Protective effects of anthocyanins against brain ischemic damage.

Anthocyanins are considered as bioactive components of plant-based diets that provide protection against ischemic cardiovascular pathologies by mechanisms dependent on their antioxidant and reductive capacities. However, it is not clear whether similar anthocyanin-mediated mechanisms can provide protection against ischemia-induced brain mitochondrial injury and cell death. In this study, we compared effects of three cyanidin-3-glycosides - glucoside (Cy3G), galactoside (Cy3Gal) and rutinoside (Cy3R), with pelargonxidin-3-glucoside (Pg3G) and found that at 10-20 µM concentrations they have no direct effect on respiratory functions of mitochondria isolated from normal or ischemia-damaged rat brain slices. However, intravenous injection of Cy3Gal and Cy3G (0,025 mg/kg or 0,05 mg/kg what matches 10 µM or 20 µM respectively) but not Cy3R in rats protected against ischemia-induced caspase activation and necrotic cell death, and reduced infarct size in cerebral cortex and cerebellum. These effects correlated with cytochrome c reducing capacity of cyanidin-3-glycosides. In contrast, intravenous injection of 0,025 mg/kg Pg3G which has the lowest cytochrome c reducing capacity among investigated anthocyanins, had no effect on ischemia-induced caspase activation and necrosis but reduced brain infarct size whereas intravenous injection of 0,05 mg/kg of Pg3G slightly promoted necrosis in the brain. Our data suggest that reductive rather than antioxidant capacities of anthocyanins may be important components in providing protection against ischemic brain damage.

Anthocyanins: From plant pigments to health benefits at mitochondrial level.

Anthocyanins are water-soluble pigments providing certain color for various plant parts, especially in edible berries. Earlier these compounds were only known as natural food colorants, the stability of which depended on pH, light, storage temperature and chemical structure. However, due to the increase of the in vitro, in vivo experimental data, as well as of the epidemiological studies, today anthocyanins and their metabolites are also regarded as potential pharmaceutical compounds providing various beneficial health effects on either human or animal cardiovascular system, brain, liver, pancreas and kidney. Many of these effects are shown to be related to the free-radical scavenging and antioxidant properties of anthocyanins, or to their ability to modulate the intracellular antioxidant systems. However, it is generally overlooked that instead of acting exclusively as antioxidants certain anthocyanins affect the activity of mitochondria that are the main source of energy in cells. Therefore, the aim of the present review is to summarize the major knowledge about the chemistry and regulation of biosynthesis of anthocyanins in plants, to overview the facts on bioavailability, and to discuss the most recent experimental findings related to the beneficial health effects emphasizing mitochondria.

Anthocyanins in cardioprotection: A path through mitochondria.

Constantly growing experimental data from in vitro, in vivo and epidemiological studies show the great potential of anthocyanin-containing fruit and berry extracts or pure individual anthocyanins as cardioprotective food components or pharmacological compounds. In general it is regarded that the cardioprotective activity of anthocyanins is related to their antioxidant properties. However there are recent reports that certain anthocyanins may protect the heart against ischemia/reperfusion-induced injury by activating signal transduction pathways and sustaining mitochondrial functions instead of acting solely as antioxidants. In this review, we summarize the proposed mechanisms of direct or indirect actions of anthocyanins within cardiac cells with the special emphasis on recently discovered their pharmacological effects on mitochondria in cardioprotection: reduction of cytosolic cytochrome c preventing apoptosis and sustainment of electron transfer between NADH dehydrogenase and cytochrome c supporting oxidative phosphorylation in ischemia-damaged mitochondria.

Protecting the heart against ischemia/reperfusion-induced necrosis and apoptosis: the effect of anthocyanins.

BACKGROUND AND OBJECTIVE: It is well known that cardiomyocyte apoptosis contributes to ischemic heart damage. There is also increasing evidence that the polyphenolic compounds of natural origin, such as anthocyanins, may attenuate ischemia/reperfusion injury though the mechanisms of such protection are not clear. Following our previous studies showing the effect of certain anthocyanins on cytochrome c redox state, mitochondrial functions, and ischemia-induced caspase activation in the heart, here we investigated whether these anthocyanins can rescue cardiac cells from death by the mechanism involving the reduction of cytosolic cytochrome c. MATERIAL AND METHODS: Before global ischemia and reperfusion, isolated rat hearts were preloaded with cyanidin-3-O-glucoside (Cy3G) that has high cytochrome c-reducing capacity or pelargonidin-3-O-glucoside (Pg3G) that possesses low reducing activity. Cell death was evaluated assessing apoptosis by the TUNEL method or necrosis measuring the release of lactate dehydrogenase into perfusate. RESULTS: The perfusion of hearts with 20-µM Cy3G prevented ischemia/reperfusion-induced apoptosis of cardiomyocytes: the number of TUNEL-positive myocytes was decreased by 73% if compared with the untreated ischemic group. The same effect was observed measuring the activity of lactate dehydrogenase as the measure of necrosis: perfusion with 20-µM Cy3G reduced the level of LDH release into the perfusate to the control level. The perfusion of hearts with 20-µM Pg3G did not prevent ischemia/reperfusion-induced apoptosis as well as necrosis. CONCLUSIONS: Cy3G protected the rat heart from ischemia/reperfusion-induced apoptosis and necrosis; meanwhile, Pg3G did not exert any protective effect. The protective effect of Cy3G may be related due to its high capacity to reduce cytosolic cytochrome c.

The Single Intra-Articular Injection of Platelet-Rich Plasma vs. Non-Steroidal Anti-Inflammatory Drugs as Treatment Options for Canine Cruciate Ligament Rupture and Patellar Luxation.

Cranial cruciate ligament rupture (CCLR) and patellar luxation (PL) are common pathologies affecting canines. Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used as a non-surgical treatment plan in these cases. Clinical usage of platelet-rich plasma (PRP) is an emerging area of interest in veterinary medicine. There is a lack of studies comparing those two different treatment methods in veterinary medicine. The main purpose of this study was to evaluate and compare the use of oral NSAIDs and single intra-articular injection of PRP on treatment outcomes in cases of canine CCLR and PL. Dogs diagnosed with CCRL (n = 12) and PL (n = 10) were subgrouped by the severity of pathologies and administered treatment: half of the CCRL and PL groups were orally administered NSAIDs and supplements for 14 days, and the other half received a single intra-articular PRP injection into affected stifle joint. We measured serum TNF-α levels and clinical outcomes (lameness scores, painfulness to manipulations, goniometry of stifle joint in flexion and extension, and muscle strength) before treatment, at day 14 and day 28 of treatment. The results of TNF-α concentration indicates a significant difference between groups of differently treated partial CCLR groups on d14 (p = 0.006). Results of group CCLR-P1 on d14 were decreased, while results of group CCLR-P2 on d14 were increased. When comparing TNF-α concentration between all CCLR cases treated with NSAIDs and treated with PRP, there was a significant difference between those groups on d14 (p = 0.001). The results of TNF-α concentration indicates a significant difference between groups of differently treated PL-III on d28 (p = 0.036). Results of group PL-III1 indicate growth of TNF-α concentration, while at the same d28, results of group PL-III2 indicate decreased levels of cytokine, comparing results between the subgroups at the same time point and within subgroups from baseline. Results indicate a significant difference in muscle strength between group CCLR-P1 and group CCLR-P2 on d28 (p = 0.007), indicating an increment in muscle strength in group CCLR-P1 up to d14 and its reduction up to d28, and muscle strength of group CCLR-P2 increasing up to d28. When comparing the muscle strength between all CCLR cases treated with NSAIDs and treated with PRP, there was a significant difference between those groups on d28 (p = 0.007). In conclusion, a single intra-articular injection of PRP has a superior effect on management of inflammatory processes, has better clinical outcomes, and longer duration of action than oral NSAIDs, in the treatment of canine CCRL or PL.

Direct effects of K(ATP) channel openers pinacidil and diazoxide on oxidative phosphorylation of mitochondria in situ.

K(ATP) channel openers protect ischemic-reperfused myocardium by mimicking ischemic preconditioning, however, the protection mechanisms have not been fully clarified yet. Since the skinned fibers technique gives an opportunity to investigate an entire population of mitochondria in their native milieu, in this study we have investigated the effects of K(ATP) channel openers pinacidil and diazoxide on the respiration rate of rat heart mitochondria in situ, oxidizing physiological substrates pyruvate and malate (6+6 mM). Respiration rates were recorded by the means of Clark-type oxygen electrode in the physiological salt solution (37 degrees C). Our results showed that both pinacidil and diazoxide (60-1250 muM) in a concentration-dependent manner increased pyruvate-malate supported State 2 respiration rate of skinned cardiac fibers (59.1 +/- 5.1 nmol O/min/mg fiber dry weight, RCI 2.6 +/- 0.2, n=4) by 15-120%. Moreover, diazoxide did not affect, whereas pinacidil (60-1250 muM) decreased the State 3 respiration rate of skinned cardiac fibers (116.6 +/- 13.6 nmol O/min/mg fiber dry weight, RCI 2.3 +/- 0.2, n=4) by 4-27%. Thus, common effect for both K(ATP) channel openers is uncoupling of pyruvate and malate oxidizing mitochondria in skinned cardiac fibers, whereas pinacidil under same conditions also inhibits mitochondrial respiratory chain. Since mitochondria in situ resemble to the great extent mitochondria in vivo, our results suggest that uncoupling and/or respiratory chain inhibition could play a role in the cardioprotection by K(ATP) channel openers.

Comparison of Effects of Metformin, Phenformin, and Inhibitors of Mitochondrial Complex I on Mitochondrial Permeability Transition and Ischemic Brain Injury.

Damage to cerebral mitochondria, particularly opening of mitochondrial permeability transition pore (MPTP), is a key mechanism of ischemic brain injury, therefore, modulation of MPTP may be a potential target for a neuroprotective strategy in ischemic brain pathologies. The aim of this study was to investigate whether biguanides-metformin and phenformin as well as other inhibitors of Complex I of the mitochondrial electron transfer system may protect against ischemia-induced cell death in brain slice cultures by suppressing MPTP, and whether the effects of these inhibitors depend on the age of animals. Experiments were performed on brain slice cultures prepared from 5-7-day (premature) and 2-3-month old (adult) rat brains. In premature brain slice cultures, simulated ischemia (hypoxia plus deoxyglucose) induced necrosis whereas in adult rat brain slice cultures necrosis was induced by hypoxia alone and was suppressed by deoxyglucose. Phenformin prevented necrosis induced by simulated ischemia in premature and hypoxia-induced-in adult brain slices, whereas metformin was protective in adult brain slices cultures. In premature brain slices, necrosis was also prevented by Complex I inhibitors rotenone and amobarbital and by MPTP inhibitor cyclosporine A. The latter two inhibitors were protective in adult brain slices as well. Short-term exposure of cultured neurons to phenformin, metformin and rotenone prevented ionomycin-induced MPTP opening in intact cells. The data suggest that, depending on the age, phenformin and metformin may protect the brain against ischemic damage possibly by suppressing MPTP via inhibition of mitochondrial Complex I.

Anthocyanins as substrates for mitochondrial complex I - protective effect against heart ischemic injury.

Anthocyanins, a subclass of flavonoids, are known to protect against myocardial ischemia; however, little is known about their direct, acute effects on mitochondria injured by the ischemic insult. In this study, the effects of delphinidin 3-O-glucoside (Dp3G), cyanidin 3-O-glucoside (Cy3G) and pelargonidin 3-O-glucoside (Pg3G) on the activity of complex I of the mitochondrial respiratory chain were studied in mitochondria isolated from normal rat hearts and rat hearts subjected to ischemia for 45 min. Cy3G and Dp3G increased the activity of complex I, measured in the presence or absence of coenzyme Q1 (CoQ1 ), in ischemia-damaged mitochondria, whereas in nonischemic mitochondria the effect was observed only in the absence of CoQ1 . Dp3G and Cy3G but not Pg3G increased state 3 respiration and ATP synthesis with NADH-dependent substrates in mitochondria after ischemia. The results suggest that certain anthocyanins can act as electron acceptors at complex I, and bypass ischemia-induced inhibition, resulting in increased ATP production after ischemia. This study provides new information on a possible role of certain anthocyanins in the regulation of energy metabolism in mammalian cells.

Anthocyanins block ischemia-induced apoptosis in the perfused heart and support mitochondrial respiration potentially by reducing cytosolic cytochrome c.

Anthocynanins, found in fruits and vegetables, have a variety of protective properties, which have generally been attributed to their antioxidant capacity. However, antioxidants are generally strong reductants, and some reductants have been found to block apoptosis by reducing cytosolic cytochrome c, which prevents caspase activation. We tested the ability of various anthocyanins: to reduce cytochrome c, to support cytochrome c-induced mitochondrial respiration and to inhibit apoptosis induced by heart ischemia. Anthocyanins such as delphinidin-3-glucoside (Dp3G) and cyanidin-3-glucoside (Cy3G) were able to reduce cytochrome c directly and rapidly, whereas pelargonidin-3-glucoside (Pg3G), malvinidin-3-glucoside (Mv3G) and peonidin-3-glucoside (Pn3G) had relatively low cytochrome c reducing activities. Dp3G and Cy3G but not Pg3G supported mitochondrial state 4 respiration in the presence of exogenous cytochrome c. Pre-perfusion of hearts with 20 µM Cy3G but not Pg3G prevented ischemia-induced caspase activation. This suggests that the ability of anthocyanins to block caspase activation may be due to their ability to reduce cytosolic cytochrome c. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.