Growth hormone secretagogues modulate inflammation and fibrosis in mdx mouse model of Duchenne muscular dystrophy.

Introduction: Growth hormone secretagogues (GHSs) exert multiple actions, being able to activate GHS-receptor 1a, control inflammation and metabolism, to enhance GH/insulin-like growth factor-1 (IGF-1)-mediated myogenesis, and to inhibit angiotensin-converting enzyme. These mechanisms are of interest for potentially targeting multiple steps of pathogenic cascade in Duchenne muscular dystrophy (DMD). Methods: Here, we aimed to provide preclinical evidence for potential benefits of GHSs in DMD, via a multidisciplinary in vivo and ex vivo comparison in mdx mice, of two ad hoc synthesized compounds (EP80317 and JMV2894), with a wide but different profile. 4-week-old mdx mice were treated for 8 weeks with EP80317 or JMV2894 (320 µg/kg/d, s.c.). Results: In vivo, both GHSs increased mice forelimb force (recovery score, RS towards WT: 20% for EP80317 and 32% for JMV2894 at week 8). In parallel, GHSs also reduced diaphragm (DIA) and gastrocnemius (GC) ultrasound echodensity, a fibrosis-related parameter (RS: ranging between 26% and 75%). Ex vivo, both drugs ameliorated DIA isometric force and calcium-related indices (e.g., RS: 40% for tetanic force). Histological analysis highlighted a relevant reduction of fibrosis in GC and DIA muscles of treated mice, paralleled by a decrease in gene expression of TGF-ß1 and Col1a1. Also, decreased levels of pro-inflammatory genes (IL-6, CD68), accompanied by an increment in Sirt-1, PGC-1α and MEF2c expression, were observed in response to treatments, suggesting an overall improvement of myofiber metabolism. No detectable transcript levels of GHS receptor-1a, nor an increase of circulating IGF-1 were found, suggesting the presence of a novel receptor-independent mechanism in skeletal muscle. Preliminary docking studies revealed a potential binding capability of JMV2894 on metalloproteases involved in extracellular matrix remodeling and cytokine production, such as ADAMTS-5 and MMP-9, overactivated in DMD. Discussion: Our results support the interest of GHSs as modulators of pathology progression in mdx mice, disclosing a direct anti-fibrotic action that may prove beneficial to contrast pathological remodeling.

Protective Effects of a Red Grape Juice Extract against Bisphenol A-Induced Toxicity in Human Umbilical Vein Endothelial Cells.

Human exposure to bisphenol A (BPA) occurs through the ingestion of contaminated food and water, thus leading to endothelial dysfunction, the first signal of atherosclerosis. Vitis vinifera L. (grape) juice is well known for its health-promoting properties, due to its numerous bioactive compounds among which are polyphenols. The aim of this study was to evaluate the protective effect of a red grape juice extract (RGJe) against the endothelial damage induced by BPA in human umbilical vein endothelial cells (HUVECs) as an in vitro model of endothelial dysfunction. Our results showed that RGJe treatment counteracted BPA-induced cell death and apoptosis in HUVECs, blocking caspase 3 and modulating p53, Bax, and Bcl-2. Moreover, RGJe demonstrated antioxidant properties in abiotic tests and in vitro, where it reduced BPA-induced reactive oxygen species as well as restored mitochondrial membrane potential, DNA integrity, and nitric oxide levels. Furthermore, RGJe reduced the increase of chemokines (IL-8, IL-1ß, and MCP-1) and adhesion molecules (VCAM-1, ICAM-1, and E-selectin), caused by BPA exposure, involved in the primary phase of atheromatous plaque formation. Overall, our results suggest that RGJe prevents BPA-induced vascular damage modulating specific intracellular mechanisms, along with protecting cells, owing to its antioxidant capability.

Inflammation and Obesity: The Pharmacological Role of Flavonoids in the Zebrafish Model.

A Mediterranean-style diet is highly encouraged thanks to its healthy food pattern, which includes valuable nutraceuticals such as polyphenols. Among these, flavonoids are associated with relevant biological properties through which they prevent or fight the onset of several human pathologies. Globally, the enhanced incidence of overweight and obese people has caused a dramatic increase in comorbidities, raising the need to provide better therapies. Therefore, the development of sophisticated animal models of metabolic dysregulation has allowed for a deepening of knowledge on this subject. Recent advances in using zebrafish (Danio rerio) as model for metabolic disease have yielded fundamental insights into the potential anti-obesity effects of flavonoids. Chronic low-grade inflammation and immune system activation seem to characterize the pathogenesis of obesity; thus, their reduction might improve the lipid profile of obese patients or prevent the development of associated metabolic illnesses. In this review, we highlight the beneficial role of flavonoids on obesity and related diseases linked to their anti-inflammatory properties. In light of the summarized studies, we suggest that anti-inflammatory therapies could have a relevant place in the prevention and treatment of obesity and metabolic disorders.

Targets Involved in the Anti-Cancer Activity of Quercetin in Breast, Colorectal and Liver Neoplasms.

Phytochemicals have long been effective partners in the fight against several diseases, including cancer. Among these, flavonoids are valuable allies for both cancer prevention and therapy since they are known to influence a large panel of tumor-related processes. Particularly, it was revealed that quercetin, one of the most common flavonoids, controls apoptosis and inhibits migration and proliferation, events essential for the development of cancer. In this review, we collected the evidence on the anti-cancer activity of quercetin exploring the network of interactions between this flavonol and the proteins responsible for cancer onset and progression focusing on breast, colorectal and liver cancers, owing to their high worldwide incidence. Moreover, quercetin proved to be also a potentiating agent able to push further the anti-cancer activity of common employed anti-neoplastic agents, thus allowing to lower their dosages and, above all, to sensitize again resistant cancer cells. Finally, novel approaches to delivery systems can enhance quercetin's pharmacokinetics, thus boosting its great potentiality even further. Overall, quercetin has a lot of promise, given its multi-target potentiality; thus, more research is strongly encouraged to properly define its pharmaco-toxicological profile and evaluate its potential for usage in adjuvant and chemoprevention therapy.

Branched-Chain Amino Acids and Di-Alanine Supplementation in Aged Mice: A Translational Study on Sarcopenia.

In age-related sarcopenia, the gradual loss of skeletal muscle mass, function and strength is underpinned by an imbalanced rate of protein synthesis/breakdown. Hence, an adequate protein intake is considered a valuable strategy to mitigate sarcopenia. Here, we investigated the effects of a 12-week oral supplementation with branched-chain amino acids (BCAAs: leucine, isoleucine, and valine) with recognized anabolic properties, in 17-month-old (AGED) C57BL/6J male mice. BCAAs (2:1:1) were formulated in drinking water, alone or plus two L-Alanine equivalents (2ALA) or dipeptide L-Alanyl-L-Alanine (Di-ALA) to boost BCAAs bioavailability. Outcomes were evaluated on in/ex vivo readouts vs. 6-month-old (ADULT) mice. In vivo hind limb plantar flexor torque was improved in AGED mice treated with BCAAs + Di-ALA or 2ALA (recovery score, R.S., towards ADULT: ≥20%), and all mixtures significantly increased hind limb volume. Ex vivo, myofiber cross-sectional areas were higher in gastrocnemius (GC) and soleus (SOL) muscles from treated mice (R.S. ≥ 69%). Contractile indices of isolated muscles were improved by the mixtures, especially in SOL muscle (R.S. ≥ 20%). The latter displayed higher mTOR protein levels in mice supplemented with 2ALA/Di-ALA-enriched mixtures (R.S. ≥ 65%). Overall, these findings support the usefulness of BCAAs-based supplements in sarcopenia, particularly as innovative formulations potentiating BCAAs bioavailability and effects.

Citrus Flavonoids and Autoimmune Diseases: A Systematic Review of Clinical Studies.

BACKGROUND: Autoimmune diseases are chronic disorders in which the immune system does not recognize and attacks one self's healthy components. In this context, although natural remedies might represent a promising therapeutic strategy, evidence regarding Citrus flavonoids is still controversial. OBJECTIVE: To summarize and critically discuss the clinical evidence on the effects of Citrus flavonoids on managing autoimmune diseases. METHOD: A systematic review of articles has been carried out independently by two authors using MEDLINE, Scopus and ISI Web of Science databases. Search terms comprised keywords related to Citrus flavonoids and autoimmune diseases. The last search was performed on the 16th of March, 2021. No language restrictions were applied. Systematic review and study selection were conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Before starting the review, the authors defined the types of articles to be included. Three reviewers independently carried out the extraction of papers. RESULTS: Ten clinical studies fulfilled the eligibility criteria and were included in the final review. CONCLUSION: The studies discussed in this review are heterogeneous. Indeed, some studies suggest using Citrus flavonoids in the frame of autoimmune disorders, whereas others discourage it. Hence, this systematic review highlights the need for further large-scale clinical studies to define the exact role of Citrus flavonoids in managing autoimmune diseases (PROSPERO number CRD42021234903).

The Anticancer Effect of a Flavonoid-Rich Extract of Bergamot Juice in THP-1 Cells Engages the SIRT2/AKT/p53 Pathway.

Novel targets are constantly sought to fight hematologic malignancies. In this regard, high levels of SIRT2 expression are associated with unfavorable prognosis of acute myeloid leukemia. The interest in the plant kingdom has allowed the identification of ever-new anti-leukemic agents. Citrus × bergamia (bergamot) was proved to possess anticancer properties, yet no evidence is available regarding leukemia. For the first time, we studied the potential anti-leukemic effect of a flavonoid-rich extract of bergamot juice (BJe) in THP-1 cells, investigating the underlying mechanisms. Our findings showed that BJe reduced THP-1 cell proliferation, without affecting that of primary PBMCs, blocking the cell cycle in S phase and inducing apoptosis. Triggering of both extrinsic and intrinsic apoptotic pathways was witnessed by cleavage of caspase-8 and -9, which in turn activated caspase-3 and PARP. Interestingly, the increased p53 acetylation in THP-1 cells underlies SIRT2 inhibition by BJe, that was proved also in the isolated enzyme. Moreover, BJe hampered SIRT2 also by lowering its gene expression. Finally, BJe reduced AKT phosphorylation, which we hypothesized being the joining link between SIRT2 and p53, that play a pivotal role in BJe-induced cell cycle arrest and apoptosis in THP-1 cells. Our results suggest BJe as a potential anti-leukemic agent, via targeting of the SIRT2/AKT/p53 pathway.

The SIRT2 Pathway Is Involved in the Antiproliferative Effect of Flavanones in Human Leukemia Monocytic THP-1 Cells.

Acute myeloid leukemia (AML) represents the most alarming hematological disease for adults. Several genetic modifications are known to be pivotal in AML; however, SIRT2 over-expression has attracted the scientific community's attention as an unfavorable prognostic marker. The plant kingdom is a treasure trove of bioactive principles, with flavonoids standing out among the others. On this line, the aim of this study was to investigate the anti-leukemic properties of the main flavanones of Citrus spp., exploring the potential implication of SIRT2. Naringenin (NAR), hesperetin (HSP), naringin (NRG), and neohesperidin (NHP) inhibited SIRT2 activity in the isolated recombinant enzyme, and more, the combination between NAR and HSP. In monocytic leukemic THP-1 cells, only NAR and HSP induced antiproliferative effects, altering the cell cycle. These effects may be ascribed to SIRT2 inhibition since these flavonoids reduced its gene expression and hampered the deacetylation of p53, known sirtuin substrate, and contextually modulated the expression of the downstream cell cycle regulators p21 and cyclin E1. Additionally, these two flavanones proved to interact with the SIRT2 inhibitory site, as shown by docking simulations. Our results suggest that both NAR and HSP may act as anti-leukemic agents, alone and in combination, via targeting the SIRT2/p53/p21/cyclin E1 pathway, thus encouraging deeper investigations.

Effects of a Novel Gel Formulation of Dog Appeasing Pheromone (DAP) on Behavioral and Physiological Stress Responses in Dogs Undergoing Clinical Examination.

The veterinary visit is necessary for safeguarding the health of dogs, but it can be stressful and threaten both the welfare of the patient and the accuracy of the examination. This randomized, triple-blind, placebo-controlled, crossover study aims at evaluating how dog appeasing pheromone (DAP) in a novel gel formulation influences the behavioral and physiological stress responses of 28 dogs undergoing a standardized clinical examination, while staying in the waiting room (WR) and visited in the examination room (ER). Behavioral responses were studied through behavioral categories and subjective scales (WR and ER). Autonomic response considered heart rate (WR and ER), blood pressure (WR and ER), respiratory rate (ER), and rectal temperature (ER). Neuroendocrine response considered salivary cortisol (WR and ER). In the waiting room, the use of DAP was associated with a significant reduction of lip licking (p = 0.0189), an increase in panting (p = 0.0276), and a reduction close to significance (p = 0.0584) of low body postures. No significant differences were observed within the physiological responses. In the examination room, neither behavioral nor physiological differences were found.

IL-13 and IL-33 Serum Levels Are Increased in Systemic Sclerosis Patients With Interstitial Lung Disease.

OBJECTIVE: Systemic sclerosis (SSc) mortality is extremely variable in its internal organ involvement. Pulmonary fibrosis occurs in up to 30% of the cases. Animal models provide evidence that IL-33 is able to induce both cutaneous and pulmonary fibrosis via increased IL-13 and in SSc patients the levels of IL-33 correlate with skin fibrosis. Our aim was to test whether both IL-33 and IL-13 are higher in patients with diffuse SSc and interstitial lung disease (SSc-ILD) compared to SSc patients without ILD and healthy controls. METHODS: Serum levels of IL-13 and IL-33 were measured in 30 SSc patients with diffuse disease and 30 healthy controls by enzyme-linked immunosorbent assay. The extent of pulmonary fibrosis was assessed according to HRCT Warrick score. Pulmonary function tests included lung diffusion capacity for carbon monoxide, forced vital capacity and total lung capacity. RESULTS: Both IL-13 and IL-33 levels were increased in SSc patients compared to controls and significantly associated each other. DLco, FVC and TLC scores were inversely associated with IL-33 and IL-13 levels. Both IL-33 and IL-13 levels were significantly associated with the Warrick severity score and higher in the group of SSc patients with reduced pulmonary function compared to SSc patients with normal pulmonary function tests. CONCLUSION: The IL-13/IL-33 axis needs to be further explored in longitudinal studies of SSc-ILD patients to assess its validity as a biomarker and future treatment target, as does downstream mediator ST2.

Pharmacology and toxicology of tannins.

Tannins are an interesting class of polyphenols, characterized, in almost all cases, by a different degree of polymerization, which, inevitably, markedly influences their bioavailability, as well as biochemical and pharmacological activities. They have been used for the process of tanning to transform hides into leather, from which their name derives. For several time, they have not been accurately evaluated, but now researchers have started to unravel their potential, highlighting anti-inflammatory, antimicrobial, antioxidant and anticancer activities, as well as their involvement in cardiovascular, neuroprotective and in general metabolic diseases prevention. The mechanisms underlying their activity are often complex, but the main targets of their action (such as key enzymes modulation, activation of metabolic pathways and changes in the metabolic fluxes) are highlighted in this review, without losing sight of their toxicity. This aspect still needs further and better-designed study to be thoroughly understood and allow a more conscious use of tannins for human health.

Oleacein Attenuates Lipopolysaccharide-Induced Inflammation in THP-1-Derived Macrophages by the Inhibition of TLR4/MyD88/NF-κB Pathway.

It is known that plant phenolic compounds exert anti-inflammatory activity through both anti-oxidant effects and modulation of pivotal pro-inflammatory factors. Recently, Olea europaea has been studied as a natural source of bioactive molecules; however, few studies have focused on the biological effect of oleacein (OLC), the most abundant secoiridoid. Therefore, the aim of this study was to investigate the potential anti-oxidant activity of OLC, as well as to study its anti-inflammatory effect in lipopolysaccharide (LPS)-stimulated THP-1-derived macrophages. LPS brought a dramatic increase of both release and gene expression of pro-inflammatory cytokines (IL-6, IL-1ß and TNF-α), as well as a decrease of anti-inflammatory ones (IL-10), the effects of which are reverted by OLC. Moreover, it reduced the levels of COX-2, NO and PGE2 elicited by LPS exposure in THP-1 macrophages. Interestingly, OLC modulated inflammatory signaling pathways through the inhibition of CD14/TLR4/CD14/MyD88 axis and the activation of NF-κB. Finally, OLC showed relevant anti-oxidant capability, assessed by abiotic assays, and reduced the intracellular amount of ROS generated by LPS exposure in THP-1 macrophages. Overall, these results suggest that the anti-oxidant activity and anti-inflammatory effect of OLC may cooperate in its protective effect against inflammatory stressors, thus being a possible alternative pharmacological strategy aimed at reducing the inflammatory process.

The chorioallantoic membrane: A novel approach to extrapolate data from a well-established method.

The chorioallantoic membrane (CAM) of the chicken embryo is a highly vascularized extra-embryonic structure that has been widely used as an in vivo model for the evaluation of angiogenesis. This study was designed to optimize data extrapolation from the most exploited experimental protocol to improve its efficiency and the reliability of the obtainable results. In our study, we followed the most common procedure for CAM assay, employing retinoic acid and vascular endothelial growth factor as standards. CAMs were photographed at t0 , t24 , and t48 ; then, the main parameters of the predefined vascular network/area were evaluated. Subsequently, their variations in each CAM were calculated comparing them within the same CAM over the course of the whole treatment (t24 and t48 ), also comparing the treated CAMs respect to the untreated ones. Thus, we provide a novel approach aimed at extrapolating data from CAM assay that allows to (i) have a greater reliability and richness of data; (ii) better estimate the potential pro- and anti-angiogenic activity of new candidate drugs; (iii) save both eggs and time for the experiments.

Cadmium-Induced Kidney Injury in Mice Is Counteracted by a Flavonoid-Rich Extract of Bergamot Juice, Alone or in Association with Curcumin and Resveratrol, via the Enhancement of Different Defense Mechanisms.

Cadmium (Cd) represents a public health risk due to its non-biodegradability and long biological half-life. The main target of Cd is considered the kidney, where it accumulates. No effective treatment for Cd poisoning is available so that several therapeutic approaches were proposed to prevent damages after Cd exposure. We evaluated the effects of a flavonoid-rich extract of bergamot juice (BJe), alone or in association with curcumin (Cur) and resveratrol (Re), in the kidney of mice exposed to cadmium chloride (CdCl2). Male mice were administered with CdCl2 and treated with Cur, Re, or BJe alone or in combination for 14 days. The kidneys were processed for biochemical, structural and morphometric evaluation. Cd treatment significantly increased urea nitrogen and creatinine levels, along with tp53, Bax, Nos2 and Il1b mRNA, while reduced that of Bcl2, as well as glutathione (GSH) content and glutathione peroxidase (GPx) activity. Moreover, Cd caused damages to glomeruli and tubules, and increased Nrf2, Nqo1 and Hmox1 gene expression. Cur, Re and BJe at 40 mg/kg significantly improved all parameters, while BJe at 20 mg/kg showed a lower protective effect. After treatment with the associations of the three nutraceuticals, all parameters were close to normal, thus suggesting a new potential strategy in the protection of renal functions in subjects exposed to environmental toxicants.

ß-Dystroglycan Restoration and Pathology Progression in the Dystrophic mdx Mouse: Outcome and Implication of a Clinically Oriented Study with a Novel Oral Dasatinib Formulation.

ROS-activated cSrc tyrosine kinase (TK) promotes the degradation of ß-dystroglycan (ß-DG), a dystrophin-glycoprotein complex component, which may reinforce damaging signals in Duchenne muscular dystrophy (DMD). Therefore, cSrc-TK represents a promising therapeutic target. In mdx mice, a 4-week subcutaneous treatment with dasatinib (DAS), a pan-Src-TKs inhibitor approved as anti-leukemic agent, increased muscle ß-DG, with minimal amelioration of morphofunctional indices. To address possible dose/pharmacokinetic (PK) issues, a new oral DAS/hydroxypropyl(HP)-ß-cyclodextrin(CD) complex was developed and chronically administered to mdx mice. The aim was to better assess the role of ß-DG in pathology progression, meanwhile confirming DAS mechanism of action over the long-term, along with its efficacy and tolerability. The 4-week old mdx mice underwent a 12-week treatment with DAS/HP-ß-CD10% dissolved in drinking water, at 10 or 20 mg/kg/day. The outcome was evaluated via in vivo/ex vivo disease-relevant readouts. Oral DAS/HP-ß-CD efficiently distributed in mdx mice plasma and tissues in a dose-related fashion. The new DAS formulation confirmed its main upstream mechanism of action, by reducing ß-DG phosphorylation and restoring its levels dose-dependently in both diaphragm and gastrocnemius muscle. However, it modestly improved in vivo neuromuscular function, ex vivo muscle force, and histopathology, although the partial recovery of muscle elasticity and the decrease of CK and LDH plasma levels suggest an increased sarcolemmal stability of dystrophic muscles. Our clinically oriented study supports the interest in this new, pediatric-suitable DAS formulation for proper exposure and safety and for enhancing ß-DG expression. This latter mechanism is, however, not sufficient by itself to impact on pathology progression. In-depth analyses will be dedicated to elucidating the mechanism limiting DAS effectiveness in dystrophic settings, meanwhile assessing its potential synergy with dystrophin-based molecular therapies.

The Second Life of Citrus Fruit Waste: A Valuable Source of Bioactive Compounds.

Citrus fruits (CF) are among the most widely cultivated fruit crops throughout the world and their production is constantly increasing along with consumers' demand. Therefore, huge amounts of waste are annually generated through CF processing, causing high costs for their disposal, as well as environmental and human health damage, if inappropriately performed. According to the most recent indications of an economic, environmental and pharmaceutical nature, CF processing residues must be transformed from a waste to be disposed to a valuable resource to be reused. Based on a circular economy model, CF residues (i.e., seeds, exhausted peel, pressed pulp, secondary juice and leaves) have increasingly been re-evaluated to also obtain, but not limited to, valuable compounds to be employed in the food, packaging, cosmetic and pharmaceutical industries. However, the use of CF by-products is still limited because of their underestimated nutritional and economic value, hence more awareness and knowledge are needed to overcome traditional approaches for their disposal. This review summarizes recent evidence on the pharmacological potential of CF waste to support the switch towards a more environmentally sustainable society.

A Flavonoid-Rich Extract of Mandarin Juice Counteracts 6-OHDA-Induced Oxidative Stress in SH-SY5Y Cells and Modulates Parkinson-Related Genes.

Parkinson's disease (PD) is a degenerative disorder of the nervous system due to unceasing impairment of dopaminergic neurons situated in the substantia nigra. At present, anti-PD drugs acting on dopamine receptors are mainly symptomatic and have only very limited neuroprotective effects, whereas drugs slowing down neurodegeneration of dopaminergic neurons and deterioration of clinical symptoms are not yet available. Given that, the development of more valuable pharmacological strategies is highly demanded. Comprehensive research on innovative neuroprotective drugs has proven that anti-inflammatory and antioxidant molecules from food sources may prevent and/or counteract neurodegenerative diseases, such as PD. The present study was aimed at the evaluation the protective effect of mandarin juice extract (MJe) against 6-hydroxydopamine (6-OHDA)-induced SH-SY5Y human neuroblastoma cell death. Treatment of differentiated SH-SY5Y cells with 6-OHDA brought cell death, and specifically, apoptosis, which was significantly inhibited by the preincubation with MJe through caspase 3 blockage and the modulation of p53, Bax, and Bcl-2 genes. In addition, it showed antioxidant properties in abiotic models as well as in vitro, where it reduced both reactive oxygen and nitrogen species induced by 6-OHDA, along with restored mitochondrial membrane potential, and prevented the oxidative DNA damage evoked by 6-OHDA. Furthermore, MJe restored the impaired balance of SNCA, LRRK2, PINK1, parkin, and DJ-1 gene levels, PD-related factors, caused by 6-OHDA oxidative stress. Overall, these results indicate that MJe exerts neuroprotective effects against 6-OHDA-induced cell death in SH-SY5Y cells by mechanisms involving both the specific interaction with intracellular pathways and its antioxidant capability. Our study suggests a novel possible strategy to prevent and/or ameliorate neurodegenerative diseases, such as PD.

A Flavonoid-Rich Extract from Bergamot Juice, Alone or in Association with Curcumin and Resveratrol, Shows Protective Effects in a Murine Model of Cadmium-Induced Testicular Injury.

It is known that cadmium damages testis structure and functionality. We examined the effects of nutraceuticals such as a flavonoid-rich extract of bergamot juice (BJe), alone or in association with curcumin (Cur) and resveratrol (Re), on mice testicular dysfunction caused by cadmium chloride (CdCl2). Controversial data on the protective effects of Cur and Re are available, while no evidence on the possible role of BJe exists. Adult male C57 BL/6J mice were administered with CdCl2 and treated with Cur, Re, or BJe alone or in combination for 14 days. Then, testes were removed and processed for molecular, structural, and immunohistochemical analyses. CdCl2 increased the mRNA of IL-1ß, TNF-α, p53, and BAX while reduced that of Bcl-2 and induced tubular lesions and apoptosis of germinal cells. Cur, Re, and BJe at 40 mg/kg significantly improved all of these parameters and events, although BJe at 20 mg/kg showed a lower protective effect. The association of Cur, Re, and BJe at both doses of 50/20/20 and 100/20/40 mg/kg brought each parameter close to those of the control. Our results indicate that the nutraceuticals employed in this study and their associations exert a positive action against Cd-induced testicular injury, suggesting a possible protection of testis functionality in subjects exposed to environmental toxicants.

Bergamottin and 5-Geranyloxy-7-methoxycoumarin Cooperate in the Cytotoxic Effect of Citrus bergamia (Bergamot) Essential Oil in Human Neuroblastoma SH-SY5Y Cell Line.

The plant kingdom has always been a treasure trove for valuable bioactive compounds, and Citrus fruits stand out among the others. Bergamottin (BRG) and 5-geranyloxy-7-methoxycoumarin (5-G-7-MOC) are two coumarins found in different Citrus species with well-acknowledged pharmacological properties. Previously, they have been claimed to be relevant in the anti-proliferative effects exerted by bergamot essential oil (BEO) in the SH-SY5Y human neuroblastoma cells. This study was designed to verify this assumption and to assess the mechanisms underlying the anti-proliferative effect of both compounds. Our results demonstrate that BRG and 5-G-7-MOC are able to reduce the proliferation of SH-SY5Y cells, inducing apoptosis and increasing cell population in sub-G0/G1 phase. Moreover, we demonstrated the pro-oxidant activity of the two coumarins that increased reactive oxygen species and impaired mitochondrial membrane potential. From a molecular point of view, BRG and 5-G-7-MOC were able to modulate apoptosis related factors at both protein and gene levels. Lastly, we evaluated the synergistic effect of their combination, finding that the highest synergy was observed at a concentration ratio similar to that occurring in the BEO, supporting our initial hypothesis. Taken together, our results deepen the knowledge regarding the effect of BRG and 5-G-7-MOC in SH-SY5Y cells, emphasizing the relevance of their cooperation in achieving this effect.

Zebrafish as a Useful Model to Study Oxidative Stress-Linked Disorders: Focus on Flavonoids.

The zebrafish is considered one of the most versatile experimental animal models. The transparency of the embryos, the small size, the rapid development and the homology with higher vertebrates have made the zebrafish a valuable model also for drug screening. Its use is closely related for the determination of bioactivity, toxicity and off-target side effects of novel drug candidates, which also allows a thorough evaluation of new targets; thus, it may represent a suitable model for drug screening and the optimization of novel candidates. Flavonoids are polyphenolic compounds widely present in fruits, vegetables and cereals. Polyphenols are important for both plants and humans, considering their involvement in defense mechanisms, particularly against oxidative stress. They protect plants from biotic and abiotic stressors and prevent or treat oxidative-based human diseases. For these reasons, polyphenols are used as nutraceuticals, functional foods and supplements by the pharmaceutical industry. Therefore, the most relevant findings on zebrafish as a useful experimental model to study oxidative stress-linked disorders, focusing on the biological activities of flavonoids, are here summarized and reviewed.