NMDA receptor inhibitor MK801 alleviated pro-inflammatory polarization of BV-2 microglia cells.

Microglia have both protective and pathogenic properties, while polarization plays a decisive role in their functional diversity. Apart from being an energetic organelle, mitochondria possess biological capabilities of signaling and immunity involving mitochondrial dynamics. The N-methyl-D-aspartate (NMDA)-type glutamate receptor displays excitatory neurotransmission, excitatory neurotoxicity and pro-inflammatory properties in a membrane location- and cell context-dependent manner. In this study, we have provided experimental evidence showing that by acting on mitochondrial dynamics, NMDA receptors displayed pro-inflammatory properties, while its non-competitive inhibitor MK801 exhibited anti-inflammatory potential in Lipopolysaccharide (LPS)-challenged BV-2 microglia cells. LPS stimulation increased the protein phosphorylation of cells regarding their NMDA receptor component subunits and Calcium/Calmodulin-dependent Protein Kinase II (CaMKII), along with mobilizing intracellular calcium. Additionally, parallel changes occurred in the activation of Transforming Growth Factor-ß (TGF-ß)-Activated Kinase 1 (TAK1), NF-κB p65 and NF-κB DNA binding activity, acquisition of pro-inflammatory M1 polarization and expression of pro-inflammatory cytokines. LPS-treated cells further displayed signs of mitochondrial dysfunction with higher expressions of the active form of Dynamin-Related Protein 1 (Drp1), NADPH Oxidase-2 (NOX2) expression and the generation of DCFDA-/MitoSOX-sensitive Reactive Oxygen Species (ROS). NMDA receptor blockade by MK801, along with CaMKII inhibitor KN93, Drp1 inhibitor Mdivi-1 and antioxidant apocynin alleviated LPS-induced pro-inflammatory changes. Other than the reported CaMKII/TAK1/NF-κB axis, our in vitro study revealed the CaMKII/Drp1/ROS/NF-κB axis being an alternative cascade for shaping pro-inflammatory phenotypes of microglia upon LPS stimulation, and MK801 having the potential for inhibiting microglia activation and any associated inflammatory damages.

Tetramethylpyrazine alleviates mitochondrial abnormality in models of cerebral ischemia and oxygen/glucose deprivation Reoxygenation.

Traditional herbal medicine Ligusticum wallichii Franchat (Chuan Xiong) is frequently prescribed and highly recommended to patients with stroke. Rodent studies have demonstrated the neuroprotective effects of its active component tetramethylpyrazine against post-stroke brain injury and highlighted its role in antioxidant, anti-inflammation, and anti-apoptosis activity. Using permanent cerebral ischemia in rats and oxygen/glucose deprivation and reoxygenation (OGDR) in rat primary neuron/glia cultures, this study sheds light on the role of mitochondria as crucial targets for tetramethylpyrazine neuroprotection. Tetramethylpyrazine protected against injury and alleviated oxidative stress, interleukin-1ß release, and caspase 3 activation both in vivo and in vitro. Reduction of mitochondrial biogenesis- and integrity-related proliferator-activated receptor-gamma coactivator-1 alpha, mitochondrial transcription factor A (TFAM), translocase of outer mitochondrial membrane 20, mitochondrial DNA, and citrate synthase activity, as well as activation of mitochondrial dynamics disruption-related Lon protease, dynamin-related protein 1 (Drp1) phosphorylation, stimulator of interferon genes, TANK-binding kinase 1 phosphorylation, protein kinase RNA-like endoplasmic reticulum kinase phosphorylation, eukaryotic initiation factor 2α phosphorylation, and activating transcription factor 4 were revealed in permanent cerebral ischemia in rats and OGDR in neuron/glia cultures. TMP alleviated those biochemical changes. Our findings suggest that preservation or restoration of mitochondrial dynamics and functional integrity and alleviation of mitochondria-oriented pro-oxidant, pro-inflammatory, and pro-apoptotic cascades are alternative neuroprotective mechanisms of tetramethylpyrazine. Additionally, mitochondrial TFAM and Drp1 as well as endoplasmic reticulum stress could be targeted by TMP to induce neuroprotection. Data of this study provide experimental base to support clinical utility and value of Chuan Xiong towards stroke treatment and highlight an alternative neuroprotective target of tetramethylpyrazine.

α7 nicotinic acetylcholine receptor agonist improved brain injury and impaired glucose metabolism in a rat model of ischemic stroke.

Vagus nerve stimulation through the action of acetylcholine can modulate inflammatory responses and metabolism. α7 Nicotinic Acetylcholine Receptor (α7nAChR) is a key component in the biological functions of acetylcholine. To further explore the health benefits of vagus nerve stimulation, this study aimed to investigate whether α7nAChR agonists offer beneficial effects against poststroke inflammatory and metabolic changes and to identify the underlying mechanisms in a rat model of stroke established by permanent cerebral ischemia. We found evidence showing that pretreatment with α7nAChR agonist, GTS-21, improved poststroke brain infarction size, impaired motor coordination, brain apoptotic caspase 3 activation, dysregulated glucose metabolism, and glutathione reduction. In ischemic cortical tissues and gastrocnemius muscles with GTS-21 pretreatment, macrophages/microglia M1 polarization-associated Tumor Necrosis Factor-α (TNF-α) mRNA, Cluster of Differentiation 68 (CD68) protein, and Inducible Nitric Oxide Synthase (iNOS) protein expression were reduced, while expression of anti-inflammatory cytokine IL-4 mRNA, and levels of M2 polarization-associated CD163 mRNA and protein were increased. In the gastrocnemius muscles, stroke rats showed a reduction in both glutathione content and Akt Serine 473 phosphorylation, as well as an elevation in Insulin Receptor Substrate-1 Serine 307 phosphorylation and Dynamin-Related Protein 1 Serine 616 phosphorylation. GTS-21 reversed poststroke changes in the gastrocnemius muscles. Overall, our findings, provide further evidence supporting the neuroprotective benefits of α7nAChR agonists, and indicate that they may potentially exert anti-inflammatory and metabolic effects peripherally in the skeletal muscle in an acute ischemic stroke animal model.

Metformin Mitigated Obesity-Driven Cancer Aggressiveness in Tumor-Bearing Mice.

Metformin may offer benefits to certain cancer populations experiencing metabolic abnormalities. To extend the anticancer studies of metformin, a tumor model was established through the implantation of murine Lewis Lung Carcinoma (LLC) cells to Normal Diet (ND)-fed and High-Fat Diet (HFD)-fed C57BL/6 mice. The HFD-fed mice displayed metabolic and pro-inflammatory alterations together with accompanying aggressive tumor growth. Metformin mitigated tumor growth in HFD-fed mice, paralleled by reductions in circulating glucose, insulin, soluble P-selectin, TGF-ß1 and High Mobility Group Box-1 (HMGB1), as well as tumor expression of cell proliferation, aerobic glycolysis, glutaminolysis, platelets and neutrophils molecules. The suppressive effects of metformin on cell proliferation, migration and oncogenic signaling molecules were confirmed in cell study. Moreover, tumor-bearing HFD-fed mice had higher contents of circulating and tumor immunopositivity of Neutrophil Extracellular Traps (NETs)-associated molecules, with a suppressive effect from metformin. Data taken from neutrophil studies confirmed the inhibitory effect that metformin has on NET formation induced by HMGB1. Furthermore, HMGB1 was identified as a promoting molecule to boost the transition process towards NETs. The current study shows that metabolic, pro-inflammatory and NET alterations appear to play roles in the obesity-driven aggressiveness of cancer, while also representing candidate targets for anticancer potential of metformin.

Preventive Intrathecal Injection of Bupivacaine Alleviated Microglia Activation and Neuropathic Pain in a Rat Model of Chronic Constriction Injury.

Spinal microglia are crucial to neuronal hyper-excitability and pain hypersensitivity. The local anesthetic bupivacaine is commonly used for both peripheral and spinal anesthesia. The pain-relief effects resulting from the peripheral and systemic administration of bupivacaine have been previously reported. In this study, the preventive effects of intrathecal bupivacaine administration against neuropathic pain were revealed in a rat model of sciatic nerve chronic constriction injury (CCI). Using a CCI rat model, pain hypersensitivity, characterized by mechanical allodynia and thermal hyperalgesia, correlated well with microglia M1 polarization, activation and pro-inflammatory cytokine expression in both spinal cord dorsal horns and sciatic nerves. Bupivacaine attenuated pain behaviors and inflammatory alternations. We further identified that the Interferon Regulatory Factor 5 (IRF5)/P2X Purinoceptor 4 (P2X4R) and High Mobility Group Box 1 (HMGB1)/Toll-Like Receptor 4 (TLR4)/NF-κB inflammatory axes may each play pivotal roles in the acquisition of microglia M1 polarization and pro-inflammatory cytokine expression under CCI insult. The relief of pain paralleled with the suppression of microglia M1 polarization, elevation of microglia M2 polarization, and inhibition of IRF5/P2X4R and HMGB1/TLR4/NF-κB in both the spinal cord dorsal horns and sciatic nerve. Our findings provide molecular and biochemical evidence for the anti-neuropathic effect of preventive bupivacaine.

Endoplasmic Reticulum Stress Contributed to Dipyridamole-Induced Impaired Autophagic Flux and Glioma Apoptosis.

Elevation of intracellular cAMP levels has been implicated in glioma cell proliferation inhibition, differentiation, and apoptosis. Inhibition of phosphodiesterase is a way to elevate intracellular cAMP levels. The present study aimed to investigate the anti-glioma potential of dipyridamole, an inhibitor of phosphodiesterase. Upon treatment with dipyridamole, human U87 glioma cells decreased cell viability, clonogenic colonization, migration, and invasion, along with Noxa upregulation, Endoplasmic Reticulum (ER) stress, impaired autophagic flux, Yes-associated Protein 1 (YAP1) phosphorylation, and YAP1 reduction. Pharmacological and genetic studies revealed the ability of dipyridamole to initiate Noxa-guided apoptosis through ER stress. Additionally, the current study further identified the biochemical role of YAP1 in communicating with ER stress and autophagy under situations of dipyridamole treatment. YAP1 promoted autophagy and protected glioma cells from dipyridamole-induced apoptotic cell death. Dipyridamole impaired autophagic flux and rendered glioma cells more vulnerable to apoptotic cell death through ER stress-inhibitable YAP1/autophagy axis. The overall cellular changes caused by dipyridamole appeared to ensure a successful completion of apoptosis. Dipyridamole also duplicated the biochemical changes and apoptosis in glioma T98G cells. Since dipyridamole has additional biochemical and pharmacological properties, further research centered on the anti-glioma mechanisms of dipyridamole is still needed.

Jak2 Inhibitor AG490 Improved Poststroke Central and Peripheral Inflammation and Metabolic Abnormalities in a Rat Model of Ischemic Stroke.

Poststroke hyperglycemia and inflammation have been implicated in the pathogenesis of stroke. Janus Kinase 2 (Jak2), a catalytic signaling component for cytokine receptors such as Interleukin-6 (IL-6), has inflammatory and metabolic properties. This study aimed to investigate the roles of Jak2 in poststroke inflammation and metabolic abnormality in a rat model of permanent cerebral ischemia. Pretreatment with Jak2 inhibitor AG490 ameliorated neurological deficit, brain infarction, edema, oxidative stress, inflammation, caspase-3 activation, and Zonula Occludens-1 (ZO-1) reduction. Moreover, in injured cortical tissues, Tumor Necrosis Factor-α, IL-1ß, and IL-6 levels were reduced with concurrent decreased NF-κB p65 phosphorylation, Signal Transducers and Activators of Transcription 3 phosphorylation, Ubiquitin Protein Ligase E3 Component N-Recognin 1 expression, and Matrix Metalloproteinase activity. In the in vitro study on bEnd.3 endothelial cells, AG490 diminished IL-6-induced endothelial barrier disruption by decreasing ZO-1 decline. Metabolically, administration of AG490 lowered fasting glucose, with improvements in glucose intolerance, plasma-free fatty acids, and plasma C Reactive Proteins. In conclusion, AG490 improved the inflammation and oxidative stress of neuronal, hepatic, and muscle tissues of stroke rats as well as impairing insulin signaling in the liver and skeletal muscles. Therefore, Jak2 blockades may have benefits for combating poststroke central and peripheral inflammation, and metabolic abnormalities.

TNF-α Receptor Inhibitor Alleviates Metabolic and Inflammatory Changes in a Rat Model of Ischemic Stroke.

Hyperglycemia and inflammation, with their augmented interplay, are involved in cases of stroke with poor outcomes. Interrupting this vicious cycle thus has the potential to prevent stroke disease progression. Tumor necrosis factor-α (TNF-α) is an emerging molecule, which has inflammatory and metabolic roles. Studies have shown that TNF-α receptor inhibitor R-7050 possesses neuroprotective, antihyperglycemic, and anti-inflammatory effects. Using a rat model of permanent cerebral ischemia, pretreatment with R-7050 offered protection against poststroke neurological deficits, brain infarction, edema, oxidative stress, and caspase 3 activation. In the injured cortical tissues, R-7050 reversed the activation of TNF receptor-I (TNFRI), NF-κB, and interleukin-6 (IL-6), as well as the reduction of zonula occludens-1 (ZO-1). In the in vitro study on bEnd.3 endothelial cells, R-7050 reduced the decline of ZO-1 levels after TNF-α-exposure. R-7050 also reduced the metabolic alterations occurring after ischemic stroke, such as hyperglycemia and increased plasma corticosterone, free fatty acids, C reactive protein, and fibroblast growth factor-15 concentrations. In the gastrocnemius muscles of rats with stroke, R-7050 improved activated TNFRI/NF-κB, oxidative stress, and IL-6 pathways, as well as impaired insulin signaling. Overall, our findings highlight a feasible way to combat stroke disease based on an anti-TNF therapy that involves anti-inflammatory and metabolic mechanisms.

Glycerol Improves Intracerebral Hemorrhagic Brain Injury and Associated Kidney Dysfunction in Rats.

In stroke patients, the development of acute kidney injury (AKI) is closely linked with worse outcomes and increased mortality. In this study, the interplay between post-stroke and AKI and treatment options was investigated in a rodent model of hemorrhagic stroke. Intrastriatal collagenase injection for 24 h caused neurological deficits, hematoma formation, brain edema, apoptosis, blood-brain barrier disruption, oxidative stress, and neuroinflammation in Sprague Dawley rats. Elevation of serum blood urea nitrogen, serum creatinine, urine cytokine-induced neutrophil chemoattractant-1, and urine Malondialdehyde, as well as moderate histological abnormality in the kidney near the glomerulus, indicated evidence of kidney dysfunction. The accumulation of podocalyxin DNA in urine further suggested a detachment of podocytes and structural deterioration of the glomerulus. Circulating levels of stress hormones, such as epinephrine, norepinephrine, corticosterone, and angiotensin II were elevated in rats with intracerebral hemorrhage. Osmotic agent glycerol held promising effects in alleviating post-stroke brain injury and kidney dysfunction. Although the detailed protective mechanisms of glycerol have yet to be determined, the intrastriatal collagenase injection hemorrhagic stroke model in rats allowed us to demonstrate the functional and structural integrity of glomerulus are targets that are vulnerable to post-stroke injury and stress hormones could be surrogates of remote communications.

Endoplasmic Reticulum Stress Contributes to Gefitinib-Induced Apoptosis in Glioma.

Adequate stress on the Endoplasmic Reticulum (ER) with the Unfolded Protein Response (UPR) could maintain glioma malignancy. Uncontrolled ER stress, on the other hand, predisposes an apoptosis-dominant UPR program. We studied here the proapoptotic actions of the Epidermal Growth Factor Receptor (EGFR) inhibitor gefitinib, with the focus on ER stress. The study models were human H4 and U87 glioma cell lines. We found that the glioma cell-killing effects of gefitinib involved caspase 3 apoptotic cascades. Three branches of ER stress, namely Activating Transcription Factor-6 (ATF6), Protein Kinase R (PKR)-Like ER Kinase (PERK), and Inositol-Requiring Enzyme 1 (IRE1), were activated by gefitinib, along with the elevation of intracellular free Ca2+, Reactive Oxygen Species (ROS), and NADPH Oxidase2/4 (NOX2/4). Specifically, elevated IRE1 phosphorylation, Tumor Necrosis Factor (TNF) Receptor-Associated Factor-2 (TRAF2) expression, Apoptosis Signal-Regulating Kinase-1 (Ask1) phosphorylation, c-Jun N-Terminal Kinase (JNK) phosphorylation, and Noxa expression appeared in gefitinib-treated glioma cells. Genetic, pharmacological, and biochemical studies further indicated an active ROS/ER stress/Ask1/JNK/Noxa axis causing the glioma apoptosis induced by gefitinib. The findings suggest that ER-stress-based therapeutic targeting could be a promising option in EGFR inhibitor glioma therapy, and may ultimately achieve a better patient response.

Exosomal HMGB1 Promoted Cancer Malignancy.

Reciprocal crosstalk between platelets and malignancies underscores the potential of antiplatelet therapy in cancer treatment. In this study, we found that human chronic myeloid leukemia K562 cell-differentiated megakaryocytes and murine platelets produced bioactive substances and these are released into the extracellular space, partly in their exosomal form. High-mobility group box 1 (HMGB1) is a type of exosomal cargo, and the antiplatelet drugs aspirin and dipyridamole interfered with its incorporation into the exosomes. Those released substances and exosomes, along with exogenous HMGB1, promoted cancer cell survival and protected cells from doxorubicin cytotoxicity. In a tumor-bearing model established using murine Lewis lung carcinoma (LLC) cells and C57BL/6 mice, the tumor suppressive effect of dipyridamole correlated well with decreased circulating white blood cells, soluble P-selectin, TGF-ß1 (Transforming Growth Factor-ß1), exosomes, and exosomal HMGB1, as well as tumor platelet infiltration. Exosome release inhibitor GW4869 exhibited suppressive effects as well. The suppressive effect of dipyridamole on cancer cell survival was paralleled by a reduction of HMGB1/receptor for advanced glycation end-products axis, and proliferation- and migration-related ß-catenin, Yes-associated protein 1, Runt-related transcription factor 2, and TGF- ß1/Smad signals. Therefore, exosomes and exosomal HMGB1 appear to have roles in platelet-driven cancer malignancy and represent targets of antiplatelet drugs in anticancer treatment.

DHA attenuated Japanese Encephalitis virus infection-induced neuroinflammation and neuronal cell death in cultured rat Neuron/glia.

Japanese Encephalitis Virus (JEV) is a neurotropic virus and its Central Nervous System (CNS) infection causes fatal encephalitis with high mortality and morbidity. Microglial activation and consequences of bystander damage appear to be the dominant mechanisms for Japanese Encephalitis and complications. Docosahexaenoic acid (DHA), an essential fatty acid and a major component of brain cell membranes, possesses additional biological activities, including anti-apoptosis, anti-inflammation, and neuroprotection. Through this study, we have provided experimental evidence showing the anti-inflammatory, neuroprotective, and anti-viral effects of DHA against JEV infection in rat Neuron/glia cultures. By Neuron/glia and Neuron cultures, DHA protected against neuronal cell death upon JEV infection and reduced JEV amplification. In Neuron/glia and Microglia cultures, the effects of DHA were accompanied by the downregulation of pro-inflammatory M1 microglia, upregulation of anti-inflammatory M2 microglia, and reduction of neurotoxic cytokine expression, which could be attributed to its interference in the Toll-Like Receptor (TLR), Mitogen-Activated Protein Kinase (MAPK), and Interferon/Janus Kinase/Signal Transducers and Activators of Transcription (Stat), along with the NF-κB, AP-1, and c-AMP Response Element Binding Protein (CREB) controlled transcriptional programs. Parallel anti-inflammatory effects against JEV infection were duplicated by G Protein-Coupled Receptor (GPR120) and GPR40 agonists and a reversal of DHA-mediated anti-inflammation was seen in the presence of GPR120 antagonist, while the GPR40 was less effectiveness. Since increasing evidence indicates its neuroprotection against neurodegenerative diseases, DHA is a proposed anti-inflammatory and neuroprotective candidate for the treatment of neuroinflammation-accompanied viral pathogenesis such as Japanese Encephalitis.

Effects of ß-Adrenergic Blockade on Metabolic and Inflammatory Responses in a Rat Model of Ischemic Stroke.

Ischemic stroke provokes an inflammatory response concurrent with both sympathetic nervous system activation and hyperglycemia. Currently, their crosstalk and consequences in stroke outcomes are of clinical attraction. We have provided experimental evidence showing the suppressive effects of the nonselective ß-adrenoreceptor antagonist propranolol on hyperglycemia, inflammation, and brain injury in a rat model experiencing cerebral ischemia. Pretreatment with propranolol protected against postischemic brain infarction, edema, and apoptosis. The neuroprotection caused by propranolol was accompanied by a reduction in fasting glucose, fasting insulin, glucose tolerance impairment, plasma C-reactive protein, plasma free fatty acids, plasma corticosterone, brain oxidative stress, and brain inflammation. Pretreatment with insulin alleviated-while glucose augmented-postischemic brain injury and inflammation. Additionally, the impairment of insulin signaling in the gastrocnemius muscles was noted in rats with cerebral ischemia, with propranolol improving the impairment by reducing oxidative stress and tumor necrosis factor-α signaling. The anti-inflammatory effects of propranolol were further demonstrated in isoproterenol-stimulated BV2 and RAW264.7 cells through its ability to decrease cytokine production. Despite their potential benefits, stroke-associated hyperglycemia and inflammation are commonly linked with harmful consequences. Our findings provide new insight into the anti-inflammatory, neuroprotective, and hypoglycemic mechanisms of propranolol in combating neurodegenerative diseases, such as stroke.

Quercetin protects against cerebral ischemia/reperfusion and oxygen glucose deprivation/reoxygenation neurotoxicity.

Beyond nutrition effect, quercetin is applied as a complement or an alternative for promoting human health and treating diseases. However, its complicated neuroprotective mechanisms have not yet been fully elucidated. This study provides evidence of an alternative target for quercetin, and sheds light on the mechanisms of its neuroprotection against cerebral ischemia/reperfusion (I/R) injury in Sprague-Dawley rats. Oral pretreatment using quercetin has alleviated cerebral I/R-induced neurological deficits, brain infarction, blood-brain barrier disruption, oxidative stress, TNF-α and IL-1ß mRNA expression, along with apoptotic caspase 3 activity. The neuroprotective, anti-oxidative, anti-inflammatory, and anti-apoptotic effects of quercetin were replicated in rat hippocampal slice cultures and neuron/glia cultures which suffered from oxygen-glucose deprivation and reoxygenation (OGDR). Biochemical studies revealed a reduction of extracellular signal-regulated kinase (ERK) and Akt phosphorylation, along with an increase in protein tyrosine and serine/threonine phosphatase activity in cerebral I/R rat cortical tissues and OGDR hippocampal slice and neuron/glia cultures. Quercetin alleviated the changes in ERK/Akt phosphorylation and protein phosphatase activities. Inhibition of ERK or Akt alone was enough to cause apoptotic cell death and cytotoxicity in hippocampal slice cultures and neuron/glia cultures, while activators of ERK or Akt alleviated OGDR-induced cytotoxicity. Taken together, our results demonstrate that quercetin alleviated the increment of protein tyrosine and serine/threonine phosphatase activity, along with the reduction of ERK and Akt phosphorylation, which may play pivotal roles in the expansion of brain injury after cerebral I/R.

Aspirin Induced Glioma Apoptosis through Noxa Upregulation.

Clinically, high cyclooxygenase-2 expression in malignant glioma correlates well with poor prognosis and the use of aspirin is associated with a reduced risk of glioma. To extend the current understanding of the apoptotic potential of aspirin in most cell types, this study provides evidence showing that aspirin induced glioma cell apoptosis and inhibited tumor growth, in vitro and in vivo. We found that the human H4 glioma cell-killing effects of aspirin involved mitochondria-mediated apoptosis accompanied by endoplasmic reticulum (ER) stress, Noxa upregulation, Mcl-1 downregulation, Bax mitochondrial distribution and oligomerization, and caspase 3/caspase 8/caspase 9 activation. Genetic silencing of Noxa or Bax attenuated aspirin-induced viability loss and apoptosis, while silencing Mcl-1 augmented the effects of aspirin. Data from genetic and pharmacological studies revealed that the axis of ER stress comprised an apoptotic cascade leading to Noxa upregulation and apoptosis. The apoptotic programs and mediators triggered by aspirin in H4 cells were duplicated in human U87 glioma cell line as well as in tumor-bearing BALB/c nude mice. The involvement of ER stress in indomethacin-induced Mcl-1 downregulation was reported in our previous study on glioma cells. Therefore, the aforementioned phenomena indicate that ER stress may be a valuable target for intervention in glioma apoptosis.

ß-Funaltrexamine Displayed Anti-inflammatory and Neuroprotective Effects in Cells and Rat Model of Stroke.

Chronic treatment involving opioids exacerbates both the risk and severity of ischemic stroke. We have provided experimental evidence showing the anti-inflammatory and neuroprotective effects of the µ opioid receptor antagonist ß-funaltrexamine for neurodegenerative diseases in rat neuron/glia cultures and a rat model of cerebral Ischemia/Reperfusion (I/R) injury. Independent of in vitro Lipopolysaccharide (LPS)/interferon (IFN-γ)-stimulated neuron/glia cultures and in vivo cerebral I/R injury in Sprague-Dawley rats, ß-funaltrexamine downregulated neuroinflammation and ameliorated neuronal degeneration. Alterations in microglia polarization favoring the classical activation state occurred in LPS/IFN-γ-stimulated neuron/glia cultures and cerebral I/R-injured cortical brains. ß-funaltrexamine shifted the polarization of microglia towards the anti-inflammatory phenotype, as evidenced by decreased nitric oxide, tumor necrosis factor-α, interleukin-1ß, and prostaglandin E2, along with increased CD163 and arginase 1. Mechanistic studies showed that the suppression of microglia pro-inflammatory polarization by ß-funaltrexamine was accompanied by the reduction of NF-κB, AP-1, cyclic AMP response element-binding protein, along with signal transducers and activators of transcription transcriptional activities and associated upstream activators. The effects of ß-funaltrexamine are closely linked with its action on neuroinflammation by switching microglia polarization from pro-inflammatory towards anti-inflammatory phenotypes. These findings provide new insights into the anti-inflammatory and neuroprotective mechanisms of ß-funaltrexamine in combating neurodegenerative diseases, such as stroke.

Endoplasmic Reticulum Stress Contributes to Indomethacin-Induced Glioma Apoptosis.

The dormancy of cellular apoptotic machinery has been highlighted as a crucial factor in therapeutic resistance, recurrence, and poor prognosis in patients with malignancy, such as malignant glioma. Increasing evidence indicates that nonsteroidal anti-inflammatory drugs (NSAIDs) confer chemopreventive effects, and indomethacin has been shown to have a novel chemotherapeutic application targeting glioma cells. To extend these findings, herein, we studied the underlying mechanisms of apoptosis activation caused by indomethacin in human H4 and U87 glioma cells. We found that the glioma cell-killing effects of indomethacin involved both death receptor- and mitochondria-mediated apoptotic cascades. Indomethacin-induced glioma cell apoptosis was accompanied by a series of biochemical changes, including reactive oxygen species generation, endoplasmic reticulum (ER) stress, apoptosis signal-regulating kinase-1 (Ask1) activation, p38 hyperphosphorylation, protein phosphatase 2A (PP2A) activation, Akt dephosphorylation, Mcl-1 and FLICE-inhibiting protein (FLIP) downregulation, Bax mitochondrial distribution, and caspases 3/caspase 8/caspase 9 activation. Data on pharmacological inhibition related to oxidative stress, ER stress, free Ca2+, and p38 revealed that the axis of oxidative stress/ER stress/Ask1/p38/PP2A/Akt comprised an apoptotic cascade leading to Mcl-1/FLIP downregulation and glioma apoptosis. Since indomethacin is an emerging choice in chemotherapy and its antineoplastic effects have been demonstrated in glioma tumor-bearing models, the findings further strengthen the argument for turning on the aforementioned axis in order to activate the apoptotic machinery of glioma cells.

Fibronectin Promotes Cell Growth and Migration in Human Renal Cell Carcinoma Cells.

The prognostic and therapeutic values of fibronectin have been reported in patients with renal cell carcinoma (RCC). However, the underlying mechanisms of malignancy in RCC are not completely understood. We found that silencing of fibronectin expression attenuated human RCC 786-O and Caki-1 cell growth and migration. Silencing of potential fibronectin receptor integrin α5 and integrin ß1 decreased 786-O cell ability in movement and chemotactic migration. Biochemical examination revealed a reduction of cyclin D1 and vimentin expression, transforming growth factor-ß1 (TGF-ß1) production, as well as Src and Smad phosphorylation in fibronectin-silenced 786-O and Caki-1 cells. Pharmacological inhibition of Src decreased 786-O cell growth and migration accompanied by a reduction of cyclin D1, fibronectin, vimentin, and TGF-ß1 expression, as well as Src and Smad phosphorylation. In 786-O cells, higher activities in cell growth and migration than in Caki-1 cells were noted, along with elevated fibronectin and TGF-ß1 expression. The additions of exogenous fibronectin and TGF-ß1 promoted Caki-1 cell growth and migration, and increased cyclin D1, fibronectin, vimentin, and TGF-ß1 expression, as well as Src and Smad phosphorylation. These findings highlight the role of fibronectin in RCC cell growth and migration involving Src and TGF-ß1 signaling.