cancer cell lines (IC 50 = 31.7–81.5 μM), whereas all of the compounds inhibited NO overproduction in LPS-stimulated BV2 cells (IC 50 = 19.0–41.9 μM). Furthermore, all of the compounds showed antimicrobial effects against Enterococcus faecalis and Candida albican s, with MIC values in the range of 32–256 μM. Our investigation contributes to clarification of the chemical constituents of the soybean-derived A. versicolor and warrants further studies of some bioactive metabolites for therapeutic uses.

Inhibition of Autotaxin and Lysophosphatidic Acid Receptor 5 Attenuates Neuroinflammation in LPS-Activated BV-2 Microglia and a Mouse Endotoxemia Model.

Increasing evidence suggests that systemic inflammation triggers a neuroinflammatory response that involves sustained microglia activation. This response has deleterious consequences on memory and learning capability in experimental animal models and in patients. However, the mechanisms connecting systemic inflammation and microglia activation remain poorly understood. Here, we identify the autotaxin (ATX)/lysophosphatidic acid (LPA)/LPA-receptor axis as a potential pharmacological target to modulate the LPS-mediated neuroinflammatory response in vitro (the murine BV-2 microglia cell line) and in vivo (C57BL/6J mice receiving a single i.p. LPS injection). In LPS-stimulated (20 ng/mL) BV-2 cells, we observed increased phosphorylation of transcription factors (STAT1, p65, and c-Jun) that are known to induce a proinflammatory microglia phenotype. LPS upregulated ATX, TLR4, and COX2 expression, amplified NO production, increased neurotoxicity of microglia conditioned medium, and augmented cyto-/chemokine concentrations in the cellular supernatants. PF8380 (a type I ATX inhibitor, used at 10 and 1 µM) and AS2717638 (an LPA5 antagonist, used at 1 and 0.1 µM) attenuated these proinflammatory responses, at non-toxic concentrations, in BV-2 cells. In vivo, we demonstrate accumulation of PF8380 in the mouse brain and an accompanying decrease in LPA concentrations. In vivo, co-injection of LPS (5 mg/kg body weight) and PF8380 (30 mg/kg body weight), or LPS/AS2717638 (10 mg/kg body weight), significantly attenuated LPS-induced iNOS, TNFα, IL-1β, IL-6, and CXCL2 mRNA expression in the mouse brain. On the protein level, PF8380 and AS2717638 significantly reduced TLR4, Iba1, GFAP and COX2 expression, as compared to LPS-only injected animals. In terms of the communication between systemic inflammation and neuroinflammation, both inhibitors significantly attenuated LPS-mediated systemic TNFα and IL-6 synthesis, while IL-1β was only reduced by PF8380. Inhibition of ATX and LPA5 may thus provide an opportunity to protect the brain from the toxic effects that are provoked by systemic endotoxemia.

Naloxone Protects against Lipopolysaccharide-Induced Neuroinflammation and Microglial Activation via Inhibiting ATP-Sensitive Potassium Channel.

Aim The aim of this study was to evaluate the anti-inflammatory effects and underlying mechanism of naloxone on lipopolysaccharide- (LPS-) induced neuronal inflammation and microglial activation. Methods LPS-treated microglial BV-2 cells and mice were used to investigate the anti-inflammatory effects of naloxone. Results The results showed that naloxone dose-dependently promoted cell proliferation in LPS-induced BV-2 cells, downregulated the expression of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) and proinflammatory enzymes iNOS and COX-2 as well as the expression of free radical molecule NO, and reduced the expression of Iba-1-positive microglia in LPS-stimulated BV-2 cells and mouse brain. Moreover, naloxone improved LPS-induced behavior degeneration in mice. Mechanically, naloxone inhibited LPS-induced activation in the ATP-sensitive potassium (KATP) channel. However, the presence of glibenclamide (Glib), an antagonist of KATP channel, ameliorated the suppressive effects of naloxone on inflammation and microglial activation. Conclusion Naloxone prevented LPS-induced neuroinflammation and microglial activation partially through the KATP channel. These findings might highlight the potential of naloxone in neuroinflammation therapy.

Gualou Guizhi Granule Suppresses LPS-Induced Inflammatory Response of Microglia and Protects against Microglia-Mediated Neurotoxicity in HT-22 via Akt/NF-κB Signaling Pathways.

Neuroinflammation plays a crucial part in the commencement and advancement of ischemic stroke. Gualou Guizhi granule (GLGZG) is known to well exhibit neuroprotective effect, but it is not known whether GLGZG can regulate the inflammatory process at the cellular level in BV2 microglia cells and protect against microglia-mediated neurotoxicity in neurons. Herein, we aimed to investigate the anti-inflammatory effects of GLGZG on BV2 microglia cells and protection against microglia-mediated neurotoxicity in neurons. Methods. The cell model of neuroinflammation was constructed by lipopolysaccharide (LPS) to observe the effect of GLGZG in the presence or absence of GLGZG. The production of nitric oxide (NO), inflammatory mediators, was detected. Moreover, potential mechanisms associated with the anti-inflammatory effect, such as inhibition of microglial activation and nuclear factor kappa B (NF-κB), were also investigated. In addition, to prove whether GLGZG protects against microglia-mediated neurotoxicity, neuronal HT-22 cells were cultured in the conditioned medium. And cell survivability and neuronal apoptosis of HT-22 were evaluated. Results. It was found that a main regulator of inflammation, NO, is suppressed by GLGZG in BV2 microglial cells. Moreover, GLGZG dose dependently decreased the mRNA and protein levels of inducible NO synthase (iNOS) in LPS-stimulated BV2 cells. Additionally, GLGZG inhibited the expression and secretion of proinflammatory cytokines in BV2 microglial cells. Also, GLGZG inhibited LPS-activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in BV2 microglial cells at the intracellular level. GLGZG significantly affected Akt phosphorylation: phosphorylated forms of Akt increased. To check whether GLGZG protects against microglia-mediated neurotoxicity, neuronal HT-22 cells were incubated in the conditioned medium. GLGZG showed a neuroprotective effect by promoting cell survivability and suppressing neuronal apoptosis. Conclusions. GLGZG exerted its potential effects on suppressing inflammatory responses in LPS-induced BV2 cells by regulating NF-κB and Akt pathways. In addition, GLGZG could protect against microglia-mediated neurotoxicity in HT-22.

Chemical constituents from Lycopodiella cernua and their anti-inflammatory and cytotoxic activities

Phytochemical investigation of the whole plants of Lycopodiella cernua resulted in the isolation and identification of three new compounds (1–3), namely lycocernuaside E (1), lycernuic ketone F (2), and lycernuic B (3) and 12 known ones (4–15). Their chemical structures were established based on 1 D/2D NMR spectroscopic and HR-ESI-MS data analyses. Compounds 5, 12, and 13 displayed NO inhibitory effects in LPS-stimulated BV2 cells, with IC50 values of 21.2 ± 1.1, 28.5 ± 1.4, and 21.9 ± 1.1 µM, respectively. In addition, cytotoxic activity of the isolated compounds against MCF7 (breast carcinoma), HepG2 (hepatocarcinoma), and SK-Mel2 (melanoma) cancer cell lines were also reported.

Isoginkgetin treatment attenuated lipopolysaccharide-induced monoamine neurotransmitter deficiency and depression-like behaviors through downregulating p38/NF-κB signaling pathway and suppressing microglia-induced apoptosis.

Background:Microglia activation-induced neuroinflammation may contribute to the etiology of depression. Podocarpus nagi containing high concentration of isoginkgetin could effectively treat mental diseases in ancient times. However, the therapeutic role, peculiarly in the brain–immune modulation in depression is still unclear. This study aimed to determine effects of isoginkgetin on lipopolysaccharide (LPS)-induced depression-like changes. Furthermore, its modulation on the p38/nuclear factor-kappa B (NF-κB) pathway in LPS-activated microglia was evaluated.Methods:Adult Kunming mice were intraperitoneally injected vehicle or isoginkgetin (4 mg/kg) daily for 14 days before saline or LPS (0.83 mg/kg) administration. Depression-like behavior, neurotransmitter levels, and markers of neuroinflammation were determined. Isoginkgetin effect on LPS-induced microglial activation was then assessed in BV2 cells. Finally, conditioned medium (CM) derived from isoginkgetin-treated BV2 cells was co-cultured with SH-SY5Y cells for 24 h. Cell viability and apoptosis were evaluated.Results:LPS significantly induced helplessness and anxiety, which were associated with decreased 5-HT, noradrenaline, and dopamine concentrations. Meanwhile, LPS increased microglia M1 hallmark Iba1 expression and serum interleukin (IL)-1β concentration. These changes were attenuated by isoginkgetin treatment. In vitro, isoginkgetin markedly suppressed the production of IL-1β, IL-6, tumor necrosis factor-alpha, cyclooxygenase-2, inducible nitric oxide, and reactive oxygen species, which are released from LPS-stimulated BV2 cells. More interestingly, CM from isoginkgetin-treated BV2 cells significantly alleviated SH-SY5Y cell apoptosis and restored cell viability compared to LPS-treated group through the inhibition of p38/NF-κB signaling pathway.Conclusion:These data demonstrate that isoginkgetin is an effective therapeutic agent for depression-like behaviors and neuropathological changes via potent anti-inflammatory property.

TCMIP v2.0 Powers the Identification of Chemical Constituents Available in Xinglou Chengqi Decoction and the Exploration of Pharmacological Mechanisms Acting on Stroke Complicated With Tanre Fushi Syndrome.

Xinglou Chengqi (XLCQ) decoction, composed of three botanical drugs and one inorganic drug, is used in clinics during the treatment of acute stroke complicated with Tanre Fushi (TRFS) syndrome in China. However, its active ingredients and the molecular mechanism have not been clarified. So, we aimed to preliminarily characterize its chemical constituents and investigate its pharmacological mechanisms using an integrative pharmacology strategy, including component analysis, network prediction, and experimental verification. We employed UPLC-QTOF-MS/MS to describe the chemical profile of XLCQ, Integrative Pharmacology-based Network Computational Research Platform of Traditional Chinese Medicine (TCMIP v2.0, http://www.tcmip.cn/), to assist in identifying the chemical components and predict the putative molecular mechanism against acute stroke complicated with TRFS, and LPS-stimulated BV-2 cells to verify the anti-neuroinflammatory effects of luteolin, apigenin, and chrysoeriol. Altogether, 197 chemical compounds were identified or tentatively characterized in the water extraction of XLCQ, 22 of them were selected as the key active constituents that may improve the pathological state by regulating 27 corresponding targets that are mainly involved in inflammation/immune-related pathways, and furthermore, luteolin, apigenin, and chrysoeriol exhibited good anti-neuroinflammatory effects from both protein and mRNA levels. In summary, it is the first time to employ an integrative pharmacology strategy to delineate 22 constituents that may improve the pathological state of stroke with TRFS by regulating 27 corresponding targets, which may offer a highly efficient way to mine the scientific connotation of traditional Chinese medicine prescriptions. This study might be a supplement for the deficiency of the basic research of XLCQ.

Mitigating Effect of Lindera obtusiloba Blume Extract on Neuroinflammation in Microglial Cells and Scopolamine-Induced Amnesia in Mice.

Lindera obtusiloba Blume (family, Lauraceae), native to Northeast Asia, has been used traditionally in the treatment of trauma and neuralgia. In this study, we investigated the neuroinflammatory effect of methanol extract of L. obtusiloba stem (LOS-ME) in a scopolamine-induced amnesia model and lipopolysaccharide (LPS)-stimulated BV2 microglia cells. LOS-ME downregulated the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, inflammatory cytokines, and inhibited the phosphorylation of nuclear factor kappa-B (NF-ĸB) and extracellular signal-regulated kinase (ERK) in LPS-stimulated BV2 cells. Male C57/BL6 mice were orally administered 20 and 200 mg/kg of LOS-ME for one week, and 2 mg/kg of scopolamine was administered intraperitoneally on the 8th day. In vivo behavioral experiments (Y-maze and Morris water maze test) confirmed that LOS-ME alleviated cognitive impairments induced by scopolamine and the amount of iNOS expression decreased in the hippocampus of the mouse brain. Microglial hyper-activation was also reduced by LOS-ME pretreatment. These findings suggest that LOS-ME might have potential in the treatment for cognitive improvement by regulating neuroinflammation.

Asiatic acid attenuates diabetic retinopathy through TLR4/MyD88/NF-κB p65 mediated modulation of microglia polarization

AimThis study aimed to evaluate the effects of Asiatic acid (AA), a naturally occurring compound of pentacyclic triterpenoid, on the pathological processes of diabetic retinopathy (DR). MethodsSD rats were induced to develop early DR by intraperitoneal injection of STZ (60 mg/kg). Four weeks after injection, the diabetic rats were orally administrated with 37.5 mg/kg or 75 mg/kg AA every day for four weeks. The integrity of blood-retinal barrier (BRB) was measured by Evans blue staining. The polarization of microglia was determined by real-time PCR, western blot, and ELISA assays. The inner BRB (iBRB) or outer BRB (oBRB) breakdown was induced in human retinal endothelial cells or APRE19 cells through co-culture with high glucose and LPS-stimulated microglia BV2 cells. The damage to the iBRB and oBRB was measured using transendothelial/transepithelial electrical resistance (TEER/TER) and FITC-conjugated dextran cell permeability assays. Key findingsResults demonstrated that AA alleviated BRB breakdown, as evidenced by decreased protein expression of occludin, claudin-5, and ZO-1. Furthermore, AA treatment suppressed inflammation and M1 polarization, while it increased M2 polarization in the retina of DR rats. In vitro, the iBRB or oBRB breakdown was alleviated by AA. LPS-induced M1-polarization of BV2 cells under high glucose condition was also repressed through AA administration. Finally, we demonstrated that AA weakened the TLR4/MyD88/NF-κB p65 signaling pathway both in vivo and in vitro. SignificanceAA ameliorated early DR by regulating microglia polarization via the TLR4/MyD88/NF-κB p65 pathway. These data indicate that AA is a potential candidate for DR treatment.

Anti-Neuroinflammatory Effects and Mechanism of Action of Fructus ligustri lucidi Extract in BV2 Microglia.

For centuries, Fructus ligustri lucidi (FLL; the fruit of Ligustrum lucidum Aiton or Ligustrum japonicum Thunb.) has been commonly used in traditional Chinese medicine for treating hepatitis and aging-related symptoms and in traditional Korean medicine to detoxify kidneys and the liver. Pharmacological research has shown FLL has antioxidant, anti-inflammatory, anticancer, anti-osteoporosis, and hepatoprotective activities. This study was undertaken to investigate the effects of FLL extract (FLLE) on neuroinflammation. After setting a non-toxic concentration using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assay data, we investigated the effects of FLLE using Western blotting, cell migration, enzyme-linked immunosorbent assay, a nitric oxide (NO) assay, and immunofluorescence staining in lipopolysaccharide (LPS)-stimulated murine BV2 microglial cells. FLLE was non-toxic to BV2 cells up to a concentration of 500 μg/mL and concentration-dependently inhibited the production of NO and prostaglandin E2 and the protein levels of inducible nitric oxide synthase and cyclooxygenase-2 under LPS-induced inflammatory conditions. It also inhibited the secretion of the inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Furthermore, FLLE pretreatment attenuated LPS-induced increases of CD68 (a marker of microglia activation) and suppressed the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) signaling pathways in LPS-stimulated BV2 cells, and significantly increased heme oxygenase (HO)-1 levels. FLLE also reduced the LPS-induced increase in the migratory ability of BV2 cells and the phosphorylation of vascular endothelial growth factor receptor 1. Collectively, FLLE effectively inhibited inflammatory response by suppressing the MAPK and NF-κB signaling pathways and inducing HO-1 in LPS-stimulated BV2 microglial cells. Our findings provide a scientific basis for further study of FLL as a candidate for preventing or alleviating neuroinflammation.