Lipopolysaccharide-stimulated Microglia Research Articles

Synthesis and pharmacological evaluation of nature-inspired phenacyl glycosides

Phenylethanoid glycosides are a well-studied class of bioactive compounds found throughout the plant kingdom. In contrast, research on the synthesis and pharmacological activity of phenacyl glycosides, a specific subgroup of phenylethanoid glycosides with a ketone functionality at the alpha position of the phenol ring, has been limited. In this study, we report the synthesis, cytotoxic, antiviral, and anti-inflammatory evaluation of a series of 18 4′-hydroxyphenacyl glycosides. These compounds consist of six different sugar residues (β-d-glucose, β-d-galactose, α-l-arabinose, β-d-xylose, α-l-rhamnose, and β-d-glucuronic acid) and display three distinct methoxylation patterns at the phenacyl ring, similar to the substitution motifs of anthocyanins. We obtained the target phenacyl glycosides in high yield and stereoselectivity through the coupling of benzoyl-protected trichloroacetimidate glycosyl donors and corresponding acetophenones. Our work represents the first total synthesis of the natural products 4′-hydroxyphenacyl-β-d-glucopyranoside (1) and 4′-hydroxy-3′-methoxyphenacyl-β-d-glucopyranoside (2). None of the phenacyl glycosides showed cytotoxicity against the tested cell lines. Notably, several of the synthesized compounds exhibited antiviral activity, with natural product 2 being the most active against herpes simplex virus type 1, while phenacyl arabinoside 9 and natural product 2 were the most active against human coronavirus OC43. Natural product 2 significantly inhibited the production of interleukin-6 in lipopolysaccharide-stimulated microglia cells. Overall, our findings highlight the importance of the sugar residue and phenacyl ring substitution pattern in modulating the antiviral activity of phenacyl glycosides. Natural product 2 and phenacyl arabinoside 9 emerge as promising leads for the development of antiviral agents.

An ALS-associated mutation dysregulates microglia-derived extracellular microRNAs in a sex-specific manner.

Evidence suggests the presence of microglial activation and microRNA (miRNA) dysregulation in amyotrophic lateral sclerosis (ALS), the most common form of adult motor neuron disease. However, few studies have investigated whether the miRNA dysregulation originates from microglia. Furthermore, TDP-43 (encoded by TARDBP), involved in miRNA biogenesis, aggregates in tissues of ∼98% of ALS cases. Thus, this study aimed to determine whether expression of the ALS-linked TDP-43M337V mutation in a transgenic mouse model dysregulates microglia-derived miRNAs. RNA sequencing identified several dysregulated miRNAs released by transgenic microglia and a differential miRNA release by lipopolysaccharide-stimulated microglia, which was more pronounced in cells from female mice. We validated the downregulation of three candidate miRNAs, namely, miR-16-5p, miR-99a-5p and miR-191-5p, by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and identified their predicted targets, which primarily include genes involved in neuronal development and function. These results suggest that altered TDP-43 function leads to changes in the miRNA population released by microglia, which may in turn be a source of the miRNA dysregulation observed in the disease. This has important implications for the role of neuroinflammation in ALS pathology and could provide potential therapeutic targets.

Eicosapentaenoic acid activates the P62/KEAP1/NRF2 pathway for the prevention of diabetes-associated cognitive dysfunction.

Diabetes-associated cognitive dysfunction (DCD) is a severe complication of diabetes mellitus (DM), threatening the life quality of the diabetic population. However, there is still a lack of effective approaches for its intervention. Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid that was not previously investigated for its effect on DCD. In this study, EPA was found to improve DCD in a mouse model of type 2 DM (T2DM) induced by streptozotocin and a high-fat diet, exhibiting profound protective effects on cognitive dysfunction, neuronal loss, and cerebral oxidative stress and inflammation. While EPA did not attenuate advanced glycation end product-induced neuron injury, we hypothesized that EPA might protect neurons by regulating microglia polarization, the effect of which was confirmed by the co-culture of neurons and lipopolysaccharide-stimulated microglia. RNA sequencing identified nuclear factor-erythroid-2-related factor 2 (NRF2) antioxidant signaling as a major target of EPA in microglia. Mechanistically, EPA increased sequestosome-1 (SQSTM1 or P62) levels that might structurally inhibit Kelch-like ECH associated protein 1 (KEAP1), leading to nuclear translocation of NRF2. P62 and NRF2 predominantly mediated EPA's effect since the knockdown of P62 or NRF2 abolished EPA's protective effect on microglial oxidative stress and inflammation and sequential neuron injuries. Moreover, the regulation of P62/KEPA1/NRF2 axes by EPA was confirmed in the hippocampi of diabetic mice. The present work presents EPA as an effective nutritional approach and microglial P62/KEAP1/NRF2 as molecular targets for the intervention of DCD.

Telmisartan inhibits microglia-induced neurotoxic A1 astrocyte conversion via PPARγ-mediated NF-κB/p65 degradation

Astrocytes are crucially involved in neuroinflammation. Activated astrocytes exhibit at least two phenotypes, A1 (neurotoxic) and A2 (neuroprotective). The A1 phenotype is the major reactive astrocyte phenotype involved in aging and neurodegenerative diseases. Telmisartan, which is an antihypertensive agent, is a promising neuroprotective agent. This study aimed to investigate the effects of telmisartan on the phenotype of reactive astrocytes. Astrocytes were activated by culturing with the conditioned medium derived from lipopolysaccharide-stimulated microglia. This conditioned medium induced early, transient A2 astrocyte conversion (within 24 h) and late, sustained A1 conversion (beginning at 24 h and lasting up to 7 days), with a concomitant increase in the production of pro-inflammatory cytokines (interleukin [IL]-1β, tumor necrosis factor [TNF]α, and IL-6) and phosphorylation of nuclear factor-κB (NF-κB)/p65. Telmisartan treatment promoted and inhibited A2 and A1 conversion, respectively. Telmisartan reduced total and phosphorylated p65 protein levels. Losartan, a specific angiotensin II type-1 receptor (AT1R) blocker, did not influence the reactive state of astrocytes. Additionally, AT1R activation by angiotensin II did not induce the expression of pro-inflammatory cytokines and A1/A2 markers, indicating that the AT1R signaling pathway is not involved in the astrocyte-mediated inflammatory response. A peroxisome proliferator-activated receptor γ (PPARγ) antagonist reversed the effects of telmisartan. Moreover, telmisartan-induced p65 downregulation was reversed by the proteasome inhibitor MG132. These results indicate that telmisartan suppresses activated microglia-induced neurotoxic A1 astrocyte conversion through p65 degradation. Our findings contribute towards the elucidation of the anti-inflammatory activity of telmisartan in brain disorders.

Functionalized retinoic acid lipid nanocapsules promotes a two-front attack on inflammation and lack of demyelination on neurodegenerative disorders.

Demyelinating disorders, with a particular focus on multiple sclerosis (MS), have a multitude of detrimental cognitive and physical effects on the patients. Current treatment options that involve substances promoting remyelination fail in the clinics due to difficulties in reaching the central nervous system (CNS). Here, the dual encapsulation of retinoic acid (RA) into lipid nanocapsules with a nominal size of 70nm, and a low PdI of 0.1, coupled with super paramagnetic iron oxide nanoparticles (SPIONs) was accomplished, and joined by an external functionalization process with a transferrin-receptor binding peptide. This nanosystem showed a 3-fold improved internalization by endothelial cells compared to the free drug, ability to interact with oligodendrocyte progenitor cells and microglia, and improvements in the permeability through the blood-brain barrier by 5-fold. The lipid nanocapsules also induced the differentiation of oligodendrocyte progenitor cells into more mature, myelin producing oligodendrocytes, as evaluated by high-throughput image screening, by 3-5-fold. Furthermore, the ability to tame the inflammatory response was verified in lipopolysaccharide-stimulated microglia, suppressing the production of pro-inflammatory cytokines by 50-70%. Overall, the results show that this nanosystem can act in both the inflammatory microenvironment present at the CNS of affected patients, but also stimulate the differentiation of new oligodendrocytes, paving the way for a promising platform in the therapy of MS.

Restoring myocardial infarction-induced long-term memory impairment by targeting the cystic fibrosis transmembrane regulator.

Cognitive impairment is a serious comorbidity in heart failure patients, but effective therapies are lacking. We investigated the mechanisms that alter hippocampal neurons following myocardial infarction (MI). MI was induced in male C57Bl/6 mice by left anterior descending coronary artery ligation. We utilised standard procedures to measure cystic fibrosis transmembrane regulator (CFTR) protein levels, inflammatory mediator expression, neuronal structure, and hippocampal memory. Using invitro and invivo approaches, we assessed the role of neuroinflammation in hippocampal neuron degradation and the therapeutic potential of CFTR correction as an intervention. Hippocampal dendrite length and spine density are reduced after MI, effects that associate with decreased neuronal CFTR expression and concomitant microglia activation and inflammatory cytokine expression. Conditioned medium from lipopolysaccharide-stimulated microglia (LCM) reduces neuronal cell CFTR protein expression and the mRNA expression of the synaptic regulator post-synaptic density protein 95 (PSD-95) invitro. Blocking CFTR activity also down-regulates PSD-95 in neurons, indicating a relationship between CFTR expression and neuronal health. Pharmacologically correcting CFTR expression invitro rescues the LCM-mediated down-regulation of PSD-95. Invivo, pharmacologically increasing hippocampal neuron CFTR expression improves MI-associated alterations in neuronal arborisation, spine density, and memory function, with a wide therapeutic time window. Our results indicate that CFTR therapeutics improve inflammation-induced alterations in hippocampal neuronal structure and attenuate memory dysfunction following MI. Knut and Alice Wallenberg Foundation [F 2015/2112]; Swedish Research Council [VR; 2017-01243]; the German Research Foundation [DFG; ME 4667/2-1]; Hjärnfonden [FO2021-0112]; The Crafoord Foundation; Åke Wibergs Stiftelse [M19-0380], NMMP 2021 [V2021-2102]; the Albert Påhlsson Research Foundation; STINT [MG19-8469], Lund University; Canadian Institutes of Health Research [PJT-153269] and a Heart and Stroke Foundation of Ontario Mid-Career Investigator Award.

Sepsis-Induced Gut Dysbiosis Mediates the Susceptibility to Sepsis-Associated Encephalopathy in Mice.

ABSTRACTSepsis-associated encephalopathy (SAE) is common in septic patients and is associated with adverse outcomes. The gut microbiota has been recognized as a key mediator of neurological disease development. However, the exact role of the gut microbiota in regulating SAE remains elusive. Here, we investigated the role of the gut microbiota in SAE and its underlying mechanisms. Cecal ligation and puncture (CLP) was conducted to induce sepsis in mice. Neurological scores were recorded to distinguish SAE-resistant (SER) (score of >6 at 36 h postoperatively) from SAE-susceptible (SES) (score of ≤6 at 36 h postoperatively) mice. 16S rRNA gene sequencing and metabolomics analyses were used to characterize the gut microbiota in the two groups. Fecal microbiota transplantation was performed to validate the role of the gut microbiota in SAE progression. The gut microbiota was more severely disrupted in SES mice than in SER mice after sepsis modeling. Interestingly, mice receiving postoperative feces from SES mice exhibited more severe cortical inflammation than mice receiving feces from SER mice. Indole-3-propionic acid (IPA), a neuroprotective molecule, was more enriched in feces from SER mice than in feces from SES mice. IPA alleviated CLP-induced anxiety and spatial memory impairment in septic mice. Moreover, IPA markedly inhibited NLRP3 inflammasome activation and interleukin-1β (IL-1β) secretion in lipopolysaccharide-stimulated microglia. These responses were attenuated after antagonizing the aryl hydrocarbon receptor. Our study indicates that the variability in sepsis-induced gut dysbiosis mediates the differential susceptibility to SAE in CLP-induced experimental sepsis mice, and microbially derived IPA is possibly involved in SAE development as a neuroprotective compound.IMPORTANCE The bidirectional interactions between the gut microbiota and sepsis-associated encephalopathy (SAE) are not well characterized. We found that the gut microbiota was more severely disturbed in SAE-susceptible (SES) mice than in SAE-resistant (SER) mice after sepsis modeling. Mice gavaged with postoperative feces from SES mice exhibited more severe neuroinflammation than mice gavaged with feces from SER mice. The gut microbiota from SER mice enriched a neuroprotective metabolite, IPA, which appeared to protect mice from SAE. The potential underlying mechanism of the protective effect of IPA may be mediated via the inhibition of NLRP3 inflammasome activation and IL-1β secretion in microglia. These anti-inflammatory effects of IPA may be regulated by aryl hydrocarbon receptors. These results enhance our understanding of the role of the intestinal microbiota in sepsis. In particular, gut microbiota-derived IPA may serve as a potential therapeutic agent to prevent neuroinflammation in SAE.

Muse cells decrease the neuroinflammatory response by modulating the proportion of M1 and M2 microglia in vitro

Neuroinflammation hinders repair of the central nervous system (CNS). Stem cell transplantation is a very promising approach for treatment of CNS injuries. However, it is difficult to select seed cells that can both facilitate nerve regeneration and improve the microenvironment in the CNS. In this study, we isolated multilineage-differentiating stress-enduring (Muse) cells from bone marrow mesenchymal stem cells. We explored the anti-inflammatory effect and mechanism of Muse cells in vitro by coculture of Muse cells with lipopolysaccharide-stimulated microglia. Our results showed that Muse cells effectively reduced the transcription and secretion of tumor necrosis factor α and interleukin-1β and increased the expression of transforming growth factor-β and interleukin-10 in microglia. In addition, Muse cells decreased the number of M1 microglia and increased the proportion of M2 microglia in an inflammatory environment more effectively than bone marrow mesenchymal stem cells. We also show that Muse cells inhibited the protein expression of toll-like receptor 4 (TLR4) and myeloid differentiation primary response protein (MyD88) and inhibited the expression of the phosphorylated forms of transcription factor p65, nuclear factor (NF)-κB inhibitor alpha, and p38 mitogen-activated protein kinase (MAPK) in microglia. Therefore, we suggest Muse cells cause antineuroinflammatory effects by inhibition of the TLR4/MyD88/NF-κB and p38 MAPK signaling pathways in microglia. Our results shed light on the function of Muse cells in relation to CNS diseases and provide insight into the selection of seed cells.

Anti-Inflammatory Effects of Dimethyl Fumarate in Microglia via an Autophagy Dependent Pathway

Dimethyl fumarate (DMF), which has been approved by the Food and Drug Administration for the treatment of relapsing-remitting multiple sclerosis, is considered to exert anti-inflammatory and antioxidant effects. Microglia maintain homeostasis in the central nervous system and play a key role in neuroinflammation, while autophagy controls numerous fundamental biological processes, including pathogen removal, cytokine production, and clearance of toxic aggregates. However, the role of DMF in autophagy induction and the relationship of this effect with its anti-inflammatory functions in microglia are not well known. In the present study, we investigated whether DMF inhibited neuroinflammation and induced autophagy in microglia. First, we confirmed the anti-neuroinflammatory effect of DMF in mice with streptozotocin-induced diabetic neuropathy. Next, we used in vitro models including microglial cell lines and primary microglial cells to examine the anti-inflammatory and neuroprotective effects of DMF. We found that DMF significantly inhibited nitric oxide and proinflammatory cytokine production in lipopolysaccharide-stimulated microglia and induced the switch of microglia to the M2 state. In addition, DMF treatment increased the expression levels of autophagy markers including microtubule-associated protein light chain 3 (LC3) and autophagy-related protein 7 (ATG7) and the formation of LC3 puncta in microglia. The anti-inflammatory effect of DMF in microglia was significantly reduced by pretreatment with autophagy inhibitors. These data suggest that DMF leads to the induction of autophagy in microglia and that its anti-inflammatory effects are partially mediated through an autophagy-dependent pathway.

Cellular uptake and anti-inflammatory effects of palm oil-derived delta (δ)-tocotrienol in microglia

Tocopherols long dominated studies on vitamin E, although interest has shifted to tocotrienols. It was previously shown that δ-tocotrienol derived from palm oil reduced nitric oxide released by BV2 microglia as early as 18 h after lipopolysaccharide stimulation. The current study measured δ-tocotrienol uptake by BV2 over a 24 h incubation period and its anti-inflammatory effects on primary microglia. Uptake of 17.5 μg/mL δ-tocotrienol by BV2 microglia began as early as 5 min and rose steeply to 21 ± 3% of the amount administered at 24 h. The amount of δ-tocotrienol retained in the lipopolysaccharide-stimulated microglia at 24 h was 14 ± 2%, with no substantial difference seen in unstimulated microglia. The same δ-tocotrienol regimen reduced nitric oxide levels by 82% at 24 h after lipopolysaccharide stimulation (p < 0.05). This was accompanied by decreased inducible nitric oxide synthase protein expression by 67 ± 5% compared to untreated controls (p < 0.05). In primary microglia, δ-tocotrienol downregulated IL-1β production, but TNF-α and IL-6 were not affected. δ-Tocotrienol also reduced prostaglandin E2 production by ~78%% and decreased transcription of COX-2 and 5-LOX, but not COX-1. This study showed the anti-inflammatory effects of δ-tocotrienol derived from palm oil and opens up interest for tocotrienol supplementation to reduce the effects of inflammatory conditions.

Mitigation of Microglia-mediated Acute Neuroinflammation and Tissue Damage by Heme Oxygenase 1 in a Rat Spinal Cord Injury Model

Acute neuroinflammation is the major detrimental factor that causes secondary tissue damage after spinal cord injury (SCI). Curbing neuroinflammation would reduce the neuronal death and benefit functional recovery. In the current study, we used a HO-1-encoding lentivirus to transduce microglia, and adoptively transferred these microglia into injured rat spinal cords. Lentivirus-induced overexpression of exogenous HO-1 significantly inhibited microglia-mediated inflammatory response after SCI, as demonstrated by lower expression of pro-inflammatory mediators in transferred microglia. In addition, the overall post-SCI neuroinflammation was also suppressed by HO-1-overexpressing microglia, as indicated by less leukocyte infiltration and lower pro-inflammatory cytokine production in the spinal cord. Consistently, the tissue damage and neuronal apoptosis were decreased in injured spinal cords, while the locomotor function was moderately improved. We further identified that adenosine 5′-monophosphate-activated protein kinase (AMPK) signaling was involved in the regulatory effect of HO-1 on microglia, because HO-1 overexpression increased the activating phosphorylation of AMPKα. Moreover, the AMPK inhibitor compound C diminished the anti-inflammatory effect of HO-1 in lipopolysaccharide-stimulated microglia in vitro. Taken together, we proved that microglial HO-1 reduced acute post-SCI neuroinflammation. Our study might provide a promising therapeutic approach to benefit SCI recovery.

Bone marrow mesenchymal stem cells upregulate PI3K/AKT pathway and down-regulate NF-κB pathway by secreting glial cell-derived neurotrophic factors to regulate microglial polarization and alleviate deafferentation pain in rats

Deafferentation pain (DP), a typical neuropathic pain, occurs due to peripheral or central sensory nerve injury, which causes abnormal discharge of the upstream neurons or C fibers. Current treatment methods for DP have multiple side effects. Bone marrow mesenchymal stem cells (BMSC) have been used to treat neuropathic pain because of their ability to regulate neuroinflammation. Glial cell-derived neurotrophic factor (GDNF) is a neurotrophic mediator that exerts neuroprotective effects in neurological diseases. In this study, we investigated whether DP could be alleviated by BMSCs and the underlying mechanism. In vitro study, microglia was stimulated by lipopolysaccharide and then co-cultured with BMSC, GDNF or siRNA GDNF-BMSC. In vivo study, BMSC or siRNA GDNF-BMSC was transplanted intramedullarily on the 21st day after DP surgery. The expression of inflammatory-related factors were detected by RT-PCR and ELISA, RT-PCR,flow cytometry and immunofluorescence staining were performed to detect the expression of microglial surface markers, and Western blot was used to detect the expression levels of p-NF-kb, pPI3K, and pAKT. The pain-related behavioral changes were detected 7 days after transplantation. ELISA and RT-PCR results showed that the production of inflammatory cytokines in lipopolysaccharide-stimulated microglia and DP model plasma was downregulated, while anti-inflammatory mediators were upregulated significantly following pretreatment with BMSCs or GDNF. Flow cytometry, immunofluorescence staining, and RT-PCR results showed that BMSCs inhibited the microglial M1 phenotype and promoted the M2 phenotype by secreting GDNF. Furthermore, modulation functions of BMSCs involve inhibiting NF-κB while promoting PI3K /AKT signaling pathway activation. We found that our in vivo DP model was completely deafferent and BMSC administration clearly alleviated symptoms of DP. This function was also, at least partly, achieved by GDNF. The present studies demonstrate that BMSC can inhibit neuroinflammation by transforming microglial destructive M1 phenotype into regenerative M2 phenotype, and thus alleviate DP,likely by suppressing the NF-κB signaling pathway while promoting the PI3K/AKT signaling pathway activation through producing GDNF. The present findings are in support of the potential therapeutic application of BMSCs and the pharmaceutical application of GDNF for DP.

CD200R Is Involved in the Anti-inflammatory Effect of Dexmedetomidine in Lipopolysaccharide-Stimulated Microglia.

Dexmedetomidine has been confirmed to have anti-inflammatory effect in numerous diseases. However, the detailed mechanism is still unknown. We explore the hypothesis that CD200R is related to the anti-inflammatory effect of dexmedetomidine. Primary microglia were treated with different concentrations of dexmedetomidine followed with lipopolysaccharide (LPS) for 24h. Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) were measured by enzyme-linked immunosorbent assay. Arginase 1 (Arg1), IL-6, and TNF-α were measured by TR-PCR. CD200R was observed by Western blot analysis. We found dexmedetomidine reduced LPS-induced IL-6 and TNF-α production and increase Arg1 in primary microglia. CD200R expression was downregulated by LPS and dexmedetomidine can attenuate the effect of LPS. Our results showed that CD200R may be related to the anti-inflammatory effect of dexmedetomidine.

Adipose-Derived Stem Cells Modulate BV2 Microglial M1/M2 Polarization by Producing GDNF.

Neuroinflammation is associated with the pathogenesis of all types of neurological disease, in which microglial cells play a critical role. In response to disturbances in the microenvironment, microglia (MG) become activated and differentiate into either an M1 phenotype, which has a proinflammatory damaging effect, or an M2 phenotype, which plays an anti-inflammatory and reparative role. Thus, modulating microglial polarization is a suitable strategy to treat neuroinflammatory disorders. Glial cell-derived neurotrophic factor (GDNF) is a neurotrophic mediator that exerts neuroprotective effects during neurological diseases. In this study, we predicted that adipose-derived stem cells (ADSCs) could produce GDNF and investigated the effects of GDNF on microglial M1/M2 polarization. Furthermore, we determined whether GDNF modulates microglial activation and polarization via the phosphoinositide-3-kinase (PI3K)/AKT signaling pathway. We found that the secretion of inflammatory cytokines in lipopolysaccharide-stimulated MG was downregulated, whereas the anti-inflammatory mediators in interleukin-4-stimulated MG were upregulated obviously, following pretreatment with ADSCs or GDNF. In addition, GDNF produced by ADSCs inhibited the MG M1 phenotype and promoted the M2 phenotype by upregulating the PI3K/ATK pathway. These results reveal that GDNF produced by ADSCs might be useful for the regulation of neuroinflammatory disorders.

Extracellular Vesicle lncRNA Metastasis-Associated Lung Adenocarcinoma Transcript 1 Released From Glioma Stem Cells Modulates the Inflammatory Response of Microglia After Lipopolysaccharide Stimulation Through Regulating miR-129-5p/High Mobility Group Box-1 Protein Axis.

Glioma stem cell (GSC)–derived extracellular vesicles (EVs) can mediate the communication between GSCs and microglia. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) expression in GSCs, EVs, and supernatant was detected by real-time PCR. The direct targeting between MALAT1 and miR-129-5p, miR-129-5p, and HMGB1 were tested with luciferase reporter analysis. The expression and secretion of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α were determined in lipopolysaccharide-stimulated microglia or miR-129-5p inhibitor transferred to microglia exposed to GSC EVs or EVs derived from siMALAT1 pre-transferred GSCs. MALAT1 was enriched in GSC EVs compared with GSCs, and up-regulated MALAT1 was also observed in microglia upon GSC EVs incubation. The relative expression and secretion of IL-6, IL-8, and TNF-α in lipopolysaccharide-stimulated microglia were up-regulated in the GSC supernatant group, which could be reversed by dimethyl amiloride (DMA) (EV secretion inhibitor) co-administration or si-MALAT1 pre-transfection of GSCs. Luciferase reporter assay testified the direct binding of MALAT1 and miR-129-5p, miR-129-5p, and HMGB1, and si-MALAT1 could up-regulate miR-129-5p expression and down-regulate HMGB1 expression in microglia cells. The concentration of IL-6, IL-8, and TNF-α in lipopolysaccharide-stimulated microglia exposed to EVs from siMALAT1 transfected GSCs could be up-regulated by miR-129-5p inhibition. EVs lncRNA MALAT1 released from GSCs could modulate the inflammatory response of microglia after lipopolysaccharide stimulation through regulating the miR-129-5p/HMGB1 axis.

SS-31 reduces inflammation and oxidative stress through the inhibition of Fis1 expression in lipopolysaccharide-stimulated microglia

SS-31 is a kind of mitochondrion-targeted peptide. Recent studies indicated significant neuroprotective effects of SS-31. In this study, we investigated that SS-31 protected the murine cultured microglial cells (BV-2) against lipopolysaccharide (LPS)-induced inflammation and oxidative stress through stabilizing mitochondrial morphology. The morphological study showed that SS-31 preserved LPS-induced mitochondrial ultrastructure by reducing the fission protein 1 (Fis1) expression. Flow cytometry and Western blot verified that SS-31 defended the BV-2 cells against LPS-stimulated inflammation and oxidative stress via suppressing Fis1. To sum up, our study represents that SS-31 preserves BV-2 cells from LPS-stimulated inflammation and oxidative stress by down-regulating the Fis1 expression.

Exosomes from adipose-derived stem cells alleviate neural injury caused by microglia activation via suppressing NF-kB and MAPK pathway

Activation of microglia cells play critical role in neuroinflammation after brain injury. Exosomes from adipose-derived stem cells (ADSC) possess immunoregulation effect similar with ADSC. We hypothesized that ADSC derived exosomes (ADSC-exosomes) could inhibit the activation of microglia cells and prevent neuroinflammation. We found that ADSC-exosomes could inhibit the activation of microglia cells by suppressing NF-kB and MAPK pathway. Production of inflammatory factors in lipopolysaccharide-stimulated microglia cells decreased significantly when pretreated with ADSC-exosomes. Furthermore, ADSC-exosomes could decrease the cytotoxicity of activated microglia. These results revealed that ADSC-exosomes might be a promising strategy for the therapy of neural injury.

Propofol attenuates inflammatory damage on neurons following cerebral infarction by inhibiting excessive activation of microglia.

The overall incidence rate of stroke is increasing worldwide. Inflammatory damage following a stroke is a leading cause for the poor prognosis and high disability rate observed in stroke patients. Microglia are considered to be the main causative agents of inflammatory injury following cerebral infarction, as they secrete various inflammatory cytokines and cytotoxic factors. The aim of the present study was to identify novel methods for attenuating inflammatory injury and improving the prognosis of stroke patients. Lipopolysaccharide-stimulated microglia were treated using propofol invitro and a transient middle cerebral artery occlusion/reperfusion model was constructed in rats. Expression of cytotoxic factors, microglia proliferation and the neuroprotective effects of propofol were measured invitro and invivo. The invitro studies demonstrated that propofol inhibits the expression of multiple cytotoxic factors, prevents structural changes to cytoskeletal proteins, and suppresses microglial migration via the adenosineA2b receptors. The results of the invivo experiments revealed that propofol inhibits the abnormal proliferation of microglia, as well as reduces the expression levels of interleukin (IL)-6, IL-1β, tumor necrosis factor α, and the cytotoxic factor nitric oxide through the A2b receptor. In conclusion, propofol inhibited the excessive activation of microglia through the A2b receptor and attenuated the inflammatory injury following cerebral infarction. The current study may provide a reliable basis for further clinical studies on propofol and its putative role in improving the prognosis of patients with cerebral infarction.

Neuronal ICAM-5 Inhibits Microglia Adhesion and Phagocytosis and Promotes an Anti-inflammatory Response in LPS Stimulated Microglia.

The intercellular adhesion molecule-5 (ICAM-5) regulates neurite outgrowth and synaptic maturation. ICAM-5 overexpression in the hippocampal neurons induces filopodia formation in vitro. Since microglia are known to prune supernumerous synapses during development, we characterized the regulatory effect of ICAM-5 on microglia. ICAM-5 was released as a soluble protein from N-methyl-D-aspartic acid (NMDA)-treated neurons and bound by microglia. ICAM-5 promoted down-regulation of adhesion and phagocytosis in vitro. Microglia formed large cell clusters on ICAM-5-coated surfaces whereas they adhered and spread on the related molecule ICAM-1. ICAM-5 further reduced the secretion of the proinflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), but on the contrary induced the secretion of the anti-inflammatory IL-10 from lipopolysaccharide (LPS) stimulated microglia. Thus, ICAM-5 might be involved in the regulation of microglia in both health and disease, playing an important neuroprotective role when the brain is under immune challenges and as a “don’t-eat-me” signal when it is solubilized from active synapses.

Anti-inflammatory mechanism of galangin in lipopolysaccharide-stimulated microglia: Critical role of PPAR-γ signaling pathway

Since microglia-associated neuroinflammation plays a pivotal role in the progression of neurodegenerative diseases, controlling microglial activation has been suggested as a potential therapeutic strategy. Here, we investigated the anti-inflammatory effects of galangin (3,5,7-trihydroxyflavone) in microglia and analyzed the underlying molecular mechanisms. Galangin inhibited the expression of inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines and enhanced the expression of anti-inflammatory interleukin (IL)-10 in lipopolysaccharide (LPS)-stimulated BV2 microglia. Galangin also suppressed microglial activation and the expression of pro-inflammatory markers in LPS-injected mouse brains. The results of mechanistic studies have shown that galangin inhibited LPS-induced phosphorylation of p38 mitogen activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), phosphatidylinositol 3-kinase (PI3K)/Akt, and nuclear factor (NF)-κB activity. On the contrary, galangin increased the activity of transcription factors, such as nuclear factor-E2-related factor 2 (Nrf2), cAMP response element-binding protein (CREB), and peroxisome proliferator-activated receptor (PPAR)-γ, known to play an anti-inflammatory role. In addition, galangin showed antioxidant effects by suppressing the expression of NADPH oxidase subunits p47phox and gp91phox, and by enhancing hemeoxygenase-1. We then investigated whether PPAR-γ was involved in the anti-inflammatory function of galangin. Pretreatment with a PPAR-γ antagonist or siRNA significantly blocked galangin-mediated upregulation of IL-10 and attenuated the inhibition of tumor necrosis factor (TNF)-α, nitric oxide (NO), and IL-6 in LPS-stimulated microglia. Moreover, the PPAR-γ antagonist reversed the effects of galangin on NF-κB, Nrf2, and CREB. Altogether, our data suggest that PPAR-γ plays a key role in mediating the anti-inflammatory effects of galangin by modulating the NF-κB and Nrf2/CREB signaling pathways.