N9 Cells Research Articles

Inhibiting the Cholesterol Storage Enzyme ACAT1/SOAT1 in Myelin Debris-Treated Microglial Cell Lines Activates the Gene Expression of Cholesterol Efflux Transporter ABCA1

Aging is the major risk factor for Alzheimer’s disease (AD). In the aged brain, myelin debris accumulates and is cleared by microglia. Phagocytosed myelin debris increases neutral lipid droplet content in microglia. Neutral lipids include cholesteryl esters (CE) and triacylglycerol (TAG). To examine the effects of myelin debris on neutral lipid content in microglia, we added myelin debris to human HMC3 and mouse N9 cells. The results obtained when using 3H-oleate as a precursor in intact cells reveal that myelin debris significantly increases the biosynthesis of CE but not TAG. Mass analyses have shown that myelin debris increases both CE and TAG. The increase in CE biosynthesis was abolished using inhibitors of the cholesterol storage enzyme acyl-CoA:cholesterol acyltransferase 1 (ACAT1/SOAT1). ACAT1 inhibitors are promising drug candidates for AD treatment. In myelin debris-loaded microglia, treatment with two different ACAT1 inhibitors, K604 and F12511, increased the mRNA and protein content of ATP-binding cassette subfamily A1 (ABCA1), a protein that is located at the plasma membrane and which controls cellular cholesterol disposal. The effect of the ACAT1 inhibitor on ABCA1 was abolished by preincubating cells with the liver X receptor (LXR) antagonist GSK2033. We conclude that ACAT1 inhibitors prevent the accumulation of cholesterol and CE in myelin debris-treated microglia by activating ABCA1 gene expression via the LXR pathway.

Neobavaisoflavone Ameliorates Memory Deficits and Brain Damage in Aβ25-35-Induced Mice by Regulating SIRT1.

Alzheimer's disease (AD) is a common chronic neurodegenerative disease in older people, and there is no specific treatment that can stop or reverse its progression. Neobavaisoflavone (NBIF) is a flavonoid that has been shown to have neuroprotective effects, but its role in AD has not been revealed. The present study investigated the role and mechanism of NBIF on Aβ25-35-induced brain injury. In this experiment, the AD mouse model was established by injection of Aβ25-35 peptides (200 μM, icv), and Donepezil (Don, 10 mg/kg/days), NBIF-L (15 mg/kg/days), and NBIF-H (30 mg/kg/days) were administered orally for 4 weeks. Learning memory, hippocampal pathological changes, pathological markers, apoptosis, oxidative stress, inflammation, immune cells were measured in mice. Network pharmacology combined with the GEO database led to the identification of SIRT1, a key target for NBIF intervention in AD, and levels of SIRT1, p-STAT3 and FOXO1 were measured. In addition, the antagonistic activity of SIRT1 transfection silencing against NBIF in Aβ25-35-induced in N9 cells and N2a-APP69 cells was investigated to assess whether the effects caused by NBIF were mediated by SIRT1. The results showed that NBIF ameliorated learning memory and hippocampal neuronal damage, reduced pathological markers, apoptosis, oxidative stress and neuroinflammation, and modulated immune cells. SIRT1 is a key target for NBIF intervention in AD, and NBIF upregulates SIRT1 and reduces the expression levels of p-STAT3 and FOXO1. Furthermore, silencing SIRT1 effectively reduced the protective effect of NBIF on Aβ25-35-induced N9 cells and N2a-APP69 cells, which indicated that the protective effect of NBIF on AD is related to SIRT1. NBIF ameliorated Aβ25-35-induced brain injury by inhibiting apoptosis, oxidative stress, and neuroinflammation, which may be mediated through SIRT1 signaling. These findings provide a rationale for NBIF in the treatment of AD and help facilitate the development of clinical therapeutic agents for AD.

Ruscogenin Exerts Anxiolytic-Like Effect via Microglial NF-κB/MAPKs/NLRP3 Signaling Pathways in Mouse Model of Chronic Inflammatory Pain.

Long-term inflammation can cause chronic pain and trigger patients' anxiety by sensitizing the central nervous system. However, effective drugs with few side effects for treating chronic pain-induced anxiety are still lacking. The anxiolytic and anti-inflammatory effects of ruscogenin (RUS), an important active compound in Ophiopogon japonicus, were evaluated in a mouse model of chronic inflammatory pain and N9 cells. RUS (5, 10, or 20 mg/kg/day, i.g.) was administered once daily for 7 days after CFA injection; pain- and anxiety-like behaviors were assessed in mice. Anti-inflammatory effect of RUS (0.1, 1, 10 μM) on N9 microglia after LPS treatment was evaluated. Inflammatory markers (TNF-α, IL-1β, IL-6, CD86, IL-4, ARG-1, and CD206) were measured using qPCR. The levels of IBA1, ROS, NF-κB, TLR4, P-IKK, P-IκBα, and P65, MAPKs (ERK, JNK, and P38), NLRP3 (caspase-1, ASC, and NLRP3) were detected by Western blotting or immunofluorescence staining. The potential target of RUS was validated by molecular docking and adeno-associated virus injection. Mice in CFA group exhibited allodynia and anxiety-like behaviors. LPS induced neuroinflammation in N9 cells. Both CFA and LPS increased the levels of IBA1, ROS, and inflammatory markers. RUS (10 mg/kg invivo and 1 μM invitro) alleviated these alterations through NF-κB/MAPKs/NLRP3 signaling pathways but had no effect on pain hypersensitivity. TLR4 strongly interacted with RUS, and TLR4 overexpression abolished the effects of RUS on anxiety and neuroinflammation. RUS exerts anti-inflammatory and anxiolytic effects via TLR4-mediated NF-κB/MAPKs/NLRP3 signaling pathways, which provides a basis for the treatment of chronic pain-induced anxiety.

MK2 inhibitor PF-3644022 shows protective effect in mouse microglial N9 cell line induced with cigarette smoke extract.

Neuroinflammation is suggested as one of the potential links between CS-induced neuronal dysfunction. Cigarette smoke (CS) is one of the significant contributors of neuroinflammation, consequently leading to cognitive impairment and neurodegeneration. Microglia are the key resident macrophage cells in the brain with cell surface TLR4 receptor for responding to various stress signals. The CS constituents promote inflammation and oxidative stress in microglia leading to cytotoxicity through the TLR4-MK2 axis. However, the role of MK2 kinase in CS-induced microglial inflammation is not yet clearly understood. Therefore, we have used an MK2 inhibitor, PF-3644022 to study modulation of CS-extract induced oxidative and inflammatory signaling in a mouse microglial cell line, Furthermore, we also evaluated the enzymatic activity of acetylcholinesterase (AChE) on a direct exposure of enzyme with CS. CS exposure led to microglial cytotoxicity and enhanced the level of oxidative stress and proinflammatory cytokine release by microglial cells. The microglial cells pretreated with MK2 inhibitor, PF-3644022 significantly reduced the levels of oxidative stress markers, proinflammatory markers, and improved the level of antioxidant proteins in these cells. In addition, direct exposure of CS showed reduction in the enzymatic activity of AChE.

Neurotoxicity Profiling of Aluminum Salt-Based Nanoparticles as Adjuvants for Therapeutic Cancer Vaccine.

Therapeutic vaccines containing aluminum adjuvants have been widely used in the treatment of tumors due to their powerful immune-enhancing effects. However, the neurotoxicity of aluminum adjuvants with different physicochemical properties has not been completely elucidated. In this study, a library of engineered aluminum oxyhydroxide (EAO) and aluminum hydroxyphosphate (EAHP) nanoparticles was synthesized to determine their neurotoxicity in vitro. It was demonstrated that the surface charge of EAHPs and size of EAOs did not affect the cytotoxicity in N9, bEnd.3, and HT22 cells; however, soluble aluminum ions trigger the cytotoxicity in three different cell lines. Moreover, soluble aluminum ions induce apoptosis in N9 cells, and further mechanistic studies demonstrated that this apoptosis was mediated by mitochondrial reactive oxygen species generation and mitochondrial membrane potential loss. This study identifies the safety profile of aluminum-containing salts adjuvants in the nervous system during therapeutic vaccine use, and provides novel design strategies for their safer applications. SIGNIFICANCE STATEMENT: In this study, it was demonstrated that engineered aluminum oxyhydroxide and aluminum hydroxyphosphate nanoparticles did not induce cytotoxicity in N9, bEnd.3, and HT22 cells. In comparation, soluble aluminum ions triggered significant cytotoxicity in three different cell lines, indicating that the form in which aluminum is presenting may play a crucial role in its safety. Moreover, apoptosis induced by soluble aluminum ions was dependent on mitochondrial damage. This study confirms the safety of engineered aluminum adjuvants in vaccine formulations.

Nanoplastics activate a TLR4/p38-mediated pro-inflammatory response in human intestinal and mouse microglia cells

The crescent presence of nanoplastics in the environment raises concerns regarding their potential impact on health. This study exposed human colon adenocarcinoma cells (HT29) and microglia cells (N9) to nanoplastics (25 nm, 50 nm, and 100 nm Polystyrene) to investigate their inflammatory responses, which are vital for body's defence. Although cytotoxicity remained generally low, HT29 cells exhibited a notable upregulation of p50 and p38 expression, concomitant with elevated TLR4 expression, in contrast with N9 cells that showed a less pronounced upregulation of these proteins. Additionally, nanoplastic exposure increased IL-1ß levels, partially attenuated by pre-exposure to TLR4 or p38 inhibitors. Intriguingly, N9 cells exposed to nanoplastics exhibited substantial increases in iNOS mRNA. This effect was entirely prevented by pre-exposure to TLR4 or p38 inhibitors, while TNF-α mRNA levels remained relatively stable. These findings underscore the potential of nanoplastics to activate inflammatory pathways, with response kinetics varying depending on the cell type.

Dichotomous effect of methylphenidate on microglia and astrocytes: Insights from in vitro and animal studies

Methylphenidate (MPH) has been used for decades to treat attention-deficit/hyperactivity disorder (ADHD) and narcolepsy. Moreover, several studies have shown that it is subject to misuse, particularly among college students and adolescents, for cognitive enhancement or as a recreational drug. This phenomenon causes concern, and it is critical to clarify better how MPH impacts brain cells. In fact, data has suggested that MPH could result in neuroinflammation and neurodegeneration across several brain regions; however, little is known about the effect of MPH on glial cells. To address this, we used microglia N9 cell line and primary cultures of cortical astrocytes that were exposed to MPH (0.01 – 2 mM), as well as Wistar Kyoto rats (WKY) chronically administered with MPH (1.5 mg/kg/day). Several parameters were analyzed, and we concluded that MPH has no significant direct effect on microglial cells, apart from cell migration impairment. On the contrary, MPH promotes astrogliosis, oxidative/nitrosative stress, and increases proinflammatory cytokine TNF levels by astrocytes, which was concordant with the results obtained in the hippocampus of WKY rats. Overall, the present results suggest that brain cells respond differently to MPH, with a more prominent direct effect on astrocytes when compared to microglia.

Regulation of microglia-mediated inflammation by host lncRNA Gm20559 upon flaviviral infection

BackgroundJapanese Encephalitis Virus (JEV) and West Nile Viruses (WNV) are neurotropic flaviviruses which cause neuronal death and exaggerated glial activation in the central nervous system. Role of host long non coding RNAs in shaping microglial inflammation upon flavivirus infections has been unexplored. This study attempted to decipher the role of lncRNA Gm20559 in regulating microglial inflammatory response in context of flaviviruses. MethodsAntisense oligonucleotide LNA Gapmers designed against lncRNA Gm20559 and non-specific site (negative control) were used for Gm20559 knockdown in JEV and WNV-infected N9 microglial cells. Upon establishing successful Gm20559 knockdown, expression of various proinflammatory cytokines, chemokines, interferon-stimulated genes (ISGs) and RIG-I were checked by qRT-PCR and cytometric bead array. Western Blotting was done to analyse the phosphorylation level of various inflammatory markers and viral non-structural protein expression. Plaque Assays were employed to quantify viral titres in microglial supernatant upon knocking down Gm20559. Effect of microglial supernatant on HT22 neuronal cells was assessed by checking expression of apoptotic protein and viral non-structural protein by Western Blotting. ResultsUpregulation in Gm20559 expression was observed in BALB/c pup brains, primary microglia as well as N9 microglia cell line upon both JEV and WNV infection. Knockdown of Gm20559 in JEV and WNV-infected N9 cell led to the reduction of major proinflammatory cytokines – IL-1β, IL-6, IP-10 and IFN-β. Inhibition of Gm20559 upon JEV infection in N9 microglia also led to downregulation of RIG-I and OAS-2, which was not the case in WNV-infected N9 microglia. Phosphorylation level of P38 MAPK was reduced in case of JEV-infected N9 microglia and not WNV-infected N9 microglia. Whereas phosphorylation of NF-κB pathway was unchanged upon Gm20559 knockdown in both JEV and WNV-infected N9 microglia. However, treating HT22 cells with JEV and WNV-infected microglial supernatant with and without Gm20559 could not trigger cell death or influence viral replication. ConclusionKnockdown studies on lncRNA Gm20559 suggests its pivotal role in maintaining the inflammatory milieu of microglia in flaviviral infection by modulating the expression of various pro-inflammatory cytokines. However, Gm20559-induced increased microglial proinflammatory response upon flavivirus infection fails to trigger neuronal death.

Lipid metabolism transcriptomics of murine microglia in Alzheimer’s disease and neuroinflammation

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) plaques followed by intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau. An unrestrained immune response by microglia, the resident cells of the central nervous system (CNS), leads to neuroinflammation which can amplify AD pathology. AD pathology is also driven by metabolic dysfunction with strong correlations between dementia and metabolic disorders such as diabetes, hypercholesterolemia, and hypertriglyceridemia. Since elevated cholesterol and triglyceride levels appear to be a major risk factor for developing AD, we investigated the lipid metabolism transcriptome in an AD versus non-AD state using RNA-sequencing (RNA-seq) and microarray datasets from N9 cells and murine microglia. We identified 52 differentially expressed genes (DEG) linked to lipid metabolism in LPS-stimulated N9 microglia versus unstimulated control cells using RNA-seq, 86 lipid metabolism DEG in 5XFAD versus wild-type mice by microarray, with 16 DEG common between both datasets. Functional enrichment and network analyses identified several biological processes and molecular functions, such as cholesterol homeostasis, insulin signaling, and triglyceride metabolism. Furthermore, therapeutic drugs targeting lipid metabolism DEG found in our study were identified. Focusing on drugs that target genes associated with lipid metabolism and neuroinflammation could provide new targets for AD drug development.

Japanese Encephalitis virus infection in astrocytes modulate microglial function: Correlation with inflammation and oxidative stress

BackgroundJapanese Encephalitis Virus (JEV) is a neurotropic virus which has the propensity to infect neuronal and glial cells of the brain. Astrocyte-microglia crosstalk leading to the secretion of various factors plays a major role in controlling encephalitis in brain. This study focused on understanding the role of astrocytic mediators that further shaped the microglial response towards JEV infection. MethodsAfter establishing JEV infection in C8D1A (mouse astrocyte cell line) and primary astrocyte enriched cultures (PAEC), astrocyte supernatant was used for preparation of conditioned media. Astrocyte supernatant was treated with UV to inactivate JEV and the supernatant was added to N9 culture media in ratio 1:1 for preparation of conditioned media. N9 microglial cells post treatment with astrocyte conditioned media and JEV infection were checked for expression of various inflammatory genes by qRT-PCR, levels of secreted cytokines in N9 cell supernatant were checked by cytometric bead array. N9 cell lysates were checked for expression of proteins – pNF-κβ, IBA-1, NS3 and RIG-I by western blotting. Viral titers were measured in N9 supernatant by plaque assays. Immunocytochemistry experiments were done to quantify the number of infected microglial cells after astrocyte conditioned medium treatment. Expression of different antioxidant enzymes was checked in N9 cells by western blotting, levels of reactive oxygen species (ROS) was detected by fluorimetry using DCFDA dye. ResultsN9 microglial cells post treatment with JEV-infected astrocyte conditioned media and JEV infection were activated, showed an upsurge in expression of inflammatory genes and cytokines both at the transcript and protein levels. These N9 cells showed a decrease in quantity of viral titers and associated viral proteins in comparison to control cells (not treated with conditioned media but infected with JEV). Also, N9 cells upon conditioned media treatment and JEV infection were more prone to undergo oxidative stress as observed by the decreased expression of antioxidant enzymes SOD-1, TRX-1 and increased secretion of reactive oxygen species (ROS). ConclusionAstrocytic mediators like TNF-α, MCP-1 and IL-6 influence microglial response towards JEV infection by promoting inflammation and oxidative stress in them. As a result of increased microglial inflammation and secretion of ROS, viral replication is lessened in conditioned media treated and JEV infected microglial cells as compared to control cells with no conditioned media treatment but only JEV infection.

Effect of aqueous extract of Corni Fructus on Aβ_(25-35)-induced brain injury and neuroinflammation in mice with Alzheimer's disease

The purpose of this study was to investigate the effect of aqueous extract of Corni Fructus on β-amyloid protein 25-35(Aβ_(25-35))-induced brain injury and neuroinflammation in Alzheimer's disease(AD) mice to provide an experimental basis for the treatment of AD by aqueous extract of Corni Fructus. Sixty C57BL/6J male mice were randomly divided into a sham group, a model group, a positive control group(huperizine A, 0.2 mg·kg~(-1)), a low-dose aqueous extract of Corni Fructus group(1.3 g·kg~(-1)), a medium-dose aqueous extract of Corni Fructus group(2.6 g·kg~(-1)), and a high-dose aqueous extract of Corni Fructus group(5.2 g·kg~(-1)). The AD model was induced by lateral ventricular injection of Aβ_(25-35) in mice except for those in the sham group, and AD model mice were treated with corresponding drugs by gavage for 24 days. The behavioral test was performed one week before animal dissection. Hematoxylin-eosin(HE) staining was performed to observe the morphology of neurons in the hippocampal region. Flow cytometry was used to detect the apoptosis level of primary hippocampal cells in mice. ELISA kits were used to detect the levels of β-amyloid protein 1-42(Aβ_(1-42)) and phosphorylated microtubule-associated protein Tau(p-Tau) in mouse brain tissues. Immunofluorescence and Western blot were used to detect the expression of related proteins in mouse brain tissues. MTT assay was used to detect the effect of compounds in aqueous extract of Corni Fructus on Aβ_(25-35)-induced N9 cell injury. Molecular docking was employed to analyze the interactions of caffeic acid, trans-p-hydroxy cinnamic acid, isolariciresinol-9'-O-β-D-glucopyranoside, esculetin, and(+)-lyoniresinol with β-amyloid precursor protein(APP), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α). Aqueous extract of Corni Fructus could improve the learning and memory abilities of Aβ_(25-35)-induced mice by increasing the duration of the autonomous activity, the rate of autonomous alternation, the preference coefficient, and the discrimination coefficient, and reduce Aβ_(25-35)-induced brain injury and neuroinflammation in mice by increasing the expression levels of interleukin-10(IL-10) and B-cell lymphoma-2(Bcl-2) in brain tissues, decreasing the expression levels of Aβ_(1-42), p-Tau, IL-6, TNF-α, cysteine aspartate-specific protease 3(caspase-3), cysteine aspartate-specific protease 9(caspase-9), and Bcl-2-associated X protein(Bax), and decreasing the number of activated glial cells in brain tissues. The results of cell experiments showed that esculetin and(+)-lyoniresinol could improve Aβ_(25-35)-induced N9 cell injury. Molecular docking results showed that caffeic acid, trans-p-hydroxy cinnamic acid, isolariciresinol-9'-O-β-D-glucopyranoside, esculetin, and(+)-lyoniresinol had good binding affinity with APP and weak binding affinity with IL-6 and TNF-α. Aqueous extract of Corni Fructus could ameliorate cognitive dysfunction and brain damage in Aβ_(25-35)-induced mice by reducing the number of apoptotic cells and activated glial cells in the brain and decreasing the expression level of inflammatory factors. Caffeic acid, trans-p-hydroxy cinnamic acid, isolariciresinol-9'-O-β-D-glucopyranoside, esculetin, and(+)-lyoniresinol may be the material basis for the anti-AD effect of aqueous extract of Corni Fructus.

Deacetyl epoxyazadiradione ameliorates BPA-induced neurotoxicity by mitigating ROS and inflammatory markers in N9 cells and zebrafish larvae

Bisphenol A (BPA) leaches from plastic products have become a major inevitable concern among the research society. Human exposure to BPA leads to deleterious effects on multiple organs by the induced hyper inflammatory and oxidative stress responses. Due to the compromised antioxidant mechanism, the brain environment was highly susceptible and required special concern to ameliorate the effects of BPA. Hence, this study investigates the potential of neem-derived semi natural deacetyl epoxyazadiradione (DEA) against the oxidative stress and inflammatory response induced by BPA exposure in N9 cells and zebrafish larvae. The results from the in vitro analyses showed a decrease in cell viability in the MTT assay and a decline in mitochondrial damage in BPA-exposed N9 cells. Further in vivo, results revealed that pre-treatment of DEA to zebrafish larvae has significantly reduced the level of superoxide anion and increased the production of antioxidant enzymes such as SOD, CAT, GST, GPx and GR. We also found a significant decrease in the production of nitric oxide (p < 0.0001) and iNOS gene expression at 150 μM concentration. Further, DEA pre-treatment improved the behaviour of zebrafish larvae by ameliorating the production of the AChE enzyme. In conclusion, DEA protected zebrafish larvae from BPA toxicity by ameliorating oxidative stress and inflammatory responses.

Effects of Jatrorrhizine on inflammatory response induced by H2O2 in microglia by regulating the MAPK/NF-κB/NLRP3 signaling pathway.

Microglia-induced neuroinflammation is a contributing factor to neurodegenerative diseases. Jatrorrhizine (JAT), an alkaloid isolated from Huanglian, has been shown to have neuroprotective effects against various neurodegenerative diseases, but its impact on microglia-induced neuroinflammation remains unclear. In this study, we investigated the role of JAT in MAPK/NF-κB/NLRP3 signaling pathway in an H2O2-induced oxidative stress model using microglia (N9 cells). We divided cells into six groups, including control, JAT, H2O2, H2O2 + 5μmol/L JAT, H2O2 + 10μmol/L JAT, and H2O2 + 20μmol/L minocycline groups. Cell viability was measured using MTT assay and TNF-α levels were detected with an ELISA Kit. Western blot was used to detect NLRP3, HMGB1, NF-κB, p-NF-κB, ERK, p-ERK, p38, p-p38, p-JNK, JNK, IL-1β, and IL-18 expressions. Our results showed that JAT intervention improved H2O2-induced cytotoxicity in N9 cells and reduced the elevated expression of TNF-α, IL-1β, IL-18, p-ERK/ERK, p-p38/p38, p-JNK/JNK, p-p65/p65, NLRP3, and HMGB1 in H2O2 group. Furthermore, treatment with ERK inhibitor SCH772984 specifically blocked ERK phosphorylation, resulting in decreased protein levels of p-NF-κB, NLRP3, IL-1β, and IL-18 in H2O2 group. These results suggest that the MAPK/NF-κB signaling pathway may regulate the protein levels of NLRP3. Overall, our study indicates that JAT may have a protective effect on H2O2-treated microglia via inhibition the MAPK/NF-κB/NLRP3 pathway and could be a potential therapeutic approach for neurodegenerative diseases.

Modified Xiaoyao San reverses lipopolysaccharide-induced depression-like behavior through suppressing microglia M1 polarization via enhancing autophagy involved in PI3K/Akt/mTOR pathway in mice

Ethnopharmacological relevanceModified Xiaoyao San (MXYS), a clinical empirical modified formula based on famous traditional Chinese herbal prescription Xiaoyao San, according to the “yu syndrome” theory of traditional Chinese medicine. MXYS has been shown to be an excellent effective therapy for depression patients in clinic, but the antidepressant mechanisms remain unclear. Aim of the studyA growing body of evidence indicates the microglia autophagy and M1 polarized microglia (proinflammatory phenotype)-mediated neuroinflammation act critical roles in the pathogenesis of depression. This study aimed to investigate whether MXYS exerts antidepressant efficacy through inhibition of M1 polarized microglia-mediated neuroinflammation and modulation of autophagy involved in PI3K/Akt/mTOR pathway. Materials and methodsIn present research, the lipopolysaccharide (LPS)-induced depressive mice and LPS-stimulated N9 microglia cell line were utilized. Behavioral tests (sucrose preference, tail suspension and open field tests) were carried out to evaluate the antidepressant effect of MXYS. The neuronal damage was measured by Nissl's staining in LPS-treated mice. The proinflammatory cytokine levels, the autophagic markers, microglia M1 polarization as well as the PI3K/Akt/mTOR pathway related proteins of MXYS treatment were analyzed by ELISA kits, Western blot and immunofluorescence staining in vivo and vitro. Finally, the influence of autophagy antagonist (3-MA) on the protective effect of MXYS-containing serum in the LPS-stimulated N9 microglia was investigated. ResultsTreatment of LPS-induced depressive mice with MXYS significantly reversed depression-like behaviors, accompanied by reduction of proinflammatory cytokine levels (TNF-α, IL-1β) and amelioration of neuronal damage in prefrontal cortex. MXYS suppressed microglia M1 polarization and promoted autophagy in prefrontal cortex and LPS-stimulated N9 cells. Importantly, the remarkable inhibitory effect of the MXYS-medicated serum on microglia M1 polarization was blocked by autophagy antagonist 3-MA in LPS-stimulated N9 cells. Meanwhile, the MXYS treatment exhibited an excellent inhibition effect of PI3K/Akt/mTOR pathway in vivo and vitro. ConclusionOur research suggests that the antidepressant effect of MXYS in LPS-induced depressive mice may be related to alleviate neuroinflammation through suppression of microglia M1 polarization via enhancing autophagy involved in inactivation of the PI3K/Akt/mTOR pathway.

Naringenin and apigenin ameliorates corticosterone-induced depressive behaviors

BackgroundDepression is a common kind of mental illness, and it becomes the main health burden in the world. PurposeThe aim of this study was to investigate the antidepressant effects of naringin and apigenin isolated from Chrysanthemum morifolium Ramatis. MethodsFirstly, 20 mg/kg corticosterone (CORT) was injected into mice to establish an in vivo model of depression. After treated with different dosages of naringenin and apigenin for 3 weeks, the mice underwent a series of behavioral experiments. Following this, all mice were sacrificed and biochemical analyses were performed. Subsequently, CORT (500 μM) induced PC12 cells was used as an in vitro model of depression, and lipopolysaccharide (LPS) (1 μg ml−1) induced N9 microglia cells was used as an in vitro model of neuroinflammation in N9 microglia cells, to investigate the neuroprotective mechanisms of naringenin and apigenin. ResultsResults showed that the naringenin and apigenin treatment ameliorated CORT-induced sucrose preference decrease and immobility time increase, elevated the 5-hydroxytryptamine(5-HT), dopamine (DA) and norepinephrine (NE) levels, and enhanced the cAMP-response element binding protein (CREB) and brain derived neurotrophic factor (BDNF) protein expressions in the hippocampus. The results showed that the naringenin and apigenin treatment improved the PC-12 cell viability through reducing apoptosis rate induced by CORT. Furthermore, naringenin and apigenin were able to inhibit the activation of N9 cells after LPS induction, and shift microglia from proinflammatory M1 microglia toward anti-inflammatory M2 microglia, as evidenced by the decreased ratio of M1 type microglia marker CD86 and M2 type microglia marker CD86. ConclusionThese results suggested that naringenin and apigenin may improve depressive behaviors through promoting BDNF and inhibiting neuroinflammation and neuronal apoptosis.

ACAT1/SOAT1 Blockade Suppresses LPS-Mediated Neuroinflammation by Modulating the Fate of Toll-like Receptor 4 in Microglia.

Cholesterol is stored as cholesteryl esters by the enzymes acyl-CoA:cholesterol acyltransferases/sterol O:acyltransferases (ACATs/SOATs). ACAT1 blockade (A1B) ameliorates the pro-inflammatory responses of macrophages to lipopolysaccharides (LPS) and cholesterol loading. However, the mediators involved in transmitting the effects of A1B in immune cells is unknown. Microglial Acat1/Soat1 expression is elevated in many neurodegenerative diseases and in acute neuroinflammation. We evaluated LPS-induced neuroinflammation experiments in control vs. myeloid-specific Acat1/Soat1 knockout mice. We also evaluated LPS-induced neuroinflammation in microglial N9 cells with and without pre-treatment with K-604, a selective ACAT1 inhibitor. Biochemical and microscopy assays were used to monitor the fate of Toll-Like Receptor 4 (TLR4), the receptor at the plasma membrane and the endosomal membrane that mediates pro-inflammatory signaling cascades. In the hippocampus and cortex, results revealed that Acat1/Soat1 inactivation in myeloid cell lineage markedly attenuated LPS-induced activation of pro-inflammatory response genes. Studies in microglial N9 cells showed that pre-incubation with K-604 significantly reduced the LPS-induced pro-inflammatory responses. Further studies showed that K-604 decreased the total TLR4 protein content by increasing TLR4 endocytosis, thus enhancing the trafficking of TLR4 to the lysosomes for degradation. We concluded that A1B alters the intracellular fate of TLR4 and suppresses its pro-inflammatory signaling cascade in response to LPS.

Effects of Marginal Zn Excess and Thiamine Deficiency on Microglial N9 Cell Metabolism and Their Interactions with Septal SN56 Cholinergic Cells

Mild thiamine deficiency aggravates Zn accumulation in cholinergic neurons. It leads to the augmentation of Zn toxicity by its interaction with the enzymes of energy metabolism. Within this study, we tested the effect of Zn on microglial cells cultivated in a thiamine-deficient medium, containing 0.003 mmol/L of thiamine vs. 0.009 mmol/L in a control medium. In such conditions, a subtoxic 0.10 mmol/L Zn concentration caused non-significant alterations in the survival and energy metabolism of N9 microglial cells. Both activities of the tricarboxylic acid cycle and the acetyl-CoA level were not decreased in these culture conditions. Amprolium augmented thiamine pyrophosphate deficits in N9 cells. This led to an increase in the intracellular accumulation of free Zn and partially aggravated its toxicity. There was differential sensitivity of neuronal and glial cells to thiamine-deficiency-Zn-evoked toxicity. The co-culture of neuronal SN56 with microglial N9 cells reduced the thiamine-deficiency-Zn-evoked inhibition of acetyl-CoA metabolism and restored the viability of the former. The differential sensitivity of SN56 and N9 cells to borderline thiamine deficiency combined with marginal Zn excess may result from the strong inhibition of pyruvate dehydrogenase in neuronal cells and no inhibition of this enzyme in the glial ones. Therefore, ThDP supplementation can make any brain cell more resistant to Zn excess.

CB2R activation ameliorates late adolescent chronic alcohol exposure-induced anxiety-like behaviors during withdrawal by preventing morphological changes and suppressing NLRP3 inflammasome activation in prefrontal cortex microglia in mice

BackgroundChronic alcohol exposure (CAE) during late adolescence increases the risk of anxiety development. Alcohol-induced prefrontal cortex (PFC) microglial activation, characterized by morphological changes and increased associations with neurons, plays a critical role in the pathogenesis of anxiety. Alcohol exposure increases NLRP3 inflammasome expression, increasing cytokine secretion by activated microglia. Cannabinoid type 2 receptor (CB2R), an essential receptor of the endocannabinoid system, regulates microglial activation and neuroinflammatory reactions. We aimed to investigate the role of CB2R activation in ameliorating late adolescent CAE-induced anxiety-like behaviors and microglial activation in C57BL/6J mice. MethodsSix-week-old C57BL/6J mice were acclimated for 7 days and then were administered alcohol by gavage (4 g/kg, 25 % w/v) for 28 days. The mice were intraperitoneally injected with the specific CB2R agonist AM1241 1 h before alcohol treatment. Anxiety-like behaviors during withdrawal were assessed by open field test and elevated plus maze test 24 h after the last alcohol administration. Microglial activation, microglia-neuron interactions, and CB2R and NLRP3 inflammasome-related molecule expression in the PFC were measured using immunofluorescence, immunohistochemical, qPCR, and Western blotting assays. Microglial morphology was evaluated by Sholl analysis and the cell body-to-total cell size index. Additionally, N9 microglia were activated by LPS in vitro, and the effects of AM1241 on NLRP3 and N9 microglial activation were investigated. ResultsAfter CAE, mice exhibited severe anxiety-like behaviors during withdrawal. CAE induced obvious microglia-neuron associations, and increased expression of microglial activation markers, CB2R, and NLRP3 inflammasome-related molecules in the PFC. Microglia also showed marked filament retraction and reduction and cell body enlargement after CAE. AM1241 treatment ameliorated anxiety-like behaviors in CAE model mice, and it prevented microglial morphological changes, reduced microglial activation marker expression, and suppressed the microglial NLRP3 inflammasome activation and proinflammatory cytokine secretion induced by CAE. AM1241 suppressed the LPS-induced increase in NLRP3 inflammasome-related molecules, IL-1β release, and M1 phenotype markers (iNOS and CD86) in N9 cell, which was reversed by CB2R antagonist treatment. ConclusionsCAE caused anxiety-like behaviors in late adolescent mice at least partly by inducing microglial activation and increasing microglia-neuron associations in the PFC. CB2R activation ameliorated these effects by preventing morphological changes and suppressing NLRP3 inflammasome activation in PFC microglia.

Echinacoside exerts antidepressant-like effects through enhancing BDNF-CREB pathway and inhibiting neuroinflammation via regulating microglia M1/M2 polarization and JAK1/STAT3 pathway.

The present study was performed to investigate the antidepressant effect of echinacoside (ECH) using chronic unpredictable mild stress (CUMS) induced depression mice and lipopolysaccharide (LPS)-stimulated N9 microglial cells. CUMS treatment was performed on C57BL/6 mice for 28days, followed by gavaging with different doses of echinacoside (15 and 60mg/kg) for 21 consecutive days. Sucrose preference test (SPT), open field test (OFT), tail suspension test (TST), and forced swimming test (FST) were measured to assess the effects of echinacoside on CUMS-Induced Depressive-Like Behaviors. After that, the pathological changes of hippocampus were determined by Hematoxylin and eosin (HE) staining and Nissl staining; the neurotransmitters, pro-inflammatory cytokines and indoleamine 2,3-dioxygenase (IDO) levels, and the hypothalamic-pituitary-adrenal (HPA) axis activity were determined by enzyme linked immunosorbent assay (ELISA); Iba 1were evaluated by Immunofluorescence assay; Key protein expression levels of CREB/BDNF signal pathway were measured by western blotting. Subsequently, N9 cells were stimulated with 1μg/ml LPS to induce N9 microglia activation, and were treated with 5-20μM of echinacoside for 24h. After that, the levels of NO, interleukin (IL)-1β, IL-6, tumor necrosis factor alpha (TNF-α), IL-4, IL-10, and transforming growth factor beta (TGF-β) in N9 cell culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA) kits; morphology and Iba 1 expression level were observed by high-content screening assay; the M1 markers of CD11b, CD86 and M2 markers of CD206 were analyzed by imaging flow cytometry. Results show that treatment with echinacoside reversed CUMS-increased immobility time in OFT, TST, FST and reversed CUMS-reduced sucrose preference in SPT. In addition, echinacoside reduced the levels of pro-inflammatory cytokines and Iba 1. Moreover, echinacoside significantly increased p-CREB/CREB ratio and BDNF level in hippocampus. Furthermore, echinacoside reduced the secretion of inflammatory factors and inhibited microglia M1 polarization in N9 cells. In conclusion, echinacoside may be beneficial for the treatment of depression diseases through regulating the microglia balance by inhibiting the polarization of microglia to M1 phenotype, and improving hippocampal neurogenesis by the CREB-BDNF signaling pathway.

Thymidine and 2′-deoxyuridine reduce microglial activation and improve oxidative stress damage by modulating glycolytic metabolism on the Aβ25-35-induced brain injury

Alzheimer's disease (AD) is a progressive disease with a long duration and complicated pathogenesis. Thymidine (Thy) and 2′-deoxyuridine (2′-De) are pyrimidines nucleotides that are associated with nervous system diseases. However, it remains unclear whether Thy and 2′-De exert neuroprotective effects in AD. Therefore, this study was conducted to explore the interventional effects and mechanisms of Thy and 2′-De on the Aβ25-35-induced brain injury. Donepezil (Do, 10 mg/kg/d), Thy (20 mg/kg/d), and 2′-De (20 mg/kg/d) were administered for 4 weeks after the injection of Aβ25-35 peptides (200 μM, i.c.v.) to mice. UPLC-MS/MS method was performed to quantify Thy and 2′-De in the hippocampus of mice brain. The cognition ability, neuronal and mitochondria damage, and levels of Aβ1-42/Aβ1-40, p-Tau, Na+ K+-ATPase, apoptosis, oxidative stress, immune cells, and Iba 1+ were measured in Aβ25-35-induced mice. The oxygen consumption (OCR) and extracellular acidification rate (ECAR) were measured using a seahorse analyzer in Aβ25-35-induced N9 cells. Moreover, 2-Deoxy-D-glucose (2-DG), a glycolysis inhibitor, was added to explore the mechanisms underlying the effects of Thy and 2′-De on Aβ25-35-induced N9 cells. The expression of Iba 1+ and levels of CD11b+ and reactive oxygen species (ROS) were measured after treatment with Thy (5 μM) and 2′-De (10 μM) against 2-DG (5 mM) in Aβ25-35-induced N9 cells. The results suggested that Do, Thy, and 2′-De improved the cognition ability, attenuated the damage to hippocampus and mitochondria, downregulated the levels of Aβ1-42/Aβ1-40, p-Tau, Na+ K+-ATPase, apoptosis, oxidative stress, and Iba 1+, and regulated the immune response induced by Aβ25-35 against the brain injury. Furthermore, Do, Thy, and 2′-De increased ATP production and inhibited glycolysis in Aβ25-35-induced N9 cells. Moreover, 2-DG enhanced the effects of drugs, reduced microglial activation, and attenuated oxidative stress to interfere with Aβ25-35-induced N9 cells. In conclusion, Thy and 2′-De reduced microglial activation and improved oxidative stress damage by modulating glycolytic metabolism on the Aβ25-35-induced brain injury.