LPS-activated BV2 Microglial Cells Research Articles

Repurposing Anakinra for Alzheimer's Disease: The In Vitro and In Vivo Effects of Anakinra on LPS- and AC-Induced Neuroinflammation.

Alzheimer's disease is a significant global health issue, and studies suggest that neuroinflammation plays a vital role in the advancement of this disease. In this study, anakinra has been shown to display a time- and concentration-dependent antineuroinflammatory effect. In the in vitro studies, it diminished the gene expressions of tumor necrosis factor-alpha (TNF-α) and nitric oxide (NO) synthase 2 stimulated by lipopolysaccharide (LPS). Anakinra also reduced the LPS-induced production of NO and reactive oxygen species. Thus, the hypertrophic state of LPS-activated BV2 microglial cells was reversed by anakinra. Furthermore, acrylamide (ACR)-induced activation of nuclear transcription factor-κB, TNF-α, and interleukin-1β was downregulated, while cAMP response element binding protein and brain-derived neurotrophic factor expression levels were markedly enhanced in ACR-treated zebrafish larvae. It was also observed that anakinra improved the uncoordinated swimming behaviors in ACR-exposed zebrafish larvae. Overall, anakinra demonstrated potential antineuroinflammatory and antioxidative effects.

Salvinorin A ameliorates pilocarpine-induced seizures by regulating hippocampal microglia polarization

Ethnopharmacological relevanceSalvia divinorum (Epling and Játiva) is a psychoactive plant traditionally used by the Latinos for various medicinal purposes. Salvinorin A (Sal A), the main bioactive constituent of S. divinorum, is a natural highly selective kappa opioid receptor (KOR) agonist. Considering the anti-inflammatory effect of S. divinorum and endogenous hippocampal dynorphin/kappa opioid receptor (KOR) system playing an anticonvulsant function, we hypothesis that Sal A can be a potential candidate to treat epilepsy. Here, we identified whether Sal A ameliorated epileptic seizures and neuronal damages in animal model and in vitro model and investigated its underlying mechanisms. Materials and methodsMice epilepsy model was induced by pilocarpine following seizures assessed by Racine classification. Hippocampus tissues were obtained for genetic, protein, and histological investigation. Furthermore, lipopolysaccharide (LPS)-activated BV2 microglial cells were utilized to validate the anti-inflammatory and microglia polarization regulation effects of Sal A. ResultsSal A treatment significantly prolonged the latency to status epileptics (SE) and shortened the duration of SE in the pilocarpine-induced model. It also alleviated neuronal damages via activation of the AMPK/JNK/p-38 MAPK pathway and inhibition of apoptosis-related protein in hippocampus tissues. Furthermore, Sal A dose-dependently reduced microglia-mediated expression of pro-inflammatory cytokines and increased anti-inflammatory factors levels in SE mice and LPS-activated BV2 microglial cells by regulating microglia polarization. In addition, the effect of Sal A in vitro was totally blocked by KOR antagonist nor-BNI. ConclusionSal A treatment protects against epileptic seizures and neuronal damages in pilocarpine-induced models by suppressing the inflammation response through regulating microglial M1/M2 polarization. This study might serve as a theoretical basis for clinical applications of Sal A and its analogs and provide a new insight into the development of anti-seizure drugs.

Nobiletin derivative, 5-acetoxy-6,7,8,3’,4’-pentamethoxyflavone, inhibits neuroinflammation through the inhibition of TLR4/MyD88/MAPK signaling pathways and STAT3 in microglia

Objective Microglia in the central nervous system regulate neuroinflammation that leads to a wide range of neuropathological alterations. The present study investigated the anti-neuroinflammatory properties of nobiletin (Nob) derivative, 5-acetoxy-6,7,8,3′,4′-pentamethoxyflavone (5-Ac-Nob), in lipopolysaccharide (LPS)-activated BV2 microglia. Materials and Methods By using the MTT assay, Griess method, flow cytometry, and enzyme-linked immunosorbent assay (ELISA), we determined the cell viability, the levels of nitric oxide (NO), reactive oxygen species (ROS), and pro-inflammatory factors (interleukin 1 beta; IL-1β, interleukin 6; IL-6, tumor necrosis factor alpha; TNF-α and prostaglandin E2; PGE2) in LPS-stimulated BV2 microglia. Toll-like receptor 4 (TLR4)-mediated myeloid differentiation primary response gene 88 (MyD88)/nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK) signaling pathway and signal transducer and activator of transcription 3 (STAT3) were measured by western blotting. Analysis of NO generation and mRNA of pro-inflammatory cytokines was confirmed in the zebrafish model. Results 5-Ac-Nob reduced cell death, the levels of NO, ROS, inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and pro-inflammatory factors in LPS-activated BV-2 microglial cells. TLR4-mediated MyD88/NF-κB and MAPK pathway (p38, ERK and JNK) after exposure to 5-Ac-Nob was also suppressed. Moreover, 5-Ac-Nob inhibited phosphorylated STAT3 proteins expression in LPS-induced BV-2 microglial cells. Furthermore, we confirmed that 5-Ac-Nob decreased LPS-induced NO generation and mRNA of pro-inflammatory cytokines in the zebrafish model. Conclusions Our findings suggest that 5-Ac-Nob represses neuroinflammatory responses by inhibiting TLR4-mediated signaling pathway and STAT3. As a result of these findings, 5-Ac-Nob has potential as an anti-inflammatory agent against microglia-mediated neuroinflammatory disorders.

Involvement of Nrf2 Activation and NF-kB Pathway Inhibition in the Antioxidant and Anti-Inflammatory Effects of Hesperetin in Activated BV-2 Microglial Cells.

Alzheimer's disease is a progressive neurodegenerative disorder leading to cognitive decline and memory loss. The incidence of this disease continues to increase due to the limited number of novel therapeutics that prevent or slow down its progression. Flavonoids have been investigated for their potential effects on cellular damage triggered by excessive reactive oxygen species (ROS) and neuroinflammatory conditions. This study investigated the effect of the flavonoid hesperetin on LPS-activated murine BV-2 microglial cells. Results show that hesperetin reduced nitric oxide levels and increased catalase, glutathione, and superoxide dismutase levels, suggesting its potential to reduce neuroinflammation and oxidative stress. Moreover, RT-PCR arrays showed that hesperetin modulated multiple genes that regulate oxidative stress. Hesperetin downregulated the mRNA expression of ERCC6, NOS2, and NCF1 and upregulated HMOX1 and GCLC. RT-PCR results showed that hesperetin-induced Nrf2 mRNA and protein expression in LPS-activated BV-2 microglial cells is involved in the transcription of several antioxidant genes, suggesting that hesperetin's antioxidant effects may be exerted via the Keap1/Nrf2 signaling pathway. Furthermore, the data demonstrated that hesperetin reduced the gene expression of PD-L1, which is upregulated as an individual ages and during chronic inflammatory processes, and inhibited the expression of genes associated with NF-kB signaling activation, which is overactivated during chronic inflammation. It was concluded from this investigation that hesperetin may have therapeutic potential to prevent or slow down the progression of neurodegenerative diseases, such as Alzheimer's disease, by reducing chronic oxidative stress and modulating neuroinflammation.

Antioxidant and Anti-Inflammatory Mechanisms of Cardamonin through Nrf2 Activation and NF-kB Suppression in LPS-Activated BV-2 Microglial Cells.

Chronic oxidative stress (OS) and inflammation are implicated in developing and progressing neurodegenerative diseases (NDs). The chronic activation of microglia cells leads to the overproduction of several substances, including nitric oxide and reactive oxygen species, which can induce neurodegeneration. Natural compounds have recently been investigated for their potential to protect cells from OS and to improve many disease-related conditions. Cardamonin (CD) is a bioactive compound in many plants, such as Alpinia katsumadai and Alpinia conchigera. The present study examined the effects of CD on LPS-activated BV-2 microglial cells. The cell viability results showed that the increasing concentrations of CD, ranging from 0.78 to 200 µM, induced BV-2 cell cytotoxicity in a dose-response manner. In the nitric oxide assay, CD concentrations of 6.25 to 25 µM reduced the release of nitric oxide in LPS-activated BV-2 cells by 90% compared to those treated with LPS only (p ≤ 0.0001). CD (6.25 µM) significantly decreased the cellular production of SOD (3-fold (p ≤ 0.05)) and increased the levels of expression of CAT (2.5-fold (p ≤ 0.05)) and GSH (2-fold (p ≤ 0.05)) in the LPS-activated BV-2 cells. Furthermore, on RT-PCR arrays, CD (6.25 µM) downregulated mRNA expression of CCL5/RANTES (5-fold), NOS2 (2-fold), SLC38A1 (3-fold), TXNIP (2-fold), SOD1 (2-fold), SOD2 (1.5-fold) and upregulated GSS (1.9-fold), GCLC (1.7-fold) and catalase (2.9-fold) expression, indicating CD efficacy in modulating genes involved in OS and inflammation. Furthermore, CD (6.25 µM) increased the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and lowered the levels of Kelch-like ECH-associated protein 1 (Keap1), indicating that this may be the signaling responsible for the elevation of antioxidant factors. Lastly, the results showed that CD (6.25 µM) modulated genes and proteins associated with the NF-kB signaling, downregulating genes related to excessive neuroinflammation. These results imply that CD may be a potential compound for developing therapeutic and preventive agents in treating neurodegeneration induced by excessive OS and inflammation.

Anti-Neuroinflammatory Potential of a Nectandra angustifolia (Laurel Amarillo) Ethanolic Extract

Microglia, the resident macrophage-like population in the CNS, plays an important role in the pathogenesis of many neurodegenerative disorders. Nectandra genus is known to produce different metabolites with anti-inflammatory, anti-oxidant and analgesic properties. Although the species Nectandra angustifolia is popularly used for the treatment of different types of inflammatory processes, its biological effects on neuroinflammation have not yet been addressed. In this study, we have investigated the role of a Nectandra angustifolia ethanolic extract (NaE) in lipopolysaccharide (LPS)-induced neuroinflammation in vitro and in vivo. In LPS-activated BV2 microglial cells, NaE significantly reduced the induced proinflammatory mediators TNF-α, IL-1β, IL-6, COX-2 and iNOS, as well as NO accumulation, while it promoted IL-10 secretion and YM-1 expression. Likewise, reduced CD14 expression levels were detected in microglial cells in the NaE+LPS group. NaE also attenuated LPS-induced ROS and lipid peroxidation build-up in BV2 cells. Mechanistically, NaE prevented NF-κB and MAPKs phosphorylation, as well as NLRP3 upregulation when added before LPS stimulation, although it did not affect the level of some proteins related to antioxidant defense such as Keap-1 and HO-1. Additionally, we observed that NaE modulated some activated microglia functions, decreasing cell migration, without affecting their phagocytic capabilities. In LPS-injected mice, NaE pre-treatment markedly suppressed the up-regulated TNF-α, IL-6 and IL-1β mRNA expression induced by LPS in brain. Our findings indicate that NaE is beneficial in preventing the neuroinflammatory response both in vivo and in vitro. NaE may regulate microglia homeostasis, not only restraining activation of LPS towards the M1 phenotype but promoting an M2 phenotype.

Hesperetin modulation of oxidative stress and inflammatory mediators in LPS‐Activated BV‐2 Microglial Cells

Neurodegeneration affects millions of Americans each year, occurring in later periods of life, where its onset is induced by neuroinflammation and oxidative stress. Flavonoids are known to protect against damages caused by reactive oxygen species (ROS) and reactive nitrogen species (RNS). This study evaluated the effect of hesperetin (HST), a major flavonoid found in oranges and mandarins, on LPS‐activated BV‐2 microglial cells. The obtained results of RT‐PCR showed that HST down‐regulated the mRNA expression of PD‐L1 in more than 50% of the LPS‐activated cells. In addition, HST induced the mRNA expression of Nrf2, which is involved in the transcription of several antioxidant genes. Moreover, the data from the oxidative stress PCR arrays showed that after 24 h, the concentration of 100 µM of HPT modulated numerous genes that regulate oxidative stress and inflammatory processes. HST down‐regulated the mRNA expression of ERCC6, ACTB, SQSTM11, NOS2, and NCF1 and up‐regulated the expression of HMOX1, which participate not only in excessive oxidative stress processes, by also in exacerbated inflammatory states. Both CAT and SOD enzymatic assays showed that treatment with HSP and LPS alone had no effect on CAT expression, but when cells were treated with the combination of HST and LPS, there was a reduction in these expressions enzymes. The quantitative glutathione assays demonstrated that only the combination treatment of HST and LPS reduced the expression of glutathione after a 24‐h treatment period. In conclusion, these data show that hesperetin modulates several genes associated with oxidative stress and neuroinflammation and may potentially slow the progression of neurodegeneration.

Antineuroinflammatory Effect of Amburana cearensis and Its Molecules Coumarin and Amburoside A by Inhibiting the MAPK Signaling Pathway in LPS-Activated BV-2 Microglial Cells.

Microglia plays an important role in the neuroinflammatory response, identified as one of the major factors in the development and progression of neurodegenerative diseases. Amburana cearensis and its bioactive compounds, including coumarin (CM), vanillic acid (VA), and amburoside A (AMB), exert antioxidant, anti-inflammatory, and neuroprotective activities, on 6-OHDA-induced neurotoxicity in rat mesencephalic cells determined by our group. The present study investigated the anti-inflammatory effect of the dry extract from A. cearensis (DEAC), CM, AMB, and VA on lipopolysaccharide- (LPS-) stimulated microglial cells and elucidated the possible molecular mechanism of action. The DEAC was characterized by HPLC-PDA (chemical markers: CM, AMB, and VA). The BV-2 microglial cell line was pretreated with increasing concentrations of DEAC, CM, AMB, or VA in the presence or absence of LPS to evaluate the toxicity and anti-inflammatory activity. The cytotoxicity of DEAC, CM, AMB, or VA on BV-2 cells was evaluated by the MTT test, the free radical scavenging activity of test drugs was investigated, and the nitric oxide (NO) production was determined using the Griess reagent, while cytokine levels were measured by ELISA. The expressions of toll-like receptor 4 (TLR-4), nuclear factor kappa B (NF-κB), MAPK members (JNK and ERK1/2), and iNOS were determined through Western blot analysis. DEAC, CM, AMB, or VA (5-100 μg/mL) did not induce any detectable cytotoxicity in BV-2 cells. All test drugs (100 μg/mL) showed free radical scavenging activity (hydroxyl and superoxide radicals); however, only DEAC, CM, and AMB (5-100 μg/mL) significantly reduced NO production. DEAC (100 μg/mL), as well as CM (50 and 100 μg/mL) and AMB (25 μg/mL), reduced at least 50% of NO produced and markedly decrease the production of TNF-α and IL-6 but they did not significantly affect IL-10 levels. Only DEAC (100 μg/mL) and AMB (25 μg/mL) reduced the expression of iNOS, and they did not affect arginase activity. DEAC (100 μg/mL) suppressed the activation of the MAPKs JNK and ERK1/2 in LPS-activated BV-2 cells but it did not suppress the expression of TLR-4 nor the phosphorylation of NF-κB. In conclusion, DEAC, CM, and AMB exerted anti-inflammatory activity in LPS-activated microglial cells as observed by the reduction in the production of inflammatory mediators and the expression of iNOS. We identified the MAPK signaling pathway as a probable mechanism of action to the anti-inflammatory effects observed.

6-methoxyflavone suppresses neuroinflammation in lipopolysaccharide- stimulated microglia through the inhibition of TLR4/MyD88/p38 MAPK/NF-κB dependent pathways and the activation of HO-1/NQO-1 signaling

Microglia-related neuroinflammation is associated with a variety of neurodegenerative diseases. Flavonoids have demonstrated different pharmacological effects, such as antioxidation, neuroprotection and anti-inflammation However, the effect of flavonoid 6-methoxyflavone (6-MeOF) on microglia-mediated neuroinflammation remain unknown. The current study aim to study the antineuroinflammatory effects of 6-MeOF in lipopolysaccharide- (LPS-) induced microglia in vitro and in vivo. Pretreatment of BV2 microglia cells with 6-MeOF for 1h then stimulated with LPS (100ng/ml) for 24h. The expression levels of pro-inflammatory factors, NO and reactive oxygen species (ROS) were performed by the enzyme-linked immunosorbent assay (ELISA), Griess assay and flow cytometry. Western blotting was used to assess MAPK, NF-κB signal transducer and antioxidant enzymes-related proteins. Analysis of ROS and microglial morphology was confirmed in the zebrafish and mice brain, respectively. Our results demonstrated that 6-MeOF dose-dependently prevent cell death and decreased the levels of pro-inflammatory mediators in LPS-stimulated BV2 microglia cells. Phosphorylated NF-κB/IκB and TLR4/MyD88/p38 MAPK/JNK proteins after exposure to 6-MeOF was suppressed in LPS-activated BV-2 microglial cells. 6-MeOF also presented antioxidant activity by reduction of NO, ROS, iNOS and COX-2 and the induction of the level of HO-1 and NQO1 expressions in LPS-activated BV2 microglial cells. Furthermore, we demonstrated that 6-MeOF inhibited LPS-induced NO generation in an experimental zebrafish model and prevent the LPS-induced microgliosis in the prefrontal cortex and substantia nigra of mice. These results explored that 6-MeOF possesses potential as anti-inflammatory and anti-oxidant agents against microglia-associated neuroinflammatory disorders.

Triterpenoids and meroterpenoids from the edible Ganoderma resinaceum and their potential anti-inflammatory, antioxidant and anti-apoptosis activities

Ganoderma resinaceum, as a traditional edible mushroom, has been widely reported to improve neurodegenerative diseases characterized by oxidative stress and inflammation. In this study, five new terpenoids, including four lanostane triterpenoids, named ganoresinoid A-D (1–4) and one meroterpenoid, named ganoresinoid E (5), along with 27 known compounds (6–32), were isolated from the fruiting bodies of edible mushroom G. resinaceum. These structures were identified by NMR, HRESIMS data analysis. All metabolites were evaluated for anti-inflammatory, antioxidative and anti-apoptosis activities. Among them, ganoresinoid A showed notably restrained nitric oxide (NO), IL-1β, IL-6 and TNF-α levels in LPS-activated BV-2 microglial cells via suppressing TLR-4/ NF-κB and MAPK signaling pathway. Simultaneously, ganoresinoid A remarkably alleviated LPS-induced apoptosis by means of the decrease of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS). In addition, ganoresinoid A demonstrated antioxidant effects in H2O2-induced SH-SY5Y cells by activating the Akt/GSK-3β/Nrf2 signaling pathway. Taken together, these results may provide a stronger theoretical basis for ganoresinoid A from G. resinaceum as nutrition intervention to alleviate neurodegenerative diseases.

Discovery of novel MIF inhibitors that attenuate microglial inflammatory activation by structures-based virtual screening and in vitro bioassays.

Macrophage migration inhibitory factor (MIF) is a pluripotent pro-inflammatory cytokine and is related to acute and chronic inflammatory responses, immune disorders, tumors, and other diseases. In this study, an integrated virtual screening strategy and bioassays were used to search for potent MIF inhibitors. Twelve compounds with better bioactivity than the prototypical MIF-inhibitor ISO-1 (IC50 = 14.41 μM) were identified by an in vitro enzymatic activity assay. Structural analysis revealed that these inhibitors have novel structural scaffolds. Compound 11 was then chosen for further characterization in vitro, and it exhibited marked anti-inflammatory efficacy in LPS-activated BV-2 microglial cells by suppressing the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs). Our findings suggest that MIF may be involved in the regulation of microglial inflammatory activation and that small-molecule MIF inhibitors may serve as promising therapeutic agents for neuroinflammatory diseases.

The Antioxidant Effects of Cardamonin in LPS‐Activated BV‐2 Microglial Cells

Chronic oxidative stress and inflammation are implicated in the development and progression of several neurodegenerative diseases. The continuous over-activation of the microglia cells produces numerous factors, including nitric oxide and reactive oxygen species, which induce neurodegeneration. Many natural compounds with anti-inflammatory and antioxidative effects can protect neurons from oxidative stress. Cardamonin is an important bioactive compound found in many plants, such as Alpinia katsumadai and Alpinia conchigera. It has been reported to have multiple therapeutic properties, which include anti-cancer, anti-inflammatory, antioxidant, antiviral antibiotic, antifungal, and antiallergic activities. The present study examined the effects of cardamonin on LPS-activated BV-2 microglial cells. In this study, cell viability results showed increasing concentrations of cardamonin, ranging from 0.78 to 200 uM induced BV-2 cells cytotoxicity in a dose-response manner. The selected concentration used for further studies was 6.25 uM based on a rate of at least 80% of cells alive. In the nitric oxide assay, cardamonin concentrations of 6.25uM significantly reduced nitric oxide release in LPS-activated BV-2 cells, compared to the cells treated with LPS only (p < 0.001). Although cardamonin was not effective in reducing the formation of H2O2 of the LPS-activated cells, it significantly decreased the cellular production of SOD 3-fold (p < 0.05). It also, increased the levels of expression of CAT 2.5-fold (p < 0.05) and glutathione 2-fold (p < 0.05) in the LPS activated BV-2 cells. The effect of cardamonin on the activity of antioxidant enzymes in BV-2 microglial cells reflects a decreased oxidative stress status in the cellular environment. These findings suggest that cardamonin may be utilized in the therapy of neurodegeneration caused by excessive oxidative stress. Cardamonin use may also help delay the onset of neurodegenerative diseases or prevent their progression in the CNS.

The Modulatory effects of Hesperitin on the Expression of Oxidative Stress Associated Genes in LPS‐Activated BV‐2 Microglial Cells

Numerous natural compounds have been reported to protect against oxidative stress and decrease the mortality related to chronic diseases. Flavonoids are known to protect against reactive oxygen species (ROS) damage. Many studies suggested a possible protective role of flavonoids in neurodegenerative and inflammatory conditions associated with excessive oxidative stress in the brain. In this study, we evaluated the effect of hesperetin, a major flavonoid found in oranges and mandarins, on LPS-activated BV-2 microglial cells. The methodology included cell viability, ELISA, RT-PCR using individual primers, and PCR arrays to screen a range of genes associated with oxidative stress. Cell viability results showed that only the highest concentration of 200 µM of hesperitin had a toxic effect on the BV-2 microglial cells, causing 20% of cell death. The combination of LPS and hesperitin also exhibited no toxicity in concentrations ranging from 0.78 to 100 µM. Still, in the concentration of 200 µM, there was a reduction of 50% of cell viability after 24 h. The ELISA and RT-PCR data demonstrated that hesperitin increased the expression of PDL-1 in the protein and transcription level in the LPS-activated cells. Also, hesperitin induced the mRNA expression of Nrf2, which is involved in the transcription of several antioxidant genes, and up-regulated the expression of MMP-9, which participates in the cleavage of extracellular matrix and plays a critical role in the central nervous system function. Also, the data from the oxidative stress PCR arrays showed that after 24 h, of cell treatment with 100 µM of hesperitin modulated numerous genes that regulate the oxidative stress process. The results show that hesperitin down-regulated the mRNA expression of Nos2, Ptgs2, Ncf1, Actb, Txnip, Ucp2, and up-regulated expression Srxn1, Prdx3, and Txn1. These genes participate not only in excessive oxidative stress processes by also in exacerbated inflammatory states. These data show that hesperitin may have a potential in the prevention and treatment of oxidative stress-related neurodegenerative diseases.

Mucuna pruriens (L.) DC. seed extract inhibits lipopolysaccharide-induced inflammatory responses in BV2 microglial cells

Ethnopharmacological relevanceInflammation caused by activated microglia is known to be associated with neurodegenerative diseases, e.g., Parkinson's disease (PD) and Alzheimer's disease (AD). Inhibiting the inflammatory process can be considered a potential strategy for the treatment of inflammation-associated diseases. Mucuna pruriens (L.) DC. (Leguminosae) has long been used in Thailand, India, China and other tropical countries to treat several diseases including PD. M. pruriens seeds have been found to possess a variety of pharmacological properties including antioxidant and anti-Parkinsonism effects. However, the anti-inflammatory effects of M. pruriens seeds during microglial activation have yet to be reported. Aim of the studyThe present study was performed to evaluate the anti-inflammatory effects of M. pruriens seed extract and elucidate its underlying mechanism using lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Materials and methodsBV2 microglial cells were pretreated with various concentrations of M. pruriens seed extract before being stimulated with LPS. The levels of inflammatory mediators were analyzed by Griess assay and enzyme-linked immunoassay (ELISA). The protein expression levels of inflammatory cytokines were determined by Western blot analysis. The translocation of nuclear factor-kappa B (NF-κB) was assessed by immunofluorescence microscopy. ResultsM. pruriens seed extract significantly inhibited the release of inflammatory mediators including nitric oxide (NO), IL-1β, IL-6, and TNF-α in LPS-stimulated BV2 microglial cells. The extract also decreased the protein expression of IL-1β, IL-6, and TNF-α. Moreover, M. pruriens seed extract inhibited the translocation of NF-κB. ConclusionsM. pruriens seed extract could suppress inflammatory responses in LPS-activated BV2 microglial cells by inhibiting the NF-κB signaling pathway. These findings support the use of M. pruriens seeds in traditional and alternative medicine for the treatment of PD and other inflammation-associated diseases.

Phyllanthus amarus prevents LPS-mediated BV2 microglial activation via MyD88 and NF-\u03baB signaling pathways

BackgroundPhyllanthus amarus has been shown to attenuate lipopolysaccharide (LPS)-induced peripheral inflammation but similar studies in the central nervous system are scarce. The aim of the present study was to investigate the neuroprotective effects of 80% ethanol extract of P. amarus (EPA) in LPS-activated BV2 microglial cells.MethodsBV2 microglial cells c for 24 h, pre-treated with EPA for 24 h prior to LPS induction for another 24 h. Surface expression of CD11b and CD40 on BV2 cells was analyzed by flow cytometry. ELISA was employed to measure the production of pro-inflammatory mediators i.e. nitric oxide (NO) and tumor necrosis factor (TNF)-α. Western blotting technique was used to determine the expression of inducible nitric oxide synthase (iNOS), myeloid differentiation protein 88 (MYD88), nuclear factor kappa B (NF-κB), caspase-1, and mitogen activated protein kinase (MAPK).ResultsQualitative and quantitative analyses of the EPA using a validated ultra-high pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method indicated the presence of phyllanthin, hypophyllanthin, niranthin, ellagic acid, corilagin, gallic acid, phyltetralin, isolintetralin and geraniin. EPA suppressed the production of NO and TNFα in LPS-activated BV2 microglial cells. Moreover, EPA attenuated the expression of MyD88, NF-κB and MAPK (p-P38, p-JNK and p-ERK1/2). It also inhibited the expression of CD11b and CD40. EPA protected against LPS-induced microglial activation via MyD88 and NF-κB signaling in BV2 microglial cells.ConclusionsEPA demonstrated neuroprotective effects against LPS-induced microglial cells activation through the inhibition of TNFα secretion, iNOS protein expression and subsequent NO production, inhibition of NF-κB and MAPKs mediated by adapter protein MyD88 and inhibition of microglial activation markers CD11b and CD40.

The inhibitory effects of Ganoderma Lucidum on NFκB related inflammatory cytokines in activated BV‐2 microglial cells

Neurodegenerative diseases have increased significantly over the last five years, and the increase in the elderly with Alzheimer’s and dementia requires increased attention and research design to combat this critical problem. Ganoderma Lucidum extract (GLE) is a potent ancient Asian remedy for extending the life and treatment of a variety of disease states. In the current study, we investigate the neuroprotective potential effects of Ganoderma Lucidum on LPS‐activated BV‐2 microglial cells. The LPS activated BV‐2 cells have been largely used as a model to illustrate the pro‐inflammatory process involved in the activation of signaling transduction pathways associated with neuronal injury. The methodology used in this work included cell viability, nitric oxide assay, screening of pro‐inflammatory cytokines using mouse cytokine arrays, individual ELISAs to validate data from the arrays, and RT‐PCR to investigate a possible signaling pathway for GLE neuroprotective effect in activated BV‐2 microglial cells. Cell viability results showed that increasing concentrations of GLE, ranging from 0.5 to 1.3 mg/ml, induced BV‐2 cytotoxicity in a dose‐response manner (IC50: 0.79 mg/ml). Also, the results obtained indicate that lower concentrations of GLE (&lt; 0.5mg/ml) showed to be potent in reducing the release of nitric oxide in activated BV‐2 cells. The results from the cytokine arrays showed that GLE decreased the expression of several pro‐inflammatory cytokines including Granulocyte Colony Stimulating Factor (G‐CSF), Interleukin 1‐alpha (IL1‐α), Monocyte chemotactic protein‐5 (MCP‐5), Macrophage Inflammatory Protein 3 Alpha (MIP3‐α), and Regulated on Activation Normal T Expressed and Secreted (RANTES). The data obtained were further validated using individual ELISA, and the results showed that GLE was able to decrease the expression of each one of the downregulated proteins in the cytokine arrays. RT‐PCR was performed using different genes that are involved in the NFκB signaling activation. The results also showed that GLE decreases the mRNA expression of CHUK, NFκB 1/p150, and IKBKE, which are genes that participate in the NFκB translocation to the nucleus, and subsequent release of pro‐inflammatory cytokines. The findings suggest that GLE may be a new potential therapeutic option for decreasing inflammatory responses seen in Alzheimer’s and dementia patients.Support or Funding InformationSupported by a NIMHD Grant U54 007582

Rhein Suppresses Neuroinflammation via Multiple Signaling Pathways in LPS‐Stimulated BV2 Microglia Cells

As a bioactive absorbed compound of rhubarb, Rhein is applied for the treatment of brain injury. However, the underlying pharmacological mechanisms remain unclear. In this study, we aimed to explore antineuroinflammatory functions and underlying mechanisms of Rhein in vitro. BV2 microglia cells were chosen and irritated by LPS. The influence of Rhein on cell viability was determined using MTT assay. We finely gauged the proinflammatory cytokines of TNF-α and IL-1β through tests of immunofluorescence staining, ELISA, RT-qPCR, and western blot. Additionally, mediators including IL-6, IL-12, iNOS, and IL-10 were surveyed by ELISA. Furthermore, protein levels of the underlying signaling pathways (PI3K/Akt, p38, ERK1/2, and TLR4/NF-κB) were tested adopting western blot. We found that Rhein reduced the secretion of pivotal indicators including TNF-α and IL-1β, effectively restraining their mRNA and protein expression in LPS-activated BV2 microglial cells. Besides, Rhein treatment demoted the production of IL-6, IL-12, and iNOS and promoted the excretion of IL-10. Subsequent mechanistic experiments revealed that Rhein obviously downregulated the phosphorylation levels of PI3K, Akt, p38, and ERK1/2 and simultaneously upregulated the PTEN expression. In addition, Rhein antagonized the increase of TLR4, p-IκBα, and NF-κB. In summary, Rhein suppresses neuroinflammation via multiple signaling pathways (PI3K/Akt, p38, ERK1/2, and TLR4/NF-κB) in LPS-stimulated BV2 microglia cells. This study highlights a natural agent for prevention and treatment of neuroinflammation.

Baicalein attenuates the neuroinflammation in LPS-activated BV-2 microglial cells through suppression of pro-inflammatory cytokines, COX2/NF-κB expressions and regulation of metabolic abnormality

Baicalein (5,6,7-trihydroxyflavone), isolated from the root of traditional Chinese herb Scutellaria baicalensis Georgi, has anti-inflammatory and anti-oxidative activities. This study explored the protective and modulatory mechanisms of baicalein on neuroinflammation, oxidative stress and metabolic abnormality in lipopolysaccharide (LPS)-activated BV-2 cells. Our results demonstrated that treatment with baicalein remarkably restrained the production of pro-inflammatory factors including nitric oxide (NO), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in LPS-activated BV-2 cells. Moreover, baicalein significantly inhibited reactive oxygen species (ROS) production, decreased cyclooxygenase-2 (COX-2) and nuclear factor-b (NF-κB)/p65 expression. 1H NMR metabolomics analysis revealed that 12 differential metabolites were regulated by baicalein, implicated in alanine, aspartate and glutamate metabolism, glutathione metabolism, arginine and proline metabolism, D-glutamine and D-glutamate metabolism. In conclusion, these results indicated that baicalein has protective and modulatory effects on neuroinflammation and oxidative stress in LPS-activated BV-2 cells.

AGK2 Alleviates Lipopolysaccharide Induced Neuroinflammation through Regulation of Mitogen-Activated Protein Kinase Phosphatase-1.

Neuroinflammation is associated with the progression of multiple neurological diseases. Many studies show that SIRT2 involves in multiple inflammatory processes. While, the mechanisms remain unclear. The purpose of this study was to explore the effect of SIRT2 inhibitor AGK2 on inflammatory responses and MAPK signaling pathways in LPS activated microglia in vitro and in vivo. The effect of AGK2 on cell viability of BV2 microglial cells was detected by CCK-8 assay. The expression of inflammatory cytokine iNOS was analyzed by western blotting and immunofluorescence. The mRNA expressions of iNOS, TNF-α, and IL-1β were detected by real-time polymerase chain reaction (RT-PCR). The SIRT2, phospho-P38, P38, phospho-JNK, JNK, phospho-ERK, ERK, α-tubulin, and acetyl-α-tubulin were analyzed by western blotting respectively. The interaction between SIRT2 and MKP-1 was measured by Co-immunoprecipitation (Co-IP) assay. Double immunofluorescent staining was performed to detect the expressions of CD11b and iNOS or SIRT2 in brain tissues. We found that AGK2 could suppress LPS-induced inflammatory cytokines (iNOS, TNF-α, and IL-1β) expression levels in BV2 microglial cells. Moreover, it could effectively reduce the expression of SIRT2 and increase the acetylation of α-tubulin in LPS activated BV2 microglial cells and LPS induced mice neuroinflammation. In addition, our results showed that AGK2 could reduce the increase of phosphorylation p38, JNK, and ERK after LPS challenge. Co-IP results showed that there was no direct interaction between MKP-1 and SIRT2. However, AGK2 by inhibition of SIRT2 could increase the expression of MKP-1. Furthermore, AGK2 could inhibit the activation of BV2 microglia and expression of iNOS and SIRT2 in LPS treated mice brain tissue. Taken together, our results suggested that AGK2 might alleviate lipopolysaccharide induced neuroinflammation through regulation of mitogen-activated protein kinase phosphatase-1. Graphical abstract.

Andalucin from Artemisia lannta suppresses the neuroinflammation via the promotion of Nrf2-mediated HO-1 levels by blocking the p65–p300 interaction in LPS-activated BV2 microglia

BackgroundNeuroinflammation plays an important role in many neurodegenerative conditions such as Alzheimer's disease (AD) and Parkinson disease (PD). Andalucin (ADL), a sesquiterpene lactone from Artemisia lannta, has been reported to exhibit NO inhibition in vitro. However, the effect of ADL on microglia-mediated neuroinflammation has not been investigated. PurposeThis study was designed to determine the anti-neuroinflammatory effect of ADL against LPS-activated BV2 microglial cells and to explore the underlying mechanisms. MethodsThe production of pro-inflammatory mediators and cytokines were measured by ELISA. The relevant mechanisms were analyzed by qRT-PCR, Luciferase assay, Western blot and Co-immunoprecipitation Assay. ResultsADL inhibited the LPS-induced release of NO, PGE2, TNF-α, IL-6 and IL-1β. In addition, ADL reduced the mRNA and protein levels of iNOS and COX-2. Mechanism studies found that ADL activated Nrf2/HO-1 signaling pathway and suppressed NF-κB signaling pathway. Further investigation showed that the stimulative effect of ADL on Nrf2 transcriptional activity and the inhibitory effect of ADL on RelA transcriptional activity were due to its regulation on p300-Nrf2/p65 interaction. ConclusionADL displayed anti-neuroinflammatory activity in LPS-activated BV2 cells. The mechanism concerns its regulatory effect on the crosstalk between Nrf2 and p65.