Lipocalin-2 inhibition alleviates neural injury by microglia ferroptosis suppression after experimental intracerebral hemorrhage in mice via enhancing ferritin light chain expression.

INTRODUCTION: Microglia play pivotal roles in post-intracerebral hemorrhage (ICH) neural injury. Iron metabolism, which is dysregulated after ICH, participates in microglial dysfunction. Previous studies have shown that iron metabolism-related lipocalin-2 (LCN2) is involved in regulating microglial function following ICH. In this study, we investigated the role of LCN2 in microglial function following ICH. METHODS: The BV2 (microglia) cell line, transfected with LCN2 for overexpression/interference, received a blood infusion from C57BL/6 mice in vitro. For the in vivo study of LCN2 function, an LCN2 knockout was conducted in mice. Liproxstatin-1 and RSL3 were used to manipulate ferroptosis and to study the effects of LCN2 on microglia after ICH. A BV2 (microglia) cell line, transfected with ferritin light chain (FTL) for overexpression/interference, was co-cultured with primary cultured neurons for a study on the mechanism of LCN2. Behavioral tests were conducted pre-ICH and on days 3, 7, and 28 post-ICH, and the brains and cultured cells were collected for protein, histological, and morphological studies. RESULTS: Brain LCN2 expression was upregulated in microglia, astrocytes, and neurons and played hazardous roles after ICH. In microglia, LCN2 promoted ferroptosis, which facilitated neural injury after ICH. LCN2-mediated FTL deficiency was shown to be responsible for microglial ferroptosis-induced neural injury. CONCLUSION: Our study suggests that LCN2-enhanced microglial ferroptosis plays a detrimental role by inducing FTL deficiency after ICH. The current study reveals a novel molecular mechanism involved in the pathophysiological progression of ICH.

Functional outcome following spontaneous intracerebral hemorrhage between ultrasound guidance endoscopic surgery and conventional craniotomy: A retrospective population-based study.

OBJECTIVES: Ultrasound guidance endoscopic surgery (ES) has been widely used in the treatment of cerebral hemorrhage in recent years, but relevant research articles are still scarce. Our study aims to investigate the effect of ES compared with conventional craniotomy (CC) on the postoperative complications, and prognosis of patients with intracerebral hemorrhage. MATERIALS AND METHODS: The clinical data of 1201 patients with ICH treated in our hospital from January 2017 to January 2020 were collected. The t-test, Chi-squared test and Fisher's exact test were used to analyze the clinical baseline data. Among 1021 spontaneous ICH patients, 193 patients who underwent hematoma evacuation were included in the present analysis. RESULTS: The Glasgow Outcome Scale (GOS) score at 6 months had a favorable prognosis in ES group (p = 0.003). ES group had fewer postoperative complications compared with CC group. Operating time and intraoperative blood loss were significantly lower in ES group than CC group (p = 0.001 and p = 0.002). CONCLUSIONS: Our study revealed that receiving ES improved the prognosis of ICH patients. Additionally, endoscopic surgery diminishes operative time, and intraoperative blood loss and reduces the incidence of postoperative complications.

Lemur tyrosine kinase 2 has a tumor-inhibition function in human glioblastoma by regulating the RUNX3/Notch pathway.

Deregulation of lemur tyrosine kinase 2 (LMTK2) is a vital determinant for the onset and progression of malignancies, yet the relationship between LMTK2 and glioblastoma (GBM) is undetermined. This study was carried out to determine the relevance of LMTK2 in GBM. Initiating investigation by assessing The Cancer Genome Atlas (TCGA) data showed LMTK2 mRNA levels were decreased in GBM tissue. Later examination of clinical specimens confirmed low levels of LMTK2 mRNA and protein in GBM tissue. The downregulated level of LMTK2 in patients with GBM was related to poor overall survival. A suppressive function of LMTK2 on the proliferative capability and metastatic potential of GBM cells was demonstrated by overexpressing LMTK2 in GBM cell lines. Moreover, the restoration of LMTK2 augmented the sensitivity of GBM cells to the chemotherapy drug temozolomide. The mechanistic investigation uncovered LMTK2 as a regulator of the runt-related transcription factor 3 (RUNX3)/Notch signaling pathway. The overexpression of LMTK2 increased the expression of RUNX3 while inhibiting the activation of Notch signaling. The silencing of RUNX3 diminished the regulatory role of LMTK2 on Notch signaling. The inhibition of Notch signaling reversed the LMTK2-silencing-elicited protumor effects. Importantly, LMTK2-overexpressed GBM cells displayed weakened tumorigenicity in xenograft models. Our findings illustrate that LMTK2 has a tumor-inhibition function in GBM by constraining Notch signaling via RUNX3. This work indicates the deregulation of the LMTK2-mediated RUNX3/Notch signaling pathway may be a novel molecular mechanism for the malignant transformation of GBMs. This work highlights the interest in LMTK2-related approaches for treating GBM.

Macrophage/Microglia Sirt3 Contributes to the Anti-inflammatory Effects of Resveratrol Against Experimental Intracerebral Hemorrhage in Mice.

Intracerebral hemorrhage (ICH) is a devastating stroke type with high mortality and disability. Inflammatory response induced by macrophages/microglia (M/Ms) activation is one of the leading causes of brain damage after ICH. The anti-inflammatory effects of resveratrol (RSV) have already been evaluated in several models of central nervous system disease. Therefore, we designed the current study to assess the role of RSV in ICH and explore its downstream mechanism related to Sirt3. The autologous artery blood injection was administrated to create an ICH mouse model. M/Ms-specific Sirt3 knockout Sirt3f/f; CX3CR1-Cre (Sirt3 cKO) mouse was used to evaluate the role of Sirt3 on RSV treatment. Neuronal function and hematoma volume were assessed to indicate brain damage. The pro-inflammatory marker (CD16) and cytokine (TNF) were measured to evaluate the inflammatory effects. Our results showed that RSV treatment alleviates neurological deficits, reduces cell death, and increases hematoma clearance on day 7 after ICH. In addition, RSV effectively suppressed CD16+ M/Ms activation and decreased TNF release. In Sirt3 cKO mice, the protective effects of RSV were abolished, indicating the potential mechanism of RSV was partially due to Sirt3 signaling activation. Therefore, RSV could be a promising candidate and therapeutic agent for ICH and Sirt3 could be a potential target to inhibit inflammation.

Stromal Interaction Molecule 1-Mediated Store-Operated Calcium Entry Promotes Autophagy Through AKT/Mammalian Target of Rapamycin Pathway in Hippocampal Neurons After Ischemic Stroke.

The pathophysiological process of neuronal injury due to cerebral ischemia is complex among which disturbance of calcium homeostasis and autophagy are two major pathogenesis. However, it remains ambiguous whether the two factors are independent. Stromal interaction molecule 1 (STIM1) is the most important Ca2+ sensor mediating the store-operated Ca2+ entry (SOCE) through interacting with Orai1 and has recently been proven to participate in autophagy in multiple cells. In this study, we aimed to investigate the potential role of STIM1-induced SOCE on autophagy and whether its regulator function contributes to neuronal injury under hypoxic conditions using in vivo transient middle cerebral artery occlusion (tMCAO) model and in vitro oxygen and glucose deprivation (OGD) primary cultured neuron model respectively. The present data indicated that STIM1 induces autophagic flux impairment in neurons through promoting SOCE and inhibiting AKT/mTOR signaling pathway. Pharmacological inhibition of SOCE or downregulation of STIM1 with siRNA suppressed the autophagic activity in neurons. Moreover, stim1 knockdown attenuated neurological deficits and brain damage after tMCAO, which could be reversed by AKT/mTOR pathway inhibitor AZD5363. Together, the modulation of STIM1 on autophagic activation indicated the potential link between Ca2+ homeostasis and autophagy which provided evidence that STIM1 could be a promising therapeutic target for ischemic stroke.

Pulsed Electromagnetic Fields Protect Against Brain Ischemia by Modulating the Astrocytic Cholinergic Anti-inflammatory Pathway.

Neuroinflammation is one of the most important pathological processes following brain ischemia. Pulsed electromagnetic fields (PEMFs) protect against brain ischemia, but their role in regulating neuroinflammation remains unclear. In the present study, we investigated the biological effects of PEMF exposure on brain ischemia-induced neuroinflammation through the astrocytic cholinergic anti-inflammatory pathway. PEMF exposure reduced the activation of astrocytes and neuroinflammation following brain ischemia by directly modulating astrocytic injury and inflammatory cytokine release. Inhibition of nicotinic acetylcholine receptor alpha 7 subunit (α7nAChR) by a specific antagonist reversed the regulatory effects of PEMF on astrocytes. Furthermore, negative regulation of signal transducer and activator of transcription 3 (STAT3) by α7nAChR was found to be an important downstream mechanism through which PEMF regulates astrocyte-related neuroinflammation. PEMF suppressed STAT3 phosphorylation and nuclear translocation by activating α7nAChR. These results demonstrate that PEMF exerts anti-inflammatory effects in the context of brain ischemia by modulating astrocytic α7nAChR/STAT3 signaling.

Protective role of TLR9-induced macrophage/microglia phagocytosis after experimental intracerebral hemorrhage in mice.

INTRODUCTION: Intracerebral hemorrhage (ICH) causes devastating morbidity and mortality, and studies have shown that the toxic components of hematomas play key roles in brain damage after ICH. Recent studies have found that TLR9 participates in regulating the phagocytosis of peripheral macrophages. The current study examined the role of TLR9 in macrophage/microglial (M/M) function after ICH. METHODS: RAW264.7 (macrophage), BV2 (microglia), and HT22# (neurons) cell lines were transfected with lentivirus for TLR9 overexpression. Whole blood from C57BL/6 or EGFPTg/+ mice was infused for phagocytosis and injury experiments, and brusatol was used for the experiments. Intraperitoneal injection of the TLR9 agonist ODN1826 or control ODN2138 was performed on days 1, 3, 5, 7, and 28 after ICH to study the effects of TLR9 in mice. In addition, clodronate was coinjected in M/M elimination experiments. The brains were collected for histological and protein experiments at different time points after ICH induction. Cellular and histological methods were used to measure hematoma/iron residual, M/Ms variation, neural injury, and brain tissue loss. Behavioral tests were performed premodeling and on days 1, 3, 7, and 28 post-ICH. RESULTS: Overexpression of TLR9 facilitated M/M phagocytosis and protected neurons from blood-derived hazards in vitro. Furthermore, ODN1826 boosted M/M activation and phagocytic function, facilitated hematoma/iron resolution, reduced brain injury, and improved neurological function recovery in ICH mice, which were abolished by clodronate injection. The experimental results indicated that the Nrf2/CD204 pathway participated in TLR9-induced M/M phagocytosis after ICH. CONCLUSION: Our study suggests a protective role for TLR9-enhanced M/M phagocytosis via the Nrf2/CD204 pathway after ICH. Our findings may serve as potential targets for ICH treatment.

Exosomes Derived From Adipose-Derived Mesenchymal Stem Cells Ameliorate Radiation-Induced Brain Injury by Activating the SIRT1 Pathway.

OBJECTIVE: Studies have shown that the therapeutic effects of mesenchymal stem cells (MSCs) are mediated in a paracrine manner, mainly through extracellular vesicles such as exosomes. Here, we designed a study to investigate whether exosomes derived from adipose-derived mesenchymal stem cells (ADMSC-Exos) had protective effects in a rat model of radiation-induced brain injury and in microglia. METHODS: Male adult Sprague-Dawley (SD) rats were randomly divided into three groups: the control group, the radiation group (30 Gy), and the radiation + exosomes group (30 Gy + 100 ug exosomes). Meanwhile, microglia were divided into four groups: the control group, the radiation group (10 Gy), the radiation + exosomes group (10 Gy + 4 ug exosomes), and radiation + exosomes + EX527 group (10 Gy + 4 ug exosomes + 100 nM EX527). Tissue samples and the levels of oxidative stress and inflammatory factors in each group were compared. RESULTS: Statistical analysis showed that after irradiation, ADMSC-Exos intervention in vivo significantly reduced the levels of caspase-3, malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), and promoted the recovery of superoxide dismutase (SOD), catalase (CAT), IL-4, and IL-10. Moreover, ADMSC-Exos intervention inhibited microglial infiltration and promoted the expression of SIRT1. Furthermore, the results in vitro showed that the above effects of ADMSC-Exos could be reversed by SIRT-1 inhibitor EX527. CONCLUSION: This study demonstrated that ADMSC-Exos exerted protective effects against radiation-induced brain injury by reducing oxidative stress, inflammation and microglial infiltration via activating the SIRT1 pathway. ADMSC-Exos may serve as a promising therapeutic tool for radiation-induced brain injury.

Antibiotics-induced intestinal dysbacteriosis caused behavioral alternations and neuronal activation in different brain regions in mice.

Antibiotics affect gut microbial composition, leading to Gut-Brain-Axis imbalance and neurobehavioral changes. However, the intestinal dysbacteriosis associated behavior changes are not consistently reported. It is not clear whether these changes are transient or permanent. The neuroprotective effect of probiotics against intestinal dysbacteriosis induced alternations needs to be determined either. In the present study, oral antibiotic mixture including Ampicillin, Streptomycin, and Clindamycin was utilized to induce intestinal dysbacteriosis in mice. Antibiotics application triggered mechanical allodynia in von frey test and spontaneous pain in open field test. It also resulted in increased anxiety and depressive-like behaviors and damaged spatial memory performance. After application of probiotics, the mechanical allodynia and spontaneous pain were alleviated significantly. The anxiety behaviors, depressive-like behaviors and recognitive performance were ameliorative as well. By using Fos protein as a marker, it is found that the sensory, emotion and memory related brain regions were activated in mice with intestinal dysbacteriosis. Our study is not only helpful for enriching our basic knowledge for understanding the changed pain responses and related brain disorders in antibiotics-induced dysbacteriosis mice, but also beneficial for providing a more comprehensive mechanistic explanation for the regulation of antibiotics and probiotics on gut microbiota and relevant alternations in animal neurological behaviors.

Iduna protects HT22 cells by inhibiting parthanatos: The role of the p53-MDM2 pathway.

Traumatic brain injury (TBI) is common and often fatal in current times. The role of poly(adenosine diphosphate-ribose) polymerase (PARP)-induced cell death (parthanatos) in TBI has not been well studied. Our past study showed that oxidative stress-induced cell death includes parthanatos by confirming the occurrence of PARP activation and nuclear translocation of apoptosis-inducing factor (AIF). As oxidative stress plays a key role in pathological progression after TBI, we believe TBI may also be alleviated by the expression of Iduna, which is the only known endogenous regulator of parthanatos. Thus, a transection model in HT-22 cells was established for present study. Downregulation of Iduna aggravated the cell damage caused by mechanical cell injury, whereas upregulation of Iduna reduced mitochondrial dysfunction induced by mechanical cell injury but exerted no effect on apoptosis associated with mitochondrial dysfunction. By contrast, Iduna prevented parthanatos by reducing PARP activation and nuclear translocation of AIF. We also investigated 2 novel p53-MDM2 pathway inhibitors, AMG 232 and Nutlin-3, which substantially reduced the protective effects of Iduna. These findings indicate that Iduna might prevent TBI by specifically inhibiting parthanatos and promoting mitochondrial function, with the p53-MDM2 pathway playing a critical role.

Pulsed electromagnetic fields regulate osteocyte apoptosis, RANKL/OPG expression, and its control of osteoclastogenesis depending on the presence of primary cilia.

Growing evidence has shown that pulsed electromagnetic fields (PEMF) can modulate bone metabolism in vivo and regulate the activities of osteoblasts and osteoclasts in vitro. Osteocytes, accounting for 95% of bone cells, act as the major mechanosensors in bone for transducing external mechanical signals and producing cytokines to regulate osteoblastic and osteoclastic activities. Targeting osteocytic signaling pathways is becoming an emerging therapeutic strategy for bone diseases. We herein systematically investigated the changes of osteocyte behaviors, functions, and its regulation on osteoclastogenesis in response to PEMF. The osteocyte-like MLO-Y4 cells were exposed to 15 Hz PEMF stimulation with different intensities (0, 5, and 30 Gauss [G]) for 2 hr. We found that the cell apoptosis and cytoskeleton organization of osteocytes were regulated by PEMF with an intensity-dependent manner. Moreover, PEMF exposure with 5 G significantly inhibited apoptosis-related gene expression and also suppressed the gene and protein expression of the receptor activator of nuclear factor κB ligand/osteoprotegerin (RANKL/OPG) ratio in MLO-Y4 cells. The formation, maturation, and osteoclastic bone-resorption capability of in vitro osteoclasts were significantly suppressed after treated with the conditioned medium from PEMF-exposed (5 G) osteocytes. Our results also revealed that the inhibition of osteoclastic formation, maturation, and bone-resorption capability induced by the conditioned medium from 5 G PEMF-exposed osteocytes was significantly attenuated after abrogating primary cilia in osteocytes using the polaris siRNA transfection. Together, our findings highlight that PEMF with 5 G can inhibit cellular apoptosis, modulate cytoskeletal distribution, and decrease RANKL/OPG expression in osteocytes, and also inhibit osteocyte-mediated osteoclastogenesis, which requires the existence of primary cilia in osteocytes. This study enriches our basic knowledge for further understanding the biological behaviors of osteocytes and is also helpful for providing a more comprehensive mechanistic understanding of the effect of electromagnetic stimulation on bone and relevant skeletal diseases (e.g., bone fracture and osteoporosis).

Differential intensity-dependent effects of pulsed electromagnetic fields on RANKL-induced osteoclast formation, apoptosis, and bone resorbing ability in RAW264.7 cells.

Pulsed electromagnetic fields (PEMF) have been proven to be effective for promoting bone mass and regulating bone turnover both experimentally and clinically. However, the exact mechanisms for the regulation of PEMF on osteoclastogenesis as well as optical exposure parameters of PEMF on inhibiting osteoclastic activities and functions remain unclear, representing significant limitations for extensive scientific application of PEMF in clinics. In this study, RAW264.7 cells incubated with RANKL were exposed to 15 Hz PEMF (2 h/day) at various intensities (0.5, 1, 2, and 3 mT) for 7 days. We demonstrate that bone resorbing capacity was significantly decreased by 0.5 mT PEMF mainly by inhibiting osteoclast formation and maturation, but enhanced at 3 mT by promoting osteoclast apoptosis. Moreover, gene expression of RANK, NFATc1, TRAP, CTSK, BAX, and BAX/BCL-2 was significantly decreased by 0.5 mT PEMF, but increased by 3 mT. Our findings reveal a significant intensity window for low-intensity PEMF in regulating bone resorption with diverse nature for modulating osteoclastogenesis and apoptosis. This study not only enriches our basic knowledge for the regulation of PEMF in osteoclastogenesis, but also may lead to more efficient and scientific clinical application of PEMF in regulating bone turnover and inhibiting osteopenia/osteoporosis. Bioelectromagnetics. 38:602-612, 2017. © 2017 Wiley Periodicals, Inc.

Early Erythrolysis in the Hematoma After Experimental Intracerebral Hemorrhage.

Erythrolysis occurs in the clot after intracerebral hemorrhage (ICH), and the release of hemoglobin causes brain injury, but it is unclear when such lysis occurs. The present study examined early erythrolysis in rats. ICH rats had an intracaudate injection of 100 µl autologous blood, and sham rats had a needle insertion. All rats had T2 and T2* magnetic response imaging (MRI) scanning, and brains were used for histology and CD163 (a hemoglobin scavenger receptor) and DARPP-32 (a neuronal marker) immunohistochemistry. There was marked heterogeneity within the hematoma on T2* MRI, with a hyperintense or isointense core and a hypointense periphery. Hematoxylin and eosin staining in the same animals showed significant erythrolysis in the core with the formation of erythrocyte ghosts. The degree of erythrolysis correlated with the severity of perihematomal neuronal loss. Perihematomal CD163 was increased by day 1 after ICH and may be involved in clearing hemoglobin caused by early hemolysis. Furthermore, ICH resulted in more severe erythrolysis, neuronal loss, and perihematomal CD163 upregulation in spontaneously hypertensive rats compared to Wistar-Kyoto rats. In conclusion, T2*MRI-detectable early erythrolysis occurred in the clot after ICH and activated CD163. Hypertension is associated with enhanced erythrolysis in the hematoma.

Unplanned Reoperations in Neurosurgical Patients Due to Postoperative Bleeding: A Single-Center Experience and Literature Review.

The aim of this study is to investigate the incidence of unplanned reoperations from all causes due to bleeding in neurosurgical patients. The medical records of patients who received neurosurgical procedures at our hospital were retrospectively reviewed and data of patients who received reoperations were extracted and summarized. A literature review was conducted of the Medline, Cochrane, EMBASE, and Google Scholar databases up to November 2013. The main outcome measure was the rate of unplanned reoperations due to bleeding. At our hospital, 68 patients with a mean age of 41.5 ±â€Š21.5 years (range, 7 months to 76 years) received an unplanned reoperation. More than 70% of the patients were older than 18 years, 64.7% were males, and 94.1% had cranial surgery. Almost 60% of the patients received >1 blood transfusion (58.8%) after the first surgery. Of the 68 patients, 35 (51.5%) received a second operation due to bleeding. Univariate logistic regression analysis only showed that an increasing time interval between the first and second surgery was associated with a decreased chance of the reoperation being performed due to bleeding (odds ratio [OR] = 0.843, 95% confidence interval [CI]: 0.720-0.987; P = .033). Of 229 studies identified, 5 retrospective reports with a total of 1375 patients were included in the analysis. The rate of reoperations for bleeding in the 5 studies ranged from 4.2% to 31.5%. Employing measures to reduce postoperative bleeding may help reduce the rate of unplanned neurosurgical reoperations.

Sirt3 attenuates hydrogen peroxide-induced oxidative stress through the preservation of mitochondrial function in HT22 cells.

Sirtuins (Sirt) are a family of phylogenetically conserved nicotinamide adenine nucleotide (NAD(+))-dependent protein deacetylases, among which Sirt3 resides primarily in the mitochondria and serves as a stress responsive deacetylase, playing a role in protecting cells from damage under stress conditions. The present study aimed to investigate the role of Sirt3 in hydrogen peroxide (H(2)O(2))-induced oxidative neuronal injury in HT22 mouse hippocampal cells. Treatment with H(2)O(2) increased the expression of Sirt3 in a dose- and time-dependent manner, and the knockdown of Sirt3 using specific small interfering RNA (siRNA) exacerbated the H(2)O(2)-induced neuronal injury. The overexpression of Sirt3 induced by lentiviral transfection significantly reduced the generation of reactive oxygen species (ROS) and lipid peroxidation following injury, whereas the activities of endogenous antioxidant enzymes were not affected. Further experiments revealed that the H(2)O(2)-induced inhibition of mitochondrial complex activity and adenosine triphosphate (ATP) synthesis, the decrease in mitochondrial Ca(2+) buffering capacity and mitochondrial swelling were all partly reversed by Sirt3. Furthermore, the overexpression of Sirt3 attenuated the release of cytochrome c, the increase in the Bax/Bcl-2 ratio, as well as caspase-9/caspase-3 activity induced by H(2)O(2), and eventually inhibited apoptotic neuronal cell death. These results suggest that Sirt3 acts as a prosurvival factor, playing an essential role in protecting HT22 cells under H(2)O(2)-induced oxidative stress, possibly by inhibiting ROS accumulation and the activation of the mitochondrial apoptotic pathway.

Activation of mGluR5 attenuates NMDA-induced neurotoxicity through disruption of the NMDAR-PSD-95 complex and preservation of mitochondrial function in differentiated PC12 cells.

Glutamate-mediated toxicity is implicated in various neuropathologic conditions, and activation of ionotropic and metabotropic glutamate receptors is considered to be the most important mechanism. It has been reported that pharmacological saturation of metabotropic glutamate receptors (mGluRs) can facilitate N-methyl-D-aspartate receptor (NMDAR) related signaling cascades, but the mechanism leading to mGluR-NMDAR interactions in excitotoxic neuronal injury has remained unidentified. In the present study, we investigated the role of mGluR5 in the regulation of N-methyl-D-aspartate (NMDA)-induced excitotoxicity in differentiated PC12 cells. We found that activation of mGluR5 with the specific agonist R,S-2-chloro-5-hydroxyphenylglycine (CHPG) increased cell viability and inhibited lactate dehydrogenase (LDH) release in a dose-dependent manner. CHPG also inhibited an increase in the Bax/Bcl-2 ratio, attenuated cleavage of caspase-9 and caspase-3, and reduced apoptotic cell death after NMDA treatment. The NMDA-induced mitochondrial dysfunction, as indicated by mitochondrial reactive oxygen species (ROS) generation, collapse of mitochondrial membrane potential (MMP), and cytochrome c release, was also partly prevented by CHPG treatment. Furthermore, CHPG blocked the NMDA-induced interaction of NMDAR with postsynaptic density protein-95 (PSD-95), but had no effects on intracellular calcium concentrations. All these results indicated that activation of mGluR5 protects differentiated PC12 cells from NMDA-induced neuronal excitotoxicity by disrupting NMDAR-PSD-95 interaction, which might be an ideal target for investigating therapeutic strategies in various neurological diseases where excitotoxicity may contribute to their pathology.

MicroRNA-34a induces apoptosis in the human glioma cell line, A172, through enhanced ROS production and NOX2 expression.

BACKGROUND: MicroRNA is a type of non-coding small RNA involved in regulating genes and signaling pathways through incomplete complementation with target genes. Recent research supports key roles of miRNA in the formation and development of human glioma. METHODS: The relative quantity of miR-34a was initially determined in human glioma A172 cells and glioma tissues. Next, we analyzed the impact of miR-34a on A172 cell viability with the MTT assay. The effects of miR-34a overexpression on apoptosis were confirmed with flow cytometry and Hoechst staining experiments. We further defined the target genes of miR-34a using immunofluorescence and Western blot. RESULTS: MiR-34a expression was significantly reduced in human glioma A172 cells and glioma tissue, compared with normal glial cells and tissue samples. Our MTT data suggest that up-regulation of miR-34a inhibits cell viability while suppression of miR-34a enhances cell viability. Flow cytometry and Hoechst staining results revealed increased rates of apoptosis in A172 human glioma cells overexpressing miR-34a. Using immunofluorescence and Western blot analyses, we identified NOX2 as a target of miR-34a in A172 cells. CONCLUSION: MiR-34a serves as a tumor suppressor in human glioma mainly by decreasing NOX2 expression.

Polymorphisms of EpCAM gene and prognosis for non-small-cell lung cancer in Han Chinese.

The epithelial cell adhesion molecule (EpCAM) is overexpressed in a wide variety of human cancers and is associated with patient prognosis, including those with lung cancer. However, the association of single nucleotide polymorphisms (SNPs) in the EpCAM gene with the prognosis for non-small-cell lung cancer (NSCLC) patients has never been investigated. We evaluated the association between two SNPs, rs1126497 and rs1421, in the EpCAM gene and clinical outcomes in a Chinese cohort of 506 NSCLC patients. The SNPs were genotyped using the Sequenom iPLEX genotyping system. Multivariate Cox proportional hazards model and Kaplan-Meier curves were used to assess the association of EpCAM gene genotypes with the prognosis of NSCLC. We found that the non-synonymous SNP rs1126497 was significantly associated with survival. Compared with the CC genotype, the CT+TT genotype was a risk factor for both death (hazard ratio, 1.40; 95% confidence interval [CI], 1.02-1.94; P = 0.040) and recurrence (hazard ratio, 1.34; 95% CI, 1.02-1.77; P = 0.039). However, the SNP rs1421 did not show any significant effect on patient prognosis. Instead, the AG+GG genotype in rs1421 was significantly associated with early T stages (T1/T2) when compared with the AA genotype (odds ratio for late stage = 0.65; 95% CI, 0.44-0.96, P = 0.029). Further stratified analysis showed notable modulating effects of clinical characteristics on the associations between variant genotypes of rs1126497 and NSCLC outcomes. In conclusion, our study indicated that the non-synonymous SNP rs1126497 may be a potential prognostic marker for NSCLC patients.

Iduna protects HT22 cells from hydrogen peroxide-induced oxidative stress through interfering poly(ADP-ribose) polymerase-1-induced cell death (parthanatos).

Oxidative stress-induced cell death is common in many neurological diseases. However, the role of poly(ADP-ribose) polymerase-1-induced cell death (parthanatos) has not been fully elucidated. Here, we found that hydrogen peroxide (H2O2) could lead to PARP-1 activation and apoptosis-inducing factor nuclear translocation in a concentration dependent manner. Iduna, as a novel regulator of parthanatos, was also induced by H2O2. Down-regulation of Iduna by genetic ablation promoted H2O2-induced cell damage. Up-regulation of Iduna reduced the loss of mitochondrial potential and ATP and NAD+ production, but did not affect the mitochondrial dysfunction-induced cytochrome c release, increase of Bax/Bcl-2 ratio, and Caspase-9/Caspase-3 activity. In contrast, overexpression of Iduna inhibited activation of PARP-1 and nuclear translocation of AIF. Further study showed that PARP-1 specific inhibitor, DPQ, blocked the protective effect of Iduna against H2O2-induced oxidative stress. Moreover, in the presence of proteasome inhibitor (MG-132) or ubiquitin E1 inhibitor (PYR-41), protective effect of Iduna was significantly weaken. These results indicate that Iduna acts as a potential antioxidant by improving mitochondrial function and inhibiting oxidative stress-induced parthanatos, and these protective effects are dependent on the involvement of ubiquitin-proteasome system.

Protective effect of Homer 1a against hydrogen peroxide-induced oxidative stress in PC12 cells.

Oxidative stress-induced cell damage is involved in many neurological diseases. Homer protein, as an important scaffold protein at postsynaptic density, regulates synaptic structure and function. Here, we reported that hydrogen peroxide (H(2)O(2)) induced the expression of Homer 1a. Down-regulation of Homer 1a with a specific small interfering RNA (siRNA) exacerbated H(2)O(2)-induced cell injury. Up-regulation of Homer 1a by lentivirus transfection did not affect the anti-oxidant activity, but significantly reduced the reactive oxygen species (ROS) production and lipid peroxidation after H(2)O(2)-induced oxidative stress. Overexpression of Homer 1a attenuated the loss of mitochondrial membrane potential (MMP) and ATP production induced by H(2)O(2), and subsequently inhibited mitochondrial dysfunction-induced cytochrome c release, increase of Bax/Bcl-2 ratio and caspase-9/caspase-3 activity. Furthermore, in the presence of BAPTA-AM, an intracellular free-calcium (Ca(2+)) chelator, overexpression of Homer 1a had no significant effects on H(2)O(2)-induced oxidative stress. These results suggest that Homer 1a has protective effects against H(2)O(2)-induced oxidative stress by reducing ROS accumulation and activation of mitochondrial apoptotic pathway, and these protective effects are dependent on the regulation of intracellular Ca(2+) homeostasis.