The mechanisms of Porphyromonas gingivalis-derived outer membrane vesicles-induced neurotoxicity and microglia activation.

Background/purpose: Periodontitis is associated with various systemic diseases, potentially facilitated by the passage of Porphyromonas gingivalis outer membrane vesicles (Pg-OMVs). Several recent studies have suggested a connection between Pg-OMVs and neuroinflammation and neurodegeneration, but the precise causal relationship remains unclear. This study aimed to investigate the mechanisms underlying these associations using in vitro models. Materials and methods: Isolated Pg-OMVs were characterized by morphology, size, and gingipain activity. We exposed SH-SY5Y neuroblastoma cells and BV-2 microglial cells to various concentrations of Pg-OMVs. Cell morphology, a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, an enzyme-linked immunosorbent assay, and Western blot analysis were used to evaluate the cellular mechanism underlying Pg-OMV-induced neurotoxicity in neuronal cells and inflammatory responses in microglial cells. Results: Exposure to Pg-OMVs induced neurotoxicity in SH-SY5Y cells, as evidenced by cellular shrinkage, reduced viability, activation of apoptotic pathways, and diminished neuronal differentiation markers. Gingipain inhibition mitigated these effects, suggesting that gingipain mediates Pg-OMVs-induced neurotoxicity in SH-SY5Y cells. Our research on neuroinflammation suggests that upon endocytosis of Pg-OMVs by BV-2 cells, lipopolysaccharide (LPS) can modulate the production of inducible nitric oxide synthase and tumor necrosis factor-alpha by activating pathways that involve phosphorylated AKT and the phosphorylated JNK pathway. Conclusion: Our study demonstrated that following the endocytosis of Pg-OMVs, gingipain can induce neurotoxicity in SH-SY5Y cells. Furthermore, the Pg-OMVs-associated LPS can trigger neuroinflammation via AKT and JNK signaling pathways in BV-2 cells.

Exploring the molecular mechanism of sepsis-associated encephalopathy by integrated analysis of multiple datasets.

BACKGROUND: We aim to deal with the Hub-genes and signalling pathways connected with Sepsis-associated encephalopathy (SAE). METHODS: The raw datasets were acquired from the Gene Expression Omnibus (GEO) database (GSE198861 and GSE167610). R software filtered the differentially expressed genes (DEGs) for hub genes exploited for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Hub genes were identified from the intersection of DEGs via protein-protein interaction (PPI) network. And the single-cell dataset (GSE101901) was used to authenticate where the hub genes express in hippocampus cells. Cell-cell interaction analysis and Gene Set Variation Analysis (GSVA) analysis of the whole transcriptome validated the interactions between hippocampal cells. RESULTS: A total of 161 DEGs were revealed in GSE198861 and GSE167610 datasets. Biological function analysis showed that the DEGs were primarily involved in the phagosome pathway and significantly enriched. The PPI network extracted 10 Hub genes. The M2 Macrophage cell decreased significantly during the acute period, and the hub gene may play a role in this biological process. The hippocampal variation pathway was associated with the MAPK signaling pathway. CONCLUSION: Hub genes (Pecam1, Cdh5, Fcgr, C1qa, Vwf, Vegfa, C1qb, C1qc, Fcgr4 and Fcgr2b) may paticipate in the biological process of SAE.

Anti-inflammatory activities of novel heat shock protein 90 isoform selective inhibitors in BV-2 microglial cells.

Heat shock protein 90 (Hsp90) is a family of chaperone proteins that consists of four isoforms: Hsp90α, Hsp90ß, glucose-regulated protein 94 (Grp94), and tumor necrosis factor type 1 receptor-associated protein (TRAP1). They are involved in modulating the folding, maturation, and activation of their client proteins to regulate numerous intracellular signaling pathways. Previous studies demonstrated that pan-Hsp90 inhibitors reduce inflammatory signaling pathways resulting in a reduction of inflammation and pain but show toxicities in cancer-related clinical trials. Further, the role of Hsp90 isoforms in inflammation remains poorly understood. This study aimed to determine anti-inflammatory activities of Hsp90 isoforms selective inhibitors on the lipopolysaccharide (LPS)-induced inflammation in BV-2 cells, a murine microglial cell line. The production of inflammatory mediators such as nitric oxide (NO), interleukin 1 beta (IL-1ß), and tumor necrosis factor-alpha (TNF-α) was measured. We also investigated the impact of Hsp90 isoform inhibitors on the activation of nuclear factor kappa B (NF-κB), nuclear factor erythroid 2-related factor 2 (Nrf2), and mitogen-activated protein kinases (MAPKs). We found that selective inhibitors of Hsp90ß reduced the LPS-induced production of NO, IL-1ß, and TNF-α via diminishing the activation of NF-κB and Extracellular signal-regulated kinases (ERK) MAPK. The Hsp90α, Grp94, TRAP1 inhibitors had limited effect on the production of inflammatory mediators. These findings suggest that Hsp90ß is the key player in LPS-induced neuroinflammation. Thereby providing a more selective drug target for development of medications involved in pain management that can potentially contribute to the reduction of adverse side effects associated with Hsp90 pan inhibitors.

PM2.5 exposure upregulates pro-inflammatory protein expression in human microglial cells via oxidant stress and TLR4/NF-κB pathway.

Exposure to ambient PM2.5 is associated with neurodegenerative disorders, in which microglia activation plays a critical role. Thus far, the underlying mechanisms for PM2.5-induced microglia activation have not been well elucidated. In this study, a human microglial cell line (HMC3) was used as the in vitro model to examine the inflammatory effect (hall marker of microglia activation) of PM2.5 and regulatory pathways. The expression of inflammatory mediators including interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) as well as the brain derived neurotrophic factor (BDNF) were determined by ELISA and/or real-time PCR, respectively. Flow cytometry was used to measure the production of intracellular reactive oxygen species (ROS). Western blot was used to measure protein levels of Toll-like receptor 4 (TLR4), NF-κB inhibitor α (IκBα) and COX-2. It was shown that PM2.5 stimulation increased IL-6 and COX-2 expression but decreased BDNF expression in a dose-dependent manner. Further studies showed that PM2.5 triggered the formation of ROS and pre-treatment with the ROS scavenger acetylcysteine (NAC) significantly suppressed PM2.5-induced IL-6 and COX-2 expression. Moreover, the nuclear factor kappa B (NF-κB) inhibitor BAY11-7085 or the TLR4 neutralizing antibody markedly blocked PM2.5-induced IL-6 and COX-2 expression. However, NAC or BAY11-7085 exhibited minimal effect on PM2.5-induced BDNF down-regulation. In addition, pre-treatment with BAY11-7085 or TLR4 neutralizing antibody reduced ROS production induced by PM2.5, and NAC pre-treatment inhibited TLR4 expression and NF-κB activation induced by PM2.5. Collectively, PM2.5 treatment induced IL-6 and COX-2 but suppressed BDNF expression. PM2.5-induced IL-6 and COX-2 expression was mediated by interactive oxidative stress and TLR4/NF-κB pathway.

Unraveling the enigma: housekeeping gene Ugt1a7c as a universal biomarker for microglia.

Background: Microglia, brain resident macrophages, play multiple roles in maintaining homeostasis, including immunity, surveillance, and protecting the central nervous system through their distinct activation processes. Identifying all types of microglia-driven populations is crucial due to the presence of various phenotypes that differ based on developmental stages or activation states. During embryonic development, the E8.5 yolk sac contains erythromyeloid progenitors that go through different growth phases, eventually resulting in the formation of microglia. In addition, microglia are present in neurological diseases as a diverse population. So far, no individual biomarker for microglia has been discovered that can accurately identify and monitor their development and attributes. Summary: Here, we highlight the newly defined biomarker of mouse microglia, UGT1A7C, which exhibits superior stability in expression during microglia development and activation compared to other known microglia biomarkers. The UGT1A7C sensing chemical probe labels all microglia in the 3xTG AD mouse model. The expression of Ugt1a7c is stable during development, with only a 4-fold variation, while other microglia biomarkers, such as Csf1r and Cx3cr1, exhibit at least a 10-fold difference. The UGT1A7C expression remains constant throughout its lifespan. In addition, the expression and activity of UGT1A7C are the same in response to different types of inflammatory activators' treatment in vitro. Conclusion: We propose employing UGT1A7C as the representative biomarker for microglia, irrespective of their developmental state, age, or activation status. Using UGT1A7C can reduce the requirement for using multiple biomarkers, enhance the precision of microglia analysis, and even be utilized as a standard for gene/protein expression.

Single-cell RNA sequencing unveils Lrg1's role in cerebral ischemia‒reperfusion injury by modulating various cells.

BACKGROUND AND PURPOSE: Cerebral ischemia‒reperfusion injury causes significant harm to human health and is a major contributor to stroke-related deaths worldwide. Current treatments are limited, and new, more effective prevention and treatment strategies that target multiple cell components are urgently needed. Leucine-rich alpha-2 glycoprotein 1 (Lrg1) appears to be associated with the progression of cerebral ischemia‒reperfusion injury, but the exact mechanism of it is unknown. METHODS: Wild-type (WT) and Lrg1 knockout (Lrg1-/-) mice were used to investigate the role of Lrg1 after cerebral ischemia‒reperfusion injury. The effects of Lrg1 knockout on brain infarct volume, blood‒brain barrier permeability, and neurological score (based on 2,3,5-triphenyl tetrazolium chloride, evans blue dye, hematoxylin, and eosin staining) were assessed. Single-cell RNA sequencing (scRNA-seq), immunofluorescence, and microvascular albumin leakage tests were utilized to investigate alterations in various cell components in brain tissue after Lrg1 knockout. RESULTS: Lrg1 expression was increased in various cell types of brain tissue after cerebral ischemia‒reperfusion injury. Lrg1 knockout reduced cerebral edema and infarct size and improved neurological function after cerebral ischemia‒reperfusion injury. Single-cell RNA sequencing analysis of WT and Lrg1-/- mouse brain tissues after cerebral ischemia‒reperfusion injury revealed that Lrg1 knockout enhances blood‒brain barrier (BBB) by upregulating claudin 11, integrin ß5, protocadherin 9, and annexin A2. Lrg1 knockout also promoted an anti-inflammatory and tissue-repairing phenotype in microglia and macrophages while reducing neuron and oligodendrocyte cell death. CONCLUSIONS: Our results has shown that Lrg1 mediates numerous pathological processes involved in cerebral ischemia‒reperfusion injury by altering the functional states of various cell types, thereby rendering it a promising therapeutic target for cerebral ischemia‒reperfusion injury.

4-methoxycinnamyl p-coumarate reduces neuroinflammation by blocking NF-κB, MAPK, and Akt/GSK-3ß pathways and enhancing Nrf2/HO-1 signaling cascade in microglial cells.

The active compound, 4-methoxycinnamyl p-coumarate (MCC), derived from the rhizome of Etlingera pavieana (Pierre ex Gagnep) R.M.Sm., has been shown to exert anti-inflammatory effects in several inflammatory models. However, its effects on microglial cells remain elusive. In the current study, we aimed to investigate the anti-neuroinflammatory activities of MCC and determine the potential mechanisms underlying its action on lipopolysaccharide (LPS)-induced BV2 microglial cells. Our results revealed that MCC significantly reduced the secretion of nitric oxide (NO) and prostaglandin E2, concomitantly inhibiting the expression levels of inducible NO synthase and cyclooxygenase-2 mRNA and proteins. Additionally, MCC effectively decreased the production of reactive oxygen species in LPS-induced BV2 microglial cells. MCC also attenuates the activation of NF-κB by suppressing the phosphorylation of IκBα and NF-κB p65 subunits and by blocking the nuclear translocation of NF-κB p65 subunits. Furthermore, MCC significantly reduced the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and protein kinase B (Akt)/glycogen synthase kinase-3ß (GSK-3ß). In addition, MCC markedly increased the expression of heme oxygenase-1 (HO-1) by upregulating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Collectively, our findings suggest that the anti-inflammatory activities of MCC could be attributed to its ability to suppress the activation of NF-κB, MAPK, and Akt/GSK-3ß while enhancing that of Nrf2-mediated HO-1. Accordingly, MCC has promising therapeutic potential to treat neuroinflammation-related diseases.

Bone marrow mesenchymal stem cell-derived exosomal microRNA regulates microglial polarization.

Objective: This study aimed to explore the effects of bone marrow mesenchymal stem cell (BMSC)-derived exosomal miR-146a-5p on microglial polarization and the potential underlying mechanisms in oxygen-glucose deprivation (OGD)-exposed microglial cells. Methods: Exosomes were isolated from BMSCs, and their characteristics were examined. The effects of BMSC-derived exosomes on microglial polarization were investigated in OGD-exposed BV-2 cells. Differentially expressed miRNAs were identified and their biological function was explored using enrichment analyses. The regulatory role of miR-146a-5p in microglial polarization was studied via flow cytometry. Finally, the downstream target gene Traf6 was validated, and the role of the miR-146a-5p/Traf6 axis in modulating microglial polarization was investigated in OGD-exposed BV-2 cells. Results: BMSC-derived exosomes were successfully isolated and characterized. A total of 10 upregulated and 33 downregulated miRNAs were identified. Exosomal treatment resulted in significant changes in microglial polarization markers. miR-146a-5p was found to be significantly downregulated in OGD-exposed microglial cells treated with exosomes. Manipulation of miR-146a-5p expression modulated microglial polarization. Moreover, the miR-146a-5p/Traf6 axis regulated microglial polarization. Conclusion: Our findings demonstrate that BMSC-derived exosomal via miR-146a-5p modulates microglial polarization by targeting Traf6, providing a potential thermal target for the treatment of neurological diseases involving microglial activation.

Lindenane sesquiterpenoid dimers from Chloranthus holostegius with anti-neuroinflammatory activities in BV-2 microglia.

Fifteen undescribed lindenane-type sesquiterpenoid dimers, designated chloranholides F-T (1-15), together with twenty-five known analogs (16-40), were isolated from the whole plants of Chloranthus holostegius. The isolate structures were elucidated by analysis of spectroscopic data and chemical methods, and their absolute configurations were determined by X-ray crystallography and electronic circular dichroism spectra. In anti-neuroinflammatory assays, all isolates were evaluated by examination of their inhibitory effect on nitric oxide (NO) in LPS-stimulated BV-2 cells, and the results showed that 21-24, 26, 30, 32 and 36 significantly inhibited the production of the inflammatory mediator NO, with IC50 values ranging from 3.18 to 11.46 µM, which was better than that of quercetin. Structure-activity relationship analysis revealed that two essential functional groups played an indispensable role in the anti-inflammatory effects. Moreover, 22 and 24 inhibited the LPS-induced upregulation of iNOS and COX-2 enzymes in BV-2 microglia at the protein level.

Microglia contribute to cognitive decline in hypercholesterolemic LDLr-/- mice.

Familial hypercholesterolemia (FH) is caused by mutations in the gene that encodes the low-density lipoprotein (LDL) receptor, which leads to an excessive increase in plasma LDL cholesterol levels. Previous studies have shown that FH is associated with gliosis, blood-brain barrier dysfunction, and memory impairment, but the mechanisms associated with these events are still not fully understood. Therefore, we aimed to investigate the role of microgliosis in the neurochemical and behavioral changes associated with FH using LDL receptor knockout (LDLr-/- ) mice. We noticed that microgliosis was more severe in the hippocampus of middle-aged LDLr-/- mice, which was accompanied by microglial morphological changes and alterations in the immunocontent of synaptic protein markers. At three months of age, the LDLr-/- mice already showed increased microgliosis and decreased immunocontent of claudin-5 in the prefrontal cortex (PFC). Subsequently, 6-month-old male C57BL/6 wild-type and LDLr-/- mice were treated once daily for 30 days with minocycline (a pharmacological inhibitor of microglial cell reactivity) or vehicle (saline). Adult LDLr-/- mice displayed significant hippocampal memory impairment, which was ameliorated by minocycline treatment. Non-treated LDLr-/- mice showed increased microglial density in all hippocampal regions analyzed, a process that was not altered by minocycline treatment. Region-specific microglial morphological analysis revealed different effects of genotype or minocycline treatment on microglial morphology, depending on the hippocampal subregion analyzed. Moreover, 6-month-old LDLr-/- mice exhibited a slight but not significant increase in IBA-1 immunoreactivity in the PFC, which was reduced by minocycline treatment without altering microglial morphology. Minocycline treatment also reduced the presence of microglia within the perivascular area in both the PFC and hippocampus of LDLr-/- mice. However, no significant effects of either genotype or minocycline treatment were observed regarding the phagocytic activity of microglia in the PFC and hippocampus. Our results demonstrate that hippocampal microgliosis, microglial morphological changes, and the presence of these glial cells in the perivascular area, but not increased microglial phagocytic activity, are associated with cognitive deficits in a mouse model of FH.

Turmeronols (A and B) from Curcuma longa have anti-inflammatory effects in lipopolysaccharide-stimulated BV-2 microglial cells by reducing NF-κB signaling.

Turmeronols (A and B), bisabolane-type sesquiterpenoids found in turmeric, reduce inflammation outside the brain in animals; however, their effects on neuroinflammation, a common pathology of various neurodegenerative diseases, are not understood. Inflammatory mediators produced by microglial cells play a key role in neuroinflammation, so this study evaluated the anti-inflammatory effects of turmeronols in BV-2 microglial cells stimulated with lipopolysaccharide (LPS). Pretreatment with turmeronol A or B significantly inhibited LPS-induced nitric oxide (NO) production; mRNA expression of inducible NO synthase; production of interleukin (IL)-1ß, IL-6, and tumor necrosis factor α and upregulation of their mRNA expression; phosphorylation of nuclear factor-κB (NF-κB) p65 proteins and inhibitor of NF-κB kinase (IKK); and nuclear translocation of NF-κB. These results suggest that these turmeronols may prevent the production of inflammatory mediators by inhibiting the IKK/NF-κB signaling pathway in activated microglial cells and can potentially treat neuroinflammation associated with microglial activation.

Testing a Polygenic Risk Score for Morphological Microglial Activation in Alzheimer's Disease and Aging.

BACKGROUND: Neuroinflammation and the activation of microglial cells are among the earliest events in Alzheimer's disease (AD). However, direct observation of microglia in living people is not currently possible. Here, we indexed the heritable propensity for neuroinflammation with polygenic risk scores (PRS), using results from a recent genome-wide analysis of a validated post-mortem measure of morphological microglial activation. OBJECTIVE: We sought to determine whether a PRS for microglial activation (PRSmic) could augment the predictive performance of existing AD PRSs for late-life cognitive impairment. METHODS: First, PRSmic were calculated and optimized in a calibration cohort (Alzheimer's Disease Neuroimaging Initiative (ADNI), n = 450), with resampling. Second, predictive performance of optimal PRSmic was assessed in two independent, population-based cohorts (total n = 212,237). Finally, we explored associations of PRSmic with a comprehensive set of imaging and fluid AD biomarkers in ADNI. RESULTS: Our PRSmic showed no significant improvement in predictive power for either AD diagnosis or cognitive performance in either external cohort. Some nominal associations were found in ADNI, but with inconsistent effect directions. CONCLUSION: While genetic scores capable of indexing risk for neuroinflammatory processes in aging are highly desirable, more well-powered genome-wide studies of microglial activation are required. Further, biobank-scale studies would benefit from phenotyping of proximal neuroinflammatory processes to improve the PRS development phase.

Effectiveness of Combinational Treatments for Alzheimer's Disease with Human Neural Stem Cells and Microglial Cells Over-Expressing Functional Genes.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. In AD patients, amyloid-ß (Aß) peptide-mediated degeneration of the cholinergic system utilizing acetylcholine (ACh) for memory acquisition is observed. Since AD therapy using acetylcholinesterase (AChE) inhibitors are only palliative for memory deficits without reversing disease progress, there is a need for effective therapies, and cell-based therapeutic approaches should fulfil this requirement. We established F3.ChAT human neural stem cells (NSCs) encoding the choline acetyltransferase (ChAT) gene, an ACh-synthesizing enzyme, HMO6.NEP human microglial cells encoding the neprilysin (NEP) gene, an Aß-degrading enzyme, and HMO6.SRA cells encoding the scavenger receptor A (SRA) gene, an Aß-uptaking receptor. For the efficacy evaluation of the cells, first, we established an appropriate animal model based on Aß accumulation and cognitive dysfunction. Among various AD models, intracerebroventricular (ICV) injection of ethylcholine mustard azirinium ion (AF64A) induced the most severe Aß accumulation and memory dysfunction. Established NSCs and HMO6 cells were transplanted ICV to mice showing memory loss induced by AF64A challenge, and brain Aß accumulation, ACh concentration and cognitive function were analyzed. All the transplanted F3.ChAT, HMO6.NEP and HMO6.SRA cells were found to survive up to 4 weeks in the mouse brain and expressed their functional genes. Combinational treatment with the NSCs (F3.ChAT) and microglial cells encoding each functional gene (HMO6.NEP or HMO6.SRA) synergistically restored the learning and memory function of AF64A-challenged mice by eliminating Aß deposits and recovering ACh level. The cells also attenuated inflammatory astrocytic (glial fibrillary acidic protein) response by reducing Aß accumulation. Taken together, it is expected that NSCs and microglial cells over-expressing ChAT, NEP or SRA genes could be strategies for replacement cell therapy of AD.

Severe Acute Liver Dysfunction Induces Delayed Hepatocyte Swelling and Cytoplasmic Vacuolization, and Delayed Cortical Neuronal Cell Death.

Liver dysfunction is the main cause of hepatic encephalopathy. However, histopathological changes in the brain associated with hepatic encephalopathy remain unclear. Therefore, we investigated pathological changes in the liver and brain using an acute hepatic encephalopathy mouse model. After administering ammonium acetate, a transient increase in the blood ammonia level was observed, which returned to normal levels after 24 h. Consciousness and motor levels also returned to normal. It was revealed that hepatocyte swelling, and cytoplasmic vacuolization progressed over time in the liver tissue. Blood biochemistry also suggested hepatocyte dysfunction. In the brain, histopathological changes, such as perivascular astrocyte swelling, were observed 3 h after ammonium acetate administration. Abnormalities in neuronal organelles, especially mitochondria and rough endoplasmic reticulum, were also observed. Additionally, neuronal cell death was observed 24 h post-ammonia treatment when blood ammonia levels had returned to normal. Activation of reactive microglia and increased expression of inducible nitric oxide synthase (iNOS) were also observed seven days after a transient increase in blood ammonia. These results suggest that delayed neuronal atrophy could be iNOS-mediated cell death due to activation of reactive microglia. The findings also suggest that severe acute hepatic encephalopathy causes continued delayed brain cytotoxicity even after consciousness recovery.

The Ketogenic Diet and Neuroinflammation: The Action of Beta-Hydroxybutyrate in a Microglial Cell Line.

The ketogenic diet (KD), a diet high in fat and protein but low in carbohydrates, is gaining much interest due to its positive effects, especially in neurodegenerative diseases. Beta-hydroxybutyrate (BHB), the major ketone body produced during the carbohydrate deprivation that occurs in KD, is assumed to have neuroprotective effects, although the molecular mechanisms responsible for these effects are still unclear. Microglial cell activation plays a key role in the development of neurodegenerative diseases, resulting in the production of several proinflammatory secondary metabolites. The following study aimed to investigate the mechanisms by which BHB determines the activation processes of BV2 microglial cells, such as polarization, cell migration and expression of pro- and anti-inflammatory cytokines, in the absence or in the presence of lipopolysaccharide (LPS) as a proinflammatory stimulus. The results showed that BHB has a neuroprotective effect in BV2 cells, inducing both microglial polarization towards an M2 anti-inflammatory phenotype and reducing migratory capacity following LPS stimulation. Furthermore, BHB significantly reduced expression levels of the proinflammatory cytokine IL-17 and increased levels of the anti-inflammatory cytokine IL-10. From this study, it can be concluded that BHB, and consequently the KD, has a fundamental role in neuroprotection and prevention in neurodegenerative diseases, presenting new therapeutic targets.

Different Contacted Cell Types Contribute to Acquiring Different Properties in Brain Microglial Cells upon Intercellular Interaction.

Microglial cells (MGs), originally derived from progenitor cells in a yolk sac during early development, are glial cells located in a physiological and pathological brain. Since the brain contains various cell types, MGs could frequently interact with different cells, such as astrocytes (ACs), pericytes (PCs), and endothelial cells (ECs). However, how microglial traits are regulated via cell-cell interactions by ACs, PCs, or ECs and how they are different depending on the contacted cell types is unclear. This study aimed to clarify these questions by coculturing MGs with ACs, PCs, or ECs using mouse brain-derived cells, and microglial phenotypic changes were investigated under culture conditions that enabled direct cell-cell contact. Our results showed that ACs or PCs dose-dependently increased the number of MG, while ECs decreased it. Microarray and gene ontology analysis showed that cell fate-related genes (e.g., cell cycle, proliferation, growth, death, and apoptosis) of MGs were altered after a cell-cell contact with ACs, PCs, and ECs. Notably, microarray analysis showed that several genes, such as gap junction protein alpha 1 (Gja1), were prominently upregulated in MGs after coincubation with ACs, PCs, or ECs, regardless of cell types. Similarly, immunohistochemistry showed that an increased Gja1 expression was observed in MGs after coincubation with ACs, PCs, or ECs. Immunofluorescent and fluorescence-activated cell sorting analysis also showed that calcein-AM was transferred into MGs after coincubation with ACs, PCs, or ECs, confirming that intercellular interactions occurred between these cells. However, while Gja1 inhibition reduced the number of MGs after coincubation with ACs and PCs, this was increased after coincubation with ECs; this indicates that ACs and PCs positively regulate microglial numbers via Gja1, while ECs decrease it. Results show that ACs, PCs, or ECs exert both common and specific cell type-dependent effects on MGs through intercellular interactions. These findings also suggest that brain microglial phenotypes are different depending on their surrounding cell types, such as ACs, PCs, or ECs.

Effects of activating mTORC2/Akt signaling pathway on dopaminergic neurons and behaviors in 6-hydroxydopamin model mice / 解剖学报

Objective To explore the effect of activation of mammalian target of rapmycin complex 2(mTORC2)/Akt signaling pathway on dopaminergic neurons and behavior in 6-hydroxydopamine (6-OHDA) model mice and its possible mechanism. Methods Selecting 36 mice which The Nestin-CreERTM and ROSA26-LacZ reporter genes were detected at the same time in 3-month-old male C57BL/6J mice weighing 20-25 g divideng them into 4 gruops, NS+ corn oil group, 6-OHDA+corn oil group, 6-OHDA+PP242 group and 6-OHDA+A-443654 group, and 6-OHDA was injected into the right striatum of the brain to replicate the Parkinson’s disease (PD) model of mice, and then daily intraperitoneal injection of mTORC2/Akt signaling pathway agonist A-443654 or inhibitor PP242. Serum interleukin-1β (IL-1β) and tumor necrosis factor-α(TNF-α)levels were measured by enzyme-linked immunosorbent assay. Immunohistochemistry and immunofluorescence staining were performed to investigate the change of microglia, dopaminergic neurons as well as neural progenitor cells (NPCs). Western blotting was used to detect the expression of related protein of mTORC2/Akt signaling pathway including rictor, p-Akt and regulated in development and DNA dgmage responses 1(REDD1) and the interaction between them were verified by immunoprecipitation. Finally, the behavioral performance of each group of mice was observed. Results With the activation of microglia and the increase of inflammatory factors in PD model mice, the number of dopaminergic neurons in the substantia nigra(SN) decreased significantly, and the motor function of the mice was impaired, but the number of NPCs increased significantly compared with the control mice, mTORC2/Akt signaling pathway related protein expression was also significantly up-regulated. A-443654 treatment further up-regulated the expression of these proteins, meanwhile the indicators mentioned above were ameliorated. However, the inhibitor PP242 treatment group showed completely opposite result with the agonist group. Conclusion A-443654 can promote the proliferation of NPCs and the number of new-born dopaminergic neurons by up-regulating related proteins of mTORC2/Akt signaling pathway, and reducing the activation of microglia and the level of inflammation factors, which ultimately lead to the amelioration of SN-striatal dopaminergic neurons and behavioral performance in PD model mice.

Quantitative magnetic susceptibility imaging sequence for intracranial inflammation in patients with optic neuromyelitis / 中国医师进修杂志

Objective:To identify the potential intracranial inflammation in neuromyelitis optica spectrum disorders(NMOSD) patients without supratentorial MRI lesions using quantitative susceptibility mapping (QSM).Methods:Seventy NMOSD patients and 35 age- and gender-matched healthy controls (NC) underwent QSM, 3D-T 1, diffusion MRI from Beijing Tiantan Hospital during June 2019 to June 2021. Susceptibility was compared among NMOSD patients with acute attack (ANMOSD), NMOSD patients in chronic phase (CNMOSD) and NC. The correlation between susceptibility in several brain regions and the cerebrospinal fluid levels of inflammatory makers were analyzed. Results:NMOSD patients showed different susceptibility in several brain regions including bilateral hippocampus, precuneus, right cuneus, putamen, superior parietal and inferior temporal ( P<0.001) and the posr-hoc showed it is higher than normal. Compared to CNMOSD patients, the ANMOSD patients showed increased susceptibility in the cuneus (0.009 ± 0.004 vs. 0.005 ± 0.004, P<0.05). There was significant positive correlations between susceptibility and CSF levels of sTREM2 which reflect the active of microglial cells ( r = 0.494, P<0.05). Conclusions:Despite the absence of supratentorial lesions on MRI, increased susceptibility suggests underlying inflammation in the cerebral cortex in both patients with ANMOSD and CNMOSD, and some of them are obviously related to inflammatory markers in CSF. QSM sequence can be used to explore the potential inflammation in NMOSD patients without obvious supratentorial lesions.

Chinese herbal formula (GCNY)-medicated serum alleviates peroxidation induced by H2O2 in human microglial cells.

Traditional Chinese herbal medicine aiming at nourishing yin formed a distinctive school of thought in history to achieve anti-aging and longevity. In the formula Gancao nourishing yin (GCNY) decoction, all of the ingredients show antioxidant properties. However, in real clinical practice, extractions of herbs are rarely applied alone but are prescribed as the integrated formula. To investigate whether GCNY possesses anti-oxidation potential, we applied GCNY to treat rats to acquire medicated serum, which was then added on H2O2 (200 µM)-modeled human microglial cell line HMC-3 in comparison with its control serum. The results revealed that GCNY-medicated serum decreased reactive oxygen species (ROS) levels. Inflammatory cytokines such as pNF-κB p65 (ser536) and IL-6 were also decreased. Nrf2 and its pathway-related molecules, such as HO1, ABCC2, GLCM, ME1, NQO1, and TKT, were activated by H2O2 modeling while declined by treating with GCNY-medicated serum, which indicated attenuated oxidative stress of GCNY. Furthermore, mRNA-seq analysis showed 58 differential expressed genes (DEGs), which were enriched in pathways including antigen processing and presentation, longevity regulation, oxidative phosphorylation, and Parkinson's disease progression. DEGs that were downregulated by H2O2 modeling but upregulated by GCNY treatment include CENPF, MKI67, PRR11, and TOP2A. Those targets were reported to be associated with the cell cycle and cell proliferation and belong to the category of growth factor genes. In conclusion, this study verified anti-oxidation effects of GCNY and indicated its promising application for cognitive degeneration and aging-related disorders.

Anti-oxidant Effect of High Dilutions of Arnica montana, Arsenicum Album, and Lachesis Mutus in Microglial Cells in Vitro.

Microglial cells play important roles in inflammatory responses. The level of oxidative stress is a well-known marker of inflammation. Homeopathic medicines are often used clinically to alleviate inflammation. We evaluated the anti-oxidative effect of high dilutions of Arnica montana (Arnica m.), Arsenicum album (Arsenicum a.), and Lachesis mutus (Lachesis m.) on production of reactive oxygen species (ROS) in inflamed microglial cells in vitro. Microglial cells, on exposure to lipopolysaccharide (LPS), have induced production of ROS compared with resting cells. The dilutions significantly reduced the oxidative stress by decreasing the level of ROS produced. Arnica m. 1C, 3C, 5C, 7C, 9C, and 30C dilutions had a range of ROS reduction between 15 and 42.1%; Arsenicum a. 3C, 5C, 7C, 15C, and 30C dilutions had a range of ROS reduction between 17.6 and 35.3%; and Lachesis m. 3C, 5C, 7C, 9C, 15C, and 30C dilutions had a range of ROS reduction between 25 and 41.7%. To summarize, the dilutions with the greatest effect were Arnica m. 1C (42.1%), Arsenicum a. 30C (35.3%), and Lachesis m. 7C (41.7%). Arnica m., Arsenicum a., and Lachesis m. did not have the same effect on ROS production and were not dose-dependent.