Pharmaceutical efficacy of novel human-origin Faecalibacterium prausnitzii strains on high-fat-diet-induced obesity and associated metabolic disorders in mice.

Introduction: Obesity and related metabolic issues are a growing global health concern. Recently, the discovery of new probiotics with anti-obesity properties has gained interest. Methods: In this study, four Faecalibacte-rium prausnitzii strains were isolated from healthy human feces and evaluated on a high-fat diet-induced mouse model for 12 weeks. Results: The F. prausnitzii strains reduced body weight gain, liver and fat weights, and calorie intake while improving lipid and glucose metabolism in the liver and adipose tissue, as evidenced by regulating lipid metabolism-associated gene expression, including ACC1, FAS, SREBP1c, leptin, and adiponectin. Moreover, the F. prausnitzii strains inhibited low-grade inflammation, restored gut integrity, and ameliorated hepatic function and insulin resistance. Interestingly, the F. prausnitzii strains modulated gut and neural hormone secretion and reduced appetite by affecting the gut-brain axis. Supplementation with F. prausnitzii strains noticeably changed the gut microbiota composition. Discussion: In summary, the novel isolated F. prausnitzii strains have therapeutic effects on obesity and associated metabolic disorders through modulation of the gut-brain axis. Additionally, the effectiveness of different strains might not be achieved through identical mechanisms. Therefore, the present findings provide a reliable clue for developing novel therapeutic probiotics against obesity and associated metabolic disorders.

New Strains of Akkermansia muciniphila and Faecalibacterium prausnitzii are Effective for Improving the Muscle Strength of Mice with Immobilization-Induced Muscular Atrophy.

Muscular atrophy is a muscle disease in which muscle mass and strength decrease due to aging, injury, metabolic disorders, or chronic conditions. Proteins in muscle tissue are degraded by the ubiquitin-proteasome pathway, and atrophy accelerates this pathway. Akkermansia muciniphila and Faecalibacterium prausnitzii strains are effective agents against metabolic and inflammatory diseases in next-generation probiotic research. In this study, we evaluated the efficacy of A. muciniphila strain EB-AMDK19 and F. prausnitzii strain EB-FPDK11 in a mouse model of muscular atrophy, since atrophy inhibits energy metabolism and immune activation. After oral administration of each strain for 4 weeks, the hind legs of the mice were fixed with a plaster cast to immobilize them for a week. As a result, the administration of EB-AMDK19 and EB-FPDK11 strains improved grip strength but did not increase muscle mass. At the molecular level, A. muciniphila and F. prausnitzii treatments decreased the expression levels of ubiquitin-proteasome genes, atrogin-1, MuRF, and cathepsin L. They increased the expression level of the mitochondrial biogenesis regulatory gene, PGC-1α. The effect of the strains was confirmed by a decrease in myostatin. Furthermore, A. muciniphila and F. prausnitzii modulated the immune function by enhancing ZO-1 and inhibiting IL-6. In particular, EB-AMDK19 promoted the expression of IL-10, an anti-inflammatory cytokine. These results suggest that A. muciniphila and F. prausnitzii may have beneficial effects on muscular atrophy, verified by newly isolated EB-AMDK19 and EB-FPDK11 as potential next-generation probiotics.

Oral administration of Faecalibacterium prausnitzii and Akkermansia muciniphila strains from humans improves atopic dermatitis symptoms in DNCB induced NC/Nga mice.

Atopic dermatitis (AD) is a common inflammatory skin disease, and its pathogenesis is closely associated with microbial homeostasis in the gut, namely the gut-skin axis. Particularly, recent metagenomics studies revealed that the abundance of two major bacterial species in the gut, Faecalibacterium prausnitzii and Akkermansia muciniphila, may play a critical role in the pathogenesis of AD, but the effect of these species in AD has not yet been elucidated. To evaluate the potential beneficial effect of F. prausnitzii or A. muciniphila in AD, we conducted an animal model study where F. prausnitzii EB-FPDK11 or A. muciniphila EB-AMDK19, isolated from humans, was orally administered to 2,5-dinitrochlorobenzene (DNCB)-induced AD models using NC/Nga mice at a daily dose of 108 CFUs/mouse for six weeks. As a result, the administration of each strain of F. prausnitzii and A. muciniphila improved AD-related markers, such as dermatitis score, scratching behavior, and serum immunoglobulin E level. Also, the F. prausnitzii and A. muciniphila treatments decreased the level of thymic stromal lymphopoietin (TSLP), triggering the production of T helper (Th) 2 cytokines, and improved the imbalance between the Th1 and Th2 immune responses induced by DNCB. Meanwhile, the oral administration of the bacteria enhanced the production of filaggrin in the skin and ZO-1 in the gut barrier, leading to the recovery of functions. Taken together, our findings suggest that F. prausnitzii EB-FPDK11 and A. muciniphila EB-AMDK19 have a therapeutic potential in AD, which should be verified in humans.

Effects of Live and Pasteurized Forms of Akkermansia from the Human Gut on Obesity and Metabolic Dysregulation.

Akkermansia muciniphila (A. muciniphila) is a promising probiotic candidate owing to its health-promoting properties. A previous study reported that the pasteurized form of A. muciniphila strains isolated from human stool samples had a beneficial impact on high-fat diet-induced obese mice. On the other hand, the differences in the probiotic effects between live and pasteurized A. muciniphila on the metabolism and immune system of the host are still inconclusive. This study examines the differences between the live and pasteurized forms of A. muciniphila strains on the lipid and glucose metabolism and on regulating the inflammatory immune responses using a HFD-fed obese mouse model. The animals were administered the live and pasteurized forms of two A. muciniphila strains five times per week for the entire study period of 12 weeks. Both forms of the bacterial strains improved the HFD-induced obesity and metabolic dysregulation in the mice by preventing body-weight gains after one week. In addition, they cause a decrease in the weights of the major adipose tissues, adipogenesis/lipogenesis and serum TC levels, improvement in glucose homeostasis and suppression of inflammatory insults. Furthermore, these treatments restored the damaged gut architecture and integrity and improved the hepatic structure and function in HFD-induced animals. On the other hand, for both bacterial strains, the pasteurized form was more potent in improving glucose tolerance than the live form. Moreover, specific A. muciniphila preparations with either live or pasteurized bacteria decreased the number and population (%) of splenic Treg cells (CD4+ Foxp3+) significantly in the HFD-fed animals, further supporting the anti-inflammatory properties of these bacteria.

Paired mechanical and electrical acupuncture of neurogenic spots induces opioid-mediated suppression of hypertension in rats.

While our recent studies have suggested that effective acupoints display neurogenic inflammation and can be identified as neurogenic spots (Neuro-Sps), the optimal stimulation conditions and the underlying mechanisms remain uncharacterized. We developed a combined mechano-electrical acupuncture device (MEA) and examined the effects of acupuncture at Neuro-Sps on systolic blood pressure (BP) in a rat model of immobilization-induced hypertension (IMH) and the mediation of endogenous opioid systems in its effect. Cutaneous neurogenic spots were found mostly in the forelimb. Electrical and mechanical acupuncture of Neuro-Sps increased 22-kHz ultrasonic vocalizations (USVs), c-Fos expression and cell excitability in the midbrain and synergistically alleviated the development of hypertension following immobilization stress, which was prevented by administration of the opioid antagonist naloxone into the rostral ventrolateral medulla (rVLM). These findings suggest that mechanical and electrical stimulation at Neuro-Sps suppresses the development of hypertension via mediation of the endogenous opioid system.

Unpleasant Sound Elicits Negative Emotion and Reinstates Drug Seeking.

Although previous studies have suggested an association between unpleasant sounds and the use of drugs, scientific evidence supporting this is lacking. This study investigated in rats (male Sprague-Dawley rats) if aversive sounds modulate dopamine (DA) transmission in the mesolimbic reward system and cocaine reinforcement. For sound stimulation, we used artificial low-frequency ultrasound (ALFUS) in the frequency ranges (22-38 kHz) which produces an aversive response in rats. Rats displayed increased anxiety-like behaviors, 22-kHz ultrasonic vocalizations (USVs), and stress responses with ALFUS. In vivo extracellular recording and immunohistochemistry revealed that ALFUS stimulation activated central amygdalar neurons and amygdalar GABAergic neurons. Amygdalar lesions prevented an increase of 22-kHz USVs by ALFUS. Dopamine levels in NAc decreased during ALFUS stimulation. In rats self-administering cocaine, ALFUS caused reinstatement of cocaine seeking after a period of extinction. Thus, ALFUS stimulation induced negative emotional states in association with a decrease in mesolimbic DA function and reinstatement of cocaine-seeking behaviors, suggesting that exposure to unpleasant sounds enhances negative emotional states and may induce relapse in addicts.

A case of a schwannoma in the nasal columella.

Schwannoma is a slow-growing, benign tumor of the nerve sheath. It rarely presents in the nasal cavity or the paranasal sinuses. Although schwannomas in the nasal septum have been reported previously, cases of this tumor in the nasal columella are rare in the literature. Here, we report on a 67-year-old woman with a schwannoma in the nasal columella that was successfully removed using a sublabial approach, along with a review of relevant literature.

Medial Forebrain Bundle Deep Brain Stimulation Reverses Anhedonic-Like Behavior in a Chronic Model of Depression: Importance of BDNF and Inflammatory Cytokines.

Deep brain stimulation (DBS) of the medial forebrain bundle (MFB) displays a promising antidepressant effects in patients with treatment-refractory depression; however, a clear consensus on underlying mechanisms is still enigmatic. Herein, we investigated the effects of MFB-DBS on anhedonic-like behavior using the Froot Loops® consumption in a chronic unpredictable mild stress (CUS) model of depression, biochemical estimation of peripheral and central inflammatory cytokines, stress hormone, and brain-derived neurotrophic factor (BDNF). Seven days of MFB-DBS significantly reversed the 42-day CUS-generated anhedonic-like phenotype (p < 0.02) indicated by an increase in Froot Loops® consumption. Gross locomotor activity and body weight remained unaffected across the different groups. A dramatic augmentation of adrenocorticotropic hormone levels was seen in the plasma and cerebrospinal fluid (CSF) samples of CUS rats, which significantly reduced following MFB-DBS treatment. However, C-reactive protein levels were found to be unaffected. Interestingly, decreased levels of BDNF in the CUS animals were augmented in the plasma, CSF, and hippocampus following MFB-DBS, but remained unaltered in the nucleus accumbens (NAc). While multiplex assay revealed no change in the neuronal levels of inflammatory cytokines including IL-1α, IL-4, IL-10, IL-12, IL-13, and IL-17 in the neuroanatomical framework of the hippocampus and NAc, increased levels of IL-1ß, IL-2, IL-5, IL-6, IL-7, IL-18, TNF-α, and INF-γ were seen in these brain structures after CUS and were differentially modulated in the presence of MFB stimulation. Here, we show that there is dysregulation of BDNF and neuroimmune mediators in a stress-driven chronic depression model, and that chronic MFB-DBS has the potential to undo these aberrations.

Amelioration of obesity-related characteristics by a probiotic formulation in a high-fat diet-induced obese rat model.

PURPOSE: Obesity is a major public health concern. Despite its multi-factorial etiology, alterations in intestinal microbiota and the immune system are frequently observed. We investigated the effect of Duolac Gold (DG), a probiotic formulation containing 2 Lactobacillus strains (L. acidophilus LA1 and L. rharmnosus LR5), 3 Bifidobacterium (B. bifidum BF3, B. lactis BL3, and B. longum BG7), and Streptococcus thermophilus ST3, on morphometric and metabolic parameters, intestinal microbiota, and intestinal immune responses in a high-fat diet (HFD)-induced obese rat model. METHODS: Rats received either a conventional balanced diet or HFD with or without water containing DG for 8 weeks. HFD-induced adiposity, intestinal microbiota, and changes in inflammatory cytokine, chemokine, and metabolite levels in serum were evaluated. RESULTS: DG administration effectively decreased HFD-induced body weight and modulated morphometric and metabolic parameters. Quantitative analysis of fecal microbiota showed that obese rats given DG exhibited significantly increased levels of Bacteroidetes, Lactobacillus, and Bifidobacterium, with significant decreases in the level of Firmicutes. Serum levels of the inflammatory cytokines and the chemokine were also altered. Serum metabolite analysis revealed that DG administration modulated HFD-induced changes in serum metabolites, including fatty acids (FA), lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylcholine (PC), and triacylglycerol (TAG). CONCLUSIONS: DG administration appears to have the potential to alleviate HDF-induced obesity through the modulation of intestinal microbiota, immune responses, and host metabolism, which supports the use of probiotics to treat obesity.

Insulin-like growth factor binding protein-6 released from human mesenchymal stem cells confers neuronal protection through IGF-1R-mediated signaling.

Human bone marrow­derived mesenchymal stem cells (hMSCs) are a desirable cell source for cell­based therapy to treat nervous system injuries due to their ability to differentiate into specific cell types. In addition to their multipotency, hMSCs render the tissue microenvironment more favorable for tissue repair by secreting various growth factors. Our previous study demonstrated that hMSCs secrete several growth factors, including several insulin­like growth factor binding proteins (IGFBPs). Among these, IGFBP­6 binds with high affinity and inhibits insulin growth factor­2 (IGF­2) to inhibit the growth of IGF­2­dependent tumors. However, the function of IGFBP­6 in the nervous system remains to be fully elucidated. The present study investigated the protective effects of IGFBP­6 secreted by hMSCs on H2O2­injured primary cortical neuron cultures and lysolecithin­injured organotypic spinal cord slice cultures. Treatment of the H2O2­injured cortical neurons with conditioned media from hMSCs (hMSC­CM) increased the phosphorylation of Akt, reduced cell death and mitochondrial translocation of Bax, and regulated extracellular levels of IGF­1 and IGF­2. MTT assay, western blot analysis and ELISA were used to detect the cell viability and protein expression levels, respectively. An inhibitory antibody against IGFBP­6 eliminated this hMSC­CM­mediated neuroprotective effect in the injured cortical neuron cultures and spinal cord slice cultures. In addition, treatment with cyclolignan picropodophyllin, an inhibitor of IGF­1 receptor (IGF­1R), significantly inhibited neuronal protection by hMSC­CM. These findings demonstrated that hMSC­CM­mediated neuroprotection was attributed to IGF­1R­mediated signaling, potentiated via the inhibition of IGF­2 by IGFBP­6. The results of the present study provide insight into the mechanism by which hMSC administration may promote recovery from nerve injury.

A multistrain probiotic formulation attenuates skin symptoms of atopic dermatitis in a mouse model through the generation of CD4+Foxp3+ T cells.

BACKGROUND: Atopic dermatitis (AD) is characterized by chronic inflammation of the skin. AD develops mainly in infants and young children. It induces skin disorders and signals the initiation of the allergic march including allergic asthma and rhinitis. Probiotics modify intestinal microbial populations in a beneficial way for human and animal hosts by reducing inflammatory cytokines. OBJECTIVE: As a result of their immunomodulatory properties, probiotics have been considered a promising therapeutic option for the prevention and treatment of AD. DESIGN: In this study, we examined the effects of GI7, a potential probiotic mixture consisting of seven strains of bifidobacteria and lactic acid bacteria, on AD in a mouse model. RESULTS: Administration of GI7 for 8 weeks reduced AD-like skin lesions and induced changes in the levels of serum markers such as immunoglobulin E and cytokines related to T helper (Th)1 and Th2 cells, and in skin barrier genes. Alleviation of AD seems to be associated with GI7-induced generation of CD4+Foxp3+ regulatory T cells. CONCLUSIONS: The probiotic mixture may have potential to improve symptoms of AD.

Epigallocatechin-3-gallate prevents oxidative stress-induced cellular senescence in human mesenchymal stem cells via Nrf2.

Human mesenchymal stem cells (hMSCs) have great therapeutic potential due to their high plasticity, immune privileged status and ease of preparation, as well as a lack of ethical barriers to their use. However, their ultimate usefulness is limited by cellular senescence occurring secondary to increased cellular levels of reactive oxygen species (ROS) during their propagation in culture. The underlying molecular mechanisms responsible for this process in hMSCs remain unclear. An antioxidant polyphenol epigallocatechin-3-gallate (EGCG) found in green tea, is known to activate nuclear factor-erythroid 2-related factor 2 (Nrf2), a master transcriptional regulator of antioxidant genes. Herein, we examined the EGCG-mediated antioxidant mechanism in hMSCs exposed to ROS which involves Nrf2 activation. The H2O2-exposed hMSCs showed cellular senescence with significantly increased protein levels of acetyl-p53 and p21 in comparison with the untreated hMSCs, and these effects were prevented by pre-treatment with EGCG. By contrast, in Nrf2-knockdown hMSCs, EGCG lost its antioxidant effect, exhibiting high levels of acetyl-p53 and p21 following EGCG pre-treatment and H2O2 exposure. This indicates that Nrf2 and p53/p21 may be involved in the anti­senescent effect of EGCG in hMSCs. Taken together, these findings indicate the important role of EGCG in preventing oxidative stress-induced cellular senescence in hMSCs through Nrf2 activation, which has applications for the massive production of more suitable hMSCs for cell-based therapy.

Ethyl pyruvate inhibits HMGB1 phosphorylation and release by chelating calcium.

Ethyl pyruvate (EP), a simple aliphatic ester of pyruvic acid, has been shown to have antiinflammatory effects and to confer protective effects in various pathological conditions. Recently, a number of studies have reported EP inhibits high mobility group box 1 (HMGB1) secretion and suggest this might contribute to its antiinflammatory effect. Since EP is used in a calcium-containing balanced salt solution (Ringer solution), we wondered if EP directly chelates Ca(2+) and if it is related to the EP-mediated suppression of HMGB1 release. Calcium imaging assays revealed that EP significantly and dose-dependently suppressed high K(+)-induced transient [Ca(2+)]i surges in primary cortical neurons and, similarly, fluorometric assays showed that EP directly scavenges Ca(2+) as the peak of fluorescence emission intensities of Mag-Fura-2 (a low-affinity Ca(2+) indicator) was shifted in the presence of EP at concentrations of ≥7 mmol/L. Furthermore, EP markedly suppressed the A23187-induced intracellular Ca(2+) surge in BV2 cells and, under this condition, A23187-induced activations of Ca(2+)-mediated kinases (protein kinase Cα and calcium/calmodulin-dependent protein kinase IV), HMGB1 phosphorylation and subsequent secretion of HMGB1 also were suppressed. (A23187 is a calcium ionophore and BV2 cells are a microglia cell line.) Moreover, the above-mentioned EP-mediated effects were obtained independent of cell death or survival, which suggests that they are direct effects of EP. Together, these results indicate that EP directly chelates Ca(2+), and that it is, at least in part, responsible for the suppression of HMGB1 release by EP.

Ankyrin repeat-rich membrane spanning (ARMS)/Kidins220 scaffold protein regulates neuroblastoma cell proliferation through p21.

Cell proliferation is tightly controlled by the cell-cycle regulatory proteins, primarily by cyclins and cyclin-dependent kinases (CDKs) in the G1 phase. The ankyrin repeat-rich membrane spanning (ARMS) scaffold protein, also known as kinase D-interacting substrate of 220 kDa (Kidins 220), has been previously identified as a prominent downstream target of neurotrophin and ephrin receptors. Many studies have reported that ARMS/Kidins220 acts as a major signaling platform in organizing the signaling complex to regulate various cellular responses in the nervous and vascular systems. However, the role of ARMS/Kidins220 in cell proliferation and cell-cycle progression has never been investigated. Here we report that knockdown of ARMS/Kidins220 inhibits mouse neuroblastoma cell proliferation by inducing slowdown of cell cycle in the G1 phase. This effect is mediated by the upregulation of a CDK inhibitor p21, which causes the decrease in cyclin D1 and CDK4 protein levels and subsequent reduction of pRb hyperphosphorylation. Our results suggest a new role of ARMS/Kidins220 as a signaling platform to regulate tumor cell proliferation in response to the extracellular stimuli.

Ethyl pyruvate inhibits HMGB1 phosphorylation and secretion in activated microglia and in the postischemic brain.

Ethyl pyruvate (EP) has been shown to have anti-inflammatory effects and confer protective effects in various pathological conditions. For example, EP inhibits secretion of high mobility group box 1 (HMGB1), which is known to be released from activated or dying cells and aggravate inflammatory pathways. In the present study, we investigated whether EP reduces HMGB1 phosphorylation and release in ischemic brain and in cultured microglia. In the postischemic brains (60 min middle cerebral artery occlusion (MCAO)), HMGB1 was released extracellularly, generating dual peaks in cerebrospinal fluid (CSF) around 1 and 7 days after ischemic insult, which were probably generated from damaged neurons and activated inflammatory cells, respectively. We showed that treatment with EP 30 min post-MCAO (5 mg/kg, i.v.), which has been shown to confer a robust neuroprotective effect in the postischemic brain, reduced both peaks. In addition, delayed EP treatment from 4 days post-MCAO reduced HMGB1 accumulation in CSF at 7 day post-MCAO in the absence of accompanying amelioration of ischemic brain damage, indicating that the suppression of HMGB1 release is a direct effect. We also found that EP markedly suppressed the LPS-induced nuclear translocations of protein kinase C alpha and calcium/calmodulin-dependent protein kinase IV, HMGB1 phosphorylation, and subsequent secretion of HMGB1 induced by LPS in BV2 cells and EP-mediated above-mentioned effects were also independent of cell death or survival. These results indicate that EP inhibits HMGB1 phosphorylation and release in activated microglia, which might be responsible for EP-mediated suppression of HMGB1 release in the postischemic brain.

Up-down regulation of HO-1 and iNOS gene expressions by ethyl pyruvate via recruiting p300 to Nrf2 and depriving It from p65.

Ethyl pyruvate (EP), a simple ester of pyruvic acid, has been shown to exert robust neuroprotection in various neuropathological conditions, such as, cerebral ischemia and KA-induced seizure animal models. The neuroprotective effect of EP is attributable to the anti-inflammatory, anti-oxidative, and anti-apoptotic effects. In the present study, we investigated convergence of anti-inflammatory and anti-oxidative functions of EP and present a novel molecular mechanism underlying anti-inflammatory effects of EP, which is conveyed by p300, a transcriptional co-activator for both Nuclear factor E2-related factor 2 (Nrf2) and p65. In BV2 cells, a microglia cell line, EP induced translocation of Nrf2 from the cytosol to the nucleus and enhanced the expression of hemeoxygenase 1 (HO-1) in a dose-dependent manner and 1h incubation with 10mM EP increased HO-1 to 4.9-fold. Nrf2 was found to translocate from the cytosol to the nucleus beginning 30 min after EP-treatment and binds to the antioxidant response element (ARE) located on HO-1 promoter. Interestingly, LPS-induced inducible NO synthase (iNOS) induction was substantially suppressed in EP-pre-treated BV2 cells and it was reverted by Nrf2 knockdown. We found that EP-induced Nrf2 accumulation in the nucleus recruits p300, a transcriptional co-activator of both Nrf2 and p65, inhibiting p65-p300 interaction. Competition between Nrf2 and p65 for p300 binding was confirmed by glutathione S-transferase (GST) pull down assay and reporter gene analysis. These results demonstrate that EP induced nuclear translocation of Nrf2 which binds to ARE along with p300 and hampers iNOS expression via depleting p300 from p65. This is a novel anti-inflammatory mechanism conveyed by EP, which enhances protective effect by converging anti-inflammatory and anti-oxidative effects and might be applicable to various Nrf2-activating agents, such as phytochemicals.

Intranasal delivery of HMGB1-binding heptamer peptide confers a robust neuroprotection in the postischemic brain.

High mobility group box 1 (HMGB1) is an endogenous danger signal molecule. In a previous report, we showed that HMGB1 is massively released during NMDA-induced acute damaging process in the postischemic brain and triggers inflammatory processes and induces neuronal apoptosis. We have also reported a robust neuroprotection of intranasally delivered HMGB1 siRNA in the postischemic rat brain (middle cerebral artery occlusion (MCAO), 60 min). In the present study, we investigated the therapeutic efficacy of intranasally delivered HMGB1 binding heptamer peptide (HBHP; HMSKPVQ), which was selected using a phage display approach, in the same stroke animal model. A pull-down assay using biotin-labeled HBHP showed that HBHP binds directly to HMGB1, specifically to HMGB1 A box, confirming HMGB1/HBHP interaction. HBHP significantly suppressed HMGB1-mediated neuronal cell death in primary cortical cultures and HMGB1/HBHP binding was detected in NMDA-conditioned culture media. However, a heptamer peptide composed of a scrambled sequence of the seven amino acids in HBHP failed to bind HMGB1 and had no protective effect. Furthermore, HBHP (300 ng) delivered intranasally at 30 min before MCAO significantly suppressed infarct volume in the postischemic rat brain (maximal reduction by 41.8±5.4%) and ameliorated neurological and behavioral deficits. In contrast, scrambled heptamer peptide had no protective effect at the same dose. Together these results suggest that intranasal HBHP ameliorates neuronal damage in the ischemic brain by binding HMGB1, which might inhibit the function of HMGB1 as an endogenous danger signal molecule.

NADPH oxidase mediates depressive behavior induced by chronic stress in mice.

Stress is a potent risk factor for depression, yet the underlying mechanism is not clearly understood. In the present study, we explored the mechanism of development and maintenance of depression in a stress-induced animal model. Mice restrained for 2 h daily for 14 d showed distinct depressive behavior, and the altered behavior persisted for >3 months in the absence of intervention. Acute restraint induced a surge of oxidative stress in the brain, and stress-induced oxidative stress progressively increased with repetition of stress. In vitro, the stress hormone glucocorticoid generated superoxide via upregulation of NADPH oxidase. Consistently, repeated restraints increased the expression of the key subunits of NADPH oxidase, p47phox and p67phox, in the brain. Moreover, stressed brains markedly upregulated the expression of p47phox to weak restress evoked in the poststress period, and this molecular response was reminiscent of amplified ROS surge to restress. Pharmacological inhibition of NADPH oxidase by the NADPH oxidase inhibitor apocynin during the stress or poststress period completely blocked depressive behavior. Consistently, heterozygous p47phox knock-out mice (p47phox(+/-)) or molecular inhibition of p47phox with Lenti shRNA-p47phox in the hippocampus suppressed depressive behavior. These results suggest that repeated stress promotes depressive behavior through the upregulation of NADPH oxidase and the resultant metabolic oxidative stress, and that the inhibition of NADPH oxidase provides beneficial antidepression effects.

Ethyl pyruvate-mediated Nrf2 activation and hemeoxygenase 1 induction in astrocytes confer protective effects via autocrine and paracrine mechanisms.

Ethyl pyruvate (EP), a simple ester of pyruvic acid, has been shown to act as an anti-inflammatory molecule under various pathological conditions, such as, during cerebral ischemia and sepsis in animal models. Here, the authors investigated the novel molecular mechanism underlying the anti-oxidative effect of EP in primary astrocyte cultures, particularly with respect to nuclear factor E2-related factor 2 (Nrf2) activation and hemeoxygenase 1 (HO-1) induction. EP was found to induce Nrf2 translocation and the inductions of various genes downstream of Nrf2 and these resulted in the amelioration of the oxidative damage of H(2)O(2). Furthermore, EP dose-dependently suppressed H(2)O(2)-induced astrocyte cell death (12h preincubation with 5mM EP increased cell survival after 1h exposure to 100 µM H(2)O(2) from 32.6±0.7% to 63±1.8%). HO-1 was markedly induced (4.9-fold) in EP-treated primary astrocyte cultures and Nrf2 was found to translocate from the cytosol to the nucleus and bind to the antioxidant response element (ARE) located on HO-1 promoter after EP treatment. siRNA-mediated HO-1 or Nrf2 knockdown and zinc protoporphyrin (ZnPP)-mediated inhibition of HO-1 activity showed that Nrf2 activation and HO-1 induction were responsible for the observed cytoprotective effect of EP, which was found to involve the ERK and Akt signaling pathways. Furthermore, EP-conditioned astrocyte culture media was found to have neuroprotective effects on primary neuronal cultures exposed to oxidative or excitotoxic stress, and this seemed to be mediated by glial cell line-derived neurotrophic factor (GDNF) and glutathione (GSH), which accumulated in EP-treated astrocyte culture media. Interestingly, we also found that in addition to HO-1, EP-induced Nrf2 activation increased the expressions of various anti-oxidant genes, including GST, NQO1, and GCLM. The study shows that EP-mediated Nrf2 activation and HO-1 induction in astrocytes act via autocrine and paracrine mechanisms to confer protective effects.

Intranasal delivery of HMGB1 siRNA confers target gene knockdown and robust neuroprotection in the postischemic brain.

Noninvasive intranasal drug administration has been noted to allow direct delivery of drugs to the brain. In the present study, the therapeutic efficacy of intranasal small interfering RNA (siRNA) delivery was investigated in the postischemic rat brain. Fluorescein isothiocyanate (FITC)-labeled control siRNA was delivered intranasally in normal adult rats using e-PAM-R, a biodegradable PAMAM dendrimer, as gene carrier. Florescence-tagged siRNA was found in the cytoplasm and processes of neurons and of glial cells in many brain regions, including the hypothalamus, amygdala, cerebral cortex, and striatum, in 1 hour after infusion, and the FITC-fluorescence was continuously detected for at least 12 hours. When siRNA for high mobility group box 1 (HMGB1), which functions as an endogenous danger molecule and aggravates inflammation, was delivered intranasally, the target gene was significantly depleted in many brain regions, including the prefrontal cortex and striatum. More importantly, intranasal delivery of HMGB1 siRNA markedly suppressed infarct volume in the postischemic rat brain (maximal reduction to 42.8 ± 5.6% at 48 hours after 60 minutes middle cerebral artery occlusion (MCAO)) and this protective effect was manifested by recoveries from neurological and behavioral deficits. These results indicate that the intranasal delivery of HMGB1 siRNA offers an efficient means of gene knockdown-mediated therapy in the ischemic brain.