LDH Assay Research Articles

Impact of Foetal Haemoglobin on Disease Severity among Sickle Cell Subjects

Objective: Haemolysis is a common complication of Sickle cell disease, exhibiting considerable variability among affected individuals. Established haemolysis markers including lactate dehydrogenase activity (LDH) and bilirubin have been reported as reliable indicators of disease severity. Foetal haemoglobin plays a pivotal role in Sickle Cell Disease by neutralizing Haemoglobin-S (HbS) polymerization, consequently extending the lifespan of red blood cells and influencing overall clinical outcomes. This study aims to investigate the impact of foetal haemoglobin on disease severity among sickle cell subjects. Method: This study involved forty (40) sickle cell subjects and thirty (30) normal subjects. Blood samples from the participants were analysed for lactate dehydrogenase activity, bilirubin concentration, and foetal haemoglobin concentration. The alkali denaturation technique was employed for HbF analysis, while diagnostic kits were used for LDH and bilirubin assays. Results: Foetal haemoglobin concentrations below 10% have been defined as low. The HbF concentration of the sickle cell subjects was relatively low (5.23%) but significantly higher than the normal subjects (1.84%). Similarly, there was a significant elevation in markers of haemolysis in the sickle cell subjects compared to the normal subjects. Subjects with HbF>10% demonstrated a noteworthy increase in haemolysis markers compared to those with HbF<5%. Additionally, a significant inverse relationship was observed between HbF concentration and haemolysis markers. Conclusion: The study highlights a significant inverse correlation between foetal haemoglobin concentration and disease severity among sickle cell subjects and also contributes valuable insights to our understanding of sickle cell disease pathology.

YTHDF1-mediated sphingosine kinase 2 upregulation alleviates bupivacaine-induced neurotoxicity via the PI3K/AKT axis.

Bupivacaine (BUP), a long-acting local anesthetic, has been widely used in analgesia and anesthesia. However, evidence strongly suggests that excessive application of BUP may lead to neurotoxicity in neurons. Sphingosine kinase 2 (SPHK2) has been reported to exert neuroprotective effects. In this study, we intended to investigate the potential role and mechanism of SPHK2 in BUP-induced neurotoxicity in dorsal root ganglion (DRG) neurons. DRG neurons were cultured with BUP to simulate BUP-induced neurotoxicityin vitro. CCK-8, LDH, and flow cytometry assays were performed to detect the viability, LDH activity, and apoptosis of DRG neurons. RT-qPCR and western blotting was applied to measure gene and protein expression. Levels. MeRIP-qPCR was applied for quantification of m6A modification. RIP-qPCR was used to analyze the interaction between SPHK2 and YTHDF1. SPHK2 expression significantly declined in DRG neurons upon exposure to BUP. BUP challenge substantially reduced the cell viability and increased the apoptosis rate in DRG neurons, which was partly abolished by SPHK2 upregulation. YTHDF1, an N6-methyladenosine (m6A) reader, promoted SPHK2 expression in BUP-treated DRG neurons in an m6A-dependent manner. YTHDF1 knockdown partly eliminated the increase in SPHK2 protein level and the protection against BUP-triggered neurotoxicity in DRG neurons mediated by SPHK2 overexpression. Moreover, SPHK2 activated the PI3K/AKT signaling to protect against BUP-induced cytotoxic effects on DRG neurons. In sum, YTHDF1-mediated SPHK2 upregulation ameliorated BUP-induced neurotoxicity in DRG neurons via promoting activation of the PI3K/AKT signaling pathway.

Considerations about Cytotoxicity of Resin-Based Composite Dental Materials: A Systematic Review.

The dental material industry is rapidly developing resin-based composites (RBCs), which find widespread use in a variety of clinical settings. As such, their biocompatibility has gained increasing interest. This literature review presents a summary of research into the cytotoxicity of methacrylate-based composites published from 2017 to 2023. Subject to analysis were 14 in vitro studies on human and murine cell lines. Cytotoxicity in the included studies was measured via MTT assay, LDH assay, and WST-1 assay. The QUIN Risk of Bias Tool was performed to validate the included studies. Included studies (based entirely on the results of in vitro studies) provide evidence of dose- and time-dependent cytotoxicity of dental resin-based composites. Oxidative stress and the depletion of cellular glutathione (GSH) were suggested as reasons for cytotoxicity. Induction of apoptosis by RBCs was indicated. While composites remain the golden standard of dental restorative materials, their potential cytotoxicity cannot be ignored due to direct long-term exposure. Further in vitro investigations and clinical trials are required to understand the molecular mechanism of cytotoxicity and produce novel materials with improved safety profiles.

Substances Secreted by Lactobacillus spp. from the Urinary Tract Microbiota Play a Protective Role against Proteus mirabilis Infections and Their Complications.

Proteus mirabilis urinary tract infections can lead to serious complications such as development of urinary stones. Lactobacillus spp., belonging to the natural microbiota of the urinary tract, exhibit a number of antagonistic mechanisms against uropathogens, including the secretion of organic acids. In this study, we determined the anti-adhesion, anti-cytotoxicity and anti-crystallization properties of the substances secreted by Lactobacillus. For this purpose, membrane inserts with a pore diameter 0.4 μm were used, which prevent mixing of cultured cells, simultaneously enabling the diffusion of metabolic products. The intensity of crystallization was assessed by measuring the levels of Ca2+, Mg2+ and NH3 and by observing crystals using microscopic methods. The cytotoxicity of the HCV-29 cell line was determined using the LDH and MTT assays, and the impact of lactobacilli on P. mirabilis adhesion to the bladder epithelium was assessed by establishing CFU/mL after cell lysis. It was shown that in the presence of L. gasseri the adhesion of P. mirabilis and the cytotoxicity of the cells decreased. The degree of crystallization was also inhibited in all experimental models. Moreover, it was demonstrated that L. gasseri is characterized by the secretion of a high concentration of L-lactic acid. These results indicate that L-lactic acid secreted by L. gasseri has a significant impact on the crystallization process and pathogenicity of P. mirabilis.

Synthesis of homogeneous spherical selenium nanoparticles through a chemical method for cancer therapy applications

Oxidative stress is associated with pathologies affecting various organs or metabolic pathways. Thus, targeting oxidative stress might represent a valid therapeutic option. Selenium nanoparticles (SeNPs) are reported to exert antioxidant effects by many mechanisms. Our purpose was to assess in vitro on normal (MRC-5) and cancer (PANC-1) cell lines the potential of SeNPs for inducing cytotoxicity and redox modulation. They were synthesized through a chemogenic method and characterized through advanced microscopy techniques. SeNPs were spherical, with 100 nm average diameters and low dimension variability. Cancer and normal cells were exposed for 24 h to different concentrations of SeNPs ranging from 1 to 25 μg/mL. According to the LDH and MTT assay results, SeNPs treatment caused a more pronounced decrease in cancer cell viability compared to normal cells, suggesting a possible therapeutic benefit on tumors, thus supporting the hypothesis of therapeutic use of SeNPs with the benefit of cell type selectivity. Neither an elevation nor an inhibition of intracellular ROS production was detected in MRC-5 cells exposed to concentrations between 1 and 25 μg/mL SeNPs. The results of this study suggest that SeNPs could represent potential candidate for treatment of cancer, especially pancreatic adenocarcinoma.

Potential Protective Effects of Spirulina (Spirulina platensis) against In Vitro Toxicity Induced by Heavy Metals (Cadmium, Mercury, and Lead) on SH-SY5Y Neuroblastoma Cells

Spirulina, a filamentous microalga, is used all over the world as a nutraceutical dietary supplement. Recent studies have focused on examining its chelating activity and antioxidant properties, especially as a candidate for protection against neurotoxicity caused by heavy metals. The MTT test and LDH assay were used to examine the viability of the SH-SY5Y cells for 24, 48, and 72 h, to Cd, Hg, and Pb, individually or in combination with Spirulina, and the effects of necrotic cell death. In comparison to the control group, the viability of SH-SY5Y cells decreased after 24 h of exposure, with Cd being more toxic than Hg and Pb being less lethal. The effects of heavy metal toxicity on cell survival were ranked in order after 72 h under identical experimental circumstances as follows: Hg, Pb, and Cd. The viability of the cells was then tested after being exposed to Spirulina at doses of 5 at 50 (%v/v) for 24, 48, and 72 h, respectively. SH-SY5Y cells that had been treated with mixtures of heavy metals and Spirulina underwent the same assay. Cell viability is considerably increased by using Spirulina treatments at the prescribed periods and doses. Instead, the same procedure, when applied to SH-SY5Y cells, caused the release of LDH, which is consistent with the reduction in cell viability. We demonstrated for the first time, considering all the available data, that Spirulina 5, 25, and 50 (%v/v) enhanced the number of viable SH-SY5Y cells utilized as a model system for brain cells. Overall, the data from the present study provide a first insight into the promising positive role of Spirulina against the potentially toxic effects of metals.

Ursolic acid induces apoptosis and pyroptosis in Reh cells by upregulating of the JNK signalling pathway based on network pharmacology and experimental validation

ObjectiveTo explore the mechanism of ursolic acid (UA) against acute B lymphoblastic leukaemia (B-ALL) based on network pharmacological analysis, molecular docking and experimental verification. MethodsThe core targets, functional processes, and biological pathways of UA in B-ALL were predicted by network pharmacology and molecular docking. The efficacy and mechanism of UA against B-ALL were verified through in vitro experiments such as cell viability assays, CCK-8 assays, LDH assays, AO/EB staining, flow cytometry, and Western blot assays. ResultsNetwork pharmacology analysis of the core targets indicated that the effects of UA on B-ALL were related to programmed cell death (apoptosis and pyroptosis). Molecular docking results showed that FOS, CASP8, MAPK8, IL-1β and JUN were the key targets of UA against B-ALL. The MTS assay showed that UA decreased the viability of Reh cells in a concentration- and time-dependent manner. Cellular and Western blot experiments found that UA induced Reh cell apoptosis and pyroptosis by upregulating the JNK signalling pathway. ConclusionsOur findings demonstrated that UA could induce Reh cell apoptosis and pyroptosis by activating the JNK signalling pathway to exert anti-B-ALL effects. This indicates that UA may become a potential drug for the effective treatment of B-ALL.

Cell Line-Dependent Adhesion and Inhibition of Proliferation on Carbon-Based Nanofilms.

Disorganisation of the extracellular matrix (ECM) is strongly connected to tumor progression. Even small-scale changes can significantly influence the adhesion and proliferation of cancer cells. Therefore, the use of biocompatible nanomaterials capable of supporting and partially replenishing degraded ECM might be essential to recover the niche after tumor resection. The objective of this study was to evaluate the influence of graphene, graphene oxide, fullerene, and diamond nanofilms on breast cancer and glioblastoma grade IV cell lines. Nanomaterials were characterized using SEM and TEM techniques; zeta potential analysis was also performed. Nanofilms of graphene, fullerene, and diamond nanoparticles were also characterized using AFM. The toxicity was tested on breast cancer MDA.MB.231 and glioblastoma grade IV U-87 MG cell lines, using LDH assay and by counting stained dead cells in bioprinted 3D models. The following parameters were analyzed: proliferation, adhesion to the nanofilm, and adhesion to particular ECM components covered with diamond nanoparticles. Our studies demonstrated that nanofilms of graphene and diamond nanoparticles are characterized by cell-specific toxicity. Those nanomaterials were non-toxic to MDA.MB.231 cells. After applying bioprinted 3D models, diamond nanoparticles were not toxic for both cell lines. Nanofilms made of diamond nanoparticles and graphene inhibit the proliferation of MDA.MB.231 cells after 48 and 72 hours. Increased adhesion on nanofilm made of diamond nanoparticles was only observed for MDA.MB.231 cells after 30 and 60 minutes from seeding the cells. However, analysis of adhesion to certain ECM components coated with diamond nanoparticles revealed enhanced adhesion to tenascin and vitronectin for both tested cell lines. Our studies show that nanofilm made of diamond nanoparticles is a non-toxic and pro-adhesive nanomaterial that might stabilize and partially replenish the niche after breast tumor resection as it enhances the adhesion of breast cancer cells and inhibits their proliferation.

C9orf72 dipeptides trigger neuroinflammation via the NLRP3 inflammasome

AbstractBackgroundThe most common cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is a repeat expansion in C9orf72. The expansion is translated to produce five toxic dipeptides (DPRs), which aggregate in patient brain. Neuroinflammation is observed in patients, with increased microglial activation and elevated levels of pro‐inflammatory cytokines in CSF. However, the causes of this inflammation are unknown. Here, we investigated whether DPRs activate immune cells and contribute to neuroinflammation.MethodPrimary macrophages or microglia were treated with synthetic C9orf72 dipeptides and levels of pro‐inflammatory cytokines released into the culture media were analysed by ELISA and Western blot. Cytotoxicity was measured by LDH assay. Mouse organotypic hippocampal slice cultures were also treated with DPRs and cytokine release measured.ResultOne of the DPRs, GR, caused secretion of the pro‐inflammatory cytokine, interleukin (IL)‐1β, from macrophages, microglia and in hippocampal slice cultures. GR also caused significant toxicity. IL‐1β secretion was prevented by pre‐treatment with MCC950, a drug which inhibits the NLRP3 inflammasome. The inflammasome is a protein complex which forms in the cytosol of immune cells is response to “danger” stimuli, resulting in cleavage of caspase‐1 and subsequent release of IL‐1β. We confirmed that caspase‐1 was cleaved in macrophages following GR treatment. In addition, knockout of NLRP3 prevented IL‐1β secretion in response to GR. Finally, we showed that several drugs which are clinically approved for use in other conditions rescued GR‐induced NLRP3 activation.ConclusionHere, we demonstrate that C9orf72 dipeptides could directly contribute to neuroinflammation in FTD/ALS via the NLRP3 inflammasome. Furthermore, we demonstrate that this can be inhibited pharmacologically, highlighting NLRP3 as a potential therapeutic target in FTD/ALS.

AM1172 (a hydrolysis-resistant endocannabinoid analog that inhibits anandamide cellular uptake) reduces the viability of the various melanoma cells, but it exerts significant cytotoxic effects on healthy cells: an in vitro study based on isobolographic analysis.

Despite great advances in our understanding of the impact of cannabinoids on human organism, many of their properties still remain undetermined, including their potential antineoplastic effects. This study was designed to assess the anti-proliferative and cytotoxic effects of AM1172 (a hydrolysis-resistant endocannabinoid analog that inhibits anandamide cellular uptake) administered alone and in combinations with docetaxel (DOCX), paclitaxel (PACX), mitoxantrone (MTX) and cisplatin (CDDP) on various human malignant melanoma A375, FM55P, SK-MEL 28 and FM55M2 cell lines. In the MTT, LDH, and BrdU assays, the potency and safety of AM1172 when administered alone and in combinations with DOCX, PACX, MTX, and CDDP were determined. The isobolographic analysis revealed that combinations of AM1172 with PACX, DOCX, MTX, and CDDP exerted additive interactions, except for a combination of AM1172 with PACX in primary melanoma A375 cell line, for which synergy was observed (*p<0.05). Nevertheless, AM1172 when administered alone produced cytotoxic effects on healthy human melanocytes (HEMa-LP) and human keratinocytes (HaCaT), which unfortunately limits its potential therapeutic utility. AM1172 cannot be used separately as a chemotherapeutic drug, but it can be combined with PACX, DOCX, MTX, and CDDP, offering additive interactions in terms of the anti-proliferative effects in various malignant melanoma cell lines.

TGF-β Inhibitor SD-208 Enhances the Anti-Tumor Efficacy of CD19 CAR-T Cells through a Reduction of CAR-Treg in Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia

Background: Acute lymphoblastic leukemia (ALL) is an incurable hematological malignancy with poor prognosis. Chimeric antigen receptor T cells (CAR-T) therapy is a milestone in the immunotherapy of relapsed or refractory B cells ALL (r/r B-ALL), but most cases are inevitably relapsed after CAR-T therapy and the causes are complex. Our preliminary work, a phase II clinical trial (#NCT02735291) involving pediatrics and adults with r/r B-ALL who were treated with CD19 CAR-T, exhibited that the overall survival (OS) and relapse-free survival (RFS) in patients with higher Treg are lower, and higher Treg may preclude the efficacy of CAR T therapy in r/r B-ALL. Further analysis of the frequencies of Treg within CD4 + cells at CD19 CAR-T therapy in r/r B-ALL patients investigated that higher circulating Treg, especially 1 week after CD19 CAR-T cells infusion, was a poor predict indicator for CD19 CAR-T therapy in r/r B-ALL patients. Methods: In the present study, we investigated the significance of CAR-Treg in CD19 CAR-T cells in clinical response and prognosis, and explored an inhibitive effect of TGF-β inhibitor SD-208 on the proliferation and lethality of CAR-Treg. Regulatory T cells (Treg) in CD19 CAR-T cells were tested in 34 patients with r/r B-ALL received CD19 CAR-T cells therapy, and then its significance on the complete remission (CR) rate, relapse-free survival (RFS) and overall survival (OS) was validated respectively. Next, TGF-β inhibitor SD-208 was added or not added in the culturing protocols of CD19 CAR-T cells, then the proportion of Treg and CAR-Treg as well as the anti-tumor ability of CD19 CAR-T cells were analyzed and compared in vitro. Results: 1) Higher proportion of Treg (≥7.2%) in CAR-T cells was a negative factor affecting RFS (p=0.0120) and OS (p=0.0162) in the r/r B-ALL patients with CAR-T therapy. 2) CD4 +CD25 +CD127 low/- Treg proliferated from 7.01% to 25.26% after anti-CD3 and anti-CD28 Abs activation. PBMCs cultured with SD-208 exhibited lower Treg levels than those of control groups (15.73% vs. 29.08%). 3) CD4 +CD25 +CD127 low/- Treg levels stimulated by SD-208 in CD19 CAR-T cells were lower than those in control groups both 24 and 48 hours (38.68% vs. 40.47% in 24 hours, 23.58% vs. 36.19% in 48 hours), and the treatment for 48 hours was most obvious. CAR +CD4 +CD25 +CD127 low/- CAR-Treg levels in CD19 CAR-T cells decreased by SD-208 stimulating compared with controls especially 48 hours (35.07% vs. 37.31% in 24 hours, 20.94% vs. 34.41% in 48 hours). The results exhibit that about 90% Treg in CD19 CAR-T cells are CAR-Treg, and inhibiting TGF-β by TGF-β/SMAD inhibitor SD-208 reduce the proliferation of Treg especially CAR-Treg in CD19 CAR-T cells. 4) To determine the function to CD19 CAR-T cell, LDH activity was utilized for tumor cell damage assay. Human Burkitt's lymphoma cell line Raji (CD19 +) was co-cultured with CD19 CAR-T cell for 24 hours before LDH assay. The percent of cytotoxicity calculated depending on the LDH activities showed that the inhibition rate of SD-208 treated CD19 CAR-T cell was significantly higher than that of control. To further determine the function of CD19 CAR-T cell Granzyme B and Perforin were tested. The results exhibited that SD-208 treated CD19 CAR-T cell had higher rates of Granzyme B and Perforin in CD3 +, CD3 +CD4 +, CD3 +CD8 + cells than controls, and achieved more functional. Conclusions: Inhibition of TGF-β by TGF-β/SMAD inhibitor SD-208 inhibit the proliferation of Treg in PBMCs of r/r B-ALL patients. TGF-β inhibitor SD-208 might improve the anti-tumor ability of CD19 CAR-T cells via decreasing Treg and CAR-Treg in r/r B-ALL.

Cytotoxicity of Anagallis arvensis L. Extracts: Implications for Traditional Healing

Anagallis arvensis L. comprises two species, specifically Anagallis foemina Mill. For centuries, the Navarra region in Spain has employed primulaceae as traditional wound healing remedies. Previous studies have established the antimicrobial and COX-inhibitory properties of this species. However, despite the known cytotoxic effects at high doses or with prolonged oral administration, there is a lack of definitive research to establish their toxicity. In this study, cytotoxicity was assessed using spectrophotometric methods, including the MTT and LDH assays, on PC12 and DHD/K12PROb cells. Notably, DHD/K12PROb cells exhibited heightened sensitivity to the extracts compared to PC12 cells, resulting in reduced cell survival and increased cell damage (LDH release). Significant reductions in cell survival were observed with methanol extracts exceeding 80 mg/ml. These findings suggest that the reported toxic effects in traditional medicine may be attributed to the cytotoxic nature of these plants.

Actinomycin D synergizes with Doxorubicin in triple-negative breast cancer by inducing P53-dependent cell apoptosis.

There are three major subtypes of breast cancer, ER+, HER2+ and triple-negative breast cancer (TNBC), namely ER-, PR-, HER2-. TNBC is the most aggressive breast cancer with poor prognosis and no target drug up to now. Actinomycin D (ActD) is a bioactive metabolite of marine bacteria that has been reported to have antitumor activity. The aim of study is to investigate whether ActD has a synergetic effect on TNBC with Doxorubicin (Dox), the major chemotherapeutic drug for TNBC, and explore the underlying mechanism. TNBC cell lines HCC1937, MDA-MB-436 and nude mice were used in the study. Drug synergy determination, LDH assay, MMP assay, Hoechst 33342 staining, Flow cytometry, Flexible docking and CESTA assay were carried out. The expression of proteins associated with apoptosis was checked by Western blot and siRNA experiments were performed to investigate the role of P53 and PUMA induced by drugs. There was much higher apoptosis rate of cells in the ActD + Dox group than that in ActD group or Dox group. Expression of MDM2 and BCL-2 was reduced while expression of P53, PUMA and BAX were increased in the groups treated with ActD + Dox or Dox compared to the control group. Furthermore, P53 siRNA or PUMA siRNA tremendously abrogated the cell apoptosis in the groups treated by ActD, Dox and ActD + Dox. Flexible docking and CESTA showed that ActD can bind MDM2. ActD had a synergetic effect on TNBC with Dox via P53-dependent apoptosis and it may be a new choice for treatment of TNBC.

PH-Dependence Cytotoxicity Evaluation of Artepillin C against Tumor Cells.

Brazilian green propolis is a well-known product that is consumed globally. Its major component, Artepillin C, showed potential as an antitumor product. This study explored the impact of Artepillin C on fibroblast and glioblastoma cell lines, used as healthy and very aggressive tumor cell lines, respectively. The focus of the study was to evaluate the pH-dependence of Artepillin C cytotoxicity, since tumor cells are known to have a more acidic extracellular microenvironment compared to healthy cells, and Artepillin C was shown to become more lipophilic at lower pH values. Investigations into the pH-dependency of Artepillin C (6.0-7.4), through viability assays and live cell imaging, revealed compelling insights. At pH 6.0, MTT assays showed the pronounced cytotoxic effects of Artepillin C, yielding a notable reduction in cell viability to less than 12% among glioblastoma cells following a 24 h exposure to 100 µM of Artepillin C. Concurrently, LDH assays indicated significant membrane damage, affecting approximately 50% of the total cells under the same conditions. Our Laurdan GP analysis suggests that Artepillin C induces autophagy, and notably, provokes a lipid membrane packing effect, contributing to cell death. These combined results affirm the selective cytotoxicity of Artepillin C within the acidic tumor microenvironment, emphasizing its potential as an effective antitumor agent. Furthermore, our findings suggest that Artepillin C holds promise for potential applications in the realm of anticancer therapies given its pH-dependence cytotoxicity.

Endothelial junctional protein associated with coronary artery disease (JCAD) as a potential therapeutic target for acute ischemic stroke

Abstract Introduction Acute ischemic stroke (AIS) is a leading cause of mortality and long-time disability worldwide. Despite the increasing availability of early reperfusion, the neurological outcome of patients with AIS is still poor. Junctional protein associated with Coronary Artery Disease (JCAD) is a novel protein, associated to coronary artery disease, arterial thrombosis, atherosclerosis and inflammation. Yet, its role in acute ischemic stroke is unclear. Purpose To investigate the role of JCAD in the pathophysiology of brain acute ischemia and to explore its potential as a pharmacological target in AIS. Methods Transient Middle Cerebral Artery Occlusion (tMCAO) was inducede in mice with either global or endothelial-specific JCAD deletion, and in littermate controls. Stroke size was assessed ex-vivo 48 hours after reperfusion. For neurological assessment, RotaRod Test and Bederson score were recorded 24 and 48 hours after reperfusion. In vivo silencing of JCAD was achieved by intravenous injection of a JCAD small interfering RNA (siRNA) after tMCAO. JCAD silencing was performed in vitro in human brain microvascular endothelial cells (HBMVECs) using siRNA transfection, followed by hypoxia/reoxygenation (H/R) injury. Cell death and monolayer integrity were measured by LDH assay and trans-endothelial electrical resistance (TEER), respectively. Molecular mechanisms were investigated in vivo by immunohistochemistry and in vitro by Western blot, respectively. JCAD plasma levels were measured by ELISA in two independent cohorts of patients with AIS. Results The expression of JCAD was up-regulated in the ipsilateral hemisphere of stroke in wild-type mice. Both global and endothelial-specific JCAD knockout mice displayed reduced stroke size after tMCAO and an improved neurological outcome, similar to mice treated with post-ischemic JCAD silencing (Figure 1). In vitro, JCADsilenced HBMVECs showed a reduced cell death and an improved monolayer integrity after H/R injury, paralleled by a reduced expression of cellular adhesion molecules (CAMs). The intracellular messenger Akt was hyperphosphorylated in JCAD-silenced HBMVECs (Figure 2). The treatment with the Akt/PI3K inhibitor Wortmannin, retrieved the non-silenced phenotype in HBMVECs, following H/R. Circulating levels of JCAD were persistently increased in patients with AIS and higher levels of JCAD at the time of hospitalization were associated with higher inflammation and an increased risk of death 90 days after the event. Conclusions JCAD expression is associated with a larger brain damage in mice in vivo and with a higher mortality in patients. Mechanistic results suggest that JCAD plays a pivotal role in regulating the integrity of endothelium after a H/R injury, inducing cellular death and inflammation through the inhibition of the Akt/PI3K pathway. Thus, in vivo silencing of JCAD may represent a therapeutic target for the treatment of acute ischemic stroke, on top of early reperfusion.Figure 1Figure 2

Synthesis and antiglycation activity of 3-phenacyl substituted thiazolium salts, new analogs of Alagebrium.

After preliminary abinitio calculations, 3-phenacyl substituted thiazolium salts, analogs of Alagebrium, were synthesized and investigated invitro as glycation reaction inhibitors. The most part of investigations focused on the potential of the title compounds to attenuate the formation of fluorescent AGEs as well on their ability to disrupt the cross-linking formation among glycated proteins. Additionally, the capability of thiazolium salts to deglycate in the reaction of early glycation products with nitroblue tetrazolium was determined. Cytotoxicological properties of the title compounds were evaluated using LDH and MTT assays. The leader compound (3-[2-(biphenyl-4-yl)-2-oxoethyl]-1,3-thiazol-3-ium bromide) in a 50 mg/kg dose (p.o. 14 days) was further tested within an invivo carbonyl stress model (rats, methylglyoxal 86.25 mg/kg/d, i.p., 14 days). As a result, the leader-molecule revealed a high effectiveness against all three examined mechanisms of glycation reaction inhibition in invitro tests and was able to suppress capacity of methylglyoxal to form AGEs invivo.

Antagonizing pathological α-synuclein-mediated neurodegeneration by J24335 via the activation of immunoproteasome

The aggregation of misfolded proteins, such as α-synuclein in Parkinson's disease (PD), occurs intracellularly or extracellularly in the majority of neurodegenerative diseases. The immunoproteasome has more potent chymotrypsin-like activity than normal proteasome. Thus, degradation of α-synuclein aggregation via immunoproteasome is an attractive approach for PD drug development. Herein, we aimed to determine if novel compound, 11-Hydroxy-1-(8-methoxy-5-(trifluoromethyl)quinolin-2-yl)undecan-1-one oxime (named as J24335), is a promising candidate for disease-modifying therapy to prevent the pathological progression of neurodegenerative diseases, such as PD. The effects of J24335 on inducible PC12/A53T-α-syn cell viability and cytotoxicity were evaluated by MTT assay and LDH assay, respectively. Evaluation of various proteasome activities was done by measuring the luminescence of enzymatic activity after the addition of different amounts of aminoluciferin. Immunoblotting and real-time PCR were employed to detect the expression of various proteins and genes, respectively. We also used a transgenic mouse model for behavioral testing and immunochemical analysis, to assess the neuroprotective effects of J24335. J24335 inhibited wild-type and mutant α-synuclein aggregation without affecting the growth or death of neuronal cells. The inhibition of α-synuclein aggregation by J24335 was caused by activation of immunoproteasome, as mediated by upregulation of LMP7, and increased cellular chymotrypsin-like activity in 20S proteasome. J24335-enhanced immunoproteasome activity was mediated by PKA/Akt/mTOR pathway activation. Moreover, animal studies revealed that J24335 treatment markedly mitigated both the loss of tyrosine hydroxylase-positive (TH-) neurons and impaired motor skill development. This is the first report to use J24335 as an immunoproteasome enhancing agent to antagonize pathological α-synuclein-mediated neurodegeneration.

Smurf2-Mediated Ubiquitination of FOXO4 Regulates Oxygen-glucose Deprivation/Reperfusion-induced Pyroptosis of Cortical Neurons.

Smad ubiquitination regulatory factor 2 (Smurf2) has been observed to alleviate ischemia-reperfusion injury. This study sought to explore the molecular mechanism of Smurf2-mediated forkhead box O4 (FOXO4) ubiquitination in oxygen-glucose deprivation/ reperfusion (OGD/R)-induced pyroptosis of cortical neurons. Human cortical neurons (HCN-2) were subjected to OGD/R to establish a cell model of cerebral stroke. Smurf2, FOXO4, and doublecortin domain containing 2 (DCDC2) expressions were determined by RT-qPCR and Western blot. LDH release, pyroptosis-related proteins NLRP3, GSDMD-N, and cleaved-caspase-3, as well as inflammatory factors IL-1β and IL-18, were assessed by LDH assay kit, Western blot, and ELISA. The ubiquitination level of FOXO4 was determined by ubiquitination assay. The bindings of Smurf2 to FOXO4 and FOXO4 to DCDC2 were testified by Co-IP, ChIP, and dual-luciferase assays. Rescue experiments were designed to validate the role of FOXO4/DCDC2 in the pyroptosis of HCN-2 cells. Smurf2 was weakly expressed, while FOXO4 and DCDC2 were prominently expressed in OGD/R-treated HCN-2 cells. Smurf2 overexpression promoted LDH release, reduced NLRP3, GSDMD-N, and cleaved-caspase-3 proteins, and decreased IL-1β and IL-18 concentrations. Sumrf2 improved the ubiquitination level of FOXO4 to downregulate its protein level. FOXO4 is bound to the DCDC2 promoter to facilitate its transcription. Overexpression of FOXO4 or DCDC2 reversed the inhibition of Smurf2 overexpression on pyroptosis of OGD/Rtreated HCN-2 cells. Smurf2 overexpression facilitated the ubiquitination of FOXO4 to reduce its protein level, thereby suppressing DCDC2 transcription and restricting OGD/R-induced pyroptosis of cortical neurons.

Perfluorooctane sulfonate (PFOS), a novel environmental pollutant, induces liver injury in mice by activating hepatocyte ferroptosis

ObjectiveWe investigated the toxicological mechanism by which perfluorooctane sulfonate (PFOS) induces liver injury via ferroptosis. MethodsPrimary mouse hepatocytes were treated with LD50 = 55 M PFOS. Their cytotoxicity was detected by CCK-8 and LDH assays, while JC-1 staining was used to identify their mitochondrial membrane potential. GSH-Px, MDA, and SOD levels were determined using kits, and Fe2 + levels were determined using the FerroOrange probe and iron ion assay kit. The DCFH-DA probe was used to examine ROS levels, while Western blot was used to examine protein expressions. After treatment with the ferroptosis inhibitor YL939 and the ROS inhibitor BABTA, the ability of PFOS to induce ferroptosis was observed. In animal experiments, we examined the liver function and the degree of hepatic ferroptosis of mice treated with PFOS as well as their histopathological changes. ResultsPFOS could induce hepatocyte ferroptosis, while YL939 and BABTA treatment could inhibit PFOS-induced ferroptosis. PFOS could induce liver injury and increase ferroptosis levels in tissue, while treatment with YL939 and BABTA could inhibit liver injury. ConclusionPFOS can induce ferroptosis in hepatocytes and cause liver injury in mice, which is its toxicological mechanism.

Neurotoxicity study of ethyl acetate extract of Zanthoxylum armatum DC. on SH-SY5Y based on ROS mediated mitochondrial apoptosis pathway

Ethnopharmacological relevanceZanthoxylum armatum DC. (ZADC) is a traditional medicinal plant with various pharmacological activities and is widely used in China, Japan, India, and other regions. Previous studies have revealed that the methanol extract of ZADC can cause neurotoxicity symptoms in rats, such as drooling, decreased appetite, decreased movement, and increased respiratory rate. However, the basis of these toxic substances and the mechanism of neurotoxicity remain unclear. Aim of the studyTo evaluate the effects of ZADC on nerve cells and their damage mechanisms and discuss the possible toxic substance basis. Materials and methodsThe ethyl acetate extract of ZADC is obtained by extracting the methanol extract of ZADC with ethyl acetate. The Q-Orbitrap LC-MS/MS method was employed to analyze the chemical composition of the EA extract of ZADC. SH-SY5Y cells were incubated with different concentrations of the ethyl acetate extract of ZADC. The cytotoxicity of the extract was evaluated using CCK-8, LDH, and ROS assays, and the oxidative stress status of cells was assessed using MDA, GSH, and SOD. Cell apoptosis was detected using flow cytometry. Damage to mitochondrial function was evaluated by labeling mitochondria, ATP, and MMP with fluorescence. Cyto-C, Caspase-3, Caspase-9, Apaf-1, Bax, and reduced Bcl2 expression were measured to evaluate the activation of the mitochondrial apoptosis pathway. Finally, NAC intervention was used to detect changes in the relevant indicators. The activation of mitochondrial apoptosis pathway was evaluated by measuring Cyto-C, Caspase-3, Caspase-9, Apaf-1, and Bax and Bcl2 expression. Finally, NAC intervention was utilized to detect changes in the relevant indicators. ResultsAfter treating SY-SY5Y cells with EA extract from ZADC, cell viability decreased significantly, and the intracellular ROS level increased in a dose-dependent manner. Meanwhile, ZADC can cause cellular oxidative stress and increase MDA and SOD concentrations while decreasing GSH concentrations. It can also shorten the mitochondrial cristae and decrease the number of mitochondria. In contrast, it can reduce ATP synthesis in the mitochondria and mitochondrial membrane potential (MMP). Furthermore, it increased the apoptosis rate and the expression of Cyto-C, Caspase-3, Caspase-9, Apaf-1, and Bax and reduced Bcl2 expression. NAC intervention alleviated the reduction in SH-SY5Y cell survival and the accumulation of reactive oxygen species induced by the EA extract in ZADC. It also inhibits signaling pathways dominated by proteins, such as Cyto-C, reducing cell apoptosis and cytotoxicity. A total of 46 compounds were identified in the extracts. ConclusionsThe results suggest that EA extract of ZADC can induce the mitochondrial apoptotic pathway by accumulating ROS in cells, leading to apoptosis. Antioxidants had a good inhibitory and protective effect against cell damage caused by the EA extract of ZADC. The neurotoxic components of ZADC may be organic acids and compounds containing amino groups.