Amentoflavone mitigates doxorubicin-induced cardiotoxicity by suppressing cardiomyocyte pyroptosis and inflammation through inhibition of the STING/NLRP3 signalling pathway.

BACKGROUND: Doxorubicin (DOX) is a potent anticancer chemotherapeutic agent whose clinical application is substantially constrained by its cardiotoxicity. The pathophysiology of DOX-induced cardiotoxicity manifests as cardiomyocyte pyroptosis and inflammation. Amentoflavone (AMF) is a naturally occurring biflavone possessing anti-pyroptotic and anti-inflammatory properties. However, the mechanism through which AMF alleviates DOX-induced cardiotoxicity remains undetermined. PURPOSE: This study aimed at investigating the role of AMF in alleviating DOX-induced cardiotoxicity. STUDY DESIGN AND METHODS: To assess the in vivo effect of AMF, DOX was intraperitoneally administered into a mouse model to induce cardiotoxicity. To elucidate the underlying mechanisms, the activities of STING/NLRP3 were quantified using the NLRP3 agonist nigericin and the STING agonist amidobenzimidazole (ABZI). Primary cardiomyocytes isolated from neonatal Sprague-Dawley rats were treated with saline (vehicle) or DOX with or without AMF and/or ABZI. The echocardiogram, haemodynamics, cardiac injury markers, heart/body weight ratio, and pathological alterations were monitored; the STING/NLRP3 pathway-associated proteins were detected by western blot and cardiomyocyte pyroptosis was analysed by immunofluorescence staining of cleaved N-terminal GSDMD and scanning electron microscopy. Furthermore, we evaluated the potential of AMF in compromising the anticancer effects of DOX in human breast cancer cell lines. RESULTS: AMF substantially alleviated cardiac dysfunction and reduced heart/body weight ratio and myocardial damage in mice models of DOX-induced cardiotoxicity. AMF effectively suppressed DOX-mediated upregulation of IL-1ß, IL-18, TNF-α, and pyroptosis-related proteins, including NLRP3, cleaved caspase-1, and cleaved N-terminal GSDMD. The levels of apoptosis-related proteins, namely Bax, cleaved caspase-3, and BCL-2 were not affected. In addition, AMF inhibited STING phosphorylation in DOX-affected hearts. Intriguingly, the administration of nigericin or ABZI dampened the cardioprotective effects of AMF. The in vitro anti-pyroptotic effect of AMF was demonstrated in attenuating the DOX-induced reduction in cardiomyocyte cell viability, upregulation of cleaved N-terminal GSDMD, and pyroptotic morphology alteration at the microstructural level. AMF exhibited a synergistic effect with DOX to reduce the viability of human breast cancer cells. CONCLUSION: AMF alleviates DOX-induced cardiotoxicity by suppressing cardiomyocyte pyroptosis and inflammation via inhibition of the STING/NLRP3 signalling pathway, thereby validating its efficacy as a cardioprotective agent.

HYDROMORPHONE MITIGATES CARDIOPULMONARY BYPASS-INDUCED ACUTE LUNG INJURY BY REPRESSING PYROPTOSIS OF ALVEOLAR MACROPHAGES.

ABSTRACT: Introduction: Acute lung injury (ALI) is a devastating pulmonary illness with diffuse inflammatory responses. Hydromorphone (Hyd) is an opioid agonist used for relieving moderate-to-severe pain. The present work investigated the effect of Hyd on cardiopulmonary bypass (CPB)-induced ALI by regulating pyroptosis of alveolar macrophages (AMs). Methods: Rats were subjected to CPB, followed by Hyd treatment. The lung injury in rat lung tissues was appraised by the ratio of lung wet/dry weight (weight), histological staining, and the total protein concentrations in bronchoalveolar lavage fluid, and lung function was assessed by oxygenation index and respiratory index, and lung macrophage pyroptosis was observed by fluorescence staining. Alveolar macrophages were separated and pyroptosis was determined by western blot assay and enzyme-linked immunosorbent assay. The expression patterns of nuclear factor erythroid 2-related factor 2/heme oxygenase 1 (Nrf2/HO-1), nod-like receptor protein 3 (NLRP3), N-terminal gasdermin-D, and cleaved caspase-1 were examined by real-time quantitative polymerase chain reaction, western blot, and immunohistochemistry assays. The impact of NLRP3 or Nrf2 on pyroptosis of AMs and CPB-induced ALI was observed after treatment of nigericin (NLRP3 agonist) or ML385 (Nrf2 inhibitor). Results: Hyd attenuated CPB-induced lung injury as manifested by reductions in lung inflammation and edema, the scores of lung injury, the ratio of lung wet/dry weight, and the total protein concentrations in bronchoalveolar lavage fluid. Besides, Hyd repressed NLRP3 inflammasome-mediated pyroptosis of AMs after CPB treatment. Hyd upregulated Nrf2/HO-1 expression levels to repress NLRP3 inflammasome-mediated pyroptosis. Treatment of nigericin or ML385 counteracted the role of Hyd in ameliorating pyroptosis of AMs and CPB-induced ALI. Conclusions: Hyd alleviated NLRP3 inflammasome-mediated pyroptosis and CPB-induced ALI via upregulating the Nrf2/HO-1 pathway, which may be achieved by AMs.

Nigericin Abrogates Maternal and Embryonic Oxidative Stress in the Streptozotocin-Induced Diabetic Pregnant Rats.

Hyperglycemic exposure in diabetic pregnancy can lead to many developmental changes, such as delayed development, fetal malformations, and fetal/embryo death. These detrimental complications are collectively known as diabetic embryopathy or teratogenesis. The current study focuses to discover the therapeutic properties of the nigericin against the STZ-stimulated diabetic embryopathy via alleviation of maternal and embryonic oxidative stress. The male and female rats at a 1:1 ratio were permitted to mate overnight to establish the course of pregnancy. The pregnant rats were distributed into four groups control, diabetic pregnant (via administering 40 mg/kg of STZ), and diabetic + 10 and 20 mg/kg of nigericin-administered (via oral gavage from days 5 to 12) groups, respectively. The glucose level, urine output, diet intake, and body weight were determined carefully. The embryo and placenta weight and implantation rates were examined, and data were tabulated. The total protein and lipid profiles were assessed using respective kits. The oxidative stress markers and antioxidant enzymes were examined using respective assay kits. The 10 and 20 mg/kg of nigericin treatment decreased the glucose level and urine output and improved the diet intake and body weight gain in diabetic pregnant rats. The nigericin also decreased the total protein, cholesterol, triglycerides, and very-low-density lipoprotein (VLDL) and improved the high-density lipoprotein (HDL) in the serum of pregnant rats. The levels of malondialdehyde (MDA), reactive oxygen species (ROS), and protein carbonyls were decreased by the nigericin in both liver and embryos of the pregnant rats. The levels of glutathione (GSH), total thiols, and activities of catalase (CAT), glutathione reductase (GR), superoxide dismutase (SOD), glutathione peroxidase (GPX), and glutathione S-transferase (GST) were improved by the nigericin in the diabetic pregnant rats. Altogether, these results provide evidence that nigericin treatment remarkably attenuates the diabetes-stimulated embryopathy in rats. The nigericin effectively decreased embryo lethality, reduced glucose and dyslipidemia, and relieves oxidative stress via upregulating the antioxidant enzyme activities. Hence, it can be a talented therapeutic agent to treat diabetic pregnancy-associated complications.

Picroside II alleviates DSS-induced ulcerative colitis by suppressing the production of NLRP3 inflammasomes through NF-κB signaling pathway.

CONTEXT: Ulcerative colitis (UC) is a common acute or chronic intestinal disease with an imbalance of inflammation. Picroside II (P-II) exerts a protective role in various inflammation-related diseases. However, the effect of P-II on UC is still unclear. OBJECTIVE: To explore the effect of P-II on UC and its potential mechanism. MATERIALS AND METHODS: Human monocytic leukemia cell line THP-1 was treated with phorbol ester (PMA) to differentiate into a macrophage. The differentiated THP-1 cells were hatched with LPS combined with ATP or Nigericin to activate the NLRP3 inflammasome in vitro. The UC model was constructed by injection of DSS into mice. RESULTS: The maximum nontoxic concentration of P-II on THP-1 cells was 60 µM. LPS combined with ATP or Nigericin stimulated the production of IL-1ß, which was antagonized by P-II treatment. Meanwhile, P-II administration interfered with the aggregation of ASC and the assembly of NLRP3 inflammasomes. Also, P-II treatment reduced the LPS and ATP-induced elevation of the relative protein expression of NLRP3, pro-caspase-1, IL-1ß and p-p65/p65, and the concentration of TNF-α and IL-6. Besides, the NF-κB specific inhibitor BAY-117082 notably repressed the LPS together with ATP-enhanced the relative protein expression of NLRP3, caspase-1 and IL-1ß. Moreover, in vivo results showed that P-II relieved the DDS-induced UC, as evidenced by the improvement of mice weight, DAI and pathological scores. In addition, P-II treatment notably decreased DDS-promoted expression of NLRP3 inflammasomes and inflammatory factors in vivo. CONCLUSION: P-II alleviated DSS-induced UC by repressing the production of NLRP3 inflammasomes via the NF-κB signaling pathway.

Evidence of nigericin as a potential therapeutic candidate for cancers: A review.

Emerging studies have shown that nigericin, an H+, K+ and Pb2+ ionophore, has exhibited a promising anti-cancer activity in various cancers. However, its anti-cancer mechanisms have not been fully elucidated. In this review, the recent progresses on the use of nigericin in human cancers have been summarized. By exchanging H+ and K+ across cell membranes, nigericin shows promising anti-cancer activities in in vitro and in vivo as a single agent or in combination with other anti-cancer drugs through decreasing intracellular pH (pHi). The underlying mechanisms of nigericin also include the inactivation of Wnt/ß-catenin signals, blockade of Androgen Receptor (AR) signaling, and activation of Stress-Activated Protein Kinase/c-Jun N-terminal Kinase (SAPK/JNK) signaling pathways. In many cancers, nigericin is proved to specifically target putative Cancer Stem Cells (CSCs), and its synergistic effects on photodynamic therapy are also reported. Other mechanisms of nigericin including influencing the mitochondrial membrane potentials, inducing an increase in drug accumulation and autophagy, controlling insulin accumulation in nuclei, and increasing the cytotoxic activity of liposome-entrapped drugs, are also discussed. Notably, the potential adverse effects such as teratogenic effects, insulin resistance and eryptosis shall not be ignored. Taken together, these reports suggest that treatment of cancer cells with nigericin may offer a novel therapeutic strategy and future potential of translation to clinics.

Salinomycin and other ionophores as a new class of antimalarial drugs with transmission-blocking activity.

The drug target profile proposed by the Medicines for Malaria Venture for a malaria elimination/eradication policy focuses on molecules active on both asexual and sexual stages of Plasmodium, thus with both curative and transmission-blocking activities. The aim of the present work was to investigate whether the class of monovalent ionophores, which includes drugs used in veterinary medicine and that were recently proposed as human anticancer agents, meets these requirements. The activity of salinomycin, monensin, and nigericin on Plasmodium falciparum asexual and sexual erythrocytic stages and on the development of the Plasmodium berghei and P. falciparum mosquito stages is reported here. Gametocytogenesis of the P. falciparum strain 3D7 was induced in vitro, and gametocytes at stage II and III or stage IV and V of development were treated for different lengths of time with the ionophores and their viability measured with the parasite lactate dehydrogenase (pLDH) assay. The monovalent ionophores efficiently killed both asexual parasites and gametocytes with a nanomolar 50% inhibitory concentration (IC50). Salinomycin showed a fast speed of kill compared to that of standard drugs, and the potency was higher on stage IV and V than on stage II and III gametocytes. The ionophores inhibited ookinete development and subsequent oocyst formation in the mosquito midgut, confirming their transmission-blocking activity. Potential toxicity due to hemolysis was excluded, since only infected and not normal erythrocytes were damaged by ionophores. Our data strongly support the downstream exploration of monovalent ionophores for repositioning as new antimalarial and transmission-blocking leads.

The influence of withdrawal time on the performance of broiler chickens fed semduramicin.

Three 49-d experiments, with a total of 6,528 male broiler chicks in floor pens, were conducted to test the hypothesis that Semduramicin feeding-time affects the body weight gain, feed consumption, and feed conversion ratio of broilers. Semduramicin ionophore was added to corn and soybean meal-based diets at the recommended level, 25 mg/kg for 0,34,39, or 42 d. Thus, three withdrawal times were employed (7, 10, and 15 d) during each experiment. Significant differences among experiments were observed for body weight gain, feed intake, and feed conversion ratio, but no significant differences due to Semduramicin were noted in body weight or feed intake. There was only one treatment by experiment interaction found for 0-to-34-d feed intake (P = 0.028), but it was not evident for 0 to 39 d (P = 0.818) or any other times. Feeding Semduramicin with a 10- or 15-d withdrawal period resulted in an improvement in feed conversion of about 0.04 units.

Effects of the ionophore anticoccidial semduramicin on broiler breeders.

Three experiments were conducted to assess the effects on broiler breeders of contamination of feed with the ionophore anticoccidial semduramicin. In Experiment 1, individually caged females received 0, 12.5, or 25 mg/kg diet for 3 wk from 48 to 50 wk of age. In Experiment 2, males and females in floor pens received 0, 12.5, or 25 mg/kg diet for 3 wk from 63 to 65 wk of age. In Experiment 3, individually caged males and females received 0, 3, 6, or 25 mg/kg diet for 1 wk at 31 wk of age and were mated by artificial insemination. There was a dose-related decrease in cumulative egg production and percentage shell in Experiment 1 after more than 1 wk exposure, but these effects were not observed in the other experiments. There was a decrease in cumulative fertile hatchability and a dose-related decrease after 3 wk exposure due to an increase in early embryonic mortality in Experiment 2, but these changes were not observed during the 1-wk exposure in Experiment 3. The data show that adverse effects of semduramicin require greater than 1 wk of exposure to be evident.

Studies on semduramicin and nutritional responses: 3. Electrolyte balance.

Four experiments were conducted to determine whether fluctuations in dietary electrolyte level (milliequivalents Na+K-Cl per kilogram) or different dietary sodium chloride levels would affect performance, water intake, or excreta moisture of chickens fed semduramicin vs nonmedicated controls. In all experiments, male commercial broiler chicks were used, and all diets were fed with and without 25 mg semduramicin/kg diet. The basal diets were based on corn, soybean meal, and poultry oil. Experiments 1, 3, and 4 were conducted using male broiler chicks in battery brooders to 18 d of age. In Experiment 1, six electrolyte levels were fed [basal (0.2% Na, 0.33% Cl, 1.10% K); basal plus 0.1% Na (from sodium chloride); basal plus 0.1% K (from potassium chloride); basal plus 0.2% Na (from sodium carbonate); basal plus 0.34% K (from potassium chloride); basal plus 0.15% Cl (from ammonium chloride)]. There were no significant changes in BW gain, feed consumption, or feed conversion ratio caused by any dietary treatment. Water consumption was not affected by the inclusion of Na, Cl, K, or electrolyte levels, but excreta moisture was affected. The highest and lowest excreta moisture levels came from chicks fed the lowest chloride levels. There were no significant dietary effects on serum Na, Cl, or K by dietary electrolytes or semduramicin. Experiment 2 was conducted with triplicate floor pens of 33 male broilers each for 42 d with four electrolyte levels [basal (0.2% Na, 0.34% Cl, 1.03% K); basal plus 0.1% Na (from sodium carbonate); basal plus 0.1% Cl (from ammonium chloride); basal plus 0.18% K (from potassium carbonate)]. Increasing electrolyte level had a significant effect on BW gain at 35 d but not at 42 d [mainly because of differences in K (1.943 kg at 35 d) vs Cl (2.013 kg at 35 d)]. At 42 d, there were no differences in growth because of N, K, Cl, or semduramicin. Potassium supplementation caused a significant increase in litter moisture (P = 0.031). Semduramicin did not affect litter moisture (P = 0.892), nor were there significant semduramicin interactions with Na, K, Cl, or semduramicin. The basal diets in Experiments 3 and 4 were identical to the basal diet in Experiment 1 except there was no sodium chloride added. The diets fed in Experiment 3 contained 0, 0.1, 0.2, 0.3, or 0.4% added sodium chloride. The diets fed in Experiment 4 contained 0.1, 0.3, 0.5, 0.7, and 0.9% added sodium chloride. The results of Experiments 3 and 4 show that about 0.4% added sodium chloride is necessary to achieve maximum growth and feed conversion. It is clear that semduramicin had no significant effect upon the variation observed in any of the variables measured.