Jianzhong Han

Protective effect of ginsenoside Rg1 on glutamate-induced lung injury

AbstractAim:To examine the possible protective effect of ginsenoside Rg1, an active component of ginseng, on lung injury caused by glutamate in vivo.Methods:The lungs of mice receiving glutamate (0.5 g/kg) and/or ginsenoside Rg1 (0.03 g/kg) via intraperitoneal administration were collected. The indexes of lung wet weight/ body weight ratios (LW/BW), lung wet/dry weight ratios (W/D), heart rate (HR), and breathing rate (BR) were determined. The activity of nitric oxide synthase (NOS), xanthine oxidase (XOD), superoxide dismutase (SOD), catalase (CAT), the content of NO, and malondialdehyde in the lung homogenate were measured.Results:Treatment with glutamate for 2 h increased LW/BW, W/D, HR, and BR. These changes were nearly abolished by pretreatment with ginsenoside Rg1 for 30 min before glutamate injection. An analysis of the lung homogenate demonstrated the protective effect as evidenced by the inhibition of NOS (12%) and XOD (50%) inactivity, the enhanced activity of SOD (20%) and CAT (25%).Conclusion:Ginsenoside Rg1 has a potential protective role in lung diseases associated with glutamate toxicity.

Antiflammin-1 attenuates bleomycin-induced pulmonary fibrosis in mice.

BackgroundAntiflammin-1 (AF-1), a derivative of uteroglobin (UG), is a synthetic nonapeptide with diverse biological functions. In the present study, we investigated whether AF-1 has a protective effect against bleomycin-induced pulmonary fibrosis.MethodsC57BL/6 mice were injected with bleomycin intratracheally to create an animal model of bleomycin-induced pulmonary fibrosis. On Day 7 and Day 28, we examined the anti-inflammatory effect and antifibrotic effect, respectively, of AF-1 on the bleomycin-treated mice. The effects of AF-1 on the transforming growth factor-beta 1 (TGF-β1)-induced proliferation of murine lung fibroblasts (NIH3T3) were examined by a bromodeoxycytidine (BrdU) incorporation assay and cell cycle analysis.ResultsSevere lung inflammation and fibrosis were observed in the bleomycin-treated mice on Day 7 and Day 28, respectively. Administration of AF-1 significantly reduced the number of neutrophils in the bronchoalveolar lavage fluid (BALF) and the levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) in the lung homogenates on Day 7. Histological examination revealed that AF-1 markedly reduced the number of infiltrating cells on Day 7 and attenuated the collagen deposition and destruction of lung architecture on Day 28. The hydroxyproline (HYP) content was significantly decreased in the AF-1-treated mice. In vitro, AF-1 inhibited the TGF-β1-induced proliferation of NIH3T3 cells, which was mediated by the UG receptor.ConclusionsAF-1 has anti-inflammatory and antifibrotic actions in bleomycin-induced lung injury. We propose that the antifibrotic effect of AF-1 might be related to its suppression of fibroblast growth in bleomycin-treated lungs and that AF-1 has potential as a new therapeutic tool for pulmonary fibrosis.

NMDA Receptor Antagonist Attenuates Bleomycin-Induced Acute Lung Injury

Background Glutamate is a major neurotransmitter in the central nervous system (CNS). Large amount of glutamate can overstimulate N-methyl-D-aspartate receptor (NMDAR), causing neuronal injury and death. Recently, NMDAR has been reported to be found in the lungs. The aim of this study is to examine the effects of memantine, a NMDAR channel blocker, on bleomycin-induced lung injury mice. Methods C57BL/6 mice were intratracheally injected with bleomycin (BLM) to induce lung injury. Mice were randomized to receive saline, memantine (Me), BLM, BLM plus Me. Lungs and BALF were harvested on day 3 or 7 for further evaluation. Results BLM caused leukocyte infiltration, pulmonary edema and increase in cytokines, and imposed significant oxidative stress (MDA as a marker) in lungs. Memantine significantly mitigated the oxidative stress, lung inflammatory response and acute lung injury caused by BLM. Moreover, activation of NMDAR enhances CD11b expression on neutrophils. Conclusions Memantine mitigates oxidative stress, lung inflammatory response and acute lung injury in BLM challenged mice.

An excessive increase in glutamate contributes to glucose-toxicity in β-cells via activation of pancreatic NMDA receptors in rodent diabetes

In the nervous system, excessive activation of NMDA receptors causes neuronal injury. Although activation of NMDARs has been proposed to contribute to the progress of diabetes, little is known about the effect of excessive long-term activation of NMDARs on β-cells, especially under the challenge of hyperglycemia. Here we thoroughly investigated whether endogenous glutamate aggravated β-cell dysfunction under chronic exposure to high-glucose via activation of NMDARs. The glutamate level was increased in plasma of diabetic mice or patients and in the supernatant of β-cell lines after treatment with high-glucose for 72 h. Decomposing the released glutamate improved GSIS of β-cells under chronic high-glucose exposure. Long-term treatment of β-cells with NMDA inhibited cell viability and decreased GSIS. These effects were eliminated by GluN1 knockout. The NMDAR antagonist MK-801 or GluN1 knockout prevented high-glucose-induced dysfunction in β-cells. MK-801 also decreased the expression of pro-inflammatory cytokines, and inhibited I-κB degradation, ROS generation and NLRP3 inflammasome expression in β-cells exposed to high-glucose. Furthermore, another NMDAR antagonist, Memantine, improved β-cells function in diabetic mice. Taken together, these findings indicate that an increase of glutamate may contribute to the development of diabetes through excessive activation of NMDARs in β-cells, accelerating β-cells dysfunction and apoptosis induced by hyperglycemia.

Antenatal blockade of N-methyl-D-aspartate receptors by Memantine reduces the susceptibility to diabetes induced by a high-fat diet in rats with intrauterine growth restriction†

Abstract Intrauterine growth retardation (IUGR) is closely related to the later development of type 2 diabetes in adulthood. Excessive activation of N-methly-D-aspartate receptors (NMDARs) causes excitatory neurotoxicity, resulting in neuronal injury or death. Inhibition of NMDARs enhances the glucose-stimulated insulin secretion and survival of islet cells in type 2 diabetic mouse and human islets. Here, we examined whether antenatal blockade of NMDARs by Memantine could decrease the risk of diabetes induced by a high-fat (HF) diet at adulthood in IUGR rats. Pregnant SD rats were assigned to four groups: control, IUGR, Memantine, and Memantine + IUGR. The pregnant rats were exposed to hypoxic conditions (FiO2 = 0.105) for 8 h/day (IUGR group) or given a daily Memantine injection (5 mg/kg, i.p.) before hypoxia exposure from embryonic day (E) 14.5 to E 20.5 (Memantine + IUGR). The offspring were fed an HF diet with 60% of the calories from age 4 to 12 weeks. We found that NMDAR mRNAs were expressed in the fetal rat pancreas. An HF diet resulted in a high rate of diabetes at adulthood in the IUGR group. Antenatal Memantine treatment decreased the risk of diabetes at adulthood of rats with IUGR, which was associated with rescued glucose tolerance, increased insulin release, improved the insulin sensitivity, and increased expression of genes related to beta-cell function in the pancreas. Together, our results suggest that antenatal blockade of NMDARs by Memantine in pregnant rats improves fetal development and reduces the susceptibility to diabetes at adulthood in offspring. Summary Sentence The excessive activation of NMDARs is involved in the restriction of fetal pancreas development induced by intrauterine hypoxia, and antenatal Memantine treatment attenuates the susceptibility to diabetes induced by an HF diet in IUGR rats.

Inhibition of lipopolysaccharide-mediated rat alveolar macrophage activation in vitro by antiflammin-1

Antiflammin‐1 (AF‐1) is a synthetic nonapeptide with a similar sequence to the conserved sequence of CC10 secreted by lung Clara cells. Studies suggest that it is potent inhibitor of inflammation. We investigated the effects and possible mechanisms of AF‐1 on LPS‐induced alveolar macrophage (AM) activation in vitro. AMs harvested from the BALF of Sprague‐Dawley (SD) rat were treated with various concentrations of AF‐1 both simultaneously and after LPS stimulation. The concentrations of the cytokines IL‐1β, IL‐6, and IL‐10 in the supernatant were detected by an enzyme‐linked immunosorbent assay. The mRNA expression levels of these cytokines in AMs were analyzed using quantitative RT‐PCR. To investigate more fully the possible mechanisms by which AF‐1 modulates the expression of cytokines, cells were pretreated with anti‐IL‐10 antibody. Toll‐like receptor‐4 (TLR‐4) expression on the cell surface was also detected using flow cytometry. The results showed that AF‐1 suppressed mRNA expression and protein production of IL‐1β and IL‐6, while it promoted IL‐10 expression in LPS‐stimulated AMs, in a dose‐dependent manner. The inhibitory effects of AF‐1 on IL‐1β were significantly decreased when endogenous production of IL‐10 was blocked. AF‐1 also showed an effect on downregulated TLR‐4 expression in LPS‐stimulated AMs. The data show for the first time that AF‐1 modulates the AM response to LPS by regulating TLR‐4 expression and upregulating IL‐10 secretion, which could be another important mechanism in the AF‐1 inhibiting effect on inflammation.

Interaction of antiflammin-1 with uteroglobin-binding protein induces phosphorylation of ERK1/2 in NIH 3T3 cells.

Previously, it has been suggested that uteroglobin (UG)-binding protein functions as a putative receptor of UG; however, the specific epitope of UG that interacts with this receptor has not yet been identified. The downstream events of UG-binding protein signaling remain unclear. Here we report that antiflammin-1 (AF-1, a bioactive C-terminal peptide of UG) specifically binds to UG-binding protein and has a cellular signaling consequence. We reduced the level of endogenous UG-binding protein expression in murine fibroblast cell line NIH 3T3 by RNA interference and found that knockdown of UG-binding protein inhibited AF-1-induced extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation. Meanwhile, the interaction between AF-1 and UG-binding protein was confirmed by flow cytometry-based binding assays and co-localization of AF-1 and enhanced green fluorescent protein (EGFP)-tagged UG-binding protein. The present study provides evidence for the first time for AF-1 binding with UG-binding protein, and preliminarily characterized UG-binding protein as a point downstream of AF-1 in mediating ERK phosphorylation.

NMDA receptor activation inhibits the anti-fibrotic effect of BM-MSCs on bleomycin-induced pulmonary fibrosis

Endogenous glutamate (Glu) release and N-methyl-d-aspartate (NMDA) receptor (NMDAR) activation are associated with lung injury in different animal models. However, the underlying mechanism is unclear. Bone marrow-derived mesenchymal stem cells (BM-MSCs), which show potential use for immunomodulation and tissue protection, play a protective role in pulmonary fibrosis (PF) process. Here, we found the increased Glu release from the BM cells of bleomycin (BLM)-induced PF mice in vivo. BLM stimulation also increased the extracellular Glu in BM-MSCs via the antiporter system xc− in vitro. The gene expression of each subunit of NMDAR was detected in BM-MSCs. NMDAR activation inhibited the proliferation, migration, and paracrine function of BM-MSCs in vitro. BM-MSCs were derived from male C57BL/6 mice, transfected with lentiviral vectors carrying the enhanced green fluorescence protein gene, pretreated with NMDA, and transplanted into the female recipient mice that were intratracheally injected with BLM to induce PF. Transplantation of NMDA-pretreated BM-MSCs significantly aggravated PF as compared with that in the normal BM-MSCs transplantation group. The sex determination gene Y chromosome and green fluorescence protein genes of BM-MSCs were detected to observe BM-MSCs homing in the fibrotic lungs. Moreover, NMDAR activation inhibited BM-MSC migration by downregulating the stromal cell-derived factor-1/C-X-C chemokine receptor type 4 signaling axis. NMDAR activation aggravated the transforming growth factor-β1-induced extracellular matrix production in alveolar epithelial cells and fibroblasts through the paracrine effects of BM-MSCs. In summary, these findings suggested that NMDAR activation-mediated Glu excitotoxicity induced by BLM in BM-MSCs abolished the therapeutic effects of normal BM-MSCs transplantation on BLM-induced PF.