Prolyl hydroxylase inhibitor FG-4592 alleviates neuroinflammation via HIF-1/BNIP3 signaling in microglia.

BACKGROUND: Neuroinflammation is responsible for neuropsychiatric dysfunction following acute brain injury and neurodegenerative diseases. This study describes how a hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) inhibitor FG-4592 prevents the lipopolysaccharide (LPS)-induced acute neuroinflammation in microglia. METHODS: The distribution of FG-4592 in mouse brain tissues was determined by collision-induced dissociation tandem mass spectrometry. Microglial activation in the hippocampus was analyzed by immunofluorescence. Moreover, we determined the activation of HIF-1 and nuclear factor-κB (NF-κB) signaling pathways, proinflammatory responses using molecular biological techniques. Transcriptome sequencing and BNIP3 silencing were conducted to explore signaling pathway and molecular mechanisms underlying FG-4592 anti-inflammatory activity. RESULTS: FG-4592 was transported into the brain tissues and LPS increased its transportation. FG-4592 promoted the expression of HIF-1α and induced the downstream gene transcription in the hippocampus. Administration with FG-4592 significantly inhibited microglial hyperactivation and decreased proinflammatory cytokine levels following LPS treatment in the hippocampus. The LPS-induced inflammatory responses and the NF-κB signaling pathway were also downregulated by FG-4592 pretreatment in microglial cells. Mechanistically, Venn diagram analysis of transcriptomic changes of BV2 cells identified that BNIP3 was a shared and common differentially expressed gene among different treatment groups. FG-4592 markedly upregulated the protein levels of BNIP3 in microglia. Importantly, BNIP3 knockdown aggravated the LPS-stimulated inflammatory responses and partially reversed the protection of FG-4592 against microglial inflammatory signaling and microglial activation in the mouse hippocampus. CONCLUSIONS: FG-4592 alleviates neuroinflammation through facilitating microglial HIF-1/BNIP3 signaling pathway in mice. Targeting HIF-PHD/HIF-1/BNIP3 axis is a promising strategy for the development of anti-neuroinflammation drugs.

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.

Metabolomic analysis of human plasma sample after exposed to high altitude and return to sea level.

When ascending to high altitude, it is a rigorous challenge to people who living in the low altitude area to acclimatize to hypoxic environment. Hypoxia exposure can cause dramatic disturbances of metabolism. This longitudinal cohort study was conducted to delineate the plasma metabolomics profile following exposure to altitude environments and explore potential metabolic changes after return to low altitude area. 25 healthy volunteers living in the low altitude area (Nor; 40m) were transported to high altitude (HA; 3,650m) for a 7-day sojourn before transported back to the low altitude area (HAP; 40m). Plasma samples were collected on the day before ascending to HA, the third day on HA(day 3) and the fourteenth day after returning to low altitude(14 day) and analyzed using UHPLC-MS/MS tools and then the data were subjected to multivariate statistical analyses. There were 737 metabolites were obtained in plasma samples with 133 significantly changed metabolites. We screened 13 differential metabolites that were significantly changed under hypoxia exposure; enriched metabolic pathways under hypoxia exposure including tryptophan metabolism, purine metabolism, regulation of lipolysis in adipocytes; We verified and relatively quantified eight targeted candidate metabolites including adenosine, guanosine, inosine, xanthurenic acid, 5-oxo-ETE, raffinose, indole-3-acetic acid and biotin for the Nor and HA group. Most of the metabolites recovered when returning to the low altitude area, however, there were still 6 metabolites that were affected by hypoxia exposure. It is apparent that high-altitude exposure alters the metabolic characteristics and two weeks after returning to the low altitude area a small portion of metabolites was still affected by high-altitude exposure, which indicated that high-altitude exposure had a long-term impact on metabolism. This present longitudinal cohort study demonstrated that metabolomics can be a useful tool to monitor metabolic changes exposed to high altitude, providing new insight in the attendant health problem that occur in response to high altitude.

Preliminary Intermittent Hypoxia Training Alleviates the Damage of Sustained Normobaric Hypoxia on Human Hematological Indexes and Cerebral White Matter.

Zhang, Guangbo, Yanzhao Zhou, Zhengtao Cao, Xiang Cheng, Xiangpei Yue, Tong Zhao, Ming Zhao, Yongqi Zhao, Ming Fan, and Lingling Zhu. Preliminary intermittent hypoxia training alleviates the damage of sustained normobaric hypoxia on human hematological indexes and cerebral white matter. High Alt Med Biol. 23:273-283, 2022. Background: We aimed to examine the effects of preliminary intermittent hypoxia training (IHT) on human hematological indexes and cerebral white matter (WM) after exposure to a simulated altitude of 4,300 m. Methods: We recruited 20 young healthy volunteers. Participants were then randomized to either the IHT group (n = 10) or the control group (n = 10). We measured the physiological function of the control group at sea level and after exposure to a simulated altitude of 4,300 m, respectively. The IHT group performed the above tests at three time points: before and after hypoxia training, and after exposure to a simulated altitude of 4,300 m, respectively. Results: We found that mean SpO2 during day 10 of hypoxia training showed a significant increase compared with mean SpO2 on day 1 (88.3% ± 1.5% vs. 90.0% ± 1.6%, p < 0.05), and erythrocyte P50 of post-training was significantly increased compared with pretraining (37.8 ± 2.9 mmHg vs. 45.9 ± 6.4 mmHg, p < 0.05). Mean SpO2 measures after acute exposure to high altitude exhibited a significant difference, with the IHT group showing significantly greater SpO2 than the control group (73.8% ± 3.7% vs. 77.4% ± 3.2%, p < 0.05), and the Lake Louise Score was also lower than the control group (2.55 ± 2.1 vs. 6.67 ± 2.5, p < 0.05). After daily IHT, brain-derived neurotrophic factor plasma levels of participants in the IHT group did not change but significantly increased in response to high-altitude hypoxia (103.5% ± 70.4% vs. 29.7% ± 73.2%, p < 0.05). Interleukin-10 (IL-10) plasma level did not change before and after IHT in the IHT group, whereas the IL-10 plasma level of the control group after high-altitude exposure was significantly higher. Furthermore, we found that fractional anisotropy values in the left corticospinal tract and splenium of the corpus callosum in the IHT group were significantly higher than those in the control group after high-altitude hypoxia. Conclusions: These results demonstrate that IHT alleviates the damage of sustained normobaric hypoxia on human hematological indexes and cerebral WM.

Intermittent hypoxia treatment alleviates memory impairment in the 6-month-old APPswe/PS1dE9 mice and reduces amyloid beta accumulation and inflammation in the brain.

BACKGROUND: Alzheimer's disease (AD) is a progressive, degenerative, and terminal disease without cure. There is an urgent need for a new strategy to treat AD. The aim of this study was to investigate the effects of intermittent hypoxic treatment (IHT) on cognitive functions in a mouse model of AD and unravel the mechanism of action of IHT. METHODS: Six-month-old APPswe/PS1dE9 (APP/PS1) male mice were exposed to hypoxic environment (14.3% O2) 4 h/day for 14 days or 28 days. Cognitive functions were measured by Morris water maze test after either 14 days or 42 days of interval. Thereafter the distribution of amyloid plaque and microglial activation were determined by mouse brain immunohistochemistry, while the amyloid beta (Aß) and inflammatory cytokines were measured by ELISA and Western Blot. Microarray was used for studying gene expressions in the hippocampus. RESULTS: IHT for 14 days or 28 days significantly improved the spatial memory ability of the 6-month-old APP/PS1 mice. The memory improvement by 14 days IHT lasted to 14 days, but not to 42 days. The level of Aß plaques and neurofilament accumulations was reduced markedly after the IHT exposure. IHT reduced the pro-inflammatory cytokines IL-1ß, IL-6 levels, and ß-secretase cleavage of APP processing which implies reduced Aß production. Microarray analysis revealed a large number of genes in the hippocampus were significantly altered which are known to be metabolism-regulated genes. CONCLUSIONS: This study provides evidence of the beneficial effect of IHT on the progression of AD by alleviating memory impairment, reducing Aß accumulation and inflammation in the brain. IHT can be developed as a novel measure to relieve the progression of AD by targeting multiple pathways in the AD pathogenesis.

Degradation of FA reduces Aß neurotoxicity and Alzheimer-related phenotypes.

Dysregulation of formaldehyde (FA) has been implicated in the development of Alzheimer's Disease (AD). Elevated FA levels in Alzheimer's patients and animal models are associated with impaired cognitive functions. However, the exact role of FA in AD remains unknown. We now identified that oxidative demethylation at serine8/26 of amyloid-beta protein (Aß) induced FA generation and FA cross-linked with the lysine28 residue in the ß-turn of Aß monomer to form Aß dimers, and then accelerated Aß oligomerization and fibrillogenesis in vitro. However, Aß42 mutation in serine8/26, lysine28 abolished Aß self-aggregation. Furthermore, Aß inhibited the activity of formaldehyde dehydrogenase (FDH), the enzyme for FA degradation, resulting in FA accumulation. In turn, excess of FA stimulated Aß aggregation both in vitro and in vivo by increasing the formation of Aß oligomers and fibrils. We found that degradation of FA by formaldehyde scavenger-NaHSO3 or coenzyme Q10 reduced Aß aggregation and ameliorated the neurotoxicity, and improved the cognitive performance in APP/PS1 mice. Our study provides evidence that endogenous FA is essential for Aß self-aggregation and scavenging FA could be an effective strategy for treating AD.

Single Point Mutation from E22-to-K in Aß Initiates Early-Onset Alzheimer's Disease by Binding with Catalase.

Amyloid-beta (Aß) is a critical etiological factor for late-onset familial Alzheimer's disease (AD). However, an early-onset AD has been found to be related with an Aß mutation in glutamic acid 22-to-lysine (Italian type E22K). Why only one single point mutation at E22 residue induces AD remains unclear. Here, we report that a Chinese familial AD pedigree with E22K mutation was associated with higher levels of serum hydrogen peroxide (H2O2) and lower activity of catalase (a H2O2 degrading enzyme) than controls. Further, we found that E22K binding with catalase caused more severe H2O2 accumulation in the brains of E22K-injected rats than Aß-injected rats. Unexpectedly, H2O2 bound with the mutation site 22K residue of E22K and elicited more rapid aggregation of E22K than Aß in vitro. Moreover, H2O2 acted with E22K synergistically to induce higher cellular toxicity than with Aß. Notably, intrahippocampal infusion of E22K led to more severe plaque deposition, neuron death, and more rapid memory decline than Aß-injected rats. However, L-cysteine, a H2O2 scavenger, not only prevented self-aggregation of E22K but also reduced H2O2-promoted E22K assembly in vitro; subsequently, it alleviated Alzheimer-related phenotypes. Hence, E22K binding with catalase promotes the early onset of familial AD, and L-cys may reverse this disease.

New insight into Alzheimer's disease: Light reverses Aß-obstructed interstitial fluid flow and ameliorates memory decline in APP/PS1 mice.

INTRODUCTION: Pharmacological therapies to treat Alzheimer's disease (AD) targeting "Aß" have failed for over 100 years. Low levels of laser light can disassemble Aß. In this study, we investigated the mechanisms that Aß-blocked extracellular space (ECS) induces memory disorders in APP/PS1 transgenic mice and addressed whether red light (RL) at 630 nm rescues cognitive decline by reducing Aß-disturbed flow of interstitial fluid (ISF). METHODS: We compared the heating effects on the brains of rats illuminated with laser light at 630, 680, and 810 nm for 40 minutes, respectively. Then, a light-emitting diode with red light at 630 nm (LED-RL) was selected to illuminate AD mice. The changes in the structure of ECS in the cortex were examined by fluorescent double labeling. The volumes of ECS and flow speed of ISF were quantified by magnetic resonance imaging. Spatial memory behaviors in mice were evaluated by the Morris water maze. Then, the brains were sampled for biochemical analysis. RESULTS: RL at 630 nm had the least heating effects than other wavelengths associated with ~49% penetration ratio into the brains. For the molecular mechanisms, Aß could induce formaldehyde (FA) accumulation by inactivating FA dehydrogenase. Unexpectedly, in turn, FA accelerated Aß deposition in the ECS. However, LED-RL treatment not only directly destroyed Aß assembly in vitro and in vivo but also activated FA dehydrogenase to degrade FA and attenuated FA-facilitated Aß aggregation. Subsequently, LED-RL markedly smashed Aß deposition in the ECS, recovered the flow of ISF, and rescued cognitive functions in AD mice. DISCUSSION: Aß-obstructed ISF flow is the direct reason for the failure of the developed medicine delivery from superficial into the deep brain in the treatment of AD. The phototherapy of LED-RL improves memory by reducing Aß-blocked ECS and suggests that it is a promising noninvasive approach to treat AD.

Illumination with 630 nm Red Light Reduces Oxidative Stress and Restores Memory by Photo-Activating Catalase and Formaldehyde Dehydrogenase in SAMP8 Mice.

AIMS: Pharmacological treatments for Alzheimer's disease (AD) have not resulted in desirable clinical efficacy over 100 years. Hydrogen peroxide (H2O2), a reactive and the most stable compound of reactive oxygen species, contributes to oxidative stress in AD patients. In this study, we designed a medical device to emit red light at 630 ± 15 nm from a light-emitting diode (LED-RL) and investigated whether the LED-RL reduces brain H2O2 levels and improves memory in senescence-accelerated prone 8 mouse (SAMP8) model of age-related dementia. RESULTS: We found that age-associated H2O2 directly inhibited formaldehyde dehydrogenase (FDH). FDH inactivity and semicarbazide-sensitive amine oxidase (SSAO) disorder resulted in endogenous formaldehyde (FA) accumulation. Unexpectedly, excess FA, in turn, caused acetylcholine (Ach) deficiency by inhibiting choline acetyltransferase (ChAT) activity in vitro and in vivo. Interestingly, the 630 nm red light can penetrate the skull and the abdomen with light penetration rates of ∼49% and ∼43%, respectively. Illumination with LED-RL markedly activated both catalase and FDH in the brains, cultured cells, and purified protein solutions, all reduced brain H2O2 and FA levels and restored brain Ach contents. Consequently, LED-RL not only prevented early-stage memory decline but also rescued late-stage memory deficits in SAMP8 mice. INNOVATION: We developed a phototherapeutic device with 630 nm red light, and this LED-RL reduced brain H2O2 levels and reversed age-related memory disorders. CONCLUSIONS: The phototherapy of LED-RL has low photo toxicity and high rate of tissue penetration and noninvasively reverses aging-associated cognitive decline. This finding opens a promising opportunity to translate LED-RL into clinical treatment for patients with dementia. Antioxid. Redox Signal. 00, 000-000.

A Sensitive and Rapid Method for Detecting Formaldehyde in Brain Tissues.

The existing methods for detecting formaldehyde (FA) in brain samples are expensive and require sophisticated experimental procedures. Here, we established a highly sensitive and selective spectrophotometric method, which is based on a reaction in which FA reacts with colorless reagent 4-amino-3-penten-2-one (Fluoral-P) to produce a yellow compound, 3,5-diacetyl-1,4-dihydrolutidine (DDL), which can be detected by a spectrophotometer at 420 nm at room temperature. The sensitive response time point was found to be at the first hour, and the optimal pH of derivative reaction was pH 6.0. The limit of detection (LOD) and the limits of quantization (LOQ) for detecting FA were 0.5 µM and 2.5 µM, respectively. Using this method, an abnormally high level of FA was detected in both the brains of FA-injected mice and autopsy hippocampus tissues from patients with Alzheimer's disease. This finding suggests that the modified Fluoral-P method is effective for measuring levels of FA in the brains.

Reduction of Endogenous Melatonin Accelerates Cognitive Decline in Mice in a Simulated Occupational Formaldehyde Exposure Environment.

Individuals afflicted with occupational formaldehyde (FA) exposure often suffer from abnormal behaviors such as aggression, depression, anxiety, sleep disorders, and in particular, cognitive impairments. Coincidentally, clinical patients with melatonin (MT) deficiency also complain of cognitive problems associated with the above mental disorders. Whether and how FA affects endogenous MT metabolism and induces cognitive decline need to be elucidated. To mimic occupational FA exposure environment, 16 healthy adult male mice were exposed to gaseous FA (3 mg/m³) for 7 consecutive days. Results showed that FA exposure impaired spatial memory associated with hippocampal neuronal death. Biochemical analysis revealed that FA exposure elicited an intensive oxidative stress by reducing systemic glutathione levels, in particular, decreasing brain MT concentrations. Inversely, intraperitoneal injection of MT markedly attenuated FA-induced hippocampal neuronal death, restored brain MT levels, and reversed memory decline. At tissue levels, injection of FA into the hippocampus distinctly reduced brain MT concentrations. Furthermore, at cellular and molecular levels, we found that FA directly inactivated MT in vitro and in vivo. These findings suggest that MT supplementation contributes to the rescue of cognitive decline, and may alleviate mental disorders in the occupational FA-exposed human populations.