Meng-Qiu Li

Troxerutin Counteracts Domoic Acid–Induced Memory Deficits in Mice by Inhibiting CCAAT/Enhancer Binding Protein β–Mediated Inflammatory Response and Oxidative Stress

The C/EBP β is a basic leucine zipper transcription factor that regulates a variety of biological processes, including metabolism, cell proliferation and differentiation, and immune response. Recent findings show that C/EBP β–induced inflammatory responses mediate kainic acid–triggered excitotoxic brain injury. In this article, we show that protein kinase C ζ enhances K-ras expression and subsequently activates the Raf/MEK/ERK1/2 pathway in the hippocampus of domoic acid (DA)–treated mice, which promotes C/EBP β expression and induces inflammatory responses. Elevated production of TNF-α impairs mitochondrial function and increases the levels of reactive oxygen species by IκB kinase β/NF-κB signaling. The aforementioned inflammation and oxidative stress lead to memory deficits in DA-treated mice. However, troxerutin inhibits cyclin-dependent kinase 1 expression, enhances type 1 protein phosphatase α dephosphorylation, and abolishes MEK/ERK1/2/C/EBP β activation, which subsequently reverses the memory impairment observed in the DA-treated mice. Thus, troxerutin is recommended as a potential candidate for the prevention and therapeutic treatment of cognitive deficits resulting from excitotoxic brain damage and other brain disorders.

Luteoloside Suppresses Proliferation and Metastasis of Hepatocellular Carcinoma Cells by Inhibition of NLRP3 Inflammasome

The inflammasome is a multi-protein complex which when activated regulates caspase-1 activation and IL-1β secretion. Inflammasome activation is mediated by NLR proteins that respond to stimuli. Among NLRs, NLRP3 senses the widest array of stimuli. NLRP3 inflammasome plays an important role in the development of many cancer types. However, Whether NLRP3 inflammasome plays an important role in the process of hepatocellular carcinoma (HCC) is still unknown. Here, the anticancer effect of luteoloside, a naturally occurring flavonoid isolated from the medicinal plant Gentiana macrophylla, against HCC cells and the underlying mechanisms were investigated. Luteoloside significantly inhibited the proliferation of HCC cells in vitro and in vivo. Live-cell imaging and transwell assays showed that the migration and invasive capacities of HCC cells, which were treated with luteoloside, were significantly inhibited compared with the control cells. The inhibitory effect of luteoloside on metastasis was also observed in vivo in male BALB/c-nu/nu mouse lung metastasis model. Further studies showed that luteoloside could significantly reduce the intracellular reactive oxygen species (ROS) accumulation. The decreased levels of ROS induced by luteoloside was accompanied by decrease in expression of NLRP3 inflammasome resulting in decrease in proteolytic cleavage of caspase-1. Inactivation of caspase-1 by luteoloside resulted in inhibition of IL-1β. Thus, luteoloside exerts its inhibitory effect on proliferation, invasion and metastasis of HCC cells through inhibition of NLRP3 inflammasome. Our results indicate that luteoloside can be a potential therapeutic agent not only as an adjuvant therapy for HCC, but also, in the control and prevention of metastatic HCC.

Ursolic acid improves domoic acid-induced cognitive deficits in mice

Our previous findings suggest that mitochondrial dysfunction is the mechanism underlying cognitive deficits induced by domoic acid (DA). Ursolic acid (UA), a natural triterpenoid compound, possesses many important biological functions. Evidence shows that UA can activate PI3K/Akt signaling and suppress Forkhead box protein O1 (FoxO1) activity. FoxO1 is an important regulator of mitochondrial function. Here we investigate whether FoxO1 is involved in the oxidative stress-induced mitochondrial dysfunction in DA-treated mice and whether UA inhibits DA-induced mitochondrial dysfunction and cognitive deficits through regulating the PI3K/Akt and FoxO1 signaling pathways. Our results showed that FoxO1 knockdown reversed the mitochondrial abnormalities and cognitive deficits induced by DA in mice through decreasing HO-1 expression. Mechanistically, FoxO1 activation was associated with oxidative stress-induced JNK activation and decrease of Akt phosphorylation. Moreover, UA attenuated the mitochondrial dysfunction and cognitive deficits through promoting Akt phosphorylation and FoxO1 nuclear exclusion in the hippocampus of DA-treated mice. LY294002, an inhibitor of PI3K/Akt signaling, significantly decreased Akt phosphorylation in the hippocampus of DA/UA mice, which weakened UA actions. These results suggest that UA could be recommended as a possible candidate for the prevention and therapy of cognitive deficits in excitotoxic brain disorders.

CERS2 Suppresses Tumor Cell Invasion and is Associated with Decreased V-ATPase and MMP-2/MMP-9 Activities in Breast Cancer

Ceramide synthase 2 (CERS2) is the gene identified from a human liver cDNA library in 2001. Our previous studies have shown higher expression of CERS2 in the breast cancer patients was associated with fewer lymph node metastases. However, the molecular mechanism of CERS2 involved is unknown. Here, we found CERS2 was heterogeneously expressed in various breast cancer cells. The mRNA and protein expression levels of CERS2 in MCF7 cells, which are poorly invasive breast cancer cells, were obviously higher than that in the highly invasive cells MDA‐MB‐231. Results showed overexpression of CERS2 in MDA‐MB‐231 cells could significantly inhibit the migration and invasion ability, whereas CERS2 knockdown in MCF7 cells could significantly increase the migration and invasion ability. Overexpression of CERS2 in MDA‐MB‐231 cells significantly reduced the V‐ATPase activity, increased the extracellular pH and decreased the pH‐dependent activity of MMP‐2 and MMP‐9 matrix metalloproteinases (MMPs). CERS2 knockdown in MCF7 cells significantly increased the V‐ATPase activity, decreased the extracellular pH and increased the activity of MMP‐2 and MMP‐9. Taken together, CERS2 can significantly inhibit breast cancer cell invasion and is associated with the decrease of the V‐ATPase activity and extracellular hydrogen ion concentration, and in turn the activation of secreted MMP‐2/MMP‐9 and degradation of extracellular matrix (ECM), which ultimately suppressed tumors invasion. Thus, CERS2 may represent a novel target for selectively disrupting V‐ATPase activity and the invasive potential of cancer cells. J. Cell. Biochem. 116: 502–513, 2015.

Small Interfering RNA–Mediated Knockdown of Protein Kinase C Zeta Attenuates Domoic Acid–Induced Cognitive Deficits in Mice

Accumulated evidence indicates that domoic acid (DA)-induced excitatory neuronal injury is associated with excessive reactive oxygen species (ROS) production. Protein kinase C zeta (PKC-ζ)/nicotinamide adenine dinucleotide phosphate (NOX) signaling regulates ROS levels and is involved in various neurodegenerative disorders including excitoneurotoxicity. Our previous studies have demonstrated that ROS-induced activation of the stress-activated protein kinase/c-jun-N-terminal kinase (SAPK/JNK) pathway plays a key role in the pathogenesis of cognitive deficits induced by DA. However, the precise biological mechanisms underlying these effects are not well understood. In this study, we investigate whether the PKC-ζ mediates DA-induced cognitive deficits and further explored the potential molecular processes. DA treatment significantly increased the expression of PI3K p85α, and PKC-ζ in the hippocampus of mice, which promoted the p47phox phosphorylation and expression, enhanced NOX activity, and increased the levels of ROS and protein carbonyls. In turn, the abnormal ROS levels in the hippocampus of DA-treated mice activated SAPK/JNK pathway, decreased FoxO1 phosphorylation, stimulated the nuclear translocation of FoxO1, activated FasL/Fas signaling, and promoted the activation of caspase-8 and caspase-3, which resulted in neuron apoptosis and cognitive deficits in mice. However, PKC-ζ knockdown reversed these changes in mice. It was further demonstrated that FoxO1 was a downstream target of SAPK/JNK signaling by FoxO1 small interfering RNA and SP600125 (an inhibitor of SAPK/JNK pathway) treatment. Additionally, SP600125 treatment or FoxO1 knockdown also blocked FasL/Fas signaling-dependent apoptosis and improved DA-induced cognitive deficits in the hippocampus of mice. These results suggest that PKC-ζ could be a possible target for the prevention or treatment of cognitive deficits in excitotoxic and other brain disorders.

Relationship Between Neonatal Vitamin D at Birth and Risk of Autism Spectrum Disorders: the NBSIB Study

Previous studies suggested that lower vitamin D might be a risk factor for autism spectrum disorders (ASDs). The aim of this study was to estimate the prevalence of ASDs in 3‐year‐old Chinese children and to examine the association between neonatal vitamin D status and risk of ASDs. We conducted a study of live births who had taken part in expanded newborn screening (NBS), with outpatient follow‐up when the children 3‐year old. The children were confirmed for ASDs in outpatient by the Autism Diagnostic Interview‐Revised and Diagnostic and Statistical Manual of Mental Disorders (DSM)‐5 criteria. Intellectual disability (ID) status was defined by the intelligence quotient (IQ < 80) for all the participants. The study design included a 1:4 case to control design. The concentration of 25‐hydroxyvitamin D3 [25(OH)D3] in children with ASD and controls were assessed from neonatal dried blood samples. A total of 310 children were diagnosed as having ASDs; thus, the prevalence was 1.11% (95% CI, 0.99% to 1.23%). The concentration of 25(OH)D3 in 310 ASD and 1240 controls were assessed. The median 25(OH)D3 level was significantly lower in children with ASD as compared to controls (p < 0.0001). Compared with the fourth quartiles, the relative risk (RR) of ASDs was significantly increased for neonates in each of the three lower quartiles of the distribution of 25(OH)D3, and increased risk of ASDs by 260% (RR for lowest quartile: 3.6; 95% CI, 1.8 to 7.2; p < 0.001), 150% (RR for second quartile: 2.5; 95% CI, 1.4 to 3.5; p = 0.024), and 90% (RR for third quartile: 1.9; 95% CI, 1.1 to 3.3; p = 0.08), respectively. Furthermore, the nonlinear nature of the ID‐risk relationship was more prominent when the data were assessed in deciles. This model predicted the lowest relative risk of ID in the 72rd percentile (corresponding to 48.1 nmol/L of 25(OH)D3). Neonatal vitamin D status was significantly associated with the risk of ASDs and intellectual disability. The nature of those relationships was nonlinear.

Protective effects of microRNA-431 against cerebral ischemia-reperfusion injury in rats by targeting the Rho/Rho-kinase signaling pathway

This study investigates the protective effects of miR‐431 against cerebral ischemia‐reperfusion injury through the Rho/Rho‐kinase signaling pathway. SD rats were randomly classified into normal, sham, and model (middle cerebral artery occluded) groups. Rho expression and cerebral infarction were visualized by immunohischemistry and TTC staining, respectively. qRT‐PCR and western blotting were used to measure mRNA and protein expression of miR‐431 and Rho/Rho‐kinase signaling pathway‐related genes. Hippocampal neurons were extracted and assigned into normal, blank, negative control (NC), miR‐431 mimics, miR‐431 inhibitors, siRNA‐Rho, and miR‐431 inhibitors + siRNA‐Rho groups. Proliferation and apoptosis were detected by MTT and flow cytometry, respectively. Compared with the normal group, the model group showed elevated Rho expression, area of cerebral infarction, and expressions of Rho/Rho‐kinase related genes but reduced miR‐431 expression. Compared with the blank group, expression of Rho, Rho‐kinase α, and Rho‐kinase β decreased and miR‐431 expression increased in the miR‐431 mimics and siRNA‐Rho groups, and the tendency reversed in the miR‐431 inhibitors group. Enhanced proliferation and inhibited apoptosis were exhibited in the miR‐431 mimics and siRNA‐Rho groups while results in the miR‐431 inhibitors group reversed. Findings obtained from this study indicated that miR‐431 confers protection against cerebral ischemia‐reperfusion injury through negatively regulating the Rho/Rho‐kinase signaling pathway.

Effects of long noncoding RNA SPRY4-IT1-mediated EZH2 on the invasion and migration of lung adenocarcinoma

We aim to investigate the interaction between the EZH2 and the long noncoding RNA (lncRNA) SPRY4‐IT1. We also explore their respective effects on human lung adenocarcinoma (LA) cell invasion and migration. Both LA and adjacent normal tissues were obtained from 256 LA patients. SPTY4‐IT expression and EZH2 mRNA expressions in tissues and cells were detected by reverse transcription quantitative polymerase chain reaction (RT‐qPCR). The siRNAs against SPRY4‐IT1 and EZH2 were co‐transfected into A549 and H1975 cells. The interaction between SPRY4‐IT1 and EZH2 was determined using a RNA pull‐down assay and a RNA immunoprecipitation (RIP) assay. A Transwell assay and scratch assay were used to evaluate the cell migration and invasion abilities. The expressions of E‐cadherin and Vimentin in the epithelial‐mesenchymal transition (EMT) and EZH2 protein expression were detected through western blotting. SPRY4‐IT1 expression was observed to be significantly lower, while the expression of EZH2 was higher in the LA tissues than in the adjacent normal tissues. Compared with the HBE cell line, expressions of SPRY4‐IT1 in each human LA cell line had decreased, with the lowest observed reduction in the A549 cell line, while EZH2 mRNA and protein expression increased in each human LA cell lines. After SPRY4‐IT1‐siRNA was transfected into A549 and H1975 cells, invasion and migration abilities were enhanced, in addition to a reduction in the expression of E‐cadherin, while expressions of Vimentin exhibited an increased rate. Consequently, we find that EZH2 promotes LA cell invasion and metastasis by inhibiting SPRY4‐IT1 expression.

LncRNA LINC00880 promotes cell proliferation, migration, and invasion while inhibiting apoptosis by targeting CACNG5 through the MAPK signaling pathway in spinal cord ependymoma

The present study was to investigate the effect of lncRNA LINC00880 targeting CACNG5 on cell proliferation, migration, invasion, and apoptosis in spinal cord ependymoma (SCE) through the MAPK signaling pathway. GEO database was used to download gene expression data related with SCE (GSE50161 and GSE66354) and annotation file. LncRNA with differential expression was predicted by Multi Experiment Matrix website (MEM). The target gene was analyzed by KEGG pathway enrichment analysis. SCE tissues and adjacent tissues were collected. The positive expression of CACNG5 protein was tested by immunohistochemistry. Expression of LINC00880, CACNG5, and MAPK signaling pathway‐related proteins was measured with qRT‐PCR and Western blotting. Cell proliferation, migration, invasion, cycle, and apoptosis were detected using MTT, Transwell assay, Scratch test, and Flow cytometry. SCE tissues showed increased LINC00880 expression. CACNG5 was a target gene of LINC00880 and correlated with MAPK signaling pathway. Compared with adjacent tissues, SCE tissues showed lower positive expression of CACNG5. Compared with the blank group, LINC00880 expression was higher in the LINC00880 vector and LINC00880 vector + CACNG5 vector groups, and lower in the si‐LINC00880 and si‐LINC00880 + si‐CACNG5 groups; in the LINC00880 vector and si‐CACNG5 groups, expression of survivin, p38MAPK, ERK1/2, JNK1/2/3 increased and CACNG5 and Bax expression reduced, the proliferation, invasion and migration of tumor cells increased, and apoptosis rate decreased. Opposite results were found in the si‐LINC00880 and CACNG5 vector groups. The findings indicate that lncRNA LINC00880 targeting CACNG5 inhibits cell apoptosis and promotes proliferation, migration, and invasion in SCE through the MAPK signaling pathway.

Troxerutin Protects Kidney Tissue against BDE-47-Induced Inflammatory Damage through CXCR4-TXNIP/NLRP3 Signaling

2,2′,4,4′-Tetrabromodiphenyl ether (BDE-47) induces oxidative stress in kidney cells, but the underlying mechanism remains poorly understood. Troxerutin, a natural flavonoid, has potential antioxidant and anti-inflammatory efficacy. In this study, we assessed the effect of troxerutin on kidney damage caused by BDE-47 and investigated the underlying mechanism. The results showed troxerutin reduced reactive oxygen species (ROS) level and urine albumin-to-creatinine ratio (ACR), decreased the activities of inflammatory factors including cyclooxygenase-2 (COX-2), induced nitric oxide synthase (iNOS) and nuclear factor kappa B (NF-κB) in the kidney tissues of BDE-47-treated mice. Furthermore, troxerutin significantly weakened the expression of kidney NLRP3 inflammasome containing NLRP3, ASC, and caspase-1, contributing to the decline of IL-1β. Additionally, troxerutin inhibited the increased protein level of stromal-derived factor-1(SDF-1), C-X-C chemokine ligand 12 receptor 4 (CXCR4), and thioredoxin interaction protein (TXNIP) caused by BDE-47. Specifically, the immunoprecipitation assay indicated that there was a direct interaction between CXCR4 and TXNIP. CXCR4 siRNA and TXNIP siRNA also decreased the inflammatory damage, which was similar to the action of troxerutin. Our data demonstrated that troxerutin regulated the inflammatory lesions via CXCR4-TXNIP/NLRP3 inflammasome in the kidney of mice induced by BDE-47.