PD-1/PD-L1 axis is involved in the interaction between microglial polarization and glioma.

The tumor microenvironment plays a vital role in glioblastoma growth and invasion. PD-1 and PD-L1 modulate the immunity in the brain tumor microenvironment. However, the underlying mechanisms remain unclear. In the present study, in vivo and in vitro experiments were conducted to reveal the effects of PD-1/PD-L1 on the crosstalk between microglia and glioma. Results showed that glioma cells secreted PD-L1 to the peritumoral areas, particularly microglia containing highly expressed PD-1. In the early stages of glioma, microglia mainly polarized into the pro-inflammatory subtype (M1). Subsequently, the secreted PD-L1 accumulated and bound to PD-1 on microglia, facilitating their polarization toward the microglial anti-inflammatory (M2) subtype primarily via the STAT3 signaling pathway. The role of PD-1/PD-L1 in M2 polarization of microglia was partially due to PD-1/PD-L1 depletion or application of BMS-1166, a novel inhibitor of PD-1/PD-L1. Consistently, co-culturing with microglia promoted glioma cell growth and invasion, and blocking PD-1/PD-L1 significantly suppressed these processes. Our findings reveal that the PD-1/PD-L1 axis engages in the microglial M2 polarization in the glioma microenvironment and promotes tumor growth and invasion.

Radiomics approach to distinguish between benign and malignant soft tissue tumors on magnetic resonance imaging.

Objective: To build a radiomics signature based on MRI images and evaluate its capability for preoperatively identifying the benign and malignant Soft tissue neoplasms (STTs). Materials and methods: 193 patients (99 malignant STTs and 94 benign STTs) were at random segmented into a training cohort (69 malignant STTs and 65 benign STTs) and a validation cohort (30 malignant STTs and 29 benign STTs) with a portion of 7:3. Radiomics features were extracted from T2 with fat saturation and T1 with fat saturation and gadolinium contrast images. Radiomics signature was developed by the least absolute shrinkage and selection operator (LASSO) logistic regression model. The receiver that operated characteristics curve (ROC) analysis was used to assess radiomics signature's prediction performance. Inner validation was performed on an autonomous cohort that contained 40 patients. Results: A radiomics was developed by a total of 16 radiomics features (5 original shape features and 11 were wavelet features) achieved favorable predictive efficacy. Malignant STTs showed higher radiomics score than benign STTs in both training cohort and validation cohort. A good prediction performance was shown by the radiomics signature in both training cohorts and validation cohorts. The training cohorts and validation cohorts had an area under curves (AUCs) of 0.885 and 0.841, respectively. Conclusions: A radiomics signature based on MRI images can be a trustworthy imaging biomarker for identification of the benign and malignant STTs, which could help guide treatment strategies.

Strategy of functional nucleic acids-mediated isothermal amplification for detection of foodborne microbial contaminants: A review.

Foodborne microbial contamination (FMC) is the leading cause of food poisoning and foodborne illness. The foodborne microbial detection methods based on isothermal amplification have high sensitivity and short detection time, and functional nucleic acids (FNAs) could extend the detectable object of isothermal amplification to mycotoxins. Therefore, the strategy of FNAs-mediated isothermal amplification has been emergingly applied in biosensors for foodborne microbial contaminants detection, making biosensors more sensitive with lower cost and less dependent on nanomaterials for signal output. Here, the mechanism of six isothermal amplification technologies and their application in detecting FMC is firstly introduced. Then the strategy of FNAs-mediated isothermal amplification is systematically discussed from perspectives of FNAs' versatility including recognition elements (Aptamer, DNAzyme), programming tools (DNA tweezer, DNA walker and CRISPR-Cas) and signal units (G-quadruplex, FNAs-based nanomaterials). Finally, challenges and prospects are presented in terms of addressing the issue of nonspecific amplification reaction, developing better FNAs-based sensing elements and eliminating food matrix effects.

Lipidomic analysis identifies long-chain acylcarnitine as a target for ischemic stroke.

INTRODUCTION: Lipid metabolism dysfunction is widely involved in the pathological process of acute ischemic stroke (AIS). The coordination of lipid metabolism between neurons and astrocytes is of great significance. However, the full scope of lipid dynamic changes and the function of key lipids during AIS remain unknown. Hence, identifying lipid alterations and characterizing their key roles in AIS is of great importance. METHODS: Untargeted and targeted lipidomic analyses were applied to profile lipid changes in the ischemic penumbra and peripheral blood of transient middle cerebral artery occlusion (tMCAO) mice as well as the peripheral blood of AIS patients. Infarct volume and neurological deficits were assessed after tMCAO. The cell viability and dendritic complexity of primary neurons were evaluated by CCK8 assay and Sholl analysis. Seahorse, MitoTracker Green, tetramethyl rhodamine methyl ester (TMRM), 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and MitoSOX were used as markers of mitochondrial health. Fluorescent and isotopic free fatty acid (FFA) pulse-chase assays were used to track FFA flux in astrocytes. RESULTS: Long-chain acylcarnitines (LCACs) were the lipids with the most dramatic changes in the ischemic penumbra and peripheral blood of tMCAO mice. LCACs were significantly elevated on admission in AIS patients and associated with poor outcomes in AIS patients. Increasing LCACs through a bolus administration of palmitoylcarnitine amplified stroke injury, while decreasing LCACs by overexpressing carnitine palmitoyltransferase 2 (CPT2) ameliorated stroke injury. Palmitoylcarnitine aggravated astrocytic mitochondrial damage after OGD/R, while CPT2 overexpression in astrocytes ameliorated cocultured neuron viability. Further study revealed that astrocytes stimulated by OGD/R liberated FFAs from lipid droplets into mitochondria to form LCACs, resulting in mitochondrial damage and lowered astrocytic metabolic support and thereby aggravated neuronal damage. CONCLUSION: LCACs could accumulate and damage neurons by inducing astrocytic mitochondrial dysfunction in AIS. LCACs play a crucial role in the pathology of AIS and are novel promising diagnostic and prognostic biomarkers for AIS.

Resolvin D1 reprograms energy metabolism to promote microglia to phagocytize neutrophils after ischemic stroke.

Neutrophil aggregation and clearance are important factors affecting neuroinflammatory injury during acute ischemic stroke. Emerging evidence suggests that energy metabolism is essential for microglial functions, especially microglial phagocytosis, which determines the degree of brain injury. Here, we demonstrate that Resolvin D1 (RvD1), a lipid mediator derived from docosahexaenic acid (DHA), promotes the phagocytosis of neutrophils by microglia, thereby reducing neutrophil accumulation in the brain and alleviating neuroinflammation in the ischemic brain. Further studies reveal that RvD1 reprograms energy metabolism from glycolysis to oxidative phosphorylation (OXPHOS), providing sufficient energy for microglial phagocytosis. Moreover, RvD1 enhances microglial glutamine uptake and stimulates glutaminolysis to support OXPHOS to boost ATP production depending on adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) activation. Overall, our results reveal that RvD1 reprograms energy metabolism to promote the microglial phagocytosis of neutrophils after ischemic stroke. These findings may guide perspectives for stroke therapy from modulating microglial immunometabolism.

[Development of a widely-targeted metabolomics method based on gas chromatography-mass spectrometry].

Gas chromatography-mass spectrometry (GC-MS) detectors are widely used detection instruments owing to their distinct advantages over other analytical techniques, including lower sample consumption, higher sensitivity, faster analysis speed, and simultaneous separation and analysis. Metabolomics is an important component of system physiology that concerns systematic studies of the metabolite spectrum in one or more biological systems, such as cells, tissues, organs, body fluids, and organisms. Unfortunately, conventional GC-MS detectors also feature low scan rates, high ion loss rates, and a narrow concentration detection range, which limit their applications in the field of metabolomics. Therefore, establishing a GC-MS-based metabolomic analysis method with wide coverage is of great importance. In this research, a widely-targeted metabolomics method based on GC-MS is proposed. This method combines the universality of untargeted metabolomics with the accuracy of targeted metabolomics to realize the qualitative and semi-quantitative detection of numerous metabolites. It does not require a self-built database and exhibits high sensitivity, good repeatability, and strong support for a wide range of metabolic substances. The proposed method was used to establish the relationship between the retention time of straight-chain fatty acid methyl esters (FAMEs) and their retention index (RI) in the FiehnLib database based on the metabolite information stored in this database. We obtained a linear relationship that could be described by the equation y=40878x-47530, r2=0.9999. We then calculated the retention times of metabolites in the FiehnLib database under the experimental conditions based on their RI. In this way, the effects of significant variations in peak retention times owing to differences in the chromatographic column, temperature, carrier gas flow rate, and so on can be avoided. The retention time of a substance fluctuates within a certain threshold because of variations in instrument performance, matrix interference, and other factors. As such, the retention time threshold of the substance must be determined. In this paper, the retention time threshold was set to 0.15 min to avoid instrument fluctuations. The optimal scan interval was optimized to 0.20 s (possible values=0.10, 0.15, 0.20, 0.25, and 0.30 s) because longer sampling periods can lead to spectral data loss and reductions in the resolution of adjacent chromatographic peaks, whereas shorter sampling periods can result in deterioration of the signal-to-noise ratio of the collected signals. The metabolite quantification ions were optimized to avoid the interference of quantification ion peak accumulation in the case of similar peak times, and a selected ion monitoring (SIM) method table was constructed for 611 metabolites, covering 65% of the metabolic pathways in the KEGG (Kyoto Encyclopedia of Genes and Genomes). The developed method covered 39 pathways, including glycolysis, the tricarboxylic acid cycle, purine metabolism, pyrimidine metabolism, amino acid metabolism, and biosynthesis. Compared with the full-scan untargeted GC-MS method, the widely-targeted GC-MS method demonstrated a 20%-30% increase in the number of metabolites detected, as well as a 15%-20% increase in signal-to-noise ratio. The results of stability tests showed that 84% of the intraday relative standard deviations (RSDs) of metabolite retention times were less than 2% and 91% of that were less than 3%; moreover, 54% of the interday RSDs of metabolite retention times were less than 2% and 76% of that were less than 3%. The detection and analysis results of common biological samples confirmed that the proposed method greatly improved the quantity and signal-to-noise ratio of the detected metabolites and is applicable to substances that are thermally stable, volatile, or volatile after derivation and have relative molecular masses lower than 600. Thus, the widely-targeted GC-MS method can expand the application scope of GC-MS in metabolomics.

Iptakalim improves cerebral microcirculation in mice after ischemic stroke by inhibiting pericyte contraction.

Pericytes are present tight around the intervals of capillaries, play an essential role in stabilizing the blood-brain barrier, regulating blood flow and immunomodulation, and persistent contraction of pericytes eventually leads to impaired blood flow and poor clinical outcomes in ischemic stroke. We previously show that iptakalim, an ATP-sensitive potassium (K-ATP) channel opener, exerts protective effects in neurons, and glia against ischemia-induced injury. In this study we investigated the impacts of iptakalim on pericytes contraction in stroke. Mice were subjected to cerebral artery occlusion (MCAO), then administered iptakalim (10 mg/kg, ip). We showed that iptakalim administration significantly promoted recovery of cerebral blood flow after cerebral ischemia and reperfusion. Furthermore, we found that iptakalim significantly inhibited pericytes contraction, decreased the number of obstructed capillaries, and improved cerebral microcirculation. Using a collagen gel contraction assay, we demonstrated that cultured pericytes subjected to oxygen-glucose deprivation (OGD) consistently contracted from 3 h till 24 h during reoxygenation, whereas iptakalim treatment (10 µM) notably restrained pericyte contraction from 6 h during reoxygenation. We further showed that iptakalim treatment promoted K-ATP channel opening via suppressing SUR2/EPAC1 complex formation. Consequently, it reduced calcium influx and ET-1 release. Taken together, our results demonstrate that iptakalim, targeted K-ATP channels, can improve microvascular disturbance by inhibiting pericyte contraction after ischemic stroke. Our work reveals that iptakalim might be developed as a promising pericyte regulator for treatment of stroke.

Oridonin prevents oxidative stress-induced endothelial injury via promoting Nrf-2 pathway in ischaemic stroke.

Oridonin, a natural diterpenoid compound extracted from a Chinese herb, has been proved to exert anti-oxidative stress effects in various disease models. The aim of the present study was to investigate the protective effects of oridonin on oxidative stress-induced endothelial injury in ischaemic stroke. We found oridonin repaired blood-brain barrier (BBB) integrity presented with upregulation of tight junction proteins (TJ proteins) expression, inhibited the infiltration of periphery inflammatory cells and neuroinflammation and thereby reduced infarct volume in ischaemic stroke mice. Furthermore, our results showed that oridonin could protect against oxidative stress-induced endothelial injury via promoting nuclear translocation of nuclear factor-erythroid 2 related factor 2 (Nrf-2). The specific mechanism could be the activation of AKT(Ser473)/GSK3ß(Ser9)/Fyn signalling pathway. Our findings revealed the therapeutic effect and mechanism of oridonin in ischaemic stroke, which provided fundamental evidence for developing the extracted compound of Chinese herbal medicine into an innovative drug for ischaemic stroke treatment.

Iptakalim alleviates synaptic damages via targeting mitochondrial ATP-sensitive potassium channel in depression.

Synaptic plasticity damages play a crucial role in the onset and development of depression, especially in the hippocampus, which is more susceptible to stress and the most frequently studied brain region in depression. And, mitochondria have a major function in executing the complex processes of neurotransmission and plasticity. We have previously demonstrated that Iptakalim (Ipt), a new ATP-sensitive potassium (K-ATP) channel opener, could improve the depressive-like behavior in mice. But the underlying mechanisms are not well understood. The present study demonstrated that Ipt reversed depressive-like phenotype in vivo (chronic mild stress-induced mice model of depression) and in vitro (corticosterone-induced cellular model). Further study showed that Ipt could upregulate the synaptic-related proteins postsynaptic density 95 (PSD 95) and synaptophysin (SYN), and alleviated the synaptic structure damage. Moreover, Ipt could reverse the abnormal mitochondrial fission and fusion, as well as the reduced mitochondrial ATP production and collapse of mitochondrial membrane potential in depressive models. Knocking down the mitochondrial ATP-sensitive potassium (Mito-KATP) channel subunit MitoK partly blocked the above effects of Ipt. Therefore, our results reveal that Ipt can alleviate the abnormal mitochondrial dynamics and function depending on MitoK, contributing to improve synaptic plasticity and exert antidepressive effects. These findings provide a candidate compound and a novel target for antidepressive therapy.

Neuronal extracellular vesicle derived miR-98 prevents salvageable neurons from microglial phagocytosis in acute ischemic stroke.

Extracellular vesicles (EVs), as a novel intercellular communication carrier transferring cargo microRNAs (miRNAs), could play important roles in the brain remodeling process after ischemic stroke. However, the detailed mechanisms involved in EVs derived miRNAs-mediated cellular interactions in the brain remain unclear. Several studies indicated that microRNA-98 (miR-98) might participate in the pathogenesis of ischemic stroke. Here, we showed that expression of miR-98 in penumbra field kept up on the first day but dropped sharply on the 3rd day after ischemic stroke in rats, indicating that miR-98 could function as an endogenous protective factor post-ischemia. Overexpression of miR-98 targeted inhibiting platelet activating factor receptor-mediated microglial phagocytosis to attenuate neuronal death. Furthermore, we showed that neurons transferred miR-98 to microglia via EVs secretion after ischemic stroke, to prevent the stress-but-viable neurons from microglial phagocytosis. Therefore, we reveal that EVs derived miR-98 act as an intercellular signal mediating neurons and microglia communication during the brain remodeling after ischemic stroke. The present work provides a novel insight into the roles of EVs in the stroke pathogenesis and a new EVs-miRNAs-based therapeutic strategy for stroke.

FTY720 Exerts Anti-Glioma Effects by Regulating the Glioma Microenvironment Through Increased CXCR4 Internalization by Glioma-Associated Microglia.

Background: Glioblastoma (GBM) is one of the most malignant and aggressive primary brain tumors. The incurability of glioblastoma is heavily influenced by the glioma microenvironment. FTY720, a potent immunosuppressant, has been reported to exert anti-tumor effects in glioblastoma. However, the impact of FTY720 on the glioma microenvironment remains unclear. Methods: We examined the effects of FTY720 on the distribution and polarization of glioma-associated microglia and macrophages (GAMs) in glioma-bearing rats using immunofluorescence staining. qRT-PCR and Western blotting were used to detect the expressions of CXCR4 and MAPK pathway-related signal molecules on microglia in the coculture system. The levels of inflammatory factors were tested via ELISA. Wound healing assay and Matrigel invasion assay were used to determine the migration and invasion of C6 glioma cells. Results: We discovered that FTY720 could inhibit the growth, migration, and invasion of glioma by targeting GAMs to impede their effect on glioma cells. Simultaneously, FTY720 could block the chemoattraction of GAMs by inhibiting MAPK-mediated secretion of IL-6 through increased internalization of CXCR4. Moreover, microglia and macrophages are polarized from pro-glioma to an anti-tumor phenotype. Conclusion: These results provide novel insights into the inhibitory effects of FTY720 on glioma by targeting GAMs-glioma interaction in the tumor microenvironment.

LncRNA RMST-mediated miR-107 transcription promotes OGD-induced neuronal apoptosis via interacting with hnRNPK.

The long noncoding RNA (lncRNA) rhabdomyosarcoma 2-associated transcript (RMST) silencing has been demonstrated to protect against ischemic brain injury in vivo and neuron injury in vitro. However, its underlying mechanisms in the progression of ischemic stroke have not been well explored. The expression of RMST in oxygen-glucose deprivation (OGD)-treated HT-22 hippocampal neuron cell line was examined using quantitative Real-Time PCR (qRT-PCR). CCK-8 cell viability and apoptotic cell detection using Annexin V-FITC and PI staining coupled with flow cytometry were performed to determine the pro-apoptotic role of RMST in HT-22 hippocampal neuron cell line. Furthermore, RNA pull-down, RNA immunoprecipitation (RIP), coimmunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP) and dual-Luciferase reporter assays were performed to determine the mechanism of RMST in OGD-induced HT-22 cell apoptosis. In the results, RMST was highly expressed in OGD-treated HT-22 cells. Altered RMST expression led to marked changes in HT-22 cell proliferation and apoptosis. Mechanistically, RMST indirectly activated p53/miR-107 signaling pathway via interacting with heterogeneous nuclear ribonucleoprotein K (hnRNPK) and fulfilled its pro-apoptotic function in HT-22 cells. In conclusion, our data indicated that the RMST/hnRNPK/p53/miR-107/Bcl2l2 axis plays an important role in regulating neuronal apoptosis.

MicroRNA-99a is a Potential Target for Regulating Hypothalamic Synaptic Plasticity in the Peri/Postmenopausal Depression Model.

Accumulating evidence has demonstrated that there is a growing trend of menopausal women suffering from depression. However, the pathogenesis of menopausal depression still remains unclear. Hence, this paper aims to reveal the pathological mechanisms involved in postmenopausal depression by using a novel peri- to postmenopausal depression model induced by a two-step ovariectomy plus chronic mild stress (CMS). The results of metabolic chambers and serum hormone/cytokine determination revealed that peri/postmenopausal depressive mice exhibited endocrine and metabolic disorders. Electrophysiological recordings indicated that the hippocampal synaptic transmission was compromised. Compared to the sham group, the microRNA-99a (miR-99a) level decreased significantly in the hypothalamus, and its target FK506-binding protein 51 (FKBP51) enormously increased; in contrast, the nuclear translocation of the progesterone receptor (PR) decreased in hypothalamic paraventricular nucleus (PVN) in the peri/postmenopausal depression mouse model. Additionally, synaptic proteins, including postsynaptic density protein 95 (PSD-95) and synaptophysin (SYN), showed a similar decrease in the hypothalamus. Accordingly, the present work suggests that miR-99a may be involved in the regulation of hypothalamic synaptic plasticity and that it might be a potential therapeutic target for peri/postmenopausal depression.

LncRNA RMST activates TAK1-mediated NF-κB signaling and promotes activation of microglial cells via competitively binding with hnRNPK.

This study aimed to explore the biological role and molecular mechanism of long noncoding RNA (lncRNA) rhabdomyosarcoma 2-associated transcript (RMST) in regulating microglial activation. Mouse microglial BV2 cells were cultured to establish the cell model of cerebral ischemic stroke by oxygen-glucose deprivation (OGD). We observed highly expressed RMST, increased expression of M1, and decreased expression of M2 markers in BV2 microglial cells stimulated with OGD. These alterations were reversed by RMST knockdown. Activation of transforming growth factor-beta-activated kinase 1 (TAK1)-mediated nuclear factor-κB (NF-κB) pathway was observed upon OGD stimulation, which was promoted by RMST through competitively binding with heterogeneous nuclear ribonucleoprotein K (hnRNPK), confirmed by RNA pull down and RNA immunoprecipitation (RIP) assays. Furthermore, RMST overexpressing-BV2 cells effectively enhanced neuronal apoptosis. In conclusion, RMST promoted OGD-induced microglial M1 polarization by competitively interacting with hnRNPK via TAK1-mediated NF-κB pathway, which will provide a basis for understanding the pathogenesis of cerebrovascular diseases.

The Intra-nuclear SphK2-S1P Axis Facilitates M1-to-M2 Shift of Microglia via Suppressing HDAC1-Mediated KLF4 Deacetylation.

Sphingosine 1-phosphate (S1P) is involved in a variety of cellular responses including microglial activation and polarization. However, the impacts of S1P on ischemia-induced microglial activation and polarization remain unclear. In the present study, Sprague-Dawley rats were selected for middle cerebral artery occlusion (MCAO) establishment and treated with S1P analog FTY720 (0.5, 1, 2 mg/kg) for 24 h. The impacts of FTY720 on oxygen-glucose deprivation (OGD)-induced microglial polarization were examined in the primary cultured microglia. FTY720 treatment could prevent ischemia-induced brain injury and neurological dysfunction, also decrease the levels of IL-1ß and TNF-α and promote M2 microglial polarization in rats. Further, we found that FTY720 inhibited the expressions of M1 markers, but increased the expressions of M2 markers in the OGD-insulted microglia. And FTY720 could enhance the phagocytic function of microglia. The sphingosine kinase 1/2 (SphK1/2) or the Sphk2 inhibitor could prevent the M1 to M2 phenotype shift improved by FTY720, but the Sphk1 inhibitor failed to affect the roles of FTY720. Furthermore, the Sphk1/2 or Sphk2 inhibitor promoted the activities of histone deacetylase (HDAC1) and inhibited the histone acetylation of the Krüppel-like factor 4 (KLF4) promoter regions, indicating that intra-nuclear pFTY720 inhibited HDAC1 activations and prevented KLF4 to interact with HDAC1, and thereby suppresses KLF4 deacetylation. Therefore, our data reveals that intra-nuclear SphK2-S1P axis might facilitate the transformation of microglial polarization from M1 to M2 phenotype, which might be intra-nuclear regulatory mechanisms of FTY720-prevented neuroinflammation.

Establishment and evaluation of a novel mouse model of peri/postmenopausal depression.

Women are believed to be more vulnerable to develop depressive symptoms during the perimenopause compared to postmenopause. The traditional bilateral ovariectomy and chronic mild stress (CMS) stimulation animal model produces a postmenopausal depressive-like state but the transition from perimenopausal period to postmenopausal period was ignored. Thus we establish a novel animal model in which the mice were stimulated by CMS for three months and removed the ovaries by two-step operation, and then evaluate whether this novel model could be much better for preclinical study used as a peri/postmenopause depressive model. The present study systemically evaluated the changes induced by two-step ovariectomy plus CMS in the mice. The depression-like behaviors, the levels of corticosterone, estrogen, pro-inflammatory factors, neurotransmitters, as well as brain-derived neurotrophic factor were determined; the changes of estrogen receptors, serotonin receptors, uterine weight and bone microarchitecture were also observed. The results show that the behaviors and biochemical indexes of mice changed gradually over time. Our study suggests that this two-step ovariectomy operation plus CMS successfully establishes a more reasonable peri/postmenopausal depression animal model which effectively simulates the clinical symptoms of peri/postmenopausal depressive women.

Antagonizing peroxisome proliferator-activated receptor γ facilitates M1-to-M2 shift of microglia by enhancing autophagy via the LKB1-AMPK signaling pathway.

Microglia-mediated neuroinflammation plays a dual role in various brain diseases due to distinct microglial phenotypes, including deleterious M1 and neuroprotective M2. There is growing evidence that the peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone prevents lipopolysaccharide (LPS)-induced microglial activation. Here, we observed that antagonizing PPARγ promoted LPS-stimulated changes in polarization from the M1 to the M2 phenotype in primary microglia. PPARγ antagonist T0070907 increased the expression of M2 markers, including CD206, IL-4, IGF-1, TGF-ß1, TGF-ß2, TGF-ß3, G-CSF, and GM-CSF, and reduced the expression of M1 markers, such as CD86, Cox-2, iNOS, IL-1ß, IL-6, TNF-α, IFN-γ, and CCL2, thereby inhibiting NFκB-IKKß activation. Moreover, antagonizing PPARγ promoted microglial autophagy, as indicated by the downregulation of P62 and the upregulation of Beclin1, Atg5, and LC3-II/LC3-I, thereby enhancing the formation of autophagosomes and their degradation by lysosomes in microglia. Furthermore, we found that an increase in LKB1-STRAD-MO25 complex formation enhances autophagy. The LKB1 inhibitor radicicol or knocking down LKB1 prevented autophagy improvement and the M1-to-M2 phenotype shift by T0070907. Simultaneously, we found that knocking down PPARγ in BV2 microglial cells also activated LKB1-AMPK signaling and inhibited NFκB-IKKß activation, which are similar to the effects of antagonizing PPARγ. Taken together, our findings demonstrate that antagonizing PPARγ promotes the M1-to-M2 phenotypic shift in LPS-induced microglia, which might be due to improved autophagy via the activation of the LKB1-AMPK signaling pathway.

S1PR3 is essential for phosphorylated fingolimod to protect astrocytes against oxygen-glucose deprivation-induced neuroinflammation via inhibiting TLR2/4-NFκB signalling.

Fingolimod (FTY720) is used as an immunosuppressant for multiple sclerosis. Numerous studies indicated its neuroprotective effects in stroke. However, the mechanism remains to be elucidated. This study was intended to investigate the mechanisms of phosphorylated FTY720 (pFTY720), which was the principle active molecule in regulating astrocyte-mediated inflammatory responses induced by oxygen-glucose deprivation (OGD). Results demonstrated that pFTY720 could protect astrocytes against OGD-induced injury and inflammatory responses. It significantly decreased pro-inflammatory cytokines, including high mobility group box 1 (HMGB1) and tumour necrosis factor-α (TNF-α). Further, studies displayed that pFTY720 could prevent up-regulation of Toll-like receptor 2 (TLR2), phosphorylation of phosphoinositide 3-kinase (PI3K) and nuclear translocation of nuclear factor kappa B (NFκB) p65 subunit caused by OGD. Sphingosine-1-phosphate receptor 3 (S1PR3) knockdown could reverse the above change. Moreover, administration of TLR2/4 blocker abolished the protective effects of pFTY720. Taken together, this study reveals that pFTY720 depends on S1PR3 to protect astrocytes against OGD-induced neuroinflammation, due to inhibiting TLR2/4-PI3K-NFκB signalling pathway.

PD149163 induces hypothermia to protect against brain injury in acute cerebral ischemic rats.

Therapeutic hypothermia is a promising strategy for acute cerebral ischemia via physical or pharmacological methods. In this study, we pharmacologically induced hypothermia on Sprague Dawley rats by intraperitoneally injecting PD149163. We found that mild hypothermia was induced by PD149163 treatment without local cerebral blood flow (LCBF) alteration. To evaluate the neuroprotective effects of PD149163, TTC staining, HE staining and Nissl's staining were performed in our study. We found that PD149163 could prevent neuronal damage, and inhibit proliferation and activation of glial cells induced by ischemia. Simultaneously, we observed PD149163 ameliorated apoptosis characterized by down-regulated caspase-3 and Bax, but elevated Bcl-2. Moreover, PD149163 dramatically reduced JNK and AMPK/mTOR signaling pathway activation, and thereby inhibited autophagy by increased P62 expression, decreased the ratio of LC3-Ⅱ to LC3-Ⅰ and the expression of Beclin. Taken together, the present findings reveal the therapeutic effects of PD149163-induced hypothermia in brain ischemia, and provide a new strategy for stroke treatment.

Rosiglitazone Exerts an Anti-depressive Effect in Unpredictable Chronic Mild-Stress-Induced Depressive Mice by Maintaining Essential Neuron Autophagy and Inhibiting Excessive Astrocytic Apoptosis.

There is increasing interest in the association between depression and the development of metabolic diseases. Rosiglitazone, a therapeutic drug used to treat type 2 diabetes mellitus, has shown neuroprotective effects in patients with stroke and Alzheimer's disease. The present study was performed to evaluate the possible roles of rosiglitazone in in vivo (unpredictable chronic mild stress-induced depressive mouse model) and in vitro (corticosterone-induced cellular model) depressive models. The results showed that rosiglitazone reversed depressive behaviors in mice, as indicated by the forced swimming test and open field test. Rosiglitazone was also found to inhibit the inflammatory response, decrease corticosterone levels, and promote astrocyte proliferation and neuronal axon plasticity in the prefrontal cortex of mice. This series of in vivo and in vitro experiments showed that autophagy among neurons was inhibited in depressive models and that rosiglitazone promoted autophagy by upregulating LKB1, which exerted neuroprotective effects. Rosiglitazone was also found to activate the Akt/CREB pathway by increasing IGF-1R expression and IGF-1 protein levels, thereby playing an anti-apoptotic role in astrocytes. Rosiglitazone's autophagy promotion and neuroprotective effects were found to be reversed by the PPARγ antagonist T0070907 in primary neurons and by PPARγ knockdown in an N2a cell line. In conclusion, we found that rosiglitazone protects both neurons and astrocytes in in vivo and in vitro depressive models, thereby playing an anti-depressive role. These findings suggest that PPARγ could be a new target in the development of anti-depressive drugs.