scholarly journals Propofol Suppresses Microglia Inflammation by Targeting TGM2/NF-κB Signaling

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Yuanyuan Hou ◽  
Xi Xiao ◽  
Wei Yu ◽  
Sihua Qi
Keyword(s):  
Microglial Activation ◽  
Neuroprotective Effect ◽  
Ectopic Expression ◽  
Transglutaminase 2 ◽  
Binding Activity ◽  
Functional Verification ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sequencing Analysis ◽  
Primary Microglia ◽  
Bv2 Cells

Background. Propofol is a known intravenous hypnotic drug used for induction and maintenance of sedation and general anesthesia. Emerging studies also reveal a neuroprotective effect of propofol in diverse diseases of neuronal injuries via modulating microglia activation. In this study, we aimed to uncover the downstream targets of propofol in this process. Methods. RNA sequencing analysis to identify genes implicated in the propofol-mediated neuroprotective effect. Quantitative real-time PCR, enzyme-linked immunosorbent assay, and Western blotting analysis were performed to analyze inflammatory gene expression, cytokine levels, and TGM2. BV2 cells and primary microglia were used for functional verification and mechanism studies. Results. The multifunctional enzyme transglutaminase 2 (TGM2) was identified as a putative functional mediator of propofol. TGM2 was significantly upregulated in lipopolysaccharide- (LPS-) primed BV2 cells. Genetic silencing of TGM2 abolished LPS-induced microglial activation. Notably, gain-of-function experiments showed that the proinflammatory effects of TGM2 were dependent on its GTP binding activity instead of transamidase activity. Then, TGM2 was revealed to activate the NF-κB signaling pathway to facilitate microglial activation. Propofol can inhibit TGM2 expression and NF-κB signaling in BV2 cells and primary microglia. Ectopic expression of TGM2 or constitutively active IKKβ (CA-IKKβ) can compromise propofol-induced anti-inflammatory effects. Conclusions. Our findings suggest that TGM2-mediated activation of NF-κB signaling is an important mechanism in the propofol-induced neuroprotective effect that prevents microglial activation.

scholarly journals Propofol Ameliorates Microglia Activation by Targeting MicroRNA-221/222-IRF2 Axis

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Xi Xiao ◽  
Yuanyuan Hou ◽  
Wei Yu ◽  
Sihua Qi
Keyword(s):  
Molecular Mechanism ◽  
Neuroprotective Effect ◽  
Ectopic Expression ◽  
Microglia Activation ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Loss Of Function ◽  
Critical Function ◽  
Bv2 Cells ◽  
Primary Mouse

Background. Propofol is a widely used intravenous anesthetic drug with potential neuroprotective effect in diverse diseases of neuronal injuries such as traumatic brain injury and ischemic stroke. However, the underlying molecular mechanism remains largely unknown. Methods. Real-time qPCR, enzyme-linked immunosorbent assay, and Western blotting were used to identify the expression pattern of miR-221/222, inflammatory genes, cytokines, and IRF2. The biological roles and mechanisms of propofol in microglia activation were determined in BV2 cells and primary microglia. Bioinformatic analysis and luciferase reporter assay were used to confirm the regulatory role of miR-221/222 in Irf2 expression. Results. We found that miR-221 and miR-222 were downstream targets of propofol and were consistently upregulated in lipopolysaccharide- (LPS-) primed BV2 cells. Gain- and loss-of-function studies revealed that miR-221 and miR-222 were profoundly implicated in microglia activation. Then, interferon regulatory factor 2 (Irf2) was identified as a direct target gene of miR-221/222. IRF2 protein levels were reduced by miR-221/222 and increased by propofol treatment. Ectopic expression of IRF2 attenuated the proinflammatory roles induced by LPS in BV2 cells. More importantly, the suppressive effects of propofol on LPS-primed activation of BV2 cells or primary mouse microglia involved the inhibition of miR-221/222-IRF2 axis. Conclusions. Our study highlights the critical function of miR-221/222, which inhibited Irf2 translation, in the anti-inflammatory effects of propofol, and provides a new perspective for the molecular mechanism of propofol-mediated neuroprotective effect.

scholarly journals Diosgenin Prevents Microglial Activation and Protects Dopaminergic Neurons from Lipopolysaccharide-Induced Neural Damage In Vitro and In Vivo

2021 ◽  
Vol 22 (19) ◽  
pp. 10361
Author(s):  
Shou-Lun Lee ◽  
Ssu-Chieh Tu ◽  
Ming-Yen Hsu ◽  
Ting-Yu Chin
Keyword(s):  
Dopaminergic Neurons ◽  
Dopaminergic Neuron ◽  
Microglial Activation ◽  
Neuroprotective Effect ◽  
Motor Dysfunction ◽  
Primary Microglia ◽  
Neural Damage ◽  
Age Related

Background: The prevention of age-related neurodegenerative disorders is an important issue in an aging society. Microglia-mediated neuroinflammation resulting in dopaminergic neuron loss may lead to the pathogenesis of Parkinson’s disease (PD). Lipopolysaccharide (LPS), an endotoxin, induces neuroinflammatory microglial activation, contributing to dopaminergic neuron damage. Diosgenin is a phytosteroid sapogenin with a wide spectrum of pharmacological activities, e.g., anti-inflammatory activity. However, the preventive effect of diosgenin on neuroinflammation is not clear. Thus, in this study, we further investigated the neuroprotective effect of diosgenin on LPS-induced neural damage in vitro and in vivo. Methods: For in vitro experiments, primary mesencephalic neuron-glia cultures and primary microglia cultures isolated from Sprague–Dawley rats were used. Cells were pretreated with diosgenin and then stimulated with LPS. The expression of proinflammatory cytokines or tyrosine hydroxylase (TH) in the cells was analyzed. In vivo, rats were fed a diet containing 0.1% (w/w) diosgenin for 4 weeks before being administered a unilateral substantia nigra (SN) injection of LPS. Four weeks after the LPS injection, the rats were assessed for lesion severity using the amphetamine-induced rotation test and TH immunohistochemistry. Results: Diosgenin pretreatment prevented LPS-induced neurite shortening in TH-positive neurons in mesencephalic neuron-glia cultures. In addition, pretreatment of primary microglia with diosgenin significantly reduced tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) expression. Moreover, diosgenin pretreatment significantly suppressed LPS-induced extracellular signal-regulated kinase (ERK) activation. In vivo, the intranigral injection of LPS in rats fed a diosgenin-containing diet significantly improved motor dysfunction and reduced TH expression in SN. Conclusion: These results support the effectiveness of diosgenin in protecting dopaminergic neurons from LPS-induced neuroinflammation.

scholarly journals BAP31 regulates IRAK1-dependent neuroinflammation in microglia

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Xia Liu ◽  
Kun Jiao ◽  
Cong-cong Jia ◽  
Guo-xun Li ◽  
Qing Yuan ◽  
...  
Keyword(s):  
Cognitive Impairment ◽  
Neuroprotective Effect ◽  
Memory Deficits ◽  
Enzyme Linked Immunosorbent Assay ◽  
Behavioral Alterations ◽  
System A ◽  
Regulatory Molecule ◽  
Y Maze ◽  
Bv2 Cells ◽  
The Central Nervous System

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

scholarly journals Downregulating expression of OPTN elevates neuroinflammation via AIM2 inflammasome- and RIPK1-activating mechanisms in APP/PS1 transgenic mice

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Long-Long Cao ◽  
Pei-Pei Guan ◽  
Shen-Qing Zhang ◽  
Yi Yang ◽  
Xue-Shi Huang ◽  
...  
Keyword(s):  
Active Form ◽  
Ectopic Expression ◽  
Experimental Models ◽  
Microglial Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Death Domain ◽  
Serine Threonine Kinase ◽  
Bv2 Cells

Abstract Background Neuroinflammation is thought to be a cause of Alzheimer’s disease (AD), which is partly caused by inadequate mitophagy. As a receptor of mitophagy, we aimed to reveal the regulatory roles of optineurin (OPTN) on neuroinflammation in the pathogenesis of AD. Methods BV2 cells and APP/PS1 transgenic (Tg) mice were used as in vitro and in vivo experimental models to determine the regulatory roles of OPTN in neuroinflammation of AD. Sophisticated molecular technologies including quantitative (q) RT-PCR, western blot, enzyme linked immunosorbent assay (ELISA), co-immunoprecipitation (Co-IP) and immunofluorescence (IF) were employed to reveal the inherent mechanisms. Results As a consequence, key roles of OPTN in regulating neuroinflammation were identified by depressing the activity of absent in melanoma 2 (AIM2) inflammasomes and receptor interacting serine/threonine kinase 1 (RIPK1)-mediated NF-κB inflammatory mechanisms. In detail, we found that expression of OPTN was downregulated, which resulted in activation of AIM2 inflammasomes due to a deficiency in mitophagy in APP/PS1 Tg mice. By ectopic expression, OPTN blocks the effects of Aβ oligomer (Aβo) on activating AIM2 inflammasomes by inhibiting mRNA expression of AIM2 and apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), leading to a reduction in the active form of caspase-1 and interleukin (IL)-1β in microglial cells. Moreover, RIPK1 was also found to be negatively regulated by OPTN via ubiquitin protease hydrolysis, resulting in the synthesis of IL-1β by activating the transcriptional activity of NF-κB in BV2 cells. As an E3 ligase, the UBAN domain of OPTN binds to the death domain (DD) of RIPK1 to facilitate its ubiquitination. Based on these observations, ectopically expressed OPTN in APP/PS1 Tg mice deactivated microglial cells and astrocytes via the AIM2 inflammasome and RIPK-dependent NF-κB pathways, leading to reduce neuroinflammation. Conclusions These results suggest that OPTN can alleviate neuroinflammation through AIM2 and RIPK1 pathways, suggesting that OPTN deficiency may be a potential factor leading to the occurrence of AD.

scholarly journals Neuronutraceuticals Modulate Lipopolysaccharide- or Amyloid-β 1-42 Peptide-Induced Transglutaminase 2 Overexpression as a Marker of Neuroinflammation in Mouse Microglial Cells

2021 ◽  
Vol 11 (12) ◽  
pp. 5718
Author(s):  
Nicola Gaetano Gatta ◽  
Andrea Parente ◽  
Francesca Guida ◽  
Sabatino Maione ◽  
Vittorio Gentile
Keyword(s):  
Microglial Cell ◽  
Amyloid Β ◽  
Transglutaminase 2 ◽  
Microglial Cells ◽  
Tissue Type ◽  
Microglial Cell Line ◽  
Bv2 Cells ◽  
Important Marker

Background: Tissue type 2 transglutaminase (TG2, E.C. 2.3.2,13) is reported to be involved in the phagocytosis of apoptotic cells in mouse microglial BV2 cells and peripheral macrophages. In this study, by using lipopolysaccharide (LPS)- or amyloid-β 1-42 (Aβ 1-42) peptide-stimulated microglial cell line BV2 and mouse primary microglial cells, we examined the effects of different neuronutraceutical compounds, such as curcumin (Cu) and N-Palmitoylethanolamine (PEA), known for their anti-inflammatory activity, on TG2 and several inflammatory or neuroprotective biomarker expressions. Methods: Mouse BV2 cells were treated with LPS or Aβ1-42 in the presence of curcumin or PEA, in order to evaluate the expression of TG2 and other inflammatory or neuroprotective markers using Real Time-PCR and Western blot analyses. Results: Curcumin and PEA were capable of reducing TG2 expression in mouse microglial cells during co-treatment with LPS or Aβ 1-42. Conclusions: The results show the role of TG2 as an important marker of neuroinflammation and suggest a possible use of curcumin and PEA in order to reduce LPS- or Aβ1-42-induced TG2 overexpression in mouse microglial cells.

scholarly journals Lipopolysaccharide induces neuroinflammation in microglia by activating the MTOR pathway and downregulating Vps34 to inhibit autophagosome formation

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Xiaoxia Ye ◽  
Mingming Zhu ◽  
Xiaohang Che ◽  
Huiyang Wang ◽  
Xing-Jie Liang ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Microglial Activation ◽  
Adaptor Protein ◽  
Mtor Pathway ◽  
Microglial Cells ◽  
Inflammatory Factors ◽  
Intraventricular Injection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Autophagic Flux

Abstract Background Microglial activation is a prominent feature of neuroinflammation, which is present in almost all neurodegenerative diseases. While an initial inflammatory response mediated by microglia is considered to be protective, excessive pro-inflammatory response of microglia contributes to the pathogenesis of neurodegeneration. Although autophagy is involved in the suppression of inflammation, its role and mechanism in microglia are unclear. Methods In the present study, we studied the mechanism by which lipopolysaccharide (LPS) affects microglial autophagy and the effects of autophagy on the production of pro-inflammatory factors in microglial cells by western blotting, immunocytochemistry, transfection, transmission electron microscopy (TEM), and real-time PCR. In a mouse model of neuroinflammation, generated by intraventricular injection of LPS (5 μg/animal), we induced autophagy by rapamycin injection and investigated the effects of enhanced autophagy on microglial activation by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. Results We found that autophagic flux was suppressed in LPS-stimulated N9 microglial cells, as evidenced by decreased expression of the autophagy marker LC3-II (lipidated form of MAP1LC3), as well as increased levels of the autophagy adaptor protein SQSTM1. LPS significantly decreased Vps34 expression in N9 microglial cells by activating the PI3KI/AKT/MTOR pathway without affecting the levels of lysosome-associated proteins and enzymes. More importantly, overexpression of Vps34 significantly enhanced the autophagic flux and decreased the accumulation of SQSTM1 in LPS-stimulated N9 microglial cells. Moreover, our results revealed that an LPS-induced reduction in the level of Vps34 prevented the maturation of omegasomes to phagophores. Furthermore, LPS-induced neuroinflammation was significantly ameliorated by treatment with the autophagy inducer rapamycin both in vitro and in vivo. Conclusions These data reveal that LPS-induced neuroinflammation in N9 microglial cells is associated with the inhibition of autophagic flux through the activation of the PI3KI/AKT/MTOR pathway, while enhanced microglial autophagy downregulates LPS-induced neuroinflammation. Thus, this study suggests that promoting the early stages of autophagy might be a potential therapeutic approach for neuroinflammation-associated diseases.

scholarly journals Drosophila Mi-2 Negatively Regulates dDREF by Inhibiting Its DNA-Binding Activity

2002 ◽  
Vol 22 (14) ◽  
pp. 5182-5193 ◽  
Author(s):  
Fumiko Hirose ◽  
Nobuko Ohshima ◽  
Eun-Jeong Kwon ◽  
Hideki Yoshida ◽  
Masamitsu Yamaguchi
Keyword(s):  
Dna Binding ◽  
Polytene Chromosomes ◽  
Ectopic Expression ◽  
Binding Activity ◽  
Mobility Shift ◽  
Interacting Protein ◽  
Reciprocal Regulation ◽  
Dna Binding Activity ◽  
Expression Of Genes ◽  
Biochemical Analyses

ABSTRACT Drosophila melanogaster DNA replication-related element (DRE) factor (dDREF) is a transcriptional regulatory factor required for the expression of genes carrying the 5′-TATCGATA DRE. dDREF has been reported to bind to a sequence in the chromatin boundary element, and thus, dDREF may play a part in regulating insulator activity. To generate further insights into dDREF function, we carried out a Saccharomyces cerevisiae two-hybrid screening with DREF polypeptide as bait and identified Mi-2 as a DREF-interacting protein. Biochemical analyses revealed that the C-terminal region of Drosophila Mi-2 (dMi-2) specifically binds to the DNA-binding domain of dDREF. Electrophoretic mobility shift assays showed that dMi-2 thereby inhibits the DNA-binding activity of dDREF. Ectopic expression of dDREF and dMi-2 in eye imaginal discs resulted in severe and mild rough-eye phenotypes, respectively, whereas flies simultaneously expressing both proteins exhibited almost-normal eye phenotypes. Half-dose reduction of the dMi-2 gene enhanced the DREF-induced rough-eye phenotype. Immunostaining of polytene chromosomes of salivary glands showed that dDREF and dMi-2 bind in mutually exclusive ways. These lines of evidence define a novel function of dMi-2 in the negative regulation of dDREF by its DNA-binding activity. Finally, we postulated that dDREF and dMi-2 may demonstrate reciprocal regulation of their functions.

scholarly journals C/EBPα Inhibits Cell Growth via Direct Repression of E2F-DP-Mediated Transcription

2000 ◽  
Vol 20 (16) ◽  
pp. 5986-5997 ◽  
Author(s):  
Beatrix A. Slomiany ◽  
Kenneth L. D'Arigo ◽  
Margaret M. Kelly ◽  
David T. Kurtz
Keyword(s):  
Cell Growth ◽  
Ectopic Expression ◽  
Simian Virus 40 ◽  
Normal Liver ◽  
Simian Virus ◽  
Binding Activity ◽  
T Antigen ◽  
S Phase ◽  
3T3 Cells ◽  
L Cells

ABSTRACT Using an inducible transcription system which allows the regulated expression of C/EBP isoforms in tissue culture cells, we have found that the ectopic expression of C/EBPα, at a level comparable to that found in normal liver tissue, has a pronounced antimitogenic effect in mouse L cells and NIH 3T3 cells. The inhibition of cell division by C/EBPα in mouse cells cannot be reversed by simian virus 40 T antigen, by oncogenic ras, or by adenovirus E1a protein. When expressed in thymidine kinase-deficient L cells or 3T3 cells, C/EBPα is detected in a protein complex which binds to the E2F binding sites found in the promoters of the genes for E2F-1 and dihydrofolate reductase (DHFR). Bacterially expressed C/EBPα has no affinity for these E2F sites, but when recombinant C/EBPα is added to nuclear extracts from mouse fibroblasts, a new E2F binding activity appears, which contains the C/EBPα protein. Using an E2F-DP1-responsive promoter linked to a reporter gene, it can be shown that C/EBPα directly inhibits the induction of this promoter by E2F-DP1 in transient-transfection assays. Furthermore, C/EBPα can be shown to inhibit the S-phase induction of the E2F and DHFR promoters in permanent cell lines. These findings delineate a straightforward mechanism for C/EBPα-mediated cell growth arrest through repression of E2F-DP-mediated S-phase transcription.

scholarly journals Drosophila kinesin-8 stabilizes the kinetochore–microtubule interaction

2018 ◽  
Vol 218 (2) ◽  
pp. 474-488 ◽  
Author(s):  
Tomoya Edzuka ◽  
Gohta Goshima
Keyword(s):  
Yeast Cell ◽  
Ectopic Expression ◽  
Cell Types ◽  
Binding Activity ◽  
S2 Cells ◽  
Tubulin Binding ◽  
Chromosome Alignment ◽  
Normal Frequency ◽  
Directed Motility

Kinesin-8 is required for proper chromosome alignment in a variety of animal and yeast cell types. However, it is unclear how this motor protein family controls chromosome alignment, as multiple biochemical activities, including inconsistent ones between studies, have been identified. Here, we find that Drosophila kinesin-8 (Klp67A) possesses both microtubule (MT) plus end–stabilizing and –destabilizing activity, in addition to kinesin-8's commonly observed MT plus end–directed motility and tubulin-binding activity in vitro. We further show that Klp67A is required for stable kinetochore–MT attachment during prometaphase in S2 cells. In the absence of Klp67A, abnormally long MTs interact in an “end-on” fashion with kinetochores at normal frequency. However, the interaction is unstable, and MTs frequently become detached. This phenotype is rescued by ectopic expression of the MT plus end–stabilizing factor CLASP, but not by artificial shortening of MTs. We show that human kinesin-8 (KIF18A) is also important to ensure proper MT attachment. Overall, these results suggest that the MT-stabilizing activity of kinesin-8 is critical for stable kinetochore–MT attachment.

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