Terminal Labeling Research Articles

Neutralizing IL-15 Inhibits Tissue-Damaging Immune Response in Ex Vivo Cultured Untreated Celiac Intestinal Mucosa

In celiac disease (CeD), interleukin 15 (IL-15) affects the epithelial barrier by acting on intraepithelial lymphocytes, promoting interferon γ (IFN-γ) production and inducing strong cytotoxic activity as well as eliciting apoptotic death of enterocytes by the Fas/Fas ligand system. This study investigates the effects of a monoclonal antibody neutralizing the effects of IL-15 (aIL-15) on tissue-damaging immune response in untreated CeD patients by using an organ culture system. Jejunal biopsies from 10 untreated CeD patients were cultured ex vivo with or without aIL-15. Epithelial expressions of CD95/Fas, HLA-E and perforin were analyzed by immunohistochemistry. Apoptosis was detected in the epithelium by using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Additionally, the surface epithelium compartment of ex vivo cultured biopsy samples was isolated by laser capture microdissection (LCM). RNA from each LCM sample was extracted and the relative expression of IFN-γ was evaluated by quantitative reverse transcriptase-PCR (qRT-PCR). Biopsies cultured with the aIL-15 antibody showed a reduction in Fas, HLA-E and perforin epithelial expression, as well as a decrease in epithelial TUNEL+ cells compared to biopsies cultured without the aIL-15 antibody. Moreover, downregulation of epithelial IFN-γ expression was recorded in biopsies incubated with aIL-15, compared to those cultured without aIL-15. Our findings suggest that neutralizing the effects of IL-15 in ex vivo cultured untreated CeD intestinal mucosa could block apoptosis by downregulating Fas and HLA-E expression and the release of cytotoxic proteins, such as perforin. Furthermore, it can dampen the hyperactive immune response by reducing IFN-γ expression. More generally, our study provides new evidence for the effects of anti-IL-15 neutralizing monoclonal antibodies in preventing or repairing epithelial damage and further supports the concept that IL-15 is a meaningful therapeutic target in CeD, or inflammatory diseases associated with the upregulation of IL-15.

Lathyrol affects the expression of AR and PSA and inhibits the malignant behavior of RCC cells

Abstract Objective To investigate how lathyrol affects aggressive behaviors and related proteins of the androgen receptor (AR) 786-O cells. Methods 786-O cells were cultured in vitro and divided into these groups at random: the dimethylsulfoxide (DMSO) control group (A group), negative control group (B group), and experimental group (C group). Cells in A group were grown in DMSO working medium (contained RPMI 1640 medium and 1% DMSO), B group cells were cultured in nilutamide working medium (contained DMSO working medium and 325 μg/mL nilutamide), while those in C group were cultured in lathyrol working medium (contained DMSO working medium and 300 μg/mL lathyrol). Cell proliferation was measured via CCK-8 assays, and cell apoptosis was examined through terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. Scratch tests and Transwell invasion tests were used to evaluate cell movement and penetration. The expression information about p-AR, AR, p-Akt, ki67, caspase3, cleaved-caspase3, Bcl-2, Bax, caspase9, cleaved-caspase9, and GAPDH proteins was investigated through western blotting. Immunocytochemistry was used to identify the 786-O cells’ secretion level of matrix metalloproteinase 2 (MMP2), MMP9, and prostate-specific antigen (PSA) proteins. Results The negative control and experimental groups’ cells exhibited reduced proliferation, migration, and invasion and increased apoptosis after 24 h treatment. Furthermore, these two group cells exhibited a notable reduction in the status of Ki67, Bcl-2, MMP2, MMP9, and p-Akt (P < 0.05) and significantly increased the expressions of AR, p-AR, Bax, cleaved-caspase3, and cleaved-caspase9 (P < 0.05). There was no statistical distance in PSA, caspase3, and caspase9 expressions among the three groups (P > 0.05). Conclusion In vitro, lathyrol and nilutamide exert notable anticancer effects by effectively suppressing the proliferation, migration, and invasion of 786-O cells while also inducing apoptosis.

HDAC4 regulates apoptosis in Acan-CreERT2;HDAC4d /d mice with osteoarthritis by downregulating ATF4.

Several studies have previously shown that histone deacetylase 4 (HDAC4) can regulate endoplasmic reticulum stress-induced apoptosis through the activating transcription factor 4 (ATF4)/CAAT/enhancer binding protein homologous (CHOP) signaling pathway, thereby affecting the progression of osteoarthritis (OA). The present study investigated the regulatory mechanism of HDAC4 in chondrocyte apoptosis in OA using Acan-CreERT2;HDAC4fl/fl gene knockout mice. Forty mice were divided into four groups: TM-DMM group [tamoxifen (TM) injection at 2 months of age and destabilization of the medial meniscus (DMM) surgery at 3 months], TM-sham group (TM injection at 2 months of age and sham surgery at 3 months), no TM-DMM group (corn oil injection at 2 months of age and DMM surgery at 3 months) and no TM-sham group (corn oil injection at 2 months of age and sham surgery at 3 months). Apoptosis and cartilage damage were assessed through imaging, histological analysis, immunohistochemistry, reverse transcriptase-PCR and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. HDAC4 knockdown resulted in increased osteophyte formation, significant narrowing of the joint space and increased articular cartilage damage. Furthermore, expression levels of key apoptosis-related markers, ATF4, CHOP, caspase-3 and caspase-9, were significantly higher in the TM groups than in their respective control groups. Taken together, our results suggest that HDAC4 deficiency leads to increased apoptosis induced by the ATF4/CHOP signaling pathway in the pathogenesis of OA. Therefore, upregulation of HDAC4 may represent a potential therapeutic strategy.

Functional characterisation of WRKY transcription factor CrWRKY48 involved in regulating seed abortion of Ponkan (Citrus reticulata).

Citrus fruits are one of the most important fruits in the world, and their seedless character is favored by consumers. WRKY is a plant-specific transcription factor family involved in all aspects of plant growth and development. However, the molecular mechanism of seedless fruit formation in citrus and the role of the WRKY gene family in seed abortion are still poorly understood. In this study, we identified 47 WRKY family genes in the citrus fruit Citrus reticulata and comprehensively characterized the WRKY gene family through gene structure and evolutionary relationships. The expression patterns and protein interaction networks of the WRKY gene family were analyzed based on citrus seed abortion transcriptome data, and several WRKY genes that may be involved in the seed abortion regulation were excavated. Furthermore, CrWRKY48 was verified to regulate seed abortion positively in Arabidopsis thaliana, and the rate of seed abortion caused by overexpression of CrWRKY48 reached 45.48%. Using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assays, DNA affinity purification sequencing and yeast-one-hybrid assays, we found that CrWRKY48 activated excessive programmed cell death by regulating the expression of programmed cell death-related genes such as SOBIR1. Our results show the potential regulation of the WRKY gene family for citrus seed abortion and provide novel insights into the role of CrWRKY48 in mediating citrus seed abortion by activating programmed cell death.

Pterostilbene Suppressed Cell Viability, Induced Apoptosis and Autophagy of Cisplatin-resistant Gastric Cancer Cells.

Gastric cancer (GC) is one of the most common cancers worldwide. Cisplatin is a key therapeutic agent for treating GC. Currently, the resistance of GC cells to cisplatin remains a major concern. Pterostilbene (PTS) is a natural phytochemical found in blueberry and grape. The anti-cisplatin-resistant GC effects and pharmacological mechanisms of PTS are unknown. We investigated the anticancer activity of PTS in cisplatin-resistant GC cells and explored its pharmacological mechanisms of action via cell viability assay, cell confluence assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, acridine orange (AO) staining, monodansylcadaverine (MDC) staining, caspase-9/-3 activity assay, and RNA sequencing (RNA-Seq) analysis. Our results showed that PTS inhibited cell viability and cell confluence of cisplatin-resistant GC cells using the CCK-8 assay and the IncuCyte S3 ZOOM System. The TUNEL assay showed that PTS promoted apoptosis in cisplatin-resistant GC cells. PTS induced apoptosis by increasing caspase-9 and caspase-3 activity. PTS promoted cell autophagy by increasing vacuole formation and acidic vesicular organelles using MDC and AO staining. We also observed an increase in the expression of LC3B in PTS-treated cisplatin-resistant GC cells. RNA-Seq analysis demonstrated that PTS induced apoptosis and autophagy in cisplatin-resistant GC cells by decreasing the expression of ATM/ATR, HIF-1, PI3K, RB1CC, TBK1, and mitochondria-related genes. Our results suggested that PTS is a promising phytochemical for GC therapy, particularly against cisplatin resistance.

Electroacupuncture improves cognitive impairment after subarachnoid hemorrhage in rats through the PI3K/AKT signaling pathway.

Cognitive impairment (CI) is highly prevalent in subarachnoid hemorrhage (SAH) patients. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway plays a critical role in neuronal survival in a variety of central nervous system injuries. This study aimed to determine whether electroacupuncture (EA) at Yintang and LI20 ameliorates SAH-CI in a rat model and to examine whether it modulates the PI3K/AKT pathway by administering a PI3K inhibitor (LY294002) versus dimethyl sulfoxide (DMSO) vehicle. Notably, 129 male Sprague-Dawley rats were divided into Blank, Sham, SAH and SAH + EA groups (Experiment 1, n = 54) and SAH, SAH + EA, SAH + LY294002, SAH + EA + LY294002 and SAH + EA + DMSO groups (Experiment 2, n = 75). Garcia scoring was used to evaluate neurological function. The moisture content of the rat brain was determined by dry‒wet method. The Morris water maze was used to assess learning and memory function. Pathological changes in neurons in the hippocampus were observed via hematoxylin-eosin (H&E) staining. The number of surviving neurons and the percentage of apoptotic cells in the hippocampus were detected via Nissl and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The expression of PI3K/AKT pathway-related proteins was detected via Western blotting. The results indicated that EA intervention after SAH reduced brain water content, enhanced Garcia scores, improved neurological function and behavioral markers of CI, and increased the number of surviving neurons in the hippocampus. Moreover, EA significantly increased the expression of AKT, phosphorylated (p)-AKT, PI3K, p-PI3K, glycogen synthase kinase (GSK)-3β, p-GSK-3β and B cell lymphoma (Bcl)-2 proteins, and decreased the expression of Bcl-2-associated X (Bax) and caspase-3. In addition, the effects of EA were abolished by LY294002. EA appeared to improve CI in a rat model of SAH through the activation of the PI3K/AKT pathway.

Di Huang Yi Zhi Fang improves cognitive function in APP/PS1 mice by inducing neuronal mitochondrial autophagy through the PINK1-parkin pathway.

Alzheimer's disease (AD) is an irreversible age-related neurodegenerative condition characterized by the deposition of amyloid-β (Aβ) peptides and neurofibrillary tangles. Di Huang Yi Zhi (DHYZ) formula, a traditional Chinese herbal compound comprising several prescriptions, demonstrates properties that improve cognitive abilities in clinical. Nonetheless, its molecular mechanisms on treating AD through improving neuron cells mitochondria function have not been deeply investigated. This study administered DHYZ to APP/PS1 mice to explore its potential therapeutic mechanisms in AD treatment. APP/PS1 transgenic mice were given DHYZ (L, M, H), donepezil, or distilled water for a consecutive 12-week period. The Morris water maze test was used to assess memory capacity, transmission electron microscopy was used to observe mitochondrial and synaptic structures, immunohistochemistry and western blot detected proteins involved in the mitochondrial autophagy pathway, ELISA measured serum Aβ content, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assessed neuronal cell apoptosis. DHYZ demonstrates a notable therapeutic impact on mice with AD, effectively improving cognitive and memory impairments. DHYZ decreases Aβ accumulation in the hippocampus by reducing BACE1 activity and enhancing Aβ clearance through the blood-brain barrier. Additionally, DHYZ significantly suppresses neuronal apoptosis, enhances synaptic structure, and increases synapse numbers, processes strongly linked to the activation of mitochondrial PINK1-Parkin autophagy. DHYZ enhances cognitive function in APP/PS1 mice by stimulating neuronal mitochondrial autophagy through the PINK1-Parkin pathway.

Gintonin-Enriched Panax ginseng Extract Induces Apoptosis in Human Melanoma Cells by Causing Cell Cycle Arrest and Activating Caspases

Gintonin, a non-saponin glycolipoprotein from Panax ginseng, acts as a lysophosphatidic acid ligand. However, its anticancer effects, especially in melanoma, remain unclear. This study investigated the anti-proliferative effects and intracellular signaling mechanisms of a gintonin-enriched fraction (GEF) from Panax ginseng in human melanoma cell lines. In vitro, GEF treatment significantly inhibited cell proliferation, reduced clonogenic potential, and delayed wound healing in melanoma cells. Flow cytometry and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining showed that GEF induced apoptosis, as evidenced by increased apoptotic cell populations and nuclear changes. GEF also caused cell cycle arrest in the G1 phase for A375 cells and the G2/M phase for A2058 cells. It triggered apoptotic signaling via activation of caspase-3, -9, poly (ADP-ribose) polymerase cleavage, and downregulation of B cell lymphoma-2 (Bcl-2). GEF treatment also raised intracellular reactive oxygen species (ROS) levels and mitochondrial stress, which were mitigated by N-acetyl cysteine (NAC), an ROS inhibitor. In vivo, GEF suppressed tumor growth in A375- and A2058-xenografted mice without toxicity. These findings suggest that GEF from Panax ginseng has potential antitumor effects in melanoma by inducing apoptosis and cell cycle arrest, presenting a promising therapeutic avenue.

Dexamethasone-loaded fibroin nanoparticles promote retinal reattachment in rats by regulating the Th17/Treg balance

Retinal detachment (RD) is a common acute blinding eye disease, and dexamethasone (DEX), an adrenocorticosteroid, shows protective effects against RD. However, its poor water solubility and low bioavailability limit its effectiveness. To address this, we developed SF@DEX nanomaterials and investigated their therapeutic potential and mechanisms in RD. The nanomaterials were successfully synthesized and characterized, achieving 90% encapsulation efficiency and releasing 60% of DEX within 12 h.In vitro, phagocytosis was measured by flow cytometry, and enzyme-linked immunosorbent assay determined interleukin-17 (IL-17) and interleukin-10 (IL-10) levels. A rat RD model was established surgically, followed by oral administration of silk fibroin (SF), SF@DEX, and DEX. Polymerase chain reaction (PCR) assessed IL-17A and forkhead box P3 (FOXP3) expression, while Western blot analysed transforming growth factor-β1 (TGF-β1), IL-10, IL-17A, and FOXP3 levels. Apoptosis of retinal ganglion cells was evaluated using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and confocal microscopy detected colocalization of IL-17A and FOXP3. SF@DEX treatment significantly reduced Th17 cells and IL-17A while increasing Tregs, FOXP3, TGF-β1, and IL-10 levels. The severity of RD in rats was notably alleviated by SF@DEX, demonstrating its anti-inflammatory effects through modulation of the Th17/Treg immune balance. These results highlight SF@DEX as a promising nano-based therapy for RD.

The Ethanol Extract of Sugemule-3 Decoction Treats Isoproterenol-induced Heart Failure in Rats via PPARγ/PGC-1 Pathway

Background The herbs Baidoukou (the fruit of Amomum compactum Sol. ex Maton), Baijusheng (the fruit of Lactuca sativa L.), and Biba ( Piper longum L.) make up the traditional Mongolian medicine known as Sugemule-3 decoction. Purpose: Heart failure (HF) is severely impacted; however, the pharmacological mechanism behind it is yet unclear. This investigation looked at the Sugemule-3 ethanol extract’s therapeutic mechanism for isoproterenol-induced HF in rats. Methods To create a HF model, isoproterenol was administered to Wistar rats. Following the model’s successful establishment, various medications were administered for 4 weeks. The electrical signals of cardiac fluctuations, cardiac morphology, and function were observed using electrocardiogram (ECG) and echocardiography. The rats were killed 4 weeks later, and the hearts were observed using an electron microscope and HE staining. Cardiomyocyte apoptosis was observed using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. The levels of markers in serum were determined using enzyme-linked immunosorbent assay (ELISA). Western blot and immunohistochemistry were used to detect the expression of proteins in rats. Results Our findings show that isoproterenol injection subcutaneously interfered with the structure and function of the left ventricle, particularly during systole. In the model group, the level of oxidative stress index malondialdehyde ( p < 0.01) increased with the decrease in anti-oxidant enzymes such as superoxide dismutase ( p < 0.01) and glutathione peroxidase ( p < 0.01). Sugemule-3 ethanol extract considerably reduced the harmful effects of isoproterenol on the rat myocardium. Additionally, isoproterenol increased the level of peroxisome proliferator-activated receptor (PPAR), carnitine palmitoyl transferase-1 (CPT-1), phosphoenolpyruvate carboxylase (PEPCK), stearyl CoA desaturase-1 (SCD-1), and ubiquitin-conjugating (UCB) expression while decreasing the level of peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1) expression, leading to cardiometabolic diseases. Sugemule-3 ethanol extract improved cardiac metabolism. Conclusion The findings demonstrate that Sugemule-3 ethanol extract may improve energy metabolism of cardiomyocytes and alleviate isoproterenol-induced HF through inhibiting the peroxisome proliferator-activated receptor gamma (PPARγ)/PGC-1 signaling pathway.

Sivelestat sodium protects against renal ischemia/reperfusion injury by reduction of NETs formation.

Ischemia-reperfusion injury (IRI) often results in renal impairment. While the presence of neutrophil extracellular traps (NETs) is consistently observed, their specific impact on IRI is not yet defined. Sivelestat sodium, an inhibitor of neutrophil elastase which is crucial for NET formation, may offer a therapeutic approach to renal IRI, warranting further research. A mouse model was established for early-stage renal IRI, confirmed by injury markers and histological assessments. The involvement of NETs in renal I/R was demonstrated using immunofluorescence and Western blot. Renal function and pathology were further evaluated through a comprehensive set of methods, including Periodic Acid-Schiff staining (PAS) and Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) staining, enzyme-linked immunosorbent assay (ELISA), Real time Glomerular Filtration Rate (RT-GFR) monitoring, Polymerase Chain Reaction (PCR), biochemical analysis, and additional Western blot and immunofluorescence assays. We firstly quantified NET expression in renal IRI mice, noting a peak at 24 hours. Subsequently, sivelestat sodium treatment was administered, resulting in decreased MPO, CitH3, and attenuated tubular damage. Moreover, it resulted in a decrease in serum levels of creatinine, blood urea nitrogen (BUN), as well as neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1). Additionally, it lowered the abundance of renal tissue inflammatory markers interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), and mitigated the levels of oxidative stress indicators malondialdehyde (MDA) and 4 Hydroxynonenal (4HNE), accompanied by a decline in renal cell apoptosis and an enhancement of GFR in renal I/R mice. Sivelestat sodium ameliorates renal IRI by downregulating neutrophil NETs, reducing inflammation, oxidative stress, and apoptosis, thereby enhancing renal function.

P0113 Maintained microglial homeostasis and adult neurogenesis despite chronic ileocolitis

Abstract Background Inflammatory bowel diseases (IBD), such as Crohn‘s disease and ulcerative colitis, are associated with neuropsychiatric comorbidities, including depression, anxiety, and Parkinson’s disease [1]. These are presumably mediated along the gut-immune-brain axis by the transition of inflammation across gastrointestinal and CNS-surrounding barriers, causing the activation of microglia [1]. Adult hippocampal neurogenesis, the generation of new neurons in the hippocampus throughout life, is impaired by peripheral inflammation [2] and linked to affective and neurodegenerative disorders [3]. In the context of IBD, adult hippocampal neurogenesis has only been investigated in chemically induced animal models mimicking ulcerative colitis, leading to heterogeneous results [1]. Methods In this study, we used the Casp8∆IEC ileocolitis mouse model which is characterized by a specific knockout of caspase 8 in intestinal epithelial cells, leading to ileocolitis that resembles Crohn’s disease [4]. We investigated the effects of ileocolitis on microglia and on adult neurogenesis in the hippocampal dentate gyrus (DG). The hippocampi of 14- and 24-week-old Casp8∆IEC mice were compared to wild-type (wt) mice using immunohistochemistry and immunofluorescence analyses. Microglial density and proliferation were assessed by quantifying ionized calcium-binding adapter molecule 1 (Iba1)+ and Iba1+Mcm2+ myeloid cells, respectively. Additionally, microglial phagocytic activation was examined by co-staining Iba1 with the lysosomal marker CD68. To evaluate adult hippocampal neurogenesis, progenitor cell proliferation and maturation were analyzed by quantifying Mcm2+ proliferating cells and doublecortin (DCX)+ neuroblasts in the DG. Cells undergoing cell death during maturation were quantified by co-staining DCX with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Results Microglial density and proliferation in the dentate gyrus region were preserved between Casp8∆IEC and wt mice at both 14 and 24 weeks of age. Likewise, microglial activation state and phagocytosis remained unchanged between the two groups at both time points Adult hippocampal neurogenesis was maintained between the two groups. The proliferation and maturation of progenitor cells, as well as the rate of cell death during maturation in the dentate gyrus, remained similar between Casp8∆IEC and wt mice. Conclusion These results demonstrate that key aspects of hippocampal immune function and adult hippocampal neurogenesis are resilient to experimental ileocolitis induced by the deletion of Casp8 in intestinal epithelial cells. References (1)Masanetz RK, Winkler J, Winner B, Günther C, Süß P. The Gut-Immune-Brain Axis: An Important Route for Neuropsychiatric Morbidity in Inflammatory Bowel Disease. Int J Mol Sci. 2022;23(19):11111. Published 2022 Sep 21. doi:10.3390/ijms231911111 (2)Ekdahl CT, Claasen JH, Bonde S, Kokaia Z, Lindvall O. Inflammation is detrimental for neurogenesis in adult brain. Proc Natl Acad Sci U S A. 2003;100(23):13632-13637. doi:10.1073/pnas.2234031100 (3)Toda T, Parylak SL, Linker SB, Gage FH. The role of adult hippocampal neurogenesis in brain health and disease. Mol Psychiatry. 2019;24(1):67-87. doi:10.1038/s41380-018-0036-2 (4)Günther C, Martini E, Wittkopf N, et al. Caspase-8 regulates TNF-α-induced epithelial necroptosis and terminal ileitis. Nature. 2011;477(7364):335-339. Published 2011 Sep 14. doi:10.1038/nature10400

Arsenic trioxide preconditioning attenuates hepatic ischemia- reperfusion injury in mice: Role of ERK/AKT and autophagy.

Arsenic trioxide (ATO) is indicated as a broad-spectrum medicine for a variety of diseases, including cancer and cardiac disease. While the role of ATO in hepatic ischemia/reperfusion injury (HIRI) has not been reported. Thus, the purpose of this study was to identify the effects of ATO on HIRI. In the present study, we established a 70% hepatic warm I/R injury and partial hepatectomy (30% resection) animal models in vivo and hepatocytes anoxia/reoxygenation (A/R) models in vitro with ATO pretreatment and further assessed liver function by histopathologic changes, enzyme-linked immunosorbent assay, cell counting kit-8, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Small interfering RNA (siRNA) for extracellular signal-regulated kinase (ERK) 1/2 was transfected to evaluate the role of ERK1/2 pathway during HIRI, followed by ATO pretreatment. The dynamic process of autophagic flux and numbers of autophagosomes were detected by Green fluorescent protein-monomeric Red fluorescent protein-LC3 (GFP-mRFP-LC3)staining and transmission electron microscope. A low dose of ATO (0.75μmol/L in vitro and 1mg/kg in vivo)significantly reduced tissue necrosis, inflammatory infiltration, and hepatocyte apoptosis during the process of hepatic I/R. Meanwhile, ATO obviously promoted the ability of cell proliferation and liver regeneration. Mechanistically, in vitrostudies have shown that nontoxic concentrations of ATO can activate both ERK and phosphoinositide 3-kinase-serine/threonine kinase (PI3K-AKT) pathways and further induce autophagy. The hepatoprotective mechanism of ATO, at least in part, relies on the effects of ATO on the activation of autophagy, which is ERK-dependent. Low, non-toxic doses of ATO can activate ERK/PI3K-AKT pathways and induce ERK-dependent autophagy in hepatocytes, protecting liver against I/R injury and accelerating hepatocyte regeneration after partial hepatectomy.

Esmolol upregulates the α7 nAChR/STAT3/NF-κB pathway by decreasing the ubiquitin and increasing the ChAT+CD4+ T lymphocyte to alleviate inflammation in septic cardiomyopathy.

Esmolol has been demonstrated to mitigate inflammation damage and T lymphocyte apoptosis in septic cardiomyopathy. It has been established that the activation of α7 nicotinic acetylcholine receptor (nAChR) by cluster of differentiation 4(CD4) + T lymphocytes expressing choline acetyltransferase (ChAT) can prevent excessive inflammation and reduce splenocyte apoptosis in septic cardiomyopathy. Given the similar anti-inflammatory effects, we hypothesized that esmolol might be associated with α7 nAChR and thereby exert its cardioprotective functions. In the cecal ligation puncture (CLP)-induced rat septic cardiomyopathy model, esmolol was found to attenuate myocardial injury as evidenced by Hematoxylin and Eosin (HE) staining, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, and the reduced concentration of interleukin (IL)-1, IL-6, and Tumor Necrosis Factor (TNF)-α detected by enzyme-linked immunosorbent assay (ELISA). Western blotting (WB) revealed that esmolol enhanced the expression of α7 nAChR, elevated the level of Phosphorylated-Signal transducer and activator of transcription 3 (P-STAT3)/STAT3, and decreased the level of Nuclear factor-κB (NF-κB), which led to the reduction of plasma IL-1, IL-6, and TNF-α. Methyl lycaconitine Citrate (MLA, an α7 nAChR inhibitor) suppressed the level of P-STAT3/STAT3, while stattic (a STAT3 inhibitor) inhibited the level of P-STAT3/STAT3 and up-regulated the expression of NF-κB. Real-time quantitative PCR (RT-qPCR) results indicated no significant difference in the mRNA level of α7 nAChR, but immunofluorescence and WB results verified the upregulation of α7 nAChR by esmolol and the reduction of ubiquitin induced by esmolol. In the spleen, esmolol decreased splenocyte apoptosis and increased the expression of α7 nAChR as shown by immunofluorescence. In isolated CD4+ T cells obtained through magnetic cell separation, esmolol enhanced the expression of ChAT mRNA. In conclusion, esmolol upregulates α7 nAChR by decreasing ubiquitin and increasing ChAT+CD4+ T lymphocytes and then increases the P-STAT3/STAT3 which inhibits NF-κB thus alleviating inflammation in septic cardiomyopathy.

Inhibition of p70 Ribosomal S6K1 Protects the Myocardium against Ischemia/Reperfusion-Induced Necrosis through Downregulation of RIP3.

Myocardial ischemia-reperfusion (I/R) injury refers to cell damage that occurs as a consequence of the restoration of blood circulation following reperfusion therapy for cardiovascular diseases, and it is a primary cause of myocardial infarction. The search for nove therapeutic targets in the context of I/R injury is currently a highly active area of research. p70 ribosomal S6 kinase (S6K1) plays an important role in I/R induced necrosis, although the specific mechanisms remain unclear. This study aims to explore the effects of inhibiting S6K1 on myocardial I/R injury and its potential mechanisms. A rat myocardial I/R model was created and treated with the S6K1-specific inhibitor PF-4708671. Hematoxylin-eosin (H&E) staining was applied to evaluate the pathological changes in cardiac tissues. 2,3,5-triphenyltetrazolium chloride (TTC) staining was used to measure the area of myocardial infarction (MI). Left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), the maximum rate of increase in left ventricular pressure (+dp/dtmax), and the maximum rate of the decrease in left ventricular pressure (-dp/dtmax) were measured using ultrasonic echocardiography. The expression levels of cardiac troponin-1 (cTn-1), lactate dehydrogenase (LDH), creatine kinase MB (CK-MB), and aspartate aminotransferase (AST) were determined by enzyme-linked immunosorbent assay (ELISA). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and propidium iodide (PI) staining were used to examine the apoptosis and necrosis of myocardial tissues. The expressions of apoptotic-related proteins, and key molecules of necrosis were detected by western blot. The relationship between S6K1 and receptor-interacting protein kinase 3 (RIP3) was analyzed by immunoprecipitation. Inhibition of S6K1 reduces I/R-induced myocardial tissue damage, improves myocardial function, and inhibits myocardial tissue necrosis (p < 0.05). In addition, RIP3 is a direct target of S6K1, and activation of RIP3 blocked the protective effect of the S6K1 inhibitor PF-4708671 against myocardial I/R injury (p < 0.05). Inhibition of S6K1 protects against myocardial I/R injury by down-regulating RIP3, suggesting that targeting S6K1 may offer a novel approach for intervention in myocardial I/R injury.

N-terminomics profiling of host proteins targeted by excretory-secretory proteases of the nematode Angiostrongylus vasorum identifies points of interaction with canine coagulation and complement cascade.

The cardiopulmonary nematode Angiostrongylus vasorum can cause severe disease in dogs, including coagulopathies manifesting with bleeding. We analysed A. vasorum excretory/secretory protein (ESP)-treated dog plasma and serum by N-terminome analysis using Terminal Amine Isotopic Labelling of Substrates (TAILS) to identify cleaved host substrates. In plasma and serum samples 430 and 475 dog proteins were identified, respectively. A total of eight dog proteins were significantly cleaved at higher levels upon exposure to A. vasorum ESP: of these, three were coagulation factors (factor II, V and IX) and three were complement proteins (complement C3, C4-A and C5). Comparison with human motif sequence orthologues revealed known cleavage sites in coagulation factor IX and II (prothrombin). These and further identified cleavage sites suggest direct or indirect activation or proteolysis of complement and coagulation components through A. vasorum ESP, which contains several proteases. Further studies are needed to validate their substrate specificity.

Thymoquinone-PLGA-PF68 Nanoparticles Induce S Phase Cell Cycle Arrest and Apoptosis, Leading to the Inhibition of Migration and Colony Formation in Tamoxifen-Resistant Breast Cancer Cells.

A biocompatible polymeric nanoparticle, TQ-PLGA-PF68, was developed through the interaction of the phytochemical thymoquinone (TQ) encapsulated in poly(L-lactide-co-glycolide)-b-poly(ethylene glycol) (PLGA-PEG) with Pluronics F68. So far, this combination has not been assessed on breast cancer cells resistant to anti-cancer drugs. Therefore, this study aimed to assess the cell death caused by TQ-PLGA-PF68 nanoparticles, particularly in resistant breast cancer cell lines expressing estrogen receptor (ER) positivity, such as TamR MCF-7. The antiproliferative activity of TQ-PLGA-PF68 nanoparticles was measured using the MTS assay. The cytotoxic effects were further evaluated through colony formation assay and scratch-wound healing assay. Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assay was performed to determine the characteristics of the apoptosis as well as cell cycle arrest induced by TQ-PLGA-PF68 nanoparticles. The localization of these nanoparticles in the cells was examined using Transmission Electron Microscopy (TEM). With a TQ concentration of 58.5 μM encapsulated within the nanoparticles, cytotoxicity analysis revealed a significant inhibition of cell proliferation (p<0.05). This finding was corroborated by the results of the colony formation assay. Treatment with TQ-PLGA-PF68 nanoparticles significantly decreased the number of surviving TamR MCF-7 cells by 35% (p<0.001) compared to untreated TamR MCF-7 cells. Concurrently, the scratch-wound healing assay indicated a closure rate of 50% versus >80% (p<0.05) in untreated TamR MCF-7 cells at 12 hours post-wounding. The TUNEL assay successfully confirmed the apoptosis characteristics associated with cell cycle arrest. TEM observation confirmed the cellular internalization of these nanoparticles, suggesting the in vitro therapeutic potential of the formulation. In this study, a significant functional change in TamR MCF-7 cells induced by the TQ nanoparticles was observed. The unique incorporation of TQ into the PLGA-PEG and Pluronics F68 formulation preserved its bioactivity, thereby reducing the migratory and proliferative traits of drug-resistant cells. This discovery may pave the way for exploring the application of biocompatible polymeric TQ nanoparticles as a novel therapeutic approach in future studies pertaining to resistant breast cancer.

Targeting P21-Activated Kinase 2 as a Novel Therapeutic Approach to Mitigate Endoplasmic Reticulum Stress in Heart Failure With Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) is linked to prolonged endoplasmic reticulum (ER) stress. P21-activated kinase 2 (Pak2) facilitates a protective ER stress response. This study explores the mechanism and role of Pak2 in HFpEF pathology. The HFpEF mouse model was established using a high-fat diet combined with the nitric oxide synthase inhibitor Nω-Nitro-l-arginine methyl ester (high-fat diet+Nω-Nitro-l-arginine methyl ester). The model exhibited the typical characteristics of HFpEF (cardiac hypertrophy, diastolic dysfunction with preserved systolic function, and lung edema) as determined by echocardiography and hemodynamic analysis. Terminal deoxynucleotidyl transferase dUTP nick end labeling and dihydroethidium staining results showed that cell death and reactive oxygen species generation were higher in the high-fat diet+Nω-Nitro-l-arginine methyl ester-treated group. Transmission electron microscopy revealed disruption of the ER subcellular structures in the HFpEF mouse model, while western blot analysis confirmed reduced Pak2 phosphorylation and impaired inositol-requiring enzyme 1/X-box binding protein 1 splicing ER stress response signaling. Furthermore, H9c2 cells subjected to the palmitic acid-mediated metabolic stress developed temporal changes in unfolded protein response proteins and Pak2 activity. The inositol requiring enzyme 1/X-box binding protein 1 splicing branch of unfolded protein response was impaired earlier than other branches. Overexpression of Pak2 by adenovirus in H9c2 cells sustained the activation of inositol requiring enzyme 1/X-box binding protein 1 splicing. Pak2 deficiency in the mouse heart accelerated the HFpEF progression, and this phenomenon occurred as early as 10 weeks in Pak2 cardiomyocyte-specific knockout mice. Conversely, adeno-associated virus serotype 9-mediated Pak2 overexpression mitigated HFpEF symptoms, underscoring its protective role against HFpEF progression. Pak2 prevents HFpEF progression, exerting cardioprotective effects against ER stress. These insights underscore the therapeutic value of Pak2 in HFpEF.

FTO Alleviates Hepatic Ischemia-Reperfusion Injury by Regulating Apoptosis and Autophagy.

Objective: Despite N6-methyladenosine (m6A) being closely involved in various pathophysiological processes, its potential role in liver injury is largely unknown. We designed the current research to study the potential role of fat mass and obesity-associated protein (FTO), an m6A demethylase, on hepatic ischemia-reperfusion injury (IRI). Methods: Wild-type mice injected with an adeno-associated virus carrying fat mass and obesity-associated protein (AAV-FTO) or adeno-associated virus carrying green fluorescent protein (GFP) (AAV-GFP) were subjected to a hepatic IRI model in vivo. Hematoxylin-eosin staining was performed to observe IRI. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to observe the cell apoptosis. Reverse transcription polymerase chain reaction (RT-PCR) was used to observe the expression of FTO. The protein levels of FTO, apoptosis, or autophagy-associated signaling proteins were detected by western blot. Reactive oxygen species (ROS) levels were determined by flow cytometry, and immunohistochemistry was used to detect the FTO and LC3-II expression. For in vitro experiments, cultured hepatocytes were subjected to hypoxia/reoxygenation (H/R) stimulation. Monodansylcadaverine (MDC) staining was used to visualize autophagic vesicles. Results: In the present study, we showed that FTO was involved in hepatic IRI, apoptosis, and autophagy. Specifically, the expression level of FTO was significantly reduced in the hepatic IRI. Besides, increasing FTO expression (AAV-FTO) ameliorated the hepatic IRI in animal models, accompanied by decreased apoptosis and autophagy. Furthermore, the FTO inhibitor (FB23-2) aggravated autophagy in hepatocytes upon H/R-induced damage. Conclusion: FTO could act as a protective effector during hepatic IRI, associated with decreased apoptosis and autophagy. FTO-mediated m6A demethylation modification may be an important therapeutic target for hepatic IRI.

Apigenin facilitates apoptosis of acute lymphoblastic leukemia cells via AMP-activated protein kinase-mediated ferroptosis.

The outcomes of pediatric patients with acute lymphoblastic leukemia (ALL) remain far less than favorable. While apigenin is an anti-cancer agent, studies on the mechanism by which it regulates ALL cell cycle progression are inadequate. Ferroptosis and AMP-activated protein kinase (AMPK) signaling are important processes for ALL patients. However, it remains unclear whether apigenin works by affecting AMPK and apoptosis. SUP-B15 and T-cell Jurkat ALL cells were treated with apigenin, and cell viability and apoptosis were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, respectively. The thiobarbituric acid-reactive substances (TBARS) assay was used to evaluate lipid peroxidation. Intracellular Fe2+ levels were measured using a commercial kit. Corresponding proteins were detected by western blotting. Results showed that apigenin reduced cell viability and the levels of Ki67 and proliferating cell nuclear antigen (PCNA) expression in a concentration-dependent manner in both types of ALL cells. Apigenin also exerted anti-apoptotic effects on SUP-B15 and Jurkat cells. Apigenin activated AMP-activated protein kinase (AMPK) signaling and induced ferroptosis, and those effects were attenuated by inhibition of AMPK. Eventually, the reduced cell proliferation and increased cell apoptosis caused by apigenin in ALL cells were partly abolished by AMPK inhibition. In summary, apigenin exerted anti-leukemia activity in ALL cells, and that effect was partially achieved by activation of AMPK signaling. Our findings suggest apigenin as a potential drug for treatment of ALL.