Gypenoside-14 Reduces Depression via Downregulation of the Nuclear Factor Kappa B (NF-kB) Signaling Pathway on the Lipopolysaccharide (LPS)-Induced Depression Model.

Neuroinflammation is a common pathogenetic sign of depression and is closely linked to the development of depression. Many clinical anti-inflammatory drugs act as antidepressants by reducing the neuroinflammatory response. Previous research found that gypenosides and their bioactive compound gypenoside-14 (GP-14) had neuroprotective effects against hypoxia-induced injury and reduced neuroinflammation-related high-altitude cerebral edema. Here we investigated the effects of GP-14 on the lipopolysaccharide (LPS)-induced depression-like behavior model. LPS (0.5 mg/kg) was injected into mice intraperitoneally for 7 consecutive days to induce depression-like behavior, which is considered a model for the exacerbation of depression. GP-14 in the amount of 100 mg/kg was simultaneously administered by gavage for 7 days. In the LPS-induced depression model, GP-14 not only attenuated depression-like behavior but also improved the anxiety-like behavior of the mice. Additionally, GP-14 treatment mitigated learning and cognitive decline in depressed mice. ELISA and immunofluorescence staining results revealed that GP-14 inhibited the upregulation of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6), and suppressed the activation of astrocytes induced with LPS, indicating its potent anti-inflammatory effect. GP-14 pretreatment in C8 cells and primary astrocytes can inhibit the activation of the NF-κB signaling pathway and downregulate the levels of pro-inflammatory factors. In summary, our findings showed that GP-14 had significant anti-inflammation and anti-depression properties; thus, GP-14 could be a promising lead compound for treating depression.

Expression profile of cytokines and chemokines in a mouse high-altitude cerebral edema model.

INTRODUCTION: High-altitude cerebral edema (HACE) is considered to be the end-stage of acute mountain sickness (AMS); however, its pathophysiological mechanism remains unknown. Increasing evidences support that inflammation is an important risk factor for the occurrence of HACE. Including our published papers, previous studies demonstrated that the levels of IL-6, IL-1ß, and TNF-α in both serum and hippocampus were increased in the mouse HACE model induced by LPS stimulation combined with hypobaric hypoxia exposure; however, the expression profile of other cytokines and chemokines remains unknown. OBJECTIVE: This study was to analyze the expression profile of cytokines and chemokines in the HACE model. METHODS: The mouse HACE model was established by LPS stimulation combined with hypobaric hypoxia exposure (LH). The mice were divided into the normoxic group, LH-6 h group, LH-1 d group, and LH-7 d group. Brain water content (BWC) was determined using the wet/dry weight ratio. The levels of 30 cytokines and chemokines in the serum and hippocampal tissue were detected using LiquiChip. The mRNA expression of cytokines and chemokines in hippocampal tissue were determined by q-PCR. RESULTS: In the current study, we found that the brain water content was increased after the combinational treatment of LPS and hypobaric hypoxia. The results of LiquiChip showed that, in the serum and hippocampal tissue, most factors in all 30 cytokines and chemokines were dramatically upregulated at 6 h, and then declined at the 1st d and 7th d. Among these factors, G-CSF, M-CSF, MCP-1, KC, MIG, Eotaxin, Rantes, IP10, IL-6, MIP-2, and MIP-1ß were all increased in both serum and hippocampal tissue at 6 h. In addition, the results of q-PCR showed the mRNA levels of G-CSF, MCP-1, KC, MIG, Eotaxin, Rantes, IP10, IL-6, MIP-2, and MIP-1ß in hippocampal tissue were dramatically upregulated at 6 h. CONCLUSION: This study showed that the dynamic expression profile of 30 cytokines and chemokines in a mouse HACE model induced by LPS plus hypobaric hypoxia. The levels of G-CSF, MCP-1, KC, MIG, Eotaxin, Rantes, IP10, IL-6, MIP-2, and MIP-1ß in both serum and hippocampus were significantly increased at 6 h, which may be involved in the occurrence and development of HACE.

[Hypoxia promotes lipopolysaccharide-induced CXCL10 expression in microglia].

This study was aimed to investigate the effect of hypoxia on lipopolysaccharide (LPS)-induced CXC-chemokine ligand-10 (CXCL10) expression and the underlying mechanism. C57BL/6J mice were randomly divided into control, hypoxia, LPS, and hypoxia combined with LPS groups. The LPS group was intraperitoneally injected with 0.5 mg/kg LPS, and the hypoxia group was placed in a hypobaric hypoxia chamber (simulated altitude of 6 000 m). The serum and hippocampal tissue samples were collected after 6 h of the treatment. The levels of CXCL10 in the serum and hippocampal tissue of mice were detected by ELISA. The microglia cell line BV2 and primary microglia were stimulated with hypoxia (1% O2) and/or LPS (100 ng/mL) for 6 h. The mRNA expression level of CXCL10 and its content in culture supernatant were detected by real-time quantitative PCR and ELISA, respectively. The phosphorylation levels of nuclear factor κB (NF-κB) signaling pathway-related proteins, p65 and IκBα, were detected by Western blot. Moreover, after NF-κB signaling pathway being blocked with a small molecular compound, PDTC, CXCL10 mRNA expression level was detected in the BV2 cells. The results showed that in the LPS-induced mouse inflammatory model, hypoxia treatment could promote LPS-induced up-regulation of CXCL10 in both serum and hippocampus. Compared with the cells treated with LPS alone, the expression of CXCL10 mRNA and the content of CXCL10 in the culture supernatant of BV2 cells treated with hypoxia combined with LPS were significantly increased. The CXCL10 mRNA level of primary microglial cells treated with hypoxia combined with LPS was significantly up-regulated. Compared with the cells treated with hypoxia or LPS alone, the phosphorylation levels of p65 and IκBα in the BV2 cells treated with hypoxia combined with LPS were significantly increased. PDTC blocked the induction of CXCL10 gene expression by LPS in the BV2 cells. These results suggest that hypoxia promotes LPS-induced expression of CXCL10 in both animal and cell models, and NF-κB signaling pathway plays an important role in this process.

Cyclooxygenase­2 contributes to the hypoxia­induced aggravation of the neuroinflammation response stimulated by lipopolysaccharide in microglia.

Hypoxia and neuroinflammation are key risk factors involved in various pathophysiological neural disorders. Hypoxia can aggravate neuroinflammation in vitro and in vivo; however, the underlying mechanisms remain unknown. In the present study, hypoxia [either 3 or 1% oxygen (O2)] increased lipopolysaccharide (LPS)-induced expression of the IL-6, IL-1ß and TNF-α proinflammatory cytokines in BV2 cells. At the molecular level, both hypoxia and FG-4592, an hypoxia inducible factor 1 pathway activator, effectively induced cyclooxygenase-2 (COX-2) expression. The COX-2 inhibitor celecoxib significantly reduced the expression of cytokines induced by LPS under hypoxic conditions. Additionally, the administration of celecoxib inhibited the activation of microglia as well as cytokine expression in mice administered with hypoxia exposure and LPS injection. The present data demonstrated that COX-2 is involved in the hypoxia-induced aggravation of neuroinflammation stimulated by LPS.

[Effects of blocking lactate production by 2-DG on hypoxic injury of HT22 neurons and its mechanisms].

OBJECTIVE: To investigate the effects of blocking lactate synthesis on the HT22 cell injuries caused by hypoxia. METHODS: 2-deoxy-D-glucose (2-DG) is a non-metabolized glucose analogue that can inhibit lactate synthesis by blocking glycolysis. HT22 cells were divided into 4 groups: Control group, 2-DG group, Hypoxia group and 2-DG+Hypoxia group. The cells in control group and 2-DG treatment group were cultured in a 37℃, 5% CO2 incubator, and thecells in hypoxia group and 2-DG + Hypoxia group were cultured in a hypoxia incubator. The concentrations of 2-DG were 2.5 and 5 mmol/L, the concentration of oxygen was 0.3%, and the treatment time was 24 h. Cell activity was detected by CCK-8 assay, the levels of lactate in cell culture medium were detected by spectrophotometry, cell morphology was observed by fluorescence staining, the level of reactive oxygen species (ROS) was detected by flow cytometry, and the activities of superoxide dismutase (SOD) and catalase (CAT) were determined by enzyme activity kits. The protein expression levels of p-p38, t-p38 and ß-actin were detected by Western blot. RESULTS: Compared with that in control group, the lactate level in culture medium and cell activity were decreased significantly (P＜0.01), the number of adherent cells was decreased, the level of ROS was increased (P＜0.01), and the enzyme activity of CAT was decreased (P＜0.05) in the 2-DG group. In the hypoxia group, the level of lactate in the culture medium was increased significantly (P＜0.01), the cell activity was decreased (P＜0.01), the number of adherent cells was decreased, the ROS levels were increased (P＜0.01), and the enzyme activities of CAT and SOD were decreased (P＜0.01 or P＜0.05). In 2-DG+Hypoxia group, the level of lactate was decreased significantly (P＜0.05), the cell viability was decreased significantly (P＜0.01), the number of cells was decreased significantly, and the ability of adhere to the wall was weakened significantly. The level of ROS was increased significantly (P＜0.01), the enzyme activities of CAT and SOD were decreased significantly (P＜0.01), the protein expression level of p-p38 was increased significantly (P＜0.05), and there was no change in t-p38. Compared with hypoxia groups, in 2-DG combined with hypoxia group, the level of lactate induced by hypoxia, the cell activity, and the enzyme activity level of CAT were decreased significantly (all P＜0.01), while the level of ROS was increased significantly (P＜ 0.01). CONCLUSION: Blocking lactate can reduce the cell activity level under hypoxia and aggravate the oxidative stress injury of HT22 cells. The mechanisms may be related to increasing ROS level and activating p38 signal pathway.