Mitochondrial Membrane Potential Assay Research Articles

Negative effect of varicocele on sperm mitochondrial dysfunction: A cross-sectional study.

Varicocele is an abnormal dilation and enlargement of the scrotal venous pampiniform plexus that impairs normal blood drainage and finally leads to infertility if not treated. This study aimed to figure out the impact of mitochondria status through the mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) assessment and its correlation with semen parameters to illuminate the impact of sperm mitochondria healthiness on normal sperm functionality. This analytical cross-sectional study was conducted with 100 men including 50 cases in the normozoospermic group (normal) and 50 in an infertile group with the non-varicocelectomy operation (varicocele) referring to Infertility Research and Treatment Center, ACECR Khuzestan, Iran. Routine semen analysis was performed according to World Health Organization guidelines, DNA fragmentation index, the MMP assay, ATP content, and apoptosis were carried out for all samples. The results showed that the concentration, progressive motility, normal morphology, MMP, and ATP contents of sperm in varicocele were significantly lower than the normal group. In addition, the sperm DNA fragmentation index was significantly higher in the varicocele group in comparison with the normal group. Reduction in MMP and ATP contents, besides the loss of sperm parameters quality and increase in sperm DNA fragmentation, were seriously implicating sperm mitochondria dysfunctionality in varicocele men.

Trimethylamine-N-oxide sensitizes chondrocytes to mechanical loading through the upregulation of Piezo1.

Mechanical strain plays a crucial role in chondrocyte apoptosis and osteoarthritis (OA) disease progression through Piezo1. Trimethylamine-N-oxide (TMAO) is a diet-derived metabolite that correlates positively with multiple chronic diseases. Herein, we explored the potential role of TMAO in sensitizing chondrocytes to Piezo1-mediated mechanotransduction. The cytotoxicity of TMAO on chondrocytes was assayed. Piezo1 expression was measured after TMAO intervention. Pathological mechanical loading or Yoda1 (a specific Piezo1 channel activator) was administered in chondrocytes. The calcium levels and cytoskeleton in chondrocytes were observed by fluorescence microscopy. Flow cytometry, western blotting, and mitochondrial membrane potential assays were utilized to evaluate apoptosis. A rat OA model was constructed by anterior cruciate ligament transection. Hematoxylin-eosin staining, Safranin-O/Fast Green staining, immunochemistry, and TUNEL were applied to estimate OA severity. TMAO intervention alone did not affect chondrocyte viability up to 600μM. TMAO significantly increased Piezo1 expression and up-regulated intracellular calcium levels, further leading to cytoskeletal damage. Mechanical strain or Yoda1 treatment significantly induced chondrocyte apoptosis. Notably, TMAO intervention further aggravated chondrocyte apoptosis and cartilage destruction under pathological mechanical loading. TMAO significantly up-regulated Piezo1 expression and sensitized chondrocytes to mechanical loading, which may be closely related to the pathogenesis of OA.

Acetaminophen causes mitochondrial dysfunction in heart cardiomyocytes

Acetaminophen (APAP) is a common over-the-counter medication used to treat pain and fever. It can be taken orally, topically, or intravenously and is considered a safe and effective drug when taken at therapeutic doses. However, recent research suggests that taking APAP regularly can result in increased blood pressure and cardiovascular risk. Previous publications found that Non-Steroidal-Anti-Inflammatory-Drugs (NSAIDs) increased Reactive Oxygen Species (ROS) in cardiomyocytes, resulting in cardiac dysfunction. Although APAP is not a NSAID, APAP likely has similar effects. We hypothesized that regular APAP usage resulted in increased ROS levels, leading to potential dysfunction in hearts and other organs. We used the embryonic rat heart cell line H9C2 to test our hypothesis. We conducted a ROS Assay using H9C2 cells treated with dichlorodihydrofluorescein diacetate (DFCDA). The cells were then treated with vehicle, various concentrations of APAP (25μM, 50μM, 100μM, and 200μM), or 100μM hydrogen peroxide for 1.5 hours before measuring the ROS levels of the cells. We also measured the cell viability of H9C2 cells. Cells were treated with 10μM Alamar Blue, then treated with vehicle, various concentrations of APAP (25μM, 50μM, 100μM, and 200μM), or 100μM or 200μM hydrogen peroxide for 48 hours before detecting the percentage of viable cells. The mitochondrial membrane potential (MMP) of cells treated with APAP was also measured. Cells were treated with vehicle, various concentrations of APAP (25μM, 50μM, 100μM, and 200μM), or 250μM p-triflouromethoxyphenylhydrazone (FCCP) for 24 hours before being incubated with JC-10 for one hour. The cells were measured using a fluorescent reader at excitation/emission 490/525 nm and 540/590 nm. For the ROS assay, the cells treated with 100μM APAP, 200μM APAP, and 100μM Hydrogen Peroxide significantly increased total ROS compared to the control. The cell viability assay resulted in a significant decrease in percent viability in cells treated with 200μM APAP, 100μM hydrogen peroxide, and 200μM hydrogen peroxide compared to the control. The MMP Assay resulted in a significant decrease in MMP at all concentrations of APAP and 250μM FCCP compared to the control. These results suggest that treatment of H9C2 cardiac cells with physiological concentrations of APAP causes an increase in intracellular ROS levels, decreased cell viability, and decreased MMP. Overall, APAP causes mitochondrial dysfunction, which is likely to cause cardiomyocyte dysfunction. This research is funded by the NIEHS/Superfund Research Program (P42 ES004699). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract LB256: Type 1 calreticulin mutations in myeloproliferative neoplasms promote glycolysis via intracellular calcium mediated upregulation of GLUT1

Abstract Myeloproliferative neoplasms (MPNs) comprise a group of cancers of the bone marrow that can transform into a post-MPN acute myeloid leukemia (AML), at which point the overall survival rate is less than 6 months. JAK2 inhibitors in MPN failed to alter disease progression, emphasizing the need for new treatments. Calreticulin (CALR) is one of the MPN driver mutations and encodes an endoplasmic reticulum (ER) calcium (Ca2+) binding protein. CALR mutations are classified as either type 1 or type 2. Only type 1 proteins exhibit loss of the C-terminal Ca2+ binding sites. Additionally, type 1 and 2 CALR mutations engender significant prognostic differences. Previously published work from our lab showed that type 1 CALR exhibits a loss of Ca2+ binding sites, rendering them unable to bind ER Ca2+ and leading to depletion of ER Ca2+ stores. We next wanted to evaluate where this Ca2+ is localized. Through imaging studies, we found that CALR type 1 expressing cells have significantly increased cytosolic Ca2+ and significantly lower mitochondrial Ca2+ compared to both CALR wildtype and CALR type 2 expressing cells. It has been well established in various cancer types that increased glycolytic activity is prompted in part by elevated cytosolic Ca2+. To test if this was the case in our model, we performed a vast array of metabolic assays in UT-7-MPL cells, a human megakaryocytic cell line, including but not limited to lactate secretion, Seahorse XF Glycolysis Stress Test assay, and metabolite tracing with 13-C labelled glucose. These all confirmed a highly glycolytic profile in UT-7-MPL cells expressing the CALR type 1 mutation. In addition, through evaluation of mitochondrial mass, membrane potential and several other assays, mitochondrial metabolism in CALR type 1 mutant cells appears severely dysfunctional. All the glycolytic and mitochondrial phenotypes are almost fully rescued upon the reintroduction of the Ca2+ binding sites of the CALRwt protein (P and C domains; P+C), indicating that perturbed Ca2+ homeostasis is the driver of these changes. Pharmacological treatment with cytosolic Ca2+ chelator BAPTA-AM or with kaempferol, an activator of the mitochondrial Ca2+ uniporter mimic the P+C rescue effect for both the glycolytic and the mitochondrial phenotypes. In our in vitro system CALR type 1 mutant cells express along with other glycolytic factors significantly higher cell surface levels of the glucose transporter GLUT1, and demonstrate enhanced sensitivity to pharmacological GLUT1 inhibition with BAY-876. Preliminary data in a transgenic mouse model as well as in primary patient samples show that GLUT1 expression is higher in CALR type 1 mutant mice and patient cells, and that the mitochondrial mass in this subset of patients is lower compared to CALR type 2 mutant patients. Finally, we validated that all these phenotypes are JAK/STAT pathway independent and that they are worsened by ruxolitinib treatment, providing a strong rationale for a combination treatment. Together, these data both expand the current knowledge of the role that Ca2+ dynamics play in metabolic reprogramming and can lead to improved targeted therapies for MPN patients. Citation Format: Michele Ciboddo, Jonathan Dowgielewicz, George Yan, Deborah Rodriguez, Hunter Blaylock, Nicole Arellano, Chad Coen, Chufan Cai, Nia Hammond, Alex Rosencrance, Elisa Rumi, Daniela Pietra, Daniele Vanni, Silvia Catricalà, Ilaria Carola Casetti, Oscar Borsani, Brandon Faubert, Shannon Elf. Type 1 calreticulin mutations in myeloproliferative neoplasms promote glycolysis via intracellular calcium mediated upregulation of GLUT1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB256.

Acrylamide induces human chondrocyte cell death by initiating autophagy‑dependent ferroptosis.

Acrylamide (ACR) is formed during heat treatment of foodstuffs and ACR may serve as a probable malignant neoplastic disease agent in all organs and tissues of the human body. However, it is unknown if ACR is associated with ankylosing spondylitis (AS) pathogenesis. Cell viability and proliferation were determined using CCK-8 assay and EdU staining. Flow cytometry was used to determine cell death and cell cycle arrest. Intracellular lipid reactive oxygen species, Fe2+ and mitochondrial membrane potential (MMP) were analyzed using a C11-BODIPY581/591 fluorescent probe, FerroOrange staining and a JC-1 MMP Assay kit, respectively. The present study showed that ACR decreased chondrocyte cell viability in a dose-dependent manner and that ACR significantly promoted chondrocyte senescence. ACR also elevated the expression of cell cycle arrest-associated proteins, including p53, cyclin-dependent kinase inhibitor 1 and cyclin-dependent kinase inhibitor protein, in human chondrocytes. Similarly, DNA damage was also enhanced following ACR treatment in chondrocytes. In addition, the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1) and the autophagy inhibitor 3-methyladenine abolished ACR-induced cell death in chondrocytes. ACR was shown to activate autophagic flux and induce mitochondrial dysfunction by increasing the MMP. Western blot analysis of ferroptosis-related proteins demonstrated that ACR decreased the expression of glutathione peroxidase 4, solute carrier family 7 member 11, transferrin receptor protein 1 and ferritin heavy chain 1 in chondrocytes whereas Fer-1 abolished these effects. ACR treatment significantly elevated the phosphorylation levels of AMP-activated protein kinase (AMPK) and serine/threonine-protein kinase ULK1 in human chondrocytes. Notably, the effect of ACR was diminished by knockdown of AMPK, as evidenced by reduced lipid reactive oxygen species accumulation and Fe2+ levels. Hence, ACR inhibited cell proliferation and contributed to cell death by inducing autophagy-dependent ferroptosis while promoting autophagy by activating AMPK-ULK1-mTOR signaling in human chondrocytes. It was hypothesized that the presence of ACR in foodstuffs may increase the risk of AS and that decreasing ACR in food products is of importance.

Abstract 1719: VHLloss enables immune checkpoint blockade therapy by boosting interferon response

Abstract Background: The von-Hippel Lindau (VHL) as a part of E3 ubiquitin ligase is a key regulator of hypoxia induced factors (HIFs). It is also characterized as a tumor suppressor gene mutated in up to 80% clear cell renal cell carcinoma (ccRCC) cases. Immune checkpoint blockade (ICB) therapy has shown improved ccRCC treatment outcomes. However, the biological roles of the VHL gene in ICB therapy are to be explored. Methods: The association between VHL gene mutation and overall survival of patients receiving ICB treatments was determined by integrating multiple human datasets. CRISPR/Cas9 was used to generate VHL knockout (KO) for in vivo tumor growth and in vitro mechanistic studies. The anti-tumor responses of genetic VHL loss in combination with anti-PD1 treatment were evaluated in three syngeneic murine tumor models. Tumor infiltrated lymphocytes and intratumoral T-cell receptor (TCR) diversity were analyzed by flow cytometry and TCR sequencing, respectively. Mitochondrial defects resulted from VHL loss were tested by JC-1 mitochondrial membrane potential assay, immunofluorescence microscopy, and qRT-PCR. VHL loss-mediated downstream signaling were further examined by western blot and bulk RNA sequencing. Results: The substantial overall survival benefits among ICB-treated patients withVHL mutations across different cancers, including RCC, in multiple human cohorts. Genetic knockout of VHL enhanced anti-PD1 therapy in three murine tumor models, including those of non-RCC origins. Moreover, VHL loss caused significant infiltration of immune effector cells and increased diversity of intratumoral TCR repertoire. VHL deficiency resulted in HIFα accumulation, increased expression of pro-mitophagy factor BNIP3, and impaired mitochondrial membrane potential that led to the cytoplasmic leakage of mitochondrial DNA (mtDNA), triggering cGAS-STING activation and type I interferon production. HIFα overexpression showed similar results as demonstrated in VHL-KO cells. In contrast, double knockdown of VHL and HIFα reversed VHL-loss induced BNIP3 upregulation, rescued mtDNA leakage to the cytoplasm, and disrupted constitutive activation of cGAS-STING signaling. Furthermore, blocking type I interferon signaling abrogated VHL-deficiency induced tumor suppression. Conclusion: Our study uncovered a novel role for VHL loss in creating an immunogenic tumor microenvironment through the activation of mtDNA-cGAS-STING pathway and type I interferon signaling in tumor cells via VHL-HIFα-BNIP3 axis, making them susceptible to ICB therapy. This finding provides a mechanistic explanation for ccRCC responsiveness to ICB therapy despite a moderate tumor mutation burden and suggests potential therapeutic strategies targeting ccRCC and other VHL-deficient tumors based on constitutive cGAS-STING and type I interferon activation. Citation Format: Meng Jiao, Xuhui Bao, Mengjie Hu, Dong Pan, Jonathan Kim, Xinjian Liu, Fang Li, Chuan-Yuan Li. VHLloss enables immune checkpoint blockade therapy by boosting interferon response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1719.

P53 negatively regulates the osteogenic differentiation in jaw bone marrow MSCs derived from diabetic osteoporosis

Patients with diabetic osteoporosis (DOP) often suffer from poor osseointegration of artificial implants, which is a challenge that affects implant outcomes. The osteogenic differentiation ability of human jaw bone marrow mesenchymal stem cells (JBMMSCs) is the key to implant osseointegration. Studies have shown that the microenvironment of hyperglycemia affects the osteogenic differentiation of mesenchymal stem cells (MSC), but the mechanism is still unclear. Therefore, the aim of this study was to isolate and culture JBMMSCs from surgically derived bone fragments from DOP patients and control patients to investigate the differences in their osteogenic differentiation ability and to elucidate its mechanisms. The results showed that the osteogenic ability of hJBMMSCs was significantly decreased in the DOP environment. Mechanism study showed that the expression of senescence marker gene P53 was significantly increased in DOP hJBMMSCs compared to control hJBMMSCs according to RNA-sequencing result. Further, DOP hJBMMSCs were found to display significant senescence using β-galactosidase staining, mitochondrial membrane potential and ROS assay, qRT-PCR and WB analysis. Overexpression of P53 in hJBMMSCs, knockdown of P53 in DOP hJBMMSCs, and knockdown followed by overexpression of P53 significantly affected the osteogenic differentiation ability of hJBMMSCs. These results suggest that MSC senescence is an important reason for decreasing osteogenic capacity in DOP patients. P53 is a key target in regulating hJBMMSCs aging, and knocking down P53 can effectively restore the osteogenic differentiation ability of DOP hJBMMSCs and promote osteosynthesis in DOP dental implants. It provided a new idea to elucidate the pathogenesis and treatment of diabetic bone metabolic diseases.

P20BAP31 induces cell apoptosis via both AIF caspase-independent and the ROS/JNK mitochondrial pathway in colorectal cancer

BackgroundDuring cell apoptosis, the C-terminus of BAP31 is cleaved by caspase-8 and generates p20BAP31, which has been shown to induce an apoptotic pathway between the endoplasmic reticulum (ER) and mitochondria. However, the underlying mechanisms of p20BAP31 in cell apoptosis remains unclear.MethodsWe compared the effects of p20BAP31 on cell apoptosis in six cell lines and selected the most sensitive cells. Functional experiments were conducted, including Cell Counting Kit 8 (CCK-8), reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) assay. Then, cell cycle and apoptosis were investigated by flow cytometry and verified by immunoblotting. Next, NOX inhibitors (ML171 and apocynin), ROS scavenger (NAC), JNK inhibitor (SP600125), and caspase inhibitor (Z-VAD-FMK) were used to further investigate the underlying mechanisms of p20BAP31 on cell apoptosis. Finally, apoptosis-inducing factor (AIF) translocation from the mitochondria to the nuclei was verified by immunoblotting and immunofluorescence assay.ResultsWe found that overexpression of p20BAP31 indeed induced apoptosis and had a much greater sensitivity in HCT116 cells. Furthermore, the overexpression of p20BAP31 inhibited cell proliferation by causing S phase arrest. Further study revealed that p20BAP31 reduced MMP, with a significant increase in ROS levels, accompanied by the activation of the MAPK signaling pathway. Importantly, the mechanistic investigation indicated that p20BAP31 induces mitochondrial-dependent apoptosis by activating the ROS/JNK signaling pathway and induces caspase-independent apoptosis by promoting the nuclear translocation of AIF.Conclusionsp20BAP31 induced cell apoptosis via both the ROS/JNK mitochondrial pathway and AIF caspase-independent pathway. Compared with antitumor drugs that are susceptible to drug resistance, p20BAP31 has unique advantages for tumor therapy.

Cytotoxicity against A549 Human Lung Cancer Cell Line via the Mitochondrial Membrane Potential and Nuclear Condensation Effects of Nepeta paulsenii Briq., a Perennial Herb.

The genus Nepeta belongs to the largest Lamiaceae family, with 300 species, which are distributed throughout the various regions of Africa, Asia, India, and America. Along with other plant families distinguished by their medicinal and therapeutic values, the Nepeta genus of Lameaceae remains relatively valuable. Hence, the phytochemicals of N. paulsenii Briq. were extracted using different plant parts, i.e., leaves, stem, roots, flowers, and the whole plant by using various solvents (ethanol, water, and ethyl acetate), obtaining 15 fractions. Each extract of dried plant material was analyzed by FT-IR and GC-MS to identify the chemical constituents. The cytotoxicity of each fraction was analyzed by MTT assay and mitochondrial membrane potential and nuclear condensation assays against lung cancer cells. Among the ethyl acetate and ethanolic extracts, the flowers showed the best results, with IC50 values of 51.57 μg/mL and 50.58 μg/mL, respectively. In contrast, among the water extracts of the various plant segments, the stem showed the best results, with an IC50 value of 123.80 μg/mL. 5-flourouracil was used as the standard drug, providing an IC50 value of 83.62 μg/mL. The Hoechst 33342 stain results indicated apoptotic features, i.e., chromatin dissolution and broken down, fragmented, and crescent-shaped nuclei. The ethanolic extracts of the flowers showed more pronounced apoptotic effects on the cells. The mitochondrial membrane potential indicated that rhodamine 123 fluorescence signals suppressed mitochondrial potential due to the treatment with the extracts. Again, the apoptotic index of the ethanolic extract of the flowers remained the highest. Hence it can be concluded that the flower part of N. paulsenii Briq. was found to be the most active against the A459 human lung cancer cell line.

Molecular characterization and expression analysis of B-cell lymphoma-2 in Trachinotus ovatus and its role in apoptotic process.

B-cell lymphoma-2 (Bcl-2) is the first identified member of the Bcl-2 family that performs an anti-apoptotic function in mammals. However, its role in teleosts is not fully understood. In this study, Bcl-2 of Trachinotus ovatus (TroBcl2) was cloned, and its role in apoptosis was investigated. In this study, Bcl-2 of Trachinotus ovatus (TroBcl2) was cloned by PCR. Quantitative real-time PCR (qRT-PCR) was used to detect its mRNA expression level in healthy condition and after LPS stimulation. Subcellular localization was performed by transfecting the pTroBcl2-N3 plasmid into golden pompano snout (GPS) cells and observed under an inverted fluorescence microscope DMi8 and further verified by immunoblotting. In vivo overexpression and RNAi knockdown method were performed to evaluate the role of TroBcl2 in apoptosis. The anti-apoptotic activity of TroBcl2 was detected by flow cytometry. The effect of TroBcl2 on the mitochondrial membrane potential (MMP) was measured by an enhanced mitochondrial membrane potential assay kit with JC-1. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method was performed to evaluate the role of TroBcl2 in the DNA fragmentation. Immunoblotting was used to verify whether TroBcl2 inhibits the release of cytochrome c from mitochondria into the cytoplasm. The Caspase 3 and Caspase 9 Activity Assay Kits were used to investigate the effect of TroBcl2 on caspase 3 and caspase 9 activities. The effects of TroBcl2 on the expression of apoptosis-related and nuclear factor- κB (NF-κB) signaling pathway-related genes in vitro were evaluated by qRT-PCR and Enzyme linked immunosorbent assay (ELISA). Luciferase reporter assay was used to evaluate the activity in NF-κB signaling pathway. The full-length coding sequence of TroBcl2 contains 687 bp and encodes a protein containing 228 amino acids. Four conserved Bcl-2 homology (BH) domains and one invariant "NWGR" motif located in BH1 were identified in TroBcl2. In healthy T. ovatus, TroBcl2 was widely distributed in the eleven tested tissues, and higher expression levels were found in immune-related tissues, such as spleen and head kidney tissues. After stimulation with lipopolysaccharide (LPS), the expression of TroBcl2 in the head kidney, spleen, and liver was significantly upregulated. In addition, subcellular localization analysis revealed that TroBcl2 was localized in both the cytoplasm and nucleus. Functional experiments showed that TroBcl2 inhibited apoptosis, possibly by reducing mitochondrial membrane potential loss, decreasing DNA fragmentation, preventing cytochrome c release into cytoplasm, and reducing the caspase 3 and caspase 9 activations. Moreover, upon LPS stimulation, overexpression of TroBcl2 suppressed the activation of several apoptosis-related genes, such as BOK, caspase-9, caspase-7, caspase-3, cytochrome c, and p53. Furthermore, knockdown of TroBcl2 significantly increased the expression of those apoptosis-related genes. In addition, TroBcl2 overexpression or knockdown induced or inhibited, respectively, the transcription of NF-κB and regulated the expression of genes (such as NF-κB1 and c-Rel) in the NF-κB signaling pathway as well as the expression of the downstream inflammatory cytokine IL-1β. Overall, our study suggested that TroBcl2 performs its conserved anti-apoptotic function via the mitochondrial pathway and may serve as an anti-apoptotic regulator in T. ovatus.

Role of ferroptosis in hypoxic preconditioning to reduce propofol neurotoxicity.

Background: An increasing number of studies have reported that neurotoxicity of propofol may cause long-term learning and cognitive dysfunction. Hypoxic preconditioning has been shown to have neuroprotective effects, reducing the neurotoxicity of propofol. Ferroptosis is a new form of death that is different from apoptosis, necrosis, autophagy and pyroptosis. However, it is unclear whether hypoxic preconditioning reduces propofol neurotoxicity associated with ferroptosis. Thus, we aimed to evaluate the effect of propofol on primary hippocampal neurons in vitro to investigate the neuroprotective mechanism of hypoxic preconditioning and the role of ferroptosis in the reduction of propofol neurotoxicity by hypoxic preconditioning. Methods: Primary hippocampal neurons were cultured for 8 days in vitro and pretreated with or without propofol, hypoxic preconditioning, agonists or inhibitors of ferroptosis. Cell counting kit-8, Calcein AM, Reactive oxygen species (ROS), Superoxide dismutase (SOD), Ferrous iron (Fe2+), Malondialdehyde (MDA) and Mitochondrial membrane potential assay kit with JC-1 (JC-1) assays were used to measure cell viability, Reactive oxygen species level, Superoxide dismutase content, Fe2+ level, MDA content, and mitochondrial membrane potential. Cell apoptosis was evaluated using flow cytometry analyses, and ferroptosis-related proteins were determined by Western blot analysis. Results: Propofol had neurotoxic effects that led to decreased hippocampal neuronal viability, reduced mitochondrial membrane potential, decreased SOD content, increased ROS level, increased Fe2+ level, increased MDA content, increased neuronal apoptosis, altered expression of ferroptosis-related proteins and activation of ferroptosis. However, hypoxic preconditioning reversed these effects, inhibited ferroptosis caused by propofol and reduced the neurotoxicity of propofol. Conclusion: The neurotoxicity of propofol in developing rats may be related to ferroptosis. Propofol may induce neurotoxicity by activating ferroptosis, while hypoxic preconditioning may reduce the neurotoxicity of propofol by inhibiting ferroptosis.

Allophylus africanus Stem Bark Extract Modulates the Mitochondrial Apoptotic Pathway in Human Stomach Cancer Cells.

The present work aimed to detail the mechanisms elicited by Allophylus africanus P. Beauv. stem bark extract in human stomach cancer cells and to identify the bioactives underlying the cytotoxicity. MTT reduction and LDH leakage assays allowed characterizing the cytotoxic effects in AGS cells, which were further detailed by morphological analysis using phalloidin and Hoechst 33258. Proapoptotic mechanisms were elucidated through a mitochondrial membrane potential assay and by assessing the impact upon the activity of caspase-9 and -3. The extract displayed selective cytotoxicity against AGS cells. The absence of plasma membrane permeabilization, along with apoptotic body formation, suggested that pro-apoptotic effects triggered cell death. Intrinsic apoptosis pathway activation was verified, as mitochondrial membrane potential decrease and activation of caspase-9 and -3 were observed. HPLC-DAD profiling enabled the identification of two apigenin-di-C-glycosides, vicenin-2 (1) and apigenin-6-C-hexoside-8-C-pentoside (3), as well as three mono-C-glycosides-O-glycosylated derivatives, apigenin-7-O-hexoside-8-C-hexoside (2), apigenin-8-C-(2-rhamnosyl)hexoside (4) and apigenin-6-C-(2-rhamnosyl)hexoside (5). Isovitexin-2″-O-rhamnoside (5) is the main constituent, accounting for nearly 40% of the total quantifiable flavonoid content. Our results allowed us to establish the relationship between the presence of vicenin-2 and other apigenin derivatives with the contribution to the cytotoxic effects on the presented AGS cells. Our findings attest the anticancer potential of A. africanus stem bark against gastric adenocarcinoma, calling for studies to develop herbal-based products and/or the use of apigenin derivatives in chemotherapeutic drug development.

Extracellular vesicles from hypoxia-pretreated adipose-derived stem cells regulate hypoxia/reoxygenation-induced human dermal microvascular endothelial apoptosis and autophagy in vitro

Recent studies suggest hypoxia can promote adipose-derived stem cells (ADSCs) to attenuate hypoxia/reoxygenation (H/R)-induced damage to human dermal microvascular endothelial cells (HDMECs). Extracellular vesicles (EVs), isolated from ADSCs, play an-important role in the fields of regenerative medicine. Here, we aimed to investigate the effect of EVs isolated from hypoxia-pretreated ADSCs (ADSC-EVs[H]) on HDMECs to attenuate ischemia/reperfusion injury of free skin flaps. First, we characterized EVs isolated from normoxia-cultured ADSCs (ADSC-EVs[N]) and ADSC-EVs(H). Experimental data indicated that EVs isolated from ADSCs consisted of lipid-bilayer vesicles that exhibited positive expression of vascular endothelial growth factor (VEGF) and marker proteins CD9, CD63 and CD81, and the mean particle size of EVs in the hypoxia-pretreated ADSCs (ADSC[H]) group was smaller (74.17 nm) than in the normoxic-cultured ADSCs (ADSC[N]) group (93.87 nm). Hypoxic pretreatment increased the number of EVs. Later, we favorably constructed the co-culture model of EVs isolated from ADSCs (ADSC-EVs) and H/R-induced HDMECs. Cell counting kit-8, Ethynyldeoxyuridine assay, western blotting and immunofluorescence staining showed that ADSC-EVs(H) promoted the survival of HDMECs and increased LC3 level. Apoptosis, reactive oxygen species (ROS) and JC-1 mitochondrial membrane potential (MMP) assays revealed that ADSC-EVs(H) reduced the apoptosis rate and ROS accumulation and increased MMP level in HDMECs, indicating that ADSC-EVs(H) effectively attenuated H/R-induced damage in HDMECs through autophagy activation and the-inhibition of apoptosis and oxidative stress. This study confirmed that ADSC-EVs(H) could effectively regulate the proliferation, apoptosis, oxidative stress, and autophagy expression of H/R-induced HDMECs in vitro, and therefore the transplantation of ADSC-EVs(H) may provide novel insights for the transplantation of free skin flaps.

Green synthesis of silver nanoparticles by root extract of Premna integrifolia L. and evaluation of its cytotoxic and antibacterial activity

An efficient, reproducible, and eco-friendly protocol has been developed for the green synthesis of silver nanoparticles using an aqueous root extract of Premna integrifolia (PE-AgNPs). The synthesized nanoparticles were characterized by different techniques to determine their formation, stability, and morphology. Nanoparticles were tested for cytotoxicity against the HepG2 cancer cell line and antibacterial activity against clinical isolates of bacteria. The cytotoxic potential of PE-AgNPs was analyzed using MTT assay, Hoechst and AO/EtBr staining, ROS measurement, mitochondrial membrane potential, clonogenic, and wound healing assays. PE-AgNPs were found strongly cytotoxic against Hep G2 cell lines during all assays. A disc diffusion assay confirmed that Pseudomonas aeruginosa was most susceptible to PE-AgNPs than Staphylococcus aureus and E. coli. It was further confirmed by MIC, MBC values, and TEM microphotographs. As a result, our findings indicate that biosynthesized silver nanoparticles could be useful as cytotoxic and antimicrobial candidates.

Wnt/GSK3β/β-catenin signaling pathway regulates calycosin-mediated anticancer effects in glioblastoma multiforme cells

Calycosin, an O-methylated isoflavone, has been widely reported to induce anticancer activity in different cancer cells in vitro. Nonetheless, the associated mechanism of calycosin in glioblastoma multiforme cells (U87) still remains unknown. To explore the anticancer effects, the apoptotic mechanism of calycosin via Wnt/GSK3β/β-catenin signaling was explored in U87 cells. Different assays including: cytotoxicity, free radical determination, SOD and CAT activity, GSH content, qPCR, mitochondrial membrane potential, caspase activity, and western blotting assays were performed. It was shown that calycosin mitigated cell viability in U87 cells, whereas it showed no apparent effect on BV2 microglial cells. Calycosin triggered apoptosis via upregulating the mitochondria-associated caspase pathway in U87 cells. Calycosin induced the reduction of the mitochondrial membrane potential, overexpression of Bax, downexpression of Bcl-2, and activation of caspase-9 and caspase-3. Calycosin-stimulated apoptosis was associated with the upregulation of free radical scavenging through the modulation of antioxidant enzymes, such as SOD and CAT as well as the level of GSH. The apoptotic activity of calycosin was mediated by suppression of pGSK-3βser9, β-catenin, and c-Myc at protein level. The present study suggested that calycosin triggers U87 cell death through an antioxidant effect mediated by Wnt/GSK3β/β-catenin signaling pathway.

The effect of thymoquinone and propranolol combination on epidermoid laryngeal carcinoma cell.

We aimed to evaluate the effects of thymoquinone and propranolol on Hep-2 cells representing laryngeal Ca cell type in comparison with cisplatin. We also evaluated their combined effects. Apoptotic effects were directly analyzed via mitochondrial membrane potential and caspase-3 assays. In addition, effects on apoptosis and cell cycle via Bcl-2, Bax, P53, and Cyclin D1 mRNA expressions and effects on angiogenesis via VEGFA mRNA expression were evaluated by RT-qPCR. According to our results, it was determined that the anticancer effects of thymoquinone on Hep-2 cells were higher than propranolol. Our JC-1 and caspase-3 results showed an effect close to cisplatin, especially for 50µM thymoquinone. Significant differences were also obtained in Bcl-2, Bax, P53, and cyclin D1 results for similar concentrations compared to the control. No effect of thymoquinone was seen for VEGFA. Propranolol alone had no significant effect on JC-1 and Caspase-3. Propranolol had an effect on Bcl-2, Bax mRNA expressions compared to the control, only at 250µM concentration. Propranolol and its combinations increased VEGFA mRNA expression-like cisplatin. Thymoquinone induced apoptosis and blocked the cell cycle in Hep-2 cells. The effects of propranolol, which was reported to have an antiangiogenesis effect in some studies, on apoptosis and cell cycle were limited except at high concentrations. For this cell line, why propranolol causes an increase in VEGFA expression should be evaluated extensively. Thymoquinone shows promise for cancer therapy, but studies need to be designed in vivo to evaluate the effects more reliably.

Circ-CPSF1 Worsens Radiation-Induced Oxidative Stress Injury in Caenorhabditis elegans.

Radioactive substances have been used in various aspects in daily life. However, high-energy radiation could cause environmental problems, which would damage the human body. Circular RNA (CircRNA) has great potential in the minimization of ionizing radiation damage. To find a potential diagnostic and therapeutic target for reducing the damage of ionizing radiation, we selected circRNA cleavage and polyadenylation specificity factor subunit 1 (circ-CPSF1) based on its up-regulated expression after X-ray radiation and explored its effect on response to ionizing radiation using Caenorhabditis elegans (C. elegans). Circ-CPSF1 was screened out and its up-regulated expression was verified. The measurement of lifespan and germ cell apoptosis showed that circ-CPSF1 RNAi treatment extended lifespan and reduced apoptotic germ cells. ROS levels were significantly reduced after the interference of circ-CPSF1 in C. elegans with radiation. Mitochondrial membrane potential assay showed that the suppression of circ-CPSF1 could alleviate mitochondrial damage after radiation. Relative genes expression showed the involvement of circ-CPSF1 in radiation mediated DNA damage response pathways and apoptosis pathways. In conclusion, circ-CPSF1 exerts deleterious effects on lifespan, eggs production and germ cell apoptosis of C. elegans through oxidative stress, the DNA damage response (DDR) pathway, and the core apoptotic pathway after ionizing radiation, indicating the potential of circ-CPSF1 to be an important therapeutic target of radiation damage.

Synthesis and biological evaluation of capsaicin analogues as antioxidant and neuroprotective agents.

Capsaicin and its analogues 3a-3q were designed and synthesized as potential new antioxidant and neuroprotective agents. Many analogues exhibited good antioxidant effects, and some showed more potent free radical scavenging activities than the positive drug quercetin (IC50 = 8.70 ± 1.75 μM for DPPH assay and 13.85 ± 2.87 μM for ABTS assay, respectively). The phenolic hydroxyl of capsaicin analogues was critical in determining antioxidant activity. Among these compounds, 3k displayed the most potent antioxidant activity. Cell vitality tests revealed that the representative compound 3k was good at protecting cells from H2O2-induced oxidative damage at low concentrations (cell viability increased to 90.0 ± 5.5% at 10 μM). In addition, the study demonstrated that 3k could reduce intracellular ROS accumulation and increase GSH levels to prevent H2O2-induced oxidative stress in SY5Y cells. In the mitochondrial membrane potential assay, 3k significantly increased the MMP level of SY5Y cells treated with H2O2 and played an anti-neuronal cell death role. These results provide a promising strategy to develop novel capsaicin analogues as potential antioxidant and neuroprotective agents.

Naphthoyl benzhydrazine-decorated binuclear arene Ru(II) complexes as anticancer agents targeting human breast cancer cells.

Breast cancer is the most dangerous type in women and its fatality rate has increased over the past decade. To develop more potent and target-specific breast cancer drugs, six arene ruthenium(II) complexes (1-6) containing naphthoyl benzhydrazine ligands (NL1-NL3) were synthesized and characterized by analytical and spectroscopic (infrared, UV-visible, NMR and HR-MS) methods. The SC-XRD analysis of 1 and 6 demonstrates the bis N^O bidentate binding nature of ligands to ruthenium ions and a pseudo-octahedral geometry around the Ru(II) ion. Solution stability studies using UV-Vis spectroscopy evidenced the instantaneous hydrolysis of the complexes to form monoaquated species in a solution of 1 : 9 (v/v) DMSO/phosphate buffer. All the complexes were screened for their in vitro antiproliferative activities against different human breast cancer cells, including MCF-7, SkBr3, MDA-MB-468, MDA-MB-231, and non-cancerous HEK-293 cells, by an MTT assay, and they displayed good cancer cell growth inhibitory capacity with low IC50 values. Notably, complexes 2 and 5 comprising methoxy and p-cymene groups exhibited excellent cytotoxicity towards SkBr3 cells compared to clinical drug cisplatin. AO-EB and HOECHST-33342 staining assays revealed apoptotic morphological changes in complex-treated cancer cells. Further, reactive oxygen species and mitochondrial membrane potential assays validated that the complexes induce apoptotic cell death via an intrinsic mitochondrial pathway with ROS production. In addition, the apoptotic induction and the quantification of late apoptosis were established with the aid of western blot and flow cytometry analysis, respectively.

Artabotrys odoratissimus Bark Extract Restores Ethanol Induced Redox Imbalance and Toxicity in Hepatocytes and In Vivo Model.

Alcohol-induced oxidative stress is a key player in the development of liver diseases, and herbal alternatives are important means of ameliorating the hepatotoxic effects. The study aimed to evaluate the hepatoprotective potentiality of Artabotrys odoratissimus, an important medicinal shrub from the family Annonaceae. The phenolic compounds from bark ethanol extract (BEE) were detected using RP-HPLC. The in vitro hepatoprotective activity against ethanol-induced damage was studied in HepG2 cells with cell viability assays, mitochondrial membrane potential (MMP) assay, reactive oxygen species (ROS) assay, double staining assay and western blotting. The in vivo mice model was used to evaluate the alcohol-induced stress with liver function enzymes, lipid profile and histopathology. All the thirteen phenolic compounds detected with HPLC were docked onto protein targets such as aspartate amino transferase (AST), alkaline phosphatase (ALP) and inducible nitric oxide synthase (NO). The RP-HPLC detected the presence of various phenolics including rutin, chlorogenic acid and catechin, amongst others. Co-administration of BEE with ethanol alleviated cell death, ROS and MMP in HepG2 cells compared to the negative control. The extract also modulated the MAP kinase/caspase-3 pathway, thereby showing protective effects in HepG2 cells. Also, pre-treatment for 14days with the extract in the mice model before a single toxic dose (5g/kg body weight) reduced the liver injury by bringing the levels of liver function enzymes, lipid profile and bilirubin to near normal. In silico analysis revealed that rutin showed the best binding affinity with all the target proteins in the study. These results provide evidence that BEE possesses significant hepatoprotective effects against ethanol-induced oxidative stress in hepatic cells and in vivo models, which is further validated with in silico analysis.