Anaplastic thyroid carcinoma (ATC) is a highly aggressive malignancy with a poor prognosis, characterized by dedifferentiation and aberrant angiogenesis. Through integrated analysis of TCGA and GEO transcriptomic data and single-cell RNA sequencing, this study identified significant enrichment of angiogenesis-related genes (ARGs), particularly sphingosine kinase 1 (SPHK1), in malignant cell subpopulations of ATC. Functional investigations revealed that alkaline ceramidase 3 (ACER3) cooperates with SPHK1 within the sphingolipid metabolic pathway to promote ATC progression. The SPHK1-specific inhibitor PF543 suppresses the activity of this key protein, thereby exhibiting potential therapeutic effects. To address the poor aqueous solubility and limited targeting ability of PF543, we constructed biomimetic nanoparticles (CMOE@PLGA@PF543) coated with S1PR1-overexpressing cancer cell membranes (CMOE), enabling tumor-specific targeting through the sphingosine-1-phosphate (S1P) and sphingosine-1-phosphate receptor 1 (S1PR1) ligand-receptor interaction. In vitro, PF543 downregulated SPHK1 expression and induced apoptosis in ATC cells. In vivo, CMOE@PLGA@PF543 exhibited enhanced tumor-targeting accumulation, excellent biosafety, and potent inhibition of tumor growth by suppressing the ACER3/SPHK1/S1P axis. These findings reveal a novel molecular mechanism driving ATC progression and offer a targeted nanotherapeutic strategy with strong potential for clinical translation.

Protein kinase C isoforms are crucial in hypertension-associated vascular dysfunction. Our previous study suggested that upregulated PKCβ2 contributed to aorta hypercontraction in spontaneously hypertensive rats (SHRs). However, its role in resistance arteries remains unclear. Considering the implications of sphingosine 1-phosphate (S1P) and its receptors (S1PRs) in hypertensive vascular dysfunction, we investigated whether PKCβ2 is regulated by S1P in SHR mesenteric arteries and elucidated its underlying mechanisms. Functional studies were performed on endothelium-denuded mesenteric arteries isolated from SHRs and Wistar-Kyoto rats. Expression of PKCβ2, S1P2, and S1P3 and phosphorylation levels of PKCβ2 and key proteins in the calcium sensitization pathway were assessed by Western blotting using mesenteric arteries and mesenteric arterial smooth muscle cells. PKCβ2 expression was significantly elevated in SHRs. LY333531, a PKCβ inhibitor, attenuated contraction induced by norepinephrine and S1P in SHRs. S1P significantly increased PKCβ2 phosphorylation in SHRs, an effect suppressed by sphingosine kinase 1 inhibitor PF-543. Inhibition or silencing of PKCβ2 significantly suppressed S1P-induced calcium sensitization. The expression levels of S1P2 and S1P3 were markedly higher in SHRs, and the inhibitory effect of LY333531 on S1P-induced contraction was not altered by JTE-013 (an S1P2 antagonist) or TY-52156 (an S1P3 antagonist). Furthermore, inhibition or silencing of S1P2 or S1P3 suppressed the activation of PKCβ2 and downstream calcium sensitization pathway. These findings demonstrate that S1P activates PKCβ2 via S1P2 and S1P3, enhancing calcium sensitization pathway and promoting hypercontraction in SHR. Thus, the S1P/PKCβ2 pathway is a potential therapeutic target for hypertensive vascular dysfunction.

Lactate accumulation exacerbates the severity of sepsis-associated acute kidney injury (SA-AKI), although the mechanism remains unclear. Since pyroptosis contributes to renal tubular epithelial cell (RTEC) death during SA-AKI, this study explores whether lactate exacerbates pathogenesis by promoting RTEC pyroptosis. The clinical correlation between lactate and SA-AKI was examined using the Medical Information Mart for Intensive Care IV (MIMIC-IV) database and patient samples. Lactate's role in RTEC pyroptosis was evaluated in lipopolysaccharide (LPS)-exposed HK-2 cells and in cecal ligation and puncture (CLP)-induced mice. Cross-analyzing bioinformatics and RNA-seq data from LPS/lactate-exposed HK-2 cells revealed pyroptosis genes associated with SA-AKI. Molecular mechanisms were explored via Western blot, ELISA, mitochondrial function assays, chromatin immunoprecipitation (ChIP), and co-immunoprecipitation (co-IP). High-throughput drugs screening was conducted to identify candidates acting on the Sphingosine kinase 1(SPHK1)/Sirtuin 1(SIRT1) axis, which were validated in vitro and in vivo. Lactate aggravated SA-AKI by promoting RTEC pyroptosis. Bioinformatic and functional studies identified SPHK1 as the key mediator. Both SPHK1 knockdown and its inhibitor PF-543 alleviated lactate-augmented pyroptosis. Drug screening identified nicotinamide adenine dinucleotide (NAD+), which simultaneously suppressed SPHK1 expression and the RTEC injury marker kidney injury molecule-1 (KIM-1). Combining NAD+ and PF-543 synergistically attenuated SA-AKI. Sepsis-induced lactate accumulation promoted P300-mediated histone H3 lysine 18 lactylation (H3K18la) at the SPHK1 promoter, epigenetically enhancing its transcription. SPHK1 then phosphorylated and degraded SIRT1, inducing peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) hyperacetylation, thereby impairing SIRT1/PGC-1α signaling and triggering NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-driven pyroptosis. Reciprocally, SIRT1 acted as a delactylase delactylase to reduce H3K18la and inhibit SPHK1 transcription, forming a SPHK1-SIRT1 negative feedback loop. The study identifies an H3K18la-mediated SPHK1-SIRT1 axis as a key factor of RTEC pyroptosis in SA-AKI. The combined pharmacological strategy of NAD+ supplementation and SPHK1 inhibition represents a promising therapeutic strategy for SA-AKI.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer often accompanied by persistent abdominal pain and stress, significantly compromising patients' quality of life. Previous studies have identified mast cells as crucial contributors to pancreatic cancer-related pain. This study aimed to investigate the role of the mast cell receptor corticotropin-releasing factor receptor 1 (CRFR1) and delineate its underlying mechanisms in mediating pancreatic pain. In the current study, we demonstrated that painful patients with PDAC exhibited markedly increased mast cell infiltration in peritumoral tissues, but not in tumor tissues, along with elevated levels of CRF and mast cell-specific CRFR1 expression, compared to asymptomatic patients. Consistently, in orthotopic PDAC mice models, flow cytometry and immunofluorescence staining confirmed increased CRFR1 expression in mast cells. Both pharmacological inhibition of CRFR1 and mast cell-specific CRFR1 knockout suppressed mast cell degranulation and alleviated cancer pain in male and female mice. Mechanistically, RNA sequencing, western blotting, and enzyme-linked immunosorbent assay indicated that CRFR1 activated mitogen-activated protein kinase (MAPK) pathway signaling, upregulated sphingosine kinases type 1 (SPHK1) expression, and increased sphingosine-1-phosphate (S1P) levels. A rescue experiment revealed that MAPK inhibition blocked CRFR1-induced SPHK1 upregulation in vitro. Importantly, the SPHK1 inhibitor PF543 reduced abdominal hyperalgesia in mice, whereas this effect was abolished in mast cell deficient mice. Moreover, SPHK1 knockdown by siRNA abolished CRFR1-induced mast cell degranulation. These findings highlight the critical role of mast cell CRFR1 in mediating abdominal hyperalgesia and identify the MAPK/SPHK1/S1P axis as an essential pathway, suggesting that targeting mast cell CRFR1 may be a promising therapeutic strategy for managing pancreatic cancer pain.

Osteopontin induces airway smooth muscle cells proliferation and migration by modulating FAK/Src/YAP axis.

Acquired therapeutic resistance in glioblastoma multiforme (GBM) constitutes a major determinant of its refractory and tumor recurrence. Both tumor-intrinsic epigenetic regulation and tumor microenvironment (TME) remodeling are now understood to play pivotal roles in this resistance; however, the synergistic mechanisms and key molecular mediators underlying this interplay remain poorly defined. In this study, we demonstrated that temozolomide (TMZ) could activate the autophagy-dependent secretory pathway to promote extracellular secretion of Alpha-enolase (ENO1). Extracellular soluble ENO1 robustly enhanced GBM cell proliferation, migration, and invasion in vitro. Clinically, serum ENO1 levels were markedly elevated in GBM patients and strongly correlated with TMZ therapeutic response, suggesting its potential as a diagnostic biomarker for predicting TMZ efficacy. Mechanistically, secreted ENO1 could bind to the Toll-like receptor 4 (TLR4) receptor on GBM cells, enhancing the PI3K/Akt pathway to promote cell invasion and proliferation. Meanwhile, ENO1/TLR4 axis activated the downstream ERK/SPHK1 signaling cascade, inducing phosphorylation and membrane translocation of SPHK1 at Ser225, thereby promoting the biosynthesis of sphingosine-1-phosphate (S1P), a critical sphingolipid metabolite. Notably, extracellular ENO1 and its downstream metabolite S1P synergistically polarized tumor-associated macrophages (TAMs) toward an M2-like phenotype, fostering an immunosuppressive tumor microenvironment (TME) and conferring chemoresistance. Importantly, in vivo studies confirmed that combined therapy with the SPHK1 inhibitor PF-543, the TLR4 antagonist TAK-242, and TMZ synergistically suppressed tumor growth and significantly enhanced the efficacy of TMZ. Collectively, these findings reveal that ENO1 mediates intercellular crosstalk between GBM cells and M2-TAMs via autophagy-dependent secretion, thereby driving TMZ chemoresistance and functioning as an oncogene in GBM. Targeting the ENO1/TLR4 signaling axis reshapes the immune microenvironment and enhances the efficacy of TMZ, offering a promising therapeutic strategy and potential combinatorial targets for precision therapy in GBM.

BackgroundBone metastasis (BM) drives therapeutic resistance and mortality in nasopharyngeal carcinoma (NPC). Tumor metabolites are crucial for NPC metastasis; however, the mechanisms by which these metabolites synergistically alter the immune microenvironment to promote BM remain unclear.MethodsIn this study, limited immune infiltration was observed in the NPC BM tumor microenvironment. Multiomics analysis has identified sphingosine kinase 1 (SPHK1) as a pivotal mediator driving BM and immune evasion in NPC, orchestrating the production of 1-phosphorylated sphingosine (S1P), which is critical for NPC pathogenesis.ResultsThe aberrant buildup of lipid metabolites, along with immune microenvironment shifts, serves as a critical driver of NPC BM. Mechanistically, S1P enhanced osteoclast recruitment via S1PR3 binding and activated the Hippo pathway, worsening bone colonization and facilitating immune evasion by expanding the exhausted CD8+ T cell population.ConclusionsThe synergy between the SPHK1 inhibitor PF543 and anti-programmed cell death protein 1 therapy amplified treatment effectiveness beyond standalone approaches. Overall, the SPHK1/S1P pathway advances NPC growth and aids in suppressing immune defense. Regulation of lipid metabolism may be a therapeutic target against BM in NPC and may improve the effectiveness of immunotherapy.

Focal adhesion kinase 2 (FAK2) is a non-receptor tyrosine kinase that orchestrates key oncogenic processes, including cell adhesion, migration, proliferation, and survival, and is frequently overexpressed in multiple cancer types. Targeting its ATP-binding and active sites has emerged as a promising therapeutic approach. Here, we performed a systematic virtual screening of 11,699 phytoconstituents from the Indian medicinal plants to identify potent FAK2 inhibitors. Docking analysis shortlisted top candidates based on binding affinity, followed by pharmacokinetic profiling (ADMET) and biological activity prediction (PASS). Two compounds, Cucurbitacin S and Kammogenin, exhibited strong binding affinities (- 9.5 and - 9.3kcal/mol) and favorable ADMET properties, with predicted anticancer and anti-inflammatory activities. Detailed interaction studies revealed stable binding to critical residues, including Asp549 in the active site. Molecular dynamics simulation for 300ns confirmed the stability and compactness of FAK2-compound complexes, with minimal structural perturbation. Essential dynamics analyses indicated reduced conformational flexibility upon ligand binding, while MM-PBSA calculations demonstrated favorable binding free energies. A comparative analysis with the reference inhibitor PF-562271 indicated the therapeutic potential of both phytochemicals, pending experimental evaluation. These findings suggest that Cucurbitacin S and Kammogenin are promising lead scaffolds for the development of plant-derived FAK2 inhibitors; however, as this is a computational, hypothesis-generating study, the results warrant further experimental validation to confirm their therapeutic potential.

Background and objectiveVenous thrombus fibrosis contributes to post-thrombotic syndrome (PTS) and chronic thromboembolic pulmonary hypertension (CTEPH). M2 macrophages promote fibrosis via TGF-β1 secretion. This study investigates whether sphingosine kinase 1 (SPHK1) promotes thrombus fibrosis by regulating M2 macrophage polarization.MethodsHistological staining and immunofluorescence (IF) were performed on thrombus tissues from patients with acute thrombosis and CTEPH. Single-cell RNA sequencing (scRNA-seq) was used to characterize immune cell heterogeneity and to identify SPHK1 expression within macrophage subsets. In vivo, a rat model of thrombus was established via inferior vena cava (IVC) ligation, and the SPHK1 inhibitor PF543 was administered to evaluate its effects on fibrosis and macrophage polarization. In vitro, bone marrow-derived macrophages (BMDMs) were subjected to M2 polarization and co-cultured with fibroblasts to assess the TGF-β1-dependent fibroblast activation.ResultsHistological analysis revealed significantly increased ECM deposition and macrophage infiltration in CTEPH thrombi compared to acute thrombi. Masson staining demonstrated extensive collagen fiber accumulation in CTEPH samples. Immunofluorescence analysis of fibrotic thrombi from a rat inferior vena cava (IVC) ligation model showed strong co-expression of SPHK1 and CD68, indicating the presence of SPHK1-expressing macrophages in thrombus remodeling. scRNA-seq analysis further revealed high SPHK1 expression in M2 macrophage subsets, particularly in the MARCO-1 cluster, and its expression was closely correlated with TGF-β1 secretion. In vivo, PF543 treatment significantly reduced collagen deposition, TGF-β1 expression, and M2 macrophage polarization in thrombus tissue. In vitro, SPHK1 knockdown markedly suppressed the expression of TGF-β1, Arg1, CD36, and FASN in BMDMs, indicating an inhibition of pro-fibrotic macrophage function. Co-culture experiments further confirmed that M2 macrophages activated fibroblasts via a TGF-β1-dependent mechanism.ConclusionThis study demonstrates that SPHK1 promotes M2 macrophage polarization and drives TGF-β1-dependent thrombus fibrosis, underscoring its critical role in the progression of CTEPH. Pharmacological inhibition of SPHK1 by PF543 effectively attenuates fibrotic remodeling and suppresses M2 macrophage polarization, suggesting that SPHK1 may serve as a promising therapeutic target for the treatment of chronic thrombus-associated fibrosis.

Gastric cancer (GC) is one of the most prevalent and heterogeneous malignancies worldwide, with treatment outcomes often hindered by delayed diagnosis and limited therapeutic options. Consequently, identifying novel targets for GC diagnosis and treatment is of significant clinical relevance. This study investigated the role of CPZ in GC progression and explored its molecular mechanisms and potential as a prognostic marker and therapeutic target. Bioinformatics analysis and clinical pathological evaluations were conducted to assess CPZ expression in GC and its correlation with clinical parameters. In vivo and in vitro experiments, including CCK8 proliferation assays, flow cytometry, and xenograft mouse models, were employed to confirm the impact of CPZ on GC malignancy. Potential molecular mechanisms through which CPZ regulates GC cell biological functions were identified and validated using immunoprecipitation mass spectrometry (IP-MS), co-immunoprecipitation (Co-IP), protein truncation assays, GST-pull down assays, and Western blotting. The CPZ inhibitor PF-573228 was identified through screening a small-molecule compound library. CPZ expression was significantly upregulated in GC (tumor tissues vs. normal tissues = 54.3% vs. 10.1%), correlating with unfavorable clinical pathological parameters and poor prognosis. CPZ directly interacts with DVL2 via its C-terminal domain (187-581aa). Its role in promoting tumorigenesis (both in vivo and in vitro) by fostering GC proliferation and regulation of cell cycle distribution is partially dependent on CPZ-DVL2 interaction, which activates the Wnt/β-catenin signaling pathway. PF-573228 specifically inhibits CPZ-mediated GC cell proliferation and tumor growth in mouse models (approximately fivefold compared to the control group). This study highlights the potential of CPZ as a novel biomarker and prognostic predictor for GC, while also elucidating its role in GC progression. The identification of PF-573228 as a specific inhibitor of CPZ provides a basis for the development of new therapeutic strategies for GC diagnosis and intervention.

A new insight into the mechanism of DEHP-induced cardiotoxicity: Triggering pyroptosis of cardiomyocytes by disrupting sphingolipid metabolism.

Alisol A 24-acetate protects against NASH-associated fibrosis via suppression of Kupffer cell-derived SPHK1/S1P axis.

Focal adhesion kinase acts on the PI3K/Akt/mTOR pathway to modulate wear particle-induced autophagy in macrophages.

Neuropathy is a common chronic complication of type 1 diabetes mellitus (T1D), an autoimmune disease in which neuroinflammation is considered a key pathological mechanism. P21-activated kinase 4 (PAK4) is a significant factor in this neuroinflammatory process. Higenamine hydrochloride (HGN) possesses anti-inflammatory properties. This study investigated the effects of HGN on T1D and its potential regulation of neuroinflammation via PAK4. We established a T1D mouse model using an intraperitoneal injection of streptozotocin (STZ), followed by HGN administration. The PAK4 inhibitor PF3758309 was given 2weeks before the experiment ended. Pathological alterations in brain tissues were examined through Nissl and H&E staining. Cognitive function and behavior were assessed using the Morris water maze, elevated plus maze, and open field tests. Molecular docking, cellular thermal shift analysis (CETSA), drug affinity responsive target stability (DARTS) assays, immunofluorescence, and immunoblotting were employed to investigate the binding of HGN to PAK4, its nuclear translocation, and microglial polarization. HGN mitigated brain damage, memory loss, anxiety, and behavioral dysfunction. It also reduced the levels of proinflammatory cytokines (TNF-α and IL-1β) and markers (CD16b, CD11b, and iNOS). Mechanistically, HGN increased PAK4 stability and inhibited M1 polarization via the CRTC2-CREB pathway. PF3758309 diminished the protective effects of HGN and elevated the serum levels of brain damage indicators (S100β and NSE) and inflammatory factors. HGN effectively reduces neuroinflammation and neurological dysfunction in T1D by targeting PAK4, suggesting its potential as a therapeutic agent for diabetes-related complications.

BackgroundFocal adhesion kinase (FAK), a cytoplasmic tyrosine kinase, plays a crucial role in focal adhesion turnover by interfacing between the extracellular space, transmembrane integrins, and actin filaments. Its significance for the progression of several malignancies, including bladder cancer, has been well-documented. However, its precise role and the implications of its inhibition in bladder cancer tissues and urothelial in vitro models has not been fully explored. This study examined FAK expression and function in human bladder cancer biopsies and in vitro bladder cancer models.Materials and methodsEx vivo analyses were performed using reverse transcription-quantitative PCR (qRT-PCR), western blotting, and immunohistochemistry to compare FAK expression between bladder cancer tissues and adjacent normal tissues. In vitro, FAK expression was assessed in low-grade (LG) human non-invasive papilloma urothelial cell line RT4 for NMIBC (Ta), high-grade (HG) human muscle-invasive cancer urothelial cell line T24 for MIBC (T2) and normal porcine urothelial (NPU) cells using qRT-PCR and western blotting, as well as flow cytometry for the quantification of FAK-positive RT4 and T24 cells. The role of FAK in cancer cell survival was explored in vitro using microRNA (miRNA) to silence FAK expression. Additionally, we used FAK inhibitors PND-1186, PF-573228 and defactinib to investigate the effects of FAK inhibition on normal compared to cancerous bladder urothelial cells.ResultsEx vivo analyses demonstrated significantly higher FAK expression in bladder cancer tissues compared to adjacent normal tissues. Similarly, in vitro analyses showed significantly higher FAK expression in RT4 and T24 cells than NPU cells. Silencing FAK using anti-FAK plasmids led to increased caspase-3-mediated apoptosis of RT4 and T24 cells and growth reduction of stably transfected T24 cells. Importantly, based on cell viability assays, treatment with 100 μM defactinib for 2 hours per day on 3 consecutive days was identified as a clinically relevant regimen. Under this treatment, the viability of differentiated NPU cells remained high at 108.4 ± 17.1%, while the viability of 2-day RT4 and 2-day T24 cells was drastically reduced to 4.1 ± 2.7% and 7.6 ± 2.9%, respectively.ConclusionsTo our knowledge, this is the first report demonstrating the role of FAK and its inhibition across both normal and cancerous bladder urothelial models. This study highlights the critical role of FAK in the progression of human bladder cancer and establishes a foundation for exploring FAK inhibition as a potential therapeutic approach in bladder cancer treatment.

Hepatic stellate cells (HSCs) transdifferentiate into myofibroblasts during liver fibrosis and exhibit increased glycolysis. Phosphorylated focal adhesion kinase (FAK) (pY397-FAK) promotes monocarboxylate transporter 1 (MCT-1) expression in HSCs to increase aerobic glycolysis and cause liver fibrosis. A combined multiomics analysis of C57BL/6 mice with tetrachloromethane (CCl4)-induced liver fibrosis was performed to identify the downstream FAK signaling pathway. The effect of the FAK inhibitor PF562271 on CCl4-induced liver fibrosis was explored by immunofluorescence of liver tissues. The migration, proliferation and aerobic glycolysis of LX-2 cells after stimulation and activation by transforming growth factor beta-1 (TGF-β1) or suppression by PF562271 was assessed in vitro. Multiomics analysis of a successfully generated CCl4-induced liver fibrosis mouse model was performed. FAK and cyclin D1 were significantly enriched in mice with CCl4-induced liver fibrosis. In vivo, the MCT-1 and alpha smooth muscle actin (α-SMA) levels were increased in mice with CCl4-induced liver fibrosis, and MCT-1 and α-SMA expression decreased after PF562271 treatment. In vitro, PF562271 alleviated TGF-β1-induced LX-2 activation. LX-2 cells showed diminished migration, proliferation, and aerobic glycolysis after PF562271 intervention. FAK promotes aerobic glycolysis in LX-2 cells through the cyclin D1/c-Myc/MCT-1 pathway, thereby increasing liver fibrosis.

Medial vascular calcification is common in chronic kidney disease patients and linked to hyperphosphatemia. Upon phosphate exposure, intricate signaling events orchestrate pro-calcific effects in the vasculature mediated by vascular smooth muscle cells (VSMCs). Sphingosine kinase 1 (SPHK1) produces sphingosine-1-phosphate (S1P) and is associated with complex effects in the vascular system. The present study investigated a possible involvement of SPHK1 in VSMC calcification. Experiments were performed in primary human aortic VSMCs under pro-calcific conditions, with pharmacological inhibition or knockdown of SPHK1 or SPNS2 (a lysolipid transporter involved in cellular S1P export), as well as in Sphk1-deficient and wild-type mice treated with cholecalciferol. In VSMCs, SPHK1 expression was up-regulated by pro-calcific conditions. Calcification medium up-regulated osteogenic marker mRNA expression and activity as well as calcification of VSMCs, effects significantly augmented by co-treatment with the SPHK1 inhibitor SK1-IN-1. SK1-IN-1 alone was sufficient to up-regulate osteogenic signaling in VSMCs during control conditions. Similarly, the SPHK1 inhibitor PF-543 and SPHK1 knockdown up-regulated osteogenic signaling in VSMCs and aggravated VSMC calcification. In contrast, co-treatment with the SPNS2 inhibitor SLF1081851 suppressed osteogenic signaling and calcification of VSMCs, effects abolished by silencing of SPHK1. In addition, Sphk1 deficiency aggravated vascular calcification and aortic osteogenic marker expression in mice after cholecalciferol overload. In conclusion, SPHK1 inhibition, knockdown, or deficiency aggravates vascular pro-calcific signaling and calcification. The reduced calcification after inhibition of S1P export suggests a possible involvement of intracellular S1P, but further studies are required to elucidate the complex roles of SPHKs and S1P signaling in calcifying VSMCs.

Myocardial ischemia-reperfusion (I/R) injury refers to cell damage that occurs as a consequence of the restoration of blood circulation following reperfusion therapy for cardiovascular diseases, and it is a primary cause of myocardial infarction. The search for nove therapeutic targets in the context of I/R injury is currently a highly active area of research. p70 ribosomal S6 kinase (S6K1) plays an important role in I/R induced necrosis, although the specific mechanisms remain unclear. This study aims to explore the effects of inhibiting S6K1 on myocardial I/R injury and its potential mechanisms. A rat myocardial I/R model was created and treated with the S6K1-specific inhibitor PF-4708671. Hematoxylin-eosin (H&E) staining was applied to evaluate the pathological changes in cardiac tissues. 2,3,5-triphenyltetrazolium chloride (TTC) staining was used to measure the area of myocardial infarction (MI). Left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), the maximum rate of increase in left ventricular pressure (+dp/dtmax), and the maximum rate of the decrease in left ventricular pressure (-dp/dtmax) were measured using ultrasonic echocardiography. The expression levels of cardiac troponin-1 (cTn-1), lactate dehydrogenase (LDH), creatine kinase MB (CK-MB), and aspartate aminotransferase (AST) were determined by enzyme-linked immunosorbent assay (ELISA). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and propidium iodide (PI) staining were used to examine the apoptosis and necrosis of myocardial tissues. The expressions of apoptotic-related proteins, and key molecules of necrosis were detected by western blot. The relationship between S6K1 and receptor-interacting protein kinase 3 (RIP3) was analyzed by immunoprecipitation. Inhibition of S6K1 reduces I/R-induced myocardial tissue damage, improves myocardial function, and inhibits myocardial tissue necrosis (p < 0.05). In addition, RIP3 is a direct target of S6K1, and activation of RIP3 blocked the protective effect of the S6K1 inhibitor PF-4708671 against myocardial I/R injury (p < 0.05). Inhibition of S6K1 protects against myocardial I/R injury by down-regulating RIP3, suggesting that targeting S6K1 may offer a novel approach for intervention in myocardial I/R injury.

Intracerebral hemorrhage (ICH) is a severe stroke subtype with high mortality and limited therapeutic options. The blood-brain barrier (BBB) breakdown post-ICH exacerbates secondary brain injury, highlighting the need for targeted therapies to preserve the BBB integrity. We aim to investigate the role of the Sphk1/S1P pathway in BBB breakdown following ICH and to evaluate the therapeutic potential of Sphk1 inhibition in mitigating this breakdown. Using a combination of human patient samples, mouse models of ICH, and in vitro cellular assays, we assessed the expression levels of Sphk1/S1P after ICH and changes of the BBB after ICH. The Sphk1 inhibitor PF543 and siRNAs were utilized to explore the pathway’s impact on BBB integrity and the underlying mechanisms. The results indicate significant upregulation of Sphk1/S1P in the peri-hematomal brain tissue after ICH, which correlates with increased BBB leakage. Pharmacological inhibition of Sphk1 with PF543 attenuates BBB leakage, reduces hematoma volume, and improves neurological outcomes in mice. At the molecular and ultrastructural level, Sphk1 inhibition protects the BBB integrity by preserving tight junction proteins and suppressing endothelial transcytosis. Furthermore, mechanistic studies reveal that Sphk1 promotes Nlrp3-mediated pyroptosis of brain endothelial cells through the ERK1/2 signaling pathway. Taken together, the Sphk1/S1P pathway plays a critical role in ICH-induced BBB breakdown, and its inhibition represents a promising therapeutic strategy for ICH management.

M64HCl, which has drug-like properties, is a water-soluble Focal Adhesion Kinase (FAK) activator that promotes murine mucosal healing after ischemic or NSAID-induced injury. Since M64HCl has a short plasma half-life in vivo (less than two hours), it has been administered as a continuous infusion with osmotic minipumps in previous animal studies. However, the effects of more transient exposure to M64HCl on monolayer wound closure remained unclear. Herein, we compared the effects of shorter M64HCl treatment in vitro to continuous treatment for 24 hours on monolayer wound closure. We then investigated how long FAK activation and downstream ERK1/2 activation persist after two hours of M64HCl treatment in Caco-2 cells. M64HCl concentrations immediately after washing measured by mass spectrometry confirmed that M64HCl had been completely removed from the medium while intracellular concentrations had been reduced by 95%. Three-hour and four-hour M64HCl (100 nM) treatment promoted epithelial sheet migration over 24 hours similar to continuous 24-hour exposure. 100nM M64HCl did not increase cell number. Exposing cells twice with 2-hr exposures of M64HCl during a 24-hour period had a similar effect. Both FAK inhibitor PF-573228 (10 μM) and ERK kinase (MEK) inhibitor PD98059 (20 μM) reduced basal wound closure in the absence of M64HCl, and each completely prevented any stimulation of wound closure by M64HCl. Rho kinase inhibitor Y-27632 (20 μM) stimulated Caco-2 monolayer wound closure but no further increase was seen with M64HCl in the presence of Y-27632. M64HCl (100 nM) treatment for 3 hours stimulated Rho kinase activity. M64HCl decreased F-actin in Caco-2 cells. Furthermore, a two-hour treatment with M64HCl (100 nM) stimulated sustained FAK activation and ERK1/2 activation for up to 16 and hours 24 hours, respectively. These results suggest that transient M64HCl treatment promotes prolonged intestinal epithelial monolayer wound closure by stimulating sustained activation of the FAK/ERK1/2 pathway. Such molecules may be useful to promote gastrointestinal mucosal repair even with a relatively short half-life.