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.

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.

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.

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.

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.

The effect of Cyanidin-3-o-glucoside on UVA-induced damage in human dermal fibroblasts.

BACKGROUND/PURPOSE: Ultraviolet-A (UVA) radiation can induce photoaging and skin cancer, but means to prevent or treat UVA-induced skin damage require further study. We investigated the effects of cyanidin-3-o-glucoside (C3G), a monomer of anthocyanin, on UVA-induced damage in primary human dermal fibroblasts (HDFs), and we identify possible mechanisms underlying the protective effects of this compound. METHODS: Primary HDFs were pretreated with 80 µmol/L C3G for 2 hours and UVA irradiated at 12 J/cm2 . The cells were then incubated with 80 µmol/L C3G for 12 hours after irradiation. HDFs were randomly divided into control, UVA treatment, C3G, and UVA treatment plus C3G pretreatment groups. RESULTS: C3G increased the cell viability of primary HDFs and decreased UVA-induced ROS production and apoptosis rate. Compared to the UVA group, the UVA plus pretreatment with C3G group displayed increased Bcl-2 expression and Bcl-2/Bax ratio, decreased cleaved caspase-3 and p-P38 levels, and increased ERK phosphorylation; no significant effect on p-JNK levels was observed. CONCLUSION: C3G reduced UVA-induced HDF oxidative damage and apoptosis, likely be related to the down-regulation of p-P38, up-regulation of ERK protein phosphorylation.

Neuroprotective Effect of Lutein on NMDA-Induced Retinal Ganglion Cell Injury in Rat Retina.

Lutein injection is a possible therapeutic approach for retinal diseases, but the molecular mechanism of its neuroprotective effect remains to be elucidated. The aim of this study was to investigate its protective effects in retinal ganglion cells (RGCs) against N-methyl-D-aspartate (NMDA)-induced retinal damage in vivo. Retinal damage was induced by intravitreal NMDA injection in rats. Each animal was given five daily intraperitoneal injections of Lutein or vehicle along with intravitreal NMDA injections. Electroretinograms were recorded. The number of viable RGCs was quantified using the retinal whole-mount method by immunofluorescence. Proteins were measured by Western blot assays. Lutein reduced the retinal damage and improved the response to light, as shown by an animal behavior assay (the black-and-white box method) in rats. Furthermore, Lutein treatment prevented the NMDA-induced reduction in phNR wave amplitude. Lutein increased RGC number after NMDA-induced retina damage. Most importantly, Bax, cytochrome c, p-p38 MAPK, and p-c-Jun were all upregulated in rats injected with NMDA, but these expression patterns were reversed by continuous Lutein uptake. Bcl-2, p-GSK-3ß, and p-Akt in the Lutein-treated eyes were increased compared with the NMDA group. Lutein has neuroprotective effects against retinal damage, its protective effects may be partly mediated by its anti-excitability neurotoxicity, through MAPKs and PI3K/Akt signaling, suggesting a potential approach for suppressing retinal neural damage.

Angiogenesis is repressed by ethanol exposure during chick embryonic development.

It is now known that excess alcohol consumption during pregnancy can cause fetal alcohol syndrome to develop. However, it is not known whether excess ethanol exposure could directly affect angiogenesis in the embryo or angiogenesis being indirectly affected because of ethanol-induced fetal alcohol syndrome. Using the chick yolk sac membrane (YSM) model, we demonstrated that ethanol exposure dramatically inhibited angiogenesis in the YSM of 9-day-old chick embryos, in a dose-dependent manner. Likewise, the anti-angiogenesis effect of ethanol could be seen in the developing vessel plexus (at the same extra-embryonic regions) during earlier stages of embryo development. The anti-angiogenic effect of ethanol was found associated with excess reactive oxygen species (ROS) production; as glutathione peroxidase activity increased while superoxide dismutase 1 and 2 activities decreased in the YSMs. We further validated this observation by exposing chick embryos to 2,2'-azobis-amidinopropane dihydrochloride (a ROS inducer) and obtained a similar anti-angiogenesis effect as ethanol treatment. Semiquantitative reverse transcription-polymerase chain reaction analysis of the experimental YSMs revealed that expression of angiogenesis-related genes, vascular endothelial growth factor and its receptor, fibroblast growth factor 2 and hypoxia-inducible factor, were all repressed following ethanol and 2,2'-azobis-amidinopropane dihydrochloride treatment. In summary, our results suggest that excess ethanol exposure inhibits embryonic angiogenesis through promoting superfluous ROS production during embryo development.

Regulation of ubiquitin-specific processing protease 8 suppresses neuroinflammation.

In our previous study, we reported that luteolin might exert neuroprotective functions by inhibiting the production of proinflammatory mediators, thereby suppressing microglial activation. In this study, we used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to study the effect of ubiquitin-specific processing protease 8 (USP8) in luteolin-treated microglia. Western blot analysis verified that USP8 expression is upregulated by luteolin. Researchers have found that USP8 markedly enhanced the stability of neuregulin receptor degradation protein-1 (Nrdp1), which in turn inhibited the production of proinflammatory cytokines in toll-like receptor-triggered macrophages. We next hypothesized that luteolin inhibits microglial inflammation by regulating USP8 gene expression. After transfecting BV2-immortalized murine microglial cells with USP8, a significant reduction in the degradation of Nrdp1 was observed. USP8 overexpression also reduced the production of lipopolysaccharide (LPS)-induced proinflammatory mediators such as inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2). We also found that USP8 siRNA blocked luteolin inhibition of pro-inflammatory gene expression such as iNOS, NO, COX-2, and PGE2. Taken together, our findings suggested that luteolin inhibits microglial inflammation by enhancing USP8 protein production. We concluded that in addition to anti-inflammatory luteolin, USP8 might represent a novel mechanism for the treatment of neuroinflammation and neurodegeneration.

ß1-adrenoceptor stimulation promotes LPS-induced cardiomyocyte apoptosis through activating PKA and enhancing CaMKII and IκBα phosphorylation.

INTRODUCTION: Caspase activation and cardiomyocyte apoptosis have been implicated in lipopolysaccharide (LPS)-induced cardiac contractile dysfunction. We have recently demonstrated that ß1-adrenoceptor (AR) activation by endogenous norepinephrine contributes to cardiomyocyte apoptosis in endotoxemic mice. Here, we further investigated the molecular mechanisms for the enhancing effect of ß1-AR activation on LPS-induced cardiomyocyte apoptosis. METHODS: The adult mouse ventricular myocytes were exposed to LPS, dobutamine, protein kinase A (PKA) inhibitor or/and nifedipine, an L-type Ca(2+) channel blocker. Male BALB/c mice were treated with LPS or/ and ß1-AR antagonist, atenolol. Cardiomyocyte apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assay and apoptosis-associated molecules were detected. RESULTS: LPS induced apoptosis in adult mouse ventricular myocytes, dobutamine (DOB), a ß1-AR agonist, promoted apoptosis, caspase-8, 9 and 3 activation and increased cytosolic Ca(2+) concentration in LPS-challenged cardiomyocytes. DOB also up-regulated TNF-α expression, decreased Bcl-2 levels, promoted Bax translocation to mitochondria, mitochondrial membrane potential loss and cytochrome c release as well as IκBα, p38 MAPK, JNK and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) phosphorylation in LPS-treated cardiomyocytes. PKA inhibitor abolished the effects of DOB on caspase-9 activation, Bcl-2 levels as well as JNK and p38 MAPK phosphorylation, but not on IκBα phosphorylation, TNF-α expression and caspase-8 activation in LPS-stimulated cardiomyocytes. Pretreatment with nifedipine not only significantly blocked the enhancing effects of DOB on LPS-induced elevation in cytosolic Ca(2+) concentration and CaMKII phosphorylation in cardiomyocytes, but also partly reversed the effects of DOB on caspase-9 and caspase-3/7 activities in LPS-treated cardiomyocytes. Furthermore, atenolol suppressed TNF-α expression, JNK, p38 MAPK and CaMKII phosphorylation, increased Bcl-2 expression, and inhibited cytochrome c release and cardiomyocyte apoptosis in the myocardium of endotoxemic mice. CONCLUSIONS: ß1-AR activation promotes LPS-induced apoptosis through activating PKA, increasing CaMKII phosphorylation as well as enhancing IκBα phosphorylation and TNF-α expression in cardiomyocytes.

Ginsenoside Rg1 relieves tert-Butyl hydroperoxide-induced cell impairment in mouse microglial BV2 cells.

Microglial activation plays an important role in neurodegenerative diseases associated with oxidative stress. tert-Butyl hydroperoxide (t-BHP), an analog of hydroperoxide, mimics the oxidative damage to microglial cells. It has been reported that ginsenoside Rg1 (G-Rg1), an active ingredient of Panax ginseng, has anti-stress and anti-inflammatory properties. The present study aims to investigate the ability of G-Rg1 to decrease the t-BHP-mediated cell damage of BV2 microglial cells. We performed flow cytometry assays to facilitate the detection of reactive oxygen species as well as Western blotting analyses and immunofluorescence assays using specific antibodies, such as antibodies against phospho-mitogen-activated protein kinases (p-MAPKs), phospho-nuclear factor-κB (p-NF-κB), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X (Bax), Caspase-3, autophagy marker light chain 3 (LC3), and Becline-1. We found that treatment with 50 µM G-Rg1 protected microglial cells against oxidative damage induced by 10 µM t-BHP.

α1 adrenoceptor activation by norepinephrine inhibits LPS-induced cardiomyocyte TNF-α production via modulating ERK1/2 and NF-κB pathway.

Cardiomyocyte tumour necrosis factor α (TNF-α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)-induced cardiomyocyte TNF-α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS-induced TNF-α production in a dose-dependent manner. α1- adrenoceptor (AR) antagonist (prazosin), but neither ß1- nor ß2-AR antagonist, abrogated the inhibitory effect of NE on LPS-stimulated TNF-α production. Furthermore, phenylephrine (PE), an α1-AR agonist, also suppressed LPS-induced TNF-α production. NE inhibited p38 phosphorylation and NF-κB activation, but enhanced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and c-Fos expression in LPS-treated cardiomyocytes, all of which were reversed by prazosin pre-treatment. To determine whether ERK1/2 regulates c-Fos expression, p38 phosphorylation, NF-κB activation and TNF-α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c-Fos expression, p38 mitogen-activated protein kinase (MAPK) phosphorylation and TNF-α production, but not NF-κB activation in LPS-challenged cardiomyocytes. In addition, pre-treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS-induced TNF-α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c-Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF-α production and prevented LPS-provoked cardiac dysfunction. Altogether, these findings indicate that activation of α1-AR by NE suppresses LPS-induced cardiomyocyte TNF-α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF-κB activation.

[Effects of antagonist and agonist of nicotinic acetylcholine receptors on injury of rat neurons induced by amyloid ß-protein].

OBJECTIVE: To explore the chronic effects of nicotinic antagonist and agonist on rat neurons injury induced by ß-amyloid protein. METHODS: The rat model of neuron injury was established by the exposure to Aß25-35 and the intervention agent was either methyllycaconitine (MLA) or nicotine (Nic). And the experimental groups were control (distilled water), Aß25-35, MLA (MLA and Aß25-35) and Nic (Nic and Aß25-35). Cellular viability was detected by methyl thiazolyl tetrazolium (MTT) chromatometry while apoptosis and necrosis were detected by flow cytometer. RESULTS: Compared with control, cellular viability decreased while the apoptotic and necrotic rates increased in Aß25-35 group(P = 0.00). The values of cellular viability at (0.75 ± 0.02) and (0.75 ± 0.09) in Aß25-35 and MLA groups respectively were significantly lower than that of Nic group (0.81 ± 0.02, P = 0.01) at Day 3 and 7. No significant differences existed in cellular viability between Aß25-35 and MLA groups. At Day 14, the differences of cellular viability were not obvious in all groups. At Day 21, cell viability of MLA group (0.64 ± 0.10) was significantly higher than those of Aß25-35 (0.57 ± 0.04, P = 0.019) and Nic groups (0.56 ± 0.04, P = 0.008). The apoptotic rate was lower than that of Aß25-35 group (3.70% ± 0.20% vs 4.70% ± 0.46%, P = 0.008) while the necrotic rate lower than that of Aß25-35 group (7.73% ± 0.86% vs 16.30% ± 1.05%, P = 0.00) and Nic group (16.03% ± 1.53%, P = 0.00). However, no significant differences existed in cellular viability or apoptotic and necrotic rate between Aß25-35 and Nic groups. CONCLUSION: With chronic treatment, the protective effect of α7 nicotinic antagonist methyllycaconitine increases whereas that of nicotinic agonist nicotine decreases.

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.

Berberine protects against lipopolysaccharide-induced intestinal injury in mice via alpha 2 adrenoceptor-independent mechanisms.

AIM: To investigate the mechanisms responsible for the protective action of berberine (Ber) against gut damage in endotoxemic mice. METHODS: Male BALB/c mice were administered intragastrically with distilled water (0.1 mL/10 g), Ber (50 mg/kg) alone, yohimbine (2 mg/kg) alone, or Ber (50 mg/kg) in combination with yohimbine (2 mg/kg) for 3 d. On the third day, lipopolysaccharide (LPS, 18 mg/kg) or normal saline was intraperitoneally injected one hour after the intragastric administration. Following the treatment, intestinal injury in the ileum was histopathologically accessed; enterocyte apoptosis was examined using TUNEL method; Toll-like receptor 4 (TLR4) mRNA expression was measured using RT-PCR assay; inhibitor protein-κBα (I-κBα) phosphorylation and myeloperoxidase content were examined using Western blloting. The macrophage inflammatory protein-2 (MIP-2) production was measured using ELISA assay. RESULTS: Mice challenged with LPS caused extensive ileum injury, including a significantly increased injury score, decreased intestinal villus height, reduced gut mucosal weight and increased intestinal permeability. Furthermore, LPS significantly induced enterocyte apoptosis, increased TLR4 mRNA expression, I-κBα phosphorylation, MIP-2 production and myeloperoxidase content in the ileum. Pretreatment with Ber significantly alleviated all the alterations in the ileum in the endotoxemic mice. Pretreatment with the α2-adrenoceptor antagonist yohimbine did not block the protective action of Ber against LPS-induced intestinal injury. In addition, treatment with yohimbine alone did not prevent LPS-induced intestinal injury. CONCLUSION: Pretreatment with Ber provides significant protection against LPS-induced intestinal injury in mice, via reducing enterocyte apoptosis, inhibiting the TLR4-nuclear factor κB-MIP-2 pathway and decreasing neutrophil infiltration that are independent of α2-adrenoceptors.

Luteolin inhibits microglial inflammation and improves neuron survival against inflammation.

Microglia activation is one of the causative factors for neuroinflammation, which results in brain damage during neurodegenerative disease. Accumulating evidence has shown that the flavonoid luteolin (Lut) possesses potent anti-inflammatory properties; however, its effect on microglia inhibition is currently unknown. Moreover, it is not clear whether Lut also has indirect neuroprotective effects by reducing inflammatory mediators and suppressing microglia activation. In this study, we examined the effects of Lut on lipopolysaccharide (LPS)-induced proinflammatory mediator production and signaling pathways in murine BV2 microglia. In addition, we cocultured microglia and neurons to observe the indirect neuroprotective effects of Lut. Lut inhibited the LPS-stimulated expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), and interleukin-1ß (IL-1ß) as well as the production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)). Moreover, Lut blocked LPS-induced nuclear factor kappa B (NF-κB) activation. Preincubation of microglia with Lut diminished the neurotoxic effects, owing to the direct anti-inflammatory effects of the compound. Taken together, our findings suggest that Lut may have a potential therapeutic application in the treatment of neuroinflammatory disorders.

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.

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.

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.