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.

[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 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.

Glycine inhibits the LPS-induced increase in cytosolic Ca2+ concentration and TNFalpha production in cardiomyocytes by activating a glycine receptor.

AIM: Previous studies have demonstrated that glycine (GLY) markedly reduces lipopolysaccharide (LPS)-induced myocardial injury.However, the mechanism of this effect is still unclear. The present study investigated the effect of GLY on cytosolic calcium concentration([Ca2+]c) and tumor necrosis factor-alpha (TNFalpha) production in cardiomyocytes exposed to LPS, as well as whether the glycine-gated chloride channel is involved in this process. METHODS: Neonatal rat cardiomyocytes were isolated, and the [Ca2+]c and TNFalpha levels were determined by using Fura-2 and a Quantikine enzyme-linked immunosorbent assay, respectively. The distribution of the GLY receptor and GLY-induced currents in cardiomyocytes were also investigated using immunocytochemistry and the whole-cell patch-clamp technique, respectively. RESULTS: LPS at concentrations ranging from 10 ng/mL to 100 microg/mL significantly stimulated TNFalpha production. GLY did not inhibit TNFalpha production induced by LPS at concentrations below 10 ng/mL but did significantly decrease TNFalpha release stimulated by 100 microg/mL LPS and prevented an LPS-induced increase in [Ca2+]c, which was reversed by strychnine, a glycine receptor antagonist. GLY did not block the isoproterenol-induced increase in [Ca2+]c, but did prevent the potassium chloride-induced increase in [Ca2+]c in cardiomyocytes.Strychnine reversed the inhibition of the KCl-stimulated elevation in [Ca2+]c by GLY. In chloride-free buffer, GLY had no effect on the dipotassium hydrogen phosphate-induced increase in [Ca2+]c. Furthermore, GLY receptor alpha1 and beta subunit-immunoreactive spots were observed in cardiomyocytes, and GLY-evoked currents were blocked by strychnine. CONCLUSION: Cardiomyocytes possess the glycine-gated chloride channel, through which GLY prevents the increase in [Ca2+]c and inhibits the TNFalpha production induced by LPS at high doses in neonatal rat cardiomyocytes.

Therapeutic strategies targeting the LPS signaling and cytokines.

Lipopolysaccharide (LPS) has been recognized as a major player in the pathogenesis of sepsis and neutralization of LPS or inhibition of its signal transduction mechanism is promising new treatment strategy in preclinical experiments. However, these therapeutic approaches have been shown unsuccessful in clinical trials. LPS activates Toll-like receptor 4 (TLR4) and induces pro-inflammatory and anti-inflammatory responses, the altered innate and adaptive immune responses eventually lead to the immunosuppressive state. The future therapeutic efforts in sepsis should focus on the immunosuppressive state. In this article, we will outline the current data on therapeutic strategies targeting LPS, TLR4 and single cytokine in sepsis and discuss the experimental and clinical evaluation of the immunomodulatory action of glycine and berberine. While we have demonstrated berberine in combination with yohimbine can modulate host immune responses in endotoxemia, it seems worthwhile to conduct clinical trials on the safe and efficacy of this new immunomodulatory therapy.

Berberine inhibits cytosolic phospholipase A2 and protects against LPS-induced lung injury and lethality independent of the alpha2-adrenergic receptor in mice.

Acute lung injury is still a significant clinical problem having a high mortality rate despite significant advances in antimicrobial therapy and supportive care made in the past few years. Our previous study demonstrated that berberine (Ber) remarkably decreased mortality and attenuated the lung injury in mice challenged with LPS, but the mechanism behind this remains unclear. Here, we report that pretreatment with Ber significantly reduced pulmonary edema, neutrophil infiltration, and histopathological alterations; inhibited protein expression and phosphorylation of cytosolic phospholipase A2; and decreased thromboxane A2 release induced by LPS. Yohimbine, an alpha2-adrenergic receptor antagonist, did not antagonize these actions of Ber. Furthermore, pretreatment with Ber decreased TNF-alpha production and mortality in mice challenged with LPS, which were enhanced by yohimbine, and Ber combined with yohimbine also improved survival rate in mice subjected to cecal ligation and puncture. Taken together, these observations indicate that Ber attenuates LPS-induced lung injury by inhibiting TNF-alpha production and cytosolic phospholipase A2 expression and activation in an alpha2-adrenoceptor-independent manner. Berberine combined with yohimbine might provide an effective therapeutic approach to acute lung injury during sepsis.

Glycine receptors contribute to cytoprotection of glycine in myocardial cells.

BACKGROUND: The classic glycine receptor (GlyR) in the central nervous system is a ligand-gated membrane-spanning ion channel. Recent studies have provided evidence for the existence of GlyR in endothelial cells, renal proximal tubular cells and most leukocytes. In contrast, no evidence for GlyR in myocardial cells has been found so far. Our recent researches have showed that glycine could protect myocardial cells from the damage induced by lipopolysaccharide (LPS). Further studies suggest that myocardial cells could contain GlyR or binding site of glycine. METHODS: In isolated rat heart damaged by LPS, the myocardial monophasic action potential (MAP), the heart rate (HR), the myocardial tension and the activities of lactate dehydrogenase (LDH) from the coronary effluent were determined. The concentration of intracellular free calcium ([Ca(2+)](i)) was measured in cardiomyocytes injured by LPS and by hypoxia/reoxygenation (H/R), which excludes the possibility that reduced calcium influx because of LPS neutralized by glycine. Immunohistochemistry was used to detect the GlyR in myocardial tissue. GlyR and its subunit in the purified cultured cardiomyocytes were identified by Western blotting. RESULTS: Although significant improvement in the MAP/MAPD(20), HR, and reduction in LDH release were observed in glycine + LPS hearts, myocardial tension did not recover. Further studies demonstrated that glycine could prevent rat mycordial cells from LPS and hypoxia/reoxygenation injury (no endotoxin) by attenuating calcium influx. Immunohistochemistry exhibited a positive green-fluorescence signaling along the cardiac muscle fibers. Western blotting shows that the purified cultured cardiomyocytes express GlyR beta subunit, but GlyR alpha1 subunit could not be detected. CONCLUSIONS: The results suggest that glycine receptor is expressed in cardiomyocytes and participates in cytoprotection from LPS and hypoxia/reoxygenation injury. Glycine could directly activate GlyR on the cardiomyocytes and prevent calcium influx into the cardiomyocytes.

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.