Extracellular vesicles derived from bone marrow mesenchymal stem cells ameliorate chronic liver damage via microRNA-136-5p.

Chronic liver damage (CLD) encompasses a spectrum of conditions and poses a significant global health challenge, affecting millions of individuals. Currently, there is a deficiency of clinically validated therapeutics with minimal side effects. Emerging evidence underscores the significant potential of extracellular vesicles derived from bone marrow mesenchymal stem cells (BMSC-EVs) as a promising therapeutic method for CLD. This study aimed to evaluate the influence of BMSC-EVs containing microRNA-136-5p (BMSC-EVs-miR-136-5p) on macrophage polarization during chronic liver injury and elucidate the mechanisms associated with the GNAS/PI3K/ERK/STAT3 axis. Surface markers of BMSCs were detected via Immunofluorescent Staining. Subsequently, EVs were harvested from the BMSC culture medium. In vivo fluorescence imaging was employed to locate the BMSC-EVs. Additionally, fluorescence microscopy was used to visualize the uptake of DIR-labeled BMSC-EVs by RAW264.7 cells. Various methods were employed to assess the impact of BMSC-EVs on the expression levels of inflammatory factors (IL-1ß, IL-6, IL-10, and TNF-α), M1/M2 macrophage markers (iNOS and Arg-1), and members of inflammation-related signaling pathways (GNAS, PI3K, ERK, and STAT3) in RAW264.7 cells co-cultured with BMSC-EVs. Loss-of-function approaches targeting miR-136-5p in RAW264.7 cells were subsequently utilized to validate the role of BMSC-EVs-miR-136-5p. The Luciferase Reporter Assay indicates that GNAS was identified to be a target of miR-136-5p, and miR-136-5p demonstrating increased within BMSC-EVs compared to Raw264.7-EVs. BMSC-EVs-miR-136-5p mitigated CCl4-induced liver inflammation and improved liver function by Suppressing the GNAS/STAT3 Signaling. Notably, miR-136-5p suppressed lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells. BMSC-EVs-miR-136-5p alleviates CLD by activating M2 polarization through the GNAS-mediated PI3K/ERK/STAT3 axis. Accordingly, the members of this axis may serve as therapeutic targets.

MMP-2-mediated Scube2 degradation promotes blood-brain barrier disruption by blocking the interaction between astrocytes and endothelial cells via inhibiting Sonic hedgehog pathway during early cerebral ischemia.

We have previously demonstrated a rapid secretion of matrix metalloproteinase-2 (MMP-2) in the ischemic brain. Since Scube2 can interact with Sonic hedgehog (Shh) to maintain blood-brain barrier (BBB) integrity via regulating the interaction between brain capillary endothelial cells (ECs) and perivascular astrocytes, and it is also a substrate of MMP-2, we hypothesized that the secreted MMP-2 could degrade Scube2 and contribute to ischemic BBB disruption. Using an in vitro ischemic model of 90-min oxygen-glucose deprivation/3-h reoxygenation (OGD/R) and an in vivo mouse stroke model of 90-min middle cerebral artery occlusion (MCAO) with 3-h reperfusion, we established an important role of MMP-2-mediated Scube2 degradation in early ischemic BBB disruption. Exposure of C8-D1A cells and bEnd.3 cells to OGD/R increased MMP secretion in both cells, and C8-D1A cells appeared to secrete more MMPs than bEnd.3 cells. Co-IP and double-immunostaining revealed that Scube2 co-localized well with MMP-2 in C8-D1A cells and could be pulled down by MMP-2 antibodies. In MCAO mice, Scube2 protein showed a drastic reduction in ischemic brain tissue, which was accompanied by suppressed expression of Shh and its downstream molecules. Of note, specific knockdown of astrocytic Scube2 with AAV-shScube2 augmented MCAO-induced Shh suppression and exacerbated BBB leakage and inflammatory reactions in the ischemic brain. Last, incubation of bEnd.3 cells with conditioned medium derived from OGD-treated C8-D1A cells led to a significant inhibition of the Shh pathway in bEnd.3 cells and degradation of VE-cadherin and ZO-1. Inhibition of MMP-2 with SB-3CT or over-expression of Scube2 with plasmids in C8-D1A cells alleviated the above effect of C8-D1A cells-derived conditioned medium. Taken together, our data indicate that ischemia-induced secretion of MMP-2 may contribute to early BBB disruption in ischemic stroke via interrupting the shared Scube2-Shh pathway between brain capillary ECs and perivascular astrocytes.

Activation of Swell1 in microglia suppresses neuroinflammation and reduces brain damage in ischemic stroke.

Cl- movement and Cl--sensitive signal pathways contributes to the survival and switch of inflammatory phenotype of microglia and are believed to play a key role in the inflammatory brain injury after ischemic stroke. Here, we demonstrated an important role of Cl- transmembrane transporter Swell1, in the survival and M2-like polarization of microglia in ischemic stroke. Knockdown or overexpression of Swell1 in cultured microglia inhibited or increased hypotonic-activated Cl- currents, respectively, and these changes were completely blocked by the volume-regulated anion channels (VRACs) inhibitor DCPIB. Swell1 conditional knock-in mice promoted microglia survival in ischemic brain region and resulted in significant reductions in neural cell death, infarction volume and neurological deficits following transient middle cerebral artery occlusion (tMCAO). Using gene manipulating technique and pharmacological inhibitors, we further revealed that Swell1 opening led to SGK1 (a Cl--sensitive kinase)-mediated activation of FOXO3a/CREB as well as WNK1 (another Cl--sensitive kinase)-mediated SPAK/OSR1-CCCs activation, which promoted microglia survival and M2-like polarization, thereby attenuating neuroinflammation and ischemic brain injury. Taken together, our results demonstrated that Swell1 is an essential component of microglia VRACs and its activation protects against ischemic brain injury through promoting microglia survival and M2-like polarization.

Correction: Machine learning encodes urine and serum metabolic patterns for autoimmune disease discrimination, classification and metabolic dysregulation analysis.

Correction for 'Machine learning encodes urine and serum metabolic patterns for autoimmune disease discrimination, classification and metabolic dysregulation analysis' by Qiuyao Du et al., Analyst, 2023, https://doi.org/10.1039/d3an01051a.

Machine learning encodes urine and serum metabolic patterns for autoimmune disease discrimination, classification and metabolic dysregulation analysis.

There is a wide variety of autoimmune diseases (ADs) with complex pathogenesis and their accurate diagnosis is difficult to achieve because of their vague symptoms. Metabolomics has been proven to be an efficient tool in the analysis of metabolic disorders to provide clues about the mechanism and diagnosis of diseases. Previous studies of the metabolomics analysis of ADs were not competent in their discrimination. Herein, a liquid chromatography tandem mass spectrometry (LC-MS) strategy combined with machine learning is proposed for the discrimination and classification of ADs. Urine and serum samples were collected from 267 subjects consisting of 127 healthy controls (HC) and 140 AD patients, including those with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), sicca syndrome (SS), ankylosing spondylitis (AS), systemic scleroderma (SSc) and connective tissue disease (CTD). Machine learning algorithms were encoded for the discrimination and classification of ADs with metabolomic patterns obtained by LC-MS, and satisfactory results were achieved. Notably, urine samples exhibited higher accuracy for disease differentiation and triage than serum samples. Apart from that, differential metabolites were selected and metabolite panels were evaluated to demonstrate their representativeness. Metabolic dysregulations were also investigated to gain more knowledge about the pathogenesis of ADs. This research provides a promising method for the application of metabolomics combined with machine learning in precision medicine.

Simultaneous quantification of total and free testosterone in human serum by LC-MS/MS.

Testosterone (TTe) and free testosterone (FTe) are clinically important indicators for the diagnosis of androgen disorders, so accurate quantitative determination of them in serum is clinically of paramount significance. Currently, there is no available method suitable for routine and simultaneous measurement of TTe and FTe. Here, we developed a new UPLC-MS/MS method to quantify serum TTe and FTe simultaneously and accurately. Rapid equilibrium dialysis was used to obtain FTe in serum followed by derivatization with hydroxylamine hydrochloride. With these strategies, TTe and FTe could be measured in single injection. After optimizing the extraction and derivatization conditions, the performance of LC-MS/MS was evaluated and applied to quantify the levels of TTe and FTe in clinical samples from 42 patients. The assays were linear for TTe within the range of 0.2-30 ng/mL and for FTe within the range of 1.5-1000 pg/mL. This improved method provided a limit of quantification for TTe of 0.2 ng/mL and for FTe of 1.5 pg/mL. The intra- and inter-run CVs were less than 4.3% and 3.6% for TTe and less than 8.2% and 6.7% for FTe, respectively. The intra- and inter-run accuracies for both TTe and FTe were in the range of 96.1-108.1%. Interference, carryover effect, and matrix effect were in acceptable range. In conclusion, our new LC-MS/MS method is simple to perform and can serve as a reliable method for simultaneous determination of TTe and FTe in clinical practice, providing important information for diagnosis, treatment, and monitoring of androgen-related diseases.

An ab initio method on large sized molecular aggregate system: Predicting absorption spectra of crystalline organic semiconducting films.

Theoretical description of electronically excited states of molecular aggregates at an ab initio level is computationally demanding. To reduce the computational cost, we propose a model Hamiltonian approach that approximates the electronically excited state wavefunction of the molecular aggregate. We benchmark our approach on a thiophene hexamer, as well as calculate the absorption spectra of several crystalline non-fullerene acceptors, including Y6 and ITIC, which are known for their high power conversion efficiency in organic solar cells. The method qualitatively predicts the experimentally measured spectral shape, which can be further linked to the molecular arrangement in the unit cell.

Intrinsic efficiency limits in low-bandgap non-fullerene acceptor organic solar cells.

In bulk heterojunction (BHJ) organic solar cells (OSCs) both the electron affinity (EA) and ionization energy (IE) offsets at the donor-acceptor interface should equally control exciton dissociation. Here, we demonstrate that in low-bandgap non-fullerene acceptor (NFA) BHJs ultrafast donor-to-acceptor energy transfer precedes hole transfer from the acceptor to the donor and thus renders the EA offset virtually unimportant. Moreover, sizeable bulk IE offsets of about 0.5 eV are needed for efficient charge transfer and high internal quantum efficiencies, since energy level bending at the donor-NFA interface caused by the acceptors' quadrupole moments prevents efficient exciton-to-charge-transfer state conversion at low IE offsets. The same bending, however, is the origin of the barrier-less charge transfer state to free charge conversion. Our results provide a comprehensive picture of the photophysics of NFA-based blends, and show that sizeable bulk IE offsets are essential to design efficient BHJ OSCs based on low-bandgap NFAs.

Modulation of α7nAchR by Melatonin Alleviates Ischemia and Reperfusion-Compromised Integrity of Blood-Brain Barrier Through Inhibiting HMGB1-Mediated Microglia Activation and CRTC1-Mediated Neuronal Loss.

The only food and drug administration (FDA)-approved drug currently available for the treatment of acute ischemic stroke is tissue plasminogen activator (tPA), yet the therapeutic benefits of this drug are partially outweighed by the increased risk of hemorrhagic transformation (HT). Analysis of the NIH trial has shown that cigarette smoking protected tPA-treated patients from HT; however, the underlying mechanism is not clear. Nicotinic acetylcholine receptors (nAChR) has shown anti-inflammatory effect and modulation nAChR could be a strategy to reduce ischemia/reperfusion-induced blood-brain barrier (BBB) damage. Since melatonin could regulate the expression of α7nAchR and melatonin's neuroprotective effect against ischemic injury is mediated via α7nAChR modulation, here, we aim to test the hypothesis that melatonin reduces ischemia and reperfusion (I/R)-induced BBB damage through modulation of α7nACh receptor (α7nAChR). Mice were subjected to 1.5 h ischemia and 24 h reperfusion and at the onset of reperfusion, mice received intraperitoneal administration (i.p.) of either drug or saline. Mice were randomly assigned into five groups: Saline; α7nAChR agonist PNU282987; Melatonin; Melatonin+Methyllycaconitine (MLA, α7nAChR antagonist), and MLA group. BBB permeability was assessed by detecting the extravasation of Evan's blue and IgG. Our results showed that I/R significantly increased BBB permeability accompanied by occludin degradation, microglia activation, and high mobility group box 1 (HMGB1) release from the neuron. In addition, I/R significantly induced neuronal loss accompanied by the decrease of CREB-regulated transcriptional coactivator 1 (CRTC1) and p-CREB expression. Melatonin treatment significantly inhibited the above changes through modulating α7nAChR. Taken together, these results demonstrate that melatonin provides a protective effect on ischemia/reperfusion-induced BBB damage, at least in part, depending on the modulation of α7nAChR.

N-Glycomic profiling reveals dysregulated glycans related to oral cancer using MALDI-MS.

Aberrant protein N-glycosylation is one of the hallmarks of malignancy, while its role in oral cancer (OC) progression is still poorly understood making it a perfect biomarker for early-stage cancer. In this work, we describe a MALDI-MS-based N-glycomic analysis with multivariate data analysis approach for exploration and classification of the patients with OC. Various statistical analyses were used to determine differential N-glycome profiles. There were 8 glycan structures significantly increased (p < 0.05) in all OC-derived  microvesicles (MVs). Importantly, the increases in these glycans were significantly greater in the early stage of OC than those in the advanced stage of OC. Additionally, N-glycomic profiles from the serum were also performed, demonstrating a significant change of 11 glycan structures (p < 0.05). Our data highlighted the effectiveness approach of MALDI-MS-based MV N-glycomic profiling on OC screening, and presented strong potential for further identifying targeted glycoproteomics signatures of the cancer.

Improved profiling of low molecular weight serum proteome for gastric carcinoma by data-independent acquisition.

Low molecular weight proteins (LMWPs) in the bloodstream participate in various biological processes and are closely associated with disease status, whereas identification of serous LMWPs remains a great technical challenge due to the wide dynamic range of protein components. In this study, we constructed an integrated LMWP library by combining the LMWPs obtained by three enrichment methods (50% ACN, 20% ACN + 20 mM ABC, and 30 kDa) and their fractions identified by the data-dependent acquisition method. With this newly constructed library, we comprehensively profiled LMWPs in serum using data-independent acquisition and reliably achieved quantitative results for 75% serous LMWPs. When applying this strategy to quantify LMWPs in human serum samples, we could identify 405 proteins on average per sample, of which 136 proteins were with a MW less than 30 kDa and 293 proteins were with a MW less than 65 kDa. Of note, pre- and post-operative gastric carcinoma (GC) patients showed differentially expressed serous LWMPs, which was also different from the pattern of LWMP expression in healthy controls. In conclusion, our results showed that LMWPs could efficiently distinguish GC patients from healthy controls as well as between pre- and post-operative statuses, and more importantly, our newly developed LMWP profiling platform could be used to discover candidate LMWP biomarkers for disease diagnosis and status monitoring.

A novel DPP-4 inhibitor Gramcyclin A attenuates cognitive deficits in APP/PS1/tau triple transgenic mice via enhancing brain GLP-1-dependent glucose uptake.

Enhancing glucagon-like peptide 1 (GLP-1) signaling with a dipeptidyl peptidase IV (DPP-4) inhibitor might exert protective effects on Alzheimer's disease (AD). We found that intragastric administration of Gramcyclin A (10, 20 and 40 mg/kg), a novel DPP-4 inhibitor, for 3 months significantly reversed cognitive decline in APP/PS1/tau triple transgenic mice in a dose-dependent manner. Gramcyclin A treatment markedly reduced Aß plaques as well as the insoluble and soluble forms of Aß40 and Aß42 in the hippocampus of APP/PS1/tau mice. Treatment with Gramcyclin A remarkedly decreased the level of microglia and suppressed neuroinflammation in the hippocampus of APP/PS1/tau mice. Moreover, Gramcyclin A treatment could increase brain glucose uptake in APP/PS1/tau mice, as detected by 18-fluoro-2-deoxyglucose (18 F-FDG) micro-positron emission tomography (micro-PET) imaging. Furthermore, Gramcyclin A significantly increased expression of glucagon-like peptide-1 (GLP-1), GLP-1R, proliferator-activated receptor gamma coactivator (PGC)-1α and glucose transporter 4 (GLUT4), and inhibited insulin receptor (IRS)-1 phosphorylation and tau hyperphosphorylation in the hippocampus of APP/PS1/tau mice. Collectively, Gramcyclin A conferred protective effects against AD via enhancing brain GLP-1-dependent glucose uptake. The DPP-4 inhibitor Gramcyclin A might be a potential therapeutic drug for AD.

Occludin degradation makes brain microvascular endothelial cells more vulnerable to reperfusion injury in vitro.

Intracerebral hemorrhage is the most dangerous complication in tPA thrombolytic therapy for ischemic stroke, which occurs as a consequence of endothelial cell death at the blood-brain barrier (BBB) during thrombolytic reperfusion. We have previously shown that cerebral ischemia-induced rapid occludin degradation and BBB disruption. Here we demonstrated an important role of occludin degradation in facilitating the evolution of ischemic endothelial cells toward death. Cultured brain microvascular endothelial cells (bEnd.3 cells) were exposed to oxygen-glucose deprivation (OGD) or incubated with occludin siRNA or occludin AAV to achieve an occludin deficiency or over-expression status before exposing to reoxygenation (R) or TNF-α treatment. Cell death was assessed by measuring lactate dehydrogenase release, TUNEL staining, and flow cytometry analysis. Inhibition of OGD-induced occludin degradation with SB-3CT or over-expression of occludin with occludin AAV both significantly attenuated OGD/R-induced apoptosis and pyroptosis in bEnd.3 cells. Consistently, knockdown of occludin with siRNA potentiated TNF-α-induced apoptosis, supporting an important role of occludin integrity in endothelial cell survival. Similar results were observed for pyroptosis, in which occludin knockdown with siRNA led to a significant augmentation of cytokines secretion, inflammasome activation, and pyroptosis occurrence in TNF-α-treated bEnd.3 cells. Lastly, up-regulation of c-Yes, PI3K/AKT, and ERK concurrently occurred with occludin degradation after OGD/R or TNF-α treatment, and the level of these proteins were further increased when inhibition of occludin degradation or over-expression of occludin. These data indicate that occludin degradation inflicted during ischemia makes BBB endothelial cells more vulnerable to reperfusion-associated stress stimuli.

Identification of prognostic values defined by copy number variation, mRNA and protein expression of LANCL2 and EGFR in glioblastoma patients.

BACKGROUND: Epidermal growth factor receptor (EGFR) and lanthionine synthetase C-like 2 (LanCL2) genes locate in the same amplicon, and co-amplification of EGFR and LANCL2 is frequent in glioblastoma. However, the prognostic value of LANCL2 and EGFR co-amplification, and their mRNA and protein expression in glioblastoma remain unclear yet. METHODS: This study analyzed the prognostic values of the copy number variations (CNVs), mRNA and protein expression of LANCL2 and EGFR in 575 glioblastoma patients in TCGA database and 100 glioblastoma patients in tumor banks of the Shenzhen Second People's Hospital and the Sun Yat-sen University Cancer Center. RESULTS: The amplification of LANCL2 or EGFR, and their co-amplification were frequent in glioblastoma of TCGA database and our tumor banks. A significant correlation was found between the CNVs of LANCL2 and EGFR (p < 0.001). CNVs of LANCL2 or EGFR were significantly correlated with IDH1/2 mutation but not MGMT promoter methylation. Multivariate analysis showed that LANCL2 amplification was significantly correlated with reduced overall survival (OS) in younger (< 60 years) glioblastoma patients of TCGA database (p = 0.043, HR = 1.657) and our tumor banks (p = 0.018, HR = 2.199). However, LANCL2 or EGFR amplification, and their co-amplification had no significant impact on OS in older (≥ 60 years) or IDH1/2-wild-type glioblastoma patients. mRNA and protein overexpression of LANCL2 and EGFR was also frequently found in glioblastoma. The mRNA expression rather than the protein expression of LANCL2 and EGFR was positively correlated (p < 0.001). However, mRNA or protein expression of EGFR and LANCL2 was not significantly correlated with OS of glioblastoma patients. The protein expression level of LANCL2, rather than EGFR, was elevated in relapsing glioblastoma, compared with newly diagnosed glioblastoma. In addition, the intracellular localization of LanCL2, not EGFR, was associated with the grade of gliomas. CONCLUSIONS: Taken together, amplification and mRNA overexpression of LANCL2 and EGFR, and their co-amplification and co-expression were frequent in glioblastoma patients. Our findings suggest that amplification of LANCL2 and EGFR were the independent diagnostic biomarkers for glioblastoma patients, and LANCL2 amplification was a significant prognostic factor for OS in younger glioblastoma patients.

Synergism between the phosphatidylinositol 3-kinase p110ß isoform inhibitor AZD6482 and the mixed lineage kinase 3 inhibitor URMC-099 on the blockade of glioblastoma cell motility and focal adhesion formation.

BACKGROUND: Glioblastoma multiforme, the most aggressive and malignant primary brain tumor, is characterized by rapid growth and extensive infiltration to neighboring normal brain parenchyma. Our previous studies delineated a crosstalk between PI3K/Akt and JNK signaling pathways, and a moderate anti-glioblastoma synergism caused by the combined inhibition of PI3K p110ß (PI3Kß) isoform and JNK. However, this combination strategy is not potent enough. MLK3, an upstream regulator of ERK and JNK, may replace JNK to exert stronger synergism with PI3Kß. METHODS: To develop a new combination strategy with stronger synergism, the expression pattern and roles of MLK3 in glioblastoma patient's specimens and cell lines were firstly investigated. Then glioblastoma cells and xenografts in nude mice were treated with the PI3Kß inhibitor AZD6482 and the MLK3 inhibitor URMC-099 alone or in combination to evaluate their combination effects on tumor cell growth and motility. The combination effects on cytoskeletal structures such as lamellipodia and focal adhesions were also evaluated. RESULTS: MLK3 protein was overexpressed in both newly diagnosed and relapsing glioblastoma patients' specimens. Silencing of MLK3 using siRNA duplexes significantly suppressed migration and invasion, but promoted attachment of glioblastoma cells. Combined inhibition of PI3Kß and MLK3 exhibited synergistic inhibitory effects on glioblastoma cell proliferation, migration and invasion, as well as the formation of lamellipodia and focal adhesions. Furthermore, combination of AZD6482 and URMC-099 effectively decreased glioblastoma xenograft growth in nude mice. Glioblastoma cells treated with this drug combination showed reduced phosphorylation of Akt and ERK, and decreased protein expression of ROCK2 and Zyxin. CONCLUSION: Taken together, combination of AZD6482 and URMC-099 showed strong synergistic anti-tumor effects on glioblastoma in vitro and in vivo. Our findings suggest that combined inhibition of PI3Kß and MLK3 may serve as an attractive therapeutic approach for glioblastoma multiforme.

D1 receptor-mediated endogenous tPA upregulation contributes to blood-brain barrier injury after acute ischaemic stroke.

Blood-brain barrier (BBB) integrity injury within the thrombolytic time window is becoming a critical target to reduce haemorrhage transformation (HT). We have previously reported that BBB damage was initially damaged in non-infarcted striatum after acute ischaemia stroke. However, the underlying mechanism is not clear. Since acute ischaemic stroke could induce a significant increase of dopamine release in striatum, in current study, our aim is to investigate the role of dopamine receptor signal pathway in BBB integrity injury after acute ischaemia using rat middle cerebral artery occlusion model. Our data showed that 2-h ischaemia induced a significant increase of endogenous tissue plasminogen activator (tPA) in BBB injury area and intra-striatum infusion of tPA inhibitor neuroserpin, significantly alleviated 2-h ischaemia-induced BBB injury. In addition, intra-striatum infusion of D1 receptor antagonist SCH23390 significantly decreased ischaemia-induced upregulation of endogenous tPA, accompanied by decrease of BBB injury and occludin degradation. More important, inhibition of hypoxia-inducible factor-1 alpha with inhibitor YC-1 significantly decreased 2-h ischaemia-induced endogenous tPA upregulation and BBB injury. Taken together, our data demonstrate that acute ischaemia disrupted BBB through activation of endogenous tPA via HIF-1α upregulation, thus representing a new therapeutic target for protecting BBB after acute ischaemic stroke.

MicroRNA-9 inhibits retinal neovascularization in rats with diabetic retinopathy by targeting vascular endothelial growth factor A.

Diabetic retinopathy (DR) is a leading cause of adult visual impairment and loss. This study aims to explore the effects of microRNA-9 (miR-9) on retinal neovascularization during DR by targeting the vascular endothelial growth factor A (VEGFA). DR rat models were successfully established. Retinal microvascular endothelial cells (RMECs) of DR rats were isolated and treated with miR-9 mimic, miR-9 inhibitor or small interfering RNA (siRNA)-VEGFA. The expressions of miR-9, VEGFA, and cluster of differentiation 31 (CD31) of the rats' tissues and cells were examined. The targeting relationship between miR-9 and VEGFA was testified. The tubule formation, the cell proliferation and the periodic distribution and apoptosis were evaluated after transfection. In the retinal tissues of DR rats, miR-9 expression decreased while the expression of VEGFA and CD31 increased. Notably, miR-9 targeted and inhibited VEGFA expression. In response to the treatment of miR-9 mimic and siRNA-VEGFA, a reduction was identified in CD31 expression, tubule formation, and proliferation of RMECs and cell ratio in the S phase, but an increase was observed in apoptosis rate of RMECs. The treatment of miR-9 inhibitor reversed the manifestations. Our study demonstrated that miR-9 could inhibit retinal neovascularization of DR and tubule formation, and promote apoptosis in RMECs by targeting VEGFA.

Inhibition of Reactive Astrocytes with Fluorocitrate Ameliorates Learning and Memory Impairment Through Upregulating CRTC1 and Synaptophysin in Ischemic Stroke Rats.

Ischemic stroke often causes motor and cognitive deficits. Deregulated glia gap junction communication, which is reflected by increased protein levels of glial fibrillary acidic protein (GFAP) and connexin 43 (Cx43), has been observed in ischemic hippocampus and has been associated with cognitive impairment in animal stroke models. Here, we tested the hypothesis that reactive astrocytes-mediated loss of synaptophysin (SYP) and CREB-regulated transcription coactivator 1 (CRTC1) contribute to dysfunction in glia gap junction communication and memory impairment after ischemic stroke. Male Sprague-Dawley rats were subjected to a 90-min middle cerebral artery occlusion (MCAO) with 7-day reperfusion. Fluorocitrate (1 nmol), the reversible inhibitor of the astrocytic tricarboxylic acid cycle, was injected into the right lateral ventricle of MCAO rats once every 2 days starting immediately before reperfusion. The Morris water maze was used to assess memory in conjunction with western blotting and immunostaining to detect protein expression and distribution in the hippocampus. Our results showed that ischemic stroke caused significant memory impairment accompanied by increased protein levels of GFAP and Cx43 in hippocampal tissue. In addition, the levels of several key memory-related important proteins including SYP, CRTC1, myelin basic protein and high-mobility group-box-1 were significantly reduced in the hippocampal tissue. Of note, inhibition of reactive astrocytes with fluorocitrate was shown to significantly reverse the above noted changes induced by ischemic stroke. Taken together, our findings demonstrate that inhibiting reactive astrocytes with fluorocitrate immediately before reperfusion may protect against ischemic stroke-induced memory impairment through the upregulation of CRTC1 and SYP.

IRGM promotes glioma M2 macrophage polarization through p62/TRAF6/NF-κB pathway mediated IL-8 production.

Alternatively activated (M2) macrophage promotes glioma progression and immune escape as the most immunocyte in glioma microenvironment. Finding out the key protein regulating M2 macrophage polarization is necessary for improving treatment. Whether immunity related GTPase M (IRGM) is involved in glioma development and M2 macrophage polarization is unknown. IRGM and M2 macrophage marker CD206 expression were examined using immunohistochemistry among 35 glioma and 11 non-cancerous brain specimens. We found IRGM scores were positively correlated with CD206 scores in glioma specimens and monocyte proportion in blood samples. A172 glioma cells transfected with either IRGM knock-down lentivirus (Lenti-IRGM) or control lentivirus (Lenti-HK) were subcutaneously injected into nude mice. In vivo, xenografted glioma size of the Lenti-IRGM group was smaller and had weaker fluorescence signal than Lenti-HK control group. Immunofluorescence results showed that there was obviously decreased IRGM, CD206, and IL-8 expression in the mice glioma of Lenti-IRGM group than Lenti-HK control group. In vitro, flow cytometry results showed that M2 polarization from THP-1 cocultured with Lenti-IRGM glioma cells decreased in contrast to that with Lenti-HK glioma cells; there were less interleukin-8 (IL-8) and macrophage inflammation protein 3-α (MIP-3α), but more interleukin-6 (IL-6) in the supernatant of Lenti-IRGM glioma cells than matched control. Western blot and immunofluorescence displayed that IRGM strongly promoted sequestosome-1 (p62/SQSTM1), necrosis factor receptor-activating factor 6 (TRAF6) expression and NF-κB transportation to the nucleus. Realtime PCR results demonstrated IRGM also promoted NF-κB downstream cytokines IL-8 and MIP-3α mRNA expression. These data suggested that IRGM could promote glioma development and M2 macrophage polarization by regulating p62/TRAF6/NF-κB pathway-mediated IL-8 production.

Furanodienone overcomes temozolomide resistance in glioblastoma through the downregulation of CSPG4-Akt-ERK signalling by inhibiting EGR1-dependent transcription.

Glioblastoma multiforme (GBM) is a highly aggressive type of brain tumour. Patients with GBM respond poorly to chemotherapy and have poor survival outcomes. Neuron-glial antigen 2 (NG2), also known as chondroitin sulphate proteoglycan 4 (CSPG4), has been shown to contribute to critical processes, such as cell survival, proliferation, and chemotherapy resistance, during glioma progression. In this study, we found that furanodienone (FUR), a diene-type sesquiterpene isolated from the rhizomes of Rhizoma curcumae, exhibited a potential cytotoxic effect on temozolomide (TMZ)-resistant GBM cells in vitro by inhibiting CSPG4 and related signalling pathways. Studies investigating the mechanism demonstrated that FUR suppressed CSPG4-Akt-ERK signalling, inflammatory responses, and cytokine levels but activated caspase-dependent pathways and mitochondrial dysfunction. Furthermore, an immunofluorescence assay and a dual-luciferase reporter assay revealed that inhibition of EGR1-mediated transcription might have contributed to the FUR-dependent blockade of CSPG4 signalling and glioma cell survival. These results established a link between FUR-induced CSPG4 inhibition and the suppression of EGR1-dependent transcription. Attenuation of ERK1/2 and cytokine signalling might have generated the EGR1-dependent negative feedback loop of the CSPG4 pathway during FUR-induced apoptosis. These findings suggested that FUR could be a therapeutic candidate for the treatment of malignant glioma via targeting CSPG4 signalling.