Cleaved Caspase-3 Levels Research Articles

FAM136A depletion induces mitochondrial stress and reduces mitochondrial membrane potential and ATP production.

FAM136A deficiency has been associated with Ménière's disease. However, the underlying mechanism of action of this protein remains unclear. We hypothesized that FAM136A functions in maintaining mitochondria, even in HepG2 cells. To better characterize FAM136A function, we analyzed the cellular response caused by its depletion. FAM136A depletion induced reactive oxygen species (ROS) and reduced both mitochondrial membrane potential and ATP production. However, cleaved caspase-9 levels did not increase significantly. We next investigated why the depletion of FAM136A reduced the mitochondrial membrane potential and ATP production but did not lead to apoptosis. Depletion of FAM136A induced the mitochondrial unfolded protein response (UPRmt) and the expression levels of gluconeogenic phosphoenolpyruvate carboxykinases (PCK1 and PCK2) and ketogenic 3-hydroxy-3-methylglutaryl-CoA synthases (HMGCS1 and HMGCS2) were upregulated. Furthermore, depletion of FAM136A reduced accumulation of holocytochrome c synthase (HCCS), a FAM136A interacting enzyme that combines heme to apocytochrome c to produce holocytochrome c. Notably, the amount of heme in cytochrome c did not change significantly with FAM136A depletion, although the amount of total cytochrome c protein increased significantly. This observation suggests that greater amounts of cytochrome c remain unbound to heme in FAM136A-depleted cells.

From Cell to Organ: Exploring the Toxicological Correlation of Organophosphorus Compounds in Living System.

Malathion is an organophosphate compound widely used as an insecticide in the agriculture sector and is toxic to humans and other mammals. Although several studies have been conducted at different level in different animal models. But there is no work has been conducted on the toxicological correlation from cellular to behavioral level on surviving species model. Addressing this gap through further research is essential for a comprehensive understanding of malathion's impact on biological systems, facilitating better risk assessment and management strategies. Current research systemically evaluated the effects of malathion on the central nervous system and peripheral immune cells using immunological techniques in the BALB/c mice models. For this, animals were placed inside an inhalation chamber containing malathion (dose of 89.5mg/ml/m3) for a specific exposure time. The group exposed for 6minutes has shown a significant change in plasma-neurotransmitter (serotonin, dopamine) levels and decreased expression of Tyrosine hydroxylase in striatum and SNPC region of brain. The depolarized mitochondria and increased level of cleaved caspase-3 level and mature neurons in DG, CA1 and CA3 were also observed in the brain. Peripheral blood analysis illustrated a decrease in total leukocyte count and an increased level of early apoptosis at the same time point. From neurobehavioral results a significant locomotor hyperactivity, restlessness, and risk-taking behavior was observed. Taken together, results from the current study indicate that exposure to malathion at prolonged time durations induces neuronal and immune cell toxicity, and its toxicity may be mediated via changes in neurotransmitter levels and metabolite concentrations.

Investigation of Anti-Apoptotic Effects and Mechanisms of Astragaloside IV in a Rat Model of Cerebral Ischemia-Reperfusion Injury.

Ischemic stroke is a prevalent and life-threatening cerebrovascular disease that is challenging to treat and associated with a poor prognosis. Astragaloside IV (AS-IV), a primary bioactive component of Astragali radix, has demonstrated neuroprotective benefits in previous studies. This study aimed to explore the mechanisms through which AS-IV may treat cerebral ischemia-reperfusion injury (CIRI). Network pharmacology was employed to identify key targets and pathways of AS-IV in CIRI therapy, combined with molecular docking to predict binding affinity. Male Sprague-Dawley rats were randomly assigned to sham, MCAO/R, AS-IV, SP600125 (JNK inhibitor), AS-IV + SP600125, and 3-n-Butylphthalide (NBP) groups. Neurobehavioral deficits were assessed, and brain tissue damage was visualized through 2,3,5-triphenyltetrazolium chloride, H&E, and TUNEL staining. Immunohistochemistry was employed to detect CytC- and caspase-3-positive cells, while Western blotting, qPCR, and ELISAs were used to analyze apoptosis-related markers. A total of 48 key targets of AS-IV predicted to be involved in the treatment of CIRI were identified, enriched in 136 pathways. AS-IV was effectively bound to the top five targets from 48 targets, and those associated with the c-Jun N-terminal kinase (JNK)/Bid pathway, with binding energy values below -5.0 kJ·mol-1. JNK inhibition reduced infarcted brain areas, improved neurological function, reduced pathological brain tissue damage, and inhibited apoptosis, with AS-IV achieving similar neuroprotective effects. Both AS-IV and SP600125 reduced p-JNK, Bid, CytC, Apaf-1, caspase-3, and cleaved caspase-3 levels in rats while decreasing CytC, caspase-3, and caspase-9 levels in serum. AS-IV may suppress apoptosis partly through the modulation of JNK/Bid signaling, exerting neuroprotective effects. These findings support the potential development of AS-IV-based therapies for stroke treatment.

2-(4-Bromobenzyl) tethered 4-amino aryl/alkyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines: design, synthesis, anticancer assessment via dual topoisomerase-I/II inhibition, and in silico studies.

A series of 2-(4-bromobenzyl) tethered 4-amino aryl/alkyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines (7a-7u) were designed, synthesized, characterized and screened against a panel of cancer cell lines. Compound 7a, in particular, emerged as a potent antiproliferative agent against FaDu cells (HTB-43) with an IC50 value of 1.73 μM. 7a induced morphological alterations in FaDu cells were observed via brightfield microscopy and DAPI staining, confirming cytotoxicity. Autophagy and apoptotic effects of 7a were confirmed by acridine orange staining, Rhodamine 123 staining, and western blot analysis, which revealed dose-dependent increases in LC3A/B and cleaved caspase-3 levels, respectively. Further, 7a impaired cell migration and colony formation, as demonstrated by scratch and clonogenic assays. Additionally, 7a reduced oxidative stress and induced G2/M phase cell cycle arrest in MCF-7 cells. 7a emerged as a dual topoisomerase I and II inhibitor, and results were supported by molecular docking and simulation studies. In anti-inflammatory studies, 7a exhibited selective inhibition of COX-2 over COX-1, supporting its dual anticancer and anti-inflammatory properties.

Carnosic acid prevents heat stress-induced oxidative damage by regulating heat-shock proteins and apoptotic proteins in mouse testis.

Heat stress impacts male reproduction in animal husbandry. Carnosic acid (CA), a potent antioxidant, mitigates oxidative stress and apoptosis. αB-crystallin, a small heat shock protein, regulates apoptosis and oxidative stress. This study examines the protective effects of CA on the testis in wild-type and αB-crystallin knockout mice under heat stress. CA pretreatment increased testosterone levels and preserved testicular structure in wild-type mice, but no changes in knockout mice. CA reduced Hsp27, Hsp70, and cleaved caspase-3 levels, while knockout mice showed increased cleaved caspase-3. These results suggest that CA protects the testis by modulating heat shock and apoptosis-related proteins.

Kidney Injury in a Murine Hemorrhagic Shock/Resuscitation Model Is Alleviated by sulforaphane's Anti-Inflammatory and Antioxidant Action.

Hemorrhagic shock/resuscitation (HS/R) can lead to acute kidney injury, mainly manifested as oxidative stress and inflammatory injury in the renal tubular epithelial cells, as well as abnormal autophagy and apoptosis. Sulforaphane (SFN), an agonist of the nuclear factor-erythroid factor 2-related factor 2 (Nrf2) signaling pathway, is involved in multiple biological activities, such as anti-inflammatory, antioxidant, autophagy, and apoptosis regulation. This study investigated the effect of SFN on acute kidney injury after HS/R in mice. Hemorrhagic shock was induced in mice by controlling the arterial blood pressure at a range of 35-45mmHg for 90min within arterial blood withdrawal. Fluid resuscitation was carried out by reintroducing withdrawn blood and 0.9% NaCl. We found that SFN suppressed the elevation of urea nitrogen and serum creatinine levels in the blood induced by HS/R. SFN mitigated pathological alterations including swollen renal tubules and renal casts in kidney tissue of HS/R mice. Inflammation levels and oxidative stress were significantly downregulated in mouse kidney tissue after SFN administration. In addition, the kidney tissue of HS/R mice showed high levels of autophagosomes as observed by electron microscopy. However, SFN can further enhance the formation of autophagosomes in the HS/R + SFN group. SFN also increased autophagy-related proteins Beclin1 expression and suppressed P62 expression, while increasing the ratio of microtubule-associated protein 1 light chain 3 (LC3)-II and LC3-I (LC3-II/LC3-I). SFN also effectively decreased cleaved caspase-3 level and enhanced the ratio of anti-apoptotic protein B cell lymphoma 2 and Bcl2-associated X protein (Bcl2/Bax). Collectively, SFN effectively inhibited inflammation and oxidative stress, enhanced autophagy, thereby reducing HS/R-induced kidney injury and apoptosis levels in mouse kidneys.

METTL14 regulates inflammation in ulcerative colitis via the lncRNA DHRS4-AS1/miR-206/A3AR axis

As a chronic inflammatory bowel disease, the pathogenesis of ulcerative colitis (UC) has not been fully elucidated. N6-methyladenosine (m6A) modification, observed in various RNAs, is implicated in inflammatory bowel diseases. Methyltransferase-like 14 (METTL14) is the major subunit of the methyltransferase complex catalyzing m6A modifications. Here, we designated to examine the regulatory effects and mechanisms of METTL14 on long non-coding RNA (lncRNA) during UC progression. METTL14 knockdown decreased cell viability, promoted apoptosis, increased cleaved PARP and cleaved Caspase-3 levels, while reducing Bcl-2 levels. METTL14 knockdown also led to a significant increase in NF-κB pathway activation and inflammatory cytokine production in the Caco-2 cells treated with TNF-α. Moreover, the suppression of METTL14 aggravated colonic damage and inflammation in our dextran sulfate sodium (DSS)-induced murine colitis model. METTL14 silencing suppressed DHRS4-AS1 expression by reducing the m6A modification of DHRS4-AS1 transcripts. Furthermore, DHRS4-AS1 mitigated inflammatory injury by targeting the miR-206/adenosine A3 receptor (A3AR) axis. DHRS4-AS1 overexpression counteracted the enhancing impact of METTL14 knockdown on TNF-α-induced inflammatory injury in Caco-2 cells. In conclusion, our findings suggest that METTL14 protects against colonic inflammatory injury in UC via regulating the DHRS4-AS1/miR-206/A3AR axis, thus representing a potential therapeutic target for UC.

Prunella vulgaris Extract Ameliorates Testosterone-Induced Benign Prostatic Hyperplasia by Regulating Androgen Levels, Cell Proliferation, and Apoptosis.

Benign prostatic hyperplasia (BPH) is a prevalent urological condition affecting elderly men. Prunella vulgaris L. (PV), a perennial herbaceous plant native to Europe and Asia, has anti-inflammatory, antioxidant, and antimicrobial effects. In this study, we determined the effect of PV extract on the development of BPH. Rats were treated via a daily hypodermic injection of testosterone propionate (TP; 3 mg/kg) for 4 weeks. Groups of BPH rats were treated with or without PV (60 or 80 mg/kg) by oral gavage. In BPH model rats, PV considerably reduced their relative prostate weight and serum concentrations of dihydrotestosterone (DHT) and testosterone. The TP-induced increases in epithelial thickness in the prostate, proliferating cell nuclear antigen (PCNA) expression, and cyclin D1 expression were remarkably reduced, whereas terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells and cleaved caspase-3 levels were increased, in PV-treated rats compared to BPH rats. The mRNA expression levels of growth factors, such as transforming growth factor-β (TGF-β), fibroblast growth factor (FGF), and insulin-like growth factor (IGF-2), were significantly reduced in PV-treated rats. Mechanistically, the TP-induced activation of c-Jun N-terminal kinase (JNK) was reduced by PV administration. These results designate that PV effectively ameliorates the development of testosterone-induced BPH through anti-androgenic, anti-proliferative, and pro-apoptotic activities, suggesting that it could be a potential therapeutic substance for BPH.

Shramahara Mahakasya, a traditional polyherbal formulation, induces anti-anxiety activity in hippocampal neurons by effectuating SOD2-mediated protection against oxidative stress

BackgroundShramahara Mahakasya (SM) is an Ayurvedic polyherbal formulation known for its anti-fatigue and anti-anxiety effects on the human body. However, its mechanism of action remains largely unexplored. In this study, we investigated the intricate mechanisms through which SM polyherbal extract exerts neuroprotective and anxiolytic effects in cultured HT-22 cells and rodent models. MethodIn this study, the chemical composition of SM was first identified using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). The antioxidant potential of SM was evaluated through antioxidant assays such as DPPH, ABTS and FRAP. The neuroprotective activity of SM (200 and 600 μg/mL) was investigated in glutamate (10 mM) assaulted HT-22 cells. The anxiolytic activity of SM (50 and 100 mg/kg) was assessed in a caffeine (50 mg/kg) induced anxiety model of Sprague Dawley rats. The possible underlying mechanisms for the neuroprotective and anxiolytic activities of SM were explored through biochemical assays and brain histology. ResultsOur findings suggest that SM has antioxidant and neuroprotective potential, as evidenced by a decrease in ROS accumulation, Ca2+ overload, mitochondrial membrane potential (MMP) loss, and apoptosis in HT-22 cells subjected to glutamate-induced oxidative stress. The extract also increased SOD2 levels and decreased cleaved caspase-3 levels across various treatment sets. The anxiolytic activity of SM was demonstrated by improved behavior of the animals in the elevated plus maze test and increased SOD activity in the brain. SM exhibited the most effective improvements in anxiety disorder at a dose of 100 mg/kg body weight in rats. ConclusionsThis study is a pioneering investigation depicting the antioxidant, neuroprotective, anti-apoptotic, and anxiolytic potential of SM formulation against glutamate/caffeine-induced oxidative stress and anxiety in both in vitro and in vivo situations. Overall, our study suggests that the regular consumption of SM formulation could effectively prevent anxiety symptoms by reducing oxidative stress in neuronal cells.

Enoxaparin Protects C6 Glioma Cells from Glutamate-Induced Cytotoxicity by Reducing Oxidative Stress and Apoptosis.

Recent studies suggest enoxaparin may protect the central nervous system (CNS) from damage. However, its specific effects on glial cells and the underlying mechanisms involving cell death and oxidative stress require further investigation. Therefore, this research investigated enoxaparin's potential to safeguard C6 glioma cells against glutamate-induced cytotoxicity, specifically focusing on its influence on oxidative stress and apoptotic mechanisms. To investigate the neuroprotective effects of enoxaparin against glutamate-induced cytotoxicity in C6 cells, four groups were established: a control group, a group exposed to 10mM glutamate, a group treated with enoxaparin at concentrations ranging from 25 to 200µM, and a group receiving both 10mM glutamate and enoxaparin at concentrations ranging from 25 to 200µM. Cell viability was measured using an XTT assay. To evaluate the effects of enoxaparin on oxidative stress, superoxide dismutase (SOD) and malondialdehyde (MDA) levels were measured using ELISA, along with total antioxidant status (TAS) and total oxidant status (TOS). Apoptosis was evaluated using flow cytometry, and caspase-3 activity, a key marker of apoptosis, was assessed using caspase-3 immunofluorescence staining. Enoxaparin at 50, 100, and 200µM markedly increased cell viability in the enoxaparin + glutamate group. Enoxaparin treatment in the enoxaparin + glutamate group also significantly elevated levels of SOD and TAS, while concurrently decreasing MDA and TOS levels. These changes indicate a reduction in oxidative stress. Enoxaparin treatment further resulted in a significant decline in cleaved caspase-3 levels, a marker of apoptosis. Enoxaparin pre-treatment reduced cell death according to flow cytometry analysis. This study suggests enoxaparin's potential to shield C6 glioma cells from glutamate-induced cell death by mitigating both oxidative stress and apoptotic pathways. More research is needed to confirm this effect.

Roasted Astragalus membranaceus Inhibits Aβ25-35-Induced Oxidative Stress in Neuronal Cells by Activating the Nrf2/HO-1 and AKT/CREB/BDNF Pathways.

(1) Background: Astragalus membranaceus (AM) has antioxidant and anti-inflammatory effects, but its specific mechanism of action in the brain is still unclear. In this study, we developed a roasting process to maximize the cognitive improvement impact of AM. We focused on enhancing physiological activity to enhance the brain neuron protection effect and alleviate neuronal damage caused by neurodegenerative diseases. (2) Methods: AM was roasted at 260 °C for 20, 30, or 40 min, and the hot water extracts were tested on HT22 cells for ROS levels, apoptosis, and antioxidant protein expression. The effect on the BDNF-AKT-CREB pathway under stress was also analyzed. (3) Results: Roasted AM decreased ROS production and the expression of apoptosis-related factors while activating the expression of antioxidant proteins in HT22 cells treated with Aβ25-35. In particular, 30 min roasting (R-AM2) significantly reduced ROS production, inhibited cell death, and increased antioxidant protein expression. The Nrf2 pathway was activated Bax, and cleaved caspase-3 levels were reduced. BDNF and p-CREB expression were increased by 20% and 50-70%, respectively. In the MAPK pathway, p-ERK levels were increased by 30%, and p-P38 levels were increased by approximately 20%. (4) Conclusions: These findings suggest that roasted AM upregulates brain-derived neurotrophic factor (BDNF) in HT22 cells, providing neuroprotective effects by activating the AKT/CREB/BDNF pathway and inhibiting neuronal apoptosis. Therefore, roasted AM shows potential as a neuroprotective agent for preventing or treating neurodegenerative diseases, such as Alzheimer's, linked to BDNF deficiency.

Microglia ameliorate delirium-like phenotypes in a murine model of acute ventilator-induced lung injury

BackgroundDelirium affects 50–85% of patients on mechanical ventilation and is associated with increased mortality, prolonged hospitalization, and a three-fold higher risk of dementia. Microglia, the resident immune cells of the brain, exhibit both neuroprotective and neurotoxic functions; however, their effects in mechanical ventilation-induced acute lung injury (VILI) are unknown. We hypothesize that in a model of short-term VILI, microglia play a neuroprotective role to ameliorate delirium-like phenotypes.MethodsMicroglia depletion (n = 18) was accomplished using an orally administered colony stimulating factor 1 receptor inhibitor, while controls received a vehicle diet (n = 18). We then compared extent of neuronal injury in the frontal cortex and hippocampus using cleaved caspase-3 (CC3) and multiple delirium-like behaviors in microglia depleted and non-microglia depleted male mice (C57BL/6 J aged 4–9 months) following VILI. Delirium-like behaviors were evaluated using the Open Field, Elevated Plus Maze, and Y-maze assays. We subsequently evaluated whether repopulation of microglia (n = 14 repopulation, 14 vehicle) restored the phenotypes.ResultsFrontal/hippocampal neuronal CC3 levels were significantly higher in microglia depleted VILI mice compared to vehicle-treated VILI controls (p < 0.01, p < 0.01, respectively). These structural changes were accompanied by worse delirium-like behaviors in microglia depleted VILI mice compared to vehicle controls. Specifically, microglia depleted VILI mice demonstrated: (1) significantly increased time in the periphery of the Open Field (p = 0.01), (2) significantly increased coefficient of variation (p = 0.02), (3) trend towards reduced time in the open arms of the Elevated Plus Maze (p = 0.09), and (4) significantly decreased spontaneous alternations on Y-maze (p < 0.01). There was a significant inverse correlation between frontal CC3 and percent spontaneous alternations (R2 = 0.51, p < 0.01). Microglia repopulation showed a near-complete return to vehicle levels of delirium like-behaviors.ConclusionsThis study demonstrates that microglia depletion exacerbates structural and functional delirium-like phenotypes after VILI, while subsequent repopulation of microglia restores these phenotypes. These findings suggest a neuroprotective role for microglia in ameliorating neuronal and functional delirium-like phenotypes and call for consideration of interventions that leverage endogenous microglia physiology to mitigate delirium.

EMP1 knockdown mitigated high glucose-induced pyroptosis and oxidative stress in rat H9c2 cardiomyocytes by inhibiting the RAS/RAF/MAPK signaling pathway.

The purpose of this study was to investigate the mechanism of EMP1 action in high glucose (HG)-induced H9c2 cardiac cell pyroptosis and oxidative injury. Rat cardiomyocytes H9c2 were exposed to 33 mM glucose for 24, 48, or 72 h to induce cytotoxicity. EMP1-siRNA, NLRP3 agonist Nigericin, and pcNDA-RAS were used to treat H9c2 cells under HG conditions. Cell Counting Kit (CCK)-8 assay showed that cell proliferation was decreased following HG induction, which was rescued by EMP1 knockdown. Our results also suggested that EMP1 siRNA transfection significantly decreased the apoptosis and pyroptosis of HG-induced cells, as indicated by the reduction of NLRP3 IL-1β, ASC, GSDMD, cleaved-caspase1 and cleaved-caspase3 levels in HG-induced H9c2 cells. In addition, EMP1 knockdown alleviated HG-induced mitochondrial damage and oxidative stress in H9c2 cells. NLRP3 activation reversed the inhibitory effects of EMP1 knockdown on pyroptosis and oxidative stress in HG-induced H9c2 cells. Mechanistically, we found that EMP1 knockdown suppressed the RAS/RAF/MAPK signaling pathway in HG-induced H9c2 cells. RAS overexpression blocked the protective effect of EMP1 knockdown on HG-induced H9c2 cell apoptosis, pyroptosis, and oxidative injury. Our findings suggest that EMP1 knockdown treatment might provide a novel therapy for diabetic cardiomyopathy.

Pseudomonas aeruginosa-Mannose Sensitive Hemagglutinin-based Treatment Strategy for Liver Cancer

Background Although it does not represent a threat to healthy humans, Pseudomonas aeruginosa is a type of aerobic, gram-negative bacteria that can cause catastrophic infections in patients with immune system abnormalities and cystic fibrosis. Purpose The current study investigated the effect of Pseudomonas aeruginosa-mannose sensitive hemagglutinin (PA-MSHA) on liver cancer cell viability, colony formation, and invasion potential, and explored the underlying mechanism. Methods Proliferation potential and clonogenic survival of the cells were studied using MTT and colony formation assays, respectively. Transwell and Western blotting assays were used for the assessment of invasive potential and protein expression, respectively. Results The results revealed that exposure to PA-MSHA led to a time-dependent suppression in HepG2 and HEP3B cell proliferation. Incubation with PA-MSHA decreased HepG2 cell proliferation to 92%, 75%, 50%, 35%, and 20%, respectively, after 12, 24, 36, 48, and 72 hours. Similarly, incubation for 12, 24, 36, 48, and 72 hours reduced the proliferation of HEP3B cells to 94%, 79%, 55%, 41%, and 26%, respectively. The rate of clonogenic survival was significantly lower in HepG2 and HEP3B cells on incubation with PA-MSHA. The cell invasion potential was also significantly reduced on incubation with PA-MSHA. In HepG2 cells, incubation with PA-MSHA significantly reduced the expression of Bcl-2 (26 kDa) protein and promoted Bax (21 kDa) level. Following incubation with PA-MSHA, HepG2 cells showed higher cleaved caspase-3 level compared to the control cells. Compared to control cells, the PA-MSHA incubation of HepG2 cells resulted in a prominent reduction in Notch3 protein expression. Conclusion In summary, PA-MSHA treatment prevents liver cancer cell proliferation and targets the survival of clonogenic cells. It reduces invasiveness, targets Notch3 protein expression in liver cancer cells, and increases the level of proapoptotic and suppresses antiapoptotic protein expression. Therefore, PA-MSHA shows encouraging anticancer effects on HepG2 and HEP3B cancer cells, suggesting that it could be developed as a viable treatment option for liver cancer.

Exploring the reduction in aquaporin-4 and increased expression of ciliary neurotrophic factor with the frontal-striatal gliosis induced by chronic high-fat dietary stress.

High-fat diet (HFD)-induced obesity induces peripheral inflammation and hypothalamic pathogenesis linking the activation of astrocytes and microglia. Clinical evidence indicates a positive correlation between obesity and psychiatric disorders, such as depression. The connectivity of the frontal-striatal (FS) circuit, involving the caudate putamen (CPu) and anterior cingulate cortex (ACC) within the prefrontal cortex (PFC), is known for its role in stress-induced depression. Thus, there is a need for a thorough investigation into whether chronic obesity-induced gliosis, characterized by the activation of astrocytes and microglia, in these brain regions of individuals with chronic obesity. The results revealed increased S100β+ astrocytes and Iba1+ microglia in the CPu and ACC of male obese mice, along with immune cell accumulation in meningeal lymphatic drainage. Activated GFAP+ astrocytes and Iba1+ microglia were observed in the corpus callosum of obese mice. Gliosis in the CPu and ACC was linked to elevated cleaved caspase-3 levels, indicating potential neural cell death by chronic HFD feeding. There was a loss of myelin and adenomatous polyposis coli (APC)+ oligodendrocytes (OLs) in the corpus callosum, an area known to be linked with injury to the CPu. Additionally, reduced levels of aquaporin-4 (AQP4), a protein associated within the glymphatic systems, were noted in the CPu and ACC, while ciliary neurotrophic factor (CNTF) gene expression was upregulated in these brain regions of obese mice. The invitro study revealed thathigh-dose CNTF causing a trend of reduced astrocytic AQP4 expression, but it significantly impaired OL maturation. This pathological evidence highlights that prolonged HFD consumption induces persistent FS gliosis and demyelination in the corpus callosum. An elevated level of CNTF appears to act as a potential regulator, leading to AQP4 downregulation in the FS areas and demyelination in the corpus callosum. This cascade of events might contribute to neural cell damage within these regions and disrupt the glymphatic flow.

Analysis of the mechanisms underlying the anticancer and biological activity of retinoic acid and chitosan nanoparticles containing retinoic acid.

Retinoic acid (RA) has been shown in earlier investigations to have anticancer properties in various cancer cells. RA's effect on breast cancer treatment remains uncertain, though. This study investigated whether RA and chitosan nanoparticles (NPs) loaded with RA could be harmful to the MCF-7 cell line. In this study, NPs with RA were used in characterization tests. Using ELISA kits, the amounts of 8-okso-2'-deoksiguanozin (8-oxo-dG), BCL-2, Bcl-2-Associated X-protein (Bax), cleaved Poly (ADP-ribose) polymerases (PARP), total oxidant and antioxidant, and cleaved caspase-3 capacities were determined. The analysis of chitosan NPs showed that their drug-release profile, encapsulation efficiency (EE), and particle size were suitable for cell culture experiment. The EE value of NPs including RA was calculated as 83.32 ± 0.04%. The IC50 value for RA was 2.89 ± 0.03µg/mL, while the IC50 value for RA-loaded NPs was significantly lower at 2.28 ± 0.02µg/mL. In ELISA testing, RA and chitosan NPs containing RA at a concentration of 2µg/mL dramatically increased the concentrations of total oxidant, cleaved caspase-3. Cleaved caspase-3 levels were quantified as 614.90 ± 3.40pg/mg protein in the control group, 826.37 ± 5.82pg/mg protein in RA-treated cells, and 863.52 ± 4.32pg/mg protein in RA-NP-treated cells. Interestingly, no substantial variations were observed in the levels of the anti-apoptotic protein BCL-2. Overall, studies revealed that RA and RA-NPs promoted apoptosis in MCF-7 cells by upregulating the expression of pro-apoptotic proteins Bax, cleaved caspase-3, and cleaved PARP.

MicroRNA-195-5p Inhibits Intracerebral Hemorrhage-Induced Inflammatory Response and Neuron Cell Apoptosis.

Intracerebral hemorrhage (ICH) is a severe condition characterized by bleeding within brain tissue. Primary brain injury in ICH results from a mechanical insult caused by blood accumulation, whereas secondary injury involves inflammation, oxidative stress, and disruption of brain physiology. miR-195-5p may participate in ICH pathology by regulating cell proliferation, oxidative stress, and inflammation. Therefore, we assessed the performance of miR-195-5p in alleviating ICH-induced secondary brain injury. ICH was established in male Sprague-Dawley rats (7 weeks old, 200-250 g) via the stereotaxic intrastriatal injection of type IV bacterial collagenase, after which miR-195-5p was administered intravenously. Neurological function was assessed using corner turn and forelimb grip strength tests. Protein expression was assessed by western blotting and ELISA. The miR-195-5p treatment significantly improved neurological function; modulated macrophage polarization by promoting anti-inflammatory marker (CD206 and Arg1) production and inhibiting pro-inflammatory marker (CD68 and iNOS) production; enhanced Akt signalling, reduced oxidative stress by increasing Sirt1 and Nrf2 levels, and attenuated inflammation by decreasing NF-κB activation; inhibited apoptosis via increased Bcl-2 and decreased cleaved caspase-3 levels; and regulated synaptic plasticity by modulating NMDAR2A, NMDAR2B, BDNF, and TrkB expression and ERK and CREB phosphorylation. In conclusion, miR-195-5p exerts neuroprotective effects in ICH by reducing inflammation and oxidative stress, inhibiting apoptosis, and restoring synaptic plasticity, ultimately restoring behavioral recovery, and represents a promising therapeutic agent that warrants clinical studies.

Cdc25A phosphatase is activated and mediates neuronal cell death by PUMA via pRb/E2F1 pathway in a model of Parkinson's disease

Parkinson's disease (PD) is a predominant movement disorder caused mainly due to selective loss of the dopaminergic neurons in the substantia nigra pars compacta of the mid brain. There is currently no cure for PD barring treatments to manage symptoms. The reasons might be due to lack of precise understanding of molecular mechanisms leading to neurodegeneration. Aberrant cell cycle activation has been implicated in neuronal death pathways of various neurodegenerative diseases including PD. This study investigates the role of cell cycle regulator Cell division cycle 25A (Cdc25A) in a PD-relevant neuron death model induced by 6-OHDA treatment. We find Cdc25A is rapidly elevated, activated and is playing a key role in neuron death by regulating Rb phosphorylation and E2F1 activity. Knockdown of Cdc25A via shRNA downregulates the levels of pro-apoptotic PUMA, an E2F1 target and cleaved Caspase-3 levels, suggesting Cdc25A may regulate neuronal apoptosis through these effectors. Our work sheds light on the intricate signaling networks involved in neurodegeneration and highlights Cdc25A as a potential therapeutic target for mitigating aberrant cell cycle re-entry underlying PD pathogenesis. These novel insights into molecular mechanisms provide a foundation for future development of neuroprotective strategies to slow or prevent progression of this debilitating disease.

Novel detoxifier of spironolactone against triptolide-induced hepatotoxicity through inhibition of RPB1 degradation

Ethnopharmacological relevanceTriptolide is a major bioactive and toxic ingredient isolated from the traditional Chinese herb Tripterygium wilfordii (T. wilfordii) Hook F. It exhibits potent antitumor, immunosuppressive, and anti-inflammatory biological activities; however, its clinical application is hindered by severe systemic toxicity. Two preparations of T. wilfordii, including T. wilfordii glycoside tablets and T. wilfordii tablets, containing triptolide, are commonly used in clinical practice. However, their adverse side effects, particularly hepatotoxicity, limit their safe use. Therefore, it is crucial to discover potent and specific detoxification medicines for triptolide. Aim of the studyThis study aimed to investigate the detoxification effects and potential mechanism of action of spironolactone on triptolide-induced hepatotoxicity to provide a potential detoxifying strategy for triptolide, thereby promoting the safe applications of T. wilfordii preparations in clinical settings. Materials and methodsCell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and crystal violet staining. Nuclear fragmentation was visualized using 4′,6-diamidino-2-phenylindole (DAPI) staining, and protein expression was analyzed by Western blotting. The inhibitory effect of spironolactone on triptolide-induced hepatotoxicity was evaluated by examining the effects of spironolactone on serum alanine aminotransferase and aspartate aminotransferase levels, as well as liver pathology in a mouse model of triptolide-induced acute hepatotoxicity. Furthermore, a survival assay was performed to investigate the effects of spironolactone on the survival rate of mice exposed to a lethal dose of triptolide. The effect of spironolactone on triptolide-induced global transcriptional repression was assessed through 5-ethynyl uridine staining. ResultsTriptolide treatment decreased the cell viability, increased the nuclear fragmentation and the cleaved caspase-3 levels in both hepatoma cells and hepatocytes. It also increased the alanine aminotransferase and aspartate aminotransferase levels, induced the hepatocyte swelling and necrosis, and led to seven deaths out of 11 mice. The above effects could be mitigated by pretreatment with spironolactone. Additionally, molecular mechanism exploration unveiled that spironolactone inhibited triptolide-induced DNA-directed RNA polymerase II subunit RPB1 degradation, consequently increased the fluorescence intensity of 5-ethynyl uridine staining for nascent RNA. ConclusionsThis study shows that spironolactone exhibits a potent detoxification role against triptolide hepatotoxicity, through inhibition of RPB1 degradation induced by triptolide and, in turn, retardation of global transcriptional inhibition in affected cells. These findings suggest a potential detoxification strategy for triptolide that may contribute to the safe use of T. wilfordii preparations.

The composition of linoleic acid and conjugated linoleic acid has potent synergistic effects on the growth and death of RAW264.7 macrophages: The role in anti-inflammatory effects

Linoleic acid (LA) is an omega-6 polyunsaturated fatty acid. Conjugated linoleic acid (CLA) is a family of LA isomers that includes both a trans fatty acid and a cis fatty acid. Both fatty acids play a nutritional role in maintaining health. Inflammation is critical in the pathogenesis of many diseases, including cancer. This study found that the combination of LA and CLA (LA/CLA), each of which had no effect, had a strong anti-synergistic effect on inflammatory macrophage RAW264.7 cells in vitro. Cells were cultured in a DMEM containing fetal bovine serum with or without either LA, CLA, or a combination of LA/CLA. The composition of LA and CLA at a comparatively lower concentration synergistically suppressed cell growth, resulting in a reduction in cell number. The underlying mechanism of this effect was based on reduced levels of Ras, PI3K, Akt, MAPK, and mTOR and elevated levels of p21, p53, and Rb, which are associated with cell growth. In addition, the combination of LA and CLA at a lower concentration stimulated potential cell death associated with increased caspase-3 and cleaved caspase-3 levels. Notably, this composition synergistically suppressed the production of TNF-α, IL-6, and PGE2, which are a major mediator of inflammation, with lipopolysaccharide stimulation in RAW264.7 cells This effect was associated with decreased levels of COX-1, COX-2, and NF-κB p65. This study may provide a useful tool for treating inflammatory conditions with the composition of LA and CLA.