Sphingosine 1-Phosphate Stimulates ER to Golgi Ceramide Traffic to Promote Survival in T98G Glioma Cells.

Glioblastoma multiforme is the most common and fatal brain tumor among human cancers. Ceramide (Cer) and Sphingosine 1-phosphate (S1P) have emerged as bioeffector molecules that control several biological processes involved in both cancer development and resistance. Cer acts as a tumor suppressor, inhibiting cancer progression, promoting apoptosis, enhancing immunotherapy and sensitizing cells to chemotherapy. In contrast, S1P functions as an onco-promoter molecule, increasing proliferation, survival, invasiveness, and resistance to drug-induced apoptosis. The pro-survival PI3K/Akt pathway is a recognized downstream target of S1P, and we have previously demonstrated that in glioma cells it also improves Cer transport and metabolism towards complex sphingolipids in glioma cells. Here, we first examined the possibility that, in T98G glioma cells, S1P may regulate Cer metabolism through PI3K/Akt signaling. Our research showed that exogenous S1P increases the rate of vesicular trafficking of Cer from the endoplasmic reticulum (ER) to the Golgi apparatus through S1P receptor-mediated activation of the PI3K/Akt pathway. Interestingly, the effect of S1P results in cell protection against toxicity arising from Cer accumulation in the ER, highlighting the role of S1P as a survival factor to escape from the Cer-generating cell death response.

Functional Insights into the Sphingolipids C1P, S1P, and SPC in Human Fibroblast-like Synoviocytes by Proteomic Analysis.

The (patho)physiological function of the sphingolipids ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), and sphingosylphosphorylcholine (SPC) in articular joints during osteoarthritis (OA) is largely unknown. Therefore, we investigated the influence of these lipids on protein expression by fibroblast-like synoviocytes (FLSs) from OA knees. Cultured human FLSs (n = 7) were treated with 1 of 3 lipid species-C1P, S1P, or SPC-IL-1ß, or with vehicle. The expression of individual proteins was determined by tandem mass tag peptide labeling followed by high-resolution electrospray ionization (ESI) mass spectrometry after liquid chromatographic separation (LC-MS/MS/MS). The mRNA levels of selected proteins were analyzed using RT-PCR. The 3sphingolipids were quantified in the SF of 18 OA patients using LC-MS/MS. A total of 4930 proteins were determined using multiplex MS, of which 136, 9, 1, and 0 were regulated both reproducibly and significantly by IL-1ß, C1P, S1P, and SPC, respectively. In the presence of IL-1ß, all 3 sphingolipids exerted ancillary effects. Only low SF levels of C1P and SPC were found. In conclusion, the 3 lipid species regulated proteins that have not been described in OA. Our results indicate that charged multivesicular body protein 1b, metal cation symporter ZIP14, glutamine-fructose-6-P transaminase, metallothionein-1F and -2A, ferritin, and prosaposin are particularly interesting proteins due to their potential to affect inflammatory, anabolic, catabolic, and apoptotic mechanisms.

Metabolomics-based search for lung cancer markers among patients with different smoking status.

Tobacco smoking is the main etiological factor of lung cancer (LC), which can also cause metabolome disruption. This study aimed to investigate whether the observed metabolic shift in LC patients was also associated with their smoking status. Untargeted metabolomics profiling was applied for the initial screening of changes in serum metabolic profile between LC and chronic obstructive pulmonary disease (COPD) patients, selected as a non-cancer group. Differences in metabolite profiles between current and former smokers were also tested. Then, targeted metabolomics methods were applied to verify and validate the proposed LC biomarkers. For untargeted metabolomics, a single extraction-dual separation workflow was applied. The samples were analyzed using a liquid chromatograph-high resolution quadrupole time-of-flight mass spectrometer. Next, the selected metabolites were quantified using liquid chromatography-triple-quadrupole mass spectrometry. The acquired data confirmed that patients' stratification based on smoking status impacted the discriminating ability of the identified LC marker candidates. Analyzing a validation set of samples enabled us to determine if the putative LC markers were truly robust. It demonstrated significant differences in the case of four metabolites: allantoin, glutamic acid, succinic acid, and sphingosine-1-phosphate. Our research showed that studying the influence of strong environmental factors, such as tobacco smoking, should be considered in cancer marker research since it reduces the risk of false positives and improves understanding of the metabolite shifts in cancer patients.

Comprehensive metabolic modulations of sphingolipids are promising severity indicators in COVID-19.

The COVID-19 pandemic, caused by SARS-CoV-2, has had a significant worldwide impact, affecting millions of people. COVID-19 is characterized by a heterogenous clinical phenotype, potentially involving hyperinflammation and prolonged tissue damage, although the exact underlying mechanisms are yet to be fully understood. Sphingolipid metabolites, which govern cell survival and proliferation, have emerged as key players in inflammatory signaling and cytokine responses. Given the complex metabolic pathway of sphingolipids, this study aimed to understand their potential role in the pathogenesis of COVID-19. We conducted a comprehensive examination of sphingolipid modulations across groups classified based on disease severity, incorporating a time-course in serum and urine samples. Several sphingolipids, including sphingosine, lactosylceramide, and hexosylceramide, emerged as promising indicators of COVID-19 severity, as validated by correlation analyses conducted on both serum and urine samples. Other sphingolipids, such as sphingosine 1-phosphate, ceramides, and deoxy-dihydroceramides, decreased in both COVID-19 patients and individuals with non-COVID infectious diseases. This suggests that these sphingolipids are not specifically associated with COVID-19 but rather with pathological conditions caused by infectious diseases. Our analysis of urine samples revealed elevated levels of various sphingolipids, with changes dependent on disease severity, potentially highlighting the acute kidney injury associated with COVID-19. This study illuminates the intricate relationship between disturbed sphingolipid metabolism, COVID-19 severity, and clinical factors. These findings provide valuable insights into the broader landscape of inflammatory diseases.

Optimized Lipidomics Extraction of Sphingosine and Sphinganine from Optic Nerve for Signaling Studies.

Interconvertible sphingolipid metabolites represent germane constituents of eukaryotic membranes and are vital in the regulation of cellular homeostasis, proliferation, survival, and induction of autophagy. This protocol describes a step-by-step method for extractions of sphingosine and sphinganine from mammalian tissue samples, particularly from the murine optic nerve. These lipids are partitioned into a binary mixture of chloroform and methanol in a modified Bligh and Dyer method. This is followed with reverse phase ultrahigh-performance liquid chromatography fractionation with a C18+ column and subsequent tandem mass spectrometry (UHPLC-MS-MS) analysis of the biological abundance. These free sphingoid bases dissociate to form structurally distinctive carbocation product ions that can be confirmed with annotations of lipidomic databases or in-house fragmentation software.

Analysis of Sphingosine and Sphinganine from the Aqueous Humor for Signaling Studies Using Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry.

Sphingolipids, including sphingosine and sphinganine, are one of the major classes of lipids. They serve as constituents of cell membranes and lipid rafts and aid in the performance of cell-cell communication and adhesion. Abnormal levels of sphingolipids in the aqueous humor can indicate impaired sphingolipid metabolism and associated ocular pathologies. Sphingolipids can be extracted from the aqueous humor by the methyl-tert-butyl ether (MTBE) lipid extraction method and subsequently analyzed by liquid chromatography-mass spectrometry (LC-MS). This chapter describes a modified protocol for an MTBE lipid extraction from the aqueous humor, followed by analysis with ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS).

Inhibiting sphingosine 1-phosphate lyase: From efficacy to mechanism.

Sphingosine-1 phosphate (S1P) is a lipid metabolite regulating diverse biological processes, including proliferation, differentiation, migration, and apoptosis, highlighting its physiological and therapeutic significance. Current S1P-based therapeutic approaches primarily focus on modulating the downstream signalling via targeting S1P receptors, however, this is challenged by incomplete receptor internalisation. Sphingosine-1-phosphate lyase (SPL) is a highly conserved enzyme that "gatekeeps" the final step of S1P degradation. Cognisant of the complex ligand and receptor interaction and dynamic metabolic networks, the selective modulation of SPL activity presents a new opportunity to regulate S1P biosynthesis and reveal its role in various systems. Over the past decade, an evolving effort has been made to identify new molecules that could block SPL activity in vitro or in vivo. This review focuses on summarising the current understanding of the reported SPL inhibitors identified through various screening approaches, discussing their efficacy in diverse model systems and the possible mechanism of action. Whilst effective modulation of S1P levels via inhibiting SPL is feasible, the specificity of those inhibitors remains inconclusive, presenting a clear challenge for future implications. Yet, none of the currently available SPL inhibitors is proven effective in elevating S1P levels within the central nervous system. This review article embraces future research focusing on investigating selective SPL inhibitors with high potency and possibly blood-brain-barrier permeability, which would aid the development of new S1P-based therapeutics for neurological disorders.

S1P Signaling Genes as Prominent Drivers of BCR-ABL1-Independent Imatinib Resistance and Six Herbal Compounds as Potential Drugs for Chronic Myeloid Leukemia.

Imatinib (IM), a breakthrough in chronic myeloid leukemia (CML) treatment, is accompanied by discontinuation challenges owing to drug intolerance. Although BCR-ABL1 mutation is a key cause of CML resistance, understanding mechanisms independent of BCR-ABL1 is also important. This study investigated the sphingosine-1-phosphate (S1P) signaling-associated genes (SphK1 and S1PRs) and their role in BCR-ABL1-independent resistant CML, an area currently lacking investigation. Through comprehensive transcriptomic analysis of IM-sensitive and IM-resistant CML groups, we identified the differentially expressed genes and found a notable upregulation of SphK1, S1PR2, and S1PR5 in IM-resistant CML. Functional annotation revealed their roles in critical cellular processes such as proliferation and GPCR activity. Their network analysis uncovered significant clusters, emphasizing the interconnectedness of the S1P signaling genes. Further, we identified interactors such as BIRC3, TRAF6, and SRC genes, with potential implications for IM resistance. Additionally, receiver operator characteristic curve analysis suggested these genes' potential as biomarkers for predicting IM resistance. Network pharmacology analysis identified six herbal compounds-ampelopsin, ellagic acid, colchicine, epigallocatechin-3-gallate, cucurbitacin B, and evodin-as potential drug candidates targeting the S1P signaling genes. In summary, this study contributes to efforts to better understand the molecular mechanisms underlying BCR-ABL1-independent CML resistance. Moreover, the S1P signaling genes are promising therapeutic targets and plausible new innovation avenues to combat IM resistance in cancer clinical care in the future.

Neurolipid systems: A new target for the treatment of dementia.

Neurolipids comprise a diverse class of bioactive lipids that include molecules capable of activating G protein­coupled receptors, thereby inducing systemic effects that contribute to the maintenance of homeostasis. Dementia, a non­specific brain disorder characterized by a common set of signs and symptoms, usually arises subsequent to brain injuries or diseases and is often associated with the aging process. Individuals affected by dementia suffer from the disruption of several neurotransmitter and neuromodulatory systems, among which neurolipids play an important role, including the endocannabinoid, lysophosphatidic acid and sphingosine 1­phosphate systems. In this review, we present an overview of the most recent and pertinent findings regarding the involvement of these neurolipidic systems in dementia, including data from a wide range of both in vitro and in vivo experiments as well as clinical trials.

Sphinganine recruits TLR4 adaptors in macrophages and promotes inflammation in murine models of sepsis and melanoma.

After recognizing its ligand lipopolysaccharide, Toll-like receptor 4 (TLR4) recruits adaptor proteins to the cell membrane, thereby initiating downstream signaling and triggering inflammation. Whether this recruitment of adaptor proteins is dependent solely on protein-protein interactions is unknown. Here, we report that the sphingolipid sphinganine physically interacts with the adaptor proteins MyD88 and TIRAP and promotes MyD88 recruitment in macrophages. Myeloid cell-specific deficiency in serine palmitoyltransferase long chain base subunit 2, which encodes the key enzyme catalyzing sphingolipid biosynthesis, decreases the membrane recruitment of MyD88 and inhibits inflammatory responses in in vitro bone marrow-derived macrophage and in vivo sepsis models. In a melanoma mouse model, serine palmitoyltransferase long chain base subunit 2 deficiency decreases anti-tumor myeloid cell responses and increases tumor growth. Therefore, sphinganine biosynthesis is required for the initiation of TLR4 signal transduction and serves as a checkpoint for macrophage pattern recognition in sepsis and melanoma mouse models.

Sphingosine kills intracellular Pseudomonas aeruginosa and Staphylococcus aureus.

Sphingosine has been previously shown to kill many strains of pathogenic bacteria including Pseudomonas aeruginosa, Staphyloccus aureus, Acinetobacter, and atypical mycobacteria. However, these studies were performed on isolated or extracellular bacteria and it is unknown whether sphingosine also targets intracellular bacteria. Here, we demonstrate that exogenously-added sphingosine directly binds to extracellular P. aeruginosa and S. aureus, but also targets and binds to intracellular bacteria. Intracellular sphingosine and bacteria were identified by sequential immunostainings. We further show that exogenously-added sphingosine also kills intracellular P. aeruginosa and S. aureus using modified gentamycin assays. Intracellular killing of P. aeruginosa and S. aureus by sphingosine is not mediated by improved phagosomal-lysosomal fusion. In summary, our data indicate that sphingosine binds to and most likely also directly kills extra- and intracellular P. aeruginosa and S. aureus.

Amelioration of Fibrosis via S1P Inhibition Is Regulated by Inactivation of TGF-ß and SPL Pathways in the Human Cornea.

Human corneal fibrosis can lead to opacity and ultimately partial or complete vision loss. Currently, corneal transplantation is the only treatment for severe corneal fibrosis and comes with the risk of rejection and donor shortages. Sphingolipids (SPLs) are known to modulate fibrosis in various tissues and organs, including the cornea. We previously reported that SPLs are tightly related to both, transforming growth factor beta (TGF-ß) signaling and corneal fibrogenesis. The aim of this study was to investigate the effects of sphingosine-1-phosphate (S1P) and S1P inhibition on specific TGF-ß and SPL family members in corneal fibrosis. Healthy human corneal fibroblasts (HCFs) were isolated and cultured in EMEM + FBS + VitC (construct medium) on 3D transwells for 4 weeks. The following treatments were prepared in a construct medium: 0.1 ng/mL TGF-ß1 (ß1), 1 µM sphingosine-1-phosphate (S1P), and 5 µM Sphingosine kinase inhibitor 2 (I2). Five groups were tested: (1) control (no treatment); rescue groups; (2) ß1/S1P; (3) ß1/I2; prevention groups; (4) S1P/ß1; and (5) I2/ß1. Each treatment was administered for 2 weeks with one treatment and switched to another for 2 weeks. Using Western blot analysis, the 3D constructs were examined for the expression of fibrotic markers, SPL, and TGF-ß signaling pathway members. Scratch assays from 2D cultures were also utilized to evaluate cell migration We observed reduced fibrotic expression and inactivation of latent TGF-ß binding proteins (LTBPs), TGF-ß receptors, Suppressor of Mothers Against Decapentaplegic homologs (SMADs), and SPL signaling following treatment with I2 prevention and rescue compared to S1P prevention and rescue, respectively. Furthermore, we observed increased cell migration following stimulation with I2 prevention and rescue groups, with decreased cell migration following stimulation with S1P prevention and rescue groups after 12 h and 18 h post-scratch. We have demonstrated that I2 treatment reduced fibrosis and modulated the inactivation of LTBPs, TGF-ß receptors, SPLs, and the canonical downstream SMAD pathway. Further investigations are warranted in order to fully uncover the potential of utilizing SphK I2 as a novel therapy for corneal fibrosis.

Sphingolipid changes in mouse brain and plasma after mild traumatic brain injury at the acute phases.

BACKGROUND: Traumatic brain injury (TBI) causes neuroinflammation and can lead to long-term neurological dysfunction, even in cases of mild TBI (mTBI). Despite the substantial burden of this disease, the management of TBI is precluded by an incomplete understanding of its cellular mechanisms. Sphingolipids (SPL) and their metabolites have emerged as key orchestrators of biological processes related to tissue injury, neuroinflammation, and inflammation resolution. No study so far has investigated comprehensive sphingolipid profile changes immediately following TBI in animal models or human cases. In this study, sphingolipid metabolite composition was examined during the acute phases in brain tissue and plasma of mice following mTBI. METHODS: Wildtype mice were exposed to air-blast-mediated mTBI, with blast exposure set at 50-psi on the left cranium and 0-psi designated as Sham. Sphingolipid profile was analyzed in brain tissue and plasma during the acute phases of 1, 3, and 7 days post-TBI via liquid-chromatography-mass spectrometry. Simultaneously, gene expression of sphingolipid metabolic markers within brain tissue was analyzed using quantitative reverse transcription-polymerase chain reaction. Significance (P-values) was determined by non-parametric t-test (Mann-Whitney test) and by Tukey's correction for multiple comparisons. RESULTS: In post-TBI brain tissue, there was a significant elevation of 1) acid sphingomyelinase (aSMase) at 1- and 3-days, 2) neutral sphingomyelinase (nSMase) at 7-days, 3) ceramide-1-phosphate levels at 1 day, and 4) monohexosylceramide (MHC) and sphingosine at 7-days. Among individual species, the study found an increase in C18:0 and a decrease in C24:1 ceramides (Cer) at 1 day; an increase in C20:0 MHC at 3 days; decrease in MHC C18:0 and increase in MHC C24:1, sphingomyelins (SM) C18:0, and C24:0 at 7 days. Moreover, many sphingolipid metabolic genes were elevated at 1 day, followed by a reduction at 3 days and an absence at 7-days post-TBI. In post-TBI plasma, there was 1) a significant reduction in Cer and MHC C22:0, and an increase in MHC C16:0 at 1 day; 2) a very significant increase in long-chain Cer C24:1 accompanied by significant decreases in Cer C24:0 and C22:0 in MHC and SM at 3 days; and 3) a significant increase of C22:0 in all classes of SPL (Cer, MHC and SM) as well as a decrease in Cer C24:1, MHC C24:1 and MHC C24:0 at 7 days. CONCLUSIONS: Alterations in sphingolipid metabolite composition, particularly sphingomyelinases and short-chain ceramides, may contribute to the induction and regulation of neuroinflammatory events in the early stages of TBI, suggesting potential targets for novel diagnostic, prognostic, and therapeutic strategies in the future.

Sphingosine d18:1 promotes nonalcoholic steatohepatitis by inhibiting macrophage HIF-2α.

Non-alcoholic steatohepatitis (NASH) is a severe type of the non-alcoholic fatty liver disease (NAFLD). NASH is a growing global health concern due to its increasing morbidity, lack of well-defined biomarkers and lack of clinically effective treatments. Using metabolomic analysis, the most significantly changed active lipid sphingosine d18:1 [So(d18:1)] is selected from NASH patients. So(d18:1) inhibits macrophage HIF-2α as a direct inhibitor and promotes the inflammatory factors secretion. Male macrophage-specific HIF-2α knockout and overexpression mice verified the protective effect of HIF-2α on NASH progression. Importantly, the HIF-2α stabilizer FG-4592 alleviates liver inflammation and fibrosis in NASH, which indicated that macrophage HIF-2α is a potential drug target for NASH treatment. Overall, this study confirms that So(d18:1) promotes NASH and clarifies that So(d18:1) inhibits the transcriptional activity of HIF-2α in liver macrophages by suppressing the interaction of HIF-2α with ARNT, suggesting that macrophage HIF-2α may be a potential target for the treatment of NASH.

The Sphingosine and Phytosphingosine Ceramide Ratio in Lipid Models Forming the Short Periodicity Phase: An Experimental and Molecular Simulation Study.

The lipids located in the outermost layer of the skin, the stratum corneum (SC), play a crucial role in maintaining the skin barrier function. The primary components of the SC lipid matrix are ceramides (CERs), cholesterol (CHOL), and free fatty acids (FFAs). They form two crystalline lamellar phases: the long periodicity phase (LPP) and the short periodicity phase (SPP). In inflammatory skin conditions like atopic dermatitis and psoriasis, there are changes in the SC CER composition, such as an increased concentration of a sphingosine-based CER (CER NS) and a reduced concentration of a phytosphingosine-based CER (CER NP). In the present study, a lipid model was created exclusively forming the SPP, to examine whether alterations in the CER NS:CER NP molar ratio would affect the lipid organization. Experimental data were combined with molecular dynamics simulations of lipid models containing CER NS:CER NP at ratios of 1:2 (mimicking a healthy SC ratio) and 2:1 (observed in inflammatory skin diseases), mixed with CHOL and lignoceric acid as the FFA. The experimental findings show that the acyl chains of CER NS and CER NP and the FFA are in close proximity within the SPP unit cell, indicating that CER NS and CER NP adopt a linear conformation, similarly as observed for the LPP. Both the experiments and simulations indicate that the lamellar organization is the same for the two CER NS:CER NP ratios while the SPP NS:NP 1:2 model had a slightly denser hydrogen bonding network than the SPP NS:NP 2:1 model. The simulations show that this might be attributed to intermolecular hydrogen bonding with the additional hydroxide group on the headgroup of CER NP compared with CER NS.

Microglia-mediated pericytes migration and fibroblast transition via S1P/S1P3/YAP signaling pathway after spinal cord injury.

Platelet-derived growth factor receptor ß positive (PDGFRß+) pericytes detach from the microvascular wall and migrate into the injury center following spinal cord injury (SCI), which has been widely regarded as the main source of fibrotic scar, but the mechanism of migration and fibroblast transition remains elusive. Here we show the associated spatiotemporal distribution between microglia and pericytes at three and seven days post-injury (dpi). The increased expression of Sphingosine kinase-1 (SPHK1) in microglia significantly raised the concentration of Sphingosine-1-phosphate (S1P) in the spinal cord, which promotes migration and fibroblast transition of pericyte. In vitro experiments, we found the elevated Sphingosine 1-phosphate receptor 3 (S1P3), the S1P/S1PR3 axis inhibited the phosphorylation of YAP and promoted its nuclear translocation, which contributed to the formation of alpha-smooth muscle actin (α-SMA) and collagen type I (COL1) protein, This process can be blocked by an S1P3 specific inhibitor TY52156 in vitro. The S1P/S1P3/YAP pathway might be a potential target for treatment in SCI.

Endothelial cell sphingosine 1-phosphate receptor 1 restrains VE-cadherin cleavage and attenuates experimental inflammatory arthritis.

In rheumatoid arthritis, inflammatory mediators extravasate from blood into joints via gaps between endothelial cells (ECs), but the contribution of ECs is not known. Sphingosine 1-phosphate receptor 1 (S1PR1), widely expressed on ECs, maintains the vascular barrier. Here, we assessed the contribution of vascular integrity and EC S1PR1 signaling to joint damage in mice exposed to serum-induced arthritis (SIA). EC-specific deletion of S1PR1 or pharmacological blockade of S1PR1 promoted vascular leak and amplified SIA, whereas overexpression of EC S1PR1 or treatment with an S1PR1 agonist delayed SIA. Blockade of EC S1PR1 induced membrane metalloproteinase-dependent cleavage of vascular endothelial cadherin (VE-cadherin), a principal adhesion molecule that maintains EC junctional integrity. We identified a disintegrin and a metalloproteinase domain 10 (ADAM10) as the principal VE-cadherin "sheddase." Mice expressing a stabilized VE-cadherin construct had decreased extravascular VE-cadherin and vascular leakage in response to S1PR1 blockade, and they were protected from SIA. Importantly, patients with active rheumatoid arthritis had decreased circulating S1P and microvascular expression of S1PR1, suggesting a dysregulated S1P/S1PR1 axis favoring vascular permeability and vulnerability. We present a model in which EC S1PR1 signaling maintains homeostatic vascular barrier function by limiting VE-cadherin shedding mediated by ADAM10 and suggest this signaling axis as a therapeutic target in inflammatory arthritis.

Kaempferol mitigates sepsis-induced acute lung injury by modulating the SphK1/S1P/S1PR1/MLC2 signaling pathway to restore the integrity of the pulmonary endothelial cell barrier.

Sepsis-induced acute lung injury (SALI) is the common complication of sepsis, resulting in high incidence and mortality rates. The primary pathogenesis of SALI is the interplay between acute inflammation and endothelial barrier damage. Studies have shown that kaempferol (KPF) has anti-sepsis properties. Sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P) signaling pathway's significance in acute lung damage and S1P receptor 1 (S1PR1) agonists potential in myosin light chain 2 (MLC2) phosphorylation are documented. Whether KPF can regulate the SphK1/S1P/S1PR1/MLC2 signaling pathway to protect the lung endothelial barrier remains unclear. This study investigates the KPF's therapeutic effects and molecular mechanisms in repairing endothelial cell barrier damage in both LPS-induced sepsis mice and human umbilical vein endothelial cells (HUVECs). KPF significantly reduced lung tissue damage and showed anti-inflammatory effects by decreasing IL-6 and TNF-α synthesis in the sepsis mice model. Further, KPF administration can reduce the high permeability of the LPS-induced endothelial cell barrier and alleviate lung endothelial cell barrier injury. Mechanistic studies showed that KPF pretreatment can suppress MLC2 hyperphosphorylation and decrease SphK1, S1P, and S1PR1 levels. The SphK1/S1P/S1PR1/MLC2 signaling pathway controls the downstream proteins linked to endothelial barrier damage, and the Western blot (WB) showed that KPF raised the protein levels. These proteins include zonula occludens (ZO)-1, vascular endothelial (VE)-cadherin and Occludin. The present work revealed that in mice exhibiting sepsis triggered by LPS, KPF strengthened the endothelial barrier and reduced the inflammatory response. The SphK1/S1P/S1PR1/MLC2 pathway's modulation is the mechanism underlying this impact.

SphK1 deficiency ameliorates the development of atherosclerosis by inhibiting the S1P/S1PR3/Rhoa/ROCK pathway.

BACKGROUND AND AIMS: S1P is an important factor regulating the function of the vascular endothelial barrier. SphK1 is an important limiting enzyme for the synthesis of S1P. However, the role of the SphK1/S1P-mediated vascular endothelial barrier function in atherosclerosis has not been fully revealed. This study explored the roles and mechanisms of SphK1 on atherosclerosis in vivo and in vitro. METHODS: In vivo, ApoE-/- and SphK1-/-ApoE-/- mice were fed a high-fat diet to induce atherosclerosis. In vitro, ox-LDL induced HUVECs to establish a cell model. Aortic histological changes were measured by H&E staining, Oil Red O staining, EVG staining, Sirius scarlet staining, immunofluorescence, and Evans Blue Assay. Western blotting was performed to explore the specific mechanism. RESULTS: We validated that deficiency of SphK1 resulted in a marked amelioration of atherosclerosis, as indicated by the decreased lipid accumulation, inflammatory factors, oxidative stress, aortic plaque area, inflammatory factor infiltration, VCAM-1 expression, and vascular endothelial permeability. Moreover, deficiency of SphK1 downregulated the expression of aortic S1PR3, Rhoa, ROCK, and F-actin. The results of administration with the SphK1 inhibitor PF-543 and the S1PR3 inhibitor VPC23019 in vitro further confirmed the conclusion that deficiency of SphK1 reduced S1P level and S1PR3 protein expression, inhibited Rhoa/ROCK signaling pathway, regulated protein expression of F-actin, improved vascular endothelial dysfunction and permeability, and exerted anti-atherosclerotic effects. CONCLUSIONS: This study revealed that deficiency of SphK1 relieved vascular endothelial barrier function in atherosclerosis mice via SphK1/S1P/S1PR signaling pathway.

N-Acetylcysteine Alleviates Impaired Muscular Function Resulting from Sphingosine Phosphate Lyase Functional Deficiency-Induced Sphingoid Base and Ceramide Accumulation in Caenorhabditis elegans.

Sphingosine-1-phosphate lyase (SPL) resides at the endpoint of the sphingolipid metabolic pathway, catalyzing the irreversible breakdown of sphingosine-1-phosphate. Depletion of SPL precipitates compromised muscle morphology and function; nevertheless, the precise mechanistic underpinnings remain elusive. Here, we elucidate a model of SPL functional deficiency in Caenorhabditis elegans using spl-1 RNA interference. Within these SPL-deficient nematodes, we observed diminished motility and perturbed muscle fiber organization, correlated with the accumulation of sphingoid bases, their phosphorylated forms, and ceramides (collectively referred to as the "sphingolipid rheostat"). The disturbance in mitochondrial morphology was also notable, as SPL functional loss resulted in heightened levels of reactive oxygen species. Remarkably, the administration of the antioxidant N-acetylcysteine (NAC) ameliorates locomotor impairment and rectifies muscle fiber disarray, underscoring its therapeutic promise for ceramide-accumulation-related muscle disorders. Our findings emphasize the pivotal role of SPL in preserving muscle integrity and advocate for exploring antioxidant interventions, such as NAC supplementation, as prospective therapeutic strategies for addressing muscle function decline associated with sphingolipid/ceramide metabolism disruption.