Ca 2+ spiking activity caused by the activation of store-operated Ca 2+ channels mediates TNF-α release from microglial cells under chronic purinergic stimulation

<P>Background: Alzheimer’s disease (AD) is a multifactorial neurodegenerative disorder that eventually leads to severe cognitive impairment. Although the exact etiologies of AD still remain elusive, increasing evidence suggests that neuroinflammation cascades mediated by microglial cells are associated with AD. Piper sarmentosum Roxb. (PS) is a medicinal plant reported to possess various biological properties, including anti-inflammatory, anti-psychotic and anti-oxidant activity. However, little is known about the anti-inflammatory activity of PS roots despite their traditional use to treat inflammatory- mediated ailments. Objective: This study aimed to evaluate the anti-inflammatory and neuroprotective properties of extracts obtained from the roots of PS against beta-amyloid (Aβ)-induced microglial toxicity associated with the production of pro-inflammatory mediators. Method: BV2 microglial cells were treated with hexane (RHXN), dichloromethane (RDCM), ethyl acetate (REA) and methanol (RMEOH) extracts of the roots of PS prior to activation by Aβ. The production and mRNA expression of pro-inflammatory mediators were evaluated by Griess reagent, ELISA kits and RT-qPCR respectively. The phosphorylation status of p38α MAPK was determined via western blot assay. BV2 conditioned medium was used to treat SH-SY5Y neuroblastoma cells and the neuroprotective effect was assessed using MTT assay. Results: PS root extracts, in particular RMEOH significantly attenuated the production and mRNA expression of IL-1β, IL-6 and TNF-α in Aβ-induced BV2 microglial cells. In addition, RHXN, REA and RMEOH extracts significantly reduced nitric oxide (NO) level and the inhibition of NO production was correlated with the total phenolic content of the extracts. Further mechanistic studies suggested that PS root extracts attenuated the production of cytokines by regulating the phosphorylation of p38α MAPK in microglia. Importantly, PS root extracts have protective effects against Aβ-induced indirect neurotoxicity either by inhibiting the production of NO, IL-1β, IL-6, and TNF-α in BV2 cells or by protecting SHSY5Y cells against these inflammatory mediators. Conclusions: These findings provided evidence that PS root extracts confer neuroprotection against Aβ- induced microglial toxicity associated with the production of pro-inflammatory mediators and may be a potential therapeutic agent for inflammation-related neurological conditions including Alzheimer’s disease (AD).</P>

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Chandipura virus dysregulates the expression of hsa-miR-21-5p to activate NF-κB in human microglial cells

Journal of Biomedical Science ◽

10.1186/s12929-021-00748-0 ◽

2021 ◽

Vol 28 (1) ◽

Author(s):

Neha Pandey ◽

Meghana Rastogi ◽

Sunit K. Singh

Keyword(s):

Expression Pattern ◽

Case Fatality ◽

Microglial Cells ◽

Luciferase Assay ◽

Western India ◽

Cellular Mirnas ◽

Over Expression ◽

Chandipura Virus ◽

Tnf Α

Abstract Background Chandipura virus (CHPV) is a negative single-stranded RNA virus of the Rhabdoviridae family. CHPV infection has been reported in Central and Western India. CHPV causes acute encephalitis with a case fatality rate of 70 % and mostly affects children below 15 years of age. CHPV infection in brain leads to neuronal apoptosis and activation of the microglial cells. The microRNAs (miRNAs) are small endogenous non-coding RNA that regulate the gene expression. Viral infections perturb the expression pattern of cellular miRNAs, which may in turn affect the expression pattern of downstream genes. This study aims to investigate hsa-miR-21-5p mediated regulation of PTEN, AKT, NF-ĸBp65, IL-6, TNF-α, and IL-1β, in human microglial cells during CHPV infection. Methods To understand the role of hsa-miR-21-5p in CHPV infection, the human microglial cells were infected with CHPV (MOI-0.1). Real-time PCR, western blotting, Luciferase assay, over-expression and knockdown techniques were used to understand the role of hsa-miR-21-5p in the regulation of PTEN, AKT and, NF-ĸBp65, IL-6, TNF-α, and IL-1β in this study. Results The hsa-miR-21-5p was found to be upregulated during CHPV infection in human microglial cells. This led to the downregulation of PTEN which promoted the phosphorylation of AKT and NF-ĸBp65. Over-expression of hsa-miR-21-5p led to the decreased expression of PTEN and promoted further phosphorylation of AKT and NF-ĸBp65 in human microglial cells. However, the inhibition of hsa-miR-21-5p using hsa-miR-21-5p inhibitor restored the expression. Conclusions This study supports the role of hsa-miR-21-5p in the regulation of pro-inflammatory genes in CHPV infected human microglial cells.

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Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

International Journal of Molecular Sciences ◽

10.3390/ijms22031205 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1205

Author(s):

Ji Sun Ha ◽

Hye-Rim Choi ◽

In Sik Kim ◽

Eun-A Kim ◽

Sung-Woo Cho ◽

...

Keyword(s):

Calcium Binding ◽

Neuronal Apoptosis ◽

Microglial Cells ◽

Molecular Pattern ◽

Extracellular Signal ◽

Tnf Α ◽

Cox 2 ◽

Necrosis Factor ◽

Microglial Inflammation ◽

Tlr4 Receptor

S100 calcium-binding protein A8 (S100A8), a danger-associated molecular pattern, has emerged as an important mediator of the pro-inflammatory response. Some S100 proteins play a prominent role in neuroinflammatory disorders and increase the secretion of pro-inflammatory cytokines in microglial cells. The aim of this study was to determine whether S100A8 induced neuronal apoptosis during cerebral hypoxia and elucidate its mechanism of action. In this study, we reported that the S100A8 protein expression was increased in mouse neuronal and microglial cells when exposed to hypoxia, and induced neuroinflammation and neuronal apoptosis. S100A8, secreted from neurons under hypoxia, activated the secretion of tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) through phosphorylation of extracellular-signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in microglia. Also, phosphorylation of ERK via the TLR4 receptor induced the priming of the NLRP3 inflammasome. The changes in Cyclooxygenase-2 (COX-2) expression, a well-known inflammatory activator, were regulated by the S100A8 expression in microglial cells. Knockdown of S100A8 levels by using shRNA revealed that microglial S100A8 expression activated COX-2 expression, leading to neuronal apoptosis under hypoxia. These results suggested that S100A8 may be an important molecule for bidirectional microglia-neuron communication and a new therapeutic target for neurological disorders caused by microglial inflammation during hypoxia.

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Manifestation of lipopolysaccharide-induced tolerance in neuro-glial primary cultures of the rat afferent somatosensory system

Inflammation Research ◽

10.1007/s00011-021-01440-7 ◽

2021 ◽

Vol 70 (4) ◽

pp. 429-444

Author(s):

Franz Nürnberger ◽

Stephan Leisengang ◽

Daniela Ott ◽

Jolanta Murgott ◽

Rüdiger Gerstberger ◽

...

Keyword(s):

Transcription Factors ◽

Nuclear Translocation ◽

Mrna Level ◽

Primary Cultures ◽

Somatosensory System ◽

Cellular Level ◽

Microglial Cells ◽

Substantia Gelatinosa ◽

Tnf Α ◽

Pre Treatment

Abstract Objective Bacterial lipopolysaccharide (LPS) may contribute to the manifestation of inflammatory pain within structures of the afferent somatosensory system. LPS can induce a state of refractoriness to its own effects termed LPS tolerance. We employed primary neuro-glial cultures from rat dorsal root ganglia (DRG) and the superficial dorsal horn (SDH) of the spinal cord, mainly including the substantia gelatinosa to establish and characterize a model of LPS tolerance within these structures. Methods Tolerance was induced by pre-treatment of both cultures with 1 µg/ml LPS for 18 h, followed by a short-term stimulation with a higher LPS dose (10 µg/ml for 2 h). Cultures treated with solvent were used as controls. Cells from DRG or SDH were investigated by means of RT-PCR (expression of inflammatory genes) and immunocytochemistry (translocation of inflammatory transcription factors into nuclei of cells from both cultures). Supernatants from both cultures were assayed for tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by highly sensitive bioassays. Results At the mRNA-level, pre-treatment with 1 µg/ml LPS caused reduced expression of TNF-α and enhanced IL-10/TNF-α expression ratios in both cultures upon subsequent stimulation with 10 µg/ml LPS, i.e. LPS tolerance. SDH cultures further showed reduced release of TNF-α into the supernatants and attenuated TNF-α immunoreactivity in microglial cells. In the state of LPS tolerance macrophages from DRG and microglial cells from SDH showed reduced LPS-induced nuclear translocation of the inflammatory transcription factors NFκB and NF-IL6. Nuclear immunoreactivity of the IL-6-activated transcription factor STAT3 was further reduced in neurons from DRG and astrocytes from SDH in LPS tolerant cultures. Conclusion A state of LPS tolerance can be induced in primary cultures from the afferent somatosensory system, which is characterized by a down-regulation of pro-inflammatory mediators. Thus, this model can be applied to study the effects of LPS tolerance at the cellular level, for example possible modifications of neuronal reactivity patterns upon inflammatory stimulation.

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Astrocyte-Derived CCL2 is Associated with M1 Activation and Recruitment of Cultured Microglial Cells

Cellular Physiology and Biochemistry ◽

10.1159/000443040 ◽

2016 ◽

Vol 38 (3) ◽

pp. 859-870 ◽

Cited By ~ 27

Author(s):

Mingfeng He ◽

Hongquan Dong ◽

Yahui Huang ◽

Shunmei Lu ◽

Shu Zhang ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Microglial Activation ◽

Culture Media ◽

Microglial Cells ◽

Migration Ability ◽

Migration Assay ◽

Tnf Α

Background/Aims: Microglia are an essential player in central nervous system inflammation. Recent studies have demonstrated that the astrocytic chemokine, CCL2, is associated with microglial activation in vivo. However, CCL2-induced microglial activation has not yet been studied in vitro. The purpose of the current study was to understand the role of astrocyte-derived CCL2 in microglial activation and to elucidate the underlying mechanism(s). Methods: Primary astrocytes were pre-treated with CCL2 siRNA and stimulated with TNF-α. The culture medium (CM) was collected and added to cultures of microglia, which were incubated with and without CCR2 inhibitor. Microglial cells were analyzed by quantitative RT-PCR to determine whether they polarized to the M1 or M2 state. Microglial migratory ability was assessed by transwell migration assay. Results: TNF-α stimulated the release of CCL2 from astrocytes, even if the culture media containing TNF-α was replaced with fresh media after 3 h. CM from TNF-α-stimulated astrocytes successfully induced microglial activation, which was ascertained by increased activation of M1 and enhanced migration ability. In contrast, CM from astrocytes pretreated with CCL2 siRNA showed no effect on microglial activation, compared to controls. Additionally, microglia pre-treated with RS102895, a CCR2 inhibitor, were resistant to activation by CM from TNF-α-stimulated astrocytes. Conclusion: This study demonstrates that the CCL2/CCR2 pathway of astrocyte-induced microglial activation is associated with M1 polarization and enhanced migration ability, indicating that this pathway could be a useful target to ameliorate inflammation in the central nervous system.

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Anti-Inflammatory Property of the Essential Oil from Cinnamomum camphora (Linn.) Presl Leaves and the Evaluation of Its Underlying Mechanism by Using Metabolomics Analysis

Molecules ◽

10.3390/molecules25204796 ◽

2020 ◽

Vol 25 (20) ◽

pp. 4796

Author(s):

Jiali Chen ◽

Cailin Tang ◽

Yang Zhou ◽

Rongfei Zhang ◽

Shaoxia Ye ◽

...

Keyword(s):

Essential Oil ◽

Mrna Expression ◽

Underlying Mechanism ◽

Treated Group ◽

Microglial Cells ◽

Cinnamomum Camphora ◽

Tnf Α ◽

Anti Inflammatory ◽

Factor Α ◽

Bv2 Microglial Cells

Cinnamomum camphora (Linn.) Presl has been widely used in traditional Chinese medicine for a variety of purposes. Our previous study indicated the antibacterial mechanism of the essential oil (EO) from C. camphora leaves; however, its anti-inflammatory activity and the underlying mechanism have not been clearly demonstrated. Thus, the present study investigated its anti-inflammatory property. Our data revealed that EO significantly decreased the release of nitric oxide (NO) and the mRNA expression of inducible NO synthase (iNOS) in lipopolysaccharide (LPS)-induced BV2 microglial cells. EO also attenuated LPS-induced increase in the mRNA expression and secretion of inflammatory cytokines including interleukin-6 (IL-6), IL-18, IL-1β and tumor necrosis factor-α (TNF-α). Furthermore, the metabolic profiles of LPS-induced BV2 microglial cells treated with or without EO were explored. Thirty-nine metabolites were identified with significantly different contents, including 21 upregulated and 18 downregulated ones. Five pathways were enriched by shared differential metabolites. Compared with the control cells, the glucose level was decreased, while the lactate level was increased, in the culture supernatant from LPS-stimulated cells, which were reversed by EO treatment. Moreover, compared to the LPS-treated group, the activities of phosphofructokinase (PFK) and pyruvate kinase (PK) in EO group were decreased. In summary, the current study demonstrated that EO from C. camphora leaves acts as an anti-inflammatory agent, which might be mediated through attenuating the glycolysis capacity of microglial cells.

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Dectin-1/Syk signaling triggers neuroinflammation after ischemic stroke in mice

10.21203/rs.2.17199/v2 ◽

2019 ◽

Author(s):

Xin-chun Ye ◽

Qi Hao ◽

Wei-jing Ma ◽

Qiu-chen Zhao ◽

Wei-wei Wang ◽

...

Keyword(s):

Ischemic Stroke ◽

Brain Injury ◽

Infarct Volume ◽

Inos Expression ◽

Microglial Cells ◽

Brain Infarct ◽

Tnf Α ◽

Bv2 Microglial Cells ◽

The Brain

Abstract Dendritic cell-associated C-type lectin-1 (Dectin-1) receptor has been reported to be involved in neuroinflammation in Alzheimer's disease and traumatic brain injury. The present study was designed to investigate the role of Dectin-1 and its downstream target spleen tyrosine kinase (Syk) in early brain injury after ischemic stroke using a focal cortex ischemic stroke model. Adult male C57BL/6J mice were subjected to a cerebral focal ischemia model of ischemic stroke. The neurological score, adhesive removal test and foot-fault test were evaluated on days 1, 3, 5 and 7 after ischemic stroke. Dectin-1, Syk, phosphorylated (p)-Syk, tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) expression was analyzed via western blotting in ischemic brain tissue after ischemic stroke and in BV2 microglial cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) injury in vitro. The brain infarct volume and Iba1-positive cells were evaluated using Nissl’s and immunofluorescence staining, respectively. The Dectin-1 antagonist laminarin (LAM) and a selective inhibitor of Syk phosphorylation (piceatannol; PIC) were used for the intervention. Dectin-1, Syk, and p-Syk expression was significantly enhanced on days 3, 5 and 7 and peaked on day 3 after ischemic stroke. The Dectin-1 antagonist LAM or Syk inhibitor PIC decreased the number of Iba1-positive cells and TNF-α and iNOS expression, decreased the brain infarct volume and improved neurological functions on day 3 after ischemic stroke. In addition, the in vitro data revealed that Dectin-1, Syk and p-Syk expression was increased following the 3-h OGD and 0, 3 and 6 h of reperfusion in BV2 microglial cells. LAM and PIC also decreased TNF-α and iNOS expression 3 h after OGD/R induction. Dectin-1/Syk signaling plays a crucial role in inflammatory activation after ischemic stroke, and further investigation of Dectin-1/Syk signaling in stroke is warranted.

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Geranylgeraniol Inhibits Lipopolysaccharide-Induced Inflammation in Mouse-Derived MG6 Microglial Cells via NF-κB Signaling Modulation

International Journal of Molecular Sciences ◽

10.3390/ijms221910543 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10543

Author(s):

Wahyu Dwi Saputra ◽

Hiroki Shono ◽

Yusuke Ohsaki ◽

Halima Sultana ◽

Michio Komai ◽

...

Keyword(s):

Nuclear Translocation ◽

Microglial Cells ◽

Side Chain ◽

Mrna Levels ◽

Inflammatory Reactions ◽

Inflammatory Conditions ◽

Tnf Α ◽

Related Proteins ◽

Microglial Inflammation ◽

Pro Inflammatory Cytokines

Persistent inflammatory reactions in microglial cells are strongly associated with neurodegenerative pathogenesis. Additionally, geranylgeraniol (GGOH), a plant-derived isoprenoid, has been found to improve inflammatory conditions in several animal models. It has also been observed that its chemical structure is similar to that of the side chain of menaquinone-4, which is a vitamin K2 sub-type that suppresses inflammation in mouse-derived microglial cells. In this study, we investigated whether GGOH has a similar anti-inflammatory effect in activated microglial cells. Particularly, mouse-derived MG6 cells pre-treated with GGOH were exposed to lipopolysaccharide (LPS). Thereafter, the mRNA levels of pro-inflammatory cytokines were determined via qRT-PCR, while protein expression levels, especially the expression of NF-κB signaling cascade-related proteins, were determined via Western blot analysis. The distribution of NF-κB p65 protein was also analyzed via fluorescence microscopy. Thus, it was observed that GGOH dose-dependently suppressed the LPS-induced increase in the mRNA levels of Il-1β, Tnf-α, Il-6, and Cox-2. Furthermore, GGOH inhibited the phosphorylation of TAK1, IKKα/β, and NF-κB p65 proteins as well as NF-κB nuclear translocation induced by LPS while maintaining IκBα expression. We showed that GGOH, similar to menaquinone-4, could alleviate LPS-induced microglial inflammation by targeting the NF-kB signaling pathway.

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Neuroprotective effect of Lithospermum officinale callus extract on inflamed primary microglial cells

Current Pharmaceutical Biotechnology ◽

10.2174/1389201021666201104145439 ◽

2020 ◽

Vol 21 ◽

Author(s):

Maryam Kheyrollah ◽

Farzaneh Sabouni ◽

Mohsen Farhadpour ◽

Kamahldin Haghbeen

Keyword(s):

Neuroprotective Effect ◽

Wistar Rat ◽

Microglial Cells ◽

Root Extract ◽

Primary Microglia ◽

Methanolic Extracts ◽

Tnf Α ◽

The Central Nervous System ◽

Anti Inflammatory ◽

Lithospermum Officinale

Background and Objective: Lithospermum officinale is a famous medicinal herb in the traditional medicine of India. However, the medicinal use of its root extract is limited due to the presence of pyrrolizidine alkaloids (PzAl). It was recently shown that PzAl are not accumulated in the cell culture of L. officinale while the biosynthetic pathway of phenolic acids remains active so that rosmarinic acid (RsA) is the main product in the proliferated callus. Considering the existing literature on the anti-inflammatory effects of caffeic acid (CfA) and its derivatives, this research was devoted to the evaluation of the anti-inflammatory capacity of methanolic extracts of L. officinale callus (LoE) on the rat microglial cells as the immune cells of the Central Nervous System, which play an essential role in the responses to neuroinflammation. Methods: primary microglia were obtained from Wistar rat, then they were subjected to various amounts of CfA and methanolic extracts of 17 and 31-day L. officinale callus prior to stimulation by LPS. In addition to HPLC analysis of the extracts, viability, nitric oxide production, evaluation of the pro-inflammatory genes and cytokines in the inflamed microglia were investigated. Results: Methanolic extract of the 17-day old callus of L. officinale exhibited anti-inflammatory effects on the LPS- stimulated microglial cells much higher than that was observed for CfA. The data was further supported by the decreased expression of NOS2, TNF-α, and Cox-2 mRNA and the suppression of TNF-α and IL-1β release in the activated microglial cells pretreated with the effective dose of LoE (0.8 mg mL-1). Conclusion: It was assumed that better anti-neuroinflammatory performance of LoE than CfA in LPS-activated primary microglia could be a result of synergism of the components of the extract and the lipophilic nature of RsA as the main phenolic acid of LoE. Considering the fact that LoE shows high antioxidant capacity and lacks PzAl, it is anticipated that LoE is considered as a reliable substitute to the extract of the natural root of L. officinale and plays a key role in the preparation of neuroprotective pharmaceutical formula.

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Neopterin and Cytokines in Non-pleocytotic Cerebrospinal Fluid in Child Patients

Pteridines ◽

10.1515/pteridines.2000.11.4.142 ◽

2000 ◽

Vol 11 (4) ◽

pp. 142-146

Author(s):

Yasuhiko Kawakami ◽

Kentaro Kuwabara ◽

Takehisa Fujita ◽

Osamu Fujino ◽

Yoshitaka Fukunaga

Keyword(s):

Cerebrospinal Fluid ◽

Immune Activation ◽

Serum Levels ◽

Microglial Cells ◽

Febrile Convulsions ◽

Exanthem Subitum ◽

Tnf Α ◽

Child Patients ◽

Ifn Γ

Abstract CSF and serum levels of neopterin and several kinds of CSF cytokines were measured in child patients with non-pleocytotic CSF. The CSF neopterin levels with febrile convulsions (FCs) were 27.4±33.0 nmol/1 and the CSF neopterinlserum neopterin ratio (C/S ratio) with FCs was 2.07±2.06. The longer the FCs patients' convulsions lasted, the higher their CSF neopterin levels tended to be come. The CSF neopterin levels with FCs were significantly higher than in those with pyrexia without convulsions (6.5±2.5 nmol/L) or convulsions without pyrexia, including epilepsy (4.9±2.9 nmol/L). The C/S ratio was also higher in patients with FCs than in those with pyrexia without convulsions (0.31±0.17) or convulsions without pyrexia (0.82±0.50).In addition, there was a tendency for CSF IFN-γ levels to be higher in patients with FCs than in those with pyrexia without convulsions or convulsions without pyrexia. However, TNF-α and IL-1α were under measurable levels in cases of non-pi eocytotic CSF. The CSF neopterin levels in patients with exanthem subitum with FCs were higher than in those without FCs. It has been reported that there is a possibility of neopterin production by microglial cells. Our finding that CSF neopterin levels were elevated in patients with FCs, whose CSF had no pleocytosis, are consistent with the possibility of neopterin production by microglia. Our results further suggest that immune activation of microgrial cells is one of the mechanisms involved in the onset of FCs.

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