LPS-induced Microglial Cells Research Articles

Systematic Characterization of Sesquiterpenes from Dendrobium nobile through Offline Two-Dimensional Chromatography Tandem Mass Spectrometry and Target Isolation.

Dendrobium nobile is a species of the genus Dendrobium that can be used as both a medicinal herb and healthy food. The sesquiterpenes in D. nobile have attracted extensive attention in recent years. In this study, Amide × RP offline two-dimensional chromatography separation tandem high-resolution mass spectrometry combined with GNPS (Global Natural Product Social Molecular Networking) was developed for the characterization of sesquiterpenes in D. nobile. After first-dimensional amide separation, the 70% ethanol extract of D. nobile was divided into 40 fractions, which were analyzed by second-dimensional reverse-phase system separation and LTQ-Orbitrap detection. The raw data was imported into the GNPS, resulting in the efficient clustering of similar substances. Finally, 594 sesquiterpene compounds were characterized, and 25 compounds were isolated based on molecular network analysis, including six new compounds. In vitro bioassays, the isolated compounds decreased NO production in the LPS-induced microglial BV-2 cell model and the content of MDA in PC12 cells, demonstrating neuroprotective activity. These findings unraveled the underlying material and provided valuable insights into the quality control of D. nobile.

Polyvinylpyrrolidone-curcumin nanoparticles with immune regulatory and metabolism regulatory effects for the treatment of experimental autoimmune uveitis

Uveitis comprises a cluster of intraocular inflammatory disorders characterized by uncontrolled autoimmune responses and excessive oxidative stress leading to vision loss worldwide. In the present study, curcumin (CUR) was conjugated with polyvinylpyrrolidone (PVP) to form PVP-CUR nanoparticles with significantly elevated solubility and outstanding multiple radical scavenging abilities. In vitro studies revealed that PVP-CUR nanoparticles markedly mitigated oxidative stress and reduced apoptosis in a H2O2-induced human retinal pigment epithelial cell line (ARPE-19) and promoted phenotypic polarization from M1 to M2 in an LPS-induced human microglial cell line (HMC3). Further in vivo studies demonstrated the prominent therapeutic effects of PVP-CUR nanoparticles on experimental autoimmune uveitis (EAU), which relieved clinical and pathological progression, improved perfusion and tomographic manifestations of retinal vessels, and reduced blood–retinal barrier (BRB) leakage; these effects may be mediated by mitigating oxidative stress and attenuating macrophage/microglia-elicited inflammation. Notably, treatment with PVP-CUR nanoparticles was shown to regulate metabolite alterations in EAU rats, providing novel insights into the underlying mechanisms involved. Additionally, the PVP-CUR nanoparticles showed great biocompatibility in vivo. In summary, our study revealed that PVP-CUR nanoparticles may serve as effective and safe nanodrugs for treating uveitis and other oxidative stress- and inflammation-related diseases.

A Synthetic Derivative SH 66 of Homoisoflavonoid from Liliaceae Exhibits Anti-Neuroinflammatory Activity against LPS-Induced Microglial Cells.

Naturally occurring homoisoflavonoids isolated from some Liliaceae plants have been reported to have diverse biological activities (e.g., antioxidant, anti-inflammatory, and anti-angiogenic effects). The exact mechanism by which homoisoflavonones exert anti-neuroinflammatory effects against activated microglia-induced inflammatory cascades has not been well studied. Here, we aimed to explore the mechanism of homoisoflavonoid SH66 having a potential anti-inflammatory effect in lipopolysaccharide (LPS)-primed BV2 murine microglial cells. Microglia cells were pre-treated with SH66 followed by LPS (100 ng/mL) activation. SH66 treatment attenuated the production of inflammatory mediators, including nitric oxide and proinflammatory cytokines, by down-regulating mitogen-activated protein kinase signaling in LPS-activated microglia. The SH66-mediated inhibition of the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome complex and the respective inflammatory biomarker-like active interleukin (IL)-1β were noted to be one of the key pathways of the anti-inflammatory effect. In addition, SH66 increased the neurite length in the N2a neuronal cell and the level of nerve growth factor in the C6 astrocyte cell. Our results demonstrated the anti-neuroinflammatory effect of SH66 against LPS-activated microglia-mediated inflammatory events by down-regulating the NLRP3 inflammasome complex, with respect to its neuroprotective effect. SH66 could be an interesting candidate for further research and development regarding prophylactics and therapeutics for inflammation-mediated neurological complications.

The Standardized Extract of Centella asiatica and Its Fractions Exert Antioxidative and Anti-Neuroinflammatory Effects on Microglial Cells and Regulate the Nrf2/HO-1 Signaling Pathway.

Neuroinflammation and oxidative stress can aggravate the progression of Alzheimer's disease (AD). Centella asiatica has been traditionally consumed for memory and cognition. The triterpenes (asiaticoside, madecassoside, asiatic acid, madecassic acid) have been standardized in the ethanolic extract of Centella asiatica (SECA). The bioactivity of the triterpenes in different solvent polarities of SECA is still unknown. In this study, the antioxidative and anti-neuroinflammatory effects of SECA and its fractions were explored on lipopolysaccharides (LPS)-induced microglial cells. HPLC measured the four triterpenes in SECA and its fractions. SECA and its fractions were tested for cytotoxicity on microglial cells using MTT assay. NO, pro-inflammatory cytokines (TNF-α, IL-6, IL-1β), ROS, and MDA (lipid peroxidation) produced by LPS-induced microglial cells were measured by colorimetric assays and ELISA. Nrf2 and HO-1 protein expressions were measured using western blotting. The SECA and its fractions were non-toxic to BV2 microglial cells at tested concentrations. The levels of NO, TNF-α, IL-6, ROS, and lipid peroxidation in LPS-induced BV2 microglial cells were significantly reduced (p < 0.001) by SECA and its fractions. SECA and some of its fractions can activate the Nrf2/HO-1 signaling pathway by significantly enhancing (p < 0.05) the Nrf2 and HO-1 protein expressions. This study suggests that the inhibitory activity of SECA and its fractions on pro-inflammatory and oxidative stress events may be the result of the activation of antioxidant defense systems. The potential of SECA and its fractions in reducing neuroinflammation and oxidative stress can be further studied as a potential therapeutic strategy for AD.

NLRX1 Inhibits LPS-Induced Microglial Death via Inducing p62-Dependent HO-1 Expression, Inhibiting MLKL and Activating PARP-1.

The activation of microglia and the production of cytokines are key factors contributing to progressive neurodegeneration. Despite the well-recognized neuronal programmed cell death regulated by microglial activation, the death of microglia themselves is less investigated. Nucleotide-binding oligomerization domain, leucine-rich repeat-containing X1 (NLRX1) functions as a scaffolding protein and is involved in various central nervous system diseases. In this study, we used the SM826 microglial cells to understand the role of NLRX1 in lipopolysaccharide (LPS)-induced cell death. We found LPS-induced cell death is blocked by necrostatin-1 and zVAD. Meanwhile, LPS can activate poly (ADP-ribose) polymerase-1 (PARP-1) to reduce DNA damage and induce heme oxygenase (HO)-1 expression to counteract cell death. NLRX1 silencing and PARP-1 inhibition by olaparib enhance LPS-induced SM826 microglial cell death in an additive manner. Less PARylation and higher DNA damage are observed in NLRX1-silencing cells. Moreover, LPS-induced HO-1 gene and protein expression through the p62-Keap1-Nrf2 axis are attenuated by NLRX1 silencing. In addition, the Nrf2-mediated positive feedback regulation of p62 is accordingly reduced by NLRX1 silencing. Of note, NLRX1 silencing does not affect LPS-induced cellular reactive oxygen species (ROS) production but increases mixed lineage kinase domain-like pseudokinase (MLKL) activation and cell necroptosis. In addition, NLRX1 silencing blocks bafilomycin A1-induced PARP-1 activation. Taken together, for the first time, we demonstrate the role of NLRX1 in protecting microglia from LPS-induced cell death. The underlying protective mechanisms of NLRX1 include upregulating LPS-induced HO-1 expression via Nrf2-dependent p62 expression and downstream Keap1-Nrf2 axis, mediating PARP-1 activation for DNA repair via ROS- and autophagy-independent pathway, and reducing MLKL activation.

Circ_0006640 transferred by bone marrow-mesenchymal stem cell-exosomes suppresses lipopolysaccharide-induced apoptotic, inflammatory and oxidative injury in spinal cord injury

BackgroundEmerging proofs have shown that differentially expressed circular RNAs (circRNAs) are closely associated with the pathophysiological process of spinal cord injury (SCI). Mesenchymal stem cell (MSC)-exosomes have been demonstrated to possess favorable therapeutic effects in diseases. Herein, this work aimed to investigate the action of circ_0006640 transferred by MSC-exosomes functional recovery after SCI.MethodsSCI animal models were established by spinal cord contusion surgery in mice and lipopolysaccharide (LPS)-stimulated mouse microglial cell line BV2. Levels of genes and proteins were detected by qRT-PCR and Western blot. Properties of BV2 cells were characterized using CCK-8 assay, flow cytometry and ELISA analysis. The oxidative stress was evaluated. Dual-luciferase reporter assay was used for verifying the binding between miR-382-5p and circ_0006640 or IGF-1 (Insulin-like Growth Factor 1). Exosome separation was conducted by using the commercial kit.ResultsCirc_0006640 expression was lower in SCI mice and LPS-induced microglial cells. Circ_0006640 overexpression protected microglial cells from LPS-induced apoptotic, inflammatory and oxidative injury. Mechanistically, circ_0006640 directly sponged miR-382-5p, which targeted IGF-1. MiR-382-5p was increased, while IGF-1 was decreased in SCI mice and LPS-induced microglial cells. Knockdown of miR-382-5p suppressed apoptosis, inflammation and oxidative stress in LPS-induced microglial cells, which were reversed by IGF-1 deficiency. Moreover, miR-382-5p up-regulation abolished the protective functions of circ_0006640 in LPS-induced microglial cells. Additionally, circ_0006640 was packaged into MSC-exosomes and could be transferred by exosomes. Exosomal circ_0006640 also had protective effects on microglial cells via miR-382-5p/IGF-1 axis.ConclusionCirc_0006640 transferred by BMSC-exosomes suppressed LPS-induced apoptotic, inflammatory and oxidative injury via miR-382-5p/IGF-1 axis, indicating a new insight into the clinical application of exosomal circRNA-based therapeutic in the function recovery after SCI.

Inhibitor of bromodomain and extraterminal domain proteins decreases transcription of Cd33 in the brain of mice subjected to systemic inflammation; a promising strategy for neuroprotection.

The neuroinflammation is a crucial component of virtually all neurodegenerative disorders, including Alzheimer's disease (AD). The bacterial lipopolysaccharide (LPS), a potent activator of the innate immune system, was suggested to influence or even trigger the neuropathological alterations in AD. LPS-induced neuroinflammation involves changes in transcription of several genes, thus controlling these molecular processes may be a potentially efficient strategy to attenuate the progression of AD. Since genome-wide association studies showed that the majority of AD-related genetic risk factors (AD-GRF) are connected to the immune system, our aim was to identify AD-GRF affected in the hippocampus by LPS-induced systemic inflammatory response (SIR). Moreover, we analysed the role of bromodomain and extraterminal domain (BET) proteins, the readers of the acetylation code, in controlling the transcription of selected AD-GRF in the brain during neuroinflammation. In our study, we used a mouse model of LPS-induced SIR and mouse microglial BV2 cells. JQ1 was used as an inhibitor of BET proteins. The level of mRNA was analysed using microarrays and qPCR. Our data demonstrated that among the established AD-GRF, only the expression of Cd33 was significantly upregulated in the hippocampus during SIR. In parallel, we observed an increase in the expression of Brd4, a BET family member. JQ1 prevented an LPS-evoked increase in Cd33 expression in the hippocampus of mice. Moreover, JQ1 reduced Cd33 expression in BV2 microglial cells stimulated with blood serum from LPS-treated mice. Our study suggests that LPS-evoked SIR may increase Cd33 gene expression in the brain, and inhibition of BET proteins through suppression of Cd33 expression could be a promising strategy in prevention or in slowing down the progression of neuroinflammation and may potentially affect the pathomechanism of AD.

GLP-1R knockdown abrogates the protective effects of liraglutide on ischaemic stroke via inhibition of M2 polarisation and activation of NLRP3 inflammasome by reducing Nrf2 activation

Liraglutide has been recently discovered to penetrate the blood–brain barrier to exert neuroprotective effects. However, relevant mechanisms of the protective effects of liraglutide on ischaemic stroke remain to be elucidated. This study examined the mechanism of GLP-1R in regulating the protective effect of liraglutide against ischaemic stroke. Middle cerebral artery occlusion (MCAO) male Sprague-Dawley rat model with/without GLP-1R or Nrf2 knockdown was established and subjected to liraglutide treatment. Then neurological deficit and brain oedema of rats was evaluated and brain tissues were subjected to TTC, Nissl, TUNEL and immunofluorescence staining. Rat primary microglial cells firstly underwent lipopolysaccharide (LPS) treatment, then GLP-1R or Nrf2 knockdown treatment, and finally Liraglutide treatment to research the NLRP3 activation. As a result, Liraglutide protected rats' brain tissues after MCAO, which attenuated brain oedema, infarct volume, neurological deficit score, neuronal apoptosis and Iba1 expression but enhanced live neurons. However, GLP-1R knockdown abrogated these protective effects of liraglutide on MCAO rats. According to in vitro experiments, Liraglutide promoted M2 polarisation, activated Nrf2 and inhibited NLRP3 activation in LPS-induced microglial cells, but GLP-1R or Nrf2 knockdown reversed these effects of Liraglutide on LPS-induced microglial cells. Further, Nrf2 knockdown counteracted the protection of liraglutide on MCAO rats, whereas sulforaphane (agonist of Nrf2) counteracted the effect of Nrf2 knockdown on liraglutide-treated MCAO rats. Collectively, GLP-1R knockdown abrogated the protection of liraglutide on MCAO rats by activating NLRP3 via inactivating Nrf2.

Smart Farming Enhances Bioactive Compounds Content of Panax ginseng on Moderating Scopolamine-Induced Memory Deficits and Neuroinflammation.

Korean ginseng (Panax ginseng) is a traditional herbal supplement known to have a variety of pharmacological activities. A smart farm system could provide potential standardization of ginseng seedlings after investigating plant metabolic responses to various parameters in order to design optimal conditions. This research was performed to investigate the effect of smart-farmed ginseng on memory improvement in a scopolamine-induced memory deficit mouse model and an LPS-induced microglial cell model. A smart farming system was applied to culture ginseng. The administration of its extract (S2 extract) under specific culture conditions significantly attenuated cognitive and spatial memory deficits by regulating AKT/ERK/CREB signaling, as well as the cortical inflammation associated with suppression of COX-2 and NLRP3 induced by scopolamine. In addition, S2 extract improved the activation of iNOS and COX-2, and the secretion of NO in LPS-induced BV-2 microglia. Based on the HPLC fingerprint and in vitro data, ginsenosides Rb2 and Rd were found to be the main contributors to the anti-inflammatory effects of the S2 extract. Our findings suggest that integrating a smart farm system may enhance the metabolic productivity of ginseng and provides evidence of its potential impact on natural bioactive compounds of medicinal plants with beneficial qualities, such as ginsenosides Rb2 and Rd.

The ethanolic extract of Curcuma longa grown in Korea exhibits anti-neuroinflammatory effects by activating of nuclear transcription factor erythroid-2-related factor 2/heme oxygenase-1 signaling pathway

BackgroundCurcuma longa has been used as spices, food preservative, coloring material, and traditional medicine. This plant also has long been used for a variety of diseases including dyslipidemia, stomach disorders, arthritis, and hepatic diseases. The aim of the present investigation was to examine the anti-neuroinflammatory effects of the 50% ethanolic extract of C. longa in lipopolysaccharide (LPS)-induced BV2 microglial cells.MethodsGriess reaction was employed to measure the production of nitric oxide (NO), and the levels of prostaglandin E2 (PGE2) and pro-inflammatory cytokines such as interleukin 1-beta (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) were determined by using profit ELISA kits. Western blotting was used to determine the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor kappa B (NF-κB), mitogen activated protein kinases (MAPKs), heme oxygenase-1 (HO-1) and nuclear factor erythroid-2-related factor 2 (Nrf2).ResultsPre-treatment with CLE inhibited the overproduction and overexpression of pro-inflammatory mediators including NO, PGE2, iNOS, COX-2, and pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α in LPS-induced BV2 cells. In addition, CLE suppressed the activation of the NF-κB and three MAPK signaling pathways. Treatment with CLE induced HO-1 protein expression by activating Nrf2 pathway, and inhibiting the HO-1 expression reversed the anti-inflammatory effect of CLE.ConclusionCLE showed anti-neuroinflammatory effects against LPS-induced microglial cells activation through the inhibition of production and expression of pro-inflammatory mediators by negative regulation of the NF-κB and MAPK signaling pathways. These anti-neuroinflammatory effects of CLE were mediated by HO-1/Nrf2 signaling pathway. Taken together, the present study suggests a potent effect of CLE to prevent neuroinflammatory diseases. It is necessary to perform additional efficacy evaluation through in vivo experiments.

Natural carbazole alkaloid murrayafoline A displays potent anti-neuroinflammatory effect by directly targeting transcription factor Sp1 in LPS-induced microglial cells

Neuroinflammation is a leading cause for neurological disorders. Carbazole alkaloids, isolated from the medicinal plants of Murraya species (Rutaceae), have exhibited wide pharmacological activities particularly for neuroinflammation. However, its underlying cellular targets and molecular mechanisms still remain unclear. In current study, we found that murrayafoline A (MA), a carbazole alkaloid obtained from Murraya tetramera, potently inhibited the production of neuroinflammation mediators, such as nitric oxide (NO), TNF-α, IL-6 and IL-1β in LPS-induced BV-2 microglial cells. Then, we performed thermal proteome profiling (TPP) strategy to identify Specificity protein 1 (Sp1) as a potential cellular target of MA. Moreover, we performed surface plasmon resonance (SPR), cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DRATS) assays to confirm the direct interaction between MA and Sp1. Furthermore, we downregulated Sp1 expression in BV2 cells using siRNA transfection, and observed that Sp1 knockdown significantly antagonized MA-mediated inhibition of neuroinflammation mediator production. Meanwhile, Sp1 knockdown also markedly reversed MA-mediated inactivation of IKKβ/NF-κB and p38/JNK MAPKs pathways. Finally, in vivo studies revealed that MA significantly suppressed the expression of Iba-1, TNF-α, and IL-6, while increased the number of Nissl bodies in the brains of LPS-induced mice. Taken together, our study demonstrated that MA exerted obvious anti-neuroinflammation effect by directly targeting Sp1, thereby inhibiting NF-κB and MAPK signaling pathways. Our findings also provided a promising direction of pharmacological targeting Sp1 for anti-neuroinflammation therapeutics as well as novel agent development.

Nigella sativa Oil Reduces LPS-Induced Microglial Inflammation: An Evaluation on M 1/M 2 Balance.

Objectives The immune system plays a critical defence role against infections, injuries, and carcinogenic stimuli. As the macrophages of the brain resides in the innate immune system, microglia and their polarisation (M1/M2) play regulatory roles in inflammation in CNS, such as Parkinson's, Alzheimer's, dementia complex, and multiple sclerosis. Nigella sativa belongs to the Ranunculaceae family and has different anti-inflammatory and antioxidant effects. We conducted this study to evaluate the anti-inflammatory and protective properties of N. sativa oil (NSO) on the microglial cells and their polarisation (M1/M2) in the presence of LPS as a model of neuroinflammation. Methods The protective effects of NSO (10–40 µg/ml) were studied on the LPS-induced microglial cells, and the levels of tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, prostaglandin E2 (PGE2), and IL-10 were evaluated using both ELISA and gene expression methods. The levels of cyclooxygenase-2 (COX-2), inducible NOS (iNOS), and arginase-1 (Arg1) were also evaluated using the real-time PCR method. In addition, nitrite oxide (NO) and urea were measured using biochemical methods. Results NSO decreased LPS-induced toxicity at all doses (P < 0.001). NSO (10–40 μg/ml) also significantly reduced the levels of TNF-α, PGE2, IL-1β, and IL-6 in the presence of LPS (P < 0.01 to 0.001). Pretreatment with NSO attenuated the levels of iNOS but increased Arg1 (P < 0.001). The ratio of iNOS/Arg1 was also decreased in the presence of NSO (P < 0.001) than that of the LPS group (P < 0.001). Conclusion NSO attenuated LPS-induced inflammation and increased microglia's anti-inflammatory status. These results may prove that NSO is potentially an immunomodulator for various neurodegenerative diseases by M1 phenotype dominancy, such as Alzheimer's and Parkinson's diseases.

Blockage of ERCC6 Alleviates Spinal Cord Injury Through Weakening Apoptosis, Inflammation, Senescence, and Oxidative Stress.

Objective: Spinal cord injury (SCI) is a devastating disease resulting in lifelong disability, but the molecular mechanism remains unclear. Our study was designed to observe the role of excision repair cross-complementing group 6 (ERCC6) following SCI and to determine the underlying mechanism. Methods: SCI mouse models and LPS-induced microglia cell models were established. ERCC6 expression was blocked by ERCC6-siRNA-carrying lentivirus. Nissl staining was utilized for detecting neuronal damage, and apoptosis was analyzed with TUNEL and Western blotting (apoptotic markers). Immunofluorescence was used for measuring macrophage markers (CD68 and F4/80) and astrocyte and microglia markers (GFAP and Iba-1). Pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) were measured via ELISA. Senescent cells were estimated via SA-β-Gal staining as well as Western blot (senescent markers p21 and p27). Oxidative stress was investigated by detecting the expression of 4-HNE, Nrf2, and Keap1, and intracellular ROS levels. Results: ERCC6 expression was remarkably upregulated both in the spinal cord of SCI mice and LPS-induced microglia cells. ERCC6 deficiency alleviated neuronal damage and apoptosis. Macrophage infiltration and inflammatory response were suppressed by si-ERCC6 treatment. Moreover, ERCC6 blockage ameliorated astrocyte and microglia activation and cell senescence in the damaged spinal cord. Excessive oxidative stress was significantly decreased by ERCC6 knockdown in SCI. Conclusion: Collectively, ERCC6 exerts crucial functions in mediating physiological processes (apoptosis, inflammation, senescence, and oxidative stress), implying that ERCC6 might act as a prospective therapeutic target against SCI.

Microglia inflammatory response contributes to chronic constriction injury-induced neuropathic pain via miR-339/PFKFB3 axis

&#x0D; &#x0D; &#x0D; &#x0D; Purpose: To investigate the effect of miR-339 on neuropathic pain.&#x0D; Methods: A rat neuropathic pain model was established through chronic constriction injury (CCI). Expression of miR-339 in spinal cord was determined 14 days later. Microglial inflammatory response was evaluated using immunofluorescence analysis of ionized calcium binding adaptor molecule 1 (Iba1), while IL-6 and TNF-α were assessed by enzyme-linked immunosorbent assay (ELISA). Pain- associated behavioral effects and microglia-related inflammation were investigated after intrathecal administration of miR-339 agomir into rats post-CCI. The target gene of miR-339 involved in neuropathic pain was evaluated by a luciferase reporter assay. Microglia cells were isolated from rats, then treated with lipopolysaccharide (LPS). The LPS-induced inflammatory response in microglia cells was determined using quantitative reverse transcription PCR analysis of IL-6 and TNF-α.&#x0D; Results: CCI decreased mechanical allodynia and thermal hyperalgesia thresholds, but increased Iba1, IL-6, and TNF-α in rats. MiR-339 was reduced in rat spinal cord after CCI induction while intrathecal injection of miR-339 agomir alleviated CCI-induced changes in mechanical and thermal hyperalgesia in rats, and reversed expression of Iba1, IL-6, and TNF-α. Furthermore, 6-phosphofructo-2- kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) was identified as a miR-339 target gene, and over- expression of miR-339 decreased the expression of PFKFB3, IL-6, and TNF-α in LPS-induced microglia cells.&#x0D; Conclusion: The miR-339/PFKFB3 axis ameliorates CCI-induced neuropathic pain by suppression of microglia inflammatory response, suggesting a novel strategy for neuropathic pain management.&#x0D; &#x0D; &#x0D; &#x0D;

Protective Effect of Mild Hypothermia on Spinal Cord Ischemia-Induced Delayed Paralysis and Spinal Cord Injury.

To explore the mechanism regarding the regulation of spinal cord ischemia (SCI) in rats by mild hypothermia. A SCI rat model was established through aorta occlusion, and in some cases, the rats were intervened with mild hypothermia, after which motor function, microglia activation, and M1/M2 polarization in rats were measured. Also, the expression of inflammatory cytokines (IL-1β, IL-6 and TNF-α) and neuronal apoptosis were examined. Lipopolysaccharide (LPS)-induced M1 microglia and IL-4-induced M2 microglia were intrathecally injected into rats to evaluate the effect of microglial polarization on SCI. In in vitro experiments, primary microglial cells were treated under hypothermic condition, in which M1/M2 polarization and microglia apoptosis, the levels of iNOS, CD86, CD206, Arg-1 and inflammatory cytokines were assessed. Western blot analysis detected the activation of the TLR4/NF-κB pathway to investigate the role of this pathway in M1/M2 polarization. SCI treatment impaired motor function, induced higher M1 microglia proportion, and increased the levels of pro-inflammatory cytokines in rats, and mild hypothermic treatment attenuated these trends. Moreover, injection of M1 microglia increased M1 microglia proportion and increased the levels of pro-inflammatory cytokines, while injection of M2 microglia induced the reverse results, i.e. decreased M1 microglia proportion and reduced pro-inflammatory cytokine levels. In LPS-induced microglial cells, mild hypothermia treatment increased M2 microglia proportion and decreased pro-inflammatory cytokine levels, relative to normothermia. Mild hypothermia inactivated the TLR4/NF-κB pathway in LPS-treated microglia. TLR4 overexpression reversed the function of mild hypothermia in LPS-stimulated microglia, and under normal condition, TLR4/NF-κB pathway suppressed microglial M2 polarization. Mild hypothermia inhibits TLR4/NF-κB pathway and promotes microglial M2 polarization, thus attenuating SCI-induced injury and inflammation.

Gα13 Contributes to LPS-Induced Morphological Alterations and Affects Migration of Microglia.

Microglia are the resident immune cells of the CNS that are activated in response to a variety of stimuli. This phenotypical change is aimed to maintain the local homeostasis, also by containing the insults and repair the damages. All these processes are tightly regulated and coordinated and a failure in restoring homeostasis by microglia can result in the development of neuroinflammation that can facilitate the progression of pathological conditions. Indeed, chronic microglia activation is commonly recognized as a hallmark of many neurological disorders, especially at an early stage. Many complex pathways, including cytoskeletal remodeling, are involved in the control of the microglial phenotypical and morphological changes that occur during activation. In this work, we focused on the small GTPase Gα13 and its role at the crossroad between RhoA and Rac1 signaling when microglia is exposed to pro-inflammatory stimulation. We propose the direct involvement of Gα13 in the cytoskeletal rearrangements mediated by FAK, LIMK/cofilin, and Rac1 during microglia activation. In fact, we show that Gα13 knockdown significantly inhibited LPS-induced microglial cell activation, in terms of both changes in morphology and migration, through the modulation of FAK and one of its downstream effectors, Rac1. In conclusion, we propose Gα13 as a critical factor in the regulation of morphological and functional properties of microglia during activation, which might become a target of intervention for the control of microglia inflammation.

Hyodeoxycholic acid rescues lipopolysaccharide-induced neuroinflammation via regulating TGR5/AKT/NF-κB pathway

Hyodeoxycholic acid (HDCA) is a natural secondary bile acid with enormous pharmacological effects. It can act as a takeda G-coupled protein receptor 5 (TGR5) agonist, activation of TGR5 is found to alleviate neuroinflammation by suppressing serine/threonine kinase (AKT)/ nuclear factor-κB (NF-κB) signaling pathway. In this study, the anti-microglial inflammatory effect of HDCA was evaluated in lipopolysaccharide (LPS)-induced BV2 microglial cells and mice (n=12/group). One-way ANOVA followed by Tukey’s post hoc analysis was conducted. p <.05 is statistically significant. HDCA was shown to inhibit the over-activation of microglia both in vitro and in vivo , evidenced by down-regulated mRNA expression of inflammatory cytokines such as interleukin 1β, decreased protein expression of inducible nitric oxide synthase and cyclooxygenase 2. Moreover, in LPS-induced microglial cells, HDCA was found to counteract the suppression of TGR5 and the increased phosphorylation of AKT, NF-κB and NF-κB inhibitor factor, as well as the nuclear translocation of NF-κB, however, the effect of HDCA onTGR5, AKT and NF-κB was abolished by the TGR5 inhibitor - triamterene. HDCA suppresses LPS-induced neuroinflammation through regulating TGR5/AKT/NF-κB signaling pathway. The novel findings suggest the therapeutic potential of HDCA in central nervous system disorders with prominent neuroinflammation.

CCR1 enhances SUMOylation of DGCR8 by up-regulating ERK phosphorylation to promote spinal nerve ligation-induced neuropathic pain.

Neuropathic pain is a somatosensory nervous system dysfunction that remains a threatening health problem globally. Recent studies have highlighted the involvement of C-C motif chemokine receptor 1 (CCR1) in neuropathic pain. Herein, the current study set out to explore the modulatory role of CCR1 in spinal nerve ligation (SNL)-induced neuropathic pain and its underlying molecular mechanism. First, it was found that CCR1 was highly expressed in spinal cord tissues and microglial cells of SNL rats. On the other hand, CCR1 knockdown attenuated nerve pain in SNL rats and repressed microglial cell activation in SNL rats and also in the LPS-induced microglial cell model of nerve injury, as evidenced by elevated microglial cell markers OX-42 and IL-1β, IL-6 and TNF-α. Mechanistically, CCR1 enhanced small ubiquitin-like modifier 1 (SUMO1) modification of DiGeorge syndrome critical region gene 8 (DGCR8) in LPS-treated microglial cells by phosphorylating ERK. Moreover, CCR1 silencing brought about elevations in mechanical withdrawal threshold and thermal withdrawal latency. To conclude, our findings indicated that CCR1 enhanced the modification of DGCR8 by SUMO1 through phosphorylation of ERK, thereby promoting the activation and inflammatory response of spinal cord microglial cells and increasing the sensitivity of SNL rats to pain. Thus, this study offers a promising therapeutic target for the management of neuropathic pain.

Ficus deltoidea: Potential inhibitor of pro-inflammatory mediators in lipopolysaccharide-induced activation of microglial cells

Ethnopharmacological relevanceFicus deltoidea Jack (FD) is widely consumed in traditional medicine as a treatment for various diseases in Malaysia. Each part of the plant such as its leave, stem, fruit and root are used traditionally to treat different types of diseases. Vitexin and isovitexin are bioactive compounds abundantly found in the leaves of FD that possessed many pharmacological properties including neuroprotection. Nonetheless, its effects on key events in neuroinflammation are unknown.Aim of the study: To determine the inhibitory properties of FD aqueous extract on pro-inflammatory mediators involved in lipopolysaccharide (LPS)-induced microglial cells. MethodsVitexin and isovitexin in the extract were quantified via high performance liquid chromatography (HPLC). The extract was evaluated for its cytotoxicity activity via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Pre-treatment with the extract on LPS-induced microglial cells was done to determine its antioxidant and anti-neuroinflammatory properties by measuring the level of reactive oxygen species (ROS), nitric oxide (NO), tumour necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) via 2′-7′-dichlorofluorescin diacetate (DCFDA) assay, Griess assay and Western blot respectively. ResultsThe extract at all tested concentrations (0.1 μg/mL, 1 μg/mL, 10 μg/mL, 100 μg/mL) were not cytotoxic as the percentage viability of microglial cells were all above ~80%. At the highest concentration (100 μg/mL), the extract significantly reduced the formation of ROS, NO, TNF-α, IL-1β and IL-6 in microglial cells induced by LPS. ConclusionThe extract showed neuroprotective effects by attenuating the levels of pro-inflammatory and cytotoxic factors in LPS-induced microglial cells, possibly by mediating the nuclear factor-kappa B (NF-κB) signalling pathway.

Early glycolytic reprogramming controls microglial inflammatory activation

BackgroundMicroglial activation-mediated neuroinflammation plays an important role in the progression of neurodegenerative diseases. Inflammatory activation of microglial cells is often accompanied by a metabolic switch from oxidative phosphorylation to aerobic glycolysis. However, the roles and molecular mechanisms of glycolysis in microglial activation and neuroinflammation are not yet fully understood.MethodsThe anti-inflammatory effects and its underlying mechanisms of glycolytic inhibition in vitro were examined in lipopolysaccharide (LPS) activated BV-2 microglial cells or primary microglial cells by enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, immunoprecipitation, flow cytometry, and nuclear factor kappa B (NF-κB) luciferase reporter assays. The anti-inflammatory and neuroprotective effects of glycolytic inhibitor, 2-deoxoy-d-glucose (2-DG) in vivo were measured in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-or LPS-induced Parkinson’s disease (PD) models by immunofluorescence staining, behavior tests, and Western blot analysis.ResultsWe found that LPS rapidly increased glycolysis in microglial cells, and glycolysis inhibitors (2-DG and 3-bromopyruvic acid (3-BPA)), siRNA glucose transporter type 1 (Glut-1), and siRNA hexokinase (HK) 2 abolished LPS-induced microglial cell activation. Mechanistic studies demonstrated that glycolysis inhibitors significantly inhibited LPS-induced phosphorylation of mechanistic target of rapamycin (mTOR), an inhibitor of nuclear factor-kappa B kinase subunit beta (IKKβ), and NF-kappa-B inhibitor alpha (IκB-α), degradation of IκBα, nuclear translocation of p65 subunit of NF-κB, and NF-κB transcriptional activity. In addition, 2-DG significantly inhibited LPS-induced acetylation of p65/RelA on lysine 310, which is mediated by NAD-dependent protein deacetylase sirtuin-1 (SIRT1) and is critical for NF-κB activation. A coculture study revealed that 2-DG reduced the cytotoxicity of activated microglia toward MES23.5 dopaminergic neuron cells with no direct protective effect. In an LPS-induced PD model, 2-DG significantly ameliorated neuroinflammation and subsequent tyrosine hydroxylase (TH)-positive cell loss. Furthermore, 2-DG also reduced dopaminergic cell death and microglial activation in the MPTP-induced PD model.ConclusionsCollectively, our results suggest that glycolysis is actively involved in microglial activation. Inhibition of glycolysis can ameliorate microglial activation-related neuroinflammatory diseases.