Simultaneous targeting of peripheral and brain tumors with a therapeutic nanoparticle to disrupt metabolic adaptability at both sites.

Brain metastasis of advanced breast cancer often results in deleterious consequences. Metastases to the brain lead to significant challenges in treatment options, as the blood-brain barrier (BBB) prevents conventional therapy. Thus, we hypothesized that creation of a nanoparticle (NP) that distributes to both primary tumor site and across the BBB for secondary brain tumor can be extremely beneficial. Here, we report a simple targeting strategy to attack both the primary breast and secondary brain tumors utilizing a single NP platform. The nature of these mitochondrion-targeted, BBB-penetrating NPs allow for simultaneous targeting and drug delivery to the hyperpolarized mitochondrial membrane of the extracranial primary tumor site in addition to tumors at the brain. By utilizing a combination of such dual anatomical distributing NPs loaded with therapeutics, we demonstrate a proof-of-concept idea to combat the increased metabolic plasticity of brain metastases by lowering two major energy sources, oxidative phosphorylation (OXPHOS) and glycolysis. By utilizing complementary studies and genomic analyses, we demonstrate the utility of a chemotherapeutic prodrug to decrease OXPHOS and glycolysis by pairing with a NP loaded with pyruvate dehydrogenase kinase 1 inhibitor. Decreasing glycolysis aims to combat the metabolic flexibility of both primary and secondary tumors for therapeutic outcome. We also address the in vivo safety parameters by addressing peripheral neuropathy and neurobehavior outcomes. Our results also demonstrate that this combination therapeutic approach utilizes mitochondrial genome targeting strategy to overcome DNA repair-based chemoresistance mechanisms.

A mitochondrion-targeted cyanine agent for NIR-II fluorescence-guided surgery combined with intraoperative photothermal therapy to reduce prostate cancer recurrence.

Poorly identified tumor boundaries and nontargeted therapies lead to the high recurrence rates and poor quality of life of prostate cancer patients. Near-infrared-II (NIR-II) fluorescence imaging provides certain advantages, including high resolution and the sensitive detection of tumor boundaries. Herein, a cyanine agent (CY7-4) with significantly greater tumor affinity and blood circulation time than indocyanine green was screened. By binding albumin, the absorbance of CY7-4 in an aqueous solution showed no effects from aggregation, with a peak absorbance at 830 nm and a strong fluorescence emission tail beyond 1000 nm. Due to its extended circulation time (half-life of 2.5 h) and high affinity for tumor cells, this fluorophore was used for primary and metastatic tumor diagnosis and continuous monitoring. Moreover, a high tumor signal-to-noise ratio (up to ~ 10) and excellent preferential mitochondrial accumulation ensured the efficacy of this molecule for photothermal therapy. Therefore, we integrated NIR-II fluorescence-guided surgery and intraoperative photothermal therapy to overcome the shortcomings of a single treatment modality. A significant reduction in recurrence and an improved survival rate were observed, indicating that the concept of intraoperative combination therapy has potential for the precise clinical treatment of prostate cancer.

VEGFR2 blockade inhibits glioblastoma cell proliferation by enhancing mitochondrial biogenesis.

BACKGROUND: Glioblastoma is an aggressive brain tumor linked to significant angiogenesis and poor prognosis. Anti-angiogenic therapies with vascular endothelial growth factor receptor 2 (VEGFR2) inhibition have been investigated as an alternative glioblastoma treatment. However, little is known about the effect of VEGFR2 blockade on glioblastoma cells per se. METHODS: VEGFR2 expression data in glioma patients were retrieved from the public database TCGA. VEGFR2 intervention was implemented by using its selective inhibitor Ki8751 or shRNA. Mitochondrial biogenesis of glioblastoma cells was assessed by immunofluorescence imaging, mass spectrometry, and western blot analysis. RESULTS: VEGFR2 expression was higher in glioma patients with higher malignancy (grade III and IV). VEGFR2 inhibition hampered glioblastoma cell proliferation and induced cell apoptosis. Mass spectrometry and immunofluorescence imaging showed that the anti-glioblastoma effects of VEGFR2 blockade involved mitochondrial biogenesis, as evidenced by the increases of mitochondrial protein expression, mitochondria mass, mitochondrial oxidative phosphorylation (OXPHOS), and reactive oxygen species (ROS) production, all of which play important roles in tumor cell apoptosis, growth inhibition, cell cycle arrest and cell senescence. Furthermore, VEGFR2 inhibition exaggerated mitochondrial biogenesis by decreased phosphorylation of AKT and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), which mobilized PGC1α into the nucleus, increased mitochondrial transcription factor A (TFAM) expression, and subsequently enhanced mitochondrial biogenesis. CONCLUSIONS: VEGFR2 blockade inhibits glioblastoma progression via AKT-PGC1α-TFAM-mitochondria biogenesis signaling cascade, suggesting that VEGFR2 intervention might bring additive therapeutic values to anti-glioblastoma therapy.

Peroxiredoxin II exerts neuroprotective effects by inhibiting endoplasmic reticulum stress and oxidative stress-induced neuronal pyroptosis.

BACKGROUND: Intracerebral hemorrhage (ICH) is a critical neurological condition with few treatment options, where secondary immune responses and specific cell death forms, like pyroptosis, worsen brain damage. Pyroptosis involves gasdermin-mediated membrane pores, increasing inflammation and neural harm, with the NLRP3/Caspase-1/GSDMD pathway being central to this process. Peroxiredoxin II (Prx II), recognized for its mitochondrial protection and reactive oxygen species (ROS) scavenging abilities, appears as a promising neuronal pyroptosis modulator. However, its exact role and action mechanisms need clearer definition. This research aims to explore Prx II impact on neuronal pyroptosis and elucidate its mechanisms, especially regarding endoplasmic reticulum (ER) stress and oxidative stress-induced neuronal damage modulation. METHODS AND RESULTS: Utilizing MTT assays, Microscopy, Hoechst/PI staining, Western blotting, and immunofluorescence, we found Prx II effectively reduces LPS/ATP-induced pyroptosis and neuroinflammation in HT22 hippocampal neuronal cells. Our results indicate Prx II's neuroprotective actions are mediated through PI3K/AKT activation and ER stress pathway inhibition, diminishing mitochondrial dysfunction and decreasing neuronal pyroptosis through the ROS/MAPK/NF-κB pathway. These findings highlight Prx II potential therapeutic value in improving intracerebral hemorrhage outcomes by lessening secondary brain injury via critical signaling pathway modulation involved in neuronal pyroptosis. CONCLUSIONS: Our study not only underlines Prx II importance in neuroprotection but also opens new therapeutic intervention avenues in intracerebral hemorrhage, stressing the complex interplay between redox regulation, ER stress, and mitochondrial dynamics in neuroinflammation and cell death management.

VDR restores the expression of PINK1 and BNIP3 in TECs of streptozotocin-induced diabetic mice.

Defective mitophagy in renal tubular epithelial cells is one of the main drivers of renal fibrosis in diabetic kidney disease. Our gene sequencing data showed the expression of PINK1 and BNIP3, two key molecules of mitophagy, was decreased in renal tissues of VDR-knockout mice. Herein, streptozotocin (STZ) was used to induce renal interstitial fibrosis in mice. VDR deficiency exacerbated STZ-induced renal impairment and defective mitophagy. Paricalcitol (pari, a VDR agonist) and the tubular epithelial cell-specific overexpression of VDR restored the expression of PINK1 and BNIP3 in the renal cortex and attenuated STZ-induced kidney fibrosis and mitochondrial dysfunction. In HK-2 cells under high glucose conditions, an increased level of α-SMA, COL1, and FN and a decreased expression of PINK1 and BNIP3 with severe mitochondrial damage were observed, and these alterations could be largely reversed by pari treatment. ChIP-qPCR and luciferase reporter assays showed VDR could positively regulate the transcription of Pink1 and Bnip3 genes. These findings reveal that VDR could restore mitophagy defects and attenuate STZ-induced fibrosis in diabetic mice through regulation of PINK1 and BNIP3.

A novel inhibitor of the mitochondrial respiratory complex I with uncoupling properties exerts potent antitumor activity.

Cancer cells are highly dependent on bioenergetic processes to support their growth and survival. Disruption of metabolic pathways, particularly by targeting the mitochondrial electron transport chain complexes (ETC-I to V) has become an attractive therapeutic strategy. As a result, the search for clinically effective new respiratory chain inhibitors with minimized adverse effects is a major goal. Here, we characterize a new OXPHOS inhibitor compound called MS-L6, which behaves as an inhibitor of ETC-I, combining inhibition of NADH oxidation and uncoupling effect. MS-L6 is effective on both intact and sub-mitochondrial particles, indicating that its efficacy does not depend on its accumulation within the mitochondria. MS-L6 reduces ATP synthesis and induces a metabolic shift with increased glucose consumption and lactate production in cancer cell lines. MS-L6 either dose-dependently inhibits cell proliferation or induces cell death in a variety of cancer cell lines, including B-cell and T-cell lymphomas as well as pediatric sarcoma. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI-1) partially restores the viability of B-lymphoma cells treated with MS-L6, demonstrating that the inhibition of NADH oxidation is functionally linked to its cytotoxic effect. Furthermore, MS-L6 administration induces robust inhibition of lymphoma tumor growth in two murine xenograft models without toxicity. Thus, our data present MS-L6 as an inhibitor of OXPHOS, with a dual mechanism of action on the respiratory chain and with potent antitumor properties in preclinical models, positioning it as the pioneering member of a promising drug class to be evaluated for cancer therapy. MS-L6 exerts dual mitochondrial effects: ETC-I inhibition and uncoupling of OXPHOS. In cancer cells, MS-L6 inhibited ETC-I at least 5 times more than in isolated rat hepatocytes. These mitochondrial effects lead to energy collapse in cancer cells, resulting in proliferation arrest and cell death. In contrast, hepatocytes which completely and rapidly inactivated this molecule, restored their energy status and survived exposure to MS-L6 without apparent toxicity.

Effect of Clemizole on Alpha-Synuclein-Preformed Fibrils-Induced Parkinson's Disease Pathology: A Pharmacological Investigation.

Parkinson's disease (PD) is a neurodegenerative disorder associated with mitochondrial dysfunctions and oxidative stress. However, to date, therapeutics targeting these pathological events have not managed to translate from bench to bedside for clinical use. One of the major reasons for the lack of translational success has been the use of classical model systems that do not replicate the disease pathology and progression with the same degree of robustness. Therefore, we employed a more physiologically relevant model involving alpha-synuclein-preformed fibrils (PFF) exposure to SH-SY5Y cells and Sprague Dawley rats. We further explored the possible involvement of transient receptor potential canonical 5 (TRPC5) channels in PD-like pathology induced by these alpha-synuclein-preformed fibrils with emphasis on amelioration of oxidative stress and mitochondrial health. We observed that alpha-synuclein PFF exposure produced neurobehavioural deficits that were positively ameliorated after treatment with the TRPC5 inhibitor clemizole. Furthermore, Clemizole also reduced p-alpha-synuclein and diminished oxidative stress levels which resulted in overall improvements in mitochondrial biogenesis and functions. Finally, the results of the pharmacological modulation were further validated using siRNA-mediated knockdown of TRPC5 channels, which also decreased p-alpha-synuclein expression. Together, the results of this study could be superimposed in the future for exploring the beneficial effects of TRPC5 channel modulation for other neurodegenerative disorders and synucleopathies.

N, N-Dimethyltryptamine, a natural hallucinogen, ameliorates Alzheimer's disease by restoring neuronal Sigma-1 receptor-mediated endoplasmic reticulum-mitochondria crosstalk.

BACKGROUND: Aberrant neuronal Sigma-1 receptor (Sig-1r)-mediated endoplasmic reticulum (ER)- mitochondria signaling plays a key role in the neuronal cytopathology of Alzheimer's disease (AD). The natural psychedelic N, N-dimethyltryptamine (DMT) is a Sig-1r agonist that may have the anti-AD potential through protecting neuronal ER-mitochondrial interplay. METHODS: 3×TG-AD transgenic mice were administered with chronic DMT (2 mg/kg) for 3 weeks and then performed water maze test. The Aß accumulation in the mice brain were determined. The Sig-1r level upon DMT treatment was tested. The effect of DMT on the ER-mitochondrial contacts site and multiple mitochondria-associated membrane (MAM)-associated proteins were examined. The effect of DMT on calcium transport between ER and mitochondria and the mitochondrial function were also evaluated. RESULTS: chronic DMT (2 mg/kg) markedly alleviated cognitive impairment of 3×TG-AD mice. In parallel, it largely diminished Aß accumulation in the hippocampus and prefrontal cortex. DMT restored the decreased Sig-1r levels of 3×TG-AD transgenic mice. The hallucinogen reinstated the expression of multiple MAM-associated proteins in the brain of 3×TG-AD mice. DMT also prevented physical contact and calcium dynamic between the two organelles in in vitro and in vivo pathological circumstances. DMT modulated oxidative phosphorylation (OXPHOS) and ATP synthase in the in vitro model of AD. CONCLUSION: The anti-AD effects of DMT are associated with its protection of neuronal ER-mitochondria crosstalk via the activation of Sig-1r. DMT has the potential to serve as a novel preventive and therapeutic agent against AD.

OTUB2 silencing promotes ovarian cancer via mitochondrial metabolic reprogramming and can be synthetically targeted by CA9 inhibition.

Ovarian cancer is an aggressive gynecological tumor characterized by a high relapse rate and chemoresistance. Ovarian cancer exhibits the cancer hallmark of elevated glycolysis, yet effective strategies targeting cancer cell metabolic reprogramming to overcome therapeutic resistance in ovarian cancer remain elusive. Here, we revealed that epigenetic silencing of Otubain 2 (OTUB2) is a driving force for mitochondrial metabolic reprogramming in ovarian cancer, which promotes tumorigenesis and chemoresistance. Mechanistically, OTUB2 silencing destabilizes sorting nexin 29 pseudogene 2 (SNX29P2), which subsequently prevents hypoxia-inducible factor-1 alpha (HIF-1α) from von Hippel-Lindau tumor suppressor-mediated degradation. Elevated HIF-1α activates the transcription of carbonic anhydrase 9 (CA9) and drives ovarian cancer progression and chemoresistance by promoting glycolysis. Importantly, pharmacological inhibition of CA9 substantially suppressed tumor growth and synergized with carboplatin in the treatment of OTUB2-silenced ovarian cancer. Thus, our study highlights the pivotal role of OTUB2/SNX29P2 in suppressing ovarian cancer development and proposes that targeting CA9-mediated glycolysis is an encouraging strategy for the treatment of ovarian cancer.

Chaga mushroom extract suppresses oral cancer cell growth via inhibition of energy metabolism.

Oral cancer stands as a prevalent maligancy worldwide; however, its therapeutic potential is limited by undesired effects and complications. As a medicinal edible fungus, Chaga mushroom (Inonotus obliquus) exhibits anticancer effects across diverse cancers. Yet, the precise mechanisms underlying its efficacy remain unclear. We explored the detailed mechanisms underlying the anticancer action of Chaga mushroom extract in oral cancer cells (HSC-4). Following treatment with Chaga mushroom extracts, we analyzed cell viability, proliferation capacity, glycolysis, mitochondrial respiration, and apoptosis. Our findings revealed that the extract reduced cell viability and proliferation of HSC-4 cells while arresting their cell cycle via suppression of STAT3 activity. Regarding energy metabolism, Chaga mushroom extract inhibited glycolysis and mitochondrial membrane potential in HSC-4 cells, thereby triggering autophagy-mediated apoptotic cell death through activation of the p38 MAPK and NF-κB signaling pathways. Our results indicate that Chaga mushroom extract impedes oral cancer cell progression, by inhibiting cell cycle and proliferation, suppressing cancer cell energy metabolism, and promoting autophagy-mediated apoptotic cell death. These findings suggest that this extract is a promising supplementary medicine for the treatment of patients with oral cancer.

Caffeic acid phenethyl ester restores mitochondrial homeostasis against peritoneal fibrosis induced by peritoneal dialysis through the AMPK/SIRT1 pathway.

Increasing evidence suggests that peritoneal fibrosis induced by peritoneal dialysis (PD) is linked to oxidative stress. However, there are currently no effective interventions for peritoneal fibrosis. In the present study, we explored whether adding caffeic acid phenethyl ester (CAPE) to peritoneal dialysis fluid (PDF) improved peritoneal fibrosis caused by PD and explored the molecular mechanism. We established a peritoneal fibrosis model in Sprague-Dawley rats through intraperitoneal injection of PDF and lipopolysaccharide (LPS). Rats in the PD group showed increased peritoneal thickness, submesothelial collagen deposition, and the expression of TGFß1 and α-SMA. Adding CAPE to PDF significantly inhibited PD-induced submesothelial thickening, reduced TGFß1 and α-SMA expression, alleviated peritoneal fibrosis, and improved the peritoneal ultrafiltration function. In vitro, peritoneal mesothelial cells (PMCs) treated with PDF showed inhibition of the AMPK/SIRT1 pathway, mitochondrial membrane potential depolarization, overproduction of mitochondrial reactive oxygen species (ROS), decreased ATP synthesis, and induction of mesothelial-mesenchymal transition (MMT). CAPE activated the AMPK/SIRT1 pathway, thereby inhibiting mitochondrial membrane potential depolarization, reducing mitochondrial ROS generation, and maintaining ATP synthesis. However, the beneficial effects of CAPE were counteracted by an AMPK inhibitor and siSIRT1. Our results suggest that CAPE maintains mitochondrial homeostasis by upregulating the AMPK/SIRT1 pathway, which alleviates oxidative stress and MMT, thereby mitigating the damage to the peritoneal structure and function caused by PD. These findings suggest that adding CAPE to PDF may prevent and treat peritoneal fibrosis.

Mitochondrial alternative oxidase pathway helps in nitrooxidative stress tolerance in germinating chickpea.

Mitochondrial alternative oxidase (AOX) is an important protein that can help in regulating reactive oxygen species and nitric oxide in plants. The role of AOX in regulation of nitro-oxidative stress in chickpea is not known. Using germinating chickpea as a model system, we investigated the role of AOX in nitro-oxidative stress tolerance. NaCl treatment was used as an inducer of nitro-oxidative stress. Treatment of germinating seeds with 150 mM NaCl led to reduced germination and radicle growth. The AOX inhibitor SHAM caused further inhibition of germination, and the AOX inducer pyruvate improved growth of the radicle under NaCl stress. Isolated mitochondria from germinated seeds under salt stress not only increased AOX capacity but also enhanced AOX protein expression. Measurement of superoxide levels revealed that AOX inhibition by SHAM can enhance superoxide levels, whereas the AOX inducer pyruvate reduced superoxide levels. Measurement of NO by gas phase chemiluminescence revealed enhanced NO generation in response to NaCl treatment. Upon NaCl treatment there was enhanced tyrosine nitration, which is an indicator of nitrosative stress response. Taken together, our results revealed that AOX induced under salinity stress in germinating chickpea can help in mitigating nitro-oxidative stress, thereby improving germination.

Neuroreceptor Inhibition by Clozapine Triggers Mitohormesis and Metabolic Reprogramming in Human Blood Cells.

The antipsychotic drug clozapine demonstrates superior efficacy in treatment-resistant schizophrenia, but its intracellular mode of action is not completely understood. Here, we analysed the effects of clozapine (2.5-20 µM) on metabolic fluxes, cell respiration, and intracellular ATP in human HL60 cells. Some results were confirmed in leukocytes of clozapine-treated patients. Neuroreceptor inhibition under clozapine reduced Akt activation with decreased glucose uptake, thereby inducing ER stress and the unfolded protein response (UPR). Metabolic profiling by liquid-chromatography/mass-spectrometry revealed downregulation of glycolysis and the pentose phosphate pathway, thereby saving glucose to keep the electron transport chain working. Mitochondrial respiration was dampened by upregulation of the F0F1-ATPase inhibitory factor 1 (IF1) leading to 30-40% lower oxygen consumption in HL60 cells. Blocking IF1 expression by cotreatment with epigallocatechin-3-gallate (EGCG) increased apoptosis of HL60 cells. Upregulation of the mitochondrial citrate carrier shifted excess citrate to the cytosol for use in lipogenesis and for storage as triacylglycerol in lipid droplets (LDs). Accordingly, clozapine-treated HL60 cells and leukocytes from clozapine-treated patients contain more LDs than untreated cells. Since mitochondrial disturbances are described in the pathophysiology of schizophrenia, clozapine-induced mitohormesis is an excellent way to escape energy deficits and improve cell survival.

Vitamin C alleviates hyperglycemic stress in retinal pigment epithelial cells.

BACKGROUND: Retinal pigment epithelial cells (RPECs) are a type of retinal cells that structurally and physiologically support photoreceptors. However, hyperglycemia has been shown to play a critical role in the progression of diabetic retinopathy (DR), which is one of the leading causes of vision impairment. In the diabetic eye, the high glucose environment damages RPECs via the induction of oxidative stress, leading to the release of excess reactive oxygen species (ROS) and triggering apoptosis. In this study, we aim to investigate the antioxidant mechanism of Vitamin C in reducing hyperglycemia-induced stress and whether this mechanism can preserve the function of RPECs. METHODS AND RESULTS: ARPE-19 cells were treated with high glucose in the presence or absence of Vitamin C. Cell viability was measured by MTT assay. Cleaved poly ADP-ribose polymerase (PARP) was used to identify apoptosis in the cells. ROS were detected by the DCFH-DA reaction. The accumulation of sorbitol in the aldose reductase (AR) polyol pathway was determined using the sorbitol detection assay. Primary mouse RPECs were isolated from adult mice and identified by Rpe65 expression. The mitochondrial damage was measured by mitochondrial membrane depolarization. Our results showed that high glucose conditions reduce cell viability in RPECs while Vitamin C can restore cell viability, compared to the vehicle treatment. We also demonstrated that Vitamin C reduces hyperglycemia-induced ROS production and prevents cell apoptosis in RPECs in an AR-independent pathway. CONCLUSIONS: These results suggest that Vitamin C is not only a nutritional necessity but also an adjuvant that can be combined with AR inhibitors for alleviating hyperglycemic stress in RPECs.

FGFR inhibition blocks NF-ĸB-dependent glucose metabolism and confers metabolic vulnerabilities in cholangiocarcinoma.

Genomic alterations that activate Fibroblast Growth Factor Receptor 2 (FGFR2) are common in intrahepatic cholangiocarcinoma (ICC) and confer sensitivity to FGFR inhibition. However, the depth and duration of response is often limited. Here, we conduct integrative transcriptomics, metabolomics, and phosphoproteomics analysis of patient-derived models to define pathways downstream of oncogenic FGFR2 signaling that fuel ICC growth and to uncover compensatory mechanisms associated with pathway inhibition. We find that FGFR2-mediated activation of Nuclear factor-κB (NF-κB) maintains a highly glycolytic phenotype. Conversely, FGFR inhibition blocks glucose uptake and glycolysis while inciting adaptive changes, including switching fuel source utilization favoring fatty acid oxidation and increasing mitochondrial fusion and autophagy. Accordingly, FGFR inhibitor efficacy is potentiated by combined mitochondrial targeting, an effect enhanced in xenograft models by intermittent fasting. Thus, we show that oncogenic FGFR2 signaling drives NF-κB-dependent glycolysis in ICC and that metabolic reprogramming in response to FGFR inhibition confers new targetable vulnerabilities.

Mitochondrial SIRT3 as a protective factor against cyclosporine A-induced nephrotoxicity.

Sirtuin3 (SIRT3), a mitochondrial deacetylase, has been shown to be involved in various kidney diseases. In this study, we aimed to clarify the role of SIRT3 in cyclosporine-induced nephrotoxicity and the associated mitochondrial dysfunction. Madin-Darby canine kidney (MDCK) cells were transfected with Flag-tagged SIRT3 for SIRT3 overexpression or SIRT3 siRNA for the inhibition of SIRT3. Subsequently, the cells were treated with cyclosporine A (CsA) or vehicle. Wild-type and SIRT3 knockout (KO) mice were randomly assigned to receive cyclosporine A or olive oil. Furthermore, SIRT3 activator, honokiol, was treated alongside CsA to wild type mice. Our results revealed that CsA treatment inhibited mitochondrial SIRT3 expression in MDCK cells. Inhibition of SIRT3 through siRNA transfection exacerbated apoptosis, impaired the expression of the AMP-activated protein kinase-peroxisome proliferator-activated receptor gamma coactivator 1 alpha (AMPK-PGC1α) pathway, and worsened mitochondrial dysfunction induced by CsA treatment. Conversely, overexpression of SIRT3 through Flag-tagged SIRT3 transfection ameliorated apoptosis, increased the expression of mitochondrial superoxide dismutase 2, and restored the mitochondrial regulator pathway, AMPK-PGC1α. In SIRT3 KO mice, CsA treatment led to aggravated kidney dysfunction, increased kidney tubular injury, and accumulation of oxidative end products indicative of oxidative stress injury. Meanwhile, SIRT3 activation in vivo significantly mitigated these adverse effects, improving kidney function, reducing oxidative stress markers, and enhancing mitochondrial health following CsA treatment. Overall, our findings suggest that SIRT3 plays a protective role in alleviating mitochondrial dysfunction caused by CsA through the activation of the AMPK-PGC1α pathway, thereby preventing further kidney injury.

Enhancement of Frozen-Thawed Human Sperm Quality with Zinc as a Cryoprotective Additive.

BACKGROUND Cryopreservation preserves male fertility, crucial in oncology, advanced age, and infertility. However, it damages sperm motility, membrane, and DNA. Zinc (Zn), an antioxidant, shows promise in improving sperm quality after thawing, highlighting its potential as a cryoprotectant in reproductive medicine. MATERIAL AND METHODS Gradient concentration of ZnSO4 (0, 12.5, 25, 50, and 100 µM) was added in the Glycerol-egg yolk-citrate (GEYC) cryopreservative medium as an extender. Alterations in sperm viability and motility parameters after cryopreservation were detected in each group. Sperm plasma membrane integrity (PMI), acrosome integrity (ACR), DNA fragment index (DFI), and changes in sperm mitochondrial function were examined, including: mitochondrial potential (MMP), sperm reactive oxygen species (ROS), and sperm ATP. RESULTS We found that 50 µM ZnSO4 was the most effective for the curvilinear velocity (VCL) and the average path velocity (VAP) of sperm after cryo-resuscitation. Compared to the Zn-free group, sperm plasma membrane integrity (PMI) was increased, DNA fragmentation index (DFI) was decreased, reactive oxygen species (ROS) was reduced, and mitochondrial membrane potential (MMP) was increased after cryorevival in the presence of 50 µM ZnSO4. CONCLUSIONS Zn ion is one of the antioxidants in the cell. The results of our current clinical study are sufficient to demonstrate that Zn can improve preserves sperm quality during cryopreservation when added to GEYC. The addition of 50 µM ZnSO4 increased curve velocity, mean path velocity, sperm survival (or plasma membrane integrity), and mitochondrial membrane potential while reducing ROS production and DNA breaks compared to GEYC thawed without ZnSO4.

Comprehensive dry eye therapy: overcoming ocular surface barrier and combating inflammation, oxidation, and mitochondrial damage.

BACKGROUND: Dry Eye Disease (DED) is a prevalent multifactorial ocular disease characterized by a vicious cycle of inflammation, oxidative stress, and mitochondrial dysfunction on the ocular surface, all of which lead to DED deterioration and impair the patients' quality of life and social functioning. Currently, anti-inflammatory drugs have shown promising efficacy in treating DED; however, such drugs are associated with side effects. The bioavailability of ocular drugs is less than 5% owing to factors such as rapid tear turnover and the presence of the corneal barrier. This calls for investigations to overcome these challenges associated with ocular drug administration. RESULTS: A novel hierarchical action liposome nanosystem (PHP-DPS@INS) was developed in this study. In terms of delivery, PHP-DPS@INS nanoparticles (NPs) overcame the ocular surface transport barrier by adopting the strategy of "ocular surface electrostatic adhesion-lysosomal site-directed escape". In terms of therapy, PHP-DPS@INS achieved mitochondrial targeting and antioxidant effects through SS-31 peptide, and exerted an anti-inflammatory effect by loading insulin to reduce mitochondrial inflammatory metabolites. Ultimately, the synergistic action of "anti-inflammation-antioxidation-mitochondrial function restoration" breaks the vicious cycle associated with DED. The PHP-DPS@INS demonstrated remarkable cellular uptake, lysosomal escape, and mitochondrial targeting in vitro. Targeted metabolomics analysis revealed that PHP-DPS@INS effectively normalized the elevated level of mitochondrial proinflammatory metabolite fumarate in an in vitro hypertonic model of DED, thereby reducing the levels of key inflammatory factors (IL-1ß, IL-6, and TNF-α). Additionally, PHP-DPS@INS strongly inhibited reactive oxygen species (ROS) production and facilitated mitochondrial structural repair. In vivo, the PHP-DPS@INS treatment significantly enhanced the adhesion duration and corneal permeability of the ocular surface in DED mice, thereby improving insulin bioavailability. It also restored tear secretion, suppressed ocular surface damage, and reduced inflammation in DED mice. Moreover, it demonstrated favorable safety profiles both in vitro and in vivo. CONCLUSION: In summary, this study successfully developed a comprehensive DED management nanosystem that overcame the ocular surface transmission barrier and disrupted the vicious cycle that lead to dry eye pathogenesis. Additionally, it pioneered the regulation of mitochondrial metabolites as an anti-inflammatory treatment for ocular conditions, presenting a safe, efficient, and innovative therapeutic strategy for DED and other inflammatory diseases.

Mitochondria-Selective Dicationic Small-Molecule Ligand Targeting G-Quadruplex Structures for Human Colorectal Cancer Therapy.

Mitochondria are important drug targets for anticancer and other disease therapies. Certain human mitochondrial DNA sequences capable of forming G-quadruplex structures (G4s) are emerging drug targets of small molecules. Despite some mitochondria-selective ligands being reported for drug delivery against cancers, the ligand design is mostly limited to the triphenylphosphonium scaffold. The ligand designed with lipophilic small-sized scaffolds bearing multipositive charges targeting the unique feature of high mitochondrial membrane potential (MMP) is lacking and most mitochondria-selective ligands are not G4-targeting. Herein, we report a new small-sized dicationic lipophilic ligand to target MMP and mitochondrial DNA G4s to enhance drug delivery for anticancer. The ligand showed marked alteration of mitochondrial gene expression and substantial induction of ROS production, mitochondrial dysfunction, DNA damage, cellular senescence, and apoptosis. The ligand also exhibited high anticancer activity against HCT116 cancer cells (IC50, 3.4 µM) and high antitumor efficacy in the HCT116 tumor xenograft mouse model (∼70% tumor weight reduction).

Ginsenoside Rg3, enriched in red ginseng extract, improves lipopolysaccharides-induced suppression of brown and beige adipose thermogenesis with mitochondrial activation.

Brown adipose tissue (BAT) which is a critical regulator of energy homeostasis, and its activity is inhibited by obesity and low-grade chronic inflammation. Ginsenoside Rg3, the primary constituent of Korean red ginseng (steamed Panax ginseng CA Meyer), has shown therapeutic potential in combating inflammatory and metabolic diseases. However, it remains unclear whether Rg3 can protect against the suppression of browning or activation of BAT induced by inflammation. In this study, we conducted a screening of ginsenoside composition in red ginseng extract (RGE) and explored the anti-adipogenic effects of both RGE and Rg3. We observed that RGE (exist 0.25 mg/mL of Rg3) exhibited significant lipid-lowering effects in adipocytes during adipogenesis. Moreover, treatment with Rg3 (60 µM) led to the inhibition of triglyceride accumulation, subsequently promoting enhanced fatty acid oxidation, as evidenced by the conversion of radiolabeled 3H-fatty acids into 3H-H2O with mitochondrial activation. Rg3 alleviated the attenuation of browning in lipopolysaccharide (LPS)-treated beige adipocytes and primary brown adipocytes by recovered by uncoupling protein 1 (UCP1) and the oxygen consumption rate compared to the LPS-treated group. These protective effects of Rg3 on inflammation-induced inhibition of beige and BAT-derived thermogenesis were confirmed in vivo by treating with CL316,243 (a beta-adrenergic receptor agonist) and LPS to induce browning and inflammation, respectively. Consistent with the in vitro data, treatment with Rg3 (2.5 mg/kg, 8 weeks) effectively reversed the LPS-induced inhibition of brown adipocyte features in C57BL/6 mice. Our findings confirm that Rg3-rich foods are potential browning agents that counteract chronic inflammation and metabolic complications.