Western Blot Analysis Research Articles

Necrostatin-1 as a Potential Anticonvulsant: Insights from Zebrafish Larvae Model of PTZ-Induced Seizures.

Epilepsy is a common neurological disorder affecting around 70 million people worldwide. Despite significant research and advancements in pharmaceutical therapies, the exact mechanisms underlying epileptogenesis remain unclear. As a result, current antiepileptic drug treatments are ineffective for approximately 30% of patients, providing only symptomatic relief. The epileptic process is influenced by the signaling of tumor necrosis factor alpha (TNFα), which affects neuronal excitability. The TNFα/TNFR1 signaling pathway and the role of RIPK1 in initiating inflammatory cell death pathways are well studied in human disorders. Dysregulation of RIPK1 is linked to inflammation and neurodegenerative diseases. Necrostatin-1 (Nec-1) selectively inhibits RIPK1 in various pathological conditions. The current study aimed to investigate the anticonvulsant properties of Nec-1 (a selective RIPK1 inhibitor) in PTZ-induced seizures in zebrafish larvae. Before the onset of seizures, zebrafish were treated with Nec-1 (15µM) for 24h at 6days post-fertilization (dpf), followed by exposure to 15mM of PTZ for 30min. Behavioral assessments were conducted to observe changes in locomotor activity. Additionally, c-Fos expression was measuredas an indicator of neuronal activation and analyzed mRNA levels of the astrocyte activation marker (GFAP) along with various inflammatory cytokines. Western blot analysis was conducted to evaluate GABAA receptor expression. Pretreatment with Nec-1 restored normal behavior and reversed the expression of c-Fos in zebrafish larvae induced by PTZ. Additionally, Nec-1 reduced the elevated mRNA expression of inflammatory cytokines and significantly suppressed TNF/TNFR1 signaling, thereby inhibiting the enhanced internalization of GABAA receptors. Our findings indicate that Nec-1 reduced the severity of PTZ-induced seizures in zebrafish by regulating behavior changes, suppressing inflammatory mediators, and enhancing the expression of GABAA receptors, suggesting potential anticonvulsant properties.

Human coronary blood single-cell sequencing combined with exosome proteomic profiling reveals the molecular mechanism of recurrent in-stent restenosis (R-ISR)

Abstract Background Percutaneous coronary intervention (PCI) is recognized as the core treatment for symptomatic myocardial ischemia and acute coronary syndrome (ACS). However, the complex pathogenesis of recurrent in-stent restenosis (R-ISR) after stent implantation is still insufficiently understood. Purpose This study reports the first single-cell RNA sequencing and proteomic profiling to characterize pathogenesis of R-ISR in humans. Methods To reveal the pathophysiology and molecular mechanism of R-ISR, we performed single-cell RNA sequencing to identify RISR-induced changes in transcriptional landscape in coronary blood mononuclear cell composition. Besides, proteomic profiling of human coronary plasma-derived exosomes was used to further screen different proteins. Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blot analysis were performed to identify exosomes. In vitro cell biology experiments were used to validate the discoveries of bioinformatics analysis. Results Compared with normal subjects, the percentage of FCGR3B high monocytes, memory T cells and plasmacytoid dendritic cells (DCs) was increased in R-ISR. Furthermore, we observed a marked increase in the expression of JUN, FOS, CXCL8, S100A8 enriched in the IL-17 signaling pathway that is known to play a crucial role in activating inflammation, mediating immune response, and even maintaining inflammation to chronicity. According to in vitro experiments, the co-cultured system demonstrated that FCGR3B high monocytes isolated from RISR patients activated the expression of inflammatory factors in endothelial cells (ECs), such as IL-1β, IL-6 and TNF-α. Inflammatory activated endothelial cells could release a large amount of exosomes, proteomic profiling of exosomes isolated from ECs observed a significant upregulation of MYLK expression, which could promote proliferation and migration of vascular smooth muscle cells (VSMCs) by activating VEGFR-2 signaling pathway. Conclusions Our study reveals a novel subset of FCGR3B high monocytes in R-ISR patients that could activate ECs inflammation via IL-17 signaling pathway. Activated ECs release exosomes rich in MYLK, which promote proliferation and migration of VSMCs, ultimately leading to repeated restenosis.

SAR1A Induces Cell Growth and Epithelial–Mesenchymal Transition Through the PI3K/AKT/mTOR Pathway in Head and Neck Squamous Cell Carcinoma: An In Vitro and In Vivo Study

Objectives: Head and neck squamous cell carcinoma (HNSCC) ranks sixth globally, with a 50% five-year survival rate. SAR1A exhibits high expression levels in various tumor types, yet its specific role in HNSCC remains to be clarified. Methods: In vitro assays, such as CCK8, EdU, colony formation, wound-healing, transwell, and Western blotting analyses, as well as in vivo assays, such as tumor xenografts and lung metastasis models, were conducted to evaluate the impacts of SAR1A on HNSCC proliferation, migration, and invasion. Transcriptome sequencing and KEGG enrichment pathway analysis revealed evident alterations in the PI3K/AKT/mTOR(PAM) pathways. LY294002 (a PI3K/AKT inhibitor) was used to investigate the role of the PAM pathway in proliferation, migration, and invasion in HNSCC. Results: Univariate and multivariate Cox regression were conducted to screen SAR1A as a gene prognostic biomarker in HNSCC, and it was validated in the Cancer Genome Atlas (TCGA) database. Functional assays demonstrated that the depletion of SAR1A leads to suppressed proliferation, migration, and invasion of HNSCC cells. This is accompanied by a decrease in the expression of epithelial–mesenchymal transition (EMT)-related markers in HNSCC cell lines. In addition, the diminished capacities of proliferation, migration, and invasion observed in SAR1A knockdown cells were reversed upon the overexpression of SAR1A. Furthermore, RNA-seq and KEGG enrichment analysis demonstrated a significant alteration in the PAM pathway following SAR1A knockdown. LY294002 effectively mitigated the increased proliferation, migration, and invasion induced by SAR1A overexpression. Conclusions: SAR1A facilitates HNSCC proliferation and EMT via the PI3K/AKT/mTOR pathway.

Human epicardial adipose tissue-derived factors promote atrial endothelial dysfunction: role of pro-inflammatory cytokines and AT1R/NADPH oxidases/SGLT2 pro-oxidant pathway

Abstract Introduction Epicardial adipose tissue (EAT) has been suggested to contribute to left atrium dysfunction via paracrine mechanisms to induce endothelial dysfunction, pro-arrhythmogenic and pro-remodeling responses thereby favoring atrial fibrillation (AF) onset. Recently, sodium-glucose co-transporter2 inhibitors (SGLT2i) have been shown to reduce AF incidence, EAT volume, differentiation and inflammation, however, the underlying mechanisms are unknown. Purpose This study investigates the impact of human EAT-derived mediators on atrial endothelial cell function and, if so, to characterize the role of SGLT2 using the inhibitor empagliflozin (EMPA). Methods Epicardial and subcutaneous adipose tissues (SAT) were collected from 70 cardiac patients at a university hospital. Conditioned medium (CM, 24 h) from fats were applied to porcine atrial endothelial cells (AECs, first passage) for 24 h. Histology of tissues was assessed by haematoxylin and eosin staining, protein expression by Western blot analysis or immunofluorescence staining, mRNA levels by RT-qPCR, formation of reactive oxygen species (ROS) and nitric oxide (NO) by fluorescent probes, and pro-inflammatory cytokine levels by ELISA. Results Compared to SAT, EATs were more vascularized with increased infiltration of M1-like macrophages, expression of proinflammatory cytokines (IL-1ß, IL-6, TNF-α), markers of ECs VCAM-1 and eNOS, fibrosis TGF-β, NADPH oxidases NOX-1 and SGLT1/2. These effects were associated with a pro-oxidant response that was inhibited by neutralizing Abs directed against IL-1ß or IL-6, and by inhibition of either NOS, NADPH oxidases, ACE, AT1R or SGLT2. Levels of SGLT2 and ROS in EATs were higher in AF compared to non-AF patients and progressively increasing with age. CM of EATs showed higher concentrations of IL-1ß, IL-6, TNF-α and MCP-1 than that of SAT. Exposure of AECs to CM of EATs increased the level of oxidative stress that was positively correlated to the level of pro-inflammatory cytokines, and reduced basal and bradykinin-induced NO formation that was prevented by EMPA. It also resulted in upregulation of p53 and SGLT2 expression, and nuclear translocation of NF-kB by CM of the top quartile inflamed EATs but not by the lowest quartile of EATs or SATs, prevented by EMPA. Conclusion The findings indicate that compared to SATs, the EATs showed higher in situ expression and release of pro-inflammatory cytokines that contributed to oxidative stress involving the AT1R/NADPH oxidases/SGLT2 pro-oxidant pathway. CMs of inflamed EATs induced AECs dysfunction, oxidative stress and activation of NF-kB involving pro-inflammatory cytokines and SGLT2. Thus, SGLT2i appear as an interesting strategy to decrease EAT inflammation and improve endothelial function contributing to prevent cardiac remodeling and fibrotic responses.

Diacylglycerol kinase γ facilitates the proliferation and migration of neural stem cells in the developing neural tube.

In this study, we aim to investigate diacylglycerol kinase (DGK) γ expression in developing neural tubes (NTs) and its effects on neural stem cell (NSC) proliferation and migration. Whole-mount in situ hybridization (WMISH) and immunohistochemistry are performed to explore DGKγ localization in developing NTs in vivo. NSCs are treated with sh-DGKγ, R59949, or PMA in vitro. Cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay and neurosphere formation assay are utilized to evaluate NSC proliferation. Neurosphere migration assay and a transwell chamber assay are used to assess NSC migration. The diacylglycerol (DAG) content is detected via enzyme-linked immunosorbent assay (ELISA). The mRNA expression of DGKγ is detected via quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression levels of DGKγ, protein kinase C (PKC) and phosphorylated PKC (p-PKC) are detected via western blot analysis. The results show that DGKγ mRNA is expressed predominantly in developing NTs. The neuroepithelium in developing NTs is positive for NSC markers, including Nestin, glial fibrillary acidic protein (GFAP), and DGKγ. DGKγ is expressed in the cytoplasm and nucleus of the neuroepithelium and is coexpressed with p-PKCγ and p-PKCδ. The proliferation of NSCs, the number of EdU-positive NSCs, and the number of neurospheres are decreased by sh-DGKγ and R59949 but increased by PMA. There is a shorter migration distance of NSCs and fewer migrated NSCs in the sh-DGKγ, R59949 and PMA groups. DAG content and the p-PKCδ/PKCδ ratio are increased by sh-DGKγ, R59949 and PMA, whereas the p-PKCγ/PKCγ ratio is decreased by PMA. Taken together, our findings indicate that DGKγ facilitates NSC proliferation and migration, which is responsible for the participation of DGK in NT development. DGKγ facilitates NSC migration via the DAG/PKCδ pathway.

PI 3-kinase/AKT/FOXO1 signaling in smooth muscle cells protects against abdominal aortic aneurysm

Abstract Background Abdominal aortic aneurysms (AAA) are characterized by loss of vascular smooth muscle cells (SMCs). Growth factors induce proliferation and cell survival of SMCs. PI 3-kinase (PI3K) is an important downstream mediator in growth factor signaling. Objective This project is based on the hypothesis that PI3K-mediated SMC proliferation can compensate for the loss of SMCs in AAA and thus inhibit AAA progression. Therefore, we determined effects of reduced PI3K activity in SMCs on AAA progression, cell proliferation as well as vascular remodeling. Methods To investigate the role of PI3K, we analyzed mice and aortic SMCs harboring a smooth muscle-specific deficiency of the PI3K catalytic subunit p110α (SM-p110α-/-). AAA development in SM-p110α-/- mice and wild-type (WT) littermates was induced by perfusion of the infrarenal aortic segment with porcine pancreatic elastase (PPE). AAA diameter was determined by echocardiography. Structural changes in the aorta were identified by (immuno)histochemical staining. SMC proliferation was measured in vitro by BrdU incorporation or real-time cell analysis. p110α induced signal transduction pathways were characterized by Western blot and transcriptome analysis. Results PPE treatment led to a massive loss of SMCs in the compromised aortic segment after 3 days in both SM-p110α-/- and WT mice. The aortic diameter was enlarged after 28 days in all PPE-treated mice. However, the increase was significantly higher in SM-p110α-/- mice (70.2±10.8%, n=9) compared to WT animals (42.4±6.0%, n=10) (p<0.05). A similar effect was obtained in aged (untreated) mice: In 24-month-old SM-p110α-/- mice, abdominal aortic lumen diameter was increased by 42% compared to 3-month-old animals, but only by 15% in corresponding WT mice. PPE-treated SM-p110α-/- mice were characterized by reduced vascular remodeling with less PCNA-positive proliferating cells compared to WT animals. In addition, proliferation of aortic p110α-/- SMCs in serum-containing medium was impaired. Mechanistic analyses showed that PDGF- and insulin-induced activation of AKT as well as AKT-dependent phosphorylation and inactivation of the transcription factors FOXO-1, -3 and -4 were significantly reduced in p110α-/- SMCs compared to WT cells. Further analyses revealed that specific inhibition of FOXO1 by AS1842856 rescued PDGF or serum stimulated proliferation of p110α-/- SMCs. In addition, expression analysis of FOXOs in patients revealed significantly increased FOXO1 expression in AAA compared to aortic samples from healthy individuals. Conclusion Deficiency of the catalytic PI3K isoform p110α in SMCs promotes the development and progression of AAA. p110α/AKT-dependent inactivation of FOXO1 induces SMC proliferation, which can ameliorate aortic wall damage. As FOXO1 inhibitors and a recently developed p110α activator are available, the PI3K/AKT/FOXO1 signaling cascade may provide a therapeutic target to influence the stability of the aortic wall in AAA.

Cardiac secreted-HSP90alpha in cardiomyocyte hypertrophy by N-Cadherin mediated signaling under pressure overload

Abstract Background Left ventricular hypertrophy (LVH) in response to pressure overload is strongly associated with adverse cardiovascular outcomes. Heat shock protein 90 (HSP90) has been reported to be involved in cardiac remodeling under stress. Aims Here, we evaluate whether serum HSP90α (an isoform of HSP90) increases and associates with cardiac hypertrophy in patients with hypertension or severe aortic stenosis (AS), and explore the potential mechanisms in experimental pressure overload mouse model. Methods Serum HSP90α levels in patients with hypertension or aortic stenosis (AS) were determined by ELISA. Pressure overload mouse model was built by a transverse aortic constriction (TAC). Cardiac function was assessed by echocardiography. Cardiac hypertrophy was examined by histology, western blot and RNA analysis. Results Serum levels of HSP90α were higher in patients with hypertension or AS than in the control group, respectively, and the levels positively correlated with LVH. HSP90α levels also increased in heart tissues of patients with obstructive hypertrophic cardiomyopathy (HCM), and mice after TAC, in parallel with the hypertrophic markers. TAC induced the enhanced expression and secretion of HSP90α from cardiomyocytes and cardiac fibroblasts. Knockdown of HSP90α or blockade of extracellular HSP90α (eHSP90α) prevent the development of LVH under pressure overload in vivo and in vitro. By Nano-HPLC-MS/MS analysis, we identified eHSP90α interacted with N-cadherin in cardiomyocyte surface, which induced the activation of β-catenin and promoted β-catenin to bind TCF7 (a member of TCF/Lef family) in nucleus, enhanced the transcription of hypertrophic genes, contributing to cardiac hypertrophy and dysfunction under pressure overload. Conclusions Our data demonstrated that cardiac secreted-HSP90α promoted myocardial hypertrophy by N-Cadherin mediated β-catenin/TCF7 signaling under pressure overload. It indicates the therapeutic potential of targeting HSP90α-initiated signaling against cardiac hypertrophy and dysfunction under pressure overload.

Epigenetic modulation of vascular calcification: Looking for comprehending the role of sirt1 and histone acetylation in VSMC phenotypic transition

In light of the complex origins of ectopic vascular calcification and its significant health implications, this study offers a comprehensive exploration of the molecular dynamics governing vascular smooth muscle cells (VSMCs). Focusing on epigenetic modulation, we investigate the transition from a contractile to a calcifying phenotype in VSMCs, with an emphasis on understanding the role of SIRT1. For this purpose, a single batch of human aortic SMCs, used at a specified passage number to maintain consistency, was subjected to calcium and phosphate overload for up to 72 h. Our findings, validated through RT q-PCR, Western blot, immunofluorescence, and DNA methylation analyses, reveal a complex interplay between acetyltransferases and deacetylases during this phenotypic transition. We highlight HAT1A's critical role in histone acetylation regulation and the involvement of HDACs, as evidenced by subcellular localization studies. Moreover, we demonstrate the modulation of SIRT1 expression, a class III deacetylase, during VSMC calcification, underscoring the influence of DNA methylation in this process. Importantly, the study addresses previously unexplored aspects of the dynamic protein expression patterns observed, providing insight into the counterintuitive expressions of key proteins such as Runx2 and osterix. This research underscores the significant impact of epigenetic mechanisms, particularly the modulation of SIRT1, in the transition from a contractile to a calcifying phenotype in VSMCs, offering potential avenues for further exploration in the context of vascular calcification.

Long non-coding RNA PANDA promotes endothelial senescence and oxidative damage through the interaction with NRF2

Abstract Background Accumulation of reactive oxygen species and inflammation are major features of diabetic vasculopathy, yet the underlying mechanisms remain elusive. LncRNAs are emerging as important players in the pathogenesis of cardiovascular disease. PANDA, a newly identified lncRNA, is a key regulator of cellular senescence, apoptosis and oxidative stress. Purpose To investigate the role and molecular mechanism of PANDA in diabetic vascular disease. Methods RNAseq was performed to aorta specimen from diabetic and no diabetic patients as well to HAECs exposed to 5 mM and 25 mM concentrations (Normal -NG- and High -HG-, respectively). PANDA depletion in HG-treated HAECs was obtained by siRNA transfection while a scrambled siRNA wass used as a negative control. RNAseq and network perturbation analysis (NPA) of treated HAECs unveiled transcriptional changes upon PANDA depletion. Expression of PANDA was assessed by real time PCR. PANDA RNA-IP was performed to check its binding to relevant transcriptional factor (such as NRF2) as well Ch-IP was performed to check the NRF2 binding on oxidative gene promoters. Cellular localization of NRF2 was investigated by Immunofluorescence. Beta-galactosidase and superoxide staining was used to detect endothelial senescence and ROS formation; while, migration and tube formation were employed to evaluate angiogenic properties of HAECs. Results PANDA expression was significantly increased in diabetic human aorta as well in HG-treated HAECs (Figure1 A-B). Transcriptomic analysis revealed dysregulation of several genes upon PANDA silencing with the antioxidant gene heme oxygenase-1 (HMOX1) as the top-ranking transcript in HG cells (Figure1 C-D). Western blot analysis reveals the restored level of HMOX1 and TFRC1 upon PANDA depletion (Figure1 E-G). NPA analysis showed a strong involvement of PANDA in senescence, DNA damage, NRF2 signaling, hypoxic stress response and proliferation. In HG, NFR2 is downregulated while PANDA silencing restores its level (Figure1 I-J). We found that in HG condition PANDA binds the transcription factor NRF2 and blocks its nuclear translocation thus impeding its binding on HMOX1 and TFRC promoters (Figure1 K-M). Silencing of PANDA in HG-treated HAECs reduced genes and cellular features of senescence (Figure2 A-B), restored the expression of anti-apoptotic genes and decreases caspase 3 activity (Figure2 C-D), improved endothelial migration and tube formation (Figure2 E and G), and reduced ROS formation (Figure2 F). Conclusions In hyperglycemia PANDA upregulation drives endothelial senescence while impairing angiogenic properties. On the other hand, PANDA depletion in HG-treated HAECs rescues maladaptive transcriptional changes through the release of NRF2 that in turn translocate into the nucleus enhancing the expression of the antioxidant gene HMOX1. The results indicate PANDA as a putative novel molecular target in the setting of diabetic vascular disease (Figure2 H).

Salivary antigens rPagSP02 and rPagSP06 are a reliable composite biomarker for evaluating exposure to Phlebotomus argentipes in Sri Lanka

Phlebotomus argentipes is the established vector of leishmaniasis in the Indian sub-continent. Antibodies to sand fly salivary antigens are biomarkers for vector-host exposure in leishmaniasis-endemic regions. Ph. argentipes transmits Leishmania donovani in Sri Lanka, primarily causing cutaneous leishmaniasis (CL). Our study compared the performance of salivary gland homogenate (SGH) from a lab-reared local strain of Ph. argentipes females to a composite recombinant salivary biomarker (rPagSP02 + rPagSP06) in a CL-endemic population. Sera from 546 healthy individuals, 30 CL patients, and 15 non-endemic individuals were collected. Western blot analysis of Ph. argentipes SGH identified immunogenic bands between 15 kDa and 67 kDa, with bands of predicted molecular weight ∼of 15 kDa (SP02) and ∼28–30 kDa (SP06) as the major antibody targets. Indirect ELISAs using SGH or rPagSP02 + rPagSP06 antigens showed high sensitivity (96.7%) and specificity (100%), detecting comparable seropositivity in endemic populations. rPagSP02 + rPagSP06 exhibited enhanced discriminatory ability, supported by a strong positive correlation (r = 0.869) with SGH. Our findings indicate that the composite rPagSP02 + rPagSP06 salivary biomarker effectively identifies Ph. argentipes exposure in individuals living in Sri Lanka, showing promising potential for use in surveillance. These findings should be further validated to confirm the epidemiological applications in leishmaniasis-endemic regions.

Topical delivery of baricitinib-impregnated nanoemulgel: a promising platform for inhibition of JAK -STAT pathway for the effective management of atopic dermatitis.

Baricitinib, an inhibitor of Janus kinase 1/2 receptors majorly involved in the dysregulation of immune responses in atopic dermatitis, is currently approved for managing atopic dermatitis in Europe. The delivery of baricitinib through oral route is associated to several adverse effects due to off-target effects. Therefore, the current study is aimed at formulation of baricitinib loaded nanoemulgel for evaluation of topical delivery potential in the treatment of atopic dermatitis. The baricitinib-loaded nanoemulsions (0.05 and 0.1% w/w) revealed an average globule size of 162.86 ± 0.37 and 173.66 ± 4.88nm respectively with narrow PDI. The optimized batch of baricitinib nanoemulsion was converted to nanoemulgel by the addition of the mixture of gel bases SEPINEO™ DERM and SEPINEO™ P 600 along with propylene glycol, resulting in pseudoplastic shear thinning behaviour. The optimized nanoemulgels have shown prominent retention of baricitinib in the skin along with permeation. The skin distribution study of coumarin-6 loaded nanoemulgel demonstrated high fluorescence in the epidermal layer. The western blot analysis revealed significant inhibition of phosphorylated signal transducers and activators of transcriptions 1 (##p < 0.01) and 3 (#p < 0.05) by application of 0.05 and 0.1% baricitinib nanoemulgel. The baricitinib nanoemulgels have shown anti-inflammatory activity by significantly reducing expressions of various inflammatory markers. Histopathological analysis of skin tissues treated with baricitinib nanoemulgel has demonstrated a marked reduction in acanthosis, hyperkeratosis, and intact outer epidermis. These results supported the potential role of baricitinib-loaded nanoemulgel in reducing the inflammation and disease severity associated with atopic dermatitis.

CircPCNXL2 promotes preeclampsia progression by suppressing trophoblast cell proliferation and invasion via miR-487a-3p/interferon regulatory factor 2 axis.

Preeclampsia (PE) has culminated in maternal and perinatal sickness and death across the world, affecting approximately 4.6% of pregnancies. Circular RNAs (circRNAs) have been linked to the biology of numerous pathologies, including PE. Here, we investigated the functional role of circPCNXL2 in the progression of PE. We employed the GEO database to get the expression profile of circPCNXL2 in patients with PE. This was followed by the detection of the expression of circPCNXL2 and miR-326 by qRT-PCR. The role of circPCNXL2 on trophoblast cell proliferation, migration, and invasion was confirmed with cell viability assays, the transwell assay, and the colony formation assay. Further, we employed dual luciferase, FISH, RNA pull-down assay and Western blot analysis to determine the interaction between the expression of circPCNXL2, miR-487a-3p, and IRF2. Findings from this study revealed that proliferation and migration of trophoblast cells were significantly increased in the HTR-8/SVneo cells after silencing circPCNXL2. Additionally, knockdown of circPCNXL2 remarkably increased miR-487a-3p expression, while IRF2 expression was remarkably reduced (P < 0.05), indicating the presence of complementary binding sequence on miR-487a-3p with which they sequester circPCNXL2. Rescue experiments revealed that interaction occurs between circPCNXL2, miR-487a-3p, and the IRF2 protein, indicating that circPCNXL2 expression elicits suppression of migration and proliferation of trophoblast cells via the miR-487a-3p/IRF2 pathway. We demonstrated that circPCNXL2 upregulation promotes pre-eclampsia by inhibiting proliferation and migration of trophoblast cells via the miR-487a-3p/IRF2 pathway or axis.

Cuproptosis-related lncRNAs emerge as a novel signature for predicting prognosis in prostate carcinoma and functional experimental validation.

Prostate cancer (PCa) is one of the most common malignancies of the urinary system. Cuproptosis, a newly discovered form of cell death. The relationship between cuproptosis-related long non-coding RNAs (ClncRNAs) related to PCa and prognosis remains unclear. This study aimed to explore the clinical significance of novel ClncRNAs in the prognostic assessment of PCa. ClncRNAs and differentially expressed mRNAs linked to these ClncRNAs were identified using Pearson's correlation and differential expression analyses. A prognostic signature (risk score) comprising three ClncRNAs was established based on multivariable Cox regression analysis. The predictive performance of this ClncRNAs signature was validated using receiver operating characteristic curves and nomograms. Finally, further in vitro cell experiments were conducted for validation, including quantitative polymerase chain reaction (qPCR), western blot (WB), cell proliferation assays, cell migration assays, cell invasion assays, apoptosis, and cell cycle analysis. We constructed a prognostic signature of ClncRNAs for PCa comprising three key differentially expressed ClncRNAs(AC010896-1, AC016394-2, and SNHG9). Multivariable Cox regression analysis indicated that clinical staging and risk scores of the ClncRNAs signature were independent prognostic factors for PCa. Compared to other clinical features, the ClncRNAs signature exhibited higher diagnostic efficiency and performed well in predicting the 1-, 3-, and 5-year progression-free intervals (PFIs) for PCa. Notably, in terms of immune activity, PCa patients with high-risk scores exhibited higher tumor mutational burden (TMB) levels, while their Tumor Immune Dysfunction and Exclusion (TIDE) scores were lower than those of PCa patients with low-risk scores. Additionally, in vitro cellular functional experiments, we knocked down SNHG9 that is the most significantly differentially expressed ClncRNA among the three key ClncRNAs. SNHG9 knockdown resulted in a significant increase in G1 phase cells and a decrease in S and G2 phases, indicating inhibition of DNA synthesis and cell cycle progression. Colony formation assays showed reduced clonogenic ability, with fewer and smaller colonies. Western blot analysis revealed the upregulation of the key cuproptosis-related mRNAs FDX1 and DLST. These findings suggested that SNHG9 promotes PCa cell proliferation, migration, and invasion. Building on the three ClncRNAs, we identified a novel prognostic signature of PCa. The ClncRNA SNHG9 can promote PCa cell proliferation, migration, and invasion.

Anti-fibrotic effects of the sirt6-activator mdl-800 in cardiac stromal cells

Abstract Background Epigenetic changes are among the molecular substrates of cellular stress responses and tissue remodeling in heart failure progression. The sirtuin family of NAD+-dependent histone and non-histone protein deacetylases exert a protective anti-fibrotic role by negatively modulating the inflammatory response and by positive regulation of autophagy-related genes. Sirtuin6 (SIRT6) stands out as a key cardiac protector that can mitigate aging-induced cardiomyocyte senescence and cardiac hypertrophy. Cardiac stromal cells (CSCs) play a pivotal role in fibrosis and remodeling, regulating the composition and stiffness of the extracellular matrix (ECM). Autophagy enhancement has anti-fibrotic effects in CSCs, but the specific role of SIRT6 modulation in CSCs remains unexplored. Purpose To investigate the biological effects of MDL-800, a synthetic allosteric SIRT6 activator, on the stress response and fibrotic activation of CSCs. Methods CSCs were isolated from 4-week-old C57Bl6J mice by explant culture and stimulated with 10ng/ml of TGF-beta1 (TGFb1) up to 72h to induce fibrotic activation. CSCs were treated with MDL-800 up to 10µM and up to 72h, analyzed for cell viability, gene and protein expression levels. Results TGFb1 treatment led to a 0.64-fold reduction in SIRT6 gene expression after 24h. The MTS assay was used to assess a viability dose-response to MDL-800 up to 72h of treatment. The non-toxic concentrations of 2.5µM and 5µM were selected for further experiments (2.5µM vs CTR 0.97-fold and 5µM vs CTR 0.92-fold, normalized OD vs t0). Co-treatment of 5µM MDL-800 with TGFb1 for 72h resulted in a dose-dependent decrease in the expression of TGFb1-induced fibroblast activation markers, as confirmed by immunofluorescence staining for aSMA (TGFb1 vs CTR 2.21-fold, p&amp;lt;0.05; 5µM vs TGFb1 0.46-fold, p&amp;lt;0.05, N=4; mean fluorescence intensity). The anti-fibrotic effect mediated by MDL-800 was further validated by real-time PCR indicating a reduction in the mRNA expression of multiple fibrotic markers, including ACTA2 (TGFb1 vs CTR 1.67-fold, 5µM vs TGFB 0.29-fold; N=2), COL1a1 (TGFb1 vs CTR 1.32-fold, 5µM vs TGFb1 0.87-fold; N=2) COL3a1 (TGFb1 vs CTR 1.06-fold, 5µM vs TGFb1 0.43-fold; N=2) and POSTN (TGFb1 vs CTR 6.37-fold, 5µM vs TGFb1 0.23-fold; N=2). Treatment with 5µM MDL-800 demonstrated an increase in SIRT6 gene expression, suggesting a transcriptional modulation (TGFb1 vs CTR 0.95-fold, 5µM vs TGFb1 1.68-fold; N=2). Western Blot analyses showed an increase in protein levels of the autophagosome marker LC3-II following treatment with 5µM MDL-800 (TGFb1 vs CTR 0.95-fold; 5µM vs TGFb1 3.01-fold), suggesting that autophagy enhancement may be a potential mechanism for the observed anti-fibrotic effects. Conclusion MDL-800 treatment in CSCs is associated with a dose-dependent reduction of TGFb1-induced fibrotic markers and to increased levels of the autophagy marker, suggesting its potential use as an anti-fibrotic molecule for the heart.

Fabry cardiomyopathy: myocardial fibrosis, inflammation and reduced mannose6phosphate receptors cause low accessibility to enzyme therapy

Abstract Background Clinical impact of enzyme replacement therapy (ERT) on advanced Fabry disease cardiomyopathy (FDCM) appears limited. Purpose Clarify the pathologic mechanisms responsible of ERT inefficacy in the advanced FDCM. Methods Ten male patients with advanced FDCM (echocardiographic maximal wall thickness 19.3 ± 2.1 mm) underwent left ventricular endomyocardial biopsy before and 4 hours after beta-agalsidase infusion. Comparative studies between pre and post infusion samples included: histology and electron microscopy; assessment of myocardial alpha-galactosidase A activity; immunohistochemistry for alpha-galactosidase A and semiquantitative evaluation (from 0 to 3) of its cardiomyocyte content; Ultrastructural immunogold analysis with anti-alpha-galactosidase A ab. Western Blot (WB) quantification of mannose-6-phosphate receptors (M6Pr). Controls were surgical left ventricular biopsies from patients with mitral stenosis. Results Histologic and Ultrastructural evaluation showed no removal of storage material while myocardial fibrosis was 9.8 ± 6.8 vs 3.8 ± 2.0 of controls and virus-negative lymphocytic inflammation was observed in 7 out of 10 patients. At Ultrastructural immunogold analysis, Myocardial alpha-galactosidase A activity increased in post infusion samples by overall 1.89-fold. Alpha-galactosidase A immunostaining in cardiomyocytes was absent at baseline in all patients and did not significantly improve in post-infusion samples. Immunogold particles increased by 1.33-fold remaining far from normal controls (86.9 ± 6.6). Protein analysis showed M6Pr in advanced FDCM to be 81% lower than in normal heart. Conclusions Our study shows a low accessibility to ERT of cardiomyocytes affected by advanced FDCM. It is sustained by myocardial fibrosis, inflammation and severe down-regulation of M6Pr. Figure Legend Histologic and ultrastructural and mannose6phosphate receptors characteristics of patients with severe Fabry Disease Cardiomyopathy. Panel A shows severe cellular glycolipid infiltration associated with diffuse interstitial fibrosis (Masson trichrome, 100x). Panel B reveals an overlapping lymphocytic CD45Ro+ myocardial inflammation with focal necrosis of adjacent cardiomyocytes. Insert(i), Ultrastructural detail showing vacuoles (black arrows) to consist of myelin bodies,( scale bar=3μm). Panel C (upper) Western blot analysis of all 10 patients of Mannose-6-phosphate Receptor, molecular weight 275 Kd. Alpha sarcomeric actin (43 kDa) was used as a loading control. (Panel C -lower) Panel D: Graphs document western blot of Mannose-6-phosphate Receptor, in all 10 patients with severe FDCM showing 3-fold lower of Mannose-6-phosphate Receptor values in patients versus normal heart (4.830 ± 1116,724 vs 14.737± 1145,386; p &amp;lt; 0.001).

MECOM and the PRDM gene family in uterine endometrial cancer: bioinformatics and experimental insights into pathogenesis and therapeutic potentials

To elucidate the expression profiles, methylation states, and clinicopathological significance of the PRDM gene family, focusing on the MECOM gene's role in uterine endometrial cancer (UCEC) and its molecular interactions with the TGF-beta signaling pathway. Our methodology combined detailed bioinformatics analyses using UALCAN and GEPIA with in vitro assessments in HEC-1-A cells. Techniques included CRISPR-Cas9 for gene editing and various cellular assays (CCK-8, flow cytometry, Transwell) to evaluate the effects of MECOM on cell proliferation, migration, and apoptosis, alongside Western blot analysis for protein regulation in the TGF-beta pathway. MECOM was upregulated in UCEC tissues, influencing tumor cell behavior significantly. Knockout studies demonstrated reduced proliferation and migration and increased apoptosis, while overexpression showed reverse effects. Mechanistically, MECOM modulated critical proteins within the TGF-beta pathway, impacting cell cycle dynamics and apoptotic processes. The PRDM gene family, particularly MECOM, plays a crucial role in the pathogenesis and progression of UCEC, suggesting its utility as a target for novel therapeutic interventions. Our findings offer valuable insights for future research and potential clinical application in managing uterine endometrial cancer.

Developing p21-activated kinase 1 (PAK1) activators to treat hypertrophic cardiomyopathy (HCM)

Abstract Despite significant progress in comprehending the genetic and metabolic underpinnings of cardiomyocyte dysfunction, there is still a pressing need for targeted treatments to address hypertrophy and progressive remodelling (e.g., fibrosis), seen in HCM1 . PAK1, a regulator of ion channels and myofilaments in cardiomyocytes, has shown promise in curbing pathological hypertrophy2 . Our hypothesis centres on the potential therapeutic benefits of pharmacologically activating PAK1 in managing hypertrophy and adverse remodelling in HCM. First, we conducted virtual screening and kinase assays to identify potent small molecule PAK1 activators, which significantly increased PAK1 activity by 3 to 5-fold, with an EC50 range between 0.5 and 2.5 μΜ. We then evaluated these compounds on cellular hypertrophy in an in vitro model using neonatal rat cardiomyocytes exposed to isoprenaline (ISO). Among those, JB2020A not only prevented ISO-induced hypertrophy, but also reversed pre-existing cellular hypertrophy induced by ISO 24 hours earlier. Subsequently, we assessed the therapeutic potential of JB2020A in a well-characterised HCM mouse model (i.e., Actc1E99K) 3 , known for its rapid disease progression. We initiated a six-week oral treatment regime at 4 weeks of age, dosing at 10 mg/kg per day. Echocardiography and fibrosis evaluation confirmed that JB2020A led to a significant reduction in cardiac hypertrophy and fibrosis with preserved contractile function. Western blotting analysis revealed increased PAK1 phosphorylation and activation in JB2020A-treated E99K hearts, along with a reduction in pro-apoptotic CHOP expression and an upregulation of protective endoplasmic reticulum (ER) response (e.g., ATF4 and Xbp1), indicating an amelioration of ER stress in HCM. These findings underscore the therapeutic potential of small molecule PAK1 activators as a novel approach for addressing hypertrophy and progressive remodelling in HCM.

EPO Deficiency Upregulates GADD45b/p38 MAPK Axis, Mediating Schizophrenia-Related Synaptic and Cognitive Impairments.

Schizophrenia (SZ) is a chronic and severe mental illness associated with psychiatric symptoms, cognitive deficits, and social dysfunction. Current clinical interventions only limit relief of psychiatric symptoms and have minimal impact on cognitive impairments. Erythropoietin (EPO), known for its role in neurogenesis and synaptic plasticity, is significantly low in SZ patients. However, the role of EPO deficiency in SZ-associated cognitive deficits remains unclear. In this study, we used the MK801-induced SZ rat model to show that EPO levels were significantly decreased, correlating with cognitive impairments. EPO supplementation mitigated apoptosis, synaptic damage, and cognitive impairments caused by MK801. RNA-sequencing and Western blot analysis revealed increased expression of growth arrest and DNA damage 45b (GADD45b) in MK801-treated rats, reversed by EPO supplementation. Moreover, overexpression of GADD45b exacerbated neuronal loss and cognitive impairments in male Sprague-Dawley rats, while downregulation of GADD45b rescued these SZ-related pathologies. Notably, the benefits of EPO supplementation on SZ pathology were blocked by GADD45b overexpression. Inhibition of p38 MAPK, a GADD45b target, reduced MK801-induced apoptosis and synaptic damage. These findings uncover a novel etiopathogenic mechanism of SZ-related cognitive impairments, driven by EPO deficiency and the activation of the GADD45b/p38 MAPK axis.

The CNP analogue vosoritide reduces arrhythmia in diabetic cardiomyopathy via cGMP-dependent PDE2 stimulation on cellular and organ level

Abstract Background Detrimental arrhythmia occurs frequently in patients with diabetic cardiomyopathy (DiabCM), but safe and effective therapies are limited. Diabetes-associated neuropathological alterations increase the sympathetic tone, leading to hyperactivation of β-adrenoceptor-mediated cAMP signalling in cardiomyocytes (CM). Increased activity of deleterious cAMP-dependent kinases causes pro-arrhythmic dysregulation of intracellular Ca2+ homeostasis. Phosphodiesterases (PDE) can reduce cAMP levels. Exceptionally, the cAMP-hydrolysing activity of PDE2 can be cGMP-dependently increased by C-type natriuretic peptide (CNP). Thus, the CNP analogue vosoritide (approved for the treatment of achondroplasia) might mediate this cGMP/cAMP crosstalk, reducing arrhythmia. Purpose Here, we aim to study whether the cGMP-dependent activation of PDE2 by vosoritide (VO) is sufficient to reduce arrhythmia and arrhythmogenic triggers in DiabCM. Methods Diabetes was induced by 5 consecutive injections of streptozotocin (50 mg/kg) in control (WT) mice and mice with a CM-specific PDE2 knockout (KO). All experiments were performed 5 weeks later. Cardiac function was characterised by echocardiography. Primary ventricular CM were isolated to assess protein expression and phosphorylation levels by Western blot (WB) and cAMP levels by ELISA. Patch-clamp and Ca2+ imaging experiments were used to investigate pro-arrhythmic triggers. Arrhythmic events were quantified in ex vivo perfused hearts after ischaemia-reperfusion (I/R) injury. Results Compared to healthy controls, diabetic animals showed impaired systolic function with a ~9 % reduction in ejection fraction. The significant increase in E/E’ ratio by ~34 % indicated a diastolic dysfunction. Interestingly, the cardiac function was further worsened in diabetic PDE2 KO. WB analysis revealed upregulation of the CNP receptor NPR-B and PDE2 in diabetic CM and altered regulation of proteins involved in cAMP-dependent Ca2+ cycling such as protein kinase A, Ca2+/calmodulin-dependent kinase II and phospholamban. Interestingly, VO significantly reduced isoprenaline (Iso)-induced cAMP levels in diabetic CM. The effect of VO was prevented by specific PDE2 inhibition with BAY 60-7550 (BAY). Furthermore, VO was able to reduce the Iso-induced increase in L-type Ca2+ current, whereas BAY or genetic PDE2 deletion counteracted this effect. Accordingly, VO significantly decreased the Iso-triggered Ca2+ spark frequency by ~40 %. Concomitant PDE2 inhibition or PDE2 KO prevented this anti-arrhythmic effect. Finally, ECG analysis revealed that VO clearly reduced arrhythmia development after I/R in ex vivo perfused diabetic hearts via PDE2 activation. Conclusion Our data indicate that vosoritide-induced PDE2 stimulation may attenuate abnormal cardiac Ca2+ cycling and thereby reduce arrhythmia in diabetic hearts. Thus, vosoritide may be repurposed as a novel anti-arrhythmic drug in DiabCM.

Lactiplantibacillus plantarum NMGL2 exopolysaccharide ameliorates DSS-induced IBD in mice mainly by regulation of intestinal tight junction and NF-κB p65 protein expression.

Treatment of inflammatory bowel disease (IBD), a common chronic intestinal disease, by exopolysaccharides (EPSs) produced by lactic acid bacteria has raised increasing concerns. Here, the EPS produced by Lactiplantibacillus plantarum NMGL2 was evaluated for its ameliorating effect on dextran sodium sulfate (DSS)-induced IBD in mice. Administration of the EPS was shown to decrease the body weight loss and the values of disease activity index (DAI) and alleviate the colon damage as evidenced by an improvement in colonic length shortening, a reduction in colonic coefficient, and a reduction in colonic mucosal architecture and inflammatory infiltration. Cytokine assay of the blood and colon tissue samples showed that the EPS could decrease the levels of pro-inflammatory TNF-α and IL-1β, and increase anti-inflammatory IL-10. Oxidative stress assay of the colon tissue showed that the nitric oxide (NO) and malondialdehyde (MDA) levels decreased significantly (p < 0.05), while superoxide dismutase (SOD) and glutathione (GSH) levels increased significantly (p < 0.05) after the EPS intervention. These results were further confirmed by the significantly (p < 0.05) down-regulated levels of NF-κB p65, p-IKKβ, and p-IκBα, and significantly (p < 0.05) enhanced expression of ZO-1 and occludin, as evaluated by Western-blot analysis of these proteins expressed in colonic tissue. The EPS produced by L. plantarum NMGL2 alleviated IBD by suppressing the NF-κB signaling pathway, suggesting its potential as a functional food agent in the prevention of IBD.