PSMC5 regulates microglial polarization and activation in LPS-induced cognitive deficits and motor impairments by interacting with TLR4.

Luteolin is a flavonoid found in high concentrations in celery and green pepper, and acts as a neuroprotectant. PSMC5 (proteasome 26S subunit, ATPase 5) protein levels were reduced after luteolin stimulation in activated microglia. We aimed to determine whether regulating PSMC5 expression could inhibit neuroinflammation, and investigate the underlying mechanisms.BV2 microglia were transfected with siRNA PSMC5 before the addition of LPS (lipopolysaccharide, 1.0 µg/ml) for 24 h in serum free DMEM. A mouse model of LPS-induced cognitive and motor impairment was established to evaluate the neuroprotective effects of shRNA PSMC5. Intracerebroventricular administration of shRNA PSMC5 was commenced 7 days prior to i.p. injection of LPS (750 µg/kg). Treatments and behavioral experiments were performed once daily for 7 consecutive days. Behavioral tests and pathological/biochemical assays were performed to evaluate LPS-induced hippocampal damage. Molecular dynamics simulation was used to confirm the interaction between PSMC5 and TLR4 (Toll-like receptor 4) in LPS-stimulated BV2 microglia. SiRNA PSMC5 inhibited BV2 microglial activation, and suppressed the release of inflammatory factors (IL-1ß, COX-2, PGE2, TNF-α, and iNOS) upon after LPS stimulation in BV2 microglia. LPS increased IκB-α and p65 phosphorylation, which was attenuated by siRNA PSMC5. Behavioral tests and pathological/biochemical assays showed that shRNA PSMC5 attenuated LPS-induced cognitive and motor impairments, and restored synaptic ultrastructure and protein levels in mice. ShRNA PSMC5 reduced pro-inflammatory cytokine (TNF-α, IL-1ß, PGE2, and NO) levels in the serum and brain, and relevant protein factors (iNOS and COX-2) in the brain. Furthermore, shRNA PSMC5 upregulated the anti-inflammatory mediators interleukin IL-4 and IL-10 in the serum and brain, and promoted a pro-inflammation-to-anti-inflammation phenotype shift in microglial polarization. Mechanistically, shRNA PSMC5 significantly alleviated LPS-induced TLR4 expression. The polarization of LPS-induced microglial pro-inflammation phenotype was abolished by TLR4 inhibitor and in the TLR-4-/- mouse, as in shRNA PSMC5 treatment. PSMC5 interacted with TLR4 via the amino sites Glu284, Met139, Leu127, and Phe283. PSMC5 site mutations attenuated neuroinflammation and reduced pro-inflammatory factors by reducing TLR4-related effects, thereby reducing TLR4-mediated MyD88 (myeloid differentiation factor 88)-dependent activation of NF-κB. PSMC5 could be an important therapeutic target for treatment of neurodegenerative diseases involving neuroinflammation-associated cognitive deficits and motor impairments induced by microglial activation.

A direct interaction between RhoGDIα/Tau alleviates hyperphosphorylation of Tau in Alzheimer's disease and vascular dementia.

RhoGDIα is an inhibitor of RhoGDP dissociation that involves in Aß metabolism and NFTs production in Alzheimer's disease (AD) by regulating of RhoGTP enzyme activity. Our previous research revealed that RhoGDIα, as the target of Polygala saponin (Sen), might alleviate apoptosis of the nerve cells caused by hypoxia/reoxygenation (H/R). To further clarify the role of RhoGDIα in the generation of NFTs, we explored the relationship between RhoGDIα and Tau. We found out that RhoGDIα and Tau can bind with each other and interact by using coimmunoprecipitation (Co-IP) and GST pulldown methods in vitro. This RhoGDIα-Tau partnership was further verified by using immunofluorescence colocalization and fluorescence resonance energy transfer (FRET) approaches in PC12 cells. Using the RNA interference (RNAi) technique, we found that the RhoGDIα may be involved in an upstream signaling pathway for Tau. Subsequently, in Aß25-35- and H/R-induced PC12 cells, forced expression of RhoGDIα via cDNA plasmid transfection was found to reduce the hyperphosphorylation of Tau, augment the expression of bcl-2 protein, and inhibit the expression of Bax protein (reducing the Bax/bcl-2 ratio) and the activity of caspase-3. In mouse AD and VaD models, forced expression of RhoGDIα via injection of a viral vector (pAAV-EGFP-RhoGDIα) into the lateral ventricle of the brain alleviated the pathological symptoms of AD and VaD. Finally, GST pulldown confirmed that the binding sites on RhoGDIα for Tau were located in the range of the ΔC33 fragment (aa 1-33). These results indicate that RhoGDIα is involved in the phosphorylation of Tau and apoptosis in AD and VaD. Overexpression of RhoGDIα can inhibit the generation of NFTs and delay the progress of these two types of dementia.

Senegenin Rescues PC12 Cells with Oxidative Damage Through Inhibition of Ferroptosis.

Oxidative stress is one of the pathological mechanisms of Alzheimer's disease (AD), and ferroptosis has been determined to be involved in neurodegenerative diseases such as AD. Senegenin (Sen) prevents oxidative damage in nerve cells via a mechanism that may be highly related to ferroptosis. However, the mechanism of ferroptosis pathway involvement in AD is unclear. In this study, we established a model of PC12 cytotoxic injury induced by Aß25-35, and we detected the level of oxidative damage, MMP, and ferroptosis-related protein expression. The results showed that, compared with control group, the level of ROS increased, GPX activities decreased, and MDA levels increased in Aß25-35 group. Aß25-35 could induce mitochondrial depolarization in PC12 cells and Fer-1 could not reverse this damage. WB revealed that Aß25-35 group had increased ACSL4 and PEBP1 proteins, and decreased GPX4 protein. After adding Sen in the model, the level of oxidative damage was reduced, and mitochondrial depolarization was reversed compared with Aß25-35 group. WB suggested that the expression of ACSL4 and PEBP1 proteins decreased, and the expression of GPX4 protein increased by Sen treatment. In conclusion, we found that Sen exhibits strong neuroprotective activity against Aß25-35 induced oxidative damage and lipid metabolic associated with ferroptosis. Inhibiting nerve cell ferroptosis might facilitate the future development of strategies to AD.

The Long-term Effect of Dobutamine on Intrinsic Myocardial Function and Myocardial Injury in Septic Rats with Myocardial Dysfunction.

ABSTRACT: Dobutamine (DOB) is recommended as an inotrope for septic patients with low cardiac output, but its long-term impact on sepsis-induced cardiomyopathy remains unclear. This study investigated the long-term effect of DOB on septic myocardial dysfunction and injury. Rats were exposed to cecal ligation and puncture (CLP), the intrinsic myocardial function, other organ functions, hemodynamics, inflammatory response, serum myocardial injury biomarkers, myocardial apoptosis, and vascular permeability were determined. At 6 h after CLP, the left ventricular ±dP/dt were significantly depressed, cardiac tumor necrosis factor-α and vascular cell adhesion molecule-1 expression were increased, but not serum cardiac troponin I (cTnI), N-terminal pro-brain natriuretic peptide (NT-proBNP), heart-type fatty acid-binding protein (H-FABP), creatinine, and urea nitrogen concentrations in CLP group compared with controls. At 9 h after CLP, hepatic dysfunction was present in CLP rats compared with controls. At 6 h after CLP, DOB treatment did not affect hemodynamics, the left ventricular ±dP/dt, cytokine levels in serum and myocardium, as well as cardiomyocyte apoptosis and cardiac vascular hyperpermeability at 20 h after CLP. However, DOB (10.0 µg/kg) increased serum IL-10 level and improved survival in septic rats. These results indicate that the intrinsic myocardial depression occurs earlier than hepatic and renal dysfunction in sepsis and serum cTnI, NT-proBNP, and H-FABP are not suitable as early biomarkers for sepsis-induced myocardial dysfunction. Although DOB treatment (10.0 µg/kg) in the presence of myocardial dysfunction improves survival in septic rats, it neither improves myocardial function and hemodynamics nor attenuates myocardial injury at the later stage of sepsis.

Astaxanthin Improved the Cognitive Deficits in APP/PS1 Transgenic Mice Via Selective Activation of mTOR.

Astaxanthin (Ast) is an effective neuroprotective and antioxidant compound used to treat Alzheimer's disease (AD); however, the underlying in vivo molecular mechanisms remain unknown. In this study, we report that Ast can activate the mammalian target of rapamycin (mTOR) pathway in the 8-month-old APP/PS1 transgenic mouse model of AD. Our results suggest that Ast could ameliorate the cognitive defects in APP/PS1 mice by activating the mTOR pathway. Moreover, mTOR activation perturbed the mitochondrial dynamics, increased the synaptic plasticity after 21 days of treatment with Ast (10 mg/kg/day), and increased the expression of Aß-degrading enzymes, mitochondrial fusion, and synapse-associated proteins and decreased the expression of mitochondrial fission proteins. Intraperitoneal injection of the mTOR inhibitor, rapamycin, abolished the effects of Ast. In conclusion, Ast activates the mTOR pathway, which is necessary for mitochondrial dynamics and synaptic plasticity, leading to improved learning and memory. Our results support the use of Ast for the treatment of cognitive deficits. Graphical abstract In summary, Ast ameliorates cognitive deficits via facilitating the mTOR-dependent mitochondrial dynamics and synaptic damage, and reducing Aß accumulation. This model supports the use of Ast for the treatment of cognitive deficits.

USP8 protects against lipopolysaccharide-induced cognitive and motor deficits by modulating microglia phenotypes through TLR4/MyD88/NF-κB signaling pathway in mice.

Ubiquitin-specific protease 8 (USP8) regulates inflammation in vitro; however, the mechanisms by which USP8 inhibits neuroinflammation and its pathophysiological functions are not completely understood. In this study, we aimed to determine whether USP8 exerts neuroprotective effects in a mouse model of lipopolysaccharide (LPS)-induced cognitive and motor impairment. We commenced intracerebroventricular USP8 administration 7 days prior to i.p. injection of LPS (750 µg/kg). All treatments and behavioral experiments were performed once per day for 7 consecutive days. Behavioral tests and pathological/biochemical assays were performed to evaluate LPS-induced hippocampal damage. USP8 attenuated LPS-induced cognitive and motor impairments in mice. Moreover, USP8 downregulated several pro-inflammatory cytokines [nitric oxide (NO), tumor necrosis factor α (TNF-α), prostaglandin E2 (PGE2), and interleukin-1ß (IL-1ß)] in the serum and brain, and the relevant protein factors [inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2)] in the brain. Furthermore, USP8 upregulated the anti-inflammatory mediators interleukin (IL)-4 and IL-10 in the serum and brain, and promoted a shift from pro-inflammatory to anti-inflammatory microglial phenotypes. The LPS-induced microglial pro-inflammatory phenotype was abolished by TLR4 inhibitor and in TLR4-/- mice; these effects were similar to those of USP8 treatment. Mechanistically, we found that USP8 increased the expression of neuregulin receptor degradation protein-1 (Nrdp1), potently downregulated the expression of TLR4 and myeloid differentiation primary response protein 88 (MyD88) protein, and inhibited the phosphorylation of IκB kinase (IKK) ß and kappa B-alpha (IκBα), thereby reducing nuclear translocation of p65 by inhibiting the activation of the nuclear factor-kappaB (NF-κB) signaling pathway in LPS-induced mice. Our results demonstrated that USP8 exerts protective effects against LPS-induced cognitive and motor deficits in mice by modulating microglial phenotypes via TLR4/MyD88/NF-κB signaling.

The Secretion from Bone Marrow Mesenchymal Stem Cells Pretreated with Berberine Rescues Neurons with Oxidative Damage Through Activation of the Keap1-Nrf2-HO-1 Signaling Pathway.

Oxidative stress is a potential pathological mechanism of Alzheimer's disease (AD). Berberine (BBR) can improve antioxidative capacity and inhibit Aß protein aggregation and tau protein hyperphosphorylation in AD, and stem cell therapy is also increasingly recognized as a therapy for AD. Bone marrow mesenchymal stem cells (BMSCs) have many advantages, as they exhibit antioxidant and anti-inflammatory activity and secrete a variety of neurotrophic factors, and play important roles in neurodegenerative disease treatment. In this study, we investigated the antioxidant effects of secretions from BMSCs pretreated with BBR on tert-butyl hydroperoxide (t-BHP)-damaged neurons. We demonstrated that BBR can enhance BMSC viability and the secretion of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), both of which are vital neurotrophic factors that maintain neuronal growth. Moreover, conditioned medium from BBR-treated BMSCs (BBR-BMSC-CM) reduced reactive oxygen species (ROS) production, attenuated a decrease in the mitochondrial membrane potential, and ameliorated neuronal apoptosis by decreasing levels of the apoptotic proteins Bax/Bcl-2, cytochrome c, and cleaved caspase-3/caspase-3. In addition, increased synaptophysin (SYP) and postsynaptic density protein 95 (PSD95) levels indicated that neuronal synaptic function was restored. Further study revealed that BBR-BMSC-CM activated the antioxidant proteins Keap1, Nrf2, and HO-1. In conclusion, our results showed that BBR-BMSC-CM attenuated apoptosis and oxidative damage in neurons by activating the Keap1-Nrf2-HO-1 signaling pathway. Taken together, these results also suggest BBR as a drug to stimulate the secretion of nutritional cytokines with the potential to treat AD.

Delivery of exogenous proteins by mesenchymal stem cells attenuates early memory deficits in a murine model of Alzheimer's disease.

A promising intervention for Alzheimer's disease (AD) would ideally target key pathological factors that are involved in AD pathogenesis. Soluble factors produced by engrafted mesenchymal stem cells (MSCs) mediate potential therapeutic effects in AD. However, these therapeutic benefits are largely hampered by the limited paracrine capacity of MSCs. In this study, we used adenovirus-mediated gene transduction of bone marrow MSCs to deliver exogenous proteins into the brain of APPswe/PSEN1dE9 (APP/PS1) mice in the early stage of impairment. We observed that engrafted MSCs carrying exogenous (C-X3-C motif) ligand 1 (CX3CL1) alone reduced the production of the inflammatory cytokine TNF-ɑ and improved synapse-related protein expression but not cognitive function. Transplantation of MSCs carrying CX3CL1 and Wnt3a (CX3CL1-Wnt3a-MSC) significantly attenuated the learning and memory impairment when compared with a control group. The improvement of neurobehavioral functions in APP/PS1 mice treated with CX3CL1-Wnt3a-MSC was related to the inhibition of microglial neurotoxicity and promotion of hippocampal neurogenesis. Transplantation of CX3CL1-Wnt3a-MSC also regulated phosphoinositide 3-kinase/activated protein kinase B (PI3K/AKT) signaling to inhibit the activity of glycogen synthase kinase 3 beta (GSK3ß). Taken together, these results indicate that the delivery of exogenous proteins via MSCs can modulate microglial function and enhance neurogenesis, thereby providing new insights into AD intervention.

The Effects of Chloroquine and Hydroxychloroquine on ACE2-Related Coronavirus Pathology and the Cardiovascular System: An Evidence-Based Review.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a serious threat to global public health and there is currently no effective antiviral therapy. It has been suggested that chloroquine (CQ) and hydroxychloroquine (HCQ), which were primarily employed as prophylaxis and treatment for malaria, could be used to treat COVID-19. CQ and HCQ may be potential inhibitors of SARS-CoV-2 entry into host cells, which are mediated via the angiotensin-converting enzyme 2 (ACE2), and may also inhibit subsequent intracellular processes which lead to COVID-19, including damage to the cardiovascular (CV) system. However, paradoxically, CQ and HCQ have also been reported to cause damage to the CV system. In this review, we provide a critical examination of the published evidence. CQ and HCQ could potentially be useful drugs in the treatment of COVID-19 and other ACE2 involved virus infections, but the antiviral effects of CQ and HCQ need to be tested in more well-designed clinical randomized studies and their actions on the CV system need to be further elucidated. However, even if it were to turn out that CQ and HCQ are not useful drugs in practice, further studies of their mechanism of action could be helpful in improving our understanding of COVID-19 pathology.

Cyanidin-3-o-glucoside inhibits UVA-induced human dermal fibroblast injury by upregulating autophagy.

BACKGROUND/PURPOSE: Ultraviolet (UV) A (315-400 nm) is the UV light that most frequently reaches the Earth's surface and can penetrate the epidermis through to the dermis, causing various issues, including skin aging and skin cancer. The results of our previous studies have shown that the flavonoid monomer cyanidin-3-o-glucoside (C3G) can effectively inhibit primary human dermal fibroblast (HDF) oxidative damage and apoptosis caused by UVA radiation. Many flavonoids can regulate the level of autophagy. However, whether C3G inhibits UVA-induced oxidative damage to primary HDFs by regulating autophagy levels remains unclear. METHODS AND RESULTS: In this study, we used different doses (0-12 J/cm2 ) of UVA to irradiate cells and showed that the expression levels of autophagy-related gene 5 (Atg5) and microtubule-associated protein 1 light chain 3 (LC3)-II in primary HDFs first increased and then decreased. The expression of Atg5 and LC3-II was significantly decreased under 12 J/cm2 (light-damage model). C3G increased the levels of Atg5 and LC3-II. Primary HDFs were pretreated with C3G, followed by treatment with the autophagy inhibitor 3-methyladenine (3-MA) after 12 J/cm2 UVA irradiation. The inhibitory effects of C3G on morphological changes, oxidative damage, and apoptosis in primary HDFs induced by UVA were significantly decreased. CONCLUSION: C3G can inhibit UVA-induced damage to primary HDFs by inducing autophagy. These results provide a theoretical basis for the application of natural compounds to resist light damage to the skin in the future.

USP8 ameliorates cognitive and motor impairments via microglial inhibition in a mouse model of sepsis-associated encephalopathy.

Sepsis-associated encephalopathy (SAE) is a common and serious complication of sepsis, which is thought to be caused by neuroinflammation. In our previous study, ubiquitin-specific protease 8 (USP8), was reported to regulate inflammation in vitro. In the current study, we investigated whether increased USP8 expression would ameliorate the cognitive and motor impairments induced by cecal ligation and puncture (CLP) in mice, a model of SAE. Male adult mice were randomly divided into four groups: control, sham, CLP, and CLP + USP8 groups. The CLP + USP8 mice showed reduced weight loss on day 4 post-CLP, with a slight increase noted on day 7. The mortality rate in the CLP group was 70% 48 h after CLP; however, USP8 significantly improved survival after CLP. USP8 modulated the neurobehavioral scores in CLP mice. Our results also indicate that USP8 attenuated the CLP-induced cognitive and motor impairments, based on the performance of mice in the Morris water maze (MWM), pole-climbing, and wire suspension tests. USP8 suppressed the release of pro-inflammatory mediators, including prostaglandin E2(PGE2) in the serum and nitric oxide (NO) in brain tissue, as well as levels of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in brain tissue. Immunofluorescence experiments revealed that USP8 inhibited CLP-induced increases in microglial size and density in the hippocampus, and protected hippocampal neurons. Our findings indicate that neuroinflammation occurs in the brains of CLP mice, and that USP8 exerts protective effects against CLP-induced neuroinflammation and cognitive and motor impairments, which may aid in the development of novel therapeutic strategies for SAE.

Neuroinflammation induced by lipopolysaccharide causes cognitive impairment in mice.

In this study, we investigated lipopolysaccharide (LPS)-induced cognitive impairment and neuroinflammation in C57BL/6J mice by using behavioral tests, immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and Western blot. We found that LPS treatment leads to sickness behavior and cognitive impairment in mice as shown in the Morris water maze and passive avoidance test, and these effects were accompanied by microglia activation (labeled by ionized calcium binding adaptor molecule-1, IBA-1) and neuronal cell loss (labeled by microtubule-associated protein 2, MAP-2) in the hippocampus. The levels of interleukin-4 (IL-4) and interleukin-10 (IL-10) in the serum and brain homogenates were reduced by the LPS treatment, while the levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), prostaglandin E2 (PGE2) and nitric oxide (NO) were increased. In addition, LPS promoted the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in the brain homogenates. The Western blot analysis showed that the nuclear factor kappa B (NF-κB) signaling pathway was activated in the LPS groups. Furthermore, VIPER, which is a TLR-4-specific inhibitory peptide, prevented the LPS-induced neuroinflammation and cognitive impairment. These data suggest that LPS induced cognitive impairment and neuroinflammation via microglia activation by activating the NF-kB signaling pathway; furthermore, we compared the time points, doses, methods and outcomes of LPS administration between intraperitoneal and intracerebroventricular injections of LPS in LPS-induced neuroinflammation and cognitive impairment, and these data may provide additional insight for researchers performing neuroinflammation research.

Lycopene protects against t-BHP-induced neuronal oxidative damage and apoptosis via activation of the PI3K/Akt pathway.

Oxidative stress is a key factor of and closely implicated in the pathogenesis of Alzheimer's disease (AD). We herein used tert-butyl hydroperoxide (t-BHP) to induce oxidative stress and mimic oxidative neurotoxicity in vitro. Lycopene is a natural antioxidant that has a strong ability to eliminate free radicals and shows effective protection in some neurodegenerative disease models. However, the effect of lycopene on t-BHP-induced neuronal damage in primary mouse neurons is unknown. This study aimed to investigate the effects of lycopene on t-BHP-induced neuronal damage and the related mechanisms. We found that lycopene pretreatment effectively enhanced the cell viability, improved the neuron morphology, increased the GSH/GSSG level, restored the mitochondrial membrane potential (ΔΨm) and decreased reactive oxygen species generation. Furthermore, lycopene reduced the ratios of Bax:Bcl-2 and cleaved caspase-3:caspase-3 and the level of cytochrome C, increased the levels of synaptophysin (SYP) and postsynaptic density 95 (PSD95) and activated the PI3K/Akt pathway. In conclusion, lycopene attenuated oxidative stress and reduced t-BHP-induced cell apoptosis, and the mechanism is likely related to activation of the PI3K/Akt pathway. Therefore, lycopene is a potential agent for preventing oxidative stress-mediated AD.

Phenylephrine Attenuated Sepsis-Induced Cardiac Inflammation and Mitochondrial Injury Through an Effect on the PI3K/Akt Signaling Pathway.

OBJECTIVE: To investigate whether phenylephrine (PE) inhibits sepsis-induced cardiac dysfunction, cardiac inflammation, and mitochondrial injury through the PI3K/Akt signaling pathway. METHODS: A rat model of sepsis was established by cecal ligation and puncture. PE and/or wortmannin (a PI3K inhibitor) were administered to investigate the role of PI3K/Akt signaling in mediating the effects of PE on inhibiting sepsis-induced cardiac dysfunction, cardiac inflammation, and mitochondrial injury. Hematoxylin-eosin staining, echocardiography, and Langendorff system were used to examine the myocardial injury and function. The concentrations of TNF-α and IL-6 were analyzed by enzyme-linked immunosorbent assay. Intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), myeloperoxidase, mitochondria-related fusion/fission proteins, and PI3K/Akt signaling pathway-associated proteins were analyzed by Western blotting. RESULTS: PE improved the cardiac function and survival in septic rats. PE decreased TNF-α, IL-6, ICAM-1, VCAM-1, and myeloperoxidase contents in the myocardium of septic rats. Meanwhile, PE increased the fusion-related proteins and decreased the fission-related proteins in the myocardial mitochondria of septic rats. On the other hand, PE activated the PI3K/Akt signaling pathway in the cecal ligation and puncture-treated rats, and all the protective effects of PE were abolished by wortmannin. CONCLUSIONS: PE attenuated sepsis-induced cardiac dysfunction, cardiac inflammation, and mitochondrial injury through the PI3K/Akt signaling pathway.

Alpha-1 Adrenergic Receptor Agonist Phenylephrine Inhibits Sepsis-Induced Cardiomyocyte Apoptosis and Cardiac Dysfunction via Activating ERK1/2 Signal Pathway.

It was demonstrated that α1 adrenergic receptor (α1-AR) activation by phenylephrine (PE) attenuated cardiac dysfunction in lipopolysaccharide (LPS)-challenged mice. However, it is unclear whether PE suppresses sepsis-induced cardiomyocyte apoptosis. Here, we investigated the effects of PE on cardiomyocyte apoptosis in LPS-treated adult rat ventricular myocytes (ARVMs) and septic rats induced by cecal ligation and puncture. Cardiomyocyte apoptosis and caspase activity were detected by TUNEL and spectrophotometrical assay, respectively. Bax, Bcl-2 and cytochrome c (Cyt c) levels as well as IκBα, ERK1/2, p38 MAPK, JNK and cardiac troponin I (cTnI) phosphorylation were analyzed by Western blotting, and TNF-α concentration was analyzed by ELISA. PE inhibited LPS-induced caspase-3 activation in ARVMs, which was reversed by prazosin (a membrane permeable α1-AR antagonist), but not by CGP12177A (a membrane impermeable α1-AR antagonist). PE upregulated phosphorylated ERK1/2 and Bcl-2 contents, decreased TNF-α and Bax levels, Cyt c release, caspase-8/-9 activities as well as IκBα, p38MAPK and JNK phosphorylation in LPS-treated ARVMs, all of which were abolished by prazosin. Treatment with U0126 (a specific ERK1/2 inhibitor) reversed the effects of PE on IκBα, p38MAPK and JNK phosphorylation as well as caspase-3/-8/-9 activation in LPS-treated ARVMs. In septic rats, PE not only inhibited myocardial apoptosis as well as IκBα, p38MAPK, and JNK phosphorylation, but also upregulated myocardial phosphorylated ERK1/2. Furthermore, PE inhibited myocardial cTnI phosphorylation and improved cardiac function in septic rats. Taken together, our data suggest that α1-AR activation by PE inhibits sepsis-induced cardiomyocyte apoptosis and cardiac dysfunction via activating ERK1/2 signal pathway.

The Secretion from Neural Stem Cells Pretreated with Lycopene Protects against tert-Butyl Hydroperoxide-Induced Neuron Oxidative Damage.

Neural stem cells (NSCs) hold great potential for the treatment of Alzheimer's disease (AD) through both cellular replacement and their secretion of trophic factors. Lycopene is a potent ß-carotenoid antioxidant that has been shown to ameliorate oxidative damage in previous studies. However, it is unclear if lycopene can interact with NSCs to induce the secretion of growth factors, and whether pretreatment with lycopene will allow NSCs to secrete enough trophic factors to reduce oxidative damage to neurons. We pretreated cultured NSCs with lycopene, then applied the lycopene-treated-NSC-conditioned media (Ly-NSC-CM) to primary neuronal cultures exposed to tert-butyl hydroperoxide (t-BHP) to induce oxidative damage. We found that lycopene promoted the secretion of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and vascular endothelial growth factor (VEGF) from NSCs. In addition, Ly-NSC-CM attenuated oxidative stress and reduced t-BHP-induced cell apoptosis. We found an antiapoptotic effect related to inhibited expression of Bax/Bcl-2, cytochrome C, and cleaved caspase-3. Moreover, Ly-NSC-CM increased the levels of synaptic proteins, including synaptophysin (SYP) and postsynaptic density 95 (PSD-95), and activated the PI3K/Akt pathway in cultured neurons. Collectively, these data indicate that Ly-NSC-CM could protect neurons from t-BHP-induced oxidative damage.

PYNOD reduces microglial inflammation and consequent neurotoxicity upon lipopolysaccharides stimulation.

PYNOD, a nod-like receptors (NLR)-like protein, was indicated to inhibit NF-κB activation, caspase-1-mediated interleukin (IL)-1ß release and cell apoptosis in a dose-dependent manner. Exogenous addition of recombinant PYNOD to mixed glial cultures may suppress caspase-1 activation and IL-1ß secretion induced by Aß. However, to the best of our knowledge, there no study has focused on the immunoregulatory effects of PYNOD specifically in microglia. The present study aimed to explore the roles of PYNOD involved in the lipopolysaccharides (LPS)-induced microglial inflammation and consequent neurotoxicity. Murine microglial BV-2 cells were transfected with pEGFP-C2-PYNOD (0-5.0 µg/ml) for 24 h and incubated with or without LPS (1 µg/ml) for a further 24 h. Cell viability was determined using MTT assay and the secretion of nitric oxide (NO), IL-1ß and caspase-1 was measured using the Griess method or ELISA. Protein expression levels of NF-κB p65 and inducible nitric oxide synthase (iNOS) were detected by immunofluorescent staining and/or western blot analysis. Co-culture of BV-2 cells with human neuroblastoma cell line SK-N-SH was performed in Transwell plates and the cell viability and apoptosis (using flow cytometry) of SK-N-SH cells were determined. Results indicated that PYNOD overexpression inhibited NO secretion and iNOS protein expression induced by LPS in BV-2 cells, with no detectable cytotoxicity. PYNOD overexpression also reduced the secretion of IL-1ß and caspase-1 from BV-2 cells upon LPS stimulation. These effects were dose-dependent. Additionally, PYNOD overexpression prevented LPS-induced nuclear translocation of NF-κB p65 in BV-2 cells. The growth-inhibitory and apoptosis-promoting effects of BV-2 cells towards SK-N-SH cells were alleviated as a result of PYNOD overexpression. In conclusion, PYNOD may mitigate microglial inflammation and consequent neurotoxicity.

A new method for neonatal rat ventricular myocyte purification using superparamagnetic iron oxide particles.

BACKGROUND: Neonatal rat ventricular myocytes (NRVMs) have proven to be an ideal research model for cardiac disease. However, the current methods to purify NRVMs have a limitation to obtain high purity. The purpose of this study was to develop a NRVM purification method by using superparamagnetic iron oxide particles (SIOP). METHODS: NRVMs were purified by using SIOP (SIOP group). The differential attachment with or without bromodeoxyuridine (BrdU) treatment served as control and BrdU groups, respectively. The Percoll gradient (Percoll) and magnetic-activated cell sorting (MACS) methods were performed to compare the purity and viability of NRVMs with SIOP method. RESULTS: The SIOP group enriched NRVMs up to 93.9 ±â€¯2.0% purity determined by flow cytometry (FCM) and 95.6 ±â€¯1.3% by immunofluorescence count (IF). In contrast, the control group gave purities of 71.9 ±â€¯2.9% (by FCM) and 66.8 ±â€¯8.9% (by IF), and the BrdU group obtained 82.0 ±â€¯1.3% (by FCM) and 83.1 ±â€¯2.4% (by IF). The purity of SIOP-isolated NRVMs was not different from that of Percoll and MACS groups. However, the cardiomyocytes separated by these methods, except SIOP protocol, were mixed with intrinsic cardiac adrenergic cells. NRVMs purified by SIOP shaped the similar three-dimensional morphology, with no difference in cell yield, viability and cytosolic Ca2+ homeostasis at 24 h after isolation compared with NRVMs in other groups. Furthermore, SIOP-purified NRVMs retained the responses to phenylephrine and lipopolysaccharide challenge. CONCLUSION: We first reported an efficient and novel method to purify NRVMs using SIOP, which may help accelerate innovative research in the field of cardiomyocyte biology.

α2A-adrenergic blockade attenuates septic cardiomyopathy by increasing cardiac norepinephrine concentration and inhibiting cardiac endothelial activation.

Cardiomyopathy is a common complication associated with increased mortality in sepsis, but lacks specific therapy. Here, using genetic and pharmacological approaches, we explored the therapeutic effect of α2A-adrenergic receptor (AR) blockade on septic cardiomyopathy. CLP-induced septic rats were treated with BRL44408 (α2A-AR antagonist), prazosin (α1-AR antagonist) and/or reserpine. CLP-induced cardiomyopathy, indicated by reduced dP/dt and increased cardiac troponin I phosphorylation, was attenuated by BRL44408, this was associated with reduced cardiac TNF-α and endothelial VCAM-1 expression, cardiomyocyte apoptosis and related signal molecule phosphorylation. BRL44408 increased cardiac norepinephrine (NE) concentration in CLP rats. Pretreatment with reserpine that exhausts cardiac NE without affecting the circulating NE concentration or with prazosin partially abolished the cardioprotection of BRL44408 and reversed its inhibitory effects on myocardial TNF-α, apoptosis and related signal molecule phosphorylation, but not on VCAM-1 expression in septic rats. These effects of BRL44408 were confirmed by α2A-AR gene deletion in septic mice. Furthermore, α2-AR agonist not only enhanced LPS-induced TNF-α and VCAM-1 expression in cardiac endothelial cells that express α2A-AR, but also enhanced LPS-induced cardiac dysfunction in isolated rat hearts. Our data indicate that α2A-AR blockade attenuates septic cardiomyopathy by promoting cardiac NE release that activates myocardial α1-AR and suppressing cardiac endothelial activation.

Baicalin administration attenuates hyperglycemia-induced malformation of cardiovascular system.

In this study, the effects of Baicalin on the hyperglycemia-induced cardiovascular malformation during embryo development were investigated. Using early chick embryos, an optimal concentration of Baicalin (6 µM) was identified which could prevent hyperglycemia-induced cardiovascular malformation of embryos. Hyperglycemia-enhanced cell apoptosis was reduced in embryos and HUVECs in the presence of Baicalin. Hyperglycemia-induced excessive ROS production was inhibited when Baicalin was administered. Analyses of SOD, GSH-Px, MQAE and GABAA suggested Baicalin plays an antioxidant role in chick embryos possibly through suppression of outwardly rectifying Cl(-) in the high-glucose microenvironment. In addition, hyperglycemia-enhanced autophagy fell in the presence of Baicalin, through affecting the ubiquitin of p62 and accelerating autophagy flux. Both Baicalin and Vitamin C could decrease apoptosis, but CQ did not, suggesting autophagy to be a protective function on the cell survival. In mice, Baicalin reduced the elevated blood glucose level caused by streptozotocin (STZ). Taken together, these data suggest that hyperglycemia-induced embryonic cardiovascular malformation can be attenuated by Baicalin administration through suppressing the excessive production of ROS and autophagy. Baicalin could be a potential candidate drug for women suffering from gestational diabetes mellitus.