Sphingolipid Signaling Pathway Research Articles

Comparative transcriptomic analysis revealed potential mechanisms regulating the hypertrophy of goose pectoral muscles

Pectoral muscle development is an important economic trait. According to the different essence, muscle development can be divided into 2 processes: embryonic muscle fiber generation and postnatal muscle fiber hypertrophy, and postnatal muscle fiber hypertrophy has a greater impact on muscle development than the number of muscle fibers formed during the embryonic phase in poultry. However, the underlying mechanisms regulating the hypertrophy of goose pectoral muscles have not been elucidated. Therefore, the purpose of the present study was to conduct transcriptome sequencing in pectoral muscles of both Landes (LD) and Sichuan White (SW) geese at 6, 10, and 30 weeks of age to reveal the molecular mechanisms regulating pectoral muscle hypertrophy through intra-breed and inter-breed bioinformatics analyses. Phenotypically, the pectoral muscle weight/index of LD and SW geese increased from 6 to 30 weeks of age, and except for the pectoral muscle index at 10 weeks of age (P = 0.962), at the same age, the pectoral muscle weight/index of LD geese were significantly higher than that of SW geese (P < 0.05). In transcriptional regulation, intra-breed bioinformatics analysis identified 3331 genes whose expression levels were opposite to the trend of pectoral muscle hypertrophy both in LD and SW geese, and the 3331 genes were mainly enriched into abundant KEGG pathways related to lipid metabolism, proliferation/apoptosis, and immune response. Moreover, 23 genes (including SLC2A10, TNFRSF1A, PRKAA1, SLC27A4, ITGB2, THY1, RHOA, MYL10, ACTB, PRKCB, PIK3R2, RAC2, DMD, LATS2, YAP1, WWTR1, SMAD7, CTGF, FGF1, AXIN2, GLI2, ID2, and CCND2) who were enriched in 6 crosstalk pathways named viral myocarditis, insulin resistance, sphingolipid signaling pathway, hippo signaling pathway, chemokine signaling pathway, and leukocyte transendothelial migration were identified as the key candidate genes regulating the hypertrophy of goose pectoral muscles. In inter-breed bioinformatics analysis, abundant different expression genes (DEGs) related to lipid metabolism, immune response, and proliferation/apoptosis were identified between LD and SW geese too, and compared with SW geese, the expression level of MYL10 in LD geese was lower, while the expression levels of GLI2/CTGF/SMAD7 in LD geese were higher. These results suggested that the hypertrophy of goose pectoral muscles might be achieved through more lipid deposition and less leukocyte infiltration to promote the proliferation of cells within the muscles, and the low expression of MYL10 and high expressions of GLI2/CTGF/SMAD7 might the keys to induce the pectoral muscle hypertrophy of LD geese from 6 to 30 weeks of age over that of SW geese. All data the present study obtained will provide new insights into the molecular mechanisms regulating the hypertrophy of goose pectoral muscles.

Tumor suppressor ACER1 correlates with prognosis and Immune Infiltration in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is notorious for poor prognoses, and effective biomarkers are urgently needed for early diagnosis of HNSCC patients. We investigate the role of alkaline ceramidase 1 (ACER1) and its relationship with immune infiltration in HNSCC. The differential expression and clinical prognostic significance of ACER1 in HNSCC patients are explored using bioinformatics methods and verified in human HNSCC samples. Genetic mutation, DNA methylation and drug sensitivity linked with ACER1 are examined. The potential biological function of ACER1 co-expression genes is assessed, and a series of functional assays are performed on ACER1in vitro. The results comprehensively reveal a relationship between ACER1 and immune infiltration in HNSCC patients. ACER1 expression is significantly downregulated in HNSCC tissues and closely correlated with better prognoses for HNSCC patients, and this prognostic significance is determined by distinct clinical characteristics. Genetic alteration and promoter hypomethylation of ACER1 are involved in progression of HNSCC, and ACER1 expression is significantly related to several drug sensitivities. Functional analysis shows that ACER1 co-expression genes are mainly enriched in the sphingolipid signaling pathway associated with inhibition of tumorigenesis, leading to better prognoses for HNSCC patients. In vitro, ACER1 overexpression inhibits proliferation and migration, induces apoptosis, and promotes adhesion of Fadu and SCC9 cells. In addition, high ACER1 expression is closely linked with infiltration levels of immune cells, and strongly associated with biomarkers of immune cells in HNSCC, suggesting the important role of ACER1 in regulating tumor immunity in HNSCC patients. In summary, ACER1 may be a useful indicator for diagnosis and prognosis, and may regulate immune infiltration in HNSCC patients, thus promising targeted immunotherapy for HNSCC.

Proteomics and Metabolomics Study on the Responses of Sertoli Cells Infected With Brucella and Its bvfA-Deletion Strains.

To investigate the potential effects of BvfA in reproductive system damage caused by Brucella. Brucella intracellular multiplication ability was determined by a gentamicin protection assay; the LDH method was used to determine the lethal effect of Brucella on TM4 cells. Afterward, Label-free proteomics and LC-MS/MS metabolomics assays were combined to reveal differential abundant proteins and metabolites of TM4 cells infected with bvfA-deletion strains and parental strains. Finally, PRM mass spectrometry and western blot analysis were carried out to confirm differential expression of proteins. This report demonstrated that bvfA-deletion strains failed to invade TM4 cells and reconstitution of invasion when a strain with gene bvfA was reintroduced to the deletion strain in 3 h. The bvfA-deletion exhibited weakened intracellular multiplication compared with parental strains in TM4 cells in 12 h; however, the death rate of TM4 cells infected with bvfA-deletion strains was higher than that of TM4 cells infected with parental strains. Combined proteomics and metabolomics analyses revealed that the differential abundant proteins and metabolites in TM4 cells infected with bvfA-deletion and parental strains mainly involved the mineral absorption-related pathway, NADH:ubiquinone oxidoreductase subunit-related mitochondrial respiratory signaling pathway, and sphingolipid signaling pathway of TM4 cells. These three signaling pathways were involved in expression changes of TRPM6/7, STEAP1, Gnaq, Trp53, Pbk, Tns2, Akt2, and the NADH:ubiquinone oxidoreductase subunit, as well as content changes of l-Valine, l-Isoleucine, l-Methionine, PC, PE DG, and SM metabolites. These results indicated that BvfA of Brucella abortus S19 affected the above proteins and metabolites in TM4 cells.

Metabolomic Profiling of Large Extracellular Vesicles in Patients Suffering from Small Cell Lung Cancer.

Large extracellular vesicles (lEV) offer a unique window into the metabolism of their cells of orign and dysregulation of lipid metabolism has been described in patients with small cell lung cancer (SCLC). Therefore, metabolomic profiling of patients' lEVs may offer insight into cancer metabolism as well as new potential biomarkers for monitoring disease progression. lEVs were isolated by differential centrifugation from the peripheral blood of SCLC patients and healthy controls. Targeted mass spectrometry was used to analyze the lipid composition of lEVs. After identifying relevant metabolites, biomarker and pathway analysis were conducted. SCLC patients exhibited a distinct metabolic profile compared to healthy controls. The metabolites TG(16:0:_38:3), TG(18:3_35:2), TG(16:0_40:7), Cer(d18:1/26:0), and CE(16:0) are not only able to discriminate between patients and control samples, but are also served as prognostic markers for survival. Patients with high concentrations of these metabolites showed significantly shorter survival times. Pathway analysis revealed alterations in 'sphingolipid metabolism', 'sphingolipid signaling pathway' and 'necroptosis'. Metabolic profiling of lEVs in SCLC patients is feasible and reveals a distinct metabolic profile. High concentrations of identified lipids are associated with poor prognosis.

The Combination of Gastrodin and Gallic Acid Synergistically Attenuates AngII-Induced Apoptosis and Inflammation via Regulation of Sphingolipid Metabolism.

Hypertension (HTN) is closely related to endothelial damage. While tianma (TM) and gouqizi (GQZ) have the potential to be effective in the treatment of HTN in traditional Chinese medicine, their main active ingredients and whether its exert synergistic effects and the underlying mechanisms of synergistic effects are still unclear. This study screened the active ingredients of TM and GQZ, investigated the synergistic effects of the active ingredients and explored possible mechanisms. The potential targets and mechanisms of TM and GQZ were screened using network pharmacology, and gastrodin (GAS) and gallic acid (GA) were identified as compounds with significant antihypertensive activity. The synergistic effects of the combination of GAS and GA was assessed by measuring biomarkers of AngII-induced human umbilical vein endothelial cell (HUVECs) dysfunction model. Furthermore, the anti-apoptotic and anti-inflammatory effects were evaluated by measuring inflammatory cytokine secretion, and apoptosis-related markers. Finally, key targets of the sphingolipid signaling pathway were experimentally validated by Western blotting. In network pharmacology, the herb-pair exerted a synergetic effect by regulating sphingolipid pathways. The GAS and GA exerted synergistic protective effects in AngII-induced HUVECs injury by improving Nitric Oxide Content (NO) levels, alleviating lactate Endothelin-1 (ET-1), and Thromboxane B2 (TX-B2) release, reducing the secretion of inflammatory factors like interleukin-6 (IL-6), interleukin-1β (IL-1β), Tumor Necrosis Factor Alpha (TNF-α)), decreasing the pro-apoptotic protein BAX, and increasing the anti-apoptotic protein BCL-2. Furthermore, the results showed that the GAS and GA combination could elevate the level of S1PR1 and inhibit the expression of ROCK2 and the phosphorylation of NF-κB, which are key targets involved in sphingolipid pathways. Our study revealed that the combination of GAS and GA could suppress inflammation and apoptosis, which are highly correlated with sphingolipid signaling pathways, making it a potential candidate for the treatment of HTN.

Olink proteomics and lipidomics analysis of serum from patients infected with non-tuberculous mycobacteria and Mycobacterium tuberculosis

BackgroundNon-tuberculous mycobacterial (NTM) and Mycobacterium tuberculosis (MTB) infections are difficult to diagnose and treat, significantly burdening global health. The host immune status is generally believed to be associated with the onset and progression of NTM and MTB infections, but its specific impact remains unclear.MethodsIn the present study, proteomics and lipidomics analysis of serum from normal controls (n = 26) and patients with MTB (n = 26), rapidly growing NTM (RGM, n = 15), and slowly growing NTM (SGM, n = 21) were conducted using the Olink technique based on a highly sensitive and specific neighborhood extension assay and the lipidomics technique.ResultsIFN-γ, CXCL9, CXCL10, CXCL11, and CXCL13, etc. were simultaneously upregulated in MTB, RGM, and SGM, while lipids FAHFA 22:3, FAHFA 26:4, FAHFA 24:4, FAHFA 20:5, FAHFA 18:2 simultaneously downregulated. IL8, CCL3, CXCL5, and MCP-2, etc. were simultaneously upregulated in RGM and SGM compared to MTB, as well as PCs, LPCs, PEs, and LPEs. Compared with RGM, IL7, CD27, CCL17, CXCL12, and LPC 28:7-SN2 were downregulated in SGM. Pathway analyses revealed that tuberculosis, sphingolipid signaling pathway, and adipocytokine signaling pathway were regulated at the protein level and metabolite level. Diagnostic panels comprising immune-associated proteins and lipids greatly enhance diagnostic specificity and sensitivity.ConclusionThis integrated multi-omics analysis provides a more comprehensive understanding of the molecular landscape of NTM and MTB, which may provide molecular targets for specialized therapies.

Lipidomic and transcriptomic characteristics of boar seminal plasma extracellular vesicles associated with sperm motility

Seminal plasma extracellular vesicles (SPEVs) play an important role in regulating sperm motility by delivering various cargoes, such as miRNAs, mRNAs, proteins and metabolites. However, information on the lipid compositions of SPEVs and their roles in semen quality is limited. Here, we performed high-throughput transcriptomic and lipidomic analysis on SPEVs isolated from 20 boars with high or low sperm motility. Then, we evaluated the lipid composition and gene expression characteristics of SPEVs and identified the specific lipids and genes related to sperm motility. As a result, a total of 26 lipid classes were identified in SPEVs, and five subclasses, CerG2, CerG3, LPE, LPS and TG, were significantly different in boars with high and low sperm motility. In addition, 195 important lipids and 334 important genes were identified by weighted gene coexpression analysis (WGCNA) and differential expression analysis. We observed that several important genes and lipids in SPEVs potentially influence sperm motility via glycerophospholipid metabolism, glycerolipid metabolism, the sphingolipid signaling pathway and the ferroptosis pathway. Furthermore, we found a significant correlation between the content of 22 lipids and the expression levels of 67 genes (|cor| > 0.8, P < 0.05). Moreover, we observed that three important gene-lipid linkages (CerG1 (d22:0/24:0) - RCAN3, Cer (d18:1/24:0) - SCFD2 and CerG1 (d18:0/24:1) - SCFD2) were strongly correlated with sperm motility. Based on the results, some genes and lipids in SPEVs may play important roles in sperm motility by interacting with sperm through important pathways.

A comprehensive strategy of lipidomics and pharmacokinetics based on ultra-high-performance liquid chromatography-mass spectrometry of Shaoyao Gancao Decoction.

Shaoyao Gancao Decoction (SGD), a traditional Chinese medicine, has been proven to have a good liver protection effect, but the mechanism and pharmacodynamic substances of SGD in the treatment of acute liver injury are still unclear. In this study, an ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) method was established to characterize 107 chemical components of SGD and 12 compounds absorbed in rat plasma samples after oral administration of SGD. Network pharmacology was applied to construct a component-target-pathway network to screen the possible effective components of SGD in acute liver injury. Using lipidomics based on UHPLC-Q-TOF-MS coupled with a variety of statistical analyses, 20 lipid biomarkers were screened and identified, suggesting that the improvement of acute liver injury by SGD was involved in cholesterol metabolism, glycerol-phospholipid metabolism, sphingolipid signaling pathways and fatty acid biosynthesis. In addition, the UHPLC-tandem MS method was established for pharmacokinetics analysis, and 10 potential active components were determined simultaneously within 12 min through the optimization of 0.1% formic acid water and acetonitrile as a mobile phase system. A Pharmacokinetics study showed that paeoniflorin, albiflorin, oxypaeoniflorin, liquiritigenin, isoliquiritigenin, liquiritin, ononin, formononetin, glycyrrhizic acid, and glycyrrhetinic acid as the potential active compounds of SGD curing acute liver injury.

Integrated multi-omics approaches reveal the neurotoxicity of triclocarban in mouse brain

Triclocarban (TCC) is an antimicrobial ingredient that commonly incorporated in many household and personal care products, raising public concerns about its potential health risks. Previous research has showed that TCC could cross the blood–brain barrier, but to date our understanding of its potential neurotoxicity at human-relevant concentrations remains lacking. In this study, we observed anxiety-like behaviors in mice with continuous percutaneous exposure to TCC. Subsequently, we combined lipidomic, proteomic, and metabolic landscapes to investigate the underlying mechanisms of TCC-related neurotoxicity. The results showed that TCC exposure dysregulated the proteins involved in endocytosis and neurodegenerative disorders in mouse cerebrum. Brain energy homeostasis was also altered, as evidenced by the perturbation of pyruvate metabolism, TCA cycle, and oxidative phosphorylation, which in turn caused mitochondrial dysfunction. Meanwhile, the changing trends of sphingolipid signaling pathway and overproduction of mitochondrial reactive oxygen species (mROS) could enhance the neural apoptosis. The in vitro approach further demonstrated that TCC exposure promoted apoptosis, accompanied by the overproduction of mROS and alteration in the mitochondrial membrane potential in N2A cells. Together, dysregulated endocytosis, mROS-related mitochondrial dysfunction and neural cell apoptosis are considered to be crucial factors for TCC-induced neurotoxicity, which may contribute to the occurrence and development of neurodegenerative disorders. Our findings provide novel perspectives for the mechanisms of TCC-triggered neurotoxicity.

Exosomal MicroRNAs modulate the cognitive function in fasudil treated APPswe/PSEN1dE9 transgenic (APP/PS1) mice model of Alzheimer’s disease

Alzheimer’s disease (AD) is characterized by cognitive decline stemming from the accumulation of beta-amyloid (Aβ) plaques and the propagation of tau pathology through synapses. Exosomes, crucial mediators in neuronal development, maintenance, and intercellular communication, have gained attention in AD research. Yet, the molecular mechanisms involving exosomal miRNAs in AD remain elusive. In this study, we treated APPswe/PSEN1dE9 transgenic (APP/PS1) mice, a model for AD, with either vehicle (ADNS) or fasudil (ADF), while C57BL/6 (control) mice received vehicle (WT). Cognitive function was evaluated using the Y-maze test, and AD pathology was confirmed through immunostaining and western blot analysis of Aβ plaques and phosphorylated tau. Exosomal RNAs were extracted, sequenced, and analyzed from each mouse group. Our findings revealed that fasudil treatment improved cognitive function in AD mice, as evidenced by increased spontaneous alternation in the Y-maze test and reduced Aβ plaque load and phosphorylated tau protein expression in the hippocampus. Analysis of exosomal miRNAs identified three miRNAs (mmu-let-7i-5p, mmu-miR-19a-3p, mmu-miR-451a) common to both ADNS vs ADF and WT vs ADNS groups. Utilizing miRTarBase software, we predicted and analyzed target genes associated with these miRNAs. Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of miRNA target genes indicated that mmu-miR-19a-3p and mmu-miR-451a are implicated in signal transduction, immune response, cellular communication, and nervous system pathways. Specifically, mmu-miR-19a-3p targeted genes involved in the sphingolipid signaling pathway, such as Pten and Tnf, while mmu-miR-451a targeted Nsmaf, Gnai3, and Akt3. Moreover, mmu-miR-451a targeted Myc in signaling pathways regulating the pluripotency of stem cells. In conclusion, fasudil treatment enhanced cognitive function by modulating exosomal MicroRNAs, particularly mmu-miR-451a and mmu-miR-19a-3p. These miRNAs hold promise as potential biomarkers and therapeutic targets for novel AD treatments.Graphical

Integrative multi-omics analysis unravels the host response landscape and reveals a serum protein panel for early prognosis prediction for ARDS

BackgroundThe multidimensional biological mechanisms underpinning acute respiratory distress syndrome (ARDS) continue to be elucidated, and early biomarkers for predicting ARDS prognosis are yet to be identified.MethodsWe conducted a multicenter observational study, profiling the 4D-DIA proteomics and global metabolomics of serum samples collected from patients at the initial stage of ARDS, alongside samples from both disease control and healthy control groups. We identified 28-day prognosis biomarkers of ARDS in the discovery cohort using the LASSO method, fold change analysis, and the Boruta algorithm. The candidate biomarkers were validated through parallel reaction monitoring (PRM) targeted mass spectrometry in an external validation cohort. Machine learning models were applied to explore the biomarkers of ARDS prognosis.ResultsIn the discovery cohort, comprising 130 adult ARDS patients (mean age 72.5, 74.6% male), 33 disease controls, and 33 healthy controls, distinct proteomic and metabolic signatures were identified to differentiate ARDS from both control groups. Pathway analysis highlighted the upregulated sphingolipid signaling pathway as a key contributor to the pathological mechanisms underlying ARDS. MAP2K1 emerged as the hub protein, facilitating interactions with various biological functions within this pathway. Additionally, the metabolite sphingosine 1-phosphate (S1P) was closely associated with ARDS and its prognosis. Our research further highlights essential pathways contributing to the deceased ARDS, such as the downregulation of hematopoietic cell lineage and calcium signaling pathways, contrasted with the upregulation of the unfolded protein response and glycolysis. In particular, GAPDH and ENO1, critical enzymes in glycolysis, showed the highest interaction degree in the protein–protein interaction network of ARDS. In the discovery cohort, a panel of 36 proteins was identified as candidate biomarkers, with 8 proteins (VCAM1, LDHB, MSN, FLG2, TAGLN2, LMNA, MBL2, and LBP) demonstrating significant consistency in an independent validation cohort of 183 patients (mean age 72.6 years, 73.2% male), confirmed by PRM assay. The protein-based model exhibited superior predictive accuracy compared to the clinical model in both the discovery cohort (AUC: 0.893 vs. 0.784; Delong test, P < 0.001) and the validation cohort (AUC: 0.802 vs. 0.738; Delong test, P = 0.008).InterpretationOur multi-omics study demonstrated the potential biological mechanism and therapy targets in ARDS. This study unveiled several novel predictive biomarkers and established a validated prediction model for the poor prognosis of ARDS, offering valuable insights into the prognosis of individuals with ARDS.

Combined Proteomic and Metabolomic Analysis Reveals Comprehensive Regulation of Somatostatin DNA Vaccine in Goats.

Somatostatin (SS) plays crucial regulatory roles in animal growth and reproduction by affecting the synthesis and secretion of growth hormone (GH). However, the mechanism by which SS regulates growth and development in goats is still unclear. In order to investigate the regulatory networks of the hypothalamus and pituitary in goats affected by SS DNA vaccines, in this study, we used a previously established oral attenuated Salmonella typhimurium SS DNA vaccine, X9241 (ptCS/2SS-asd), to treat wethers. We analyzed the protein changes in hypothalamic and pituitary tissues using a TMT-based proteomics approach. Additionally, we examined the metabolic profiles of the serum of control and immunized wethers through untargeted metabolomics using liquid chromatography-mass spectrometry (LC-MS). Key signaling pathways were identified based on differentially expressed metabolites (DEMs) and differentially expressed proteins (DEPs). Furthermore, the effect of critical DEPs on signaling pathways was confirmed through Western blotting (WB) experiments, which elucidated the mechanism of active SS immunization in wethers. A proteomics analysis revealed that the expression of 58 proteins in the hypothalamus and 124 in the pituitary gland was significantly altered following SS vaccine treatment (fold change > 1.2 or < 0.83, p < 0.05). In the hypothalamus, many DEPs were associated with gene ontology (GO) terms related to neuronal signaling. In contrast, most DEPs were associated with metabolic pathways. In the pituitary gland, the DEPs were largely related to immune and nutrient metabolism functions, with significant enrichment in KEGG pathways, particularly those involving the metabolic pathway, sphingolipid signaling, and the cGMP-PKG signaling pathway. A metabolomic analysis further showed that active SS immunization in wethers led to significant alterations in seven serum metabolites. Notably, the sphingolipid signaling pathway, secondary bile acid synthesis, sphingolipid metabolism, and lysine synthesis were significantly disrupted. SS vaccines induced marked changes in hypothalamic-pituitary proteins in wethers, facilitating alterations in their growth processes. This study not only provides insights into the mechanism of the SS gene in regulating GH secretion in wethers but also establishes a basis for hormone immunoregulation technology to enhance livestock production performance.

Transcriptome analysis revealed the characteristics and functions of long non-coding RNAs in the hypothalamus during sexual maturation in goats

The hypothalamus is an essential neuroendocrine area in animals that regulates sexual development. Long non-coding RNAs (lncRNAs) are hypothesized to regulate physiological processes related to animal reproduction. However, the regulatory mechanism by which lncRNAs participate in sexual maturity in goats is poorly known, particularly from birth to sexual maturation. In this study, RNAseq analysis was conducted on the hypothalamus of four developmental stages (1day (D1, n = 5), 2 months (M2, n = 5), 4 months (M4, n = 5), and 6 months (M6, n = 5)) of Jining grey goats. The results showed that a total of 237 differentially expressed lncRNAs (DELs) were identified in the hypothalamus. Among these, 221 DELs exhibited cis-regulatory effects on 693 target genes, while 24 DELs demonstrated trans-regulatory effects on 63 target genes. The target genes of these DELs are mainly involved in biological processes related to energy metabolism, signal transduction and hormone secretion, such as sphingolipid signaling pathway, adipocytokine signaling pathway, neurotrophic signaling pathway, glutamatergic synapse, P53 signaling pathway and GnRH signaling pathway. In addition, XR_001918477.1, TCONS_00077463, XR_001918760.1, and TCONS_00029048 and their potential target genes may play a crucial role in the process of goat sexual maturation. This study advances our understanding of lncRNA in hypothalamic tissue during sexual maturation in goats and will give a theoretical foundation for improving goat reproductive features.

Pathway analysis of peripheral blood CD8+ T cell transcriptome shows differential regulation of sphingolipid signaling in multiple sclerosis and glioblastoma.

Multiple sclerosis (MS) and glioblastoma (GBM) are CNS diseases in whose development and progression immune privilege is intimately important, but in a relatively opposite manner. Maintenance and strengthening of immune privilege have been shown to be an important mechanism in glioblastoma immune evasion, while the breakdown of immune privilege leads to MS initiation and exacerbation. We hypothesize that molecular signaling pathways can be oppositely regulated in peripheral blood CD8+ T cells of MS and glioblastoma patients at a transcriptional level. We analyzed publicly available data of the peripheral blood CD8+ T cell MS vs. control (MSvsCTRL) and GBM vs. control (GBMvsCTRL) differentially expressed gene (DEG) contrasts with Qiagen's Ingenuity pathway analysis software (IPA). We have identified sphingolipid signaling pathway which was significantly downregulated in the GBMvsCTRL and upregulated in the MSvsCTRL. As the pathway is important for the CD8+ T lymphocytes CNS infiltration, this result is in line with our previously stated hypothesis. Comparing publicly available lists of differentially expressed serum exosomal miRNAs from MSvsCTRL and GBMvsCTRL contrasts, we have identified that hsa-miR-182-5p has the greatest potential effect on sphingolipid signaling regarding the number of regulated DEGs in the GBMvsCTRL contrast, while not being able to find any relevant potential sphingolipid signaling target transcripts in the MSvsCTRL contrast. We conclude that the sphingolipid signaling pathway is a top oppositely regulated pathway in peripheral blood CD8+ T cells from GBM and MS, and might be crucial for the differences in CNS immune privilege maintenance of investigated diseases, but further experimental research is necessary.

Antifungal bioactivity of Sarcococca hookeriana var. digyna Franch. against fluconazole-resistant Candida albicans in vitro and in vivo

Ethnopharmacological relevanceSarcococca hookeriana var. digyna Franch. has been widely utilized in folk medicine by the Miao people in the southwestern region of China for treating skin sores which may be associated with microbial infection. Aim of the studyTo investigate the antifungal bioactivity of S. hookeriana var. digyna against fluconazole-resistant Candida albicans in vitro and in vivo, as well as its underlying mechanism and the key bioactive component. Materials and methodsThe antifungal bioactivity of 80% ethanol extract of S. hookeriana var. digyna (SHE80) was investigated in vitro using the broth microdilution method, time-growth curve, and time-kill assay. Its key functional component and antifungal mechanism were explored with combined approaches including UPLC-Q-TOF-MS, network pharmacology and metabolomics. The antifungal pathway was further supported via microscopic observation of fungal cell morphology and examination of its effects on fungal biofilm and cell membranes using fluorescent staining reagents. In vivo assessment of antifungal bioactivity was conducted using a mouse model infected with C. albicans on the skin. ResultsS. hookeriana var. digyna suppressed fluconazole-resistant C. albicans efficiently (MIC = 16 μg/mL, MFC = 64 μg/mL). It removed fungal biofilm, increased cell membrane permeability, induced protein leakage, reduced membrane fluidity, disrupted mitochondrial membrane potential, induced the release of reactive oxygen species, promoted cell apoptosis, and inhibited the transformation of fungi from the yeast state to the hyphal state significantly. In terms of mechanism, it affected sphingolipid metabolism and signaling pathway. Moreover, the predicted bioactive component, sarcovagine D, was supported by antifungal bioactivity evaluation in vitro (MIC = 4 μg/mL, MFC = 16 μg/mL). Furthermore, S. hookeriana var. digyna promoted wound healing, reduced the number of colony-forming units, and reduced inflammation effectively in vivo. ConclusionsThe traditional use of S. hookeriana var. digyna for fungal skin infections was supported by antifungal bioactivity investigated in vitro and in vivo. Its mechanism and bioactive component were predicted and confirmed by experiments, which also provided a new antifungal agent for future research.

High fat diet increases the severity of collagen-induced arthritis in mice by altering the gut microbial community

ObjectivesResearch has demonstrated that obesity may be associated with rheumatoid arthritis (RA). In addition, gut microbiota and its metabolites contribute to the occurrence and development of RA and obesity. However, the mechanism by which obesity affects RA remains unclear. In this study, we aimed to investigate whether gut microbiota and their metabolites alter the effects of high fat diet (HFD) on the severity of collagen-induced arthritis (CIA) in mice.MethodsBriefly, mice were divided into normal group (N), CIA model group (C), HFD group (T), and HFD CIA group (CT). Hematoxylin and Eosin staining(HE) and Safranin O-fast green staining were conducted, and levels of blood lipid and inflammatory cytokines were measured. 16S rDNA sequencing technique and liquid chromatography-mass spectrometry (LC-MS)-based metabolomics were performed to explore changes in the microbiota structure to further reveal the pathomechanism of HFD on CIA.ResultsHFD aggravated the severity of CIA in mice. The CT group had the highest proportion of microbial abundance of Blautia, Oscillibacter, Ruminiclostridium-9, and Lachnospiraceae UCG 006 at the genus level, but had a lower proportion of Alistipes. Additionally, the fecal metabolic phenotype of the combined CT group shows significant changes, with differential metabolites enriched in 9 metabolic pathways, including primary bile acid biosynthesis, arginine biosynthesis, sphingolipid metabolism, purine metabolism, linoleic acid metabolism, oxytocin signaling pathway, aminoacyl-tRNA biosynthesis, the pentose phosphate pathway, and sphingolipid signaling pathway. Correlation analysis revealed that some of the altered gut microbiota genera were strongly correlated with changes in fecal metabolites, total cholesterol (TC), triglyceride (TG), and inflammatory cytokine levels.ConclusionsThis study shows that HFD may aggravate inflammatory reaction in CIA mice by altering the gut microbiota and metabolic pathways.

#911 A mice model of SHPT with bone abnormalities in CKD and potential functional role of sphingolipid in renal osteodystrophy

Abstract Background and Aims Renal osteodystrophy (ROD) is the skeletal disease of chronic kidney disease associated mineral and bone disorder (CKD-MBD). ROD is characterized by abnormal bone mineralization and impaired bone remodeling, leading to increased risk of fractures and mortality in patients with CKD. To date, the pathology of ROD remains poorly understood due to the lack of a suitable animal model. Hence, we successfully established a stable mouse model of ROD using an optimized adenine diet and explored the possible signaling based on RNA-seq expression analyses. Method Blood biochemical analysis, micro-computed tomography (micro-CT) and bone histomorphometry were performed to analyze the skeletal characteristics. RNA sequencing (RNA-seq) was used to investigate the mechanisms involved in ROD. The functions of differentially expressed genes (DEGs) were evaluated by Gene Ontology (GO) analyses, Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses and Gene Set Enrichment Analyses (GSEA). DEGs were validated by quantitative real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results The mice with ROD induced by optimized adenine diet showed severe abnormalities in serum creatinine (Scr) and blood urea nitrogen (BUN) along with marked hyperparathyroidism and hyperphosphatemia. The bone mineral density (BMD) of femurs was significantly lower in the ROD mice than that in the control (CTL) mice. The trabecular and cortical bone parameters of femurs significantly showed bone loss and severe cortical porosity. ROD mice showed high bone turnover with increased osteoid, eroded, osteoblast, and osteoclast perimeters as well as increased bone formation rate and mineral apposition rate. Transcriptomic profiling identified 1286 genes were upregulated in the femurs of ROD mice compared to the CTL mice and 333 genes that were downregulated. GO, KEGG and GSEA pathway analyses indicated that these genes were highly enriched in functions related to lipid metabolism pathway, sphingolipid signaling pathway, among others. The differential expression of alkaline ceramidase 1(Acer1), ceramide synthase 4 (Cers4) sphingomyelin phosphodiesterase 3 (Smpd3), sphingosine-1-phosphate phosphatase 2 (Sgpp2) in skeletal tissue and serum of CKD patients were validated by qRT-PCR. Since sphingolipid pathways have been recently implicated in bone metabolism, these genes are potential target genes in the settings of ROD. Conclusion In conclusion, our findings demonstrated that the CKD mice with bone abnormalities induced by optimized adenine diet may serve as a useful model for skeletal analysis in ROD. Based on the RNA-seq, we identified that sphingolipid might play a crucial role in the pathogenesis of ROD and provided new therapeutic targets for future study.

Hepatic-Metabolic Activity of α-Lipoic Acid-Its Influence on Sphingolipid Metabolism and PI3K/Akt/mTOR Pathway in a Rat Model of Metabolic Dysfunction-Associated Steatotic Liver Disease.

Excessive lipid deposition affects hepatic homeostasis and contributes to the development of insulin resistance as a crucial factor for the deterioration of simple steatosis to steatohepatitis. So, it is essential to search for an effective agent for a new therapy for hepatic steatosis development before it progresses to the more advanced stages. Our study aimed to evaluate the potential protective effect of α-lipoic acid (α-LA) administration on the intrahepatic metabolism of sphingolipid and insulin signaling transduction in rats with metabolic dysfunction-associated steatotic liver disease (MASLD). The experiment was conducted on male Wistar rats subjected to a standard diet or a high-fat diet (HFD) and an intragastrically α-LA administration for eight weeks. High-performance liquid chromatography (HPLC) was used to determine sphingolipid content. Immunoblotting was used to measure the expression of selected proteins from sphingolipid and insulin signaling pathways. Multiplex assay kit was used to assess the level of the phosphorylated form of proteins from PI3K/Akt/mTOR transduction. The results revealed that α-LA decreased sphinganine, dihydroceramide, and sphingosine levels and increased ceramide level. We also observed an increased the concentration of phosphorylated forms of sphingosine and sphinganine. Changes in the expression of proteins from sphingolipid metabolism were consistent with changes in sphingolipid pools. Treatment with α-LA activated the PI3K/Akt/mTOR pathway, which enhanced the hepatic phosphorylation of Akt and mTOR. Based on these data, we concluded that α-lipoic acid may alleviate glucose intolerance and may have a protective influence on the sphingolipid metabolism under HFD; thus, this antioxidant appears to protect from MASLD development and steatosis deterioration.

Chronic exposure to tire rubber-derived contaminant 6PPD-quinone impairs sperm quality and induces the damage of reproductive capacity in male mice

It has been reported that N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine-quinone (6PPD-Q), a derivative of the tire antioxidant, N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD), exhibits acute toxicity towards organisms. However, the possible reproductive toxicity of 6PPD-Q in mammals has rarely been reported. In this study, the effects of 6PPD-Q on the reproductive toxicity of C57Bl/6 male mice were assessed after exposure to 6PPD-Q for 40 days at 4 mg/kg body weight (bw). Exposure to 6PPD-Q not only led to a decrease in testosterone levels but also adversely affected semen quality and in vitro fertilization (IVF) outcomes, thereby indicating impaired male fertility resulting from 6PPD-Q exposure. Additionally, transcriptomic and metabolomic analyses revealed that 6PPD-Q elicited differential expression of genes and metabolites primarily enriched in spermatogenesis, apoptosis, arginine biosynthesis, and sphingolipid metabolism in the testes of mice. In conclusion, our study reveals the toxicity of 6PPD-Q on the reproductive capacity concerning baseline endocrine disorders, sperm quality, germ cell apoptosis, and the sphingolipid signaling pathway in mice. These findings contribute to an enhanced understanding of the health hazards posed by 6PPD-Q to mammals, thereby facilitating the development of more robust safety regulations governing the utilization and disposal of rubber products.

Adipose tissue place of origin and obesity influence sphingolipid signaling pathway in the adipocytes differentiated from ADMSCs isolated from morbidly obese women

Adipose derived mesenchymal stem cells (ADMSCs) are a component of adipose tissue that in recent years has gained on importance. The progenitor cells serve as an essentially unlimited source of new adipocytes and therefore are considered to be an important determinant of the tissue’s physiology. In this paper we investigated mature adipocytes differentiated from ADMSCs obtained from subcutaneous/visceral fat of patients with different metabolic status (lean, obese without and with metabolic syndrome). We focused our interests on the sphingolipid signaling pathway, i.e.a signal transduction system indispensable for cells functioning, but also implicated in the development of medical conditions associated with obesity. We observed that the cells derived from visceral tissue had significantly greater levels of almost all the examined sphingolipids (especially Cer, dhCer, SM). Moreover, obesity and metabolic syndrome present in donor patients was associated with an increased level of sphingosine kinase (SPHK) and the product of its reaction sphingosine-1-phosphate (S1P). Moreover, the condition appeared to display a tissue specific pattern. Namely, the adipocytes of subcutaneous provenance had an increased activation of ceramide de novo synthesis pathway when the donors of ADMSCs had metabolic syndrome. The above translated into greater accumulation of ceramide in the cells. To our knowledge this is the first study that demonstrated altered sphingolipid profile in the mature adipocytes differentiated from ADMSCs with respect to the stem cells tissue of origin and the donor patient metabolic status.