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 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.

Sevoflurane Preconditioning Prevents Septic Myocardial Dysfunction in Lipopolysaccharide-Challenged Mice.

Myocardial dysfunction accompanied by severe sepsis could significantly increase the mortality rate of septic patients. This study investigated the effects and the potential mechanisms of sevoflurane preconditioning on septic myocardial dysfunction, which was induced by lipopolysaccharide (LPS; from Escherichia coli O55:B5; 18 mg/kg) in mice. Results indicated that 1 hour after the administration, LPS induced a significant increase in cell-surface Toll-like receptor 4 (TLR4), cytoplasmic IKKα protein expression, and nuclear translocation of nuclear factor kappa-B (NF-κB) protein (P < 0.05), which was attenuated by preconditioning with sevoflurane. Two hours after the administration, inhalation of sevoflurane significantly reduced the serum levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1ß, and IL-10 (P < 0.05). Twelve hours after administration, LPS caused pathological damage to the heart and elevated the serum levels of lactate dehydrogenase (LDH) and creatine kinase-MB (P < 0.05). Echocardiography indicated that sevoflurane preconditioning significantly improved systolic and diastolic function. The inhalation of sevoflurane inhibited increases in myeloperoxidase (MPO), macrophage inflammatory protein-2 (MIP-2), TNF-α, and IL-1ß levels (P < 0.05) induced by endotoxemia, whereas IL-6 release was facilitated. Sevoflurane attenuated the myocardial levels of nitric oxide (P < 0.05) without an apparent influence on malondialdehyde (MDA) or superoxide dismutase (P > 0.05). In conclusion, our study indicates that exposure to 2% sevoflurane before LPS challenge is protective against myocardial dysfunction. Sevoflurane preconditioning may attenuate neutrophil infiltration and the release of inflammatory mediators during endotoxemia.

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.

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.

AHSA1 regulates proliferation, apoptosis, migration, and invasion of osteosarcoma.

Activator of 90kDa heat shock protein ATPase homolog 1 (AHSA1) is a chaperone of heat shock 90kDa (HSP90) and stimulates ATPase activity of HSP90. The function of AHSA1 in osteosarcoma (OS) has not been reported yet. A previous study showed AHSA1 was overexpressed in OS cells. In this study, we investigated the role of AHSA1 in OS cells by silencing AHSA1. We report that silencing AHSA1 inhibited cell growth, migration, and invasion, and increased apoptosis of MG-63 and Saos2 cells. We also found that silencing AHSA1 decreased the ATPase activity of HSP90 in OS cells. In addition, silencing AHSA1 increased the levels of negative regulators of Wnt/ß-catenin signalling pathway, Axin-2 and GSK3ß, and decreased the levels of two key members of Wnt/ß-catenin signalling pathway, namely, Wnt-5a and ß-catenin. In conclusion, silencing AHSA1 regulates cell growth, apoptosis, migration, and invasion by regulating Wnt/ß-catenin signalling pathway and their negative regulators.

Effects of Senegenin against hypoxia/reoxygenation-induced injury in PC12 cells.

OBJECTIVE: To investigate the effect and the potential mechanism of Senegenin (Sen) against injury induced by hypoxia/reoxygenation (H/R) in highly differentiated PC12 cells. METHODS: The cultured PC12 cells were treated with H/R in the presence or absence of Sen (60 µmol/L). Four groups were included in the experiment: control group, H/R group, H/R+Sen group and Sen group. Cell viability of each group and the level of lactate dehydrogenase (LDH) in culture medium were detected for the pharmacological effect of Sen. Hoechst 33258 staining and annexin V/propidium iodide double staining were used to analyze the apoptosis rate. Moreover, mitochondrial membrane potential (△Ψm), reactive oxygen species (ROS) and intracellular free calcium ([Ca(2+)]i) were measured by fluorescent staining and flow cytometry. Cleaved caspase-3 and activity of NADPH oxidase (NOX) were determined by colorimetric protease assay and enzyme linked immunosorbent assay, respectively. RESULTS: Sen significantly elevated cell viability (P<0.05), decreased the leakage of LDH (P<0.05) and apoptosis rate (P<0.05) in H/R-injured PC12 cells. Sen maintained the value of △Ψm (P<0.05) and suppressed the activity of caspase-3 (P<0.05). Moreover, Sen reduced ROS accumulation P<0.05) and [Ca(2+)]i increment (P<0.05) by inhibiting the activity of NOX (P<0.05). CONCLUSION: Sen may exert cytoprotection against H/R injury by decreasing the levels of intracellular ROS and [Ca(2+)]i, thereby suppressing the mitochondrial pathway of cellular apoptosis.

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.

The synergistic effects of heat shock protein 70 and ginsenoside Rg1 against tert-butyl hydroperoxide damage model in vitro.

Neural stem cells (NSCs) transplanted is one of the hottest research to treat Alzheimer's disease (AD), but cholinergic neurons from stem cells were also susceptible to cell death which Heat shock protein 70 (HSP70) was affirmed to reverse. Related to cognitive impairment, cholinergic nervous cells should be investigated and ginsenoside Rg1 (G-Rg1) was considered to increase them. We chose tert-butyl hydroperoxide (t-BHP) damage model to study in vitro. Functional properties of our recombination plasmid pEGFP-C2-HSP70 were affirmed by SH-SY5Y cells. To opposite the transitory appearance of HSP70, NSCs used as the vectors of HSP70 gene overexpressed HSP70 for at least 7 days in vitro. After transfection for 3 days, G-Rg1 pretreatment for 4 hours, and coculture for 3 days, the expression of acetylcholinesterase (ChAT), synaptophysin, and the ratio of NeuN and GFAP were assessed by western blot; Morphological properties were detected by 3D reconstruction and immunofluorescence. ChAT was markedly improved in the groups contained G-Rg1. In coculture system, the ratio of neurons/astrocytes and the filaments of neurons were increased; apoptosis cells were decreased, compared to monotherapy (P < 0.05). In conclusion, we demonstrated that, as a safe cotreatment affirmed in vitro, overexpression of HSP70 in NSCs plus G-Rg1 promoted nervous cells regeneration from chronic oxidative damage.

Senegenin Inhibits Hypoxia/Reoxygenation-Induced Neuronal Apoptosis by Upregulating RhoGDIα.

Neuronal apoptosis is an important event in hypoxia/reoxygenation (H/R)-induced neuronal injury. Senegenin (Sen), the predominant and most active component in Radix Polygalae root extracts, displays anti-apoptotic and anti-oxidative properties. Sen protects against H/R-induced neuronal apoptosis of highly differentiated PC12 cells and primary cortical neurons. Sen has also been investigated as a source of potential therapeutic targets. In this study, a proteomic approach was used to identify Sen-regulated proteins in PC12 cells. We found that Sen protected against H/R-induced neuronal apoptosis by upregulating RhoGDIα protein expression. The regulatory functions of RhoGDIα were investigated by knocking down RhoGDIα expression in PC12 cells using small interfering RNA (siRNA), followed by quantification of apoptosis and then altering the expression levels of apoptosis-related proteins. Our data show that after silencing RhoGDIα, the neuroprotective effects of Sen on H/R-induced PC12 cell apoptosis were absent. Furthermore, RhoGDIα silencing alleviated the Sen-mediated inhibition of the JNK pathway. Therefore, these findings indicated that Sen attenuates H/R-induced neuronal apoptosis by upregulating RhoGDIα expression and inhibiting the JNK pathway. In addition to the mechanism underlying neuroprotective effects of Sen, RhoGDIα was identified as a putative target of Sen based on a primary rat cortical neuron model of H/R-induced injury.

[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.

Berberine inhibits norepinephrine-induced apoptosis in neonatal rat cardiomyocytes via inhibiting ROS-TNF-α-caspase signaling pathway.

OBJECTIVE: To determine the effect of berberine (Ber) on norepinephrine (NE)-induced apoptosis in neonatal rat cardiomyocytes. METHODS: The cultured neonatal rat cardiomyocytes were treated with NE in the presence or absence of Ber. The activity of lactate dehydrogenase (LDH) in the culture medium was examined, and apoptosis of cardiomyocytes was assessed by Hoechst 33258, isothiocyanate (FITC)-conjugated annexin-V, and propidine iodide (PI) staining. In addition, the activities of caspases-2 and-3 were measured by a fluorescent assay kit. The level of secreted tumor necrosis factor α (TNF-α) and production of intracellular reactive oxygen species (ROS) were also determined. RESULTS: NE at a concentration of 50 µ mol/L induced an obvious increase in the activity of LDH in the culture medium (P<0.05), which was inhibited by coincubation with 0.5, 1.0, or 2.0 µ mol/L Ber (P<0.05). Ber also significantly attenuated NE-induced apoptosis in a dose-dependent manner (P<0.01). Moreover, Ber at a dose of 2 µ mol/L markedly decreased the ROS and TNF-α productions (P <0.05) and inhibited the activation of caspases-2 and -3 in cardiomyocytes exposed to NE (P<0.05)h. CONCLUSION: The present study suggested that Ber could reduce NE-induced apoptosis in neonatal rat cardiomyocytes through inhibiting the ROS-TNF-α-caspase signaling pathway.

Berberine inhibits doxorubicin-triggered cardiomyocyte apoptosis via attenuating mitochondrial dysfunction and increasing Bcl-2 expression.

Cardiomyocyte apoptosis is an important event in doxorubicin (DOX)-induced cardiac injury. The aim of the present study was to investigate the protection of berberine (Ber) against DOX- triggered cardiomyocyte apoptosis in neonatal rat cardiomyocytes and rats. In neonatal rat cardiomyocytes, Ber attenuated DOX-induced cellular injury and apoptosis in a dose-dependent manner. However, Ber has no significant effect on viability of MCF-7 breast cancer cells treated with DOX. Ber reduced caspase-3 and caspase-9, but not caspase-8 activity in DOX-treated cardiomyocytes. Furthermore, Ber decreased adenosine monophosphate-activated protein kinase α (AMPKα) and p53 phosphorylation at 2 h, cytosolic cytochrome c and mitochondrial Bax levels and increased Bcl-2 level at 6 h in DOX-stimulated cardiomyocytes. Pretreatment with compound C, an AMPK inhibitor, also suppressed p53 phosphorylation and apoptosis in DOX-treated cardiomyocytes. DOX stimulation for 30 min led to a loss of mitochondrial membrane potential and a rise in the AMP/ATP ratio. Ber markedly reduced DOX-induced mitochondrial membrane potential loss and an increase in the AMP/ATP ratio at 1 h and 2 h post DOX exposure. In in vivo experiments, Ber significantly improved survival, increased stroke volume and attenuated myocardial injury in DOX-challenged rats. TUNEL and Western blot assays showed that Ber not only decreased myocardial apoptosis, caspase-3 activation, AMPKα and p53 phosphorylation, but also increased Bcl-2 expression in myocardium of rats exposed to DOX for 84 h. These findings indicate that Ber attenuates DOX-induced cardiomyocyte apoptosis via protecting mitochondria, inhibiting an increase in the AMP/ATP ratio and AMPKα phosphorylation as well as elevating Bcl-2 expression, which offer a novel mechanism responsible for protection of Ber against DOX-induced cardiomyopathy.

[Supportive effects of conditioned culture media of human umbilical cord mesenchymal stem cells on hematopoiesis in vitro].

This study was aimed to explore whether the conditioned culture medium of human umbilical cord-derived mesenchymal stem cells (hUC-MSC) has supportive effects on hematopoiesis in vitro. hUC-MSC were cultured in 75 cm(2) culture flasks at a concentration of 2×10(6) cells per flask. After 48 h, the conditioned culture medium was harvested. CD34(+) cells were isolated with the human cord blood CD34 positive selection kit. The CD34(+) cells were plated in three different culture systems: the culture supernatant from hUC-MSC added into incomplete methylcellulose without recombinant human cytokines as conditioned culture medium; the complete methylcellulose medium with recombinant human cytokines as positive control medium; incomplete methylcellulose adding DMEM/F12 with 10% FBS instead of conditioned culture medium as the negative control medium. After 14 days of culture, colonies containing ≥ 50 cells were scored and types of colonies were classified under inverted microscope. The immunophenotypes of cells which were collected from the colonies were detected by flow cytometry. The results showed that conditioned culture medium of hUC-MSC supported the differentiation of CD34(+) cells into CFU-G (47.67 ± 0.58), CFU-GM (48.67 ± 4.73) and CFU-M (3.00 ± 2.00) in vitro, while the CFU-E, BFU-E or CFU-GEMM were absent. Comparatively, in the positive control medium all kinds of CFU were observed. Interestingly, the percentage of CD45(+)cells of CFU in conditioned culture medium (97.43 ± 2.15)% was more than CD45(+)cells in positive control medium (39.69 ± 0.96)% (P < 0.05). It is concluded that the conditioned culture medium of hUC-MSC has been confirmed to have ability to support hematopoiesis separately in vitro. Besides, it enhances the differentiation of CD34(+) cells into myeloid cells except cells of erythroid lineage.

Rhynchophylline prevents cardiac dysfunction and improves survival in lipopolysaccharide-challenged mice via suppressing macrophage I-κBα phosphorylation.

Myocardial dysfunction is a common complication during sepsis and significantly contributes to the mortality of patients with septic shock. However, none of the available therapeutic strategies proven to be effective in patients with severe sepsis are designed specifically to target myocardial dysfunction. The purpose of the present study is to investigate the effect of rhynchophylline (Rhy) on LPS-induced myocardial dysfunction in mice. We found that pretreatment with Rhy significantly improved cardiac systolic dysfunction, increased stroke volume and cardiac output in mice challenged with LPS. LPS induced cardiac inhibitor-κBα (I-κBα) phosphorylation, tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) mRNA expression, and in turn increased cardiac TNF-α and IL-1ß protein production, all of which were attenuated by pretreatment with Rhy. Immunohistochemistry revealed that TNF-α was found in infiltrated macrophages (F4/80(+)) and myocardium, and Rhy reduced TNF-α immunostaining in cardiac infiltrated macrophages in LPS-challenged mice. Furthermore, Rhy inhibited LPS-induced I-κBα phosphorylation and TNF-α production in cultured mouse peritoneal macrophages, but not in neonatal mouse cardiomyocytes. Pretreatment with Rhy significantly decreased the mortality of LPS-challenged mice. These results indicate that Rhy reduces cardiac dysfunction and improves survival via suppression of macrophage I-κBα phosphorylation in LPS-challenged mice, and suggest that Rhy may be a potential agent for the treatment of septic cardiac dysfunction.

Chlorogenic acid inhibits LPS-induced microglial activation and improves survival of dopaminergic neurons.

Pro-inflammatory factors released by activated microglia may contribute to the progression of neurodegenerative diseases. As a natural phenolic acid, chlorogenic acid (CGA) has been shown to have anti-inflammatory properties. However, it is unclear whether CGA has the ability to mediate microglial activation. The present study investigated the role of CGA in lipopolysaccharide (LPS)-stimulated microglia. Our data demonstrated that CGA significantly suppressed NO production and TNF-α release in LPS-stimulated primary microglia. In addition, CGA decreased LPS-stimulated phosphorylation and degradation of inhibitory kappa B-alpha (IκBα), and prevented translocation of nuclear factor-kappaB (NF-κB). Furthermore, CGA prevented neurotoxicity caused by microglial activation and ultimately improved survival of dopaminergic (DA) neuron. Finally, in vivo data showed that CGA pretreatment attenuated LPS-induced IL-1ß and TNF-α release in substantia nigra (SN). Our results suggested that the pretreatment of CGA significantly inhibits the microglial activation, and CGA may be neuroprotective for pro-inflammatory factor-mediated neurodegenerative disorders.

Yohimbine enhances protection of berberine against LPS-induced mouse lethality through multiple mechanisms.

Sepsis remains a major cause of mortality in intensive care units, better therapies are urgently needed. Gram-negative bacterial lipopolysaccharide (LPS) is an important trigger of sepsis. We have demonstrated that berberine (Ber) protects against lethality induced by LPS, which is enhanced by yohimbine (Y) pretreatment, and Ber combined with Y also improves survival in septic mice. However, the precise mechanisms by which Y enhances protection of Ber against LPS-induced lethality remain unclear. The present study confirmed that simultaneously administered Y also enhanced protection of Ber against LPS-induced lethality. Ber or/and Y attenuated liver injury, but not renal injury in LPS-challenged mice. Ber or/and Y all inhibited LPS-stimulated IκBα, JNK and ERK phosphorylation, NF-κB activation as well as TNF-α production. Ber also increased IL-10 production in LPS-challenged mice, which was enhanced by Y. Furthermore, Ber or/and Y all suppressed LPS-induced IRF3, TyK2 and STAT1 phosphorylation, as well as IFN-ß and IP-10 mRNA expression in spleen of mice at 1 h after LPS challenge. Especially, Y enhanced the inhibitory effect of Ber on LPS-induced IP-10 mRNA expression. In vitro experiments further demonstrated that Y significantly enhanced the inhibitory effect of Ber on TNF-α production in LPS-treated peritoneal macrophages, Ber combined with Y promoted LPS-induced IL-10 production and LPS-stimulated IκBα, JNK, ERK and IRF3 phosphorylation and NF-κB activation were also suppressed by Ber or/and Y pretreatment in peritoneal macrophages. Taken together, these results demonstrate that Y enhances the protection of Ber against LPS-induced lethality in mice via attenuating liver injury, upregulating IL-10 production and suppressing IκBα, JNK, ERK and IRF3 phosphorylation. Ber combined with Y may be an effective immunomodulator agent for the prevention of sepsis.

Luteolin reduces primary hippocampal neurons death induced by neuroinflammation.

OBJECTIVES: This study examined whether luteolin may exert an anti-inflammatory effect in microglia and may be neuroprotective by regulating microglia activation. METHODS: We treated BV2 microglia with 1.0 µg/ml lipopolysaccharide (LPS) after incubation with luteolin for 1 hour, the nitric oxide (NO) levels were determined by a Griess reaction, the inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-alpha), and interleukin 1beta (IL-1beta) mRNA expression were determined by real-time PCR analysis, the iNOS and COX-2 protein induction were determined by Western blot analysis, and the levels of prostaglandin E(2) (PGE(2)), TNF-alpha, and IL-1beta were determined by enzyme-linked immunosorbent assay (ELISA) kits. Rat primary hippocampal neurons were co-cultured with LPS-activated BV2 microglia with 20 µM luteolin for 24 hours, the hippocampal neurons viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the number of apoptotic hippocampal neurons was determined by immunofluorescence detection. RESULTS: Luteolin significantly inhibited the expression of iNOS and COX-2 in LPS-induced BV2 microglia. Moreover, the compound down-regulated the proinflammatory cytokines (TNF-alpha and IL-1beta) as well as the production of NO and PGE(2) in these cells. When hippocampal neurons were co-cultured with LPS-stimulated BV2 microglia, the administration of 20 µM luteolin increased the neurons viability and reduced the number of apoptotic neurons. CONCLUSION: These data demonstrate that anti-inflammatory activity of luteolin in microglia contributes to its neuroprotective effect and suggest that it may have a potential therapeutic application in the treatment of neurodegenerative diseases.

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.