Effects Of Inhibitors Research Articles

Regulatory role and molecular mechanism of MST1 in MPP+-induced Parkinson's disease cell model.

Parkinson's disease (PD) is a multi-factorial degenerative disease in the elder. Given the involvement of mammalian sterile 20-like kinase 1 (MST1) in PD, this paper was to illustrate the mechanism of MST1 in 1-methyl-4-phenylpyridinium ion (MPP+)-induced PD cell model. Cells were treated with different concentrations of MPP+ to establish a PD cell model. RT-qPCR and Western blot revealed that MST1 expression and iron ion concentration increased but cellular activity decreased with MPP+ concentration. Inhibition of MST1 decreased ferroptosis, increased cellular viability, iron ion content, and levels of glutathione peroxidase 4, and decreased reactive oxygen species, lactate dehydrogenase release. Upregulation of ferroptosis levels using ferroptosis agonist Erastin reduced the protective effect of MST1 inhibition on PD cells. Mechanistically, dual-luciferase analysis identified that miR-23b-3p targeted MST1 and inhibited its expression. Overexpression of miR-23b-3p inhibited MST1 levels, thereby reducing cellular ferroptosis and attenuating MPP+-induced cell injury. Collectively, MST1 expression increased with increasing MPP+ concentration and miR-23b-3p targeted MST1 to reduce ferroptosis and MPP+-induced cell injury.

Comparative Analysis of Acetylated Flavonoids’ Chemopreventive Effects in Different Cancer Cell Lines

Flavonoids, a class of natural compounds with anticancer activity, exhibit varying biological activities and potencies based on their structural differences. Acylation, including acetylation of flavonoids, generally increases their structural diversity, which is closely related to the diversity of bioactivity within this group of compounds. However, it remains largely unknown how acetylation affects the bioactivity of many flavonoids. Based on our previous findings that O-acetylation enhances quercetin’s bioactivity against various cancer cells, we synthesized 12 acetylated flavonoids, including seven novel compounds, to investigate their anticancer activities in the MDA-MB-231, HCT-116, and HepG2 cell lines. Our results showed that acetylation notably enhanced the cell proliferation inhibitory effect of quercetin and kaempferol across all cancer cell lines tested. Interestingly, while the 5,7,4′-O-triacetate apigenin (3Ac-A) did not show an enhanced the effect of inhibition of cell proliferation through acetylation, it exhibited significantly strong anti-migration activity in MDA-MB-231 cells. In contrast, the 7,4′-O-diacetate apigenin (2Ac-Q), which lacks acetylation at the 5-position hydroxy group, showed enhanced cell proliferation inhibitory effect but had weaker anti-migration effects compared to 3Ac-A. These results indicated that acetylated flavonoids, especially quercetin, kaempferol, and apigenin derivatives, are promising for anticancer applications, with 3Ac-A potentially having unique anti-migration pathways independent of apoptosis induction. This study highlights the potential application of flavonoids in novel chemopreventive strategies for their anti-cancer activity.

Extrachromosomal circular DNA promotes prostate cancer progression through the FAM84B/CDKN1B/MYC/WWP1 axis

BackgroundExtrachromosomal circular DNA (eccDNA), a kind of circular DNA that originates from chromosomes, carries complete gene information, particularly the oncogenic genes. This study aimed to examine the contributions of FAM84B induced by eccDNA to prostate cancer (PCa) development and the biomolecules involved.MethodsThe presence of eccDNA in PCa cells and the FAM84B transcripts that eccDNA carries were verified by outward and inward PCR. The effect of inhibition of eccDNA synthesis on FAM84B expression in PCa cells was analyzed by knocking down Lig3. The impact of FAM84B on the growth and metastases of PCa cells was verified by Cell Counting Kit-8 (CCK8), EdU, transwell assays, and a xenograft mouse model. Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) and dual-luciferase reporter assays were carried out to examine the effect of FAM84B/MYC on WWP1 transcription, and a co-immunoprecipitation (Co-IP) assay was conducted to verify the modification of CDKN1B by WWP1. The function of this molecular axis in PCa was explored by rescue assays.ResultsThe inhibited eccDNA synthesis significantly downregulated FAM84B in PCa cells, thereby attenuating the growth and metastasis of PCa. FAM84B promoted the transcription of WWP1 by MYC by activating the expression of MYC coterminous with the 8q24.21 gene desert in a beta catenin-dependent approach. WWP1 transcription promoted by MYC facilitated the ubiquitination and degradation of CDKN1B protein and inversely attenuated the repressive effect of CDKN1B on MYC expression. Exogenous overexpression of CDKN1B blocked FAM84B-activated MYC/WWP1 expression, thereby inhibiting PCa progression.ConclusionsFAM84B promoted by eccDNA mediates degradation of CDKN1B via MYC/WWP1, thereby accelerating PCa progression.

Targeting CSF1R in myeloid-derived suppressor cells: insights into its immunomodulatory functions in colorectal cancer and therapeutic implications

ObjectiveThis study aimed to investigate the critical role of MDSCs in CRC immune suppression, focusing on the CSF1R and JAK/STAT3 signaling axis. Additionally, it assessed the therapeutic efficacy of LNCs@CSF1R siRNA and anti-PD-1 in combination.MethodsSingle-cell transcriptome sequencing data from CRC and adjacent normal tissues identified MDSC-related differentially expressed genes. RNA-seq analysis comprehensively profiled MDSC gene expression in murine CRC tumors. LNCs@CSF1R siRNA nanocarriers effectively targeted and inhibited CSF1R. Flow cytometry quantified changes in MDSC surface markers post-CSF1R inhibition. RNA-seq and pathway enrichment analyses revealed the impact of CSF1R on MDSC metabolism and signaling. The effect of CSF1R inhibition on the JAK/STAT3 signaling axis was validated using Colivelin and metabolic assessments. Glucose and fatty acid uptake were measured via fluorescence-based flow cytometry. The efficacy of LNCs@CSF1R siRNA and anti-PD-1, alone and in combination, was evaluated in a murine CRC model with extensive tumor section analyses.ResultsCSF1R played a significant role in MDSC-mediated immune suppression. LNCs@CSF1R siRNA nanocarriers effectively targeted MDSCs and inhibited CSF1R. CSF1R regulated MDSC fatty acid metabolism and immune suppression through the JAK/STAT3 signaling axis. Inhibition of CSF1R reduced STAT3 activation and target gene expression, which was rescued by Colivelin. Combined treatment with LNCs@CSF1R siRNA and anti-PD-1 significantly slowed tumor growth and reduced MDSC abundance within CRC tumors.ConclusionCSF1R via the JAK/STAT3 axis critically regulates MDSCs, particularly in fatty acid metabolism and immune suppression. Combined therapy with LNCs@CSF1R siRNA and anti-PD-1 enhances therapeutic efficacy in a murine CRC model, providing a strong foundation for future clinical applications.

Spermine oxidase regulates liver inflammation and fibrosis through β-catenin pathway

BackgroundSpermine oxidase (SMOX), an inducible enzyme involved in the catabolic pathway of polyamine, was found to be upregulated in hepatocellular carcinoma and might be an important oncogene of it in our previous studies. This study attempted to further investigate its relationship with liver inflammation and fibrosis both in vitro and in vivo. MethodsThe effect of SMOX inhibition on LPS-induced inflammatory response in mouse liver cell line AML12 was validated by using small interfering RNA or SMOX inhibitor MDL72527. Western blotting and immunofluorescence were utilized to verify whether LPS could induce β-catenin to transfer into the nucleus and whether it could be reversed by interfering with the expression of SMOX or using SMOX inhibitor. Then, the SMOX inhibitor MDL72527 and SMOX knockout mice were used to verify the hypothesis above in vivo. ResultsThe expression of SMOX could be induced by LPS in AML12 cells. The inhibition of SMOX could inhibit LPS-induced inflammatory response in AML12 cells. LPS could induce β-catenin transfer from cytoplasm to nucleus, while SMOX downregulation or inhibition could partially reverse this process. In vivo intervention with SMOX inhibitor MDL72527 or SMOX knockout mice could significantly improve the damage of liver function, reduce intrahepatic inflammation, inhibit the nuclear transfer of β-catenin in liver tissue, and alleviate carbon tetrachloride-induced liver fibrosis in mice. ConclusionSMOX can promote the inflammatory response and fibrosis of hepatocytes. It provides a new therapeutic strategy for hepatitis and liver fibrosis, inhibiting early liver cancer.

Metabolic profiling and enzyme inhibitory activity of the essential oil of citrus aurantium fruit peel

BackgroundBitter orange (Citrus aurantium) is a fruiting shrub native to tropical and subtropical countries around the world and cultivated in many regions due to its nutraceutical value. The current study investigated the metabolic profiling and enzyme inhibitory activities of volatile constituents derived from the C. aurantium peel cultivated in Egypt by three different extraction methods.MethodsThe volatile chemical constituents of the peel of C. aurantium were isolated using three methods; steam distillation (SD), hydrodistillation (HD), and microwave-assisted hydrodistillation (MAHD), and then were investigated by GC-MS. The antioxidant potential was evaluated by different assays such as DPPH, ABTS, FRAP, CUPRAC, and phosphomolybdenum and metal chelating potential. Moreover, the effect of enzyme inhibition of the three essential oils was tested using BChE, AChE, tyrosinase, glucosidase, as well as amylase assays.ResultsA total of six compounds were detected by GC/MS analysis. The major constituent obtained by all three extraction methods was limonene (98.86% by SD, 98.68% by HD, and 99.23% by MAHD). Differences in the composition of the compounds of the three oils were observed. The hydrodistillation technique has yielded the highest number of compounds, notably two oxygenated monoterpenes: linalool (0.12%) and α-terpineol acetate (0.1%).ConclusionIn our study differences in the extraction methods of C. aurantium peel oils resulted in differences in the oils’ chemical composition. Citrus essential oils and their components showed potential antioxidant, anticholinesterase, antimelanogenesis, and antidiabetic activities. The presence of linalool and α-terpineol acetate may explain the superior activity observed for the oil isolated by HD in both radical scavenging and AChE inhibition assays, as well as in the enzyme inhibition assays.

Metabolic profiling and enzyme inhibitory activity of the essential oil of citrus aurantium fruit peel

BackgroundBitter orange (Citrus aurantium) is a fruiting shrub native to tropical and subtropical countries around the world and cultivated in many regions due to its nutraceutical value. The current study investigated the metabolic profiling and enzyme inhibitory activities of volatile constituents derived from the C. aurantium peel cultivated in Egypt by three different extraction methods.MethodsThe volatile chemical constituents of the peel of C. aurantium were isolated using three methods; steam distillation (SD), hydrodistillation (HD), and microwave-assisted hydrodistillation (MAHD), and then were investigated by GC-MS. The antioxidant potential was evaluated by different assays such as DPPH, ABTS, FRAP, CUPRAC, and phosphomolybdenum and metal chelating potential. Moreover, the effect of enzyme inhibition of the three essential oils was tested using BChE, AChE, tyrosinase, glucosidase, as well as amylase assays.ResultsA total of six compounds were detected by GC/MS analysis. The major constituent obtained by all three extraction methods was limonene (98.86% by SD, 98.68% by HD, and 99.23% by MAHD). Differences in the composition of the compounds of the three oils were observed. The hydrodistillation technique has yielded the highest number of compounds, notably two oxygenated monoterpenes: linalool (0.12%) and α-terpineol acetate (0.1%).ConclusionIn our study differences in the extraction methods of C. aurantium peel oils resulted in differences in the oils’ chemical composition. Citrus essential oils and their components showed potential antioxidant, anticholinesterase, antimelanogenesis, and antidiabetic activities. The presence of linalool and α-terpineol acetate may explain the superior activity observed for the oil isolated by HD in both radical scavenging and AChE inhibition assays, as well as in the enzyme inhibition assays.

Impact of homologous recombination repair/BReast CAncer (BRCA) gene alterations on survival in a real-world setting of metastatic prostate cancer.

To investigate alterations of homologous recombination repair (HRR) and especially BReast CAncer 1/2 (BRCA1/2) gene on overall survival (OS). Moreover, to explore the effect of inhibition of poly(ADP-ribose)-polymerase (PARPi) as systemic therapy for metastatic castration-resistant prostate cancer (mCRPC). Of all HRR-screened patients with metastatic prostate cancer, baseline characteristics were sampled. Kaplan-Meier estimates and multivariable Cox regression models predicted the effect of HRR/BRCA1/2 alterations on OS. Of 196 eligible patients, 61 (31%) harboured any HRR and 40 (20%) BRCA1/2 alterations. Of HRR alterations, 40 (66%) vs six (10%) vs five (8.2%) vs four (6.6%) vs two (3.3%) vs four (6.6%) were BRCA1/2 vs Ataxia-telangiectasia mutated kinase (ATM) vs checkpoint kinase 2 (CHEK2) vs cyclin-dependent kinase 12 (CDK12) vs Fanconi anaemia complementation Group A (FANCA) vs positive for other mutations. Of these, 30% received a PARPi. OS differed significantly between HRR-positive vs -negative patients. Specifically in hormone-sensitive prostate cancer, the median OS was 63 (HRR positive) vs 57 (BRCA1/2 positive) vs 113 months (HRR negative) (P ≤ 0.01). In mCRPC, OS was 42 (HRR positive) vs 41 (BRCA1/2 positive) vs 70 months (HRR negative) (P ≤ 0.01). HRR and BRCA1/2 alterations were associated with worse OS after multivariable adjustment. Finally, patients with mCRPC with BRCA1/2 mutation treated without PARPi harboured worse OS than patients with BRCA1/2 mutation and PARPi therapy (median OS: 33 vs 48 months, P < 0.03). Incidence of HRR alteration in a clinical real-world setting is high when using blood- and tissue-based tests. Patients with HRR/BRCA alterations have worse outcomes resulting in significant OS differences between HRR/BRCA-positive patients with mCRPC with and without PARPi usage vs HRR/BRCA-negative patients.

Enhancement of the structure and biochemical function of cyclomaltodextrinase from the Anoxybacillus flavithermus ZNU-NGA with site-directed mutagenesis.

This study was conducted to examine the role of the central domain of cyclomaltodextrinase in terms of stability, substrate specificity, becoming dodecameric form, and enzyme activity. To this end, H403R/L309V double-point mutation and T280Q single-point mutation were performed at the central domain and (β/α)8-barrel. The results indicated that the activity of the H403R/L309V mutant at the optimal pH and temperature increased by about 25% and 40%, respectively. Plus, the irreversible thermal inactivation of the H403R/L309V mutant at 60°C and 160min was approximately twice of the enzyme without mutation. Both mutants underwent significant structural change relative to the wild enzyme and subsequently a significant catalytic activity. However, the catalytic efficiency (kcat/Km) of the H403R/L309V mutant increased in the presence of beta- and gamma-cyclomaltodextrin substrates compared to the wild enzyme and T280Q mutant. As a result, by applying the L309V mutant and given the smaller size of the valine, leucine spatial inhibition in the wild protein seems to decline, and also it facilitates the substrate access to active site amino acids. Moreover, as gamma substrate is larger, eliminating the effect of spatial inhibition on this substrate has a greater effect on improving the catalytic activity of this enzyme.

Death associated protein like 1 acts as a novel tumor suppressor in melanoma by increasing the stability of P21 protein.

Melanoma is a primary malignant tumor with high lethality, which occurs in the skin and eye tissues, while the molecular mechanisms of melanomagenesis remain largely unknown. Here, we show that death-associated protein-like 1 (DAPL1) expression is lower in melanoma tissues than in paracancerous tissues or nevus tissues, and Uveal melanoma patients with lower DAPL1 expression have a poorer survivalrate than those with higher expression of DAPL1. Overexpression of DAPL1 inhibits proliferation of cultured melanoma cells, whereas knockdown of DAPL1 increases cell proliferation. Tumor transplantation experiment results also demonstrate that DAPL1 inhibits tumorigenesis of melanoma cells both in subretinal and subcutaneous tissues of nude mice in vivo. Finally, DAPL1 inhibits proliferation of melanoma cells by increasing the protein level of P21 via decreasing the ubiquitin mediated degradation of P21 and promoting its stability. Conversely, knockdown of P21 neutralizes the effects of inhibition of DAPL1 on melanoma cell proliferation and enhances the severity of melanoma tumorigenesis. These results suggest that DAPL1 is a novel melanoma tumor suppressor gene and thus a potential therapeutic target for melanoma.

Effects of sodium-glucose transporter-2 inhibition on systemic hemodynamics, renal function, and intra-renal oxygenation in sepsis-associated acute kidney injury

BackgroundPeople with type 2 diabetes mellitus treated with sodium-glucose transporter-2 inhibitors (SGLT2i) have lower rates of acute kidney injury (AKI). Sepsis is responsible for the majority of AKI in critically ill patients. This study investigated whether SGLT2i is renoprotective in an ovine model of Gram-negative septic AKI.MethodsSixteen healthy merino ewes were surgically instrumented to enable measurement of mean arterial pressure, cardiac output, renal blood flow, renal cortical and medullary perfusion, and oxygenation. After a 5-day recovery period, sepsis was induced via slow and continuous intravenous infusion of live Escherichia coli. Twenty-three hours later, sheep were randomized to receive an intravenous bolus of 0.2 mg/kg empagliflozin (n = 8) or a fluid-matched vehicle (n = 8).ResultsEmpagliflozin treatment did not significantly reduce renal medullary hypoperfusion or hypoxia, improve kidney function, or induce histological changes. Renal cortical oxygenation during the intervention period was 47.6 ± 5.9 mmHg in the empagliflozin group compared with 40.6 ± 8.2 mmHg in the placebo group (P = 0.16). Renal medullary oxygenation was 28.0 ± 18.5 mmHg in the empagliflozin compared with 25.7 ± 16.3 mmHg (P = 0.82). Empagliflozin treatment did not result in significant between-group differences in renal blood flow, kidney function, or renal histopathological changes.ConclusionIn a large mammalian model of septic AKI, a single dose of empagliflozin did not improve renal microcirculatory perfusion, oxygenation, kidney function, or histopathology.

Complement Factor B Inhibition or Deletion Is Not Sufficient to Prevent Neurodegeneration in a Murine Model of Glaucoma.

Purpose: Activation of the classical complement pathway is thought to contribute to the development and progression of glaucoma. The role of alternative complement or amplification pathways in glaucoma is not well understood. We evaluated complement factor B (FB) expression in postmortem human ocular tissues with or without glaucoma and the effect of FB inhibition and deletion in a mouse ocular hypertensive model of glaucoma induced by photopolymerized hyaluronic acid glycidyl methacrylate (HAGM). Methods: Human CFB mRNA in human eyes was assessed by RNAscope and TaqMan. HAGM model was performed on C57BL6/J mice. The effect of FB in HAGM model was evaluated with an oral FB inhibitor and Cfb-/- mice. Complement mRNA and proteins in mouse eyes were assessed by TaqMan and western blot, respectively. Results: CFB mRNA in human glaucomatous macular neural retina and optic nerve head was upregulated. Cfb mRNA is also upregulated in the HAGM model. Oral FB inhibitor, ED-79-GX17, dosed daily at 200 mg/kg for 3 days after intraocular pressure (IOP) induction in wild-type mice showed complement inhibition in ocular tissues and significantly inhibited systemic complement levels. Daily dosing of ED-79-GX17 for 30 days or Cfb deletion was also unable to prevent retinal ganglion cell or axon loss 30 days after IOP induction in mice. Conclusion: The alternative complement component FB may not substantially contribute to RGC loss in the HAGM mouse glaucoma model despite upregulation of Cfb expression and activation of the alternative pathway. The relevance of these findings to human glaucoma remains to be determined.

Complement Factor B Inhibition or Deletion Is Not Sufficient to Prevent Neurodegeneration in a Murine Model of Glaucoma.

Purpose: Activation of the classical complement pathway is thought to contribute to the development and progression of glaucoma. The role of alternative complement or amplification pathways in glaucoma is not well understood. We evaluated complement factor B (FB) expression in postmortem human ocular tissues with or without glaucoma and the effect of FB inhibition and deletion in a mouse ocular hypertensive model of glaucoma induced by photopolymerized hyaluronic acid glycidyl methacrylate (HAGM). Methods: Human CFB mRNA in human eyes was assessed by RNAscope and TaqMan. HAGM model was performed on C57BL6/J mice. The effect of FB in HAGM model was evaluated with an oral FB inhibitor and Cfb-/- mice. Complement mRNA and proteins in mouse eyes were assessed by TaqMan and western blot, respectively. Results: CFB mRNA in human glaucomatous macular neural retina and optic nerve head was upregulated. Cfb mRNA is also upregulated in the HAGM model. Oral FB inhibitor, ED-79-GX17, dosed daily at 200 mg/kg for 3 days after intraocular pressure (IOP) induction in wild-type mice showed complement inhibition in ocular tissues and significantly inhibited systemic complement levels. Daily dosing of ED-79-GX17 for 30 days or Cfb deletion was also unable to prevent retinal ganglion cell or axon loss 30 days after IOP induction in mice. Conclusion: The alternative complement component FB may not substantially contribute to RGC loss in the HAGM mouse glaucoma model despite upregulation of Cfb expression and activation of the alternative pathway. The relevance of these findings to human glaucoma remains to be determined.

Effects of sodium-glucose transporter-2 inhibition on systemic hemodynamics, renal function, and intra-renal oxygenation in sepsis-associated acute kidney injury.

People with type 2 diabetes mellitus treated with sodium-glucose transporter-2 inhibitors (SGLT2i) have lower rates of acute kidney injury (AKI). Sepsis is responsible for the majority of AKI in critically ill patients. This study investigated whether SGLT2i is renoprotective in an ovine model of Gram-negative septic AKI. Sixteen healthy merino ewes were surgically instrumented to enable measurement of mean arterial pressure, cardiac output, renal blood flow, renal cortical and medullary perfusion, and oxygenation. After a 5-day recovery period, sepsis was induced via slow and continuous intravenous infusion of live Escherichia coli. Twenty-three hours later, sheep were randomized to receive an intravenous bolus of 0.2 mg/kg empagliflozin (n = 8) or a fluid-matched vehicle (n = 8). Empagliflozin treatment did not significantly reduce renal medullary hypoperfusion or hypoxia, improve kidney function, or induce histological changes. Renal cortical oxygenation during the intervention period was 47.6 ± 5.9mmHg in the empagliflozin group compared with 40.6 ± 8.2mmHg in the placebo group (P = 0.16). Renal medullary oxygenation was 28.0 ± 18.5mmHg in the empagliflozin compared with 25.7 ± 16.3mmHg (P = 0.82). Empagliflozin treatment did not result in significant between-group differences in renal blood flow, kidney function, or renal histopathological changes. In a large mammalian model of septic AKI, a single dose of empagliflozin did not improve renal microcirculatory perfusion, oxygenation, kidney function, or histopathology.

Inhibition of Mast Cell Degranulation by Novel Small Molecule MRGPRX2 Antagonists

BackgroundMas-related G-protein coupled receptor X2 (MRGPRX2) is a promiscuous receptor on mast cells that mediates IgE-independent degranulation and has been implicated in multiple mast cell-mediated disorders, including chronic urticaria, atopic dermatitis, and pain disorders. Although it is a promising therapeutic target, few potent, selective, small molecule antagonists have been identified, and functional effects of human MRGPRX2 inhibition have not been evaluated in vivo. ObjectiveWe identified and characterized novel, potent, and selective orally active small molecule MRGPRX2 antagonists for potential treatment of mast cell-mediated disease. MethodsAntagonists were identified using multiple functional assays in cell lines overexpressing human MRGPRX2, LAD2 mast cells, human peripheral stem cell-derived mast cells, and isolated skin mast cells. Skin mast cell degranulation was evaluated in Mrgprb2em(-/-) knockout (KO) and Mrgprb2em(MRGPRX2) transgenic human MRGPRX2 knock-in (KI) mice by assessment of agonist-induced skin vascular permeability. Ex vivo skin mast cell degranulation and associated histamine release was evaluated by microdialysis of human skin tissue samples. ResultsMRGPRX2 antagonists potently inhibited agonist-induced MRGPRX2 activation and mast cell degranulation in all mast cell types tested, in an IgE-independent manner. Orally administered MRGPRX2 antagonists also inhibited agonist-induced degranulation and resulting vascular permeability in MRGPRX2 KI mice. In addition, antagonist treatment dose dependently inhibited agonist-induced degranulation in ex vivo human skin. ConclusionMRGPRX2 small molecule antagonists potently inhibited agonist-induced mast cell degranulation in vitro and in vivo as well as ex vivo in human skin, supporting potential therapeutic utility as a novel treatment for multiple human diseases involving clinically relevant mast cell activation.

Pharmacologic inhibition of dipeptidyl peptidase 1 (cathepsin C) does not block in vitro granzyme-mediated target cell killing by CD8 T or NK cells

Recently developed small-molecule inhibitors of the lysosomal protease dipeptidyl peptidase 1 (DPP1), also known as cathepsin C (CatC), can suppress suppurative inflammation in vivo by blocking the processing of zymogenic (pro-) forms of neutrophil serine proteases (NSPs), including neutrophil elastase, proteinase 3, and cathepsin G. DPP1 also plays an important role in activating granzyme serine proteases that are expressed by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Therefore, it is critical to determine whether DPP1 inhibition can also cause off-target suppression of CTL/NK-cell-mediated killing of virus-infected or malignant cells. Herein, we demonstrate that the processing of human granzymes A and B, transitioning from zymogen to active proteases, is not solely dependent on DPP1. Thus, the killing of target cells by primary human CD8+ T cells, NK cells, and gene-engineered anti-CD19 CAR T cells was not blocked in vitro even after prior exposure to high concentrations of the reversible DPP1 inhibitor brensocatib. Consistent with this observation, the turnover of model granzyme A/B peptide substrates in the human CTL/NK cell lysates was not significantly reduced by brensocatib. In contrast, preincubation with brensocatib almost entirely abolished (&amp;gt;90%) both the cytotoxic activity of mouse CD8+ T cells and granzyme substrate turnover. Overall, our finding that the effects of DPP1 inhibition on human cytotoxic lymphocytes are attenuated in comparison to those of mice indicates that granzyme processing/activation pathways differ between mice and humans. Moreover, the in vitro data suggest that human subjects treated with reversible DPP1 inhibitors, such as brensocatib, are unlikely to experience any appreciable deficits in CTL/NK-cell-mediated immunities.

Effect of Early Inhibition of Toll-Like Receptor 4 on Hippocampal Plasticity in a Neonatal Rat Model of Hypoxic-Ischemic Brain Damage.

Hippocampal plasticity is closely related to physiological brain functions such as learning and memory. However, the effect of toll-like receptor 4 (TLR4) activation on hippocampal plasticity after neonatal hypoxic-ischaemic brain damage (HIBD) remains unclear. In our study, seven-day-old rat pups were randomly categorised into three groups: control, hypoxic-ischemia (HI), and HI + TAK-242 (TAK-242). The pups were ligated in the left common carotid artery and then subjected to hypoxia to establish the neonatal HIBD model.The expression of the TLR4 in the left hippocampus of the HI group was increased compared to the control group, while TAK-242 reduced the expression level at 3 days after HIBD. Additionally, TAK-242 reversed the increased Zea-Longa score, increased the left/right hippocampal weight ratio, and increased the number of Nissl-positive neurons in the hippocampal CA1 region compared to HI group at 3 days after HIBD. Pre-injection of TAK-242 alleviated the decrease in PSD95, Aggrecan and NR1, BDNF, CREB, and pCREB expression in the hippocampus at 24 h after HIBD. It also alleviated the decrease in PSD95, BDNF, and NR2A/NR1 expression in the hippocampus at 7 days after HIBD. Furthermore, Pre-injection of TAK-242 alleviated the decrease in NR2A/NR1 expression at 21 days after HIBD. Finally,TAK-242 increased the percentage of third-grade dendritic mushroom spines processes in the basal and apical segments of neurons in the hippocampal CA1 region at 21 days after HIBD.Therefore, we conclude that preinhibition of TLR4 prior to neonatal HIBD improved the plasticity of the hippocampus.

Repression of BRD4 mitigates NLRP3 inflammasome-mediated pyroptosis in Mycobacterium-infected macrophages by repressing endoplasmic reticulum stress

Tuberculosis (TB) is the leading cause of human death worldwide due to Mycobacterium tuberculosis (Mtb) infection. Multiple lines of evidences have illuminated the emerging role of NLRP3 inflammasome-mediated pyroptosis in the clearance of pathogenic infection. In the current study, we sought to investigate the functional role and feasible potential mechanism of BRD4 in Mtb-infected macrophages. We observed that BRD4 was distinctly ascended in THP-1 macrophages upon Mtb infection. Functionally, intervention of BRD4 or pretreated with JQ1 obviously restricted Mtb-triggered cell pyroptosis, as evidenced by declination of protein level of the specific pyroptosis markers including Cleaved Caspase 1, gasdermin D (GSDMD-N) and Cleaved-IL-1β. In the meanwhile, disruption of BRD4 or JQ1 application remarkably prohibited excessive inflammatory responses as characterized by reduce the production of the inflammatory factors such as IL-1β and IL-18. Concomitantly, disruption of BRD4 or administrated with JQ1 manifestly repressed Mtb-aroused Nod-like receptor family pyrindomain-containing 3 (NLRP3) inflammasome activation, as witnessed by attenuation of protein levels of NLRP3, Pro-Caspase1 and apoptosis-associated speck-like protein (ASC). The above findings clearly demonstrated that suppression of BRD4 exerted great influence on regulating Mtb-elicited inflammatory response by coordinating NLRP3 inflammasome-mediated pyroptosis. More importantly, perturbation of BRD4 or JQ1 employment notably restrained endoplasmic reticulum (ER) stress triggered by Mtb-infection, as reflected by noticeably lessened the levels of GRP78, CHOP and ATF6. In terms of mechanism, ER stress agonist tunicamycin profoundly abrogated the favorable effects of BRD4 inhibition on Mtb-triggered pyroptosis, inflammation reaction and inflammasome activation. Collectively, these preceding outcomes strongly illuminated that inhibition of BRD4 targeted ER stress to retard NLRP3 inflammasome activation and subsequent cell pyroptosis and prevention of inflammatory response in Mtb-infected macrophages, highlighting that blocking BRD4 might serve as a promising candidate for protection against Mtb-triggered inflammatory injury.

Molecular mechanism of HDAC6-mediated pyroptosis in neurological function recovery after cardiopulmonary resuscitation in rats

Brain injury after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) is the leading cause of neurological dysfunction and death. This study aimed to explore the mechanism of histone deacetylase 6 (HDAC6) in neurofunctional recovery following CA/CPR in rats. A rat model was established by CA/CPR treatment. Adenovirus-packaged sh-HDAC6 was injected into the tail vein. To evaluate the neurofunction of rats, survival time, neurofunctional scores, serum NSE/S100B, and brain water content were measured and Morris water maze test was performed. HDAC6, microRNA (miR)-138-5p, Nod-like receptor protein 3 (NLRP3), and pyroptotic factors levels were determined by real-time quantitative polymerase chain reaction or Western blot assay. HDAC6 and H3K9ac enrichment on miR-138-5p promoter were examined by chromatin immunoprecipitation. miR-138-5p-NLRP3 binding was analyzed by dual-luciferase reporter assay. NLRP3 inflammasome was activated with nigericin sodium salt. After CPR treatment, HDAC6 was highly expressed, while miR-138-5p was downregulated. HDAC6 downregulation improved neurofunction and reduced pyroptosis. HDAC6 enrichment on the miR-138-5p promoter deacetylated H3K9ac, inhibiting miR-138-5p, and promoting NLRP3-mediated pyroptosis. Downregulating miR-138-5p partially reversed the protective effect of HDAC6 inhibition after CPR. In Conclusion, HDAC6 enrichment on miR-138-5p promoter deacetylated H3K9ac, inhibiting miR-138-5p expression and promoting NLRP3-mediated pyroptosis, worsening neurological dysfunction in rats after CPR.

Targeting CCR3 with antagonist SB 328437 sensitizes 5‑fluorouracil‑resistant gastric cancer cells: Experimental evidence and computational insights.

Gastric cancer (GC) ranks fifth globally in cancer diagnoses and third for cancer-related deaths. Chemotherapy with 5-fluorouracil (5-FU), a primary treatment, faces challenges due to the development of chemoresistance. Tumor microenvironment factors, including C-C motif chemokine receptor 3 (CCR3), can contribute to chemoresistance. The present study evaluated the effect of CCR3 receptor inhibition using the antagonist SB 328437 and the molecular dynamics of this interaction on resistance to 5-FU in gastric cancer cells. The 5-FU-resistant AGS cell line (AGS R-5FU) demonstrated notable tolerance to higher concentrations of 5-FU, with a 2.6-fold increase compared with the parental AGS cell line. Furthermore, the mRNA expression levels of thymidylate synthase (TS), a molecular marker for 5-FU resistance, were significantly elevated in AGS R-5FU cells. CCR3 was shown to be expressed at significantly higher levels in these resistant cells. Combining SB 328437 with 5-FU resulted in a significant decrease in cell viability, particularly at higher concentrations of 5-FU. Furthermore, when SB 328437 was combined with 5-FU at a high concentration, the relative mRNA expression levels of CCR3 and TS decreased significantly. Computational analysis of CCR3 demonstrated dynamic conformational changes, especially in extracellular loop 2 region, which indicated potential alterations in ligand recognition. Docking simulations demonstrated that SB 328437 bound to the allosteric site of CCR3, inducing a conformational change in ECL2 and hindering ligand recognition. The present study provides comprehensive information on the molecular and structural aspects of 5-FU resistance and CCR3 modulation, highlighting the potential for therapeutic application of SB 328437 in GC treatment.