Pharmacological Evaluation of Newly Synthesized Cannabidiol Derivates on H9c2 Cells.

Cannabidiol (CBD) is a nonpsychoactive phytocannabinoid that can be found in Cannabis sativa and possesses numerous pharmacological effects. Due to these promising effects, CBD can be used in a wide variety of diseases, for instance cardiovascular diseases. However, CBD, like tetrahydrocannabinol (THC), has low bioavailability, poor water solubility, and a variable pharmacokinetic profile, which hinders its therapeutic use. Chemical derivatization of CBD offers us potential ways to overcome these issues. We prepared three new CBD derivatives substituted on the aromatic ring by Mannich-type reactions, which have not been described so far for the modification of cannabinoids, and studied the protective effect they have on cardiomyocytes exposed to oxidative stress and hypoxia/reoxygenation (H/R) compared to the parent compound. An MTT assay was performed to determine the viability of rat cardiomyocytes treated with test compounds. Trypan blue exclusion and lactate dehydrogenase (LDH) release assays were carried out to study the effect of the new compounds in cells exposed to H2O2 or hypoxia/reoxygenation (H/R). Direct antioxidant activity was evaluated by a total antioxidant capacity (TAC) assay. To study antioxidant protein levels, HO-1, SOD, catalase, and Western blot analysis were carried out. pIC50 (the negative log of the IC50) values were as follows: CBD1: 4.113, CBD2: 3.995, CBD3: 4.190, and CBD: 4.671. The newly synthesized CBD derivatives prevented cell death induced by H/R, especially CBD2. CBD has the largest direct antioxidant activity. The levels of antioxidant proteins were increased differently after pretreatment with synthetic CBD derivatives and CBD. Taken together, our newly synthesized CBD derivatives are able to decrease cytotoxicity during oxidative stress and H/R. The compounds have similar or better effects than CBD on H9c2 cells.

Effectiveness of Anthocyanin-Rich Sour Cherry Extract on Gliadin-Induced Caco-2 Barrier Damage.

Several types of gluten-related disorders are known, in which the common starting point is gluten-induced zonulin release. Zonulin results in varying degrees of increased permeability in certain gluten-related disorders but is largely responsible for the development of further pathogenic processes and symptoms. Therefore, it is important to know the barrier-modulating role of individual nutritional components and to what extent the antioxidant substance supports the protection of gliadin-induced membrane damage with its radical scavenging capacity. We investigated the pH dependence of the gliadin-anthocyanin interaction using UV photometry, during which a concentration-dependent interaction was observed at pH 6.8. The barrier modulatory effect of the anthocyanin-rich sour cherry extract (AC) was analyzed on Caco-2 cell culture with pepsin-trypsin-resistant gliadin (PT-gliadin) exposure by TEER measurement, zonula occludens-1 (ZO-1), and Occludin immunohistochemistry. In addition to the TEER-reducing and TJ-rearranging effects of PT-gliadin, NF-κB activation, an increase in cytokine (TNF-α, IFN-γ, and IL-8) release, and mitochondrial ROS levels were observed. We confirmed the anti-inflammatory, stabilizing, and restoring roles of AC extract during gliadin treatment on the Caco-2 monolayer. The extract was able to significantly reduce cytokine and ROS levels despite the known interaction of the main components of the extract with PT-gliadin.

In Vitro and In Vivo Efficacy of Topical Dosage Forms Containing Self-Nanoemulsifying Drug Delivery System Loaded with Curcumin.

The external use of curcumin is rare, although it can be a valuable active ingredient in the treatment of certain inflammatory diseases. The aim of our experimental work was to formulate topical dosage forms containing curcumin for the treatment of atopic dermatitis. Curcumin has extremely poor solubility and bioavailability, so we have tried to increase it with the usage of self-emulsifying drug delivery systems. Creams and gels were formulated using penetration-enhancing surfactants and gelling agents. The release of the drug from the vehicle and its penetration through the membrane were determined using a Franz diffusion cell. An MTT cytotoxicity and in vitro antioxidant assays were performed on HaCaT cell line. The in vivo anti-inflammatory effect of the preparations was tested by measuring rat paw edema. In addition, we examined the degree of inflammation induced by UV radiation after pretreatment with the cream and the gel on rats. For the gels containing SNEDDS, the highest penetration was measured after half an hour, while for the cream, it took one hour to reach the maximum concentration. The gel containing Pemulen TR-1 showed the highest drug release. It was determined that the curcumin-containing preparations can be safely applied on the skin and have antioxidant effects. The animal experiments have proven the effectiveness of curcumin-containing topical preparations.

Rapamycin treatment increases survival, autophagy biomarkers and expression of the anti-aging klotho protein in elderly mice.

Previous investigations have demonstrated that treatment of animals with rapamycin increases levels of autophagy, which is a process by which cells degrade intracellular detritus, thus suppressing the emergence of senescent cells, whose pro-inflammatory properties, are primary drivers of age-associated physical decline. A hypothesis is tested here that rapamycin treatment of mice approaching the end of their normal lifespan exhibits increased survival, enhanced expression of autophagic proteins; and klotho protein-a biomarker of aging that affects whole organism senescence, and systemic suppression of inflammatory mediator production. Test groups of 24-month-old C57BL mice were injected intraperitoneally with either 1.5 mg/kg/week rapamycin or vehicle. All mice administered rapamycin survived the 12-week course, whereas 43% of the controls died. Relative to controls, rapamycin-treated mice experienced minor but significant weight loss; moreover, nonsignificant trends toward decreased levels of leptin, IL-6, IL-1ß, TNF-α, IL-1α, and IGF-1, along with slight elevations in VEGF, MCP-1 were observed in the blood serum of rapamycin-treated mice. Rapamycin-treated mice exhibited significantly enhanced autophagy and elevated expression of klotho protein, particularly in the kidney. Rapamycin treatment also increased cardiomyocyte Ca2+ -sensitivity and enhanced the rate constant of force re-development, which may also contribute to the enhanced survival rate in elderly mice.

BGP-15 Protects against Doxorubicin-Induced Cell Toxicity via Enhanced Mitochondrial Function.

Doxorubicin (DOX) is an efficacious and commonly used chemotherapeutic agent. However, its clinical use is limited due to dose-dependent cardiotoxicity. Several mechanisms have been proposed to play a role in DOX-induced cardiotoxicity, such as free radical generation, oxidative stress, mitochondrial dysfunction, altered apoptosis, and autophagy dysregulation. BGP-15 has a wide range of cytoprotective effects, including mitochondrial protection, but up to now, there is no information about any of its beneficial effects on DOX-induced cardiotoxicity. In this study, we investigated whether the protective effects of BGP-15 pretreatment are predominantly via preserving mitochondrial function, reducing mitochondrial ROS production, and if it has an influence on autophagy processes. H9c2 cardiomyocytes were pretreated with 50 µM of BGP-15 prior to different concentrations (0.1; 1; 3 µM) of DOX exposure. We found that BGP-15 pretreatment significantly improved the cell viability after 12 and 24 h DOX exposure. BGP-15 ameliorated lactate dehydrogenase (LDH) release and cell apoptosis induced by DOX. Additionally, BGP-15 pretreatment attenuated the level of mitochondrial oxidative stress and the loss of mitochondrial membrane potential. Moreover, BGP-15 further slightly modulated the autophagic flux, which was measurably decreased by DOX treatment. Hence, our findings clearly revealed that BGP-15 might be a promising agent for alleviating the cardiotoxicity of DOX. This critical mechanism appears to be given by the protective effect of BGP-15 on mitochondria.

Enhanced Antioxidant and Anti-Inflammatory Effects of Self-Nano and Microemulsifying Drug Delivery Systems Containing Curcumin.

Turmeric has been used for decades for its antioxidant and anti-inflammatory effect, which is due to an active ingredient isolated from the plant, called curcumin. However, the extremely poor water-solubility of curcumin often limits the bioavailability of the drug. The aim of our experimental work was to improve the solubility and thus bioavailability of curcumin by developing self-nano/microemulsifying drug delivery systems (SN/MEDDS). Labrasol and Cremophor RH 40 as nonionic surfactants, Transcutol P as co-surfactant and isopropyl myristate as the oily phase were used during the formulation. The average droplet size of SN/MEDDS containing curcumin was between 32 and 405 nm. It was found that the higher oil content resulted in larger particle size. The drug loading efficiency was between 93.11% and 99.12% and all formulations were thermodynamically stable. The curcumin release was studied at pH 6.8, and the release efficiency ranged between 57.3% and 80.9% after 180 min. The results of the MTT cytotoxicity assay on human keratinocyte cells (HaCaT) and colorectal adenocarcinoma cells (Caco-2) showed that the curcumin-containing preparations were non-cytotoxic at 5 w/v%. According to the results of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide dismutase (SOD) assays, SNEDDS showed significantly higher antioxidant activity. The anti-inflammatory effect of the SN/MEDDS was screened by enzyme-linked immunosorbent assay (ELISA). SNEDDS formulated with Labrasol as surfactant, reduced tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) levels below 60% at a concentration of 10 w/w%. Our results verified the promising use of SN/MEDDS for the delivery of curcumin. This study demonstrates that the SN/MEDDS could be promising alternatives for the formulation of poorly soluble lipophilic compounds with low bioavailability.

Beta-Carotene Affects the Effects of Heme Oxygenase-1 in Isolated, Ischemic/Reperfused Rat Hearts: Potential Role of the Iron.

Beta-carotene (BC) is a well-known antioxidant. However, increasing evidence shows that under severe oxidative conditions, BC can become pro-oxidant, an effect that may be enhanced in the presence of iron (II). In our earlier studies, we observed that despite increasing heme oxygenase-1 (HO-1) levels in the heart, the protective effects of BC have been lost when it was used at a high concentration. Since iron releases from heme as a consequence of HO-1 activity, we hypothesized that the application of an iron-chelator (IC) would reverse the lost cardiac protection associated with an elevated HO-1 level. Thus, in the present study, we investigated the effects of desferrioxiamine (DFO) in isolated, ischemic/reperfused rat hearts after long-term treatment with vehicle or high-dose (HD) BC. Vehicle or 150 mg/bw kg daily doses of BC were administered to the rats for 4 weeks, and then their hearts were removed and subjected to 30 min of global ischemia (ISA) followed by 120 min of reperfusion (REP). During the experiments, cardiac function was registered, and at the end of the REP period, infarct size (IS) and HO-1 expression were measured. The results show that DFO treatment alone during REP significantly ameliorated postischemic cardiac function and decreased IS, although HO-1 expression was not increased significantly. In hearts isolated from BC-treated rats, no cardioprotective effects, despite an elevated HO-1 level, were observed, while DFO administration after ISA resulted in a mild improvement in heart function and IS. Our results suggest that iron could have a role whether BC exerts antioxidant or pro-oxidant effects in ISA/REP-injured hearts.

Concentration-Dependent Antibacterial Activity of Chitosan on Lactobacillus plantarum.

The antimicrobial effect of chitosan and synthetic chitosan derivatives has been confirmed on many Gram-positive and Gram-negative bacteria and fungi. The tests were carried out on pathogenic microorganisms, so the mechanism and concentration dependence of the inhibitory effect of chitosan were revealed. We conducted our tests on a probiotic strain, Lactobacillus plantarum. Commercially available chitosan derivatives of different molecular weights were added to L. plantarum suspension in increasing concentrations. The minimum inhibitory concentration (MIC) value of chitosan was determined and confirmed the viability decreasing effect at concentrations above the MIC with a time-kill assay. The release of bacterium cell content was measured at 260 nm after treatment with 0.001-0.1% concentration chitosan solution. An increase in the permeability of the cell membrane was observed only with the 0.1% treatment. The interaction was also investigated by zeta potential measurement, and the irreversible interaction and concentration dependence were established in all concentrations. The interaction of fluorescein isothiocyanate (FITC) labeled low molecular weight chitosan and bacterial cells labeled with membrane dye (FM® 4-64) was confirmed by confocal microscopy. In conclusion, the inhibitory effect of chitosan was verified on a probiotic strain, which is an undesirable effect in probiotic preparations containing chitosan additives, while the inhibitory effect experienced with pathogenic strains is beneficial.

Nicotinic Amidoxime Derivate BGP-15, Topical Dosage Formulation and Anti-Inflammatory Effect.

BGP-15 is a Hungarian-developed drug candidate with numerous beneficial effects. Its potential anti-inflammatory effect is a common assumption, but it has not been investigated in topical formulations yet. The aim of our study was to formulate 10% BGP-15 creams with different penetration enhancers to ensure good drug delivery, improve bioavailability of the drug and investigate the potential anti-inflammatory effect of BGP-15 creams in vivo. Since the exact mechanism of the effect is still unknown, the antioxidant effect (tested with UVB radiation) and the ability of BGP-15 to decrease macrophage activation were evaluated. Biocompatibility investigations were carried out on HaCaT cells to make sure that the formulations and the selected excipients can be safely used. Dosage form studies were also completed with texture analysis and in vitro release with Franz diffusion chamber apparatus. Our results show that the ointments were able to reduce the extent of local inflammation in mice, but the exact mechanism of the effect remains unknown since BGP-15 did not show any antioxidant effect, nor was it able to decrease LPS-induced macrophage activation. Our results support the hypothesis that BGP-15 has a potential anti-inflammatory effect, even if it is topically applied, but the mechanism of the effect remains unclear and requires further pharmacological studies.

Basic Pharmacological Characterization of EV-34, a New H2S-Releasing Ibuprofen Derivative.

BACKGROUND: Cardioprotective effects of H2S are being suggested by numerous studies. Furthermore, H2S plays a role in relaxation of vascular smooth muscle, protects against oxidative stress, and modulates inflammation. Long-term high-dose use of NSAIDs, such as ibuprofen, have been associated with enhanced cardiovascular risk. The goal of the present work is the synthesis and basic pharmacological characterization of a newly designed H2S-releasing ibuprofen derivative. METHODS: Following the synthesis of EV-34, a new H2S-releasing derivative of ibuprofen, oxidative stability assays were performed (Fenton and porphyrin assays). Furthermore, stability of the molecule was studied in rat serum and liver lysates. H2S-releasing ability of the EC-34 was studied with a hydrogen sulfide sensor. MTT (3-(4,5-dimethylthiazol 2-yl)-2,5-(diphenyltetrazolium bromide)) assay was carried out to monitor the possible cytotoxic effect of the compound. Cyclooxygenase (COX) inhibitory property of EV-34 was also evaluated. Carrageenan assay was carried out to compare the anti-inflammatory effect of EV-34 to ibuprofen in rat paws. RESULTS: The results revealed that the molecule is stable under oxidative condition of Fenton reaction. However, EV-34 undergoes biodegradation in rat serum and liver lysates. In cell culture medium H2S is being released from EV-34. No cytotoxic effect was observed at concentrations of 10, 100, 500 µM. The COX-1 and COX-2 inhibitory effects of the molecule are comparable to those of ibuprofen. Furthermore, based on the carrageenan assay, EV-34 exhibits the same anti-inflammatory effect to that of equimolar amount of ibuprofen (100 mg/bwkg). CONCLUSION: The results indicate that EV-34 is a safe H2S releasing ibuprofen derivative bearing anti-inflammatory properties.

Prophylactic, single-drug cardioprotection in a comparative, experimental study of doxorubicin-induced cardiomyopathy.

BACKGROUND: Cardiomyopathy is a common side effect of doxorubicin (DOX) chemotherapy. Despite intensive research efforts in the field, there is still no evidence available for routine cardioprotective prophylaxis to prevent cardiotoxicity in the majority of oncological patients at low risk of cardiovascular disease. We have recently demonstrated the advantages of a prophylactic, combined heart failure therapy in an experimental model of DOX-induced cardiomyopathy. In the current work, we focus on individually applied prophylactic medications studied in the same translational environment to clarify their distinct roles in the prevention of DOX cardiotoxicity. METHODS: Twelve-week-old male Wistar rats were divided into 5 subgroups. Prophylactic ß-blocker (BB, bisoprolol), angiotensin-converting enzyme inhibitor (ACEI, perindopril) or aldosterone antagonist (AA, eplerenone) treatments were applied 1 week before DOX administration, then 6 cycles of intravenous DOX chemotherapy were administered. Rats receiving only intravenous DOX or saline served as positive and negative controls. Blood pressure, heart rate, body weight, and echocardiographic parameters were monitored in vivo. Two months after the last DOX administration, the animals were sacrificed, and their heart and serum samples were frozen in liquid nitrogen for histological, mechanical, and biochemical measurements. RESULTS: All prophylactic treatments increased the survival of DOX-receiving animals. The lowest mortality rates were seen in the BB and ACEI groups. The left ventricular ejection fraction was only preserved in the BB group. The DOX-induced increase in the isovolumetric relaxation time could not be prevented by any prophylactic treatment. A decreased number of apoptotic nuclei and a preserved myocardial ultrastructure were found in all groups receiving prophylactic cardioprotection, while the DOX-induced fibrotic remodelling and the increase in caspase-3 levels could only be substantially prevented by the BB and ACEI treatments. CONCLUSION: Primary prophylaxis with cardioprotective agents like BB or ACEI has a key role in the prevention of DOX-induced cardiotoxicity in healthy rats. Future human studies are necessary to implement this finding in the clinical management of oncological patients free of cardiovascular risk factors.

The Role of Autophagy and Death Pathways in Dose-dependent Isoproterenolinduced Cardiotoxicity.

BACKGROUND: Isoproterenol (ISO) is a non-selective ß-adrenergic agonist. Our aims were to investigate the autophagy and cell death pathways including apoptosis and necrosis in ISO-induced cardiac injury in a dosedependent manner. METHODS: Male Sprague-Dawley rats were treated for 24 hours with I. vehicle (saline); II. 0.005 mg/kg ISO; III. 0.05 mg/kg ISO; IV. 0.5 mg/kg ISO; V. 5 mg/kg ISO; VI. 50 mg/kg ISO, respectively. Hearts were isolated and infarct size was measured. Serum levels of Troponin T (TrT), lactate dehydrogenase (LDH), creatine kinase isoenzyme MB (CK-MB) were measured. TUNEL assay was carried out to monitor apoptotic cell death and Western blot was performed to evaluate the level of autophagic and apoptotic markers. RESULTS: Survival rate of animals was dose-dependently decreased by ISO. Serum markers and infarct size revealed the development of cardiac toxicity. Level of Caspase-3, and results of TUNEL assay, demonstrated that the level of apoptosis was dose-dependently increased. They reached the highest level in ISO 5 and it decreased slightly in ISO 50 group. Focusing on autophagic proteins, we found that level of Beclin-1 was increased in a dose-dependent manner, but significantly increased in ISO 50 treated group. Level of LC3B-II and p62 showed the same manner, but the elevated level of p62 indicated that autophagy was impaired in both ISO 5 and ISO 50 groups. CONCLUSION: Taken together these results suggest that at smaller dose of ISO autophagy may cope with the toxic effect of ISO; however, at higher dose apoptosis is initiated and at the highest dose substantial necrosis occurs.

Advantages of prophylactic versus conventionally scheduled heart failure therapy in an experimental model of doxorubicin-induced cardiomyopathy.

BACKGROUND: Chemotherapy-induced left ventricular dysfunction represents a major clinical problem, which is often only recognised at an advanced stage, when supportive therapy is ineffective. Although an early heart failure treatment could positively influence the health status and clinical outcome, there is still no evidence of routine prophylactic cardioprotection for the majority of patients without previous cardiovascular history awaiting potentially cardiotoxic chemotherapy. In this study, we set out to investigate whether a prophylactic cardioprotective therapy relative to a conventionally scheduled heart failure treatment is more effective in preventing cardiotoxicity in a rodent model of doxorubicin (DOX)-induced cardiomyopathy. METHODS: Male Wistar rats (n = 7-11 per group) were divided into 4 subgroups, namely negative controls receiving intravenous saline (CON), positive controls receiving intravenous DOX (6 cycles; D-CON), and DOX-treated animals receiving either prophylactic (PRE, started 1 week before DOX) or conventionally applied (POST, started 1 month after DOX) combined heart failure therapy of oral bisoprolol, perindopril and eplerenone. Blood pressure, heart rate, body weight and echocardiographic parameters were monitored in vivo, whereas myocardial fibrosis, capillarisation, ultrastructure, myofilament function, apoptosis, oxidative stress and mitochondrial biogenesis were studied in vitro. RESULTS: The survival rate in the PRE group was significantly improved compared to D-CON (p = 0.0207). DOX increased the heart rate of the animals (p = 0.0193), while the blood pressure (p ≤ 0.0105) and heart rate (p = 0.0029) were significantly reduced in the PRE group compared to D-CON and POST. The ejection fraction remained preserved in the PRE group compared to D-CON or POST (p ≤ 0.0237), while none of the treatments could prevent the DOX-induced increase in the isovolumetric relaxation time. DOX decreased the rate of the actin-myosin cross-bridge cycle, irrespective of any treatment applied (p ≤ 0.0433). The myocardium of the D-CON and POST animals displayed pronounced ultrastructural damage, which was not apparent in the PRE group (p ≤ 0.033). While the DOX-induced apoptotic activity could be reduced in both the PRE and POST groups (p ≤ 0.0433), no treatment was able to prevent fibrotic remodelling or the disturbed mitochondrial biogenesis. CONCLUSION: For attenuating DOX-induced adverse myocardial effects, prophylactic cardioprotection has many advantages compared to a late-applied treatment.

Electrically-Induced Ventricular Fibrillation Alters Cardiovascular Function and Expression of Apoptotic and Autophagic Proteins in Rat Hearts.

BACKGROUND: The pathological heart contractions, called arrhythmias, especially ventricular fibrillation (VF), are a prominent feature of many cardiovascular diseases leading to sudden cardiac death. The present investigation evaluates the effect of electrically stimulated VF on cardiac functions related to autophagy and apoptotic mechanisms in isolated working rat hearts. METHODS: Each group of hearts was subjected to 0 (Control), 1, 3, or 10 min of spacing-induced VF, followed by 120 min of recovery period and evaluated for cardiac functions, including aortic flow (AF), coronary flow (CF), cardiac output (CO), stroke volume (SV), and heart rate (HR). Hearts were also evaluated for VF effects on infarcted zone magnitude and Western blot analysis was conducted on heart tissue for expression of the apoptotic biomarker cleaved-caspase-3 and the autophagy proteins: p62, P-mTOR/mTOR, LC3BII/LC3BI ratio, and Atg5-12 complexes. RESULTS: Data revealed that VF induced degradation in AF, CF, CO, and SV, which prominently included-variable post-VF capacity for recovery of normal heart rhythm; increased extent of infarcted heart tissue; altered expression of cleaved-caspase-3 suggesting potential for VF-mediated amplification of apoptosis. VF influence on expression of p62, LC3BII/LC3BI, and Atg5-12 proteins was complex, possibly due to differential effects of VF-induced expression on proteins comprising the autophagic program. CONCLUSIONS: VF was observed to cause time-dependent changes in autophagy processes, which with additional analysis under ongoing investigations, likely to yield novel therapeutic targets for the prevention of VF and sudden cardiac death.

Inhibited autophagy may contribute to heme toxicity in cardiomyoblast cells.

Several groups have demonstrated that induction of heme-oxygenase-1 (HO-1) could protect the myocardium against ischemic events; however, heme accumulation could lead to toxicity. The aim of the present study was to investigate the role of autophagy in heme toxicity. H9c2 cardiomyoblast cells were treated with different dose of hemin or cobalt-protoporphyrin IX (CoPPIX) or vehicle. Cell viability was measured by MTT assay. DCF and MitoSOX staining was employed to detect reactive oxygen species. Western blot analysis was performed to analyse the levels of HO-1, certain autophagy related proteins and pro-caspase-3 as an apoptosis marker. To study the autophagic flux, CytoID staining was carried out and cells were analyzed by fluorescence microscope and flow cytometry. Decreased cell viability was detected at high dose of hemin and CoPPIX treated H9c2 cells in a dose-dependent manner. Furthermore, at concentration of the inducers used in the present study a significantly enhanced level of ROS were detected. As it was expected both treatments induced a robust elevation of HO-1 level. In addition, the Beclin-1- independent autophagy was significantly increased, but caused a defective autophagic flux with triggered activation of caspase-3. In conclusion, these results suggest that overexpression of HO-1 by high dose of hemin and CoPPIX can induce cell toxicity in H9c2 cells via enhanced ROS level and impaired autophagy.

The Impact of Moderate Chronic Hypoxia and Hyperoxia on the Level of Apoptotic and Autophagic Proteins in Myocardial Tissue.

The redox imbalance and the consequent oxidative stress have been implicated in many pathological conditions, including cardiovascular diseases. The lack or the excess of O2 supply can alter the redox balance. The aim of the present study was to understand the heart responses to prolonged hypoxia or hyperoxia and how such situations may activate survival mechanisms or trigger cell death. Seven-week-old Foxn1 mice were exposed to hypoxia (10% O2), normoxia (21% O2), or hyperoxia (30% O2) for 28 days, then the heart tissue was excised and analyzed. The alterations in redox balance, housekeeping protein levels, and autophagic and apoptotic process regulation were studied. The D-ROM test demonstrated an increased oxidative stress in the hypoxic group compared to the hyperoxic group. The level of hypoxia inducible factor-1 (HIF-1α) was increased by hypoxia while HIF-2α was not affected by treatments. Chronic hypoxia activated the biochemical markers of autophagy, and we observed elevated levels of Beclin-1 while LC3B-II and p62 were constant. Nevertheless, we measured significantly enhanced number of TUNEL-positive cells and higher Bax/Bcl2 ratio in hyperoxia with respect to hypoxia. Surprisingly, our results revealed alterations in the level of housekeeping proteins. The expression of α-tubulin, total-actin, and GAPDH was increased in the hypoxic group while decreased in the hyperoxic group. These findings suggest that autophagy is induced in the heart under hypoxia, which may serve as a protective mechanism in response to enhanced oxidative stress. While prolonged hypoxia-induced autophagy leads to reduced heart apoptosis, low autophagic level in hyperoxia failed to prevent the excessive DNA fragmentation.

Aged (Black) versus Raw Garlic against Ischemia/Reperfusion-Induced Cardiac Complications.

Recent evidence from studies suggests that aged black garlic also has an effect on health. The major aim of the present study is to compare the effect of raw and aged black garlic on postischemic cardiac recovery. Male Sprague Dawley rats were randomly divided into three groups. Animals of the first group were fed with raw garlic, animals of the second group received aged black garlic, while the third group served as vehicle-treated controls. Upon conclusion of the treatment, isolated hearts were undertaken to ischemia/reperfusion. Heart function and infarct size were measured and the level of HO-1 and iNOS were studied. Superior postischemic cardiac function and reduced infarct size in both garlic treated groups compared to the drug-free control group, indicated cardioprotective effects. However, no significant differences between the garlic treated groups were observed. Western blot analysis revealed that raw garlic enhanced the level of HO-1 before ischemia, while in ischemic samples, we found elevated HO-1 expression in both garlic treated groups. The level of iNOS was the same before ischemia in all groups, however, a markedly reduced iNOS level in ischemic/reperfused hearts originating from control and raw garlic treated animals was observed. Samples from aged black garlic treated animals demonstrated that the level of iNOS was not significantly reduced after ischemia/reperfusion. Taken together these results indicate that not only raw but also aged black garlic possess a cardioprotective effect.

Effects of Momordica charantia (Bitter Melon) on Ischemic Diabetic Myocardium.

Objective: A rat model is here used to test a hypothesis that Momordica charantia (Bitter melon (BM)) extract favorably alters processes in cardiovascular tissue and is systemically relevant to the pathophysiology of type 2 diabetes (T2DM) and related cardiovascular disease. Methods: Male Lean and Zucker Obese (ZO) rats were gavage-treated for six weeks with 400 mg/kg body weight bitter melon (BM) extract suspended in mucin-water vehicle, or with vehicle (Control). Animals were segregated into four treatment groups, 10 animals in each group, according to strain (Lean or ZO) and treatment (Control or BM). Following six-week treatment periods, peripheral blood was collected from selected animals, followed by sacrifice, thoracotomy and mounting of isolated working heart setup. Results: Body mass of both Lean and ZO rats was unaffected by treatment, likewise, peripheral blood fasting glucose levels showed no significant treatment-related effects. However, some BM treatment-related improvement was noted in postischemic cardiac functions when Lean, BM-treated animals were compared to vehicle treated Lean control rats. Treatment of Lean, but not ZO, rats significantly reduced the magnitude of infarcted zone in isolated hearts subjected to 30 min of ischemia followed by 2 h of working mode reperfusion. Immunohistochemical demonstration of caspase-3 expression by isolated heart tissues subjected to 30 min of ischemia followed by 2 h of reperfusion, revealed significant correlation between BM treatment and reduced expression of this enzyme in hearts obtained from both Lean and ZO animals. The hierarchy and order of caspase-3 expression from highest to lowest was as follows: ZO rats receiving vehicle > ZO rats receiving BM extract > Lean rats treated receiving vehicle > Lean rats administered BM extract. Outcomes of analyses of peripheral blood content of cardiac-related analytics: with particular relevance to clinical application was a significant elevation in blood of ZO and ZO BM-treated, versus Lean rats of total cholesterol (high density lipoprotein HDL-c + low density lipoprotein LDL-c), with an inferred increase in HDL-c/LDL-c ratio-an outcome associated with decreased risk of atherosclerotic disease. Conclusions: BM extract failed to positively affect T2DM- and cardiovascular-related outcomes at a level suggesting use as a standalone treatment. Nevertheless, the encouraging effects of BM in enhancement of cardiac function, suppression of post-ischemic/reperfused infarct size extent and capacity to modulate serum cholesterol, will likely make it useful as an adjuvant therapy for the management of T2DM and related cardiovascular diseases.

Cardioprotection afforded by sour cherry seed kernel: the role of heme oxygenase-1.

Cardiovascular diseases are primary cause of death worldwide, particularly among populations with sedentary lifestyles and diets rich in animal products and processed foods. Currently, public health countermeasures to these disorders focus on costly and often marginally effective interventions administered only after the development of disease. These countermeasures are mainly palliative and fail to address the underlying causes of cardiac pathologies. Previously, the authors of this report have demonstrated that sour cherry seed kernel extract (SCSE), a nontoxic low-cost plant material, strongly preserves tissues through induction of heme oxygenase-1 (HO-1), a critical host antioxidant defense enzyme. This investigation seeks to characterize underlying mechanisms of SCSE-mediated tissue protection. Isolated hearts from Sprague-Dawley rats fed 30 mg·kg·d SCSE for 8 weeks, and untreated controls were mounted in a "working heart" apparatus and subjected to ischemia and reperfusion. A panel of cardiac functional evaluations was conducted on each heart. Infarct size assessments were made along with Western blot and immunohistochemical analysis for selected proteins involved in cardiovascular homeostasis. SCSE treatment was observed to improve postischemic cardiac functions and suppress infarct size. Analysis of the outcomes produced by this study is consistent with SCSE cardioprotection that involve interaction of Bcl-2 and HO-1.