Role of mitochondrial dysfunction in combined bile acid-induced cytotoxicity: the switch between apoptosis and necrosis

The goal of this investigation was to determine whether chenodeoxycholic acid (CDCA)-induced apoptosis is prevented by ursodeoxycholic acid (UDCA) or tauroursodeoxycholic acid (TUDC) and to characterize the involvement of mitochondria in the process. Cultured human HepG2 cells were treated in a dose- and time-dependent protocol in order to establish a sufficiently low exposure to CDCA that causes apoptosis but not necrosis. Low-dose CDCA induced an S-phase block and G2 arrest of the cell cycle, as determined by flow cytometry. As a result, cell proliferation was inhibited. CDCA-induced apoptosis, as determined by fluorescence microscopy of Hoechst 33342-stained nuclei, was evident upon coincubation with TUDC. Additionally, after exposure to UDCA plus CDCA, the cell membrane was permeable to fluorescent dyes. Caspase-9-like activity, poly(ADP-ribose) polymerase (PARP) cleavage, and extensive DNA fragmentation were detected in CDCA-exposed cells and in cells coincubated with TUDC, but not UDCA. CDCA caused a decrease in mitochondrial membrane potential and depletion of ATP, both of which were potentiated by UDCA but not TUDC. The results suggest that UDCA potentiates CDCA cytotoxicity, probably at the level of induction of the mitochondrial permeability transition (MPT). Consequently, as suggested by the lack of the main hallmarks of the apoptotic pathway, in the presence of UDCA, CDCA-induced apoptosis is not properly executed but degenerates into necrosis

Impact of carvedilol on the mitochondrial damage induced by hypoxanthine and xantine oxidase: what role in myocardial ischemia and reperfusion?

OBJECTIVES: The cardioprotective effects of carvedilol (CV) may be explained in part by interactions with heart mitochondria. The objective of this work was to study the protection afforded by CV against oxidative stress induced in isolated heart mitochondria by hypoxanthine and xanthine oxidase (HX/XO), a well-known source of reactive oxygen species (ROS) in the cardiovascular system. METHODS: Mitochondria were isolated from Wistar rat hearts (n = 8) and incubated with HX/XO in the presence and in the absence of calcium. Several methods were used to assess the protection afforded by CV: evaluation of mitochondrial volume changes (by measuring changes in the optical density of the mitochondrial suspension), calcium uptake and release (with a fluorescent probe, Calcium Green 5-N) and mitochondrial respiration (with a Clark-type oxygen electrode). RESULTS: CV decreased mitochondrial damage associated with ROS production by HX and XO, as verified by the reduction of mitochondrial swelling and increase in mitochondrial calcium uptake. In the presence of HX and XO, CV also ameliorated mitochondrial respiration in the active phosphorylation state and prevented decrease in the respiratory control ratio (p < 0.05) and in mitochondrial phosphorylative efficiency (p < 0.001). CONCLUSIONS: The data indicate that CV partly protected heart mitochondria from oxidative damage induced by HX and XO, which may be useful during myocardial ischemia and reperfusion. It is also suggested that mitochondria may be a priority target for the protective action of some compounds

Impact of Carvedilol on the Mitochondrial Damage Induced by Hypoxanthine and Xantine Oxidase - What Role in Myocardial Ischemia and Reperfusion?

Objectivo: Os efeitos cardioprotectores do carvedilol (CV) poderão ser explicados, em parte, por interacções ao nível da mitocôndria cardíaca. O objectivo deste trabalho visou o estudo do efeito protector do CV em mitocôndrias cardíacas durante danos oxidativos induzidos por hipoxantina e xantina oxidase (HX/XO), uma conhecida fonte de estresse oxidativo no sistema cardiovascular. Métodos: As mitocôndrias foram isoladas a partir de corações de ratos Wistar (n=8) e incubadas com o par HX/XO, na presença e na ausência de cálcio. Vários métodos foram utilizados de modo a verificar a acção protectora do CV: avaliação das alterações de volume mitocondrial (variação da densidade óptica da suspensão mitocondrial), tomada e libertação de cálcio mitocondrial (com uma sonda fluorescente, Calcium Green-5N) e respiração mitocondrial (com um eléctrodo de oxigénio). Resultados: O CV reduziu os danos mitocondriais associados à produção de espécies reactivas de oxigénio (ERO) pelos prooxidantes, como verificado pela redução no entumescimento mitocondrial e aumento da capacidade de retenção do cálcio pela mitocôndria. O CV melhorou ainda a capacidade respiratória mitocondrial associada ao estado fosforilativo e aumentou o índice de controlo respiratório (p<0.05) e o quociente ADP/O (p<0.001) das mitocôndrias cardíacas sob estresse oxidativo induzido por HX/XO. Conclusões: Os dados indicam que o CV protegeu parcialmente a mitocôndria cardíaca de danos oxidativos induzidos por HX/XO, o que poderá ser de grande utilidade em situações de isquémiareperfusão do miocárdio. Os resultados também sugerem que a mitocôndria poderá ser um alvo prioritário para a acção benéfica de alguns fármacos cardioprotectores.Objectives: The cardioprotective effects of carvedilol (CV) may be explained in part by interactions with heart mitochondria. The objective of this work was to study the protection afforded by CV against oxidative stress induced in isolated heart mitochondria by hypoxanthine and xanthine oxidase (HX/XO), a well-known source of reactive oxygen species (ROS) in the cardiovascular system. Methods: Mitochondria were isolated from Wistar rat hearts (n=8) and incubated with HX/XO in the presence and in the absence of calcium. Several methods were used to assess the protection afforded by CV: evaluation of mitochondrial volume changes (by measuring changes in the optical density of the mitochondrial suspension), calcium uptake and release (with a fluorescent probe, Calcium Green 5-N) and mitochondrial respiration (with a Clark-type oxygen electrode). Results: CV decreased mitochondrial damage associated with ROS production by HX and XO, as verified by the reduction of mitochondrial swelling and increase in mitochondrial calcium uptake. In the presence of HX and XO, CV also ameliorated mitochondrial respiration in the active phosphorylation state and prevented decrease in the respiratory control ratio (p<0.05) and in mitochondrial phosphorylative efficiency (p<0.001). Conclusions: The data indicate that CV partly protected heart mitochondria from oxidative damage induced by HX and XO, which may be useful during myocardial ischemia and reperfusion. It is also suggested that mitochondria may be a priority target for the protective action of some compounds

Advantages in the use of carvedilol versus propranolol for the protection of cardiac mitochondrial function

BACKGROUND: Carvedilol is a neurohormonal antagonist of multiple action which is used in clinical practice for the treatment of congestive heart failure, mild to moderate hypertension and myocardial infarction. Previous results from our group have demonstrated that one of the main targets for the protective effect of carvedilol is the cardiac mitochondrial network. In-this work, we compare the effect of carvedilol with propranolol in different models of mitochondrial dysfunction and in the generation of transmembrane electric potential (EP). We further tested if carvedilol was able to inhibit the mitochondrial permeability transition (MPT) induced by doxorubicin and calcium-dependent cytochrome c release, a phenomenon frequently associated with apoptotic cell death. METHODS: Cardiac mitochondria were isolated by differential centrifugation. Oxygen consumption and mitochondrial EP were determined using an oxygen electrode and a tetraphenylphosphonium-sensitive electrode, respectively. Changes in mitochondrial volume and the release of cytochrome c were measured with spectrophotometric techniques. RESULTS: Propranolol, compared with carvedilol, had only a marginal effect, not only in protection against MPT induction, but also against oxygen consumption linked to the oxidation of external NADH, a process that is considered by several authors as key in the cardiotoxicity of doxorubicin. Regarding EP generation, propranolol had no effect, in contrast to carvedilol, which was confirmed to act as a protonophore. For the first time we also show that carvedilol inhibits the MPT induced by doxorubicin and calcium-dependent cytochrome c release. CONCLUSIONS: With this work, we further support the notion that carvedilol is effective in several models of mitochondrial dysfunction, particularly those involving oxidative stress. The results demonstrate that for some pathological conditions, carvedilol and propranolol have different mechanisms of action at the sub-cellular level, as propranolol seems to lack effectiveness in the protection of cardiac mitochondria

Vaginal sheets with Thymbra capitata essential oil for the treatment of bacterial vaginosis: design, characterization and in vitro evaluation of efficacy and safety

We aimed to incorporate Thymbra capitata essential oil (TCEO), a potent antimicrobial natural product against bacterial vaginosis (BV)-related bacteria, in a suitable drug delivery system. We used vaginal sheets as dosage form to promote immediate relief of the typical abundant vaginal discharge with unpleasant odour. Excipients were selected to promote the healthy vaginal environment reestablishment and bioadhesion of formulations, while the TCEO acts directly on BV pathogens. We characterized vaginal sheets with TCEO in regard to technological characterization, predictable in vivo performance, in vitro efficacy and safety. Vaginal sheet D.O (acid lactic buffer, gelatine, glycerine, chitosan coated with TCEO 1% w/w) presented a higher buffer capacity and ability to absorb vaginal fluid simulant (VFS) among all vaginal sheets with EO, showing one of the most promising bioadhesive profiles, an excellent flexibility and structure that allow it to be easily rolled for application. Vaginal sheet D.O with 0.32 µL/mL TCEO was able to significantly reduce the bacterial load of all in vitro tested Gardnerella species. Although vaginal sheet D.O presented toxicity at some concentrations, this product was developed for a short time period of treatment, so this toxicity can probably be limited or even reversed when the treatment ends.This work supported by the Portuguese Foundation for Science and Technology within the research project PTDC/BIA-MIC/28271/2017 under the scope of COMPETE 2020 (POCI-01- 0145-FEDER-028271) including an individual scholarship and general funding. This work was also developed within the scope of the CICS-UBI projects UIDB/00709/2020 and UIDP/00709/2020, financed by national funds through the Portuguese Foundation for Science and Technology/MCTES.info:eu-repo/semantics/publishedVersio

Inhibitory effect of carvedilol in the high-conductance state of the mitochondrial permeability transition pore

The mitochondrial permeability transition is a widely studied, but poorly understood, phenomenon in mitochondrial bioenergetics. It has been recognised that this phenomenon is related to the opening of a protein pore in the inner mitochondrial membrane, and that opening of this pore is the cause of some forms of mitochondrial dysfunction. In this work, we propose that carvedilol, a multi-role cardioprotective compound, may act as an inhibitor of the high-conductance state of the mitochondrial permeability transition pore, a conclusion supported by the finding that carvedilol provides differential protection against mitochondrial swelling in sucrose and KCl-based media, and that it is unable to protect against calcium-induced depolarisation of the mitochondrial membrane. We also show that carvedilol inhibits the oxidation of mitochondrial thiol groups and that, beyond causing a slight depression of the membrane potential, it has no inhibitory effect on mitochondrial calcium uptake.http://www.sciencedirect.com/science/article/B6T1J-4292HK0-5/1/3f9b42626ac2f0c2ab80880219b5d9c

Carvedilol: Relation Between Antioxidant Activity and Inhibition of the Mitochondrial Permeability Transition

Objectivos: A transição de permeabilidade mitocondrial (TPM) é um evento associado a estresse oxidativo severo (por exemplo, durante isquémia e reperfusão do miocárdio) e acumulação excessiva de cálcio mitocondrial, podendo mesmo levar a morte celular. Neste estudo comparou-se o efeito do Carvedilol (CV) na TPM cardíaca induzida por cálcio/fosfato (Ca/Pi) e cálcio/carboxiatractilato (Ca/Catr). Para a indução da TPM por Ca/Pi, o estresse oxidativo tem um papel importante, levando a oxidação de grupos tiólicos proteicos mitocondriais, em contraste com o efeito do Ca/Catr, onde essa oxidação é secundária à indução da TPM e não é motivada por estresse oxidativo. Materiais e métodos: As mitocôndrias foram isoladas a partir do coração de rato e avaliaram-se parâmetros relacionados com a indução da TPM (n=5 para cada indutor): entumescimento mitocondrial e oxidação dos grupos tiólicos proteicos (ambos por espectrofotometria). Resultados: Com Ca/Pi, o CV protegeu a mitocôndria da indução da TPM, nomeadamente na sua forma deletéria de alta condutância. Este efeito evidenciou-se pela diminuição do entumescimento mitocondrial. Este efeito foi simultâneo com a inibição da oxidação dos grupos tiólicos proteicos carmitocondriais (p<0.001). O CV não mostrou efeitos protectores com Ca/Catr. Conclusões: O CV protegeu a mitocôndria cardíaca da TPM, mas apenas quando a oxidação dos grupos tiólicos proteicos foi causa e não consequência da TPM. Estes resultados mostram claramente que, durante agressões ao miocárdio (durante a isquémia/reperfusão, por exemplo), o efeito protector do CV é primariamente devido a um efeito antioxidante, inibindo a produção e os efeitos das espécies reactivas de oxigénio.Objectives: The mitochondrial permeability transition (MPT) is an event related to severe oxidative stress (for example, during myocardial ischemia and reperfusion) and excessive mitochondrial calcium accumulation, also being implicated in cell death. In this study, we compared the effect of carvedilol on the cardiac MPT induced by calcium and phosphate (Ca/Pi) and calcium/carboxyatractyloside (Ca/Catr). Oxidative stress plays a major role in MPT induction by Ca/Pi, leading to the oxidation of protein thiol groups, in contrast with Ca/Catr, where such oxidation is secondary to MPT induction and is not caused by oxidative stress. Materials and methods: Mitochondria were isolated from rat hearts and parameters related to MPT induction were evaluated (n=5 for each inducer): mitochondrial swelling and oxidation of protein thiol groups (both measured by spectrophotometry). Results: Using Ca/Pi, carvedilol protected mitochondria from MPT induction, particularly in its high conductance form. Its effect was demonstrated by analyzing the decrease in mitochondrial swelling amplitude. Simultaneously, we observed inhibition of protein thiol group oxidation (p<0.001). By contrast, carvedilol did not show any protective effect with Ca/Catr. Conclusions: Carvedilol was only effective against the MPT when the oxidation of protein thiol groups was the cause and not the consequence of the MPT phenomenon. The results clearly show that during myocardial aggressions (ischemia and reperfusion, for example), the protective effect of carvedilol is primarily due to an antioxidant mechanism, inhibiting the production and effects of reactive oxygen species

Regulation of the mPTP by SIRT3-mediated deacetylation of CypD at lysine 166 suppresses age-related cardiac hypertrophy

Cardiac failure is a leading cause of age-related death, though its root cause remains unknown. Mounting evidence implicates a decline in mitochondrial function due to increased opening of the mitochondrial permeability transition pore (mPTP). Here we report that the NAD+-dependent deacetylase SIRT3 deacetylates the regulatory component of the mPTP, cyclophilin D (CypD) on lysine 166, adjacent to the binding site of cyclosporine A, a CypD inhibitor. Cardiac myocytes from mice lacking SIRT3 exhibit an age-dependent increase in mitochondrial swelling due to increased mPTP opening, a phenotype that is rescued by cyclosporine A. SIRT3 knockout mice show accelerated signs of aging in the heart including cardiac hypertrophy and fibrosis at 13 months of age. SIRT3 knockout mice are also hypersensitive to heart stress induced by transverse aortic constriction (TAC), as evidenced by cardiac hypertrophy, fibrosis, and increased mortality. Together, these data show for the first time that SIRT3 activity is necessary to prevent mitochondrial dysfunction and cardiac hypertrophy during aging and shed light on new pharmacological approaches to delaying aging and treating diseases in cardiac muscle and possibly other post-mitotic tissues

Polyphenols characterization and toxicological evaluation of pterospartum tridentatum leaf water extracts

Pterospartum tridentatum Willk. (prickled broom) is an autochthonous and common plant in Portugal. Leaves and stems are normally used in cooking, to flavour rice, roast meat or hunting animals. Leaves are also used as a condiment in fresh salads and, despite of its traditional use, no toxicological evaluation has been performed. P. tridentatum leaves aqueous extract ESI-MS spectrum revealed the presence of several luteolin and isorhamnetin derived phenolic compounds, which can be associated to the health benefits claimed for this plant species. Still, P. tridentatum leaves extract (up to 100 µg plant extract.mg-1 protein) stimulated state 4 and FCCP-stimulated liver mitochondria respiratory rates and inhibited the state 3 respiratory rate. Respiratory control ratio was diminished, indicating a decrease in phosphorylative efficiency due to inner mitochondrial membrane induced by P. tridentatum leaves extract. Nevertheless, previous results, cytotoxicity evaluation by MTT assay (50 and 125 µg plant extract) showed no significant decrease on HepG2 cell viability. Overall, the present study suggests that the consumption of P. tridentatum leaves should be regarded as safe

Lack of Additive Effects of Resveratrol and Energy Restriction in the Treatment of Hepatic Steatosis in Rats

The aims of the present study were to analyze the effect of resveratrol on liver steatosis in obese rats, to compare the effects induced by resveratrol and energy restriction and to research potential additive effects. Rats were initially fed a high-fat high-sucrose diet for six weeks and then allocated in four experimental groups fed a standard diet: a control group, a resveratrol-treated group, an energy restricted group and a group submitted to energy restriction and treated with resveratrol. We measured liver triacylglycerols, transaminases, FAS, MTP, CPT1a, CS, COX, SDH and ATP synthase activities, FATP2/FATP5, DGAT2, PPAR alpha, SIRT1, UCP2 protein expressions, ACC and AMPK phosphorylation and PGC1 alpha deacetylation. Resveratrol reduced triacylglycerols compared with the controls, although this reduction was lower than that induced by energy restriction. The mechanisms of action were different. Both decreased protein expression of fatty acid transporters, thus suggesting reduced fatty acid uptake from blood stream and liver triacylglycerol delivery, but only energy restriction reduced the assembly. These results show that resveratrol is useful for liver steatosis treatment within a balanced diet, although its effectiveness is lower than that of energy restriction. However, resveratrol is unable to increase the reduction in triacylglycerol content induced by energy restriction.The aims of the present study were to analyze the effect of resveratrol on liver steatosis in obese rats, to compare the effects induced by resveratrol and energy restriction and to research potential additive effects. Rats were initially fed a high-fat high-sucrose diet for six weeks and then allocated in four experimental groups fed a standard diet: a control group, a resveratrol-treated group, an energy restricted group and a group submitted to energy restriction and treated with resveratrol. We measured liver triacylglycerols, transaminases, FAS, MTP, CPT1a, CS, COX, SDH and ATP synthase activities, FATP2/FATP5, DGAT2, PPAR alpha, SIRT1, UCP2 protein expressions, ACC and AMPK phosphorylation and PGC1 alpha deacetylation. Resveratrol reduced triacylglycerols compared with the controls, although this reduction was lower than that induced by energy restriction. The mechanisms of action were different. Both decreased protein expression of fatty acid transporters, thus suggesting reduced fatty acid uptake from blood stream and liver triacylglycerol delivery, but only energy restriction reduced the assembly. These results show that resveratrol is useful for liver steatosis treatment within a balanced diet, although its effectiveness is lower than that of energy restriction. However, resveratrol is unable to increase the reduction in triacylglycerol content induced by energy restriction