Microglial Apoptosis Research Articles

Baihe Extracts Reduce the Activation and Apoptosis of Microglia in the Hippocampus of Mice with Depression-like Behaviors by Downregulating MYC.

The purpose of our investigation is to identify the potential effects and key molecular targets of Baihe extracts in depression treatment. Network meta-analysis was applied for the synthesis of efficacy outcomes of fluoxetine and three traditional Chinese medicine Baihe prescriptions in depression. Depression-related target genes were screened using "GeneCards" database and "Comparative Toxicogenomics Database (CTD)". The major active components and targets of Baihe were screened using the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. The identified depression-related genes and the target genes of Baihe were intersected, an interaction network was constructed using the "String" database, and key target genes were determined based on their degree value. Functional enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) profiles was performed using the "ClusterProfiler" R package. A mouse model with depression-like behaviors was constructed to verify the putative roles of the in silico identified key genes. Microglia were isolated from the mouse hippocampus, and the effects of Baihe extract-containing serum on microglia activation and apoptosis by targeting the key genes were examined in vitro. The meta-analysis results revealed no obvious differences in depression treatment efficacy between fluoxetine and the three Baihe prescriptions, suggesting Baihe extracts as a safe and effective alternative treatment for depression. Using network pharmacology and bioinformatics analysis, Baihe extracts were found to modulate depression by regulating 15 key genes, with MYC as the key gene. Subsequent animal experiments demonstrated that Baihe extracts reduced depression-related behavior, microglial activation, and inflammatory mediator release in mice by inhibiting MYC. Serum containing Baihe extracts could inhibit the activation of microglia and the release of inflammatory mediators by downregulating MYC. In summary, Baihe extracts were found to diminish MYC expression to reduce microglial activation and inflammatory factor release, thereby exerting antidepressant effects.

LncRNA GAS5/miR-137 Is a Hypoxia-Responsive Axis Involved in Cardiac Arrest and Cardiopulmonary Cerebral Resuscitation.

BackgroundCardiac arrest/cardiopulmonary resuscitation (CA/CPR) represents one of the devastating medical emergencies and is associated with high mortality and neuro-disability. Post-cardiac arrest syndrome (PCAS) is mechanistically ascribed to acute systemic ischemia/reperfusion(I/R) injury. The lncRNA/microRNA/mRNA networks have been found to play crucial roles in the pathogenesis of the hypoxia-responsive diseases. Nonetheless, the precise molecular mechanisms by which lncRNA/miRNA/mRNA axes are involved in the astrocyte–microglia crosstalk in CA/CPR have not been fully elucidated.MethodsWe collected and purified the exosomes from the blood of CA/CPR patients and supernatant of OGD/R-stimulated astrocytes. On the basis of microarray analysis, bioinformatic study, and luciferase activity determination, we speculated that lncRNA GAS5/miR-137 is implicated in the astrocyte–microglia crosstalk under the insult of systemic I/R injury. The regulation of lncRNA GAS5/miR-137 on INPP4B was examined by cellular transfection in OGD/R cell culture and by lateral ventricle injection with miR-137 agomir in CA/CPR mice model. Flow cytometry and immunofluorescence staining were performed to detect the microglial apoptosis, M1/M2 phenotype transformation, and neuroinflammation. Neurological scoring and behavior tests were conducted in CA/CPR group, with miR-137 agomir lateral-ventricle infusion and in their controls.ResultsIn all the micRNAs, miR-137 was among the top 10 micRNAs that experienced greatest changes, in both the blood of CA/CPR patients and supernatant of OGD/R-stimulated astrocytes. Bioinformatic analysis revealed that miR-137 was sponged by lncRNA GAS5, targeting INPP4B, and the result was confirmed by Luciferase activity assay. qRT-PCR and Western blotting showed that lncRNA GAS5 and INPP4B were over-expressed whereas miR-137 was downregulated in the blood of CA/CPR patients, OGD/R-stimulated astrocytes, and brain tissue of CA/CPR mice. Silencing lncRNA GAS5 suppressed INPP4B expression, but over-expression of miR-137 negatively modulated its expression. Western blotting exhibited that PI3K and Akt phosphorylation was increased when lncRNA GAS5 was silenced or miR-137 was over-expressed. However, PI3K and Akt phosphorylation was notably suppressed in the absence of miR-137, almost reversing their phosphorylation in the silencing lncRNA GAS5 group. Then we found that GAS5 siRNA or miR-137 mimic significantly increased cell viability and alleviated apoptosis after OGD/R injury. Furthermore, over-expression of miR-137 attenuated microglial apoptosis and neuroinflammation in CA/CPR mice model, exhibiting significantly better behavioral tests after CA/CPR.ConclusionLncRNA GAS5/miR-137 may be involved in the astrocyte–microglia communication that inhibits PI3K/Akt signaling activation via regulation of INPP4B during CA/CPR.

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.

LncRNA KCNQ1OT1 promotes the apoptosis and inflammatory response of microglia by regulating the miR-589-5p/NPTN axis after spinal cord injury.

Spinal cord injury (SCI) is a devastating traumatic condition accompanied with excessive inflammatory response and apoptosis of microglia. Long noncoding RNAs (lncRNAs) have been confirmed to be key regulators of cell inflammatory response. Nevertheless, the role of lncRNA KCNQ1OT1 in microglia apoptosis or inflammatory response after SCI remains to be explored. Our study focused on exploring the role and mechanism of KCNQ1OT1 in microglia after SCI. RT-qPCR showed that SCI induced the increase of KCNQ1OT1 level in mice spinal cord. Inhibition of KCNQ1OT1 suppressed the inflammatory response and apoptosis of microglia. In addition, KCNQ1OT1 was proved to bind with miR-589-5p, and NPTN was directly targeted by miR-589-5p. Furthermore, KCNQ1OT1 was negatively correlated with miR-589-5p and positively associated with NPTN. Rescue assays indicated that NPTN overexpression reversed the anti-inflammatory and anti-apoptosis effects of KCNQ1OT1 silencing. In summary, these data revealed that KCNQ1OT1 promoted inflammatory response and apoptosis of microglia by regulating the miR-589-5p/NPTN axis after SCI, which may offer a novel promising therapeutic target for SCI.

MiR-191-5p alleviates microglial cell injury by targeting Map3k12 (mitogen-activated protein kinase kinase kinase 12) to inhibit the MAPK (mitogen-activated protein kinase) signaling pathway in Alzheimer’s disease

ABSTRACT Alzheimer’s disease (AD) is a progressive neurodegenerative disease. Multiple reports have elucidated that microRNAs are promising biomarkers for AD diagnosis and treatment. Herein, the effect of miR-191-5p on microglial cell injury and the underlying mechanism were explored. APP/PS1 transgenic mice were utilized to establish mouse model of AD. Amyloid-β protein 1–42 (Aβ1-42)-treated microglia were applied to establish in vitro cell model of AD. MiR-191-5p expression in hippocampus and microglia was measured by reverse transcription quantitative polymerase chain reaction. The viability and apoptosis of microglia were evaluated by Cell Counting Kit-8 assays and flow cytometry analyses, respectively. The binding relationship between miR-191-5p and its downstream target mitogen-activated protein kinase kinase kinase 12 (Map3k12) was determined by luciferase reporter assays. Pathological degeneration of hippocampus was tested using hematoxylin-eosin staining and Nissl staining. Aβ expression in hippocampus was examined via immunohistochemistry. In this study, miR-191-5p was downregulated in Aβ1-42-stimulated microglia and hippocampal tissues of APP/PS1 mice. MiR-191-5p overexpression facilitated cell viability and inhibited apoptosis rate of Aβ1-42-treated microglia. Mechanically, miR-191-5p targeted Map3k12 3ʹ-untranslated region to downregulate Map3k12 expression. MiR-191-5p inhibited Aβ1-42-induced microglial cell injury and inactivated the MAPK signaling by downregulating Map3k12. Overall, miR-191-5p alleviated Aβ1-42-induced microglia cell injury by targeting Map3k12 to inhibit the MAPK signaling pathway in microglia.

MicroRNA miR-27b-3p regulate microglial inflammation response and cell apoptosis by inhibiting A20 (TNF-α-induced protein 3)

ABSTRACT Inflammatory reaction exerts a pivotal role in secondary damage after cerebral hemorrhage and spinal cord injury. miRNAs can both promote and inhibit inflammatory actions among microglial cells. The objective of the present paper was to figure out whether miR-27b-3p produced regulatory effects during processes of microglial inflammation. Lipopolysaccharides (LPS) were used to prepare microglial activation models. Following miR-27b-3p overexpression and interference, the RNA and protein levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β were subjected to real-time fluorescent quantitative PCR (qPCR) and western blot assays, respectively. Cellular apoptosis was subjected to flow cytometry and miR-27b-3p target genes were visualized using a dual luciferase reporter system for verification. The levels of TNF-α, IL-6, and IL-1β mRNA in miR-27b-3p-overexpressed microglial cells were markedly increased compared to the control. Apoptosis of microglial cells was increased markedly in the overexpressed miR-27b-3p group compared to the negative control. Conversely, a different result was presented in the microglial transfected with miR-27b-3p inhibitors. The downregulation of A20, a miR-27b-3p target gene, mediated levels of TNF-α, IL-6, and IL-1β. Furthermore, A20 reduced microglial apoptosis. These data revealed that miR-27b-3p could mediate not only microglia activation but also neuroinflammation via downregulating A20 expression. Thus, miR-27b-3p is regarded as gene therapy in treating cerebral hemorrhage and spinal cord injury.

NeuroD1 induces microglial apoptosis and cannot induce microglia-to-neuron cross-lineage reprogramming

The regenerative capacity of neurons is limited in the central nervous system (CNS), with irreversible neuronal loss upon insult. In contrast, microglia exhibit extraordinary capacity for repopulation. Matsuda etal. (2019) recently reported NeuroD1-induced microglia-to-neuron conversion, aiming to provide an "unlimited" source to regenerate neurons. However, the extent to which NeuroD1 can exert cross-lineage reprogramming of microglia (myeloid lineage) to neurons (neuroectodermal lineage) is unclear. In this study, we unexpectedly found that NeuroD1 cannot convert microglia to neurons in mice. Instead, NeuroD1 expression induces microglial cell death. Moreover, lineage tracing reveals non-specific leakage of similar lentiviruses as previously used for microglia-to-neuron conversion, which confounds the microglia-to-neuron observation. In summary, we demonstrated that NeuroD1 cannot induce microglia-to-neuron cross-lineage reprogramming. We here propose rigid principles for verifying glia-to-neuron conversion. This Matters Arising paper is in response to Matsuda etal. (2019), published in Neuron.

Salidroside inhibits NLRP3 inflammasome activation and apoptosis in microglia induced by cerebral ischemia/reperfusion injury by inhibiting the TLR4/NF-κB signaling pathway.

BackgroundThe NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is an important mediator of neuroinflammatory responses that regulates inflammatory injury following cerebral ischemia and may be a potential target. Salidroside (Sal) has good anti-inflammatory effects; however, it remains unclear whether Sal can regulate NLRP3 inflammasome activation through the Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling pathway after cerebral ischemia to alleviate inflammatory injury.MethodsWe established an oxygen-glucose deprivation and reoxygenation (OGD/R) model of BV2 cells and a middle cerebral artery occlusion/reperfusion (MCAO/R) rat model. Cell Counting Kit-8 (CCK-8), flow cytometry and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay were used to detect the viability and apoptosis of BV2 cells. Enzyme-linked immunosorbent assay (ELISA) was used to detect the level of inflammatory factors. 2,3,5-triphenyltetrazolium chloride (TTC) staining and modified Neurological Severity Score (mNSS) were used to detect cerebral infarction volume and neurological deficit in rats. Western blot, immunohistochemistry and immunofluorescence staining were used to detect the protein expression levels.ResultsOur results showed that Sal increased viability, inhibited lactate dehydrogenase (LDH) release, and reduced apoptosis in OGD/R-induced BV2 cells. Sal reduced the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-8. Following induction by OGD/R, BV2 cells exhibited NLRP3 inflammasome activation and increased protein levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, IL-1β, and IL-18. Protein levels of key TLR4 signaling pathway elements, such as TLR4, myeloid differentiation primary response 88 (MyD88), and phosphorylated nuclear factor kappa B p65 (p-NF-κB p65)/NF-κB p65 were upregulated. Interestingly, it was revealed that Sal could reverse these changes. In addition, TAK242, a specific inhibitor of TLR4, had the same effect as Sal treatment on BV2 cells following induction by OGD/R. In the MCAO/R rat model, Sal was also observed to inhibit NLRP3 inflammasome activation in microglia, reduce cerebral infarction volume, and inhibit apoptosis.ConclusionsIn summary, we found that Sal inhibited NLRP3 inflammasome activation and apoptosis in microglia induced by cerebral ischemia/reperfusion injury by inhibiting the TLR4/NF-κB signaling pathway, thus playing a protective role. Therefore, Sal may be a promising drug for the clinical treatment of ischemic stroke.

Microglial phagocytosis of polystyrene microplastics results in immune alteration and apoptosis in vitro and in vivo

The remarkable increase in plastic usage and widespread microplastic (MP) pollution has emerged as a substantial concern today. Many recent studies have revealed MPs as potentially hazardous substances in mammals. Despite several reports on the impact of small MPs in the brain and behaviors in aquatic animals, it is still unclear how small MPs affect the brain and its underlying cellular physiology in terrestrial animals. In this study, we investigated the accumulation of polystyrene MPs (PS-MPs) in mouse brain after oral treatment using three types of fluorescent PS-MPs of different sizes (0.2,2 and 10 μm). We found that PS-MPs were deposited in microglial cells of the brain. Following differential treatment of PS-MPs in human microglial HMC-3 cells, we identified changes in cellular morphology, immune responses, and microglial apoptosis induced by phagocytosis of 0.2 and 2 μm PS-MPs. By analyzing the PS-MP-treated HMC-3 cell transcriptome, we showed that PS-MPs treatment altered the expression of clusters of immune response genes, immunoglobulins, and several related microRNAs. In addition, we confirmed alterations in microglial differentiation marker expression with the activation of NF-κB, pro-inflammatory cytokines and apoptotic markers in PS-MP-treated human microglial cells and in mouse brain. Our findings suggest a potential risk of small PS-MPs in microglial immune activation, which leads to microglial apoptosis in murine and human brains.

Mitochondrial Dynamics in SARS-COV2 Spike Protein Treated Human Microglia: Implications for Neuro-COVID.

Emerging clinical data from the current COVID-19 pandemic suggests that ~ 40% of COVID-19 patients develop neurological symptoms attributed to viral encephalitis while in COVID long haulers chronic neuro-inflammation and neuronal damage result in a syndrome described as Neuro-COVID. We hypothesize that SAR-COV2 induces mitochondrial dysfunction and activation of the mitochondrial-dependent intrinsic apoptotic pathway, resulting in microglial and neuronal apoptosis. The goal of our study was to determine the effect of SARS-COV2 on mitochondrial biogenesis and to monitor cell apoptosis in human microglia non-invasively in real time using Raman spectroscopy, providing a unique spatio-temporal information on mitochondrial function in live cells. We treated human microglia with SARS-COV2 spike protein and examined the levels of cytokines and reactive oxygen species (ROS) production, determined the effect of SARS-COV2 on mitochondrial biogenesis and examined the changes in molecular composition of phospholipids. Our results show that SARS- COV2 spike protein increases the levels of pro-inflammatory cytokines and ROS production, increases apoptosis and increases the oxygen consumption rate (OCR) in microglial cells. Increases in OCR are indicative of increased ROS production and oxidative stress suggesting that SARS-COV2 induced cell death. Raman spectroscopy yielded significant differences in phospholipids such as Phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylcholine (PC), which account for ~ 80% of mitochondrial membrane lipids between SARS-COV2 treated and untreated microglial cells. These data provide important mechanistic insights into SARS-COV2 induced mitochondrial dysfunction which underlies neuropathology associated with Neuro-COVID.Graphical

Small extracellular vesicles encapsulating CCL2 from activated astrocytes induce microglial activation and neuronal apoptosis after traumatic spinal cord injury

BackgroundSpinal cord injury (SCI) is a severe traumatic disease which causes high disability and mortality rates. The molecular pathological features after spinal cord injury mainly involve the inflammatory response, microglial and neuronal apoptosis, abnormal proliferation of astrocytes, and the formation of glial scars. However, the microenvironmental changes after spinal cord injury are complex, and the interactions between glial cells and nerve cells remain unclear. Small extracellular vesicles (sEVs) may play a key role in cell communication by transporting RNA, proteins, and bioactive lipids between cells. Few studies have examined the intercellular communication of astrocytes through sEVs after SCI. The inflammatory signal released from astrocytes is known to initiate microglial activation, but its effects on neurons after SCI remain to be further clarified.MethodsElectron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting were applied to characterize sEVs. We examined microglial activation and neuronal apoptosis mediated by astrocyte activation in an experimental model of acute spinal cord injury and in cell culture in vitro.ResultsOur results indicated that astrocytes activated after spinal cord injury release CCL2, act on microglia and neuronal cells through the sEV pathway, and promote neuronal apoptosis and microglial activation after binding the CCR2. Subsequently, the activated microglia release IL-1β, which acts on neuronal cells, thereby further aggravating their apoptosis.ConclusionThis study elucidates that astrocytes interact with microglia and neurons through the sEV pathway after SCI, enriching the mechanism of CCL2 in neuroinflammation and spinal neurodegeneration, and providing a new theoretical basis of CCL2 as a therapeutic target for SCI.

The Synergistic Effect of TRPV1 on Oxidative Stress-Induced Autophagy and Apoptosis in Microglia.

Stroke mostly including ischemic stroke is the second leading mortality and disability worldwide. Oxidative stress injury occurred during ischemic stroke treatment generally. A high amount of reactive oxygen species (ROS) is involved in oxidative stress induction. Transient receptor potential vanilloid 1 (TRPV1) has been shown to regulate oxidative stress and apoptosis in microglia; however, the detailed mechanisms remain unclear. We aimed to explore whether autophagy-regulated oxidative stress and apoptosis are associated with TRPV1. The model of oxygen and glucose deprivation (OGD/R) in microglia was established. The siRNA of Atg5 and inhibitors and agonists of both autophagy and TRPV1 were involved in our study. Autophagy-related markers Atg5, LC3II/LC3I, and Beclin-1 were measured, and the autophagosome was observed under a transmission electron microscope (TEM). Caspase 3 was detected using ELISA. ROS and JC-1 were detected using flow cytometry. Apoptosis was observed by TUNEL. The results indicated that oxidative stress-induced injury and apoptosis may be impeded by the increasing autophagy, and TRPV1 inhibition could suppress the OGD/R-induced autophagy of microglia. However, the effect of TRPV1's inhibitor on oxidative stress and apoptosis was not obvious when the autophagy was blocked. These findings suggested that TRPV1 may exhibit antioxidative and antiapoptosis effect on OGD/R-induced microglia. However, the experimental results do not fully demonstrate that the TRPV1-mediated antioxidative and antiapoptosis effect is through the affecting autophagy entirely.

MSCs\u2011derived exosomes attenuate ischemia-reperfusion brain injury and inhibit microglia apoptosis might via exosomal miR-26a-5p mediated suppression of CDK6

BackgroundThis study aimed to explore the role of mesenchymal stromal cells (MSCs)-derived exosomes (MSCs-Exo) in the cerebral ischemia–reperfusion (I/R) injury.MethodsExosomes were isolated from MSCs of adult C57BL/6J mice by the gradient centrifugation method. The expression of miR-26a-5p and CDK6 in MSCs-Exo and mice brain tissues were evaluated by qRT-PCR and western blot. miR-26a-5p mimics and miR-NC were transfected into MSCs, and exosomes were isolated from the MSCs stably expressing miR-26a-5p. Then MSCs-Exo-miR-26a-5p mimics or MSCs-Exo-miR-NC was injected into mice through the tail vein, or added into medium to stimulate BV-2 cells. Cell viability was evaluated by CCK-8 assay. Cell apoptosis was detected by flow cytometry. The apoptosis in brain tissues was evaluated by TUNEL staining assay. Bioinformatics analysis and luciferase reporter assay were performed to determine the binding relationship between miR-26a-5p and CDK6.ResultsmiR-26a-5p was downregulated and CDK6 was upregulated in MSCs-Exo of MCAO-mice and OGD-induced MSCs. MSCs-Exo-miR-26a-5p mimics significantly reduced cell apoptosis of OGD-injured BV-2 cells. MSCs-Exo-miR-26a-5p mimics significantly reduced the infarct volume of MCAO-induced mice. Luciferase reporter assay revealed that CDK-6 was a target of miR-26a-5p. In addition, MSCs-Exo-miR-26a-5p mimics significantly decreased the expression of CDK6 in both OGD-induced BV-2 cells and the brain tissues of MCAO-treated mice.ConclusionOur results indicated that MSCs‑Exo attenuated I/R injury in mice by inhibiting microglia apoptosis might via exosomal miR-26a-5p mediated suppression of CDK6. Our study shed light on the application of MSC-Exo as a potential therapeutic tool for cerebral I/R injury.

The Regulatory NOD-Like Receptor NLRC5 Promotes Ganglion Cell Death in Ischemic Retinopathy by Inducing Microglial Pyroptosis.

Retinal ischemia is a common pathological event that can result in retinal ganglion cell (RGC) death and irreversible vision loss. The pathogenic mechanisms linking retinal ischemia to RGC loss and visual deficits are uncertain, which has greatly hampered the development of effective treatments. It is increasingly recognized that pyroptosis of microglia contributes to the indirect inflammatory death of RGCs. In this study, we report a regulatory NOD-like receptor, NOD-, LRR- and CARD-containing 5 (NLRC5), as a key regulator on microglial pyroptosis and the retinal ischemia process. Through an in-depth analysis of our recently published transcriptome data, we found that NLRC5 was significantly up-regulated in retina during ischemia–reperfusion injury, which were further confirmed by subsequent detection of mRNA and protein level. We further found that NLRC5 was upregulated in retinal microglia during ischemia, while NLRC5 knockdown significantly ameliorated retinal ischemic damage and RGC death. Mechanistically, we revealed that knockdown of NLRC5 markedly suppressed gasdermin D (GSDMD) cleavage and activation of interleukin-1β (IL-1β) and caspase-3, indicating that NLRC5 promotes both microglial pyroptosis and apoptosis. Notably, we found that NLRC5 directly bound to NLRP3 and NLRC4 in inflammasomes to cooperatively drive microglial pyroptosis and apoptosis mediating retinal ischemic damage. Overall, these findings reveal a previously unidentified key contribution of NLRC5 signaling to microglial pyroptosis under ischemia or hypoxia conditions. This NLRC5-dependent pathway may be a novel therapeutic target for treatment of ischemic retinopathy.

Upregulation of lncRNA147410.3 in the Brain of Mice With Chronic Toxoplasma Infection Promoted Microglia Apoptosis by Regulating Hoxb3.

Toxoplasma gondii is neurotropic and affects the function of nerve cells, while the mechanism is unclear. LncRNAs are abundantly enriched in the brain and participated in the delicate regulation of the central nervous system (CNS) development. However, whether these lncRNAs are involved in the regulation of microglia activation during the process of T. gondii infection is largely unknown. In this study, the upregulation of a novel lncRNA147410.3 (ENSMUST00000147410.3) was identified as a key factor to influence this process. The target gene of lncRNA147410.3 was predicted and identified as Hoxb3. The localization of lncRNA147410.3 in the brain and cells was proved in the nucleus of neuroglia through FISH assay. Furthermore, the function of lncRNA147410.3 on neuronal cell was confirmed that lncRNA147410.3 could affect proliferation, differentiation, and apoptosis of mouse microglia by positively regulating Hoxb3. Thus, our study explored the modulatory action of lncRNA147410.3 in T. gondii infected mouse brain, providing a scientific basis for using lncRNA147410.3 as a therapeutic target to treat neurological disorder induced by T. gondii.

Tetrahydrocurcumin ameliorates Alzheimer's pathological phenotypes by inhibition of microglial cell cycle arrest and apoptosis via Ras/ERK signaling

1,7-bis(4-hydroxy-3-methoxyphenyl)heptane-3,5-dione (tetrahydrocurcumin, THC) is a major bioactive metabolite of curcumin, demonstrating the potential anti-inflammatory, antioxidant and neuroprotective properties, etc. In this study, it was found that Aβ induced decreased cell viability, cell cycle arrest and apoptosis in BV-2 cells, which were ameliorated by THC. In vivo, THC administration rescued learning and memory, and reduced Aβ burden in the hippocampus of APP/PS1 mice. By proteomic analysis of the hippocampus of mice, 157 differentially expressed proteins were identified in APP/PS1 mice treated with THC (comparing with APP/PS1 mice), which also suggested that the effects of THC on the cell cycle and apoptosis were mostly related to the “Ras signaling pathway”, etc. In APP/PS1 mice, the down-regulation of Gab2 and K-Ras, and the up-regulation of caspase-3, TGF-β1 and TNF-ɑ were observed; THC attenuated the abnormal expression of Gab2, K-Ras, caspase-3 and TNF-ɑ, and up-regulated TGF-β1 and Bag1 expression. In BV-2 cells, Aβ induced the down-regulation of Gab2, K-Ras and TGF-β1, and the overexpression of caspase-3, PARP1, cleaved-PARP1 and TNF-ɑ, which were restored by THC. Moreover, THC up-regulated Bag1 expression in Aβ-treated BV-2 cells. The decreased transcriptional expression of Ccnd2 and Cdkn1a were also observed in Aβ-treated BV-2 cells, and THC alleviated the down-regulation of Ccnd2. For the first time, we identified that the action of THC in preventing AD was associated with inhibition of cell cycle arrest and apoptosis of microglia via the Ras/ERK signaling pathway, shedding new light on the role of THC in alleviating the progression of AD.

Doxycycline Induces Apoptosis of Brucella Suis S2 Strain-Infected HMC3 Microglial Cells by Activating Calreticulin-Dependent JNK/p53 Signaling Pathway.

Neurobrucellosis is a chronic complication of human brucellosis that is caused by the presence of Brucella spp in the central nervous system (CNS) and the inflammation play a key role on the pathogenesis. Doxycycline (Dox) is a widely used antibiotic that induces apoptosis of bacteria-infected cells. However, the mechanisms of Brucella inhibition of microglial apoptosis and Dox induction of apoptosis are still poorly understood. In this study, we found that Brucella suis S2 strain (B. suis S2) increased calreticulin (CALR) protein levels and inhbited HMC3 cell apoptosis. Hence, we constructed two HMC3 cell line variants, one with stable overexpression (HMC3-CALR) and one with low expression of CALR (HMC3-sh-CALR). CALR was found to decrease levels of p-JNK and p-p53 proteins, as well as suppress apoptosis in HMC3 cells. These findings suggest that CALR suppresses apoptosis by inhibiting the JNK/p53 signaling pathway. Next, we treated HMC3, HMC3-CALR and HMC3-sh-CALR cell lines with B. suis S2 or Dox. Our results demonstrate that B. suis S2 restrains the JNK/p53 signaling pathway to inhibit HMC3 cell apoptosis via increasing CALR protein expression, while Dox plays the opposite role. Finally, we treated B. suis S2-infected HMC3 cells with Dox. Our results confirm that Dox induces JNK/p53-dependent apoptosis in B. suis S2-infected HMC3 cells through inhibition of CALR protein expression. Taken together, these results reveal that CALR and the JNK/p53 signaling pathway may serve as novel therapeutic targets for treatment of neurobrucellosis.

Baicalin and Geniposide Inhibit Polarization and Inflammatory Injury of OGD/R-Treated Microglia by Suppressing the 5-LOX/LTB4 Pathway

Cerebral ischemia causes severe neurological disorders and neuronal dysfunction. Baicalin (BC), geniposide (GP), and their combination (BC/GP) have been shown to inhibit post-ischemic inflammatory injury by inhibiting the 5-LOX/CysLTs pathway. The aims of this study were to observe the inhibitory effects of BC/GP on the activation of microglial cells induced by oxygen glucose deprivation and reoxygenation (OGD/R) and to investigate whether the 5-LOX/LTB4 pathway was involved in these effects. Molecular docking showed that BC and GP exhibited considerable binding activity with LTB4 synthase LTA4H. BV-2 microglia were transfected with a 5-LOX overexpression lentiviral vector, and then OGD/R was performed. The effects of different concentrations of BC, GP, and BC/GP (6.25 μM, 12.5 μM, and 25 μM) on cell viability and apoptosis of microglia were evaluated by MTT and flow cytometry. The expression of TNF-α, IL-1β, NF-κB, and pNF-κB also was measured by ELISA, Western blots and immunofluorescence. Western blots and qRT-PCR analysis were used to determine the levels of CD11b, CD206, and 5-LOX pathway proteins. Results showed that BC, GP, and BC/GP reduced the apoptosis caused by OGD/R in a dose-dependent manner, and cell viability was significantly increased at a concentration of 12.5 μM. OGD/R significantly increased the release of TNF-α, IL-1β, NF-κB, pNF-κB, and CD11b. These effects were suppressed by BC, GP, and BC/GP, and the OGD/R-induced transfer of NF-κB p65 from the ctytoplasm to the nucleus was inhibited in microglia. Interestingly, the LTB4 inhibitor, U75302, exhibited the same effect. Also, BC, GP, and BC/GP significantly reduced the expression of 5-LOX pathway proteins. These results demonstrated that BC/GP inhibited OGD/R-induced polarization in BV2 microglia by regulating the 5-LOX/LTB4 signaling pathways and attenuating the inflammatory response. Our results supported the theoretical basis for additional in-depth study of the function of BC/GP and the value of determining its unique target, which might provide a new therapeutic strategy for ischemic cerebrovascular disease.

Flavonoids Alleviate Peripheral Neuropathy Induced by Anticancer Drugs.

Simple SummaryChemotherapy-induced peripheral neuropathy (CIPN) is a debilitating condition that severely reduces the quality of life of a considerable proportion of cancer patients. There is no cure for CIPN to date. Here, we explore the potential of flavonoids as pharmacological agents in combating CIPN. Flavonoids alleviate CIPN by reducing oxidative stress, inflammation, and neuronal damage, among other mechanisms. Future research should evaluate the efficacy and side effects of flavonoids in human models of CIPN.Purpose: This study aimed to assess the potential of flavonoids in combating CIPN. Methods: PubMed and Google Scholar were used, and studies that investigated flavonoids in models of CIPN and models of neuropathic pain similar to CIPN were included. Only studies investigating peripheral mechanisms of CIPN were used. Results: Flavonoids inhibit several essential mechanisms of CIPN, such as proinflammatory cytokine release, astrocyte and microglial activation, oxidative stress, neuronal damage and apoptosis, mitochondrial damage, ectopic discharge, and ion channel activation. They decreased the severity of certain CIPN symptoms, such as thermal hyperalgesia and mechanical, tactile, and cold allodynia. Conclusions: Flavonoids hold immense promise in treating CIPN; thus, future research should investigate their effects in humans. Specifically, precise pharmacological mechanisms and side effects need to be elucidated in human models before clinical benefits can be achieved.

Melatonin prevents diabetes-associated cognitive dysfunction from microglia-mediated neuroinflammation by activating autophagy via TLR4/Akt/mTOR pathway.

Cognitive dysfunction often occurs in diabetes mellitus patients. This study aimed to investigate the efficacy of melatonin (MLT) in improving diabetes-associated cognitive decline and the underlying mechanism involved. Type 2 diabetic mice and palmitic acid (PA)-stimulated BV-2 cells were treated by MLT, and the potential mechanisms among MLT, cognition, and autophagy were explored. The results showed that type 2 diabetic mice showed obvious learning and memory impairments in the Morris water maze test compared with normal controls, which could be ameliorated by MLT treatment. Meanwhile, MLT administration significantly improved neuroinflammation and regulated microglial apoptosis. Furthermore, autophagy inhibitor 3-methyladenine (3-MA) increased the microglial inflammation and apoptosis, indicating that the treatment effect of MLT was mediated by autophagy. Lastly, MLT treatment significantly decreased the levels of toll-like receptors 4 (TLR4), phosphorylated-protein kinase B (Akt), and phosphorylated-mechanistic target of rapamycin (mTOR), indicating that blocking TLR4/Akt/mTOR pathway might be an underlying basis for the anti-inflammatory and anti-apoptosis effects of MLT. Collectively, our study suggested that MLT could improve learning and memory in type 2 diabetic mice by activating autophagy via the TLR4/Akt/mTOR pathway, thereby inhibiting neuroinflammation and microglial apoptosis.