Dose-dependent neuroprotective effects of adipose-derived mesenchymal stem cells on amyloid ß-induced Alzheimer's disease in rats.

AIM: Mesenchymal stem cells (MSCs) have emerged as an intriguing candidate in cell therapy for treating neurodegenerative diseases, including Alzheimer's disease (AD). To achieve the maximum efficiency of cell therapy, determining the optimal dose of MSCs is essential. This study was conducted to assess the dose-dependent therapeutic response of MSCs against pathological and behavioral AD-associated alterations. METHODS: Aß1-42 was injected intrahippocampally to establish an AD rat model. The MWM test was utilized to evaluate the animal's behavioral functions after receiving low and high doses of MSCs in the hippocampus region. ELISA and RT-qPCR were also employed to assess the concentration of markers related to antioxidant activity and inflammation and the gene expression related to apoptosis in the hippocampus region, respectively. RESULTS: Low-dose MSC transplantation by increasing the concentrations of the antioxidant GSH, the anti-inflammatory cytokine IL-10, as well as by lowering the concentrations of TNF-α, and the expression levels of apoptotic factors (Bax and caspase 3), exerted a neuroprotective effect in the hippocampus of AD rats and relatively ameliorated spatial learning and memory impairments. However, increasing the dose of MSCs decreased the therapeutic benefits of these cells and had no significant effect on the recovery of behavioral disorders. CONCLUSION: Our findings reveal the dose-dependent neuroprotective effect of MSCs in AD. The therapeutic response of MSCs to ameliorate the pathological and behavioral alterations associated with AD is attenuated when the dosage of MSCs is increased.

Preconditioning adipose-derived mesenchymal stem cells with dimethyl fumarate promotes their therapeutic efficacy in the brain tissues of rats with Alzheimer's disease.

AIM: Transplantation of mesenchymal stem cell (MSC) has been suggested to be a promising method for treating neurodegenerative conditions, including Alzheimer's disease (AD). However, the poor survival rate of transplanted MSCs has limited their therapeutic application. This study aimed to evaluate whether preconditioning MSCs with dimethyl fumarate (DMF), a Nrf2 inducer, could enhance MSC therapeutic efficacy in an amyloid-ß (Aß1-42)-induced AD rat model. METHODS: The survival and antioxidant capacity of MSCs treated with DMF were assessed in vitro. Aß1-42 intrahippocampal injection was used to create a rat model of AD. Following the transplantation of MSCs preconditioned with DMF and using the Morris blue maze test, spatial learning and memory were assessed. Using RT-qPCR, we evaluated the gene expression related to apoptosis and neurotrophins in the hippocampus region. RESULTS: Treatment with DMF enhanced cell survival and Nrf2 protein expression in MSCs in vitro. Preconditioning with DMF also enhanced the efficacy of transplanted MSCs in rescuing learning and spatial memory deficits in Aß-AD rats. Besides, DMF preconditioning enhanced the neuroprotective effect of transplanted MSCs in the hippocampus of rats treated with Aß1-42 by decreasing the expression of apoptotic markers (Bax, caspase 3, and cytochrome c), and elevating the expression of the anti-apoptotic marker Bcl2 and neurotrophins, including BDNF and NGF. CONCLUSION: Preconditioning MSCs with DMF boosted the therapeutic efficacy of these cells; therefore, it could serve as a targeted strategy for increasing the therapeutic efficacy of MSCs in treating neurodegenerative disorders, including AD.

Effective inhibition of breast cancer stem cell properties by quercetin-loaded solid lipid nanoparticles via reduction of Smad2/Smad3 phosphorylation and ß-catenin signaling pathway in triple-negative breast cancer.

BACKGROUND: The presence of cancer stem cells (CSCs) is a major cause of resistance to cancer therapy and recurrence. Triple-negative breast cancer (TNBC) is a subtype that responds poorly to therapy, making it a significant global health issue. Quercetin (QC) has been shown to affect CSC viability, but its low bioavailability limits its clinical use. This study aims to increase the effectiveness of QC in inhibiting CSC generation by using solid lipid nanoparticles (SLNs) in MDA-MB231 cells. MATERIALS AND METHODS: After treating MCF-7 and MDA-MB231 cells with 18.9 µM and 13.4 µM of QC and QC-SLN for 48 h, respectively, cell viability, migration, sphere formation, protein expression of ß-catenin, p-Smad 2 and 3, and gene expression of EMT and CSC markers were evaluated. RESULTS: The QC-SLN with particle size of 154 nm, zeta potential of -27.7 mV, and encapsulation efficacy of 99.6% was found to be the most effective. Compared to QC, QC-SLN significantly reduced cell viability, migration, sphere formation, protein expression of ß-catenin and p-Smad 2 and 3, and gene expression of CD44, zinc finger E-box binding homeobox 1 (ZEB1), vimentin, while increasing the gene expression of E-cadherin. CONCLUSIONS: Our findings demonstrate that SLNs improve the cytotoxic effect of QC in MDA-MB231 cells by increasing its bioavailability and inhibiting epithelial-mesenchymal transition (EMT), thereby effectively inhibiting CSC generation. Therefore, SLNs could be a promising new treatment for TNBC, but more in vivo studies are needed to confirm their efficacy.

Quercetin-loaded solid lipid nanoparticles exhibit antitumor activity and suppress the proliferation of triple-negative MDA-MB 231 breast cancer cells: implications for invasive breast cancer treatment.

BACKGROUND: Quercetin (QC) is a naturally occurring flavonoid found in abundance in fruits and vegetables. Its anti-cancer and anti-inflammatory properties have been previously demonstrated, but its low bioavailability hampers its clinical use. Triple-negative breast cancer is a subtype of breast cancer with a poor response to chemotherapy. This study investigates the anti-cancer effects of quercetin-solid lipid nanoparticles (QC-SLN) on the triple-negative breast cancer cell line MDA-MB231. MATERIALS AND METHODS: MCF-7 and MDA-MB231 cells were treated with 18.9 µM of QC and QC-SLN for 48 h. Cell viability, apoptosis, colony formation assay, and the anti-angiogenic effects of the treatment were evaluated. RESULTS: QC-SLN displayed optimal properties (particle size of 154 nm, zeta potential of -27.7 mV, encapsulation efficiency of 99.6%, and drug loading of 1.81%) and exhibited sustained release of QC over 72 h. Compared to the QC group, the QC-SLN group showed a significant decrease in cell viability, colony formation, angiogenesis, and a substantial increase in apoptosis through the modulation of Bax and Bcl-2 at both gene and protein levels. The augmentation in the proportion of cleaved-to-pro caspases 3 and 9, as well as poly (ADP-ribose) polymerase (PARP), under the influence of QC-SLN, was conspicuously observed in both cancer cell lines. CONCLUSIONS: This study showcases quercetin-solid lipid nanoparticles (QC-SLN) as a promising therapy for triple-negative breast cancer. The optimized QC-SLN formulation improved physicochemical properties and sustained quercetin release, resulting in reduced cell viability, colony formation, angiogenesis, and increased apoptosis in the MDA-MB231 cell line. These effects were driven by modulating Bax and Bcl-2 expression, activating caspases 3 and 9, and poly (ADP-ribose) polymerase (PARP). Further in vivo studies are needed to confirm QC-SLN's efficacy and safety.

The Effect of Platelet-Rich Plasma on the Osteoblastic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stromal Cells.

BACKGROUND: Mesenchymal stem cells (MSCs) are cell populations that have the potential to proliferate and differentiate. The process of stem cell differentiation from pluripotent cells to bone cells requires general changes in their pattern of gene expression, the most well-known of which are changes in miRNA-dependent settings. Platelet-enriched plasma (PRP) releases growth factors that are mitogenic to mesenchymal cells and can accelerate the process of osteogenic differentiation. The aim of this study was to investigate the effect of PRP on the expression changes of Let-7a, mir-27a, mir-31, mir-30c, mir-21, and mir-106a during osteogenic differentiation. METHODS: MSCs were isolated from adipose tissue after abdominoplasty and evaluated by flow cytometry. The ef-fect of PRP (10%) on the process of osteogenic differentiation was determined by measuring the expression of Let-7a, mir-27a, mir-31, mir-30c, mir-21, and mir-106a using the real-time polymerase chain reaction (PCR) technique. RESULTS: The increase in Let-7a expression was significant on the 14th day compared to the 3rd day. mir-27a expression rose significantly on the 3rd day. The expression of mir-30 exhibited a significant increase on the 14th day. mir-21 expression was significantly enhanced on the 3rd day and was downregulated on the 14th day. mir-106a expression showed a significant decreasing tendency between days 3 and 14 in a time-dependent pattern. CONCLUSIONS: These findings indicate that PRP probably accelerates the process of differentiation into bone. PRP, as a biological catalyst, showed a clear and distinct impact on the miRNAs regulating bone differentiation of human mesenchymal cells.

Dimethyl itaconate reprograms neurotoxic to neuroprotective primary astrocytes through the regulation of NLRP3 inflammasome and NRF2/HO-1 pathways.

The activation of neurotoxic reactive astrocytes contributes to the pathogenesis of many neurodegenerative diseases. Itaconate, a product of cellular metabolism, is released from activated macrophage/microglia and has been shown to regulate inflammatory responses in several mammalian cells. This study was designed to investigate the impact of cell-permeable dimethyl itaconate (DI) on reactive astrocyte-dependent neurotoxicity. Primary murine astrocyte cells were isolated and stimulated with lipopolysaccharide (LPS) to generate reactive astrocytes. Treating these activated cells with DI was able to diminish the neurotoxic phenotype of reactive astrocytes, as we found reduced LPS-induced Nod-like receptor protein 3 (NLRP3) inflammasome activation and interleukin-1ß (IL-1ß) secretion. DI reduced the level of inflammasome components, attenuated inflammasome assembly and subsequently reduced caspase-1 cleavage and IL-1ß levels. Additionally, DI attenuated nuclear factor-kappa B (NF-κB) phosphorylation in LPS-activated astrocytes and also protected astrocytes from LPS-induced cytotoxicity, including a lowering of Bax and caspase3. DI-treated reactive astrocytes showed an elevated GSH/GSSG ratio and improved antioxidant defense factors including catalase and superoxide dismutase, while lipid peroxidation was reduced. We found that DI activated the nuclear factor 2 (NRF2) and heme oxygenase-1 (HO-1) pathway in astrocytes and thereby potentially control redox-regulation and the inflammatory state of astrocytes. Collectively, these results indicate the neuroprotective role of DI by reprogramming astrocytes from neurotoxic A1 to neuroprotective A2 states and thereby reveal a novel potential strategy for the treatment of neurodegenerative diseases.

Effects of UHDR and Conventional Irradiation on Behavioral and Cognitive Performance and the Percentage of Ly6G+ CD45+ Cells in the Hippocampus.

We assessed the effects of conventional and ultra-high dose rate (UHDR) electron irradiation on behavioral and cognitive performance one month following exposure and assessed whether these effects were associated with alterations in the number of immune cells in the hippocampus using flow cytometry. Two-month-old female and male C57BL/6J mice received whole-brain conventional or UHDR irradiation. UHDR mice were irradiated with 9 MeV electrons, delivered by the Linac-based/modified beam control. The mice were irradiated or sham-irradiated at Dartmouth, the following week shipped to OHSU, and behaviorally and cognitively tested between 27 and 41 days after exposure. Conventional- and UHDR-irradiated mice showed impaired novel object recognition. During fear learning, conventional- and UHDR-irradiated mice moved less during the inter-stimulus interval (ISI) and UHDR-irradiated mice also moved less during the baseline period (prior to the first tone). In irradiated mice, reduced activity levels were also seen in the home cage: conventional- and UHDR-irradiated mice moved less during the light period and UHDR-irradiated mice moved less during the dark period. Following behavioral and cognitive testing, infiltrating immune cells in the hippocampus were analyzed by flow cytometry. The percentage of Ly6G+ CD45+ cells in the hippocampus was lower in conventional- and UHDR-irradiated than sham-irradiated mice, suggesting that neutrophils might be particularly sensitive to radiation. The percentage of Ly6G+ CD45+ cells in the hippocampus was positively correlated with the time spent exploring the novel object in the object recognition test. Under the experimental conditions used, cognitive injury was comparable in conventional and UHDR mice. However, the percentage of CD45+ CD11b+ Ly6+ and CD45+ CD11b+ Ly6G- cells in the hippocampus cells in the hippocampus was altered in conventional- but not UHDR-irradiated mice and the reduced percentage of Ly6G+ CD45+ cells in the hippocampus might mediate some of the detrimental radiation-induced cognitive effects.

Enhancement of Radiation Therapy through Blockade of the Immune Checkpoint, V-domain Ig Suppressor of T Cell Activation (VISTA), in Melanoma and Adenocarcinoma Murine Models.

Radiation therapy (RT) has recently demonstrated promise at stimulating an enhanced immune response. The recent success of immunotherapies, such as checkpoint inhibitors, CART cells, and other immune modulators, affords new opportunities for combination with radiation. The aim of this study is to evaluate whether and to what extent blockade of VISTA, an immune checkpoint, can potentiate the tumor control ability of radiation therapy. Our study is novel in that it is the first comparison of two VISTA-blocking methods (antibody inhibition and genetic knockout) in combination with RT. VISTA was blocked either through genetic knockout (KO) or an inhibitory antibody and combined with RT in two syngeneic murine flank tumor models (B16 and MC38). Selected mRNA, immune cell infiltration, and tumor growth delay were used to assess the biological effects. When combined with a single 15Gy radiation dose, VISTA blockade via genetic knockout in the B16 model and via anti-VISTA antibodies in the MC38 model significantly improved survival compared to RT alone by an average of 5.5 days and 6.3 days, respectively (p < 0.05). The gene expression data suggest that the mechanism behind the enhanced tumor control is primarily a result of increased apoptosis and immune-mediated cytotoxicity. VISTA blockade significantly enhances the anti-tumor effect of a single dose of 15Gy radiation through increased expression and stimulation of cell-mediated apoptosis pathways. These results suggest that VISTA is a biologically relevant immune promoter that has the potential to enhance the efficacy of a large single radiation dose in a synergic manner.

Endothelin-1 mediated glycosaminoglycan synthesizing gene expression involves NOX-dependent transactivation of the transforming growth factor-ß receptor.

G protein-coupled receptor (GPCR) agonist endothelin-1 (ET-1) through transactivation of the transforming growth factor (TGF) ß receptor (TGFBR1) stimulates glycosaminoglycan (GAG) elongation on proteoglycans. GPCR agonists thrombin and lysophosphatidic acid (LPA) via respective receptors transactivate the TGFBR1 via Rho/ROCK dependent pathways however mechanistic insight for ET-1 transactivation of the TGFBR1 remains unknown. NADPH oxidase (NOX) generates reactive oxygen species (ROS) and is a signalling entity implicated in the pathogenesis of many diseases including atherosclerosis. If implicated in this pathway, NOX/ROS would be a potential therapeutic target. In this study, we investigated the involvement of NOX in ET-1/ET receptor-mediated transactivation of TGFBR1 to stimulate mRNA expression of GAG chain synthesizing enzymes chondroitin 4-O-sulfotransferase 1 (C4ST-1) and chondroitin sulfate synthase 1 (ChSy-1). The invitro model used vascular smooth muscle cells that were treated with pharmacological antagonists in the presence and absence of ET-1 or TGF-ß. Proteins and phosphoproteins isolated from treated cells were quantified by western blotting and quantitative real-time PCR was used to assess mRNA expression of GAG synthesizing enzymes. In the presence of diphenyliodonium (DPI) (NOX inhibitor), ET-1 stimulated phospho-Smad2C levels were inhibited. ET-1 mediated mRNA expression of GAG synthesizing enzymes C4ST-1 and ChSy-1 was also blocked by TGBFR1 antagonists, SB431542, broad spectrum ET receptor antagonist bosentan, DPI and ROS scavenger N-acetyl-L-cysteine. This work shows that NOX and ROS play an important role in ET-1 mediated transactivation of the TGFBR1 and downstream gene targets associated with GAG chain elongation. As ROS is involved in GPCR to protein tyrosine kinase receptor transactivation, the NOX/ROS axis presents as the first common biochemical target in all GPCR to kinase receptor transactivation signalling.

A single injection of vitamin D3 improves insulin sensitivity and ß-cell function but not muscle damage or the inflammatory and cardiovascular responses to an acute bout of resistance exercise in vitamin D-deficient resistance-trained males.

Vitamin D deficiency is now a recognised problem affecting multiple physiological functions. The aim of the present study was to evaluate the effect of a single dose of vitamin D3 injection on the inflammatory, muscular damage, metabolic and cardiovascular responses to an acute bout of resistance exercise (RE) in vitamin D-deficient resistance-trained males. Blood samples from fourteen vitamin D-deficient resistance-trained males were obtained during two separate trials: lower vitamin D (LVD) and higher vitamin D (HVD, after vitamin D3 injection). Metabolic, inflammatory, muscle damage and cardiovascular markers were evaluated at baseline, immediately and 1 h after RE. There were significant trial-by-time interactions for insulin and homeostatic model assessment of insulin resistance (HOMA-IR) which significantly (P < 0·05) declined for 1 h after RE in the HVD trial compared with the LVD trial. Homeostasis model assessment of ß-cell function (HOMA-ß) declines at 1 h post-RE in the HVD trial. There was also a time effect for blood sugar which significantly (P < 0·05) decreased and for creatine kinase, lactate dehydrogenase and IL-6 which increased significantly 1 h post-RE in both trials. There were no significant changes in other inflammatory and cardiovascular markers following both trials. A single injection of vitamin D3 improved insulin resistance and ß-cell function following RE in previously vitamin D-deficient resistance-trained males. Conversely, the injection did not change muscle damage and the inflammatory response to acute RE. Intramuscular vitamin D replacement may have key implications for the promotion of glucose metabolism and lowering the risk of diabetes in vitamin D-deficient individuals.

Signaling Crosstalk of FHIT, p53, and p38 in etoposide-induced apoptosis in MCF-7 cells.

Fragile histidine trail (FHIT) is a tumor suppressor in response to DNA damage which has been deleted in various tumors. However, the signaling mechanisms and interactions of FHIT with regard to apoptotic proteins including p53 and p38 in the DNA damage-induced apoptosis are not well described. In the present study, we used etoposide-induced DNA damage in MCF-7 as a model to address these crosstalks. The time course study showed that the expression of FHIT, p53, and p38MAPK started after 1 hour following etoposide treatment. FHIT overexpression led to increase p53 expression, p38 activation, and augmented apoptosis following etoposide-induced DNA damage compared to wild-type cells. However, FHIT knockdown blocked p53 expression, delayed p38 activation, and completely inhibited etoposide-induced apoptosis. Inhibition of p38 activity prevented induction of p53, FHIT, and apoptosis in this model. Thus, activation of p38 upon etoposide treatment leads to increase in FHIT and p53 expression. In p53 knockdown MCF-7, the FHIT induction was hampered but p38 activation was induced in lower doses of etoposide. In p53 knockdown cells, inhibition of p38 induced FHIT expression and apoptosis. Our data demonstrated that the exposure of MCF-7 cells to etoposide increases apoptosis through a mechanism involving the activation of the p38-FHIT-p53 pathway. Moreover, our findings suggest signaling interaction for these pathways may represent a promising therapy for breast cancer.

Reactive Oxygen Species and p38MAPK Have a Role in the Smad2 Linker Region Phosphorylation Induced by TGF-ß.

Background: Transforming growth factor-ß (TGF-ß) in addition to the C-terminal region can phosphorylate receptor-regulated Smads (R-Smads) in their linker region. The aim of the present study was to evaluate the role of signaling mediators such as NAD(P)H oxidases (reactive oxygen species [ROS] generators), ROS, and ROS-sensitive p38 mitogen-activated protein kinase (p38MAPK) in this signaling pathway in cultured human vascular smooth muscle cells (VSMCs). Methods: The present in vitro study was performed on human VSMCs. Proteins were detected by western blotting utilizing an anti-phospho-Smad2 (Ser245/250/255) rabbit polyclonal antibody and a horseradish peroxidase-labeled secondary antibody. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. The phospho-Smad2 linker region (pSmad2L) was detected in all the experimental groups: a control group (untreated group), a group treated with TGF-ß (2 ng/mL), and a group treated with TGF-ß plus different inhibitors. The data were normalized and presented as mean±SEM. The statistical analyses were performed using SPSS, version 16.0, and the nonparametric Kruskal-Wallis test. A P value smaller than 0.05 was considered statistically significant. Results: The VSMCs treated with TGF-ß (2 ng/mL) showed a time-dependent increase in the pSmad2L level. The highest level was observed at 15 minutes (P=0.03). The inhibitors of NAD(P)H oxidases (diphenyleneiodonium and apocynin) (P=0.04), ROS scavenger (N-acetylcysteine) (P=0.04), and p38MAPK inhibitor (SB-202190) (P=0.04) were able to reduce the increased level of the pSmad2L by TGF-ß. Conclusion: Our results suggested that NAD(P)H oxidases played an important role in the Smad2L phosphorylation in the human VSMCs. Furthermore, our results confirmed that ROS and p38MAPK were involved in this signaling pathway. Thus, TGF-ß via a ROS-dependent mechanism can transmit its signals to the pSmad2L.

Involvement of cdc42/Rho kinase in apoA-I-mediated cholesterol efflux through interaction between cytosolic lipid-protein particles and microtubules in rat astrocytes.

Activation of cdc42 reportedly enhances apoA-I-mediated cholesterol release through ATP-binding cassette transporter A1 (ABCA1). We examined the involvement of cdc42 and Rho kinase in intracellular cholesterol transport for release of cholesterol after the interaction between apoA-I and ABCA1 in astrocytes. Exogenously added apoA-I increased the GTP-bound form of cdc42 and enhanced Rho kinase activity in rat astrocytes. Suppression of ABCA1 expression by siRNA substantially repressed both cellular level of GTP-bound cdc42 and Rho kinase activity, indicating that these reactions require ABCA1. ApoA-I-mediated lipid release and Rho kinase activation were inhibited by not only Rho kinase inhibitor but also cdc42 siRNA. These findings suggest that cdc42 is activated by the interaction between apoA-I and ABCA1 and enhances cholesterol release through the activation of Rho kinase. ApoA-I increased the binding of Rock1, one of the Rho kinases, to reconstituted microtubule-like filaments (rMT). Y-27632 suppressed not only the association of rMT with the cytosolic lipid-protein particles (CLPP)-related proteins and lipids but also the intracellular transport of newly synthesized cholesterol to the plasma membrane in rat astrocytes treated with apoA-I without inhibiting cholesterol synthesis. Finally, cdc42 siRNA reduced apoA-I-induced interaction between rMT and major players in intracellular cholesterol trafficking, such as caveolin-1 and Rock1, suggesting a regulatory role of Rho family proteins in the apoA-I-mediated intracellular cholesterol transport. We conclude that ABCA1/cdc42/Rho kinase signaling is involved in apoA-I-induced intracellular cholesterol transport and apoA-I-mediated cholesterol release in rat astrocytes.

Carbon nanotubes induce cytotoxicity and apoptosis through increasing protein levels of Bax and ROS in mouse skin fibroblasts.

Background and purpose: Carbon nanotubes (CNTs) are a significant discovery in nanotechnology, with widespread applications in modern technology. However, there are concerns about their potential toxicity, particularly in skin cells. This study aimed to investigate the mechanisms by which CNTs induced cytotoxicity and apoptosis in mouse skin fibroblasts. Experimental approach: The mice skin fibroblasts were isolated and exposed to two types of CNTs at various concentrations and then analyzed for changes in viability, reactive oxygen species (ROS) production, the levels of Bcl-2-associated X protein (Bax), and lactate production. Findings/Results: The results demonstrated that CNTs reduced cell viability and increased ROS production in a dose-dependent manner. Additionally, the current study found that CNTs increased the protein levels of Bax, a pro-apoptotic protein, in mouse skin fibroblasts. Furthermore, it was observed a significant decrease in lactate production in cells exposed to CNTs. Conclusion and implications: The findings concluded that CNTs have the potential to be toxic substances for skin fibroblasts, which serve as the body's first line of defense. This is evidenced by their ability to increase the production of ROS and the protein levels of Bax, as well as reduce lactic acid levels. As lactic acid has been reported to have beneficial effects on skin collagen production, further studies are needed to fully understand the impact of carbon nanotube exposure on human skin health.

Paradoxical effect of Aß on protein levels of ABCA1 in astrocytes, microglia, and neurons isolated from C57BL/6 mice: an in vitro and in silico study to elucidate the effect of Aß on ABCA1 in the brain cells.

Impaired cholesterol metabolism has been reported in Alzheimer's disease. Since ABCA1 is one of the main players in the brain's cholesterol homeostasis, here we used the in-vitro and in-silico experiments to investigate the effect of Aß on ABCA1 protein levels in microglia, astrocytes, and neurons in mice. Microglia, astrocytes, and neurons were cultured and exposed to beta amyloid. ABCA1 in cell lysates was determined by Western blotting, and cholesterol efflux was measured in the conditioned media. Molecular docking, molecular dynamics simulations, and MM-GBSA analysis were conducted to gain a better understanding of the effects of Aß on ABCA1. In response to Aß, the protein levels of ABCA1 increase significantly in microglia, astrocytes, and neurons; however, its ability to enhance cholesterol efflux is diminished. Aß inhibited the function of ABCA1 by obstructing the extracellular tunnel that transports lipids outside the cell, as determined by molecular docking. MD simulation analysis validated these findings. Our results demonstrated that Aß could increase ABCA1 protein levels in various brain cells, regardless of cell type. Molecular docking, molecular dynamics simulation, and MM-GBSA studies indicate that Aß has a significant effect on the structural conformation of ABCA1, possibly interfering with its function. We believe that the conformational changes of ABCA1 will inhibit its ability to subsequently release cellular cholesterol. Aß may obstruct the extracellular tunnel of ABCA1, rendering it less accessible to proteases such as the calpain family, which may explain the increase in ABCA1 levels but decrease in its function.Communicated by Ramaswamy H. Sarma.

Investigating the Effect of Aspirin on apoAI-Induced ATP Binding Cassette Transporter 1 Protein Expression and Cholesterol Efflux in Human Astrocytes.

Background: Neurons need a high amount of cholesterol to maintain the stability of their membrane-rich structures. Astrocytes synthesize and distribute cholesterol to neurons, and ABCA1 is a key mediator of cholesterol efflux to generate HDL for cholesterol transport in the brain. Several studies imply the effect of aspirin on ABCA1 expression in peripheral cells such as macrophages. Here, we compared the effect of aspirin with apoA-I on ABCA1 protein expression and cholesterol efflux in human astrocytes. Materials and Methods: Human astrocytes were cultured, and the effects of aspirin on the expression and protein levels of ABCA1 were investigated through RT-PCR and Western blot analysis. Additionally, the effect of co-treatment with apoA-I and aspirin on ABCA1 protein level and cholesterol efflux was evaluated. Results: Dose and time-course experiments showed that the maximum effect of aspirin on ABCA1 expression occurred at a concentration of 0.5 mM after 12 h of incubation. RT-PCR and western blot data showed that aspirin upregulates ABCA1 expression by up to 4.7-fold and its protein level by 67%. Additionally, co-treatment with aspirin and apoA-I increased cholesterol release from astrocytes, indicating an additive effect of aspirin on apoAI-mediated cholesterol efflux. Conclusions: The results suggest a potential role of aspirin in increasing ABCA1 expression and cholesterol efflux in astrocytes, similar to the effect of apoA-I. This indicates that aspirin could potentially regulate brain cholesterol balance and can be considered in certain neurological diseases, in particular in some neurological disorders related to cholesterol accumulation such as Alzheimer's disease.

Anesthetic Oxygen Use and Sex Are Critical Factors in the FLASH Sparing Effect.

Purpose: Ultra High Dose-Rate (UHDR) radiation has been reported to spare normal tissue, compared with Conventional Dose-Rate (CDR) radiation. However, important work remains to be done to improve the reproducibility of the FLASH effect. A better understanding of the biologic factors that modulate the FLASH effect may shed light on the mechanism of FLASH sparing. Here, we evaluated whether sex and/or the use of 100% oxygen as a carrier gas during irradiation contribute to the variability of the FLASH effect. Methods and Materials: C57BL/6 mice (24 male, 24 female) were anesthetized using isoflurane mixed with either room air or 100% oxygen. Subsequently, the mice received 27 Gy of either 9 MeV electron UHDR or CDR to a 1.6 cm2 diameter area of the right leg skin using the Mobetron linear accelerator. The primary postradiation endpoint was time to full thickness skin ulceration. In a separate cohort of mice (4 male, 4 female), skin oxygenation was measured using PdG4 Oxyphor under identical anesthesia conditions. Results: Neither supplemental oxygen nor sex affected time to ulceration in CDR irradiated mice. In the UHDR group, skin damage occured earlier in male and female mice that received 100% oxygen compared room air and female mice ulcerated sooner than male mice. However, there was no significant difference in time to ulceration between male and female UHDR mice that received room air. Oxygen measurements showed that tissue oxygenation was significantly higher when using 100% oxygen as the anesthesia carrier gas than when using room air, and female mice showed higher levels of tissue oxygenation than male mice under 100% oxygen. Conclusions: The skin FLASH sparing effect is significantly reduced when using oxygen during anesthesia rather than room air. FLASH sparing was also reduced in female mice compared to male mice. Both tissue oxygenation and sex are likely sources of variability in UHDR studies. These results suggest an oxygen-based mechanism for FLASH, as well as a key role for sex in the FLASH skin sparing effect.

24-Hydroxycholesterol Moderates the Effects of Amyloid-ß on Expression of HMG-CoA Reductase and ABCA1 Proteins in Mouse Astrocytes.

Background: Elevated brain cholesterol increases the risk of Alzheimer's disease. Production of 24-hydroxycholesterol (24s-OHC) by neurons prevents cholesterol accumulation in the brain. In this study, we investigated the effect of 24s-OHC on the HMG-COA reductase and ABCA1 which are involved in the brain cholesterol homeostasis with or without ß-amyloid in astrocytes. Methods and Materials: Astrocytes were treated with 24s-OHC with or without Aß. Western blot and real-time polymerase chain reaction were done to detect protein and gene expression of ß-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) and ABCA1, respectively. Cholesterol release was determined using a quantitation kit. Results: Protein levels of HMGCR and ABCA1 were significantly increased by Aß; however, the 24s-OHC was able to restore their levels and diminish the effect of amyloid-ß. Aß did not have a significant effect on HMGCR expression, while 24s-OHC reduced it by 68%. Aß-induced ABCA1 expression did not increase cholesterol efflux as the lower levels of cholesterol in conditioned medium of Aß-treated cells were found. Conclusion: Our novel findings show that Aß affects two key elements in the brain cholesterol homeostasis, HMGCR and ABCA1, which are crucial in cholesterol synthesis and efflux. Since 24s-OHC could suppress the Aß effects on enhancement of HMGCR and ABCA1, therefore the cytochrome P450 46A1 (Cyp46A1), which is exclusively expressed in the central nervous system and responsible for producing of 24s-OHC, could consider as a therapeutic target in the cholesterol-related neurodegenerative diseases such as Alzheimer's disease.

Adipose Tissue-Derived Mesenchymal Stem Cells Alter Metabolites of Brain Cholesterol Homeostasis in An Alzheimer's Model.

OBJECTIVE: Disruption of cholesterol homeostasis in Alzheimer's disease (AD) plays a crucial role in disease pathogenesis, making it a potential therapeutic target. Mesenchymal stem cells (MSCs) show promise in treating cognitive impairment and provide a novel therapeutic approach. This study aims to investigate the effects of MSCs on specific metabolites associated with brain cholesterol homeostasis in an AD rat model. MATERIALS AND METHODS: In this experimental study, animals were divided into three groups: control, AD, and AD+MSCs. AD was induced using amyloid beta (Aß) and confirmed through the Morris water maze (MWM) behavioural test and Congo red staining. MSCs were extracted, characterised via flow cytometry, subjected to osteoblast and adipose differentiation, and injected intraventricularly. The cholesterol metabolite levels were measured using gas chromatography-mass spectrometry (GC)-MS and compared among the groups. RESULTS: Treatment with MSCs significantly improved memory function in the AD+MSCs group compared to the AD group and the number of beta-amyloid plaques decreased according to histological assessment. Disturbances in the brain cholesterol metabolites that included desmosterol, 7-dehydrocholesterol, 24S-hydroxycholesterol, 27-hydroxycholesterol and cholesterol were observed in the AD group compared to the control group. Treatment with MSCs resulted in significant alterations in the levels of these metabolites. CONCLUSION: The findings indicate that MSC therapy has the potential to improve AD by modulating brain cholesterol homeostasis and promoting the differentiation of stem cells into nerve cells. The results emphasize the importance of investigating the role of cholesterol metabolites in the context of MSC therapy to gain deeper insights into underlying mechanisms of the therapeutic efficacy of MSCs in AD.

Mesenchymal Stem Cells Therapy Led to the Improvement of Spatial Memory in Rats with Alzheimer's disease Through Changing the Expression of LncRNA TUSC7/ miR-449a/ PPARγ and CD36 Genes in the Brain Tissue.

Mesenchymal stem cells (MSCs) have the ability to phagocytize amyloid beta (Aß) plaques and lower inflammation through the activity of microglia. Peroxisome proliferator-activated receptor gamma (PPARγ) is a protein involved in reducing inflammation through the activity of microglia and the phagocytosis of Aß plaques by scavenger receptor CD36, in this study, the effect of MSCs therapy on memory function and plaques was investigated. A total of 24 adult male Wistar rats were randomly divided into three groups:1) the control group, 2) the Aß-treated group (Alzheimer's disease (AD)), and 3) the MSC-treated group (AD + MSC). After the treatment with Aß and MSCs, western blotting and real-time polymerase chain reaction (PCR) techniques were used to assess protein and gene expression levels, respectively. MSCs improved spatial learning and memory in the AD group (p ≤0.05). The expression levels of PPARγ, lncRNA TUSC7, and CD36 genes were significantly elevated in the group receiving MSCs compared to the AD group (p≤0.0001). Also, the expression level of miR-449a significantly decreased in the AD + MSC group (p≤0.0001). Moreover, western blot analysis revealed that PPARγ and CD36 protein levels were enhanced in the AD + MSC group compared to the AD group (p≤0.0001). MSC treatment led to the positive regulation of the PPARγ gene and its protein expression by ncRNAs, which could have a beneficial impact on CD36 protein levels, and subsequently, reduce the number of plaques in the cell recipient.