Targeting senescence induced by age or chemotherapy with a polyphenol-rich natural extract improves longevity and healthspan in mice.

Accumulating senescent cells within tissues contribute to the progression of aging and age-related diseases. Botanical extracts, rich in phytoconstituents, present a useful resource for discovering therapies that could target senescence and thus improve healthspan. Here, we show that daily oral administration of a standardized extract of Salvia haenkei (Haenkenium (HK)) extended lifespan and healthspan of naturally aged mice. HK treatment inhibited age-induced inflammation, fibrosis and senescence markers across several tissues, as well as increased muscle strength and fur thickness compared with age-matched controls. We also found that HK treatment reduced acutely induced senescence by the chemotherapeutic agent doxorubicin, using p16LUC reporter mice. We profiled the constituent components of HK by mass spectrometry, and identified luteolin-the most concentrated flavonoid in HK-as a senomorphic compound. Mechanistically, by performing surface plasmon resonance and in situ proximity ligation assay, we found that luteolin disrupted the p16-CDK6 interaction. This work demonstrates that administration of HK promotes longevity in mice, possibly by modulating cellular senescence and by disrupting the p16-CDK6 interaction.

A multifactorial study of in situ antioxidant activity of modified GrO in myocardial reperfusion injury using the Langerdorff model.

Carbon nanomaterials possess antioxidant properties that can be applied in biomedicine and clinics for the development of new highly effective treatments against oxidative stress-induced diseases like ischemic heart disease. We previously reported the usage of graphene oxide (GrO) as a precursor for the elaboration of such prototypes. The promising findings led to the development of two new modifications of GrO: nitrogen-doped (N-GrO) and l-cysteine functionalized (S-GrO) derivatives as possible antioxidant agents in ischemia-reperfusion (I/R) conditions. In this study, the cardioprotective and antioxidant potential of modified GrO as a pre-treatment in rats was evaluated for the first time. In Langendorff isolated rat heart I/R model, the left ventricle developed pressure (LVDP), the end-diastolic pressure (EDP), the maximal (dP/dtmax) and minimal (dP/dtmin) value of the first derivative of LVDP, and heart rate (HR) were measured. The oxidative-nitrosative markers, in particular, the rate of O2*- and H2O2 generation, the content of malonic dialdehyde, diene conjugates, and leukotriene as well as cNOS and iNOS activity were estimated. Obtained results show a significant restoration of cadiodynamic parameters at the reperfusion period. Simultaneously, all samples significantly reduced the rate of reactive oxygen species (ROS) and lipid peroxidation markers in cardiac homogenates and preserved cNOS activity at the preischemic level. This evidence makes GrO derivatives promising candidates for the correction of reperfusion disorders affecting myocardial function.

Upregulation of ATP-Sensitive Potassium Channels as the Potential Mechanism of Cardioprotection and Vasorelaxation Under the Action of Pyridoxal-5-Phosphate in Old Rats.

Background: The aging process is accompanied by the weakening of the protective systems of the organism, in particular by the decrease in the expression of ATP-sensitive potassium (KATP) channels and in the synthesis of H2S. The aim of our work was to investigate the role of KATP channels in the cardioprotection induced by pyridoxal-5-phosphate (PLP) in aging. Methods: Experiments were performed on adult and old (aged 24 months) male Wistar rats, which were divided into 3 groups: adults, old, and old PLP-treated rats. PLP was administered orally once a day for 14 days at a dose of 0.7 mg/kg. The levels of mRNA expression of subunits KATP channels were determined by reverse transcription and real-time polymerase chain reaction analysis. Protein expression levels were determined by the Western blot. Cardiac tissue morphology was determined using transverse 6 µm deparaffinized sections stained with picrosirius red staining. Vasorelaxation responses of isolated aortic rings and the function of Langendorff-perfused isolated hearts during ischemia-reperfusion, H2S levels, and markers of oxidative stress were also studied. Results: Administration of PLP to old rats reduces cardiac fibrosis and improves cardiac function during ischemia-reperfusion and vasorelaxation responses to KATP channels opening. At the same time, there was a significant increase in mRNA and protein expression of SUR2 and Kir6.1 subunits of KATP channels, H2S production, and reduced markers of oxidative stress. The specific KATP channel inhibitor-glibenclamide prevented the enhancement of vasodilator responses and anti-ischemic protection in PLP-treated animals. Conclusions: We suggest that this potential therapeutic effect of PLP in old animals may be a result of increased expression of KATP channels and H2S production.

Glutathione Upregulates the Expression of KATP Channels and Vasorelaxation Responses and Inhibits mPTP Opening and Oxidative Stress in the Heart Mitochondria of Old Rats.

Background: In the present work, we investigated the effect of exogenous glutathione in old rats on the expression of ATP-sensitive potassium (KATP) channels, the mitochondrial permeability transition pore (mPTP) opening in the heart, and the vasorelaxation responses of isolated aortic rings to activation of KATP channels. Methods: Experiments were performed on adult (6 months) and old (24 months) male Wistar rats, which were divided into three groups: adult, old, and glutathione-treated old rats. Glutathione was injected intraperitoneally at a dose of 52 mg/kg 1 hour before the studies. The mRNA expression of KATP channels was determined using reverse transcription and real-time polymerase chain reaction analysis. The effect of glutathione administration on mPTP opening, relaxation responses of isolated aortic rings, and oxidative stress markers was studied. Results: It was shown that the expression levels of Kir6.1, Kir6.2, and SUR1 subunits of KATP channels and levels of reduced glutathione were significantly increased in glutathione-treated old rats (by 8.3, 2.8, 13.1, and 1.5-fold, respectively), whereas the levels of oxidative stress markers (hydrogen peroxide, diene conjugates, malondialdehyde, and rate of superoxide generation) in heart mitochondria and mPTP opening were significantly reduced. Relaxation of aortic rings was significantly increased in response to the actions of KATP channel openers flocalin and pinacidil in glutathione-treated animals, which was prevented by glibenclamide. Conclusions: Thus, the administration of exogenous glutathione to old rats resulted in a significant increase in the expression levels of the Kir6.1, Kir6.2, and SUR1 subunits of KATP channels and a decrease in oxidative stress. This was accompanied by inhibition of mPTP opening and enhancement of vasorelaxation responses to activation of KATP channels.

Exercise restores endogenous H2 S synthesis and mitochondrial function in the heart of old rats.

BACKGROUND: Ageing is accompanied by a decrease in endogenous hydrogen sulphide (H2 S) synthesis and the development of mitochondrial dysfunction. The aim of our work was to study the possible participation of exercise training-induced regulation of endogenous H2 S production in the restoration of mitochondrial function in old rats. MATERIALS AND METHODS: Male rats were divided into three groups: adult, old and exercise-trained old. Exercise training of old rats was performed for 4 weeks. The mRNA expression cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) were determined using reverse transcription and real-time polymerase chain reaction analysis. Mitochondrial dysfunction was determined by mPTP opening, which was investigated by spectrophotometric registration of the swelling of mitochondria isolated from the rat heart. We also studied the effect of exercise on H2 S content, oxidative stress and mtNOS activity. RESULTS: Exercise training in old animals significantly increased the expression of H2 S-synthesizing enzymes CSE and 3-MST and restored endogenous H2 S production in cardiac tissue and cardiac mitochondria to levels of adult animals. In addition, the training significantly reduced oxidative stress in old rats, in particular the rate of formation of •O2 - and H2 O2 , diene conjugates and malondialdehyde levels in the mitochondria of the heart. Simultaneously, in the hearts of these animals, resistance of mPTP to the inducer of its opening of calcium ions was increased. CONCLUSIONS: Thus, exercise training restores endogenous H2 S production, and significantly reduces oxidative stress in cardiac mitochondria of old rats that are associated with the inhibition of calcium-induced mPTP opening as an indicator of mitochondrial dysfunction.

Graphene oxide nanoflakes prevent reperfusion injury of Langendorff isolated rat heart providing antioxidative activity in situ.

Carbon materials possess powerful antioxidant activity that might be promising for the development of new generation treatment of cardiovascular diseases, ischemic conditions, and reperfusion injury. The present study aimed to characterize the structure of nanosized graphene oxide (GrO) sample and evaluate the antioxidant efficacy of GrO in situ models of oxidative stress widely used in pre-clinical studies. The structure and surface chemistry of the initial samples were analyzed via LDS, RAMAN, LDI, TPD-MS, and FTIR methods. The GrO showed a strong ability to scavenge DPPH, hydroxyl, and superoxide anion free radicals and have a total antioxidant capacity. The DFT quantum-chemical calculation demonstrated the radical scavenging effect of GrO proceeding due to the physical adsorption of the free radical on the surface. For evaluation of the antioxidant effect of GrO in situ, we used the model of ischemia-reperfusion (I/R) of Langendorff isolated rat heart. We revealed that intravenous pretreatment of Wistar male rats with GrO significantly increased resistance of myocardium to I/R, improved restoration of heart function, prevented non-effective oxygen utilization, and I/R-induced reactive oxygen species production in cardiac tissue. Thus, our data demonstrate the perspective of further use of GrO for the development of antiischemic therapy.

Stimulation of mitochondrial hydrogen sulfide and glutathione production improves the Frank-Starling response of the rat heart via a nitric oxide-dependent pathway.

The Frank-Starling response of the heart is known to be mediated by nitric oxide (NO) signaling, which is regulated by reduced glutathione (GSH) and hydrogen sulfide (H2S). We hypothesized that stimulation of endogenous H2S or GSH synthesis would improve the Frank-Starling response. Wistar male rats were injected with propargylglycine (PAG; 11.3 mg/kg, 40 min, n = 12), an inhibitor of H2S-producing enzyme (cystationine-γ-lyase), and l-cysteine (121 mg/kg, 30 min, n = 20), a precursor of H2S and GSH. Pretreatment with PAG or l-cysteine separately slightly improved the pressure-volume (P-V) dependence of the isolated rat heart, but the combination of PAG and l-cysteine (n = 12) improved heart contractile activity. H2S content, Ca2+-dependent NOS activity (cNOS) activity, nitrate reductase activity, and nitrite content increased by 2, 3.83, 2.5, and 1.3 times in cardiac mitochondria, and GSH and oxidized glutathione (GSSG) levels increased by 2.24 and 1.86 times in the heart homogenates of the PAG + l-cysteine group compared with the control (all P < 0.05). Inhibition of glutathione with DL-buthionine-sulfoximine (BSO; 22.2 mg/kg, 40 min, n = 6) drastically decreased Frank-Starling response of the heart and prevented PAG + l-cysteine-induced increase of GSH and GSSG levels (BSO + PAG + l-cysteine, n = 9). Inhibition of NOS, N-nitro-l-arginine-methylester hydrochloride (l-NAME; 40 min, 27 mg/kg) abolished positive inotropy induced by PAG+l-cysteine pretreatment (l-NAME + PAG + l-cysteine, n = 7). Thus, PAG + l-cysteine administration improves the Frank-Starling response by upregulating mitochondrial H2S, glutathione, and NO synthesis, which may be a promising approach in the treatment of myocardial dysfunction.

Pyridoxal-5-phosphate induced cardioprotection in aging associated with up-expression of cystathionine-γ-lyase, 3-mercaptopyruvate sulfurtransferase, and ATP-sensitive potassium channels.

BACKGROUND: In the present work, we investigated the cardioprotective potential of pyridoxal-5-phosphate (PLP) in old rats as a cofactor of enzymes that synthesize hydrogen sulphide (H2 S). MATERIALS AND METHODS: PLP was administered per os in a dose of 0.7 mg per kg daily for 2 weeks. Rats were divided into three groups (adult, old and old +PLP) of 20 animals. The cardiac mRNA levels of genes encoding H2 S-synthesizing enzymes cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), uncoupling proteins (UCP3), subunits of ATP-sensitive potassium (KATP ) channels were determined using real-time polymerase chain reaction analysis. We also studied the effect of PLP-administration on the content of H2 S, oxidative stress, the activities of inducible and constitutive NO-synthase (iNOS, cNOS), arginase and nitrate reductase in the heart homogenates as well as cardiac resistance to ischemia-reperfusion in Langendorff-isolated heart model. RESULTS: It was shown that PLP restored mRNA levels of CSE, 3-MST and UCP3 genes, and H2 S content and also significantly increased the expression of SUR2 and Kir6.1 (2.2 and 3.3 times, respectively) in the heart of old rats. PLP significantly reduced the formation of superoxide, malondialdehyde, diene conjugates as well as the activity of iNOS and arginase. PLP significantly increased constitutive synthesis of NO and prevented reperfusion disturbances of the heart function after ischemia. CONCLUSIONS: Thus, PLP-administration in old rats was associated with up-expression of CSE, 3-MST, UCP3 and SUR2 and Kir6.1 subunits of KATP channels, and also increased cNOS activity and reduced oxidative stress and prevented reperfusion dysfunction of the heart in ischemia-reperfusion.

Glutathione restores the mitochondrial redox status and improves the function of the cardiovascular system in old rats.

Introduction: Aging is accompanied by cardiovascular disorders which is associated with an imbalance of pro- and antioxidant systems, the mitochondrial dysfunction, etc. Glutathione (GSH) plays a critical role in protecting cells from oxidative damage. The aim of the work was to study the effect of exogenous glutathione on the redox status of mitochondria, the content of H2S and the function of the cardiovascular system in old rats. Methods: Experiments were performed on adult (6 months) and old (24 months) Wistar rats divided into three groups: adult, old and glutathionetreated old rats. Glutathione was injected intraperitoneally at a dose of 52 mg/kg. We investigated glutathione redox balance, H2S levels, oxidative stress, the opening of the mitochondrial permeability transition pore (mPTP), the resistance of isolated heart to ischemia/reperfusion in Langendorff model, endothelium-dependent vasorelaxation of isolated aortic rings, and cardiac levels of 3-MST, CSE, and UCP3 mRNA were determined using real-time PCR analysis. Results: Our data shows that in old rats treated with glutathione, the balance of its oxidized and reduced form changes in the direction of a significant increase (by 53.6%) of the reduced form. Glutathione pretreatment significantly increased the H2S levels, mtNOS activity, and UCP3 expression which considered as protective protein, and conversely, significantly decreased oxidative stress markers (the rate of O2•- generation, the levels of H2O2, diene conjugates and malone dialdehyde, in 2.5, 2.3, 2, and 1.6 times, respectively) in heart mitochondria. This was associated with the inhibition mitochondrial permeability transition pore opening and increased resistance of the isolated heart to ischemia/reperfusion in these animals. At the same time, in glutathione-treated old rats, we also observed restoration of endothelium-dependent vasorelaxation responses to acetylcholine, which were almost completely abolished by the NO-synthase inhibitor L-NAME. Conclusion: Thus, the pretreatment of old rats with glutathione restores the mitochondrial redox status and improves the function of the cardiovascular system.

Induction of Glutathione Synthesis Provides Cardioprotection Regulating NO, AMPK and PPARa Signaling in Ischemic Rat Hearts.

Glutathione (GSH) is essential for antioxidant defence, and its depletion is associated with tissue damage during cardiac ischemia-reperfusion (I/R). GSH is synthesized by the glutamate-cysteine ligase enzyme (GCL) from L-cysteine, which alternatively might be used for hydrogen sulfide production by cystathionine-gamma-lyase (CSE). Here, we have investigated whether in vivo treatment with L-cysteine and an inhibitor of CSE,D,L-propargylglycine (PAG), can modulate cardiac glutathione and whether this treatment can influence heart resistance to I/R in a Langendorff isolated rat hearts model. Pretreatment with PAG + L-cysteine manifested in pronounced cardioprotection, as there was complete recovery of contractile function; preserved constitutive NOS activity; and limited the production of reactive oxygen and nitrogen species in the ischemized myocardium. Cardiac GSH and GSSG levels were increased by 3.5- and 2.1-fold in PAG + L-cysteine hearts and were 3.3- and 3.6-fold higher in PAG + L-cysteine + I/R compared to I/R heart. The cardioprotective effect of PAG + L-cysteine was completely abolished by an inhibitor of GCL, DL-buthionine-(S,R)-sulfoximine. Further analysis indicated diminished fatty acid ß-oxidation, increased glucose consumption and anaerobic glycolysis, and promoted OXPHOS proteins and SERCA2 in PAG + L-cysteine + I/R compared to the I/R group. PAG + L-cysteine inhibited PPARα and up-regulated AMPK signalling in the heart. Thus, induction of glutathione synthesis provided cardioprotection regulating NO, AMPK and PPARa signaling in ischemic rat hearts.