Autophagy involvement in T lymphocyte signalling induced by nickel with quantitative phosphoproteomic analysis.

Nickel-induced allergic contact dermatitis (ACD) is a common skin disease. The mechanism by which nickel causes ACD is not clear. There is no treatment for it, only symptomatic therapy. However, due to the lifetime sensitization characteristics, the recurrence rate in patients is high. T lymphocytes play a key role in nickel-induced ACD. Elucidating the potential mechanism underlying nickel-induced T lymphocyte signalling might make it possible to achieve targeted treatment of nickel-induced ACD. In our study, a phosphoproteomic approach based on tandem mass tag (TMT) labelling and LCMS/MS analyses was employed. An animal model of nickel allergy was established. Splenic T lymphocytes were purified for quantitative phosphoproteomic analysis. The numbers of phosphoproteins, phosphopeptides and phosphosites identified in this study were 3072, 7977 and 10,200, respectively. Comprehensive gene ontology (GO) analysis combined with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that nickel can significantly affect the phosphorylation of the mTOR signalling pathway in T lymphocytes. Western blotting analysis was used to detect changes in the expression of autophagy-related proteins (Beclin 1, LC3II, and p62). Nickel allergy changed autophagy-related protein expression (p < 0.05). It has been demonstrated that nickel causes autophagy of T lymphocytes in the spleen. Using autophagy inhibitors to intervene, it was found that Th1 differentiation was inhibited, and the expression of Th1-related inflammatory factors was downregulated. Overall, the identification of relevant signalling pathways yielded new insights into the molecular mechanisms underlying nickel allergy and might help in the discovery and development of mechanism-based drugs.

Activation of forkhead box O3a by mono(2-ethylhexyl)phthalate and its role in protection against mono(2-ethylhexyl)phthalate-induced oxidative stress and apoptosis in human cardiomyocytes.

Mono(2-ethylhexyl)phthalate (MEHP), the active metabolite of di(2-ethylhexyl)phthalate (DEHP), is known to exert cardiotoxicity. The aim of the present study was to investigate the role of forkhead box O3a (FOXO3a) in MEHP-induced human AC16 cardiomyocyte injuries. MEHP reduced cell viability and mitochondrial membrane potential (ΔΨm), whereas it increased lactate dehydrogenase (LDH) leakage, production of reactive oxygen species (ROS), and apoptosis in cardiomyocytes. The expression of FOXO3a and its target genes, mitochondrial superoxide dismutase (Mn-SOD) and apoptosis repressor with caspase recruitment domain (ARC), increased after MEHP exposure, but the expression of p-FOXO3a protein was decreased. Overexpression of FOXO3a decreased the production of ROS and the apoptosis rate induced by MEHP, and the expression of Mn-SOD and ARC was further increased after MEHP exposure. In contrast, knockdown of FOXO3a resulted in increased ROS production and apoptosis and suppressed the expression of Mn-SOD and ARC in the presence of MEHP. However, overexpression or knockdown of FOXO3a did not affect MEHP-induced loss of ΔΨm. In conclusion, the loss of ΔΨm and apoptosis are involved in MEHP-induced cardiomyocyte toxicity. Activation of FOXO3a defends against MEHP-induced oxidative stress and apoptosis by upregulating the expression of Mn-SOD and ARC in AC16 cardiomyocytes.

Electromagnetic field exposure-induced depression features could be alleviated by heat acclimation based on remodeling the gut microbiota.

BACKGROUND: Electromagnetic pollution cannot be ignored. Long-term low-dose electromagnetic field (EMF) exposure can cause central nervous system dysfunction without effective prevention. MATERIALS/METHODS: Male C57BL/6J mice (6-8 weeks, 17-20 g) were used in this study. Depression-like and anxiety-like behaviors detected by behavioral experiments were compared among different treatments. 16S rRNA gene sequencing and non-targeted liquid chromatography-mass spectrometry (LC-MS) metabolomics were used to explore the relationship between EMF exposure and heat acclimation (HA) effects on gut microbes and serum metabolites. RESULTS: Both EMF and HA regulated the proportions of p_Firmicutes and p_Bacteroidota. EMF exposure caused the proportions of 6 kinds of bacteria, such as g_Butyricicoccus and g_Anaerotruncus, to change significantly (p < 0.05). HA restored the balance of gut microbes that was affected by EMF exposure and the proportion of probiotics (g_Lactobacillus) increased significantly (p < 0.01). Serum metabolite analysis suggested that HA alleviated the disturbance of serum metabolites (such as cholesterol and D-mannose) induced by EMF exposure. Both the metabolic KEGG pathways and PICRUSt functional analysis demonstrated that tryptophan metabolism, pyrimidine metabolism and amino acid biosynthesis were involved. CONCLUSIONS: EMF exposure not only led to depression-like neurobehavioral disorders, but also to gut microbiota imbalance. HA alleviated the depression features caused by EMF exposure. Based on the analysis of gut microbiota associated with serum metabolites, we speculated that gut microbiota might play a vital role in the cross-tolerance provided by HA.

Astragaloside IV alleviates heatstroke brain injury and neuroinflammation in male mice by regulating microglial polarization via the PI3K/Akt signaling pathway.

Heatstroke is a condition caused by overheating of the body that leads to severe central nervous system dysfunction. Although there have been numerous studies on the pathological process of heatstroke, effective treatment methods are lacking. Astragaloside IV can protect the brain from inflammation and brain damage in various inflammation-related diseases, but it has not yet been used clinically for the treatment of heatstroke. Therefore, the aim of this study was to explore the neuroprotective effect of Astragaloside IV on heatstroke-induced central nervous system damage and its mechanism. Brain injury model under heatstroke was established using artificial climate simulation cabin. By scoring neurological deficits, performing histological and immunofluorescence staining of microglia, and detecting cytokine levels, we determined that Astragaloside IV alleviated brain injury and neuroinflammation. To further explore the potential molecular mechanism, RNA sequencing was performed to investigate the differences in the brain. The results revealed that the PI3K/AKT pathway is involved. In vitro experiments further confirmed that Astragaloside IV can abrogate the phenotypic changes in microglia induced by heatstroke. Moreover, Astragaloside IV promotes the polarization of M2 microglia by activating the PI3K/AKT pathway. In summary, these results indicate that Astragaloside IV alleviates neuroinflammation and brain injury induced by heatstroke through the PI3K/AKT pathway. Astragaloside IV is a commonly used therapeutic agent in the clinic, but its use in the treatment of heatstroke-induced brain injury has not been explored. This study reveals that Astragaloside IV may be a new therapeutic agent for the treatment of heatstroke-induced brain injury.

Transcriptome sequencing reveals neurotoxicity in embryonic neural stem/progenitor cells under heat stress.

Neural tube malformation is a common kind of human birth defect. High temperature is one of the most common physical teratogenic factors. Several studies have suggested that heat stress may cause neurotoxicity during brain development, but more studies are warranted to reveal the mechanism and draw consistent conclusions. The current study used a cell model of primary mouse embryonic neural stem/progenitor cells (NSPCs) subjected to heat stress of 43 °C for 20 min. Our study investigated the changes in the NSPCs transcriptome under heat stress using high-throughput mRNA-seq. The NSPCs showed remarkably altered genes associated with cell growth, proliferation, cell cycle, and survival when exposed to heat stress. Heat stress reduced cell viability, proliferation, and neurosphere formation and caused cell cycle arrest and apoptosis in cultured NSPCs. PCR arrays confirmed that the TNF receptor family plays an important role in the apoptosis of NSPCs during heat stress. The results of real-time PCR confirmed that heat stress affects the expression of critical genes. We provide transcriptomic insight into heat stress-induced developmental neurotoxic effects and the underlying mechanisms.

Intestinal alkaline phosphatase improves intestinal permeability and alleviates multiple organ dysfunction caused by heatstroke.

Objective: Heatstroke (HS) is a severe acute disease related to gastrointestinal barrier dysfunction, systemic inflammation and multiple organ injury. Many of the functions of Intestinal alkaline phosphatase (IAP) have been linked to gut homeostasis, gut barrier function and inflammation. However, the protective effect of IAP on heatstroke is not fully elucidated. This study aims to explore the protective effect of IAP on heatstroke by maintaining intestinal barrier and improving permeability. Methods: Male C57BL/6 mice were placed in a controlled climate chamber (ambient temperature: 40.0 ± 0.5 °C; humidity: 60 ± 5 %) until the maximum core temperature (Tc, max) reached 42.7 °C (the received criterion of HS). Then heat exposed mice (n = 195) were divided into three groups: 0.2 mL of 0.9 % physiological saline (HS) or vehicle (HS + Vehicle) or 300 IU IAP (HS + IAP) by gavage at 0, 24, and 48 h after onset. Control group mice (Con) (n = 65) were not exposed to heat and were gavaged with 0.9 % physiological saline of the same volume at the same time. Results: IAP treatment significantly reduced the levels of endotoxin, FD4, and D-lactate in the blood of heatstroke mice, reduced intestinal permeability and maintained the integrity of the intestinal barrier by increasing the expression of tight junction proteins. Meanwhile, IAP treatment alleviated liver and kidney damage caused by heatstroke, reduced serum levels of inflammatory cytokines, and thus improved survival rate of mice after heatstroke. Conclusion: This study indicates that IAP can improve the intestinal barrier function and intestinal permeability by increasing intestinal tight junctions, reduce systemic inflammation and multiple organ injury and improving the survival rate of heatstroke. Therefore, we consider IAP may be added to enteral nutrition formulas as a potential means for diseases characterized by intestinal permeability disorders, including heatstroke.

Holistic view of heat acclimation alleviated intestinal lesion in mice with heat stroke based on microbiome-metabolomics analysis.

The severity of heat stroke (HS) is associated with intestinal injury, which is generally considered an essential issue for HS. Heat acclimation (HA) is considered the best strategy to protect against HS. In addition, HA has a protective effect on intestinal injuries caused by HS. Considering the essential role of gut microbes in intestinal structure and function, we decided to investigate the potential protective mechanism of HA in reducing intestinal injury caused by HS. HA model was established by male C57BL/6J mice (5-6 weeks old, 17-19 g) were exposed at (34 ± 0.7)°C for 4 weeks to establish an animal HA model. The protective effect of HA on intestinal barrier injury in HS was investigated by 16S rRNA gene sequencing and nontargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics. According to the experimental results, HA can change the composition of the gut microbiota, which increases the proportion of lactobacilli, faecal bacteria, and urinobacteria but decreases the proportion of deoxycholic acid. Moreover, HA can reduce liver and kidney injury and systemic inflammation caused by HS and reduce intestinal injury by enhancing the integrity of the intestinal barrier. In addition, HA regulates inflammation by inhibiting NF-κB signalling and increasing tight junction protein expression in HS mice. HA induces changes in the gut microbiota, which may enhance tight junction protein expression, thereby reducing intestinal inflammation, promoting bile acid metabolism, and ultimately maintaining the integrity of the intestinal barrier. In conclusion, HA induced changes in the gut microbiota. Among the gut microbiota, lactobacilli may play a key role in the potential protective mechanism of HA.

Increased miR-155 in Microglial Exosomes Following Heat Stress Accelerates Neuronal Autophagy via Their Transfer Into Neurons.

Background: Heat stroke is the outcome of excessive heat stress, which results in core temperatures exceeding 40°C accompanied by a series of complications. The brain is particularly vulnerable to damage from heat stress. In our previous studies, both activated microglia and increased neuronal autophagy were found in the cortices of mice with heat stroke. However, whether activated microglia can accelerate neuronal autophagy under heat stress conditions is still unknown. In this study, we aimed to investigate the underlying mechanism that caused neuronal autophagy upregulation in heat stroke from the perspective of exosome-mediated intercellular communication. Methods: In this study, BV2 and N2a cells were used instead of microglia and neurons, respectively. Exosomes were extracted from BV2 culture supernatants by ultracentrifugation and then characterized via transmission electron microscopy, nanoparticle tracking analysis and Western blotting. N2a cells pretreated with/without miR-155 inhibitor were cocultured with microglial exosomes that were treated with/without heat stress or miR-155 overexpression and subsequently subjected to heat stress treatment. Autophagy in N2a cells was assessed by detecting autophagosomes and autophagy-related proteins through transmission electron microscopy, immunofluorescence, and Western blotting. The expression of miR-155 in BV2 and BV2 exosomes and N2a cells was measured using real-time reverse transcription polymerase chain reaction. Target binding analysis was verified via a dual-luciferase reporter assay. Results: N2a autophagy moderately increased in response to heat stress and accelerated by BV2 cells through transferring exosomes to neurons. Furthermore, we found that neuronal autophagy was positively correlated with the content of miR-155 in microglial exosomes. Inhibition of miR-155 partly abolished autophagy in N2a cells, which was increased by coculture with miR-155-upregulated exosomes. Mechanistic analysis confirmed that Rheb is a functional target of miR-155 and that microglial exosomal miR-155 accelerated heat stress-induced neuronal autophagy mainly by regulating the Rheb-mTOR signaling pathway. Conclusion: Increased miR-155 in microglial exosomes after heat stroke can induce neuronal autophagy via their transfer into neurons. miR-155 exerted these effects by targeting Rheb, thus inhibiting the activity of mTOR signaling. Therefore, miR-155 could be a promising target for interventions of neuronal autophagy after heat stroke.

Modulation of microglial phenotypes by dexmedetomidine through TREM2 reduces neuroinflammation in heatstroke.

No FDA approved pharmacological therapy is available to reduce neuroinflammation following heatstroke. Previous studies have indicated that dexmedetomidine (DEX) could protect against inflammation and brain injury in various inflammation-associated diseases. However, no one has tested whether DEX has neuro-protective effects in heatstroke. In this study, we focused on microglial phenotypic modulation to investigate the mechanisms underlying the anti-inflammatory effects of DEX in vivo and in vitro. We found that DEX treatment reduced the expression of CD68, iNOS, TNF-α, and IL-1ß, and increased the expression of CD206, Arg1, IL-10 and TGF-ß in microglia, ameliorating heatstroke induced neuroinflammation and brain injury in mice. TREM2, whose neuro-protective function has been validated by genetic studies in Alzheimer's disease and Nasu-Hakola disease, was significantly promoted by DEX in the microglia. TREM2 esiRNA reversed the DEX-induced activation of PI3K/Akt signalling. Overall these findings indicated that DEX may serve, as a potential therapeutic approach to ameliorate heatstroke induced neuroinflammation and brain injury via TREM2 by activating PI3K/Akt signalling.

Lack of lymphocytes exacerbate heat stroke severity in male mice through enhanced inflammatory response.

Though it has long been thought that the immune system is implicated in the pathophysiology of heat stroke, the underlying mechanisms are still poorly understood. As it has been reported in the literature that lymphocyte disturbance occurs in heat stroke patients or animals, we attempted to seek experimental evidence to define the role of lymphocytes in the pathophysiology of heat stroke. In our study, we used male Balb/c mice to establish a passive heat stroke model. We found that lymphocyte-deficient Severe combined immunodeficient (SCID) mice exposed to heat stress exhibited exacerbated heat stroke severity, which could be indicated by increased rates of mortality and serum levels of inflammatory cytokines compared to wildtype control mice. We further showed, through the depletion of T lymphocytes in wildtype mice and the transfer of wildtype lymphocytes into SCID mice, respectively, that T lymphocytes were both necessary and sufficient to alleviate the severity of heat stroke by inhibiting the early inflammatory response. Moreover, we found that the severity of heat injuries in heat-stressed wildtype mice showed great inter-individual variability, and the early number of T lymphocytes could be negatively associated with the severity of heat stroke. Our results suggest that lack of T lymphocytes could exacerbate the severity of heat stroke by augmenting inflammatory response, and the early circulating T lymphocytes may serve as a potential biomarker for the diagnosis of heat stroke.

The involvement of microglial CX3CR1 in heat acclimation-induced amelioration of adult hippocampal neurogenesis impairment in EMF-exposed mice.

Microglial CX3C chemokine receptor 1 (CX3CR1) has been implicated in numerous cellular mechanisms, including signalling pathways that regulate brain homoeostasis and adult hippocampal neurogenesis. Specific environmental conditions can impair hippocampal neurogenesis-related cognition, learning and memory. However, the role of CX3CR1 in the neurogenic alterations resulting from the cross-tolerance protection conferred by heat acclimation (HA) against the effects of electromagnetic field (EMF) exposure is less well understood. Here, we investigated the role of microglial CX3CR1 signalling in adult hippocampal neurogenesis induced by HA in EMF-exposed mice. We found that EMF exposure significantly decreased the number of proliferating and differentiating cells in the dentate gyrus (DG) of the hippocampus, resulting in a reduced neurogenesis rate. Moreover, alterations in the phenotypes of activated microglia and decreased expression levels of CX3CR1, but not sirtuin 1 (SIRT1), were observed in the brains of EMF-exposed mice. Remarkably, HA treatment improved microglial phenotypes, restored the expression of CX3CR1, and ameliorated the decrease in the adult hippocampal neurogenesis rate following EMF exposure. Moreover, pharmacological inhibition of CX3CR1 and SIRT1 failed to restore CX3CR1 expression and ameliorate hippocampal neurogenesis impairment following HA plus EMF stimulation. These results indicate that microglial CX3CR1 is involved in the cross-tolerance protective effect of HA on adult hippocampal neurogenesis upon EMF exposure.

TREM2 Regulates Heat Acclimation-Induced Microglial M2 Polarization Involving the PI3K-Akt Pathway Following EMF Exposure.

The function of triggering receptor expressed on myeloid cells-2 (TREM2) has been described within microglia with a beneficial activated phenotype. However, the role of TREM2 underlying microglial phenotypic alterations in the cross-tolerance protection of heat acclimation (HA) against the inflammatory stimuli electromagnetic field (EMF) exposure is less well known. Here, we investigated the TREM2-related signaling mechanism induced by HA in EMF-stimulated N9 microglial cells (N9 cells). We found that EMF exposure significantly increased the production of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α, IL-1ß, and IL-6), and the expression of M1 markers (CD11b and CD86); meanwhile, decreased the levels of anti-inflammatory cytokines (IL-4 and IL-10) and the expression of M2 markers (CD206 and Arg1) in N9 cells. Clearly, HA treatment decreased the secretion of TNF-α, IL-1ß and IL-6 and the expression of CD11b and CD86, and enhanced the production of IL-4 and IL-10 and the expression of CD206 and Arg1. Moreover, TREM2 esiRNA and selective inhibitor of PI3K clearly decreased anti-inflammatory cytokines production, M2 markers expression, and phosphorylation of PI3K and Akt following HA plus EMF stimulation. These results indicate that TREM2 and PI3K-Akt pathway are involved in the cross-tolerance protective effect of HA in microglial polarization towards the EMF exposure. This finding inspires future studies that aim to explore the non-drug approaches underlying EMF stimulation and other central nervous system (CNS) inflammatory diseases.

Derivatization method for the quantification of lactic acid in cell culture media via gas chromatography and applications in the study of cell glycometabolism.

Lactic acid represents an important metabolite that reflects mitochondria function and may further serve as energy source for cancer cells. In light of this physiological and pathological significance, we developed a novel and sensitive gas chromatography method to detect lactic acid in cell culture media. Here, ethyl chloroformate was selected as derivative reagent and the derivatization process was further optimized in terms of number of reagents and reaction time as well as extraction reagents. Under optimal conditions, good linearity was achieved in the tested calibration range. The limit of detection (LOD) was determined to be 0.67 µmol/L, the recovery rates were 99.6%-106% and the precision rate RSD was <5.49%. Furthermore, this method has been applied to quantify the secretion of lactic acid in cells exposed to mono­2­ethylhexyl phthalate at different doses and in cancer cells over time. Taken in concert, our method proved to be both sensitive and reliable and may be applied for studies on mitochondrial function and cell glycolysis conditions.