Mito-tempo alleviates ox-LDL-provoked foam cell formation by regulating Nrf2/NLRP3 signaling.

Our previous studies have demonstrated that Mito-Tempol (also known as 4-hydroxy-Tempo), a mitochondrial reactive oxygen species (ROS) scavenger, alleviates oxidized low-density lipoprotein (ox-LDL)-triggered foam cell formation. Given the effect of oxidative stress on activating the NOD-, LRR-, and pyrin domain-containing 3 (NLRP3) inflammasome, which promotes foam cell formation, we aimed to explore whether Mito-Tempo inhibits ox-LDL-triggered foam cell formation by regulating NLRP3 inflammasome. The results revealed that Mito-Tempo re-activated Nrf2 and alleviated macrophage foam cell formation induced by ox-LDL, whereas the effects were reversed by ML385 (a specific Nrf2 inhibitor). Mito-Tempo restored the expression and nuclear translocation of Nrf2 by decreasing ox-LDL-induced ubiquitination. Furthermore, Mito-Tempo suppressed ox-LDL-triggered NLRP3 inflammasome activation and subsequent pyroptosis, whereas the changes were blocked by ML385. Mito-Tempo decreased lipoprotein uptake by inhibiting CD36 expression and suppressed foam cell formation by regulating the NLRP3 inflammasome. Taken together, Mito-Tempo exhibits potent anti-atherosclerotic effects by regulating Nrf2/NLRP3 signaling.

The evaluation effect of nanoliposome-loaded Mito-Tempo on sperm parameters during human sperm cryopreservation.

AIM: The aim of this study is the evaluation effect of nanoliposome-loaded Mito-Tempo on sperm parameters during human sperm cryopreservation. METHODS: Semen samples of 50 Asthenoteratozoospermia men (random) were collected. Sperm parameters were analyzed based on World Health Organization (WHO, 2010) criteria (2021) and each sample was divided into 5 groups (E1-E5). E1 (control group): the sperm was cryopreserved without nanoliposome, and Mito-Tempo. E2: sperm cryopreservation with Mito-Tempo-loaded nanoliposome (Mito-Tempo 0.1 mM) + freezing medium. E3: sperm cryopreservation with Mito-Tempo-loaded nanoliposome (Mito-Tempo 0.2 mM) + freezing medium. E4: in this group, the cryopreservation sperm with Mito-Tempo 0.3 mM + freezing medium. E5: the cryopreservation sperm with Mito-Tempo 0.2 mM + freezing medium. RESULTS: The result of this study indicated that sperm parameters and total antioxidant capacity (TAC) significantly increase in E3 and E4 groups, compared to E1, E2, and E5 groups respectively (P < 0.05). The percentage of abnormal morphology, DNA fragmentation index (DFI), malondialdehyde (MDA), and the levels of ROS significantly decrease in E3 and E4 groups, compared to E1, E2, and E5 groups (P < 0.05). In addition, the sperm parameters and stress oxidative factors significantly improve in E3 group compared to other groups (P < 0.05). CONCLUSIONS: In conclusion, the combination of Mito-Tempo with nanoliposome due to its ability to cooperate with lipid layers may lead to significant performance in reducing oxidative stress damage and increasing the quality of sperm parameters.

Mitofilin in cardiovascular diseases: Insights into the pathogenesis and potential pharmacological interventions.

The impact of mitochondrial dysfunction on the pathogenesis of cardiovascular disease is increasing. However, the precise underlying mechanism remains unclear. Mitochondria produce cellular energy through oxidative phosphorylation while regulating calcium homeostasis, cellular respiration, and the production of biosynthetic chemicals. Nevertheless, problems related to cardiac energy metabolism, defective mitochondrial proteins, mitophagy, and structural changes in mitochondrial membranes can cause cardiovascular diseases via mitochondrial dysfunction. Mitofilin is a critical inner mitochondrial membrane protein that maintains cristae structure and facilitates protein transport while linking the inner mitochondrial membrane, outer mitochondrial membrane, and mitochondrial DNA transcription. Researchers believe that mitofilin may be a therapeutic target for treating cardiovascular diseases, particularly cardiac mitochondrial dysfunctions. In this review, we highlight current findings regarding the role of mitofilin in the pathogenesis of cardiovascular diseases and potential therapeutic compounds targeting mitofilin.

The effect of the mitochondria-targeted antioxidant Mito-tempo during sperm ultra-rapid freezing.

During the freeze-thaw process, human spermatozoa are susceptible to oxidative stress, which may cause cryodamage and reduce sperm quality. As a novel mitochondria-targeted antioxidant, Mito-tempo has been used for sperm cryopreservation. However, it is currently unknown what role it will play in the process of sperm ultra-rapid freezing. The purpose of this study was to investigate whether Mito-tempo can improve sperm quality during ultra-rapid freezing. In this study, samples with the addition of Mito-tempo (0, 5, 10, 20, and 40 µM) to sperm freezing medium were selected to evaluate the changes in sperm quality, antioxidant capacity and ultrastructure after ultra-rapid freezing. After ultra-rapid freezing, the quality and antioxidant function of the spermatozoa were significantly reduced and the spermatozoa ultrastructure was destroyed. The addition of 10 µM Mito-tempo significantly increased post thaw sperm motility, viability, plasma membrane integrity and mitochondrial membrane potential (P < 0.05). Moreover, the DNA fragmentation index (DFI), ROS levels and MDA content were reduced, and the antioxidant enzyme (CAT and SOD) activities were enhanced in the 10 µM Mito-tempo group (P < 0.05). Moreover, Mito-tempo protected sperm ultrastructure from damage. In conclusion, Mito-tempo improved the quality and antioxidant function of sperm after ultra-rapid freezing while reducing freezing-induced ultrastructural damage.

Mito-TEMPO Improves the Meiosis Resumption and Mitochondrial Function of Vitrified Sheep Oocytes via the Recovery of Respiratory Chain Activity.

Vitrification is a crucial method for preserving animal germ cells. Considering the increased oxidative stress and organelle damage incurred, it is still necessary to make the process more efficient for oocytes. As the energy source of oocytes, mitochondria are the most abundant organelle in oocytes and play a crucial role in their maturation. Here, we found that Mito-TEMPO, a mitochondria-targeted antioxidant, could efficaciously improve the oxidative stress injury of vitrified oocytes by recovering mitochondrial function via the mitochondrial respiratory chain. It was observed that Mito-TEMPO not only improves oocyte viability and meiosis but also maintains spindle structure. A subsequent study indicated that Mito-TEMPO effectively rescued mitochondrial dysfunction and attenuated vitrification-induced oxidative stress. Further investigation revealed that Mito-TEMPO regulates vitrified oocytes' intracellular Ca2+ homeostasis and ATP content and provides strong antioxidant properties. Additionally, an analysis of the transcriptome at the single-cell level revealed that the respiratory chain mediates the beneficial effect of Mito-TEMPO on vitrified oocytes. Overall, our findings indicate that supplementing oocytes with Mito-TEMPO is an effective method to shield them from the damage caused by vitrification. In addition, the beneficial effects of Mito-TEMPO on vitrified sheep oocytes could inspire further investigations of the principles underlying oocyte cryobiology in other animals.

Arsenic-Induced Ferroptosis in Chicken Hepatocytes via the Mitochondrial ROS Pathway.

Arsenic has been shown to be highly toxic and can cause liver damage. Previous studies have shown that arsenic causes severe liver damage and induces accumulation of reactive oxygen species (ROS). This study aimed to investigate the effects of ferroptosis on the liver in arsenic trioxide (ATO) and to explore the underlying mechanisms. We confirmed the hepatotoxic effects of arsenic by in vivo and in vitro experiments. After 28 days of administration of arsenic trioxide (4-mg/kg, 8-mg/kg) by gavage, chickens exhibited body weight loss and liver damage in a dose-dependent manner. In addition, in vivo and in vitro western blot and real-time fluorescence quantitative PCR analyses simultaneously indicated that ferroptosis might be the main pathway of arsenic-induced liver injury. Finally, Mito-TEMPO effectively eliminated the ROS accumulation in mitochondria, significantly attenuating the process of cellular ferroptosis. In summary, the hepatotoxic effects of arsenic are related to ferroptosis, and the hepatic ferroptosis process of arsenic is regulated by mitochondrial ROS (MtROS). Our study reveals new mechanisms of arsenic toxicity to the liver, which may deepen our understanding of arsenic toxicology.

Neurovascular dysfunction associated with erectile dysfunction persists after long-term recovery from simulations of weightlessness and deep space irradiation.

There has been growing interest within the space industry for long-duration manned expeditions to the Moon and Mars. During deep space missions, astronauts are exposed to high levels of galactic cosmic radiation (GCR) and microgravity which are associated with increased risk of oxidative stress and endothelial dysfunction. Oxidative stress and endothelial dysfunction are causative factors in the pathogenesis of erectile dysfunction, although the effects of spaceflight on erectile function have been unexplored. Therefore, the purpose of this study was to investigate the effects of simulated spaceflight and long-term recovery on tissues critical for erectile function, the distal internal pudendal artery (dIPA), and the corpus cavernosum (CC). Eighty-six adult male Fisher-344 rats were randomized into six groups and exposed to 4-weeks of hindlimb unloading (HLU) or weight-bearing control, and sham (0Gy), 0.75 Gy, or 1.5 Gy of simulated GCR at the ground-based GCR simulator at the NASA Space Radiation Laboratory. Following a 12-13-month recovery, ex vivo physiological analysis of the dIPA and CC tissue segments revealed differential impacts of HLU and GCR on endothelium-dependent and -independent relaxation that was tissue type specific. GCR impaired non-adrenergic non-cholinergic (NANC) nerve-mediated relaxation in the dIPA and CC, while follow-up experiments of the CC showed restoration of NANC-mediated relaxation of GCR tissues following acute incubation with the antioxidants mito-TEMPO and TEMPOL, as well as inhibitors of xanthine oxidase and arginase. These findings indicate that simulated spaceflight exerts a long-term impairment of neurovascular erectile function, which exposes a new health risk to consider with deep space exploration.

Mito-Tempo improves acrosome integrity of frozen-thawed epididymal spermatozoa in tomcats.

Introduction: In tomcats, epididymal spermatozoa provide an additional source of male gametes available for cryopreservation. While this procedure is feasible, the survival rate and motility of epididymal cat spermatozoa are both low after thawing. Cryopreservation is known to induce oxidative stress in spermatozoa, with mitochondria and the plasma membrane being the two major generation sites, and an imbalanced presence of free radicals is a possible cause for this low survival rate. Different antioxidants have been tested before for their effect on cryopreserved cat spermatozoa quality, with varying results. Here, we used Mito-Tempo, which is a synthetic mitochondria-targeted antioxidant and a specific scavenger of the mitochondrial superoxide system. By supplementing Mito-Tempo with the freezing extender, we aimed to improve the sperm quality of frozen-thawed cat epididymal spermatozoa. Methods: Epididymal spermatozoa obtained from twelve tomcats were assessed for motility and concentration. Prior to freezing, samples were diluted in TRIS buffered extender with egg yolk and glycerol and divided into five aliquots supplemented with 0 (control), 0.5, 5, 50, and 1005M of Mito-Tempo. After thawing, sperm motility, concentration, morphology, plasma membrane integrity, acrosome integrity, and mitochondrial membrane potential were evaluated. A Friedman rank sum test with a Bonferroni post-hoc test was used to determine statistical in-between group differences in post-thaw semen parameters. Results and discussion: The results indicated a slight improvement in acrosome integrity across all groups that were supplemented with Mito-Tempo, with the group that received 55M of Mito-Tempo showing the greatest improvement [(median of 67.99%, IQR of 5.55) compared to the control group (median of 65.33%, IQR of 7.75; P = 0.05)]. For all other sperm parameters, no significant differences (P > 0.05) were detected between different Mito-Tempo concentrations. These findings highlight the protective effect of Mito-Tempo on acrosome integrity and suggest that 55M is the most effective concentration for maintaining acrosome integrity. Since Mito-Tempo has shown a positive effect on multiple sperm parameters in other species, such as men, boars, roosters, rams, and bulls, we need to conclude that species-specificity may play a role here.

Mito-TEMPO protects preimplantation porcine embryos against mitochondrial fission-driven apoptosis through DRP1/PINK1-mediated mitophagy.

AIMS: Mdivi-1 (Md-1) is a well-known inhibitor of mitochondrial fission and mitophagy. The mitochondrial superoxide scavenger Mito-TEMPO (MT) exerts positive effects on the developmental competence of pig embryos. This study aimed to explore the adverse effects of Md-1 on developmental capacity in porcine embryos and the protective effects of MT against Md-1-induced injury. MAIN METHODS: We exposed porcine embryos to Md-1 (10 and 50 µM) for 2 days after in vitro fertilization (IVF). MT (0.1 µM) treatment was applied for 4 days after exposing embryos to Md-1. We assessed blastocyst development, DNA damage, mitochondrial superoxide production, and mitochondrial distribution using TUNEL assay, Mito-SOX, and Mito-tracker, respectively. Subsequently, the expression of PINK1, DRP1, and p-DRP1Ser616 was evaluated via immunofluorescence staining and Western blot analysis. KEY FINDINGS: Md-1 compromised the developmental competence of blastocysts. Apoptosis and mitochondrial superoxide production were significantly upregulated in 50 µM Md-1-treated embryos, accompanied by a downregulation of p-DRP1Ser616, PINK1, and LC3B levels and lower mitophagy activity at the blastocyst stage. We confirmed the protective effects of MT against the detrimental effect of Md-1 on blastocyst developmental competence, mitochondrial fission, and DRP1/PINK1-mediated mitophagy activation. Eventually, MT recovered DRP1/PINK1-mediated mitophagy and mitochondrial fission by inhibiting superoxide production in Md-1-treated embryos. SIGNIFICANCE: MT protects against detrimental effects of Md-1 on porcine embryos by suppressing superoxide production. These findings expand available scientific knowledge on improving outcomes of IVF.

Necroptosis signaling and NLRP3 inflammasome cross-talking in epithelium facilitate Pseudomonas aeruginosa mediated lung injury.

Pseudomonas aeruginosa induced acute lung injury is such a serious risk to public health, but the pathological regulation remains unclear. Here, we reported that PA mediated epithelial necroptosis plays an important role in pathological process. Pharmacological and genomic ablation of necroptosis signaling ameliorate PA mediated ALI and pulmonary inflammation. Our results further proved NLRP3 inflammasome to involve in the process. Mechanism investigation revealed the cross-talking between inflammasome activation and necroptosis that MLKL-dependent necroptosis signaling promotes the change of mitochondrial membrane potential for the release of reactive oxygen species (ROS), which is the important trigger for functional inflammasome activation. Furthermore, antioxidants such as Mito-TEMPO was confirmed to significantly restrain inflammasome activation in epithelium, resulting in a reduction in PA induced pulmonary inflammation. Taken together, our findings revealed that necroptosis-triggered NLRP3 inflammasome in epithelium plays a crucial role in PA mediated injury, which could be a potential therapeutic target for pulmonary inflammation.

Flow cytometry study of post-thawed buck spermatozoa: Mito-TEMPO improves cryopreservation performance by controlling apoptosis rate, DNA fragmentation and ROS production.

Sperm cryopreservation is used to spread qualified semen for artificial insemination, but the freezing process reduces sperm quality. This study assessed the efficacy of Mito-TEMPO on post-thawed goat sperm quality. Semen samples divided to five equal groups and after dilution, received different doses of Mito-TEMPO (0, 1, 10, 100 and 1000 µM), and cryopreserved in liquid nitrogen. After thawing, flow cytometry analysis was performed to evaluate sperm mitochondria membrane potential, viability, apoptotic-like changes, DNA fragmentation and ROS concentration. According to the results, Mito-TEMPO (10 and 100 µM) improved (P ≤ 0.05) sperm viability and decreased (P ≤ 0.05) apoptotic-like changes and ROS concentration compared to the other groups. Mitochondria membrane potential was higher (P ≤ 0.05) in groups received 1, 10 and 100 µM Mito-TEMPO. The lowest (P ≤ 0.05) DNA fragmentation was observed in group received 10 µM Mito-TEMPO. In conclusion, mitochondria-targeted antioxidant Mito-TEMPO could be an efficient cryo-additive to enhance flowcytometric quality parameters of post-thawed buck semen.

Mito-TEMPO, a Mitochondria-Targeted Antioxidant, Improves Cognitive Dysfunction due to Hypoglycemia: an Association with Reduced Pericyte Loss and Blood-Brain Barrier Leakage.

Hypoglycemia is associated with cognitive dysfunction, but the exact mechanisms have not been elucidated. Our previous study found that severe hypoglycemia could lead to cognitive dysfunction in a type 1 diabetes (T1D) mouse model. Thus, the aim of this study was to further investigate whether the mechanism of severe hypoglycemia leading to cognitive dysfunction is related to oxidative stress-mediated pericyte loss and blood-brain barrier (BBB) leakage. A streptozotocin T1D model (150 mg/kg, one-time intraperitoneal injection), using male C57BL/6J mice, was used to induce hypoglycemia. Brain tissue was extracted to examine for neuronal damage, permeability of BBB was investigated through Evans blue staining and electron microscopy, reactive oxygen species and adenosine triphosphate in brain tissue were assayed, and the functional changes of pericytes were determined. Cognitive function was tested using Morris water maze. Also, an in vitro glucose deprivation model was constructed. The results showed that BBB leakage after hypoglycemia is associated with excessive activation of oxidative stress and mitochondrial dysfunction due to glucose deprivation/reperfusion. Interventions using the mitochondria-targeted antioxidant Mito-TEMPO in both in vivo and in vitro models reduced mitochondrial oxidative stress, decreased pericyte loss and apoptosis, and attenuated BBB leakage and neuronal damage, ultimately leading to improved cognitive function.

ROS scavengers decrease γH2ax spots in motor neuronal nuclei of ALS model mice in vitro.

Background: Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by the loss of motor neurons in cerebral cortex, brainstem and spinal cord. Numerous studies have demonstrated signs of oxidative stress in postmortem neuronal tissue, cerebrospinal fluid, plasma and urine of ALS patients, without focusing on the specific processes within motor neurons. Thus, we aimed to investigate the relevance of reactive oxygen species (ROS) detoxification mechanisms and its consequences on the formation of toxic/lethal DNA double strand breaks (DSBs) in the ALS model of the Wobbler mouse. Methods: Live cell imaging in dissociated motor neuronal cultures was used to investigate the production of ROS using Dihydroethidium (DHE). The expression levels of ROS detoxifying molecules were investigated by qPCR as well as Western blots. Furthermore, the expression levels of DNA damage response proteins p53bp1 and H2ax were investigated using qPCR and immunofluorescence staining. Proof-of-principle experiments using ROS scavengers were performed in vitro to decipher the influence of ROS on the formation of DNA double strand breaks quantifying the γH2ax spots formation. Results: Here, we verified an elevated ROS-level in spinal motor neurons of symptomatic Wobbler mice in vitro. As a result, an increased number of DNA damage response proteins p53bp1 and γH2ax in dissociated motor neurons of the spinal cord of Wobbler mice was observed. Furthermore, we found a significantly altered expression of several antioxidant molecules in the spinal cord of Wobbler mice, suggesting a deficit in ROS detoxification mechanisms. This hypothesis could be verified by using ROS scavenger molecules in vitro to reduce the number of γH2ax foci in dissociated motor neurons and thus counteract the harmful effects of ROS. Conclusion: Our data indicate that maintenance of redox homeostasis may play a key role in the therapy of the neurodegenerative disease ALS. Our results underline a necessity for multimodal treatment approaches to prolong the average lifespan of motor neurons and thus slow down the progression of the disease, since a focused intervention in one pathomechanism seems to be insufficient in ALS therapy.

Inhibition of mitochondrial superoxide promotes the development of hiPS-CMs during differentiation.

The redox state is a crucial determinant of the maturation transition of cardiomyocytes in vivo. Mitochondria, the primary site of superoxide generation, are very sensitive to various stimulations, including oxygen and nutrient supply. How mitochondrial superoxide affects the differentiation and development of induced pluripotent stem cell (iPSC)-derived cardiac myocytes (iPS-CMs) is not completely clear. To address the questions, we monitored the superoxide level during the differentiation and development of human iPS-CMs using MitoSOX. Mitochondria-targeted antioxidant Mito-TEMPO was used to treat hiPS-CMs in the differentiation period. We found that mitochondrial superoxide generation was dramatically enhanced during the differentiation and early development of iPS-CMs. Increased oxidative stress induced oxidative damage to macromolecules in iPS-CMs, such as lipids, proteins, and DNA. Mito-TEMPO protected mitochondrial functions, alleviated oxidative damage to lipids, proteins, and DNA and improved cellular structure and fatty acid utilization. Our findings confirmed that iPS-CM suffered from oxidative stress during differentiation and that mitochondrial-targeted antioxidant is beneficial for the maturation of iPS-CMs.

Targeting glutamine utilization to block metabolic adaptation of tumor cells under the stress of carboxyamidotriazole-induced nutrients unavailability.

Tumor cells have unique metabolic programming that is biologically distinct from that of corresponding normal cells. Resetting tumor metabolic programming is a promising strategy to ameliorate drug resistance and improve the tumor microenvironment. Here, we show that carboxyamidotriazole (CAI), an anticancer drug, can function as a metabolic modulator that decreases glucose and lipid metabolism and increases the dependency of colon cancer cells on glutamine metabolism. CAI suppressed glucose and lipid metabolism utilization, causing inhibition of mitochondrial respiratory chain complex I, thus producing reactive oxygen species (ROS). In parallel, activation of the aryl hydrocarbon receptor (AhR) increased glutamine uptake via the transporter SLC1A5, which could activate the ROS-scavenging enzyme glutathione peroxidase. As a result, combined use of inhibitors of GLS/GDH1, CAI could effectively restrict colorectal cancer (CRC) energy metabolism. These data illuminate a new antitumor mechanism of CAI, suggesting a new strategy for CRC metabolic reprogramming treatment.

Supplementation of Mito TEMPO and acetovanillone in semen extender improves freezability of buffalo spermatozoa.

BACKGROUND: Oxidative stress is one of the leading factors responsible for poor post-thaw semen quality because of overproduction of reactive oxygen species (ROS) over neutralizing antioxidants present in semen. Mainly two ROS generation sites are present in spermatozoa, that is, mitochondria and plasma membrane. Therefore, the idea of targeting these specific sites for minimization of ROS production with the compounds having known mechanism of actions was built up as a core for this research. OBJECTIVE: Present study was done to investigate the effects of Mito TEMPO and acetovanillone individually and in combination on freezability of buffalo spermatozoa. MATERIALS AND METHODS: For the experiment, semen extender was supplemented with Mito TEMPO (50 µM), acetovanillone (50 µM), and a combination of Mito TEMPO + acetovanillone (50 µM+ 50 µM), designated as Group II, Group III, and Group IV, respectively. Control group without any supplementation was designated as Group I. A total of 24 ejaculates with individual progressive motility (IPM) of ≥70% were selected for the study. After final dilution, filling-sealing of straws, equilibration, and freezing were done as per the standard procedure. Semen samples were evaluated for IPM, plasma membrane integrity, lipid peroxidation, total antioxidant capacity (TAC), and cholesterol to phospholipids (C/P) ratio at both fresh and post-thaw stages. Evaluation of ROS, mitochondrial membrane potential (MMP), capacitation status (CTC assay), and in vitro fertility potential were conducted only on frozen-thawed samples. RESULTS: The addition of Mito TEMPO (50 µM) and acetovanillone (50 µM) individually and in combination significantly (p < 0.05) improved post-thaw semen quality in terms of IPM, plasma membrane integrity, TAC, cholesterol content, C/P ratio, MMP, Chlortetracycline (CTC)-Full (F) pattern, and zona binding ability of buffalo spermatozoa, while significantly (p < 0.05) reduced ROS production, lipid peroxidation, and capacitation like changes as compared to the control group. DISCUSSION: As Mito TEMPO acts as an SOD mimetic and also detoxifies ferrous iron at the mitochondria level, it aids in neutralization of excessive ROS production and minimizes oxidative stress-related damages that enhances the antioxidant potential of sperm mitochondria. Earlier studies also indicated improved post-thaw semen quality in 50 µM supplemented group. The improvement observed in acetovanillone (50 µM) group might be because of inhibition of Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as this enzyme activation by various physical/chemical inducers during cryopreservation process leads to activation of CatSper channel resulting in calcium influx, premature capacitation, and acrosomal reaction like changes through activation of adenylate cyclase and cAMP/PKA-mediated tyrosine phosphorylation of sperm proteins. Acetovanillone also prevents NADPH oxidase-mediated inhibition of glutathione reductase activity, which has a vital role in protecting the structural and functional integrity of sperm plasma membrane. CONCLUSION: Results indicated beneficial effects of supplementation of Mito TEMPO and acetovanillone on sperm freezability and individual supplementation was as efficient as the combination group for sustaining post-thaw semen quality.

Supplementation of ram's semen extender with Mito-TEMPO II: Quality evaluation and flow cytometry study of post-thawed spermatozoa.

Cryopreservation is an effective method to spread qualified ram spermatozoa for reproductive goals in different farms, but cryopreservation's shocks reduce sperm quality. This study investigated the efficacy of the new mitochondria-targeted antioxidant Mito-TEMPO on post-thawed quality of spermatozoa in sheep. Collected samples were divided into five groups and after dilution, received different doses of Mito-TEMPO (0, 0.5, 5, 50 and 500 µM), and frozen. Thawed sperm motility parameters, malondialdehyde content, membrane functionality, abnormal morphology, mitochondria activity, acrosome integrity, DNA fragmentation, ROS concentration, viability and apoptotic-like changes, were evaluated. According to the results, Mito-TEMPO (5 and 50 µM) improved (p ≤ 0.05) motility parameters, average path velocity, membrane functionality, mitochondria activity and viability compared with the other groups. Moreover, apoptotic-like changes, lipid peroxidation and ROS concentration were lower (p ≤ 0.05) in groups received 5 and 50 µM Mito-TEMPO. Mito-TEMPO showed no effect (p > 0.05) on sperm acrosome integrity, morphology and DNA fragmentation. In conclusion, Mito-TEMPO as a targeted antioxidant could be an efficient cryo-additive to enhance quality parameters of post-thawed ram semen.

Flow cytometry study of post-thawed bulk spermatozoa: Mito-TEMPO improves cryopreservation performance by controlling apoptosis rate, DNA fragmentation and ROS production.

Sperm cryopreservation is used to spread qualified semen for artificial insemination, but the freezing process reduces sperm quality. This study assessed the efficacy of Mito-TEMPO on post-thawed goat sperm quality. Semen samples divided to five equal groups and after dilution, received different doses of Mito-TEMPO (0, 1, 10, 100 and 1000 µM), and cryopreserved in liquid nitrogen. After thawing, flow cytometry analysis was performed to evaluate sperm mitochondria membrane potential, viability, apoptotic-like changes, DNA fragmentation and ROS concentration. According to the results, Mito-TEMPO (10 and 100 µM) improved (P ≤ 0.05) sperm viability and decreased (P ≤ 0.05) apoptotic-like changes and ROS concentration compared to the other groups. Mitochondria membrane potential was higher (P ≤ 0.05) in groups received 1, 10 and 100 µM Mito-TEMPO. The lowest (P ≤ 0.05) DNA fragmentation was observed in group received 10 µM Mito-TEMPO. In conclusion, mitochondria-targeted antioxidant Mito-TEMPO could be an efficient cryo-additive to enhance flowcytometric quality parameters of post-thawed bulk semen.

Fertility and flow cytometry evaluations of ram frozen semen in plant-based extender supplemented with Mito-TEMPO.

Semen cryopreservation is an effective strategy for distributing spermatozoa for artificial insemination, but this process reduces the fertility potential of post-thawed spermatozoa. The current study was conducted to assess effects of the novel mitochondria-targeted antioxidant "Mito-TEMPO" on ram sperm quality and fertility potential during the freeze-thawing process in a plant-based extender. Semen samples were diluted in extenders supplemented with 0, 0.5, 5, 50 and 500 µM Mito-TEMPO and then frozen using a standard protocol. Motility, abnormal morphology, acrosome integrity, membrane integrity, mitochondria membrane potential, viability, apoptotic-like changes, lipid peroxidation, DNA fragmentation, H2O2 concentration and fertility potential were assessed after thawing. Results indicated that with the 5 and 50 µM Mito-TEMPO there was a greater (P ≤ 0.05) percentage of sperm total motility, progressive motility, acrosome integrity and viability as well as less (P ≤ 0.05) lipid peroxidation and late apoptotic-like changes. Membrane integrity and mitochondria membrane potential were greater (P ≤ 0.05) with the 50 µM Mito-TEMPO extender supplementation. Furthermore, with 0.5, 5 and 50 µM Mito-TEMPO supplementations there was a greater (P ≤ 0.05) average path velocity and lesser (P ≤ 0.05) percentages of spermatozoa with DNA fragmentation and relatively greater H2O2 concentration. Results from the fertility experiment indicated the 5 and 50 µM Mito-TEMPO treatments resulted in greater pregnancy, parturition and lambing rates. It, therefore, is concluded that Mito-TEMPO may enhance quality and fertility potential of post-thawed semen of rams.

Mito-Tempo suppresses autophagic flux via the PI3K/Akt/mTOR signaling pathway in neuroblastoma SH-SY5Y cells.

The generation of excessive mitochondrial reactive oxygen species (mtROS) is associated with glutamate-stimulated neurotoxicity and pathogenesis of Alzheimer's disease (AD). Impaired mitochondrial function is accompanied with oxidative stress that is a significant contributor to initiate autophagy, but the underlying mechanisms are not fully understood. The present study aimed to investigate the neuroprotective effects of Mito-Tempo on glutamate-induced neuroblastoma SH-SY5Y cell toxicity. SH-SY5Y cells were treated with 100 µM glutamate in the presence or absence of 50 and 100 µM Mito-Tempo for 24 h. Changes in cell viability were measured by MTT assay. Cytotoxicity and intracellular ROS accumulation were also evaluated using lactate dehydrogenase (LDH) activity assay and 2,7-dichlorofluorescein diacetate (DCFDA) Reactive Oxygen Species Assay kit, respectively. Mitochondrial membrane potential was analyzed by tetraethylbenzimidazoly-lcarbocyanine iodide (JC-1) staining. Expression of PI3K/AKT/mTOR pathway and autophagy markers, including LC3 (LC3-I/-II) and p62 (SQSTM1) were performed using Western blot analysis. Our results demonstrated that glutamate-exposed cells significantly increased cellular oxidative stress by enhancing ROS production. Glutamate treatment also increased LDH release follows the loss of mitochondrial membrane potential, caused cell viability loss. Treatment with Mito-Tempo not only attenuated the generation of ROS and improved mitochondrial membrane potential but also reduced the neurotoxicity of glutamate in a concentration-dependent manner, which leads to increased cell viability and decreased LDH release. Mito-Tempo has a greater protective effect by enhancing superoxide dismutase (SOD) activity and PI3K/AKT/mTOR phosphorylation. Moreover, Mito-Tempo treatment altered the autophagy process resulting in the decline in the ratio of the autophagy markers LC3-I/-II and p62 (SQSTM1). We propose that Mito-Tempo can improve neuronal properties against glutamate cytotoxicity through its direct free radical scavenging activity and inhibit excessive autophagy signaling pathway, therefore, allow for further studies to investigate the therapeutic potentials of Mito-Tempo in animal disease models and human.