Pretreatment Of Cells Research Articles

Investigation of Thiazolidine-2,4-Dione Derivatives as Acetylcholinesterase Inhibitors: Synthesis, In Vitro Biological Activities and In Silico Studies.

The inhibition of acetylcholinesterase (AChE), an enzyme responsible for the inactivation and decrease in acetylcholine in the cholinergic pathway, has been considered an attractive target for small-molecule drug discovery in Alzheimer's disease (AD) therapy. In the present study, a series of TZD derivatives were designed, synthesized, and studied for drug likeness, blood-brain barrier (BBB) permeability, and adsorption, distribution, metabolism, excretion, and toxicity (ADMET). Additionally, docking studies of the designed compounds were performed on AChE. Additionally, all the TZD derivatives (CHT1-5) showed an acceptable affinity for AChE inhibition, and the results showed convincing binding modes in the active site of AChE. Among them, 5-(4-methoxybenzylidene) thiazolidine-2,4-dione (CHT1) was identified as the most potent AChE inhibitor (IC50 of 165.93 nM) with the highest antioxidant activity. Following the exposure of PC12 cells to Aβ1-42 (100 μM), a marked reduction in cell survival was observed. Pretreatment of PC12 cells with TZD derivatives had a neuroprotective effect and significantly enhanced cell survival in response to Aβ-induced toxicity. Western blotting analysis revealed that CHT1 (5 and 8 μM) downregulated p-Tau and HSP70 expression levels. The results indicate that CHT1 is a promising and effective AchE-I that could be utilized as a powerful candidate against AD.

The potency of aloe emodin‐loaded nanoparticles in conjunction with IFN‐γ for the pretreatment of mesenchymal stem cells with class II transactivator silence to alleviate severe acute pancreatitis

AbstractMesenchymal stem cells (MSCs) have a moderate impact on the therapy of severe acute pancreatitis. This study seeks to improve the therapeutic effectiveness of MSCs. By preconditioning them via the upregulation of critical anti‐inflammatory molecules, so diminishing immune rejection, we are creating a path for more effective treatments. Aloe emodin (AE), a natural active monomer with low‐toxicity, in conjunction with interferon gamma (IFN‐γ) (I‐AE), markedly upregulated immunosuppressive molecules indoleamine 2,3‐dioxygenase and programmed cell death‐Ligand 1 in MSCs, thereby pharmacologically modulating the inhibition of CD4 − T cell activation in vitro effectively. Transient transfection of small interfering RNA silenced the class II transactivator (CIITA) gene expression of umbilical cord mesenchymal stem cells (UMSCs) interfering with human leukocyte antigen class II expression to avert immune rejection. AE‐loaded nanoparticles efficiently maintained proliferation inhibition of MSCs within a manageable range by sustained release. UMSCs pretreated by I‐AE with CIITA silencing preserved pancreatic structure as evidenced by diminished acinar cell death, reduced pancreatic edema and inflammation, and significantly lowered serum amylase levels The encouraging potential of UMSCs with CIITA gene silencing combined with AE and IFN‐γ pretreatment offers optimism for clinical application in pancreatitis therapy.

Protective Effects of Heat-Killed Lactobacilli against Plasma-Induced Neurotoxicity in Multiple Sclerosis.

Heat-killed lactobacilli seem to have protective effects against oxidative stress and neurotoxicity. This study aimed to evaluate the antioxidant properties of specific heat-killed lactobacilli extracts and determine their neuroprotective effects against the neurotoxicity induced by blood plasma from people with multiple sclerosis (MS). The antioxidant activity of the three heat-killed lactobacilli was measured using the DPPH assay. For neuroprotective evaluations of lactobacilli, human neuroblastoma cells (SK-N-SH) were exposed to plasma from individuals with relapsing-remitting MS (RRMS) and healthy controls, with or without pre-treatment of heat-killed lactobacilli including Lactiplantibacillus plantarum (L. plantarum), Levilactobacillus brevis (L. brevis), and Lacticaseibacillus rhamnosus (L. rhamnosus). The morphological changes of SK-N-SH cells associated with plasma-induced apoptosis were observed using an inverted microscope. The neurotoxic effects of plasma samples were assessed using flow cytometry as the percentage of apoptosis in neuronal cells treated with plasma from RRMS patients and healthy controls. The neuroprotective effects of the lactobacilli were also evaluated using flow cytometry, which showed an increased viability percentage in cells pretreated with heat-killed lactobacilli extracts compared to those without pre-treatment. Compared to plasma from healthy controls, plasma from RRMS patients caused morphological changes characteristic of apoptosis such as rounding, detachment, and shrinkage in SK-N-SH cells on microscopy observations. Significant apoptosis in MS plasma-treated neuronal cells was identified by flow cytometry analysis compared to cells treated with plasma from healthy controls (p < 0.01). Heat-killed lactobacilli extracts showed antioxidant activity above 50% in the DPPH radical scavenging assay. Pre-treatment of cells with heat-killed lactobacilli significantly reduced the morphological changes and apoptosis percentage in neuronal cells induced by MS plasma samples. L. plantarum and L. rhamnosus had considerable neuroprotective effects (p < 0.001), followed by L. brevis (p < 0.05). These findings demonstrate that heat-killed lactobacilli extracts as bacterial fractions free of live microorganisms, are suitable safe candidates for adjunctive therapy with potential antioxidant and neuroprotective properties in MS.

Tetrabromobisphenol A induced p38-MAPK/AMPKα activation downstream-triggered CHOP signal contributing to neuronal apoptosis and death

Tetrabromobisphenol A (TBBPA), a brominated flame retardant (BFR), has been implicated as the neurotoxic effects in mammalian. However, the exact mechanisms underlying TBBPA-induced neurotoxicity remain unclear. In the present study, Neuro-2a cells, a mouse neural crest-derived cell line, were used to examine the mechanism of TBBPA-induced neuronal cytotoxicity. TBBPA exposure caused alterations in cell viability and mitochondrial membrane potential (MMP) and induction of apoptotic events, such as increased apoptotic cell population and cleaved caspase-3, -7, -9, and poly (ADP-ribose) polymerase (PARP) protein expression). TBBPA exposure triggered CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) activation. Transfection with CHOP-specific small interfering RNA (siRNA) obviously prevented the expression of CHOP protein and markedly attenuated MMP loss, and caspase-3 and -7 activation in TBBPA-exposed Neuro-2a cells. In addition, TBBPA exposure significantly evoked the phosphorylation of c-Jun N-terminal kinase (JNK), extracellular-signal regulated kinase1/2 (ERK1/2), p38-mitogen-activated protein kinase (p38-MAPK), and AMP-activated protein kinase (AMPK)α proteins. Pretreatment of cells with pharmacological inhibitors of p38-MAPK (SB203580) and AMPK (compound C), but not inhibitors of JNK (SP600125) or ERK1/2 (PD98059), effectively prevented the increase in caspase-3 activity, MMP loss, and activated CHOP and cleaved caspase-3 and -7 protein expression in TBBPA-treated cells. Notably, transfection with either p38α-MAPK- or AMPKα1/2-specific siRNAs markedly attenuated the expression of CHOP, and cleaved caspase-3 and -7. Interestingly, transfection with each siRNA significantly reduced the TBBPA-induced phosphorylation of p38-MAPK and AMPKα proteins. Collectively, these findings suggest that CHOP activation-mediated mitochondria-dependent apoptosis contributes to TBBPA-induced neurotoxicity. An interdependent p38-MAPK and AMPKα signaling-regulated apoptotic pathway may provide new insights into the mechanism understanding TBBPA-elicited neurotoxicity.

Insurmountable antagonism of human mu opioid receptors by buprenorphine is due to hemi-equilibrium.

Buprenorphine is an FDA approved drug for the treatment of opioid use disorder and is a long-lasting, low efficacy (partial) agonist of the μ opioid receptor. As a partial agonist, buprenorphine can act as either an agonist or an antagonist depending on the efficiency of the cellular signaling system. Here we investigated the antagonist properties of buprenorphine using a genetically-encoded biosensor to monitor cAMP levels in real time in HEK293 cells expressing a relatively low density of the human μ receptor. Pre-treatment of cells with buprenorphine decreased the maximal response (Emax) of the μ receptor agonists DAMGO and fentanyl, consistent with expectations of a long-lasting partial agonist in these cells. However, buprenorphine pretreatment also reduced the potency of these agonists. In a Schild analysis, the reduction in Emax saturated with increasing concentrations of buprenorphine, while the reduction in potency did not. This unexpected behavior of buprenorphine on potency was mediated through the orthosteric site, as pretreatment with the opioid receptor antagonist naloxone prevented buprenorphine antagonism. Computation simulations using a model of hemi-equilibrium indicated that buprenorphine's slow receptor dissociation could account for both effects of antagonism. In support of this kinetic explanation, buprenorphine antagonism became surmountable with additional agonist incubation time. In this system, buprenorphine appeared to behave both as a competitive and non-competitive (insurmountable) antagonist due to its slow dissociation resulting in partial re-equilibration (hemi-equilibrium). As fast-acting biosensors become more prevalent, it will be important to consider kinetically-mediated effects such as hemi-equilibrium when characterizing the pharmacological properties of antagonists.

Antibodies to the RBD of SARS-CoV-2 spike mediate productive infection of primary human macrophages

AbstractThe role of myeloid cells in the pathogenesis of SARS-CoV-2 is well established, in particular as drivers of cytokine production and systemic inflammation characteristic of severe COVID-19. However, the potential for myeloid cells to act as bona fide targets of productive SARS-CoV-2 infection, and the specifics of entry, remain unclear. Using a panel of anti-SARS-CoV-2 monoclonal antibodies (mAbs) we performed a detailed assessment of antibody-mediated infection of monocytes/macrophages. mAbs with the most consistent potential to mediate infection were those targeting a conserved region of the receptor binding domain (RBD; group 1/class 4). Infection was closely related to the neutralising concentration of the mAbs, with peak infection occurring below the IC50, while pre-treating cells with remdesivir or FcγRI-blocking antibodies inhibited infection. Studies performed in primary macrophages demonstrated high-level and productive infection, with infected macrophages appearing multinucleated and syncytial. Infection was not seen in the absence of antibody with the same quantity of virus. Addition of ruxolitinib significantly increased infection, indicating restraint of infection through innate immune mechanisms rather than entry. High-level production of pro-inflammatory cytokines directly correlated with macrophage infection levels. We hypothesise that infection via antibody-FcR interactions could contribute to pathogenesis in primary infection, systemic virus spread or persistent infection.

Hyaluronic acid coating of poly(β-amino ester)/mRNA polyplexes enables ultra-high transfection efficiency.

mRNA holds significant potential for a wide range of biomedical applications, efficient and safe delivery of mRNA into target cells is essential to realize its therapeutic benefits. Linear poly(β-amino ester)s (LPAEs) have been utilized for mRNA delivery, yet there is a need to enhance their transfection efficiency and safety profile. In this study, a novel LPAE-based ternary mRNA delivery system with ultra-high transfection efficiency is developed by coating hyaluronic acid (HA) onto LPAE/mRNA binary polyplexes. Results demonstrate that across a broad range of HA doses, mRNA transfection and cell viability can be significantly enhanced. Intriguingly, pre-treating cells with HA further boosts the transfection efficiency up to 99.2%. Mechanistic studies reveal that HA coating impacts the size, Zeta potential of the binary polyplexes, enhancing their interaction with the cell membrane and facilitating cellular uptake. Leveraging the unique biocompatibility and biodegradability of HA, this ternary mRNA delivery system emerges as a promising option for practical applications. The approach of coating binary polyplexes with natural biomacromolecules can be extended to other non-viral mRNA delivery vectors to achieve superior transfection efficiency and safety profiles.

Cell therapy using anti-NKG2A pretreated natural killer cells in patients with hepatocellular carcinoma

Background: The activities and functions of natural killer (NK) cells are controlled by a limited repertoire of activating and inhibitory NK receptors. Therefore, blocking inhibitory receptors such as NKG2A using monoclonal antibodies (mAbs) enhances tumor immunity. Method: In this study, we investigated the safety of anti-NKG2A-pretreated NK cells in improving ADCC function to manage hepatocyte carcinoma (HCC). After a conditioning regimen, we initiated a pilot study of expanded donor haploidentical NK cell infusion. The goals were to determine the safety and feasibility. Patients received a fludarabine/cyclophosphamide conditioning followed by adoptive immunotherapy with IL2–activated haploidentical NK cells. Anti-NKG2A pretreated NK cells were infused on days 0, +5, and +10 post-conditioning regimens at a dose of 7 × 108 cells (n=3). The median follow-up was 4 months for all patients. Results: Although all patients were alive at the last follow-up, two of them showed progressive disease and an increase in tumor size. In addition, all patients had a relative decrease in the expression level of an alpha-fetoprotein (AFP) after one month. Conclusion: This study demonstrated the safety and feasibility of infusing high doses of ex vivo expanded NK cells after conditioning with transient side effects.

The Mitochondrial Fusion Promoter M1 Mitigates Cigarette Smoke-Induced Airway Inflammation and Oxidative Stress via the PI3K-AKT Signaling Pathway.

This study investigated the efficacy and underlying mechanism of the mitochondrial fusion promoter M1 in mitigating cigarette smoking (CS)-induced airway inflammation and oxidative stress both in vitro and in vivo models. Cigarette smoke extract (CSE)-treated airway epithelial cells (BEAS-2B) and CS-exposed mice were pretreated with M1, followed by the measurement of proinflammatory cytokines, oxidative stress, mitochondrial fusion proteins (MFN2 and OPA1) and fission proteins (DRP1 and MFF). Molecular pathways were elucidated through transcriptomic analysis and Western blotting. M1 pretreatment in CSE-treated cells significantly reduced the release of inflammatory cytokines (interleukin (IL)-6, IL-8 and tumor necrosis factor (TNF)-α); reduced malondialdehyde (MDA) and reactive oxygen species (ROS) levels; increased superoxide dismutase (SOD) activity; protected mitochondrial function by increasing the expression of mitochondrial fusion proteins (MFN2 and OPA1) and decreasing the expression of mitochondrial fission proteins (DRP1 and MFF). M1 attenuated CS-induced lung histologic damage and mucus hypersecretion in mice, relieved high oxidative stress and reduced the release of IL-6 and IL-8 in BALF. Similarly, it also protected mitochondrial function by regulating the CS-induced imbalance of mitochondrial dynamic proteins. Transcriptome sequencing and Western blotting showed that M1 inhibited CSE- or CS-induced activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT) signaling pathway. M1 plays a protective role in inflammation, oxidative stress and mitochondrial dynamics dysfunction caused by CS by inhibiting the PI3K-AKT signaling pathway; thus, it has therapeutic potential for the treatment of CS-induced airway disorders.

Protective effect of Auraptene, a novel acetylcholinesterase inhibitor, on hydrogen peroxide-induced cell toxicity in PC12 cells.

Alzheimer's disease (ad) is a progressive and degenerative disorder of the central nervous system that is associated with cognitive and memory impairment. The main factors which have been implicated in neurodegeneration of ad are oxidative stress and cholinergic neurons dysfunction. Here, we examined the effects of auraptene, a novel acetylcholinesterase (AChE) inhibitor, on hydrogen peroxide (H2O2)-induced cell death in PC12 cells. Thereby, we measured cell viability, intracellular reactive oxygen species (ROS) production, AChE inhibitory activity, cell damage and apoptosis with AlmarBlue, 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA), Ellman method, lactate dehydrogenase (LDH) release, propidium iodide (PI) staining and western blot analysis, respectively. H2O2 (150μM) resulted in the cell death and apoptosis while, pretreatment with auraptene (10, 20 and 50μM) significantly increased the viability (P<0.01), and at 5-50μM decreased ROS amount (P<0.05 and P<0.001). Pretreatment with auraptene (10, 20 and 50μM) lessened AChE activity (P<0.001), and at 20 and 50μM reduced the release of LDH (P<0.001), and at (10, 20 and 50μM) diminished the percentage of apoptotic cells (P<0.001). Also, pretreatment with auraptene at 10,20 and 50μM prevented from poly (ADP-ribose) polymerase (PARP) cleavage (P<0.001), and cytochrome c release (P<0.01 and P<0.001). The amount of caspase 3 activity (P<0.001) and survivin (P<0.001) were elevated after pretreatment of cells with auraptene at 10-50μM and 10 and 50μM. It seems that auraptene has the ability to slow down or stop H2O2-induced nerve cells death by reducing the activity of AChE and suppression of internal pathway of apoptosis.

Effect of imatinib on lipopolysaccharide‑induced acute lung injury and endothelial dysfunction through the P38 MAPK and NF-κB signaling pathways in vivo and in vitro.

The primary purpose of this study was to demonstrate the preventive effects of imatinib (IMA) on lipopolysaccharide (LPS)-induced inflammation in a mouse model of acute lung injury (ALI) and human umbilical vascular endothelial cells. LPS stimulation for 24h induced ALI and cell inflammation. The pathological results of the lungs were evaluated using the wet/dry weight ratio, pulmonary vascular permeability measurements, and myeloperoxidase immunohistochemistry. The expression of pro-inflammatory mediators was analyzed using RT-PCR and enzyme-linked immunosorbent assay. Protein levels were analyzed using western blotting. The structure of cell junctions was detected using immunofluorescence. IMA improved LPS-induced pulmonary pathological damage and reduced the lung wet/dry weight ratio and myeloperoxidase expression in the lung tissue. IMA decreased bronchoalveolar lavage fluid inflammatory cell count and the release of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and monocyte chemotactic protein 1 (MCP-1) in the blood. Pretreatment of human umbilical vascular endothelial cells with IMA significantly attenuated LPS-induced actin stress fiber formation and vascular endothelial-cadherin disruption. In addition, IMA downregulated the mRNA abundances of vascular cell adhesion molecule 1, intercellular adhesion molecule 1, IL-1β, IL-6, and tumor necrosis factor-α(TNF-α) expression. The phosphorylation of p65, nuclear factor-kappa B inhibitor alpha (IκBα), p38, extracellular signal-regulated kinase, and Jun N-terminal kinase induced by LPS were attenuated after IMA treatment in vivo and in vitro. IMA modulates the nuclear factor-kappa B and mitogen-activated protein kinase signaling pathways and the production of pro-inflammatory cytokines to prevent cellular damage due to LPS infection. These results indicate that IMA may be a potential modulator of LPS-induced ALI.

The actions of varenicline on alkaloids from Conium maculatum (poison hemlock), Lupinus sulphureus (sulphur lupine) and Nicotiana glauca (tree tobacco).

Evidence-based therapies to manage the clinical signs of intoxication caused by toxic plants in livestock are lacking. For that reason, the aim of this work was to develop a drug-based intervention for the management of clinical signs of piperidine alkaloid intoxication in livestock. The actions of anabasine, coniine, γ-coniceine, and two total alkaloid extracts from Lupinus sulphureus were compared in the presence and absence of the nicotinic acetylcholine receptor partial agonist varenicline in RD cells, mice and goats. Pretreatment of RD cells with 10.0μM varenicline significantly shifted the anabasine fifty percent effective concentration (EC50) value to a greater concentration and blocked the response of the cells to coniine. γ-coniceine did not have any effect on RD cells as measured by membrane potential sensing dye. Swiss Webster mice median lethal dose (LD50) values for anabasine, coniine, γ-coniceine were 1.5, 5.5, and 3.7mg/kg respectively, and pretreatment with 10.0mg/kg i. p. dosed varenicline shifted the LD50 values to 4.2, 9.1, and 4.3mg/kg respectively. The rodent LD50 value of the Pendelton, WA L. sulphureus quinolizidine alkaloid extract was shifted to a lesser concentration by varenicline while the Ritzville, WA L. sulphureus piperidine alkaloid extract was shifted to a greater concentration by varenicline. The clinical signs of intoxication in goats orally dosed with Conium maculatum were exacerbated by 0.5, 1.0 and 10.0mg/kg i. v. dosed varenicline. These results suggest that varenicline was effective at shifting piperidine alkaloid EC50 values in RD cells and increasing piperidine but not quinolizidine alkaloid LD50 values in mice and was not useful at managing the clinical signs of poison hemlock intoxication in goats.

Comprehensive transcriptomic analysis identifies cholesterol transport pathway as a therapeutic target of porcine epidemic diarrhea coronavirus

Porcine epidemic diarrhea virus (PEDV) is a highly contagious virus that poses a serious threat to the global pig industry. Despite extensive efforts, the mechanism underlying virus entry for PEDV remains elusive. In this study, we first identified PEDV-susceptible and non-susceptible cell lines by using PEDV spike pseudotyped vesicular stomatitis virus. Subsequently, we conducted a comprehensive transcriptomic analysis on these cell lines. Through integrating differential expression gene analysis with weighted gene co-expression network analysis, we identified the key pathways that are correlated with the PEDV entry. Our analysis revealed a strong correlation between cholesterol, sterols, and lipid transport with PEDV entry, suggesting a potential role for cholesterol transport in the PEDV entry. For further investigation, we treated Huh7, Vero and LLC-PK1 cells with a cholesterol transport inhibitor, ezetimibe, and observed a significant inhibition of PEDV entry and subsequent viral replication in these cells. Interestingly, pre-treating Huh7 cells with ezetimibe resulted in an increase in the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV) pseudoviruses. Moreover, we found that cholesterol could facilitate the entry of PEDV into Huh7 and Vero cells, and this promoting effect can be blocked by ezetimibe. These findings suggest that targeting cholesterol transport specifically inhibits PEDV entry into susceptible cells. Our study offers novel insights into the mechanism of PEDV entry and the development of new therapeutic strategies against this economically important virus.

Study of Host-Guest Interaction and In Vitro Neuroprotective Potential of Cinnamic Acid/Randomly Methylated β-Cyclodextrin Inclusion Complex.

Cinnamic acid (CA) has many beneficial effects on human health. However, its poor water solubility (0.23 g/L, at 25 °C) is responsible for its poor bioavailability. This drawback prevents its clinical use. To overcome the solubility limits of this extraordinary natural compound, in this study, we developed a highly water-soluble inclusion complex of CA with randomly methylated-β-cyclodextrin (RAMEB). The host-guest interaction was explored in liquid and solid states by UV-Vis titration, phase solubility analysis, FT-IR spectroscopy, and 1H-NMR. Additionally, molecular modeling studies were carried out. Both experimental and theoretical studies revealed a 1:1 CA/RAMEB inclusion complex, with a high apparent stability constant equal to 15,169.53 M-1. The inclusion complex increases the water solubility of CA by about 250-fold and dissolves within 5 min. Molecular modeling demonstrated that CA inserts its phenyl ring into the RAMEB cavity with its propyl-2-enoic acid tail leaning from the wide rim. Finally, a biological in vitro study of the inclusion complex, compared to the free components, was performed on the neuroblastoma SH-SY5Y cell line. None of them showed cytotoxic effects at the assayed concentrations. Of note, the pretreatment of SH-SY5Y cells with CA/RAMEB at 10, 30, and 125 µM doses significantly counteracted the effect of the neurotoxin MPP+, whilst CA and RAMEB alone did not show any neuroprotection. Overall, our data demonstrated that inclusion complexes overcome CA solubility problems, supporting their use for clinical applications.

Human Umbilical Cord Mesenchymal Stem Cells Alleviate Diabetic Nephropathy by Inhibiting Ferroptosis via the JNK/KEAP1/NRF2 Signaling Pathway.

Aims: Diabetic nephropathy (DN) is a major cause of end-stage renal disease, with no therapeutic interventions available to control its progression. Ferroptosis, an iron-dependent regulated cell death characterized by lipid peroxidation, plays a pivotal role in the pathogenesis of DN. Human umbilical cord mesenchymal stem cells (hUCMSCs) are an effective treatment modality for DN; however, the underlying mechanism of action remains unclear. The aim of the present study was to investigate whether hUCMSCs alleviate DN via inhibiting ferroptosis and its molecular mechanisms in type 2 diabetic mice and high-glucose and palmitate-stimulated human renal tubular epithelial cell (HK-11) models. Results: Our findings revealed that hUCMSCs improved the renal structure and function and tubular injuries. HUCMSC treatment can inhibit ferroptosis by decreasing iron content, reducing reactive oxygen species, malondialdehyde and 4-hydroxynonenal generation, decreasing the expression of positive ferroptosis mediator transferrin receptor 1 and long-chain acyl-CoA synthetase 4, and enhancing the expression of negative ferroptosis mediators (i.e., ferritin heavy chain, glutathione peroxidase 4, and system Xc-cystine/glutamate reverse transporter). Mechanistically, hUCMSC treatment inhibited c-Jun N-terminal kinase (JNK) and Kelch-like ECH-associated protein 1 (KEAP1) activation while increasing the expression of nuclear factor erythroid 2-related factor 2 (NRF2). Furthermore, pretreatment of HK-11 cells with NRF2 siRNA, the JNK inhibitor SP600125, or the JNK agonist anisomycin demonstrated the regulation of the JNK/KEAP1/NRF2 signaling pathway by hUCMSCs. Innovation and Conclusion: HUCMSCs inhibit ferroptosis in DN via the JNK/KEAP1/NRF2 signaling pathway, providing a new perspective and scientific evidence for treating DN. Antioxid. Redox Signal. 00, 000-000.

Two Disaccharide-Bearing Polyethers, K-41B and K-41Bm, Potently Inhibit HIV-1 via Mechanisms Different from That of Their Precursor Polyether, K-41A.

The screening of novel antiviral agents from marine microorganisms is an important strategy for new drug development. Our previous study found that polyether K-41A and its analog K-41Am, derived from a marine Streptomyces strain, exhibit anti-HIV activity by suppressing the activities of HIV-1 reverse transcriptase (RT) and its integrase (IN). Among the K-41A derivatives, two disaccharide-bearing polyethers-K-41B and K-41Bm-were found to have potent anti-HIV-1IIIB activity in vitro. This study aimed to clarify whether K-41B and K-41Bm have inhibitory effects on different HIV-1 strains or whether these two derivatives have mechanisms of action different from that of their precursor, K-41A. An anti-HIV-1 assay indicated that K-41B and K-41Bm have potent anti-HIV-1BaL activity, with low 50% inhibitory concentrations (IC50s) (0.076 and 0.208 μM, respectively) and high selective indexes (SIs) (58.829 and 31.938, respectively) in the peripheral blood mononuclear cell (PBMC)-HIV-1BaL system. The time-of-addition (TOA) assay indicated that K-41B and K-41Bm may exert antiviral effects by activating multiple stages of HIV-1 replication. A cell protection assay indicated that the pretreatment of cells with K-41B or K-41Bm has almost no inhibitory effect on HIV-1 infection. A virus inactivation assay indicated that pretreatment of the virus with K-41B or K-41Bm inhibits HIV-1 infection by 60%. A cell-cell fusion assay showed that K-41B and K-41Bm blocked the cell fusion mediated by viral envelope proteins. The HIV-1 key enzyme experiment also indicated that both compounds have certain inhibitory effects on HIV-1 IN. Furthermore, molecular docking showed that K-41B and K-41Bm interact with several viral and host proteins, including HIV-1 IN, an envelope protein (gp120), a transmembrane protein (gp41), and cell surface receptors (CD4, CCR5, and CXCR4). Overall, in addition to having a similar anti-HIV-1 mechanism of inhibiting HIV-1 IN like the precursor polyether K-41A, the disaccharide-bearing polyether derivatives K-41B and K-41Bm may also inhibit viral entry. This suggests that they display anti-HIV-1 mechanisms that are different from those of their precursor polyethers.

2′-Hydroxycinnamaldehyde induces ROS-mediated apoptosis in cancer cells by targeting PRX1 and PRX2

2′-Hydroxycinnamaldehyde (HCA) is a component of the commonly used spice cinnamon, which has beneficial effects on cancer, allergies, bacterial/viral infections, and Alzheimer’s disease. Our previous study showed that HCA induced reactive oxygen species (ROS) and apoptosis in cancer cells, and pretreatment of cancer cells with antioxidants abolished HCA-mediated ROS production and apoptosis. This indicates that ROS are critical effector for HCA activity. However, the molecular target of HCA for ROS induction has not been identified. In the present study, we identified peroxiredoxin 1 (PRX1) and peroxiredoxin 2 (PRX2) as target proteins of HCA using affinity chromatography, and further confirmed these association using a cellular thermal shift assay (CETSA). In addition, we used mutagenesis to identify important cysteine residues in PRX1 for HCA binding. PRX1 has four cysteines (Cys52, Cys71, Cys83, and Cys173), and when Cys173 (but not the other cysteine sites) was mutated to serine, it was unable to bind biotin-conjugated HCA, suggesting that Cys173 is important for HCA binding. Treatment of SW620 cancer cells transfected by control vector with 20 μM HCA increased ROS levels by 5.2-fold compared to DMSO-treated cells. However, downregulation of target proteins PRX1 and PRX2 using shRNAs (short hairpin RNA) significantly reduced HCA-mediated ROS induction (1.6-fold), supporting that PRX1 and PRX2 are targets of HCA for ROS elevation. Additionally, intraperitoneal injection of 50 mg/kg HCA inhibited SW620 tumor growth, resulting in a 59.9 % reduction in tumor volume. CETSA analysis of tumor tissues showed that PRX1 and PRX2 were bound and thus inactivated by HCA in a mouse xenograft model. These findings demonstrate that PRX1 and PRX2 are molecular target proteins responsible for HCA-induced ROS elevation and cancer cell death.

Evaluation of the Immunoadjuvant Effects of miR-155-Chitosan Polyplex on Leishmania major Infected Mice

ABSTRACT Background MicroRNAs have gained attention as key immunomodulators, with miR-155 specifically shown in various studies to drive macrophage polarization toward the classical phenotype. This polarization is crucial, as classical macrophages play a well-recognized role in differentiating type-1 immune responses and resisting Leishmania infection. Objective The present study aims to evaluate the anti-leishmanial immunoadjuvant effects of the miR-155 chitosan polyplex (miR-155 CP). Methods The anti-leishmanial immunoadjuvant activity of miR-155 CP synthesized by the coacervation method was assessed against L. major (MRHO/IR/75/ER) by analyzing the infectivity rate on RAW 264.7 cells in vitro.MiR-155 CP as an adjuvant co-administrated with soluble Leishmania antigen (SLA) for immunization of BALB/c mice, then the challenge was performed by subcutaneous injection of 1 × 106 L. major promastigotes. Eight weeks following the challenge, lesion size, parasite load, cytokine assay, and nitric oxide production were evaluated. Results The nanoparticles were produced with a size of 233.87 ± 8 nm and a zeta potential of + 22.6 ± 2 mV with good transfection efficiency. The mean infection index among pretreated cells with miR-155 CP (72±1.1) decreased significantly compared to the control group (420 ± 2.8). The parasite burden and the size of the lesions were significantly reduced in the immunized infected mice. Vaccination by miR-155 CP/SLA triggered the production of IFN-γ and NO and changed the cytokine profile of antigen-specific cells.Conclusion:The effectiveness of the SLA vaccine can be enhanced by including miR-155 CP as an adjuvant. SLA and miR-155 CP co-administration improve the type-1 immune response. This enhanced immune response helps prevent severe leishmaniasis.

Dendrobium nobile Lindl. alkaloids protect CCl4-induced acute liver injury via upregulating LAMP1 expression and activating autophagy flux.

Dendrobium nobile Lindl. alkaloids (DNLA) are considered important active ingredients of Dendrobium, which have a variety of pharmacological functions. Recent studies indicate that DNLA has beneficial activity in acute liver injury. However, the specific mechanism by which DNLA produces liver protective effects is stills unclear. This study was designed to determine whether regulation of autophagy is involved in the mode of action of DNLA in liver protection. Using CCl4-induced acute liver injury (ALI) and cell culture models, the molecular mechanism of DNLA-mediated autophagy regulation was studied. The results showed that DNLA significantly improved CCl4-induced liver damage and oxidative stress, which was confirmed in AML-12 cells. DNLA promoted autophagy in cells treated with CCl4, manifested by reduced protein expressions of p62 and LC3-II. Fluorescence imaging showed a decrease in the number of autophagosomes in AML-12 cells transfected with mCherry-GFP-LC3B. In addition, DNLA inhibited lysosomal membrane permeabilization by upregulating lysosomal associated membrane protein-1 (LAMP1), thereby promoting autophagy, preventing CCl4-induced mitochondrial dysfunction, and reducing the production of mitochondrial reactive oxygen species (ROS). While pretreatment of cells with lysosomal inhibitor chloroquine weakened mitochondrial protection elicited by DNLA, overexpression of mitochondrial-targeted SOD2 in AML-12 cells significantly blocked CCl4 induced downregulation of LAMP1, thereby improving lysosome integrity and promoting lysosome dependent autophagy, suggesting that there may exist a bidirectional regulation between mitochondrial ROS and lysosome-autophagy activation. Collectively, these results demonstrated that DNLA can protect the liver injury mediated by dysregulation of lysosome-autophagy process through promoting ROS-lysosome-autophagy axis and improving mitochondrial damage.

STEM-09. A GROUP 3 MEDULLOBLASTOMA STEM CELL PROGRAM IS MAINTAINED BY OTX2-MEDIATED ALTERNATIVE SPLICING

Abstract Group 3 medulloblastomas (MBs) are highly metastatic pediatric brain tumors with limited therapeutic options. The transcription factor orthodenticle homeobox 2 (OTX2) is a known driver and molecular hallmark of Group 3 MB. We previously demonstrated that OTX2 regulates Group 3 MB cell fate decisions that favor self-renewal or stemness and the retention of a primitive cerebellar rhombic lip signature. Here, we show that OTX2-regulated alternative splicing (AS) is a major driver of Group 3 MB progression. Surprisingly, the functional role of post-transcriptional events, such as AS, in Group 3 MB development has never been explored. This gene regulatory layer represents an untapped source of therapeutic targets. Using a combination of TurboID, RNA-sequencing and single cell RNA-seq, we characterized the Group 3 primary MB AS landscape with a focus on genes that control MB stem cells. OTX2 associates with the multimeric Large Assembly of Splicing Regulators (LASR) complex through protein-protein interactions and can directly or indirectly bind RNA. Together, OTX2 and LASR complex proteins oversee a pro-tumorigenic splicing program that is mirrored in the human cerebellar rhombic lip origins and is associated with Group 3γ MBs that exhibit the worst prognosis. Among our core set of OTX2-regulated spliced genes, periphilin 1 (PPHLN1) is expressed in the most primitive cerebellar rhombic lip stem cells, and mimicking PPHLN1 AS by pre-treating cells with a splice blocking antisense morpholino reduces tumor growth while increasing survival in vivo. We propose that OTX2-mediated AS is a key determinant of cell fate decisions favoring the maintenance of the stem cell state. Targeting transcription factors like OTX2 is notoriously difficult; thus, our work offers fresh opportunities to exploit MB-specific AS events therapeutically to abrogate tumor progression.