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Supplemental Digital Content is available in the text., Memantine has demonstrated beneficial effects on several types of brain insults via therapeutic mechanisms mainly related to its activity as a receptor antagonist of N-methyl-d-aspartate. However, the influences of memantine on intracerebral hemorrhage (ICH) remain obscure. This research probed into the neurovascular protective mechanisms of memantine after ICH and its impacts on neuronal nitric oxide synthase (nNOS) ser1412 phosphorylation. ICH model was established by employing intrastriatal collagenase injection in rats. After modeling, rats were then allocated randomly into sham-operated (sham), vehicle-treated (ICH+V), and memantine-administrated (ICH+M) groups. Memantine (20 mg/kg/day) was intraperitoneally administered 30 min after ICH and thenceforth once daily. Rats were dedicated at 0.25, 6, 12, 24 h, 3 and 7 d post-ICH for measurement of corresponding indexes. Behavioral changes, brain edema, levels of nNOS ser1412 phosphorylation, peroxynitrite, matrix metalloproteinase (MMP)-9, NLRP3, IL-1β and numbers of dying neurons, as well as the cellular localization of gelatinolytic activity, were detected among the groups. Memantine improved the neurologic deficits and mitigated brain water content, levels of MMP-9, NLRP3, IL-1β and dying neurons. Additionally, treatment with memantine also reduced nNOS ser1412 phosphorylation and peroxynitrite formation compared with the ICH+V group at 24 h after ICH. In situ zymography simultaneously revealed that gelatinase activity was primarily colocalized with vessel walls and neurons. We concluded that memantine ameliorated blood–brain barrier disruption and neurologic dysfunction in an ICH rat model. The underlying mechanism might involve repression of nNOS ser1412 phosphorylation, as well as peroxynitrite-related MMP-9 and NLRP3 inflammasome activation.

Background & Aims Enterotoxigenic Bacteroides fragilis (ETBF) is strongly associated with the occurrence of inflammatory bowel disease (IBD), colitis-associated colorectal cancer, and colorectal cancer (CRC). However, the mechanism of ETBF-induced intestinal inflammation and tumorigenesis remains unclear. Methods microRNA sequencing was used to detect the differentially expressed microRNAs in both ETBF-treated cells and exosomes derived from ETBF-inoculated cells. Cell Counting Kit 8 assays were used to evaluate the effect of ETBF and exosomes on CRC cell proliferation. The biological role and mechanism of ETBF-mediated miR-149-3p in colitis and colon carcinogenesis were determined both in vitro and in vivo. Results ETBF promoted CRC cell proliferation by down-regulating miR-149-3p both in vitro and in vivo. ETBF–down-regulated miR-149-3p depended on METTL14-mediated N6-methyladenosine methylation. As the target gene of miR-149-3p, PHF5A transactivated SOD2 through regulating KAT2A messenger RNA alternative splicing after ETBF treatment in CRC cells. miR-149-3p could be released in exosomes and mediated intercellular communication by modulating T-helper type 17 cell differentiation. The level of plasma exosomal miR-149-3p was gradually decreased from healthy control individuals to patients with IBD and CRC. miR-149-3p, existing in plasma exosomes, negatively correlated with the abundance of ETBF in patients with IBD and CRC. Conclusions Exosomal miR-149-3p derived from ETBF-treated cells facilitated T-helper type 17 cell differentiation. ETBF-induced colorectal carcinogenesis depended on down-regulating miR-149-3p and further promoting PHF5A-mediated RNA alternative splicing of KAT2A in CRC cells. Targeting the ETBF/miR-149-3p pathway presents a promising approach to treat patients with intestinal inflammation and CRC with a high amount of ETBF.

Background: As tumor microenvironment (TME) play an indispensable role in tumorigenesis of colorectal cancer, this study performs a bunch of bioinformatics analysis to identify the indicator of the status of TME in Colorectal cancer (CRC). Results: In the presented study, we applied CIBERSORT and ESTIMATE computational methods to calculate the proportion of tumor-infiltrating immune cells (TICs) and the amount of immune and stromal components in 444 COAD-READ cases from The Cancer Genome Atlas (TCGA) database. The differentially expressed genes (DEGs) were analyzed by COX regression analysis and protein–protein interaction (PPI) network construction. Then, fatty acid-binding protein four ( FABP4 ) was determined as a predictive factor by the intersection analysis of univariate COX and PPI. Further analysis revealed that FABP4 expression was positively correlated with the clinical pathologic characteristics (clinical stage, distant metastasis) and negatively correlated with the survival of CRC patients. Gene Set Enrichment Analysis (GSEA) showed that the genes in the high-expression FABP4 group were mainly enriched in immune-related activities. In the low-expression FABP4 group, the genes were enriched in metabolic pathways. CIBERSORT analysis for the proportion of TICs revealed that NK cell, CD4 + T cells and CD8 + T cells were negatively correlated with FABP4 expression, suggesting that FABP4 might be a potential prognostic factor of CRC patients. Conclusion: Our study has developed a new biomarker (FABP4) that can predict the status of tumor microenvironment in Colorectal cancer. Keywords: FABP4, tumor microenvironment, ESTIMATE, CIBERSORT, colorectal cancer

ObjectiveColorectal cancer (CRC) is highly malignant and cancer metastasis remains the predominant cause of CRC death. The potential molecular mechanism of long non-coding RNA (lncRNAs) in CRC malignance is still poorly elucidated.MethodsCCMAlnc expression was analyzed by using the Sequence ReadArchive (SRA) database. Target gene expression was examined by real-time PCR and Western blotting. The biological function of CCMAlnc and miR-5001-5p was detected by cell invasion, CCK8 proliferation, and colony formation assays in loss of function and gain of function experiments in vitro. A luciferase assay was performed to validate the target site of miR-5001-5p on the 3′-UTR of HES6 mRNA.ResultsCCMAlnc was identified as a novel functional lncRNA in CRC. Elevated CCMAlnc was detected in CRC cells as well as in clinical CRC tissue samples, and the expression of this lncRNA positively correlated with the poor prognosis of CRC patients. Functional validation assays revealed that downregulation of CCMAlnc impaired CRC cell proliferation and invasion in vitro, but upregulation of CCMAlnc reversed this effect. Moreover, CCMAlnc was validated to act as a competing endogenous RNA (ceRNA) that stabilizes the expression of HES6 by downregulating miR-5001-5p.ConclusionCCMAlnc/miR-5001-5p/HES6 signaling is strongly activated to promote CRC malignance. CCMAlnc is defined as a potential candidate biomarker for metastasis prediction in CRC patients and as a potential therapeutic target for CRC treatment.

Cerebral ischemia is a leading cause of death and disability. A previous study indicated that remote ischemic postconditioning (RIP) in the treatment of cerebral ischemia reduces ischemia/reperfusion (I/R) injury. However, the underlying mechanism is not well understood. In the present study, the authors hypothesized that the protective effect of RIP on neurological damage is mediated by exosomes that are released by endothelial cells in femoral arteries. To test this, right middle cerebral artery occlusion/reperfusion with RIP was performed in rats. In addition, an I/R injury cell model was tested that included human umbilical vein endothelial cells (HUVECs) and SH-SY5Y cells. Both the in vivo and in vitro models were examined for injury. Markers of exosomes (CD63, HSP70 and TSG101) were assessed by immunohistochemistry, western blot analysis and flow cytometry. Exosomes were extracted from both animal serum and HUVEC culture medium and identified by electron microscopy. They investigated the role of endothelial cell-derived exosomes in the proliferation, apoptosis, cell cycle, migration and invasion of I/R-injured SH-SY5Y cells. In addition, apoptosis-related molecules caspase-3, Bax and Bcl-2 were detected. RIP was determined to increase the number of exosomes and the expression levels of CD63, HSP70 and TSG101 in plasma, but not in brain hippocampal tissue. The size of exosomes released after I/R in HUVECs was similar to the size of exosomes released in rats subjected to RIP. Endothelial cell-derived exosomes partly suppressed the I/R-induced cell cycle arrest and apoptosis, and inhibited cell proliferation, migration and invasion in SH-SY5Y nerve cells. Endothelial cell-derived exosomes directly protect nerve cells against I/R injury, and are responsible for the protective role of RIP in I/R.

The widespread use of combined anti-retroviral therapy (cART) has not decreased the prevalence of HIV-1-associated neurocognitive disorder (HAND), a type of neurodegenerative disease, even though cART effectively inhibits virus colonization in the central nervous system. Therefore, anti-retroviral agents cannot be fully excluded from the pathogenesis of HAND. Our previous study reported that long-term nucleoside analogue (NA) exposure induced mitochondrial toxicity in the cortical neurons of HAND patients and mice, but the exact mechanism of NA-associated neurotoxicity has remained unclear. Alteration of autophagy can result in protein aggregation and the accumulation of dysfunctional organelles, which are hallmarks of some neurodegenerative diseases. In this study, we first found increased autophagy in cortical autopsy specimens of AIDS patients. We then found that a low dose of NAs could stimulate autophagy in primary cultured neurons, while a high dose of NAs could induce only neuronal apoptosis. The level of NA-induced Bcl-2 and Bax expressions determined whether neuronal autophagy or apoptosis occurred. Furthermore, the level of NA-induced neuronal apoptosis correlated with the dysfunction of cellular DNA polymerase gamma. Damage-regulated autophagy modulator (DRAM) overexpression was also involved in NA-induced neuronal autophagy. p53 played a role in the regulation of NA-induced neuronal apoptosis, but its role in NA-associated neuronal autophagy was uncertain. Our results suggest that DRAM is involved in the regulation of NA-induced neuronal autophagy in a p53-independent manner. Further research is needed to investigate the underlying mechanism.

Glioblastoma is a malignant brain tumor originating in the central nervous system. Successfully therapy of this disease required the efficient delivery of therapeutic agents to the tumor cells and tissues. Delivery of anticancer drugs using novel nanocarriers is promising in glioma treatment.Polymeric nanoparticles (PNPs), solid lipid nanoparticles (SLNs), and nanostructured lipid carriers (NLCs) were constructed for the delivery of temozolomide (TMZ). The anti-tumor effects of the three kinds of nanocarriers were compared to provide the optimum choice for gliomatosis cerebri treatment.TMZ-loaded PNPs (T-PNPs), SLNs (T-SLNs), and NLCs (T-NLCs) were formulated. Their particle size, zeta potential, drug encapsulation efficiency (EE), and drug loading (DL) capacity were evaluated. Anti-tumor efficacies of the three kinds of nanocarriers were evaluated on U87 malignant glioma cells (U87 MG cells) and mice-bearing malignant glioma model.T-NLCs displayed the best anti-tumor activity than other formulations in vivo and in vitro. The most significantly glioma inhibition was observed on NLCs formulations than PNPs and SLNs.This work demonstrates that NLCs can deliver TMZ into U87MG cells more efficiently, with higher inhibition efficacy than PNPs and SLNs. T-NLCs could be an excellent drug delivery system for glioblastoma chemotherapy.

Jicai Zhang, Xiang Xiao, Jianming Zhu, Ziyun Gao, Xianliang Lai, Xingen Zhu, Guohua Mao Department of Neurosurgery, Second Affiliated Hospital, Nanchang University, Nanchang, Jiangxi, China Purpose: Glioblastoma multiforme (GBM) is the most common malignant brain tumor originating in the central nervous system in adults. Based on nanotechnology such as liposomes, polymeric nanoparticles, and lipid nanoparticles, recent research efforts have been aimed to target drugs to the brain. Methods: In this study, lactoferrin- and arginine–glycine–aspartic acid (RGD) dual-ligand-comodified, temozolomide and vincristine-coloaded nanostructured lipid carriers (L/R-T/V-NLCs) were introduced for GBM combination therapy. The physicochemical properties of L/R-T/V-NLCs such as particle size, zeta potential, and encapsulated efficiency are measured. The drug release profile, cellular uptake, cytotoxicity, tissue distribution, and antitumor activity of L/R-T/V-NLCs are further investigated in vitro and in vivo. Results: L/R-T/V-NLCs were stable with nanosize and high drug encapsulation efficiency. L/R-T/V-NLCs exhibited sustained-release behavior, high cellular uptake, high cytotoxicity and synergy effects, increased drug accumulation in the tumor tissue, and obvious tumor inhibition efficiency with low systemic toxicity. Conclusion: L/R-T/V-NLCs could be a promising drug delivery system for glioblastoma chemotherapy. Keywords: gliomatosis cerebri, combination therapy, nanostructured lipid carriers, lactoferrin, arginine–glycine–aspartic acid peptide, vincristine, temozolomide

The purpose of the present study was to investigate the expression of matrix metalloproteinase-9 (MMP-9) and interleukin-6 (IL-6) in patients with subarachnoid hemorrhage (SAH) and the clinical significance thereof. Forty-three patients with SAH and 23 healthy individuals were enrolled in this study. Patients were divided into the cerebral vasospasm (CVS) and non-cerebral vasospasm (non-CVS) groups, or the good and poor prognosis groups. Serum levels of MMP-9 and IL-6 were detected by ELISA. Expression levels of MMP-9 and IL-6 mRNAs were detected by RT-qPCR. Expression levels of MMP-9 and IL-6 were elevated with the increase of grades as determined by the Hunt-Hess grading method. Serum levels of MMP-9 and IL-6 in the CVS and poor prognosis groups were significantly higher than those in the control group. Expression levels of MMP-9 and IL-6 in the CVS group were significantly higher than those in the non-CVS group (P

Skull base chordomas are invasive tumors, with high rate of local recurrence even when totally extracted. The aggressive biological behavior in chordoma remains unclear. The purpose of this study was to investigate the relationship between fascin expression and tumor biological behavior in skull base chordoma. Using immunohistochemical techniques, we investigated the expression of fascin in 34 patients with skull base chordomas (19 primary tumors and 20 recurrent tumors). Correlation between fascin expression and clinicopathological factors such as patient’s age, sex, tumor locations, and fascin expression in recurrent tumor and in tumor with dura mater erosion was analyzed. Various extent of fascin expression was observed in all tumors. There was a higher fascin expression in recurrent chordoma than in primary chordoma, and the difference was statistically significant (p = 0.031). No difference of fascin expression was found between histology types. Interestingly, in 8 tumors where the cranial base dura was eroded, there was a high level of fascin expression (p = 0.047). These immunohistochemical findings suggest that fascin expression was correlated with tumor recurrence and high invasiveness, and that fascin overexpression may play an important role in the biologic behavior of skull base chordomas.

Nucleoside analogue reverse transcriptase inhibitor (NRTI), an integral component of highly active antiretroviral therapy (HAART), was widely used to inhibit HIV replication. Long-term exposure to NRTIs can result in mitochondrial toxicity which manifests as lipoatrophy, lactic acidosis, cardiomyopathy and myopathy, as well as polyneuropathy. But the cerebral neurotoxicity of NRTIs is still not well known partly due to the restriction of blood-brain barrier (BBB) and the complex microenvironment of the central nervous system (CNS). In this study, the Balb/c mice were administered 50 mg/kg stavudine (D4T), 100 mg/kg zidovudine (AZT), 50 mg/kg lamivudine (3TC) or 50 mg/kg didanosine (DDI) per day by intraperitoneal injection, five days per week for one or four months, and primary cortical neurons were cultured and exposed to 25 µM D4T, 50 µM AZT, 25 µM 3TC or 25 µM DDI for seven days. Then, single neuron was captured from mouse cerebral cortical tissues by laser capture microdissection. Mitochondrial DNA (mtDNA) levels of the primary cultured cortical neurons, and captured neurons or glial cells, and the tissues of brains and livers and muscles were analyzed by relative quantitative real-time PCR. The data showed that mtDNA did not lose in both NRTIs exposed cultured neurons and one month NRTIs treated mouse brains. In four months NRTIs treated mice, brain mtDNA levels remained unchanged even if the mtDNA levels of liver (except for 3TC) and muscle significantly decreased. However, mtDNA deletion was significantly higher in the captured neurons from mtDNA unchanged brains. These results suggest that long-term exposure to NRTIs can result in mtDNA deletion in mouse cortical neurons.