Expression Of Caspase-3 Research Articles

Chrysophanol Induces Cell Death and Inhibits Invasiveness through Alteration of Calcium Levels in HepG2 Human Liver Cancer Cells.

To investigate the effect of chrysophanol, a phytochemical derived from Radix et Rhizoma Rhei on HepG2 liver cancer cells. HepG2 cell line was treated with different concentrations chrysophanol (0-100 μmol/L) for 24 h. The cell counting kit 8 assay was employed to assess cell viability. Intracellular calcium levels were examined using Fluo-4 AM and Mag-fluo-4 AM staining, followed by flow cytometry analysis. Mitochondrial membrane potential was measured with JC-1 assay kit. Additionally, the expressions of key proteins such as p-JNK, Bax, cytochrome c (Cyt C), cleaved caspase-3 (cCaspase-3), and caspase-8 were analyzed by Western blot. The inhibitory effects of chrysophanol on the invasion of cells were determined using a Transwell assay. Analysis of invasiveness was conducted by wound healing assay. Chrysophanol significantly reduced the proliferation of HepG2 liver cancer cells by affecting intracellular calcium distribution, diminishing mitochondrial membrane potential, and enhancing the expressions of p-JNK, Bax, Cyt C, cCaspase-3, and caspase-8 in the groups treated with 75 or 100 μmol/L chrysophanol compared to the control group (P<0.05). Additionally, 75 and 100 μmol/L chrysophanol exhibited inhibitory effects on cell migration and wound healing. Chrysophanol demonstrates potential against HepG2 liver cancer cells, suggesting its potential use as a therapeutic agent for liver cancer treatment.

Effect of crowding stress on liver health, gut permeability and gut microbiota of genetically improved farmed tilapia (GIFT, Oreochromis niloticus)

This study investigated the effects of crowding stress on liver and gut health in genetically improved farmed tilapia (GIFT, Oreochromis niloticus). Fish averaging 18.90 ± 0.01 g were reared at low (LD, 5 g/L) or high densities (HD, 100 g/L) for 14 days. The analysis revealed that the HD group significantly increased serum cortisol, glucose and adrenocorticotropic hormone (ACTH) levels in GIFT (P < 0.05). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were elevated in the HD group (P < 0.05). TUNEL staining of liver tissues revealed that the fluorescence intensity indicating apoptosis was significantly higher in the HD group (P < 0.05). Additionally, the HD group exhibited increased expression of p53 and Caspase3 in the liver (P < 0.05). In comparison to the LD group, the HD group showed a significant increase in gut superoxide dismutase (SOD) and catalase (CAT) activities, as well as malondialdehyde (MDA) content (P < 0.05). The expressions of inflammatory-related genes (tnfα, il-1β, il-8, and il-6) and pro-apoptotic genes (p53 and Caspase7) were significantly upregulated in the gut of the HD group (P < 0.05). Serum contents of D-lactate and diamine oxidase (DAO) were significantly elevated in HD group as comparison to LD group (P < 0.05), and significantly higher serum FITC-CM-dextran was observed in HD group versus LD group following oral gavage (P < 0.05). Furthermore, the HD group down-regulated the expressions of gut tight junction proteins (tjp2a, hif-1a, and zo-1) (P < 0.05), and significantly up-regulated the protein expression of phosphorylated myosin light chain 2 (P-MLC2) (P < 0.05). The 16S rDNA gene sequencing results indicated that the relative abundance of Cetobacterium was significantly lower (P < 0.05), and Enterovibrio and Weissella were significantly higher in the HD group (P < 0.05). Overall, crowding stress negatively affects GIFT liver and gut health, which is caused by inducing hepatic and intestinal apoptosis, impairing intestinal barrier function, and disrupting the gut microbiota.

Lactiplantibacillus plantarum exerts anticancer effects and increase the chemosensitivity of 5-fluorouracil against oral cancer cells in vitro

Introduction: Probiotics are used to provide health benefits and can improve the immune response. They can also target cancer cells directly with anticancer effects through various mechanisms. In this study, Lactiplantibacillus plantarum (basonym: Lactobacillus plantarum) strain MCC 3016 and its postbiotic metabolites/cell free supernatant (CFS) were used against Cal27 oral cancer cells in vitro. Methods: Standard assays were employed to investigate the effect of Lpb. plantarum on cell viability, proliferation, migration, and clonogenicity of Cal27 cells. The mechanism of action was assessed by measuring the levels of reactive oxygen species (ROS), interleukins (IL)-6 and IL-8, tumor necrosis factor-α (TNF-α), as well as the expression of Ki67, vascular endothelial growth factor (VEGF), p53 and caspase-3. Further, the effect of Lpb. plantarum and its CFS on the cytotoxicity of chemotherapy drug 5-fluorouracil (5-FU) was evaluated using cell viability assays. Results: Cal27 cells treated with Lpb. plantarum and its CFS showed a significant decrease (P &lt; 0.01) in cell viability, proliferation, migration, and clonogenicity, along with increased levels of ROS and induced apoptosis. It significantly reduced IL-6, IL-8, TNF-α, and VEGF levels and upregulated p53 and caspase-3 expression. The postbiotic metabolites also showed similar effects on Cal27 cells. Furthermore, the cytotoxic effect of 5-FU on Cal27 cells was enhanced by Lpb. plantarum and its CFS treatment. Conclusion: Lpb. plantarum MCC 3016 and its postbiotic metabolites exhibited promising anticancer effects on oral cancer cells and improved drug efficacy, demonstrating their potential therapeutic value in oral cancer therapy.

β-D-Glucose-Reduced Silver Nanoparticles Remodel the Tumor Microenvironment in a Murine Model of Triple-Negative Breast Cancer.

Breast cancer is the most diagnosed type of cancer worldwide and the second cause of death in women. Triple-negative breast cancer (TNBC) is the most aggressive, and due to the lack of specific targets, it is considered the most challenging subtype to treat and the subtype with the worst prognosis. The present study aims to determine the antitumor effect of beta-D-glucose-reduced silver nanoparticles (AgNPs-G) in a murine model of TNBC, as well as to study its effect on the tumor microenvironment. In an airbag model with 4T1 tumor cell implantation, the administration of AgNPs-G or doxorubicin showed antitumoral activity. Using immunohistochemistry it was demonstrated that treatment with AgNPs-G decreased the expression of PCNA, IDO, and GAL-3 and increased the expression of Caspase-3. In the tumor microenvironment, the treatment increased the percentage of memory T cells and innate effector cells and decreased CD4+ cells and regulatory T cells. There was also an increase in the levels of TNF-α, IFN-γ, and IL-6, while TNF-α was increased in serum. In conclusion, we suggest that AgNPs-G treatment has an antitumor effect that is demonstrated by its ability to remodel the tumor microenvironment in mice with TNBC.

From beverage to anticancer agent: The repurposing of green coffee bean extract loaded in solid lipid nanoparticles

Green coffee bean extract (GCBE) captivated the world with its various health benefits. However, its topical use was limited by the hampered penetration of hydrophilic GCBE through the skin. So, this study aimed to incorporate GCBE-loaded solid lipid nanoparticles (SLNs) into water-in-oil (W/O) cream, to explore its repurposed application as an anticancer drug. The anticancer activity was assessed in vitro using MTT assay against human breast cancer cell lines (MCF-7) and in vivo using the Ehrlich ascites carcinoma (EAC) model in mice compared to free GCBE followed by immunohistochemical analysis of caspase-3 and estrogen receptor. The optimized GCBE-SLNs results of the MTT assay exhibited a concentration-dependent reduction in the viability of MCF-7 with approximately 36 times more toxic effect against MCF-7 compared to free GCBE. The results of the in vivo Ehrlich ascites carcinoma model revealed that GCBE-SLNs cream exhibited statistically significant higher expression of caspase-3, lower expression of estrogen receptor, and increased necrotic index compared to the control group. Conclusively, GCBE-SLNs cream seems to have reassuring promises as anticancer remediation in both in vivo and in vitro studies. The present work is considered the first pre-clinical evidence for the potential merits of GCBE-SLNs in cancer management.

Abstract Mo004: The Protective Effects of Empagliflozin in Carfilzomib-induced Cardiotoxicity in Mice: Unveiling the Crosstalk between Oxidative Stress, Inflammation, Endoplasmic Reticulum Stress, and Autophagy Axes.

Introduction: Multiple myeloma ranks as the second most prevalent hematologic cancer in the US. Survival rates have significantly improved owing to advances in chemotherapy treatment. Carfilzomib, an irreversible proteasome inhibitor, has been used globally for the treatment of multiple myeloma. Unfortunately, its clinical use has been linked to cardiovascular complications. Empagliflozin is a sodium glucose co-transporter-2 inhibitor with potential cardioprotective activities, for which it is approved for heart failure management. Aims: The current study determined the beneficial effects of Empagliflozin against Carfilzomib-induced cardiotoxicity in C57BL/6 mice and the possible underlying mechanisms. Methods: Thirty-two mice were randomly divided into four groups and treated for two days as follows: Control (vehicle), Empagliflozin (10 mg/kg, oral), Carfilzomib (8 mg/kg, i.p.), and Carfilzomib + Empagliflozin groups. Results: The impact of Carfilzomib and Empagliflozin treatment on markers of cardiotoxicity and heart tissue architecture was assessed. Empagliflozin corrected histological alterations, CK-MB activity, and troponin I level induced by Carfilzomib. Additionally, Empagliflozin mitigated Carfilzomib-induced oxidative deficits via its effect on catalase activity, reduced glutathione level, and superoxide dismutase activity, restoring the balance of redox circuit. Moreover, Empagliflozin averted Carfilzomib-induced inflammatory response as evidenced by reducing nuclear factor-κB (p65), tumor necrosis factor-α, and interleukin-1β levels. Mechanistically, a substantial amelioration in the activating transcription factor 6-mediated endoplasmic reticulum stress was demonstrated with Empagliflozin treatment owing to its effect on glucose regulated protein-78, activating transcription factor-6, and C/EBP homologous protein. Intriguingly, the induction of autophagy by Carfilzomib was notably amplified following Empagliflozin administration as evidenced by higher mRNA expression of LC3B and beclin-1, and lower expression of p62. Accordingly, the apoptotic marker cleaved caspase-3 expression was markedly reduced following Empagliflozin treatment. Importantly, Empagliflozin did not alter the cytotoxic effect of Carfilzomib on human U266B1 myeloma cell line. Conclusion: This research suggests Empagliflozin could offer cardioprotection against Carfilzomib-induced cardiotoxicity in multiple myeloma patients.

External pressure induces the dysfunction of spermatogonia via triggering the intrinsic pathway of apoptosis.

Cryptorchidism, the failure of testes to descend into the scrotum, exposes the testes to higher temperature and external pressure. Scholars from Razi University found through research conducted at different pressure gradients (0, 25, 50, and 100 mmHg) and time gradients (2 and 4 h) that high hydrostatic pressure may lead to sperm apoptosis. In this work, we investigated the effect of external pressure on spermatogonia, exploring a new mechanism of male infertility caused by cryptorchidism. Various pressure gradients (0, 25, 50, and 100 mmHg) were applied to spermatogonia for different durations (0, 2, and 4 h) in the Cell Counting Kit-8 (CCK8) experiment. Morphological changes, cell ultrastructure, apoptosis rates, and the expression of apoptosis-related proteins (bax, bcl-2, caspase-3, and caspase-9) were assessed through immunofluorescence, electron microscopy, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, flow cytometry, immunohistochemistry, real-time quantitative polymerase chain reaction (qPCR), and western blot. The cell viability assay showed that higher external pressure had a greater negative time-dependent impact on cell viability. Immunofluorescence results indicated that external pressure stimuli altered the morphology of spermatogonia. The results of TUNEL assay and flow cytometry demonstrated that external pressure stimuli induced apoptosis in spermatogonia. Transmission electron microscopy (TEM) observations showed the generation of apoptotic bodies, mitochondrial swelling, vacuolization, and mitochondrial cristae fusion. The results of immunohistochemistry indicated that pressure induced the expression of caspase-3 and caspase-9 proteins. qPCR and western blot analyses revealed an increased ratio of bax/bcl-2 and expression of caspase-3 and caspase-9. Methazolamide (cytochrome C inhibitor) blocked the pressure-induced cell apoptosis and inhibited the activation of caspase-3 while Z-IETD-FMK (caspase-8 inhibitor) did not. External pressure promotes spermatogonia apoptosis through the intrinsic apoptosis pathway, which may be one of the mechanisms of male infertility induced by cryptorchidism.

Hexavalent chromium damages intestinal cells and coelomocytes and impairs immune function in the echiuran worm Urechis unicinctus by causing oxidative stress and apoptosis

Hexavalent chromium (Cr(VI)) is a common pollutant in the marine environment, which impairs immunity and causes reproductive and heredity disorders in organisms. To clarify the immunotoxic effects of Cr (VI) on the marine worm Urechis unicinctus, we analyzed tissue damage and immune dysfunction caused by Cr (VI) in this organism at histopathologic, zymologic, apoptotic and molecular levels. The results indicated that the bioaccumulation of Cr (VI) bioaccumulation levels in coelomocytes was significantly higher than in the intestines and muscles. Pathological observation showed that Cr (VI) caused damage to the respiratory intestine, stomach and midgut. Cr (VI) also increased the replication of goblet cells and a reduction in the replication of epithelial cells. Meanwhile, Cr (VI) induced apoptosis of intestinal cells and coelomocytes, accompanied by an increase in the expression of Caspase-3, COX-2, and MyD88 in the intestine and coelomocytes. At the same time, Cr (VI) significantly affected the activities of antioxidant enzymes such as SOD, ACP, CAT, CAT, and GST, and increased H2O2 and MDA contents in U. unicinctus. Moreover, Cr (VI) exposure also up-regulated the transcription of hsc70, mt and jnk genes but decreased that of sod in the intestines. In contrast, Cr (VI) down-regulated the expression of sod, hsc70, mt, and jnk genes in coelomocytes. Collectively, Cr (VI) bioaccumulated in U. unicinctus cells and tissues, causing several histopathological changes, oxidative stress, and apoptosis of several cells in the organism, resulting in intestinal and coelomocyte damage and immune dysfunctioning.

SIGMAR1 targets AMPK/ULK1 pathway to inhibit SH-SY5Y cell apoptosis by regulating endoplasmic reticulum stress and autophagy.

Distal hereditary motor neuropathy (dHMN) is a progressive neurological disease characterized by distal limb muscle weakness and amyotrophy. Sigma 1 receptor (σ1R), a gene product of SIGMAR1, mutations have been reported to induce dHMN, but its mechanism remains unknown. This study aims to explore the effect of C238T and 31_50del mutations in σ1R on neuronal SH-SY5Y cell functions. The SH-SY5Y cells that overexpressed σ1R, C238T mutant σ1R (σ1RC238T) or 31_50del mutant σ1R (σ1R31_50del) were constructed by pEGFPN1 vectors. We used Western blot (WB) and immunofluorescence (IF) staining to detect the expression of σ1R and green fluorescent proteins (GFP). Then, we evaluated the impact of σ1R mutation on apoptosis, autophagy, endoplasmic reticulum stress, and the involvement of the unfolded protein response (UPR) pathway in SH-SY5Y cells. We found that σ1RC238T and σ1R31_50del downregulated σ1R and promoted the apoptosis of SH-SY5Y cells. σ1RC238T and σ1R31_50del increased p-PERK, p-eIF2α, p-JNK, BIP, ATF4, CHOP, ATF6, XBP1, Caspase3, Caspase12 expressions and Ca2+ concentration, whereas decreased ATP content in SH-SY5Y cells. Besides, the expressions of LC3B, Lamp1, ATG7, Beclin-1 and phosphorylation of AMPK and ULK1 were increased, while the p62 level decreased after C238T or 31_50del mutation of σ1R. Additionally, AMPK knockdown abolished the apoptosis mediated by σ1RC238T or σ1R31_50del in SH-SY5Y cells. Our results indicated that C238T or 31_50del mutation in σ1R promoted motor neuron apoptosis through the AMPK/ULK1 pathway in dHMN. This study shed light on a better understanding of the neurons pathological mechanisms mediated by σ1R C238T and σ1R 31-50del in dHMN.

Chemical, Antibacterial, and Cytotoxic Properties of Four Different Endodontic Sealer Leachates Over Time

IntroductionThe management of apical periodontitis involves the elimination of bacteria to achieve healing of the periapical tissues. To this end, understanding of the antimicrobial properties and cytotoxicity of root canal sealers over time is important. This study aimed to assess the cytocompatibility and antibacterial activity of leachates obtained from selected endodontic sealers. MethodsFour sealers were used in this study; AH Plus, an epoxy resin-based sealer, and three hydraulic calcium silicate-based sealers: AH Plus Bioceramic, BioRoot RCS, and BioRoot Flow. Sealer chemistry was assessed by scanning electron microscopy and energy dispersive spectrum analysis. Leachates of the tested sealers were prepared by immersing sealer discs in Hank’s balanced salt solution for 28 and 90 days. Element release was assessed using inductively coupled plasma mass spectroscopy. Enterococcus faecalis and Fusobacterium nucleatum were exposed to the leachates followed by colony-forming unit determination, and the human osteoblast-like cell line Saos-2 was assessed with regard to cell death, caspase expression and activity of alkaline phosphatase (ALP) after stimulation with the leachates. ResultsCalcium leaching was detected in the tricalcium silicate-based sealers, which increased the leachate pH significantly (P < .0001). AH Plus Bioceramic leachate displayed antimicrobial activity at 28 days against E. faecalis (P < .01). ALP levels were reduced in response to AH Plus Bioceramic and BioRoot RCS 1-day leachates (P < .05). High cell viability was observed after exposure to all sealers. ConclusionsThe leachates of the tested sealers were cytocompatible; however, their antimicrobial activity as well as their potential to induce the bone formation marker ALP was minimal.

Pterostilbene, a Resveratrol Derivative, Improves Ovary Function by Upregulating Antioxidant Defenses in the Aging Chickens via Increased SIRT1/Nrf2 Expression.

Oxidative stress is recognized as a prominent factor contributing to follicular atresia and ovarian aging, which leads to decreased laying performance in hens. Reducing oxidative stress can improve ovarian function and prolong the laying period in poultry. This study investigates the impact of Pterostilbene (PTS), a natural antioxidant, on ovarian oxidative stress in low-laying chickens. Thirty-six Hy-Line White laying chickens were evenly divided into four groups and fed diets containing varying doses of PTS for 15 consecutive days. The results showed that dietary supplementation with PTS significantly increased the laying rate, with the most effective group exhibiting a remarkable 42.7% increase. Furthermore, PTS significantly enhanced the antioxidant capacity of aging laying hens, as evidenced by increased levels of glutathione, glutathione peroxidase, superoxide dismutase, catalase, and total antioxidant capacity in the ovaries, livers, and serum. Subsequent experiments revealed decreased expressions of Bax, Caspase-3, and γ-H2AX, along with an increased expression of BCL-2 in the ovaries and livers of laying hens. PTS supplementation also positively affects fat metabolism by reducing abdominal fat accumulation and promoting fat transfer from the liver to the ovary. To elucidate the mechanism underlying the effects of PTS on ovarian function, a series of in vitro experiments were conducted. These in vitro experiments revealed that PTS pretreatment restored the antioxidant capacity of D-galactose-induced small white follicles by upregulating SIRT1/Nrf2 expression. This protective effect was inhibited by EX-527, a specific inhibitor of SIRT1. These findings suggest that the natural antioxidant PTS has the potential to regulate cell apoptosis and fat metabolism in laying chickens by ameliorating oxidative stress, thereby enhancing laying performance.

Trimethyltin chloride induces oxidative damage and apoptosis in chicken liver

Trimethyltin chloride (TMT) is widespread in the environment and is harmful to both humans and animals. In order to investigate the toxicity mechanism of TMT exposure on chicken liver, We established an in vivo experimental model by giving chickens oral administration of different concentrations of TMT dilution solution and vitro experiments of treating leghorn male hepatoma (LMH) cells for 12 h. The results showed that Albumin (ALB), total protein (TP) and alanine aminotransferase (ALT) in the blood of TMT-treated chickens, as well as ALT and aspartate aminotransferase (AST) in the liver, were dose-dependently increased, and different degrees of necrosis of hepatocytes were observed in histology. Meanwhile, TMT exposure led to a significant decrease in glutathione (GSH) content in chicken liver tissues and LMH cells, what's more a significant increase in malondialdehyde (MDA) content in cell supernatants. The expression of apoptosis-related genes Caspase8, Caspase3 and Caspase9 were increased in chicken liver tissues and LMH cells after treated by TMT, and an increased in the percentage of late apoptosis in LMH cells. This suggests that TMT can cause oxidative stress and apoptosis in chicken livers and cells, resulting in liver injury.

Tongqiao Yizhi granule repress the nuclear factor kappa-b/nucleotide oligomerization domain-like receptors 3/caspase-1 pyroptosis pathway in the hippocampus to counter vascular dementia in rats.

To explore the mechanism by which Tongqiao Yizhi granule (, TQYZKL) intervenes pyroptosis to treat vascular dementia (VaD) in a rat model. The rat model of VaD was established by two-vessel occlusion (2VO). The rats were randomly divided into Sham group, Model group, Nimodipine group, TQYZKL (6.2 g?kg-1?d-1), TQYZKL (12.4 g?kg-1?d-1), TQYZKL (24.8 g?kg-1?d-1). The Morris water maze (MWM) test was carried out to test the learning and memory function; Hematoxylin-eosin staining and transmission electron microscopy (TEM) to observe the pathological damage in the hippocampus; Tunel fluorescence staining to detect neuronal pyroptosis in the hippocampus. The expression levels of pyroptosis-related proteins, namely Golgi peripheral membrane protein p65 (P65), nucleotide oligomerization domain-like receptors 3 (NLRP3), caspase-1 and Gasdermin D (GSDMD), were detected using Western blotting and reverse transcription polymerase chain reaction. Moreover, the serum levels of interleukin-1β (IL-1β) and interleukin-18 (IL-18) were determined through the enzyme-linked immunosorbent assay. The study revealed that TQYZKL effectively improved the ability of VaD ratsto learn and memorize, relieved the pathological damage in the hippocampus, restored neuronal morphology, and reduced the expression of pyroptosis-related proteins P65, NLRP3, caspase-1, GSDMD-N, IL-18 and IL-1β (P < 0.05). TQYZKL inhibits neuronal pyroptosis in the hippocampus of VaD rats by regulating nuclear factor kappa-B/NLRP3/caspase-1 signaling pathway, thus exerting a therapeutic effect on VaD in the rats.

Sulodexide protects endothelial cells against 4-hydroxynonenal-induced oxidative stress and glutathione-dependent redox imbalance by modulation of sestrin2/nuclear factor erythroid 2-related factor 2 pathway.

The lipid peroxidation product 4-hydroxynonenal (HNE) may be involved in vascular endothelial cell damage by induction of oxidative stress, apoptosis, and loss of redox homeostasis. There is evidence that stimulation of endothelial cells with 4-HNE induces the activation of the nuclear factor erythroid 2-related factor 2/Kelch-like ECH-associated protein 1 (Nrf2/Keap-1) pathway. Sestrin2 protein (SESN2) is one of the key regulators of Nrf2 and is involved in the cellular response to oxidative stress. However, the function of SESN2 in HNE-induced endothelial injury is not yet understood. Sulodexide (SDX) is a mixture of glycosaminoglycans used in clinical practice in the treatment of chronic venous and arterial diseases. While SDX has well-documented endothelial protective properties, little is known about its antioxidant effects. The aim of this study was to elucidate the molecular mechanisms activated by SDX in human umbilical endothelial cells (HUVECs) under HNE-induced oxidative stress. In this experimental model, we decided to evaluate the anti-apoptotic and antioxidant potential of SDX and its effect on the SESN2/Nrf2/GSH pathway. HUVECs were treated with 25 _M HNE or HNE combined with 0.5 LRU/mL SDX for 4 hours. Cell viability, apoptosis and intracellular reactive oxygen species (ROS) production were assessed by MTT assay and fluorescence microscopy. The expressions of Bax, cleaved caspase-3, Keap-1 and Nrf2 were determined by Western blot analysis. The intracellular concentrations of reduced glutathione (GSH) and oxidized glutathione (GSSG) were measured by colorimetric assay. SESN2, glutamate-cysteine ligase catalytic subunit (GCLc) and glutathione synthase (GSS) were assessed using ELISA. RT-qPCR was performed to detect Nrf2, GCLc and GSS mRNA levels. Transient Nrf2 silencing was obtained by short interfering RNA (siRNA). We have demonstrated that SDX can reduce the negative impact of HNE on HUVECs. SDX significantly protected HNE-treated HUVECs from apoptosis (p<0.001) and oxidative stress (p<0.001). SDX treatment significantly reduced Bax (p<0.05) and cleaved caspase-3 (p<0.01) expression. Co-administration of HNE and SDX increased GSH content (p<0.001) and GSH:GSSG ratio (p<0.001) as well as decreased SESN2 concentration (p<0.001) and Nrf2 (p<0.01), GCLc (p<0.05) and GSS (p<0.01) gene expression compared with the HNE group. Moreover, we revealed a negative correlation between SESN2 levels and GSH concentrations (p<0.001). Nrf2 silencing significantly decreased the effect of HNE and SDX on the induction of GCLc and GSS genes. SDX also significantly ameliorated the increase of nuclear Nrf2 in response to HNE (p<0.05). The results confirmed that SDX may protect against HNE-induced endothelial damage through its antioxidant effect and modulation of the SESN2/Nrf2/GSH signaling pathway.

The ameliorative effect of Naringenin on fenamiphos induced hepatotoxicity and nephrotoxicity in a rat model: Oxidative stress, inflammatory markers, biochemical, histopathological, immunohistochemical and electron microscopy study

Fenamiphos (FNP) is an organophospate pesticide that causes many potential toxicities in non-target organisms. Naringenin (NAR) has protective properties against oxidative stress. In this study, FNP (0.76 mg/kg bw) toxicity and the effect of NAR (50 mg/kg bw) on the liver and kidney of rats were investigated via biochemical, oxidative stress, immunohistochemical, cytopathological and histopathologically. As a result of biochemical studies, FNP caused oxidative stress in tissues with a change in total antioxidant/oxidant status. After treatment with FNP, hepatic and renal levels of AChE were significantly reduced while 8-OHdG and IL-17 levels, caspase-3 and TNF-α immunoreactivity increased compared to the control group. It also changed in serum biochemical markers such as ALT, AST, BUN, creatinine. Exposure to FNP significantly induced cytopathological, histopathological and immunohistochemical changes through tissue damage. NAR treatment restored biochemical parameters, renal/hepatic AChE, ultrastructural, histopathological and immunohistochemical changes modulated and blocked the increasing effect of FNP on tissues caspase-3 and TNF-α expressions, 8-OHdG and IL-17 levels. In electron microscopy studies, swelling was observed in the mitochondria of the cells in both tissues of the FNP-treated rats, while less ultrastructural changes in the FNP plus NAR-treated rats.

MiR-21 attenuated inflammation targeting MyD88 in human chondrocytes stimulated with Hyaluronan oligosaccharides

Inflammation is the body's response to injuries, which depends on numerous regulatory factors. Among them, miRNAs have gained much attention for their role in regulating inflammatory gene expression at multiple levels. In particular, miR-21 is up-regulated during the inflammatory response and reported to be involved in the resolution of inflammation by down-regulating pro-inflammatory mediators, including MyD88. Herein, we evaluated the regulatory effects of miR-21 on the TLR-4/MyD88 pathway in an in vitro model of 6-mer HA oligosaccharides-induced inflammation in human chondrocytes.The exposition of chondrocytes to 6-mer HA induced the activation of the TLR4/MyD88 pathway, which culminates in NF-kB activation. Changes in miR-21, TLR-4, MyD88, NLRP3 inflammasome, IL-29, Caspase1, MMP-9, iNOS, and COX-2 mRNA expression of 6-mer HA-stimulated chondrocytes were examined by qRT-PCR. Protein amounts of TLR-4, MyD88, NLRP3 inflammasome, p-ERK1/2, p-AKT, IL-29, caspase1, MMP-9, p-NK-kB p65 subunit, and IKB-a have been evaluated by ELISA kits. NO and PGE2 levels have been assayed by colorimetric and ELISA kits, respectively.HA oligosaccharides induced a significant increase in the expression of the above parameters, including NF-kB activity. The use of a miR-21 mimic attenuated MyD88 expression levels and the downstream effectors. On the contrary, treatment with a miR-21 inhibitor induced opposite effects. Interestingly, the use of a MyD88 siRNA confirmed MyD88 as the target of miR-21 action.Our results suggest that miR-21 expression could increase in an attempt to reduce the inflammatory response, targeting MyD88.

AB020. Flagellin synergistically enhances anti-tumor effect of EGFRvIII peptide in a glioblastoma-bearing mouse brain tumor model.

Glioblastoma (GBM) is the most aggressive type of brain tumor with heterogeneity and strong invasive ability. Treatment of GBM has not improved significantly despite the progress of immunotherapy and classical therapy. Epidermal growth factor receptor variant III (EGFRvIII), one of GBM-associated mutants, is regarded as an ideal therapeutic target in EGFRvIII-expressed GBM patients because it is a tumor-specific receptor expressed only in tumors. Flagellin B (FlaB) originated from Vibrio vulnificus, is known as a strong adjuvant that enhances innate and adaptive immunity in various vaccine models. This study investigated whether FlaB synergistically could enhance the anti-tumor effect of EGFRvIII peptide (PEGFRvIII). EGFRvIII-GL261/Fluc cells were used for GBM-bearing mouse brain model. Cell-bearing mice were inoculated with phosphate-buffered saline (PBS), FlaB alone, PEGFRvIII alone, and PEGFRvIII plus FlaB. Tumor growth based on magnetic resonance imaging (MRI) and the survival rate was investigated. T cell population was examined by flow cytometry analysis. Both cleaved caspase-3 and CD8+ lymphocytes were shown by immunohistochemistry (IHC) staining. The PEGFRvIII plus FlaB group showed delayed tumor growth and increased survival rate when compared to other treatment groups. As evidence of apoptosis, cleaved caspase-3 expression and DNA disruption were more increased in the PEGFRvIII plus FlaB group than in other groups. In addition, the PEGFRvIII plus FlaB group showed more increased CD8+ T cells and decreased Treg cells than other treatment groups in the brain. FlaB can enhance the anti-tumor effect of PEGFRvIII by increasing CD8+ T cell response in a mouse brain GBM model.

Grp78 regulates NLRP3 inflammasome and participates in Sjogren’s syndrome

ObjectiveThe purpose of the present study was to potential effects of forsythiaside A (FA) on Sjogren’s syndrome (SS). MethodsEnzyme linked immunosorbent assay for detecting cytokines and Western blotting was used for detecting related protein expression. ResultsFA effectively reduced the secretion of inflammatory cytokines, the expression of Caspase-1 and NLRP3 proteins and the expression of p65 in SS. FA also effectively inhibited the high expression of Grp78 in SS. When Grp78 expression was silenced, it effectively reduced the secretion of inflammatory cytokines, the expression of Caspase-1 and NLRP3 proteins and the expression of p65 in the nucleus in SS. FA effectively inhibit the secretion of inflammatory cytokines induced by overexpression of Grp78, the expression of Caspase-1 and NLRP3 proteins and the expression of p65 in the nucleus in SS. ConclusionFA induces the degradation of Grp78 protein, regulates the NF-κB signaling pathway in SS and inhibited NLRP3 inflammasome activation and reduced the release of inflammatory cytokines to alleviate SS.

Esaxerenone Attenuates Aldosterone-Induced Mitochondrial Damage-Mediated Pyroptosis in Mouse Aorta and Rat Vascular Smooth Muscle Cells.

Vascular smooth muscle cell (VSMC) injury caused by the inflammatory response plays a key role in cardiovascular disease (CVD), and the vasoprotective effects of mineralocorticoid receptor blockers (MRBs) support the role of mineralocorticoid receptor (MR) activation. C57BL/6 mice and VSMCs isolated from rats were treated with aldosterone and esaxerenone. Caspase-1, GSDMD-N, IL-1β, and NR3C2 expression and DNA damage in aortic VSMCs were detected using immunohistochemistry, Western blotting, and TUNEL staining. Mitochondrial changes were detected by transmission electron microscopy (TEM). Reactive oxygen species (ROS), MitoTracker, JC-I, mitochondrial respiratory chain complexes I-V, and NR3C2 were detected using immunofluorescence and flow cytometry. Pyroptosis was detected with scanning electron microscopy (SEM). After aldosterone treatment, the number of TUNEL-positive cells increased significantly, and the expression of caspase-1, GSDMD-N, and IL-1β increased. TEM revealed mitochondrial damage, and SEM revealed specific pyroptotic changes, such as cell membrane pore changes and cytoplasmic extravasation. Increased ROS levels and nuclear translocation of NR3C2 were also observed. These pyroptosis-related changes were reversed by esaxerenone. Aldosterone activates the MR and mediates mitochondrial damage, thereby inducing pyroptosis in VSMCs via the NLRP3/caspase-1 pathway. Esaxerenone inhibits MR activation and reduces mitochondrial damage and oxidative stress, thereby inhibiting pyroptosis.

Naringin and temozolomide combination suppressed the growth of glioblastoma cells by promoting cell apoptosis: network pharmacology, in-vitro assays and metabolomics based study.

Introduction: Glioblastoma, which affects a large number of patients every year and has an average overall lifespan of around 14.6months following diagnosis stands out as the most lethal primary invasive brain tumor. Currently, surgery, radiation, and chemotherapy with temozolomide (TMZ) are the three major clinical treatment approaches. However, the ability to treat patients effectively is usually limited by TMZ resistance. Naringin, a bioflavonoid with anti-cancer, antioxidant, metal-chelating, and lipid-lowering effects, has emerged as a promising therapeutic option. Methods: To explore the targets and pathways of naringin and TMZ in glioblastoma network pharmacology, cell line-based ELISA, flow cytometry, immunocytochemistry, western blotting, and LC-HRMS based metabolomics study were used. Results: The findings through the network pharmacology suggested that the key targets of naringin in the chemosensitization of glioblastoma would be Poly [ADP-ribose] polymerase 1 (PARP-1), O-6-Methylguanine-DNA Methyltransferase (MGMT), and caspases. The functional enrichment analysis revealed that these targets were significantly enriched in important pathways such as p53 signaling, apoptosis, and DNA sensing. Further, the results of the in-vitro study in U87-MG and T98-G glioblastoma cells demonstrated that TMZ and naringin together significantly reduced the percentage of viability and inhibited the DNA repair enzymes PARP-1 and MGMT, and PI3K/AKT which led to chemosensitization and, in turn, induced apoptosis, which was indicated by increased p53, caspase-3 expression and decreased Bcl2 expression. Additionally, a metabolomics study in T98-G glioblastoma cells using liquid chromatography high-resolution mass spectrometry (LC-HRMS) revealed downregulation of C8-Carnitine (-2.79), L-Hexanoylcarnitine (-4.46), DL-Carnitine (-2.46), Acetyl-L-carnitine (-3.12), Adenine (-1.3), Choline (-2.07), Propionylcarnitine (-1.69), Creatine (-1.33), Adenosine (-0.84), Spermine (-1.42), and upregulation of Palmitic Acid (+1.03) and Sphingosine (+0.89) in the naringin and TMZ treatment groups. Discussion: In conclusion, it can be said that naringin in combination with TMZ chemosensitized TMZ antiglioma response and induced apoptosis in tumor cells.