Increased Cell Survival Research Articles

Knockdown of KLF6 ameliorates myocardial infarction by regulating autophagy via transcriptional regulation of PTTG1.

Krüppel-like factor 6 (KLF6) knockdown provides protection against kidney ischemia/reperfusion (I/R) injury and ischemic stroke. However, it is unclear whether it plays a role in myocardial infarction (MI) remains unknown. Here, the expression of KLF6 was analyzed using the GEO database and determined in patients with MI. The impact of KLF6 knockdown was further confirmed in in vivo and >in vitro models of MI. The interaction between KLF6 and PTTG1 was also evaluated. According to the GEO database, KLF6 expression was found to be upregulated in mouse hearts after MI compared to sham-operated mice. The upregulation of KLF6 in hearts from mice post-MI and in patients with MI was confirmed. KLF6 knockdown was found to alleviate myocardial injury, diminish infarct size, and suppress apoptosis and autophagy in mice with MI. Additionally, inactivation of the AMPK/mTOR signaling was observed after KLF6 knockdown in mice with MI. In an in vitro model of MI, knockdown of KLF6 increased cell survival and inhibited autophagy through the AMPK/mTOR pathway. Additionally, KLF6 interacted with the promoter of PTTG1 and negatively regulated its expression. Knockdown of PTTG1 abolished the function of KLF6 knockdown in vitro. This study demonstrates the protective effect of KLF6 knockdown against MI, which is attributed to the elevation of PTTG1 expression and inhibition of the AMPK/mTOR pathway. These findings provide a novel insight into MI treatment.

Dihydromyricetin Suppresses Lipopolysaccharide-Induced Intestinal Injury Through Reducing Reactive Oxygen Species Generation and NOD-Like Receptor Pyrin Domain Containing 3 Inflammasome Activation.

As an integral component of the gram-negative bacterial cellular envelope, excess production of lipopolysaccharide (LPS) regularly precipitates causing intestinal damage and barrier dysfunction in avian species. Dihydromyricetin (DHM), a naturally occurring constituent in rattan tea, exhibits protective characteristics against various tissue injuries. However, the intervention mechanism of DHM on intestinal injury induced by LPS in chickens has not been determined. Consequently, this study aimed to elucidate the mechanisms through which DHM mitigates LPS-induced intestinal damage in chickens through the reactive oxygen species (ROS)-NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome. Primary intestinal epithelial cells (IECs) were isolated and cultured from 14-day-old specific pathogen free (SPF) chicken embryos, and DHM ranging from 20 to 320 μmol/L increased cell survival rates. Additionally, DHM at 20 and 40 μmol/L demonstrated reduction in oxidative stress and ROS accumulation, mirroring the impact of ROS inhibitor (2.5 mmol/L NAC). DHM efficiently regulated ROS production, thereby augmenting ZO-1, occludin and claudin-1 expression to enhance barrier function; upregulating bcl-2 expression and downregulating bax and caspase-3 expression to regulate apoptosis and suppressing inflammation in IECs. Suppression of ROS subsequently attenuates NLRP3 inflammasome activation, leading to a remarkable downregulation of IL-1β, IL-18 and lactate dehydrogenase (LDH) secretion, consistent with direct inactivation of NLRP3 inflammasome (10 μmol/L MCC950). Notably, DHM diminished IL-1β and IL-18 levels and LDH activity via suppression of ROS-regulated NLRP3 and caspase-1 expression and activation. In summary, DHM prevents LPS-induced intestinal impairment by modulating ROS generation and NLRP3 inflammasome activation.

Gb3 trisaccharide-bearing exosomes as a novel neutralizer for Shiga toxin type 1

Shiga toxin types 1 (Stx1) and 2 (Stx2), produced by Shiga toxin-producing Escherichia coli (STEC) and Shigella dysenteriae, are key virulence factors responsible for severe foodborne diseases, such as hemorrhagic colitis and hemolytic uremic syndrome (HUS). The receptors for Stxs are Gb3 and P1 glycotope, which contain the Galα1→4Galβ epitope and are synthesized by human α1,4-galactosyltransferase (A4galt). Stx-related infections pose a global public health challenge, owing to the limited therapeutic options due to the restricted use of antibiotics. Therefore, there is an urgent need to develop novel therapeutic strategies. This study proposes an innovative strategy utilizing exosomes derived from CHO-Lec2 cells, which were modified with Functional-Spacer-Lipid (FSL) conjugates bearing the Gb3 carbohydrate epitope (exo-Gb3-FSL). Flow cytometry analysis confirmed the presence of Galα1→4Galβ disaccharides on exo-Gb3-FSL constructs, enabling them to bind Stx1. Moreover, using CHO-Lec2 cells verified the ability of exo-Gb3-FSL agents to bind Stx1 and protect these cells from Stx1-mediated cytotoxicity. For Stx1-treated CHO-Lec2 cells, increased cell survival was observed when using 25 μM exo-Gb3-FSL constructs, compared to control cells. These findings highlight the potential of exosome-based anti-Stx1 agents as promising alternatives to conventional therapies. This innovative strategy may provide novel directions for studies on Stx1 neutralization, offering a valuable strategy for the treatment of Stx-related diseases.

TMOD-26. GENERATION OF A CHROMOSOME 8 GAIN;NF1-/- HIPSC-DERIVED SCHWANN CELL PRECURSOR MODEL

Abstract Neurofibromatosis type 1 (NF1) is an autosomal dominant cancer predisposition syndrome caused by a germline mutation in the NF1 gene. The loss of the second copy of NF1 within Schwann cell precursors (SCPs) contributes to the formation of plexiform neurofibromas (PNs) found in 30-50% of NF1 patients. PNs can progress into malignant peripheral sheath tumors (MPNSTs), an aggressive form of cancer and a leading cause of mortality in NF1 patients. Our lab previously demonstrated frequent chromosome 8 (Chr8) gain in NF1-MPNST patient-derived xenografts (PDX) and patient tumors. To investigate Chr8 gain’s role, we created isogenic NF1-/- iPSC cell lines with and without Chr8 gain. Trisomy 8 fibroblasts were obtained and single-cell cloned to establish wildtype (WT) and Chr8 gain populations. Washington University’s Genome Engineering & Stem Cell Center (GESC@MGI) reprogrammed these cells into human induced pluripotent stem cells (hiPSCs). Using synthetic gRNA and CRISPR/Cas9, NF1 knockout lines were generated. Four iPSC cell lines were differentiated into SCPs, and cell survival and proliferation were assessed using Incucyte cell survival and CellTiter-Glo® Assay. Fluorescence in situ hybridization (FISH) and karyotyping validated the human iPSCs. qPCR revealed high expression of iPSC markers OCT3/4 and SOX2 in all iPSC lines. After differentiation, SCP markers (GAP43 and ITGA4) were upregulated in WT, NF1-/-, and Chr8 gain;NF1-/- SCP lines, demonstrating successful differentiation. Furthermore, Chr8 gain; NF1-/- SCPs displayed enhanced proliferation and increased cell survival compared to WT and NF1-/- SCPs, suggesting improved characteristics in the presence of Chr8 gain. In summary, our research provides insights into Chr8 gain and NF1 loss effects on SCP differentiation, proliferation, and survival. Understanding these molecular mechanisms may contribute to developing targeted therapies for NF1.

Stress survival and longevity of Caenorhabditis elegans lacking NCS-1.

Although dysfunctional Ca2+ signaling can trigger biochemical reactions that lead to cell death, the role of calcium-binding proteins (CBPs) in this process is still a topic of debate. Neuronal calcium sensor 1 (NCS-1) is a CBP that is highly conserved and has been shown to increase cell survival against various types of injuries. As such, we hypothesized that NCS-1 could also be a stress-responsive protein with potential effects on survival and longevity. To explore this possibility, we conducted experiments to examine how Caenorhabditis elegans ncs-1 mutant nematodes fared under three different stress conditions: hyperosmotic, thermal, and chemical oxidant challenges. Our results showed that while the lack of NCS-1 had no effect on survival responses to hyperosmotic and thermal stresses, ncs-1 worms demonstrated remarkable resistance to the oxidant paraquat in a dose-dependent manner. Based on these findings, we conclude that C. elegans may employ adaptive mechanisms in the absence of NCS-1 to survive specific oxidative stress stimuli.

LncRNA AGAP2-AS1 stabilizes ATG9A to promote autophagy in endothelial cells - Implications for burn wound healing

Deep second- or mixed-degree burn lesions are difficult to heal due to the impaired dermis supporting of epidermis renewal and nutrition delivery. Early dermis debridement and preservation speed healing and enhance results, emphasizing the need of knowing processes that promote burn-denatured dermis recovery, notably endothelial cell angiogenesis and autophagy. Integrative bioinformatics investigations identified AGAP2-AS1 as a highly elevated lncRNA in burn tissues. Pearson's correlation study connected AGAP2-AS1 to 112 differently co-expressed protein-coding genes involved in burn healing processes such cell cycle and TGF-beta receptor signaling. Experimental validation showed that heat damage elevated AGAP2-AS1 in HUVECs and HDMECs. Functionally, AGAP2-AS1 overexpression in heat-denatured HUVECs and HDMECs increased cell survival, migration, invasion, and angiogenesis. In addition, AGAP2-AS1 overexpression increased endothelial cell autophagy. Additional investigation showed AGAP2-AS1's association with ATG9A, stabilizing it. Post-heat damage, ATG9A knockdown drastically reduced HUVEC and HDMEC survival, migration, invasion, angiogenesis, and autophagy. More notably, ATG9A knockdown drastically reduced the benefits of AGAP2-AS1 overexpression on endothelial cell functions and autophagy. The positive association between AGAP2-AS1 and ATG9A expression in burn tissue samples highlights their crucial roles in endothelial cell response to heat injury, indicating that targeting this axis may aid burn wound healing. The research found that lncRNA AGAP2-AS1 stabilizes ATG9A and promotes autophagy in endothelial cells. These results imply that targeting the AGAP2-AS1/ATG9A axis may improve angiogenesis and tissue regeneration in burn injuries, revealing burn wound healing molecular pathways.

Interplay between the SAFE and the sphingolipid pathway for cardioprotection

AimActivation of both the Survivor Activating Factor Enhancement (SAFE) pathway (including Tumor Necrosis Factor-alpha (TNF-α) and Signal Transducer and Activator of Transcription-3 (STAT-3)) and the sphingolipid signalling pathway (including sphingosine kinase-1 (SK1) and sphingosine-1 phosphate (S1P)) play a key role in promoting cardioprotection against ischemia-reperfusion injury (IRI). We investigated whether the activation of the SAFE pathway by exogenous S1P is dependent on the activation of SK1 for cardioprotection. Materials and methodsIsolated cardiomyocytes from TNF-α knockout (KO) mice, cardiomyocyte-specific STAT-3 KO mice and their wild-type (WT) littermates were exposed to simulated ischemia in the presence of a trigger of the SAFE pathway (S1P) and SK1 inhibitor (SK1-I). Similarly, isolated perfused hearts from adult TNF-α KO, STAT-3 KO and WT mice were subjected to IRI with S1P and/or SK1-I. Cell viability, infarct size (IS) and SK1 activity were assessed. Key findingsIn isolated cardiomyocytes and in isolated hearts subjected to simulated ischemia/IRI, S1P pretreatment decreased cell death in WT mice, an effect that was abrogated in the presence of SK1-I. S1P failed to reduce cell death after simulated ischemia/IRI in both cardiomyocytes or hearts isolated from TNF-α KO and STAT-3 KO mice. Interestingly, S1P pretreatment increased SK1 activity in WT and STAT-3 KO mice, with no changes in TNF-α KO mice. SignificanceOur data strongly suggest SK1 as a key component to activate STAT-3 downstream of TNF-α in the SAFE pathway, paving the way for the development of novel cardioprotective strategies that may target SK1 to modulate the SAFE pathway and increase cell survival following IRI.

Zhachong Shisanwei pill drug-containing serum protects H2O2-Induced PC12 cells injury by suppressing apoptosis, oxidative stress via regulating the MAPK signaling pathway.

Zhachong Shisanwei Pill (ZSP) is a classical Mongolian formula that combines 13 types of Chinese medicinal materials and has been used for treating ischemic stroke (IS) for centuries. However, the underlying molecular mechanisms have yet to be fully elucidated. The aim of this study is to explore potential mechanism of ZSP on nerve cells in cerebral ischemic injury. To simulate the pathological process of oxidative stress following IS, an injury model using PC12 cells was induced with hydrogen peroxide (H2O2). Afterward, PC12 cells were treated with ZSP medicated serum at low, medium, and high doses. Various assays were conducted to assess cell viability and oxidative stress indicators, including lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), reactive oxygen species (ROS), and mitochondrial membrane potential (MMP). Cell apoptosis was evaluated through morphological assessment and flow cytometry. Additionally, the expression levels of apoptosis-related proteins (Bcl-2, Bax, Caspase-9, Caspase-3, PARP) and signaling pathway proteins (JNK, phosphorylated JNK, ERK, phosphorylated ERK, p38, and phosphorylated p38) were measured using automated Western blotting. Our findings indicate that ZSP medicated serum preconditioning improves the condition of PC12 cells injured by H2O2. Specifically, it increased cell survival rates and reduced LDH release. Additionally, ZSP treatment decreased ROS levels and MDA content, while enhancing the activity of SOD and CAT in the injured PC12 cells. ZSP also reversed the depolarization of mitochondrial membrane potential and protected cells from apoptosis by modulating the expression of apoptosis-related proteins, including Bcl-2, Bax, Caspase-9, Caspase-3, and PARP. Furthermore, the overactivation of the MAPK signaling pathway due to H2O2-induced injury was inhibited, as evidenced by the downregulation of phosphorylated JNK, ERK, and p38 levels. Mongolian medicine ZSP demonstrates protective effects against H2O2-induced oxidative stress and apoptosis in PC12 cells. The underlying mechanism may involve the inhibition of the MAPK signaling pathway, enhancement of antioxidant enzyme activity, reduction of intracellular peroxidation levels, and suppression of intrinsic apoptosis pathways.

Functional Relationships between L1CAM, LC3, ATG12, and Aβ.

Abnormal protein accumulations in the brain are linked to aging and the pathogenesis of dementia of various types, including Alzheimer's disease. These accumulations can be reduced by cell indigenous mechanisms. Among these is autophagy, whereby proteins are transferred to lysosomes for degradation. Autophagic dysfunction hampers the elimination of pathogenic protein aggregations that contribute to cell death. We had observed that the adhesion molecule L1 interacts with microtubule-associated protein 1 light-chain 3 (LC3), which is needed for autophagy substrate selection. L1 increases cell survival in an LC3-dependent manner via its extracellular LC3 interacting region (LIR). L1 also interacts with Aβ and reduces the Aβ plaque load in an AD model mouse. Based on these results, we investigated whether L1 could contribute to autophagy of aggregated Aβ and its clearance. We here show that L1 interacts with autophagy-related protein 12 (ATG12) via its LIR domain, whereas interaction with ubiquitin-binding protein p62/SQSTM1 does not depend on LIR. Aβ, bound to L1, is carried to the autophagosome leading to Aβ elimination. Showing that the mitophagy-related L1-70 fragment is ubiquitinated, we expect that the p62/SQSTM1 pathway also contributes to Aβ elimination. We propose that enhancing L1 functions may contribute to therapy in humans.

Exceptional stability of the sugammadex-solasodine complex: Insights from experimental and theoretical studies

Sugammadex (SGM) is the first cyclodextrin (CD)-based selective relaxant binding agent. We investigated its ability to capture natural aminosteroid phytotoxins, and assessed its potential as an antidote for intoxication. Solasodine (SS), a toxic alkaloid from the Solanaceae family, was chosen as the model compound. Complexation was studied using nuclear magnetic resonance (NMR) spectroscopy, molecular modelling, and isothermal titration calorimetry (ITC). NMR in various D2O/DMSO‑d6 media revealed a particularly stable inclusion-type complex, identifying a slow exchange process between the CD and the aminosteroid along with a less significant fast exchange between DMSO and SGM. Using various NMR techniques, the structure and kinetic/thermodynamic parameters of the inclusion complex were explored. Theoretical calculations showed the secondary amino head of SS near the carboxylate ends of the SGM sidechains, facilitating intermolecular ionic interactions. ITC experiments in an aqueous environment provided Ka stability constants of 7.03 × 106 M−1 and 4.17 × 106 M−1 at 25 °C and 37 °C, respectively, similar to previously reported SGM complexes with aminosteroid neuromuscular blockers. Finally, SGM significantly increased cell survival and reduced SS toxicity in mHippoE-14 mouse hippocampal embryonic cells, supporting the hypothesis that SGM could act as an antidote to SS's toxic effects.

Insights into Advances and Applications of Biomaterials for Nerve Tissue Injuries and Neurodegenerative Disorders.

The incidence of nerve tissue injuries, such as peripheral nerve injury, spinal cord injury, traumatic brain injury, and various neurodegenerative diseases (NDs), is continuously increasing because of stress, physical and chemical trauma, and the aging population worldwide. Restoration of the damaged nervous system is challenging because of its structural and functional complexity and limited regenerative ability. Additionally, there is no cure available for NDs except for medications that provide symptomatic relief. Stem cells offer an alternative approach for promoting damage repair, but their efficacy is limited by a compromised survival rate and neurogenesis process. To address these challenges, neural tissue engineering has emerged as a promising strategy in which stem cells are seeded or encapsulated within a suitable biomaterial construct, increasing cell survival and neurogenesis. Numerous biomaterials are utilized to create different types of constructs for this purpose. Researchers are trying to develop ideal scaffolds that combine biomaterials, cells, and molecules that exactly mimic the biological and mechanical properties of the tissue to achieve functional recovery associated with neurological dysfunction. This review focuses on exploring the development and applications of different biomaterials for their potential use in the diagnosis, therapy, nerve tissue regeneration, and treatment of neurological disorders.

Evaluating the Reparative Potential of Secretome from Patient-Derived Induced Pluripotent Stem Cells during Ischemia–Reperfusion Injury in Human Cardiomyocytes

During a heart attack, ischemia causes losses of billions of cells; this is especially concerning given the minimal regenerative capability of cardiomyocytes (CMs). Heart remuscularization utilizing stem cells has improved cardiac outcomes despite little cell engraftment, thereby shifting focus to cell-free therapies. Consequently, we chose induced pluripotent stem cells (iPSCs) given their pluripotent nature, efficacy in previous studies, and easy obtainability from minimally invasive techniques. Nonetheless, using iPSC secretome-based therapies for treating injured CMs in a clinical setting is ill-understood. We hypothesized that the iPSC secretome, regardless of donor health, would improve cardiovascular outcomes in the CM model of ischemia–reperfusion (IR) injury. Episomal-generated iPSCs from healthy and dilated cardiomyopathy (DCM) donors, passaged 6–10 times, underwent 24 h incubation in serum-free media. Protein content of the secretome was analyzed by mass spectroscopy and used to treat AC16 immortalized CMs during 5 h reperfusion following 24 h of hypoxia. IPSC-derived secretome content, independent of donor health status, had elevated expression of proteins involved in cell survival pathways. In IR conditions, iPSC-derived secretome increased cell survival as measured by metabolic activity (p < 0.05), cell viability (p < 0.001), and maladaptive cellular remodelling (p = 0.052). Healthy donor-derived secretome contained increased expression of proteins related to calcium contractility compared to DCM donors. Congruently, only healthy donor-derived secretomes improved CM intracellular calcium concentrations (p < 0.01). Heretofore, secretome studies mainly investigated differences relating to cell type rather than donor health. Our work suggests that healthy donors provide more efficacious iPSC-derived secretome compared to DCM donors in the context of IR injury in human CMs. These findings illustrate that the regenerative potential of the iPSC secretome varies due to donor-specific differences.

Adipogenic-Myogenic Signaling in Engineered Human Muscle Grafts used to Treat Volumetric Muscle Loss.

Tissue-engineered muscle grafts (TEMGs) are a promising treatment for volumetric muscle loss (VML). In this study, human myogenic progenitors (hMPs) cultured on electrospun fibrin microfiber bundles and evaluated the therapeutic potential of engineered hMP TEMGs in the treatment of murine tibialis anterior (TA) VML injuries is employed. In vitro, the hMP TEMGs express mature muscle markers by 21 days. Upon implantation into VML injuries, the hMP TEMGs enable remarkable regeneration. To further promote wound healing and myogenesis, human adipose-derived stem/stromal cells (hASCs) as fibroadipogenic progenitor (FAP)-like cells with the potential to secrete pro-regenerative cytokines are incorporated. The impact of dose and timing of seeding the hASCs on in vitro myogenesis and VML recovery using hMP-hASC TEMGs are investigated. The hASCs increase myogenesis of hMPs when co-cultured at 5% hASCs: 95% hMPs and with delayed seeding. Upon implantation into immunocompromised mice, hMP-hASC TEMGs increase cell survival, collagen IV deposition, and pro-regenerative macrophage recruitment, but result in excessive adipose tissue growth after 28 days. These data demonstrate the interactions of hASCs and hMPs enhance myogenesis in vitro but there remains a need to optimize treatments to minimize adipogenesis and promote full therapeutic recovery following VML treatment.

Customizable gene sensing and response without altering endogenous coding sequences.

Synthetic biology aims to modify cellular behaviors by implementing genetic circuits that respond to changes in cell state. Integrating genetic biosensors into endogenous gene coding sequences using clustered regularly interspaced short palindromic repeats and Cas9 enables interrogation of gene expression dynamics in the appropriate chromosomal context. However, embedding a biosensor into a gene coding sequence may unpredictably alter endogenous gene regulation. To address this challenge, we developed an approach to integrate genetic biosensors into endogenous genes without modifying their coding sequence by inserting into their terminator region single-guide RNAs that activate downstream circuits. Sensor dosage responses can be fine-tuned and predicted through a mathematical model. We engineered a cell stress sensor and actuator in CHO-K1 cells that conditionally activates antiapoptotic protein BCL-2 through a downstream circuit, thereby increasing cell survival under stress conditions. Our gene sensor and actuator platform has potential use for a wide range of applications that include biomanufacturing, cell fate control and cell-based therapeutics.

Experimental study of radioprotective properties of lithium pyruvate in vitro

Radiotoxicity is a serious problem for patients undergoing radiotherapy, so the search for new radioprotective drugs to mitigate its effects is highly relevant. Radioprotectors should have a number of properties, including direct antioxidant action, reduction of oxidative stress, ability to induce DNA repair or inhibit apoptosis, and at the same time not cause their own side effects. Antioxidants based on lithium salts look promising in terms of their properties. The aim of study was to study the radioprotective properties of lithium pyruvate in vitro. Material and Methods. Relatively radiosensitive blood mononuclear cells and relatively radioresistant fibroblasts of 3T3L1 line were used as biomodels for x-ray exposure. Cells were incubated and irradiated in 96-well plates. Lithium pyruvate was used at a final concentration of 1.2 mM. Cells were irradiated at a dose rate of 15 mGy/s in the absorbed-dose range from 0 to 5 Gy using an x-ray unit (anode voltage: 160 kV, average current: 3.5 mA). Cell viability was assessed by MTT test and resazurin test. The evaluation of cell death variants and the level of oxidative stress were determined by cytofluorimetric method. Results. The cytoprotective effect of lithium pyruvate was established. Cytoprotection was manifested in the increased cell survival and decreased oxidative stress level under lithium pyruvate after x-ray in a wide range of absorbed doses. Relatively high efficiency was shown in relation to blood mononuclear cells with an increase in the viable fraction by 5–7 % and a decrease in oxidative stress level during irradiation in the range of 1.0–3.0 Gy. Apoptosis was found to be the main mechanism of cell death after irradiation. Lithium pyruvate reduced the level of apoptosis in cell population under irradiation and chemically induced oxidative stress. Conclusion. Radioprotective effect of lithium pyruvate under x-ray irradiation in vitro has been shown. Reduction of oxidative stress under the action of pyruvate provides a pathogenetic basis for the potential use of this compound as a radioprotector, which requires further studies on in vivo models.

Evaluation of SK-N-SH Cells as a Model for NMDA Receptor Induced Toxicity.

Over the years, the number of patients with neurodegenerative diseases is constantly rising illustrating the need for new neuroprotective drugs. A promising treatment approach is the reduction of excitotoxicity induced by rising (S)-glutamate levels and subsequent NMDA receptor overactivation. To facilitate the search for new NMDA receptor inhibitors neuronal cell models are needed. In this study, we evaluated the suitability of human SK-N-SH cells to serve as a cell model for neurodegeneration induced by NMDA receptor overstimulation. The cytoprotective effect of the unselective NMDA receptor blocker ketamine as well as the GluN2B-selective inhibitor WMS14-10 was evaluated utilizing different cell viability assays, such as endpoint (LDH, CCK-8, DAPI/FACS) and time dependent methods (bioimpedance). Non-differentiated as well as differentiated SK-N-SH cells express GluN1 and GluN2B subunits. Furthermore, 50 mM (S)-glutamate led to an instantaneous decrease in cell survival. Only application of unselective channel blocker ketamine could protect differentiated cells against this effect, while the selective inhibitor WMS14-10 did not significantly increase cell survival. SK-N-SH cells show an increased sensitivity to (S)-glutamate mediated cytotoxicity with higher differentiation level, that is only partially induced by NMDA receptor overstimulation. Furthermore, we showed that only unselective NMDA receptor inhibition can partially reverse (S)-glutamate-induced toxicity.

Pigment Epithelium-Derived Factor Binding to VEGFR-1 (Flt-1) Increases the Survival of Retinal Neurons.

The purpose of this study was to examine possible involvement of vascular endothelial growth factor (VEGF) receptor (VEGFR)-1/Flt-1 in pigment epithelium-derived factor (PEDF)-promoted survival of retinal neurons. Survival of growth factor-deprived retinal ganglion cells (RGCs) and R28 cells and activation of ERK-1/-2 MAP kinases were assessed in the presence of PEDF, placental growth factor (PlGF), and VEGF using cell cultures, viability assays and quantitation of ERK-1/-2 phosphorylation. VEGFR-1/Flt-1 expression was determined using quantitative PCR (qPCR) and Western blotting. VEGFR-1/Flt-1 was knocked down in R28 cells by small interfering RNA (siRNA). Binding of a PEDF-IgG Fc fusion protein (PEDF-Fc) to retinal neurons, immobilized VEGFR-1/Flt-1 and VEGFR-1/Flt-1-derived peptides was studied using binding assays and peptide scanning. PEDF in combination with PlGF stimulated increased cell survival and ERK-1/-2 MAP kinase activation compared to effects of either factor alone. VEGFR-1/Flt-1 expression in RGCs and R28 cells was significantly upregulated by hypoxia, VEGF, and PEDF. VEGFR-1/Flt-1 ligands (VEGF and PlGF) or soluble VEGFR-1 (sflt-1) competed with PEDF-Fc for binding to R28 cells. Depleting R28 cells of VEGFR-1/Flt-1 resulted in reduced PEDF-Fc binding when comparing VEGFR-1/Flt-1 siRNA- and control siRNA-treated cells. PEDF-Fc interacted with immobilized sflt-1, which was specifically blocked by VEGF and PlGF. PEDF-Fc binding sites were mapped to VEGFR-1/Flt-1 extracellular domains D3 and D4. Peptides corresponding to D3 and D4 specifically inhibited PEDF-Fc binding to R28 cells. These peptides and sflt-1 significantly inhibited PEDF-promoted survival of R28 cells. These results suggest that PEDF can target VEGFR-1/Flt-1 and this interaction plays a significant role in PEDF-mediated neuroprotection in the retina.

Role of Notch 1 signaling and glycolysis in the pathogenic mechanism of adenomyosis

To investigate the expressions of glycolysis-related factors and changes in Notch1 signaling in endometrial tissues of adenomyosis (AM) and Ishikawa cells to explore the pathogenesis of AM. Eutopic endometrial tissues were collected from 8 patients with AM and 8 patients with uterine fibroids matched for clinical characteristics (control group). The expressions of Notch1 signaling proteins and glycolysis-related factors in the collected tissues were detected using qRT-PCR and Western blotting, and the levels of glucose and lactic acid were determined. An Ishikawa cell model with lentivirus-mediated stable Notch1 overexpression was established for assessing cell survival rate with CCK-8 assay, cell migration and invasion abilities with Transwell migration and invasion assays, and glycolytic capacity by determining the extracellular acidification rate. Compared with those in the control group, the endometrial tissues in AM group showed significantly increased expression level of carbohydrate antigen 125 (CA125), increased mRNA expression levels of Notch1, HK2 and PDHA and protein expressions of Notch1, GLUT1, HK2, PKM and PDHA, lowered glucose level and increased lactate level. The Ishikawa cell models with stable Notch1 overexpression exhibited significantly increased cell survival rate with attenuated cell migration and invasion abilities and decreased glycolysis capacity and reserve. The Notch1 signaling pathway participates in the pathogenesis of AM possibly by regulating the proliferation, migration, invasion and glycolysis of endometrial cells.

Sphingosine 1-phosphate protective effect on human proximal tubule cells submitted to an in vitro ischemia model: the role of JAK2/STAT3.

Acute kidney injury is a serious public health problem worldwide, being ischemia and reperfusion (I/R) the main lesion-aggravating factor that contributes to the evolution towards chronic kidney disease. Nonetheless, intervention approaches currently available are just considered palliative options. In order to offer an alternative treatment, it is important to understand key factors involved in the development of the disease including the rescue of the affected cells and/or the release of paracrine factors that are crucial for tissue repair. Bioactive lipids such as sphingosine 1-phosphate (S1P) have significant effects on the modulation of signaling pathways involved in tissue regeneration, such as cell survival, proliferation, differentiation, and migration. The main objective of this work was to explore the protective effect of S1P using human kidney proximal tubule cells submitted to a mimetic I/R lesion, via ATP depletion. We observed that the S1P pre-treatment increases cell survival by 50% and preserves the cell proliferation capacity of injured cells. We showed the presence of different bioactive lipids notably related to tissue repair but, more importantly, we noted that the pre-treatment with S1P attenuated the ischemia-induced effects in response to the injury, resulting in higher endogenous S1P production. All receptors but S1PR3 are present in these cells and the protective and proliferative effect of S1P/S1P receptors axis occur, at least in part, through the activation of the SAFE pathway. To our knowledge, this is the first time that S1PR4 and S1PR5 are referred in these cells and also the first indication of JAK2/STAT3 pathway involvement in S1P-mediated protection in an I/R renal model.

The preventative effects of Lactococcus Lactis metabolites against LPS-induced sepsis.

Sepsis is a syndrome of organ dysfunction caused by a dysregulated host response to infection and septic shock. Currently, antibiotic therapy is the standard treatment for sepsis, but it can lead to drug resistance. The disturbance of the gut microbiota which is affected by sepsis could lead to the development of organ failure. It is reported that probiotics could shape the gut microbiota, potentially controlling a variety of intestinal diseases and promoting whole-body health. In this study, we evaluated the preventive effects of intra- and extracellular products of probiotics on sepsis. The extracellular products of Lactococcus lactis (L. lactis) were identified through the in vivo cell experiments. The preventive effect and mechanism of L. lactis extracellular products on mouse sepsis were further explored through HE staining, mouse survival rate measurement, chip analysis, etc. L. lactis extracellular products increase cell survival and significantly reduce inflammatory factors secreted in a cellular sepsis model. In in vivo experiments in mice, our samples attenuated sepsis-induced pulmonary edema and inflammatory infiltrates in the lungs of mice, and reduced mortality and inflammatory factor levels within the serum of mice. Finally, the mechanism of sepsis prevention by lactic acid bacteria is suggested. Extracellular products of L. lactis could effectively prevent sepsis episodes. In animal experiments, we reported that extracellular products of L. lactis can effectively prevent sepsis, and preliminarily discussed the pathological mechanism, which provides more ideas for the prevention of sepsis. In the future, probiotics may be considered a new way to prevent sepsis.