MicroRNA-155-5p promotes neuroinflammation and central sensitization via inhibiting SIRT1 in a nitroglycerin-induced chronic migraine mouse model

Abstract Background Previous studies have confirmed that the microglial activation and subsequent inflammatory responses in the trigeminal nucleus caudalis (TNC) are involved in the central sensitization of chronic migraine (CM). MicroRNA-155-5p has been shown to modulate the polarization of microglia and participate in inflammatory processes in a variety of neurological diseases. However, its role in CM remains unclear. The purpose of this study was to determine the precise role of miR-155-5p in CM. Methods A model of CM in C57BL/6 mice was established by recurrent intraperitoneal injection of nitroglycerin (NTG). Mechanical and thermal hyperalgesia were evaluated by Von Frey filaments and radiant heat. The expression of miR-155-5p was examined by qRT-PCR, and the mRNA and protein levels of silent information regulator 1(SIRT1) were measured by qRT-PCR, Western blotting (WB) and immunofluorescence (IF) analysis. The miR-155-5p antagomir, miR-155-5p agomir, SRT1720 (a SIRT1 activator) and EX527 (a SIRT1 inhibitor) were administered to confirm the effects of miR-155-5p and SIRT1 on neuroinflammation and the central sensitization of CM. ELISA, WB and IF assays were applied to evaluate the expression of TNF-α, myeloperoxidase (MPO), IL-10, p-ERK, p-CREB, calcitonin gene-related peptide (CGRP), c-Fos and microglial activation. The cellular localization of SIRT1 was illustrated by IF. Results After the NTG-induced mouse model of CM was established, the expression of miR-155-5p was increased. The level of SIRT1 was decreased, and partly colocalized with Iba1 in the TNC. The miR-155-5p antagomir and SRT1720 downregulated the expression of p-ERK, p-CREB, CGRP, and c-Fos, alleviating microglial activation and decreasing inflammatory substances (TNF-α, MPO). The administration of miR-155-5p agomir or EX527 exacerbated neuroinflammation and central sensitization. Importantly, the miR-155-5p agomir elevated CGRP and c-Fos expression and microglial activation, which could subsequently be alleviated by SRT1720. Conclusions These data demonstrate that upregulated miR-155-5p in the TNC participates in the central sensitization of CM. Inhibiting miR-155-5p alleviates neuroinflammation by activating SIRT1 in the TNC of CM mice.

Download Full-text

P2X7R-mediated Autophagic Impairment Contributes to Central Sensitization in a chronic Migraine Model with Recurrent Nitroglycerin Stimulation in Mice

10.21203/rs.3.rs-67855/v1 ◽

2020 ◽

Author(s):

Li Jiang ◽

Yixin Zhang ◽

Feng Jing ◽

Ting Long ◽

Guangcheng Qin ◽

...

Keyword(s):

Chronic Migraine ◽

Central Sensitization ◽

Cellular Localization ◽

Microglial Activation ◽

Purinergic Receptor ◽

Radiant Heat ◽

Underlying Mechanism ◽

Autophagic Flux ◽

Pathophysiological Mechanism

Abstract Background: Central sensitization is an important pathophysiological mechanism of chronic migraine (CM). According to our previous studies, microglial activation and subsequent inflammation in the trigeminal nucleus caudalis (TNC) contribute to the central sensitization. The P2X7 receptor (P2X7R) is a purinergic receptor expressed in microglia and participates in central sensitization in chronic pain, but its role in CM is unclear. Numerous studies have shown that P2X7R regulates the level of autophagy and that autophagy affects the microglial activation and inflammation. Recently, autophagy has been shown to be involved in neuropathic pain, but there is no information about autophagy in CM. Therefore, the current study investigated the role of P2X7R in CM and its underlying mechanism, focusing on autophagy regulation.Methods: The CM model was established by repeated intraperitoneal injection of nitroglycerin (NTG) in mice. A Von Frey filament and radiant heat were used to assess the mechanical and thermal hypersensitivity. Western blotting and immunofluorescence assays were performed to detect the expression of P2X7R, autophagy-related proteins, and the cellular localization of P2X7R. To determine the role of P2X7R and autophagy in CM, we detected the effects of the autophagy inducer, rapamycin (RAPA) and P2X7R antagonist, Brilliant Blue G (BBG), on pain behavior and the expression of calcitonin gene-related peptide (CGRP) and c-fos. In addition, the effect of RAPA and BBG on microglial activation and subsequent inflammation were investigated.Results: The expression of P2X7R was increased and was mainly colocalized with microglia in the TNC following recurrent NTG administration. The autophagic flux was blocked in CM, which was characterized by up-regulated LC3-II, and accumulated autophagy substrate protein, p62. RAPA significantly improved the basal rather than acute hyperalgesia. BBG alleviated both basal and acute hyperalgesia. BBG activated the level of autophagic flux. RAPA and BBG inhibited the activation of microglia, limited the inflammatory response, and reduced the expression of CGRP and c-fos. Conclusions: Our results demonstrate the dysfunction of the autophagic process in CM. Activated autophagy may have a preventive effect on migraine chronification. P2X7R contributes to central sensitization through mediating autophagy regulation and might become a potential target for CM.

Download Full-text

Activation of microglial GLP-1R in the trigeminal nucleus caudalis suppresses central sensitization of chronic migraine after recurrent nitroglycerin stimulation

The Journal of Headache and Pain ◽

10.1186/s10194-021-01302-x ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Feng Jing ◽

Qian Zou ◽

Yangyang Wang ◽

Zhiyou Cai ◽

Yong Tang

Keyword(s):

Chronic Migraine ◽

Western Blotting ◽

Central Sensitization ◽

Microglial Activation ◽

Morphological Changes ◽

Trigeminal Nucleus ◽

Trigeminal Nucleus Caudalis ◽

Tnf Α

Abstract Background Central sensitization is considered a critical pathogenic mechanism of chronic migraine (CM). Activation of microglia in the trigeminal nucleus caudalis (TNC) contributes to this progression. Microglial glucagon-like peptide-1 receptor (GLP-1R) activation can alleviate pain; however, whether it is involved in the mechanism of CM has not been determined. Thus, this study aims to investigate the precise role of GLP-1R in the central sensitization of CM. Methods Repeated nitroglycerin injection-treated mice were used as a CM animal model in the experiment. To identify the distribution and cell localization of GLP-1R in the TNC, we performed immunofluorescence staining. Changes in the expression of GLP-1R, Iba-1, PI3K and p-Akt in the TNC were examined by western blotting. To confirm the effect of GLP-1R and PI3K/Akt in CM, a GLP-1R selective agonist (liraglutide) and antagonist (exendin(9–39)) and a PI3K selective antagonist (LY294002) were administered. Mechanical hypersensitivity was measured through von Frey filaments. To investigate the role of GLP-1R in central sensitization, calcitonin gene-related peptide (CGRP) and c-fos were determined using western blotting and immunofluorescence. To determine the changes in microglial activation, IL-1β and TNF-α were examined by western blotting, and the number and morphology of microglia were measured by immunofluorescence. We also confirmed the effect of GLP-1R on microglial activation in lipopolysaccharide-treated BV-2 microglia. Results The protein expression of GLP-1R was increased in the TNC after nitroglycerin injection. GLP-1R was colocalized with microglia and astrocytes in the TNC and was fully expressed in BV-2 microglia. The GLP-1R agonist liraglutide alleviated basal allodynia and suppressed the upregulation of CGRP, c-fos and PI3K/p-Akt in the TNC. Similarly, the PI3K inhibitor LY294002 prevented nitroglycerin-induced hyperalgesia. In addition, activating GLP-1R reduced Iba-1, IL-1β and TNF-α release and inhibited TNC microglial number and morphological changes (process retraction) following nitroglycerin administration. In vitro, the protein levels of IL-1β and TNF-α in lipopolysaccharide-stimulated BV-2 microglia were also decreased by liraglutide. Conclusions These findings suggest that microglial GLP-1R activation in the TNC may suppress the central sensitization of CM by regulating TNC microglial activation via the PI3K/Akt pathway.

Download Full-text

P2X7R-mediated autophagic impairment contributes to central sensitization in a chronic migraine model with recurrent nitroglycerin stimulation in mice

Journal of Neuroinflammation ◽

10.1186/s12974-020-02056-0 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Li Jiang ◽

Yixin Zhang ◽

Feng Jing ◽

Ting Long ◽

Guangcheng Qin ◽

...

Keyword(s):

Chronic Migraine ◽

Central Sensitization ◽

Cellular Localization ◽

Microglial Activation ◽

Purinergic Receptor ◽

Radiant Heat ◽

Underlying Mechanism ◽

Autophagic Flux ◽

Pathophysiological Mechanism

Abstract Background Central sensitization is an important pathophysiological mechanism of chronic migraine (CM). According to our previous studies, microglial activation and subsequent inflammation in the trigeminal nucleus caudalis (TNC) contribute to the central sensitization. The P2X7 receptor (P2X7R) is a purinergic receptor expressed in microglia and participates in central sensitization in chronic pain, but its role in CM is unclear. Numerous studies have shown that P2X7R regulates the level of autophagy and that autophagy affects the microglial activation and inflammation. Recently, autophagy has been shown to be involved in neuropathic pain, but there is no information about autophagy in CM. Therefore, the current study investigated the role of P2X7R in CM and its underlying mechanism, focusing on autophagy regulation. Methods The CM model was established by repeated intraperitoneal injection of nitroglycerin (NTG) in mice. A Von Frey filament and radiant heat were used to assess the mechanical and thermal hypersensitivity. Western blotting and immunofluorescence assays were performed to detect the expression of P2X7R, autophagy-related proteins, and the cellular localization of P2X7R. To determine the role of P2X7R and autophagy in CM, we detected the effects of the autophagy inducer, rapamycin (RAPA) and P2X7R antagonist, Brilliant Blue G (BBG), on pain behavior and the expression of calcitonin gene-related peptide (CGRP) and c-fos. In addition, the effect of RAPA and BBG on microglial activation and subsequent inflammation were investigated. Results The expression of P2X7R was increased and was mainly colocalized with microglia in the TNC following recurrent NTG administration. The autophagic flux was blocked in CM, which was characterized by upregulated LC3-II, and accumulated autophagy substrate protein, p62. RAPA significantly improved the basal rather than acute hyperalgesia. BBG alleviated both basal and acute hyperalgesia. BBG activated the level of autophagic flux. RAPA and BBG inhibited the activation of microglia, limited the inflammatory response, and reduced the expression of CGRP and c-fos. Conclusions Our results demonstrate the dysfunction of the autophagic process in CM. Activated autophagy may have a preventive effect on migraine chronification. P2X7R contributes to central sensitization through mediating autophagy regulation and might become a potential target for CM.

Download Full-text

P2X7R-mediated autophagic impairment contributes to central sensitization in a chronic migraine model with recurrent nitroglycerin stimulation in mice

10.21203/rs.3.rs-97711/v1 ◽

2020 ◽

Author(s):

Li Jiang ◽

Yixin Zhang ◽

Feng Jing ◽

Ting Long ◽

Guangcheng Qin ◽

...

Keyword(s):

Chronic Migraine ◽

Central Sensitization ◽

Cellular Localization ◽

Microglial Activation ◽

Purinergic Receptor ◽

Radiant Heat ◽

Underlying Mechanism ◽

Autophagic Flux ◽

Pathophysiological Mechanism

Download Full-text

LncRNA HOTAIR Increases Inflammatory Responses by Activating NF-κB Pathway in LPS-Induced Acute Lung Injury

10.21203/rs.3.rs-49969/v1 ◽

2020 ◽

Author(s):

XiaoMei Huang ◽

ZeXun Mo ◽

YuJun Li ◽

Hua He ◽

KangWei Wang ◽

...

Keyword(s):

Acute Lung Injury ◽

Lung Injury ◽

Noncoding Rna ◽

Nuclear Translocation ◽

Inflammatory Responses ◽

A549 Cells ◽

Enzyme Linked Immunosorbent Assay ◽

Qrt Pcr ◽

Tnf Α ◽

Lncrna Hotair

Abstract Background Nuclear factor kappa-B (NF-κB) activation increased the expression of cytokines and further lead to lung injury was considered the main mechanism of acute lung injury (ALI). Here, we focus on exploring the potential regulatory mechanism between long noncoding RNA (LncRNA) HOX transcript antisense RNA (HOTAIR) and NF-κB on LPS-induced ALI. Methods A549 cells were then divided into 4 groups: HOTAIR group, NC group, si-HOTAIR group and si-NC group. These 4 groups were then treated with 1μg/mL lipopolysaccharides (LPS) or without LPS at 37°C for 24 h. The expression level of cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6) and LncRNA HOTAIR were evaluated by quantitative Real Time Polymerase Chain Reaction (qRT-PCR) and Enzyme-linked immunosorbent assay (ELISA). Western Blot analysis was adopted for evaluating the level of p-IκBα/IκBα and p-p65/p65. Nuclear translocation of p65 was observed by immunofluorescence staining. Results qRT-PCR and ELISA assay showed that the expression of cytokines (IL-1β, IL-6 and TNF-α) and inflammatory gene HOTAIR was remarkably increased with LPS treatment (p < 0.01). Over-expression of HOTAIR significantly increased the expression of cytokines (including IL-1β, IL-6 and TNF-α) and NF-κB pathway associated proteins (including p-IκBα/IκBα and p-p65/p65), while knockdown of HOTAIR had the opposite effect (p < 0.01). The immunofluorescence assay showed that the level of p65 in the nucleus was significantly higher in the HOTAIR group and significantly lowers in the si-HOTAIR group (p < 0.01). Conclusion HOTAIR may play a pro-inflammatory response through NF-κB pathway in LPS-induced ALI, which may provide a perspective for further understanding the pathogenic mechanism of ALI.

Download Full-text

Chunghyuldan attenuates brain microglial inflammatory response

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y09-028 ◽

2009 ◽

Vol 87 (6) ◽

pp. 448-454 ◽

Cited By ~ 6

Author(s):

Kyong Nyon Nam ◽

Hoon-Ji Jung ◽

Mi-Hyun Kim ◽

Chulhun Kang ◽

Woo-Sang Jung ◽

...

Keyword(s):

Rat Brain ◽

Microglial Activation ◽

Inflammatory Responses ◽

Small Vessel Disease ◽

Inhibitory Effect ◽

Stroke Recurrence ◽

Pcr Analysis ◽

Prostaglandin E ◽

Tnf Α ◽

Proinflammatory Molecules

Microglial cells are the prime effectors in immune and inflammatory responses of the central nervous system (CNS). During pathological conditions, the activation of these cells helps restore CNS homeostasis. However, chronic microglial activation endangers neuronal survival through the release of various proinflammatory molecules and neurotoxins. Thus, negative regulators of microglial activation have been considered as potential therapeutic candidates to target stroke and neurodegenerative diseases. Chunghyuldan, a combinatorial drug consisting of Scutellariae Radix, Coptidis Rhizoma, Phellodendri Cortex, Gardeniae Fructus, and Rhei Rhizoma, has an inhibitory effect on stroke recurrence in patients with small-vessel disease. It has also been reported to confer antihypertensive, antihyperlipidemic, and antiinflammatory effects. The aim of this study was to examine whether Chunghyuldan suppresses microglial activation. Chunghyuldan was effective at inhibiting LPS-induced nitric oxide (NO) release from rat brain microglia. Real-time reverse transcriptase PCR analysis revealed that pretreatment of rat brain microglia with Chunghyuldan attenuated the LPS-induced expression of mRNAs encoding inducible NO synthase, tumor necrosis factor (TNF)-α, interleukin-1β, and cyclooxygenase-2. In rat brain microglia, Chunghyuldan reduced the LPS-stimulated production of TNF-α and prostaglandin E2. In addition, Chunghyuldan significantly decreased LPS-induced phosphorylation of the ERK1/2 and p38 signaling proteins. These results suggest that Chunghyuldan provide neuroprotection by reducing the release of various proinflammatory molecules from activated microglia.

Download Full-text

THE MODULATORY ROLE PLAYED BY TNF-α AND IL-1β IN THE INFLAMMATORY RESPONSES INDUCED BY CARRAGEENAN IN THE MOUSE MODEL OF PLEURISY

Cytokine ◽

10.1006/cyto.2000.0816 ◽

2001 ◽

Vol 13 (3) ◽

pp. 162-168 ◽

Cited By ~ 57

Author(s):

Tânia S. Fröde ◽

Glória E.P. Souza ◽

João B. Calixto

Keyword(s):

Mouse Model ◽

Inflammatory Responses ◽

Tnf Α

Download Full-text

Repeated oxytocin prevents central sensitization by regulating synaptic plasticity via oxytocin receptor in a chronic migraine mouse model

The Journal of Headache and Pain ◽

10.1186/s10194-021-01299-3 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Yunfeng Wang ◽

Qi Pan ◽

Ruimin Tian ◽

Qianwen Wen ◽

Guangcheng Qin ◽

...

Keyword(s):

Synaptic Plasticity ◽

Mouse Model ◽

Chronic Migraine ◽

Central Sensitization ◽

Antinociceptive Effect ◽

Adenosine Monophosphate ◽

Oxytocin Receptor ◽

Receptor Subtype ◽

Potential Candidate ◽

Associated Proteins

Abstract Background Central sensitization is one of the characters of chronic migraine (CM). Aberrant synaptic plasticity can induce central sensitization. Oxytocin (OT), which is a hypothalamic hormone, plays an important antinociceptive role. However, the antinociceptive effect of OT and the underlying mechanism in CM remains unclear. Therefore, we explored the effect of OT on central sensitization in CM and its implying mechanism, focusing on synaptic plasticity. Methods A CM mouse model was established by repeated intraperitoneal injection of nitroglycerin (NTG). Von Frey filaments and radiant heat were used to measure the nociceptive threshold. Repeated intranasal OT and intraperitoneal L368,899, an oxytocin receptor (OTR) antagonist, were administered to investigate the effect of OT and the role of OTR. The expression of calcitonin gene-related peptide (CGRP) and c-fos were measured to assess central sensitization. N-methyl D-aspartate receptor subtype 2B (NR2B)-regulated synaptic-associated proteins and synaptic plasticity were explored by western blot (WB), transmission electron microscope (TEM), and Golgi-Cox staining. Results Our results showed that the OTR expression in the trigeminal nucleus caudalis (TNC) of CM mouse was significantly increased, and OTR was colocalized with the postsynaptic density protein 95 (PSD-95) in neurons. Repeated intranasal OT alleviated the NTG-induced hyperalgesia and prevented central sensitization in CM mouse. Additionally, the OT treatment inhibited the overexpression of phosphorylated NR2B and synaptic-associated proteins including PSD-95, synaptophysin-1 (syt-1), and synaptosomal-associated protein 25 (snap25) in the TNC of CM mouse and restored the abnormal synaptic structure. The protective effect of OT was prevented by L368,899. Furthermore, the expression of adenylyl cyclase 1 (AC1)/ protein kinase A (PKA)/ phosphorylation of cyclic adenosine monophosphate response element-binding protein (pCREB) pathway was depressed by OT and restored by L368,899. Conclusions Our findings demonstrate that repeated intranasal OT eliminates central sensitization by regulating synaptic plasticity via OTR in CM. The effect of OT has closely associated with the down-regulation of AC1/PKA/pCREB signaling pathway, which is activated in CM model. Repeated intranasal OT may be a potential candidate for CM prevention.

Download Full-text

Oxytocin Suppresses Inflammatory Responses Induced by Lipopolysaccharide through Inhibition of the eIF-2α–ATF4 Pathway in Mouse Microglia

Cells ◽

10.3390/cells8060527 ◽

2019 ◽

Vol 8 (6) ◽

pp. 527 ◽

Cited By ~ 9

Author(s):

Takayuki Inoue ◽

Hajime Yamakage ◽

Masashi Tanaka ◽

Toru Kusakabe ◽

Akira Shimatsu ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Proinflammatory Cytokines ◽

Inflammatory Responses ◽

Neurological Diseases ◽

The Novel ◽

Activated Microglia ◽

Effective Strategies ◽

Tnf Α ◽

Brain Homeostasis

Microglia maintain brain homeostasis and modulate neuroinflammation and are implicated in the pathogenesis of various neurological diseases such as Alzheimer’s disease. In this study, we found that in lipopolysaccharide (LPS)-stimulated microglia, the endoplasmic reticulum (ER) stress-related eIF-2α–ATF4 pathway plays significant roles in TNF-α and IL-6 production, as well as in the inflammasome-mediated production of IL-1β. Furthermore, our analysis revealed that oxytocin (OT), a nonapeptide synthesized in the hypothalamus, suppressed the production of these proinflammatory cytokines by inhibiting activation of the eIF-2α–ATF4 pathway. Our findings therefore suggest a novel anti-inflammatory axis of OT in activated microglia, which would be helpful for developing the novel effective strategies for regulating microglia-associated neuroinflammation.

Download Full-text

Melatonin attenuates microglial activation and improves neurological functions in rat model of collagenase-induced intracerebral hemorrhage

Melatonin Research ◽

10.32794/mr112500100 ◽

2021 ◽

Vol 4 (2) ◽

pp. 360-376

Author(s):

Joseph Wai-Hin Leung ◽

Raymond Tak Fai Cheung

Keyword(s):

Intracerebral Hemorrhage ◽

Cell Viability ◽

Microglial Activation ◽

Inflammatory Responses ◽

Neurological Diseases ◽

Brain Parenchyma ◽

Neurological Deficits ◽

Neuroprotective Effects ◽

Permanent Disability

Intracerebral hemorrhage (ICH) is a severe form of stroke with a high mortality rate. It is also an important cause of permanent disability. Apart from hematoma growth and edema development, inflammatory responses and oxidative stress are responsible for poor outcomes after ICH. Due to its antioxidant, anti-inflammatory and anti-apoptotic properties, melatonin is a neuroprotective molecule against different neurological diseases. The protective roles of melatonin on ICH, particularly in the collagenase-induced ICH model, have not been well studied. The present study aims to explore neuroprotective effects of melatonin against ICH. At 24 hours after ICH induction, rats exhibited neurological deficits with mild loss in body weight (BW). Hematoma was found in the brain parenchyma with ED-1+ activated microglia and TUNEL+ apoptotic cells in the perihematomal region. As an in vitro model of ICH, SH-SY5Y cells were treated with red blood cell lysate. This treatment significantly reduced cell viability; however, melatonin (10-5 M) restored the cell viability. At 72 hours after ICH, rats treated with melatonin (50 mg/kg) at 2, 24 and 48 hours had reduced perihematomal microglial activation. However, there was no effect on hematoma size or perihematomal apoptosis. We further treated rats with 50 mg/kg melatonin starting at 2 hours and repeating at 24-hour intervals for two or seven more days. Both melatonin treatments improved post-ICH neurological functions, and the effect was most pronounced at 4 days after ICH. Since studies regarding the protective roles of melatonin on ICH remain very limited, our study advances our understanding of the potential use of melatonin as a treatment for ICH.

Download Full-text