Activation of Sirtuin3 by honokiol ameliorates alveolar epithelial cell senescence in experimental silicosis via the cGAS-STING pathway.

BACKGROUND: Silicosis, characterized by interstitial lung inflammation and fibrosis, poses a significant health threat. ATII cells play a crucial role in alveolar epithelial repair and structural integrity maintenance. Inhibiting ATII cell senescence has shown promise in silicosis treatment. However, the mechanism behind silica-induced senescence remains elusive. METHODS: The study employed male C57BL/6 N mice and A549 human alveolar epithelial cells to investigate silicosis and its potential treatment. Silicosis was induced in mice via intratracheal instillation of crystalline silica particles, with honokiol administered intraperitoneally for 14 days. Silica-induced senescence in A549 cells was confirmed, and SIRT3 knockout and overexpression cell lines were generated. Various analyses were conducted, including immunoblotting, qRT-PCR, histology, and transmission electron microscopy. Statistical significance was determined using one-way ANOVA with Tukey's post-hoc test. RESULTS: This study elucidates how silica induces ATII cell senescence, emphasizing mtDNA damage. Notably, honokiol (HKL) emerges as a promising anti-senescence and anti-fibrosis agent, acting through sirt3. honokiol effectively attenuated senescence in ATII cells, dependent on sirt3 expression, while mitigating mtDNA damage. Sirt3, a class III histone deacetylase, regulates senescence and mitochondrial stress. HKL activates sirt3, protecting against pulmonary fibrosis and mitochondrial damage. Additionally, HKL downregulated cGAS expression in senescent ATII cells induced by silica, suggesting sirt3's role as an upstream regulator of the cGAS/STING signaling pathway. Moreover, honokiol treatment inhibited the activation of the NF-κB signaling pathway, associated with reduced oxidative stress and mtDNA damage. Notably, HKL enhanced the activity of SOD2, crucial for mitochondrial function, through sirt3-mediated deacetylation. Additionally, HKL promoted the deacetylation activity of sirt3, further safeguarding mtDNA integrity. CONCLUSIONS: This study uncovers a natural compound, HKL, with significant anti-fibrotic properties through activating sirt3, shedding light on silicosis pathogenesis and treatment avenues.

The protective effects of orexin-A in alleviating cell senescence against interleukin-1ß (IL-1ß) in chondrocytes.

Osteoarthritis (OA) is one of the most important causes of global disability, and dysfunction of chondrocytes is an important risk factor. The treatment of OA is still a challenge. Orexin-A is a hypothalamic peptide, and its effects in OA are unknown. In this study, we found that exposure to interleukin-1ß (IL-1ß) reduced the expression of orexin-2R, the receptor of orexin-A in TC-28a2 chondrocytes. Importantly, the senescence-associated ß-galactosidase (SA-ß-gal) staining assay demonstrated that orexin-A treatment ameliorates IL-1ß-induced cellular senescence. Importantly, the presence of IL-1ß significantly reduced the telomerase activity of TC-28a2 chondrocytes, which was rescued by orexin-A. We also found that orexin-A prevented IL-1ß-induced increase in the levels of Acetyl-p53 and the expression of p21. It is shown that orexin-A mitigates IL-1ß-induced reduction of sirtuin 3 (SIRT3). Silencing of SIRT3 abolished the protective effects of orexin-A against IL-1ß-induced cellular senescence. These results imply that orexin-A might serve as a promising therapeutic agent for OA.

Polydatin: a potential NAFLD therapeutic drug that regulates mitochondrial autophagy through SIRT3-FOXO3-BNIP3 and PINK1-PRKN mechanisms - a network pharmacology and experimental investigation.

Non-alcoholic fatty liver disease (NAFLD) is a prevalent chronic liver disorder that is linked to metabolic syndrome, mitochondrial dysfunction and impaired autophagy. Polydatin (PD), a natural polyphenol from Polygonum cuspidatum, exhibits various pharmacological effects and protects against NAFLD. The aim of this study was to reveal the molecular mechanisms and therapeutic potential of PD for NAFLD, with a focus on the role of mitochondrial autophagy mediated by sirtuin 3 (SIRT3), fork-head box O3 (FOXO3) and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), and by PTEN-induced putative kinase 1 (PINK1) and parkin (PRKN). We combined network pharmacology analysis, animal models and cell culture experiments to show that PD could regulate the mitochondrial autophagy pathway by modulating several key genes related to mitochondrial function, and ameliorate the liver function, histopathology and mitochondrial biogenesis of NAFLD mice and hepatocytes by activating the SIRT3-FOXO3-BNIP3 axis and the PINK1-PRKN-dependent mechanism of mitochondrial autophagy. We also identified the core targets of PD, including SIRT3, FOXO3A, CASP3, PARKIN, EGFR, STAT3, MMP9 and PINK, and confirmed that silencing SIRT3 could significantly attenuate the beneficial effect of PD. This study provided novel theoretical and experimental support for PD as a promising candidate for NAFLD treatment, and also suggested new avenues and methods for investigating the role of mitochondrial autophagy in the pathogenesis and intervention of NAFLD.

SIRT3 facilitates mitochondrial structural repair and functional recovery in rats after ischemic stroke by promoting OPA1 expression and activity.

BACKGROUND: Optical atrophy 1 (OPA1), a protein accountable for mitochondrial fusion, facilitates the restoration of mitochondrial structure and function following cerebral ischemia/reperfusion (I/R) injury. The OPA1-conferred mitochondrial protection involves its expression and activity, which can be improved by SIRT3 in non-cerebral ischemia. Nevertheless, it remains obscure whether SIRT3 enhances the expression and activity of OPA1 after cerebral I/R injury. METHODS: Mature male Sprague Dawley rats were intracranially injected with adeno-associated viral-Sirtuin-3(AAV-SIRT3) and AAV-sh_OPA1, followed by a 90-min temporary blockage of the middle cerebral artery and subsequent restoration of blood flow. Cultured cortical neurons of rats were transfected with LV-SIRT3 or LV-sh_OPA1 before a 2-h oxygen-glucose deprivation and reoxygenation. The rats and neurons were subsequently treated with a selective OPA1 activity inhibitor (MYLS22). The interaction between SIRT3 and OPA1 was assessed by molecular dynamics simulation technology and co-immunoprecipitation. The expression, function, and specific protective mechanism of SIRT3 were examined by various analyses. RESULTS: SIRT3 interacted with OPA1 in the rat cerebral cortex before and after cerebral I/R. After cerebral I/R damage, SIRT3 upregulation increased the OPA1 expression, which enhanced deacetylation and OPA1 activity, thus alleviating cerebral infarct volume, neuronal apoptosis, oxidative pressure, and impairment in mitochondrial energy production; SIRT3 upregulation also improved neuromotor performance, repaired mitochondrial ultrastructure and membrane composition, and promoted the mitochondrial biogenesis. These neuroprotective effects were partly reversed by OPA1 expression interference and OPA1 activity inhibitor MYLS22. CONCLUSION: In rats, SIRT3 enhances the expression and activity of OPA1, facilitating the repair of mitochondrial structure and functional recovery following cerebral I/R injury. These findings highlight that regulating SIRT3 may be a promising therapeutic strategy for ischemic stroke.

The neuroprotective effects of SFGDI on sirtuin 3-related oxidative stress by regulating the Sirt3/SOD/ROS pathway and energy metabolism in BV2 cells.

Recently, the investigation of neuroprotective peptides has gained attention in addressing memory impairment and cognitive decline. Although the potential neuroprotective peptide Serine-Phenylalanine-Glycine-Aspartic acid-Isoleucine (SFGDI) has been identified from sea cucumber, the molecular mechanisms remain unclear. This study was conducted to explore the neuroprotection of SFGDI against 3-TYP-induced oxidative stress in BV2 cells. The results showed a retention rate of 76.70% during in vitro simulated gastrointestinal digestion and an absorption rate of 10.41% in a rat-everted gut sac model for SFGDI. Two hours following the administration of SFGDI via gavage in mice, a notable fluorescence was observed in the brain, indicating a potential neuroprotection of SFGDI through its interactions with nerve cells. By utilizing a model of oxidative stress injury induced by 3-TYP in BV2 cells, it was determined that pretreatment with SFGDI (50-200 µg mL-1) resulted in a dose-dependent reduction in the acetylated SOD level, leading to enhanced SOD activity and reduced levels of ROS and MDA. In addition, this pretreatment triggered an increase in unsaturated lipid levels, which helped maintain the intracellular lipid metabolism balance and preserve the mitochondrial function and glycolysis levels to regulate energy metabolism. The results of this study indicate that SFGDI demonstrates neuroprotective properties through its modulation of the Sirt3/SOD/ROS pathway, regulation of lipid metabolism, and enhancement of energy metabolism in BV2 cells. These findings suggest potential novel therapeutic approaches for addressing Sirt3-related memory deficits and neurodegenerative disorders.

Plasma Proteomics Identifies B2M as a Regulator of Pulmonary Hypertension in Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Pulmonary hypertension (PH) represents an important phenotype in heart failure with preserved ejection fraction (HFpEF). However, management of PH-HFpEF is challenging because mechanisms involved in the regulation of PH-HFpEF remain unclear. METHODS: We used a mass spectrometry-based comparative plasma proteomics approach as a sensitive and comprehensive hypothesis-generating discovery technique to profile proteins in patients with PH-HFpEF and control subjects. We then validated and investigated the role of one of the identified proteins using in vitro cell cultures, in vivo animal models, and independent cohort of human samples. RESULTS: Plasma proteomics identified high protein abundance levels of B2M (ß2-microglobulin) in patients with PH-HFpEF. Interestingly, both circulating and skeletal muscle levels of B2M were increased in mice with skeletal muscle SIRT3 (sirtuin-3) deficiency or high-fat diet-induced PH-HFpEF. Plasma and muscle biopsies from a validation cohort of PH-HFpEF patients were found to have increased B2M levels, which positively correlated with disease severity, especially pulmonary capillary wedge pressure and right atrial pressure at rest. Not only did the administration of exogenous B2M promote migration/proliferation in pulmonary arterial vascular endothelial cells but it also increased PCNA (proliferating cell nuclear antigen) expression and cell proliferation in pulmonary arterial vascular smooth muscle cells. Finally, B2m deletion improved glucose intolerance, reduced pulmonary vascular remodeling, lowered PH, and attenuated RV hypertrophy in mice with high-fat diet-induced PH-HFpEF. CONCLUSIONS: Patients with PH-HFpEF display higher circulating and skeletal muscle expression levels of B2M, the magnitude of which correlates with disease severity. Our findings also reveal a previously unknown pathogenic role of B2M in the regulation of pulmonary vascular proliferative remodeling and PH-HFpEF. These data suggest that circulating and skeletal muscle B2M can be promising targets for the management of PH-HFpEF.

Autophagy-deficient macrophages exacerbate cisplatin-induced mitochondrial dysfunction and kidney injury via miR-195a-5p-SIRT3 axis.

Macrophages (Mφ) autophagy is a pivotal contributor to inflammation-related diseases. However, the mechanistic details of its direct role in acute kidney injury (AKI) were unclear. Here, we show that Mφ promote AKI progression via crosstalk with tubular epithelial cells (TECs), and autophagy of Mφ was activated and then inhibited in cisplatin-induced AKI mice. Mφ-specific depletion of ATG7 (Atg7Δmye) aggravated kidney injury in AKI mice, which was associated with tubulointerstitial inflammation. Moreover, Mφ-derived exosomes from Atg7Δmye mice impaired TEC mitochondria in vitro, which may be attributable to miR-195a-5p enrichment in exosomes and its interaction with SIRT3 in TECs. Consistently, either miR-195a-5p inhibition or SIRT3 overexpression improved mitochondrial bioenergetics and renal function in vivo. Finally, adoptive transfer of Mφ from AKI mice to Mφ-depleted mice promotes the kidney injury response to cisplatin, which is alleviated when Mφ autophagy is activated with trehalose. We conclude that exosomal miR-195a-5p mediate the communication between autophagy-deficient Mφ and TECs, leading to impaired mitochondrial biogenetic in TECs and subsequent exacerbation of kidney injury in AKI mice via miR-195a-5p-SIRT3 axis.

FNDC5 prevents oxidative stress and neuronal apoptosis after traumatic brain injury through SIRT3-dependent regulation of mitochondrial quality control.

Mitochondrial dysfunction and oxidative stress are important mechanisms for secondary injury after traumatic brain injury (TBI), which result in progressive pathophysiological exacerbation. Although the Fibronectin type III domain-containing 5 (FNDC5) was reported to repress oxidative stress by retaining mitochondrial biogenesis and dynamics, its possible role in the secondary injury after TBI remain obscure. In present study, we observed that the level of serum irisin (the cleavage product of FNDC5) significantly correlated with the neurological outcomes of TBI patients. Knockout of FNDC5 increased the lesion volume and exacerbated apoptosis and neurological deficits after TBI in mice, while FNDC5 overexpression yielded a neuroprotective effect. Moreover, FNDC5 deficiency disrupted mitochondrial dynamics and function. Activation of Sirtuin 3 (SIRT3) alleviated FNDC5 deficiency-induced disruption of mitochondrial dynamics and bioenergetics. In neuron-specific SIRT3 knockout mice, FNDC5 failed to attenuate TBI-induced mitochondrial damage and brain injuries. Mechanically, FNDC5 deficiency led to reduced SIRT3 expression via enhanced ubiquitin degradation of transcription factor Nuclear factor erythroid 2-related factor 2 (NRF2), which contributed to the hyperacetylation and inactivation of key regulatory proteins of mitochondrial dynamics and function, including OPA1 and SOD2. Finally, engineered RVG29-conjugated nanoparticles were generated to selectively and efficiently deliver irisin to the brain of mice, which yielded a satisfactory curative effect against TBI. In conclusion, FNDC5/irisin exerts a protective role against acute brain injury by promoting SIRT3-dependent mitochondrial quality control and thus represents a potential target for neuroprotection after TBI.

Ferrostatin-1 specifically targets mitochondrial iron-sulfur clusters and aconitase to improve cardiac function in Sirtuin 3 cardiomyocyte knockout mice.

AIMS: Ferroptosis is a form of iron-regulated cell death implicated in ischemic heart disease. Our previous study revealed that Sirtuin 3 (SIRT3) is associated with ferroptosis and cardiac fibrosis. In this study, we tested whether the knockout of SIRT3 in cardiomyocytes (SIRT3cKO) promotes mitochondrial ferroptosis and whether the blockade of ferroptosis would ameliorate mitochondrial dysfunction. METHODS AND RESULTS: Mitochondrial and cytosolic fractions were isolated from the ventricles of mice. Cytosolic and mitochondrial ferroptosis were analyzed by comparison to SIRT3loxp mice. An echocardiography study showed that SIRT3cKO mice developed heart failure as evidenced by a reduction of EF% and FS% compared to SIRT3loxp mice. Comparison of mitochondrial and cytosolic fractions of SIRT3cKO and SIRT3loxp mice revealed that, upon loss of SIRT3, mitochondrial, but not cytosolic, total lysine acetylation was significantly increased. Similarly, acetylated p53 was significantly upregulated only in the mitochondria. These data demonstrate that SIRT3 is the primary mitochondrial deacetylase. Most importantly, loss of SIRT3 resulted in significant reductions of frataxin, aconitase, and glutathione peroxidase 4 (GPX4) in the mitochondria. This was accompanied by a significant increase in levels of mitochondrial 4-hydroxynonenal. Treatment of SIRT3cKO mice with the ferroptosis inhibitor ferrostatin-1 (Fer-1) for 14 days significantly improved preexisting heart failure. Mechanistically, Fer-1 treatment significantly increased GPX4 and aconitase expression/activity, increased mitochondrial iron­sulfur clusters, and improved mitochondrial membrane potential and Complex IV activity. CONCLUSIONS: Inhibition of ferroptosis ameliorated cardiac dysfunction by specifically targeting mitochondrial aconitase and iron­sulfur clusters. Blockade of mitochondrial ferroptosis may be a novel therapeutic target for mitochondrial cardiomyopathies.

Mitochondrial GPX4 acetylation is involved in cadmium-induced renal cell ferroptosis.

Increasing evidences demonstrate that environmental stressors are important inducers of acute kidney injury (AKI). This study aimed to investigate the impact of exposure to Cd, an environmental stressor, on renal cell ferroptosis. Transcriptomics analyses showed that arachidonic acid (ARA) metabolic pathway was disrupted in Cd-exposed mouse kidneys. Targeted metabolomics showed that renal oxidized ARA metabolites were increased in Cd-exposed mice. Renal 4-HNE, MDA, and ACSL4, were upregulated in Cd-exposed mouse kidneys. Consistent with animal experiments, the in vitro experiments showed that mitochondrial oxidized lipids were elevated in Cd-exposed HK-2 cells. Ultrastructure showed mitochondrial membrane rupture in Cd-exposed mouse kidneys. Mitochondrial cristae were accordingly reduced in Cd-exposed mouse kidneys. Mitochondrial SIRT3, an NAD+-dependent deacetylase that regulates mitochondrial protein stability, was reduced in Cd-exposed mouse kidneys. Subsequently, mitochondrial GPX4 acetylation was elevated and mitochondrial GPX4 protein was reduced in Cd-exposed mouse kidneys. Interestingly, Cd-induced mitochondrial GPX4 acetylation and renal cell ferroptosis were exacerbated in Sirt3-/- mice. Conversely, Cd-induced mitochondrial oxidized lipids were attenuated in nicotinamide mononucleotide (NMN)-pretreated HK-2 cells. Moreover, Cd-evoked mitochondrial GPX4 acetylation and renal cell ferroptosis were alleviated in NMN-pretreated mouse kidneys. These results suggest that mitochondrial GPX4 acetylation, probably caused by SIRT3 downregulation, is involved in Cd-evoked renal cell ferroptosis.

Mitochondrial ACSS1-K635 acetylation knock-in mice exhibit altered metabolism, cell senescence, and nonalcoholic fatty liver disease.

Acetyl-CoA synthetase short-chain family member 1 (ACSS1) uses acetate to generate mitochondrial acetyl-CoA and is regulated by deacetylation by sirtuin 3. We generated an ACSS1-acetylation (Ac) mimic mouse, where lysine-635 was mutated to glutamine (K635Q). Male Acss1K635Q/K635Q mice were smaller with higher metabolic rate and blood acetate and decreased liver/serum ATP and lactate levels. After a 48-hour fast, Acss1K635Q/K635Q mice presented hypothermia and liver aberrations, including enlargement, discoloration, lipid droplet accumulation, and microsteatosis, consistent with nonalcoholic fatty liver disease (NAFLD). RNA sequencing analysis suggested dysregulation of fatty acid metabolism, cellular senescence, and hepatic steatosis networks, consistent with NAFLD. Fasted Acss1K635Q/K635Q mouse livers showed increased fatty acid synthase (FASN) and stearoyl-CoA desaturase 1 (SCD1), both associated with NAFLD, and increased carbohydrate response element-binding protein binding to Fasn and Scd1 enhancer regions. Last, liver lipidomics showed elevated ceramide, lysophosphatidylethanolamine, and lysophosphatidylcholine, all associated with NAFLD. Thus, we propose that ACSS1-K635-Ac dysregulation leads to aberrant lipid metabolism, cellular senescence, and NAFLD.

Oral supplementation with resveratrol improves hormonal profile and increases expression of genes associated with thermogenesis in oophorectomy mice.

Menopause causes important bodily and metabolic changes, which favor the increased occurrence of cardiovascular diseases, obesity, diabetes, and osteoporosis. Resveratrol exerts proven effects on body metabolism, improving glucose and lipid homeostasis and reducing inflammation and oxidative stress in various organs and tissues. Accordingly, this study evaluates the effects of resveratrol supplementation on the expression of markers associated with thermogenesis in brown adipose tissue, and on the body, metabolic and hormonal parameters of female mice submitted to bilateral oophorectomy. Eighteen female mice were randomized into three groups: G1: control (CONTROL), G2: oophorectomy (OOF), and G3: oophorectomy + resveratrol (OOF + RSV); the animals were kept under treatment for twelve weeks, being fed a standard diet and treated with resveratrol via gavage. Body, biochemical, hormonal, and histological parameters were measured; in addition to the expression of markers associated with thermogenesis in brown adipose tissue. The results showed that animals supplemented with resveratrol showed reduced body weight and visceral adiposity, in addition to glucose, total cholesterol, and triglyceride levels; decreased serum FSH levels and increased estrogen levels were observed compared to the OOF group and mRNA expression of PRDM16, UCP1, and SIRT3 in brown adipose tissue. The findings of this study suggest the important role of resveratrol in terms of improving body, metabolic, and hormonal parameters, as well as modulating markers associated with thermogenesis in brown adipose tissue of female mice submitted to oophorectomy.

Crithmum maritimum Extract Restores Lipid Homeostasis and Metabolic Profile of Liver Cancer Cells to a Normal Phenotype.

Hepatocellular carcinoma (HCC) is an alarming epidemiological clinical problem worldwide. Pharmacological approaches currently available do not provide adequate responses due to poor effectiveness, high toxicity, and serious side effects. Our previous studies have shown that the wild edible plant Crithmum maritimum L. inhibits the growth of liver cancer cells and promotes liver cell differentiation by reducing lactic acid fermentation (Warburg effect). Here, we aimed to further characterise the effects of C. maritimum on lipid metabolism and markers of cellular metabolic health, such as AMP-activated protein kinase (AMPK), Sirtuin 1 (SIRT1), and Sirtuin 3 (SIRT3), as well as the insulin signalling pathway. To better mimic the biological spectrum of HCC, we employed four HCC cell lines with different degrees of tumorigenicity and lactic acid fermentation/Warburg phenotype. Lipid accumulation was assessed by Oil Red O (ORO) staining, while gene expression was measured by real-time quantitative PCR (RT-qPCR). The activation of AMPK and insulin signalling pathways was determined by Western blotting. Results indicate that C. maritimum prevents lipid accumulation, downregulates lipid and cholesterol biosynthesis, and modulates markers of metabolic health, such as AMPK, SIRT1 and SIRT3. This modulation is different amongst HCC cell lines, revealing an important functional versatility of C. maritimum. Taken together, our findings corroborate the importance of C. maritimum as a valuable nutraceutical, reinforcing its role for the improvement of metabolic health.

SGLT2 inhibitor promotes mitochondrial dysfunction and ER-phagy in colorectal cancer cells.

BACKGROUND: Sodium-glucose transporter 2 (SGLT2) inhibitors (iSGLT2) are approved medications for type 2 diabetes. Recent studies indicate that iSGLT2 inhibit the growth of some cancer cells. However, the mechanism(s) remains to be fully elucidated. METHODS: The SGLT2 levels were determined in normal colon CCD 841 CoN and, HCT 116, HT-29, SW480 and LoVo colorectal cancer (CRC) cell lines by quantitative real-time PCR and western blot. The effect of iSGLT2 canagliflozin on cell proliferation was examined using CCK-8, as its role on CRC cells metabolism and tumorigenesis has been evaluated by XF HS Seahorse Bioanalyzer and flow cytometric analyses. Transient gene silencing experiments and analysis of protein-protein interaction network were conducted to evaluate the SGLT2 molecular targets in CRC cells. RESULTS: Data showed that the treatment with iSGLT2 (50 µM) for 72 h induced cell cycle arrest (p < 0.001), impaired glucose and energetic metabolism (p < 0.001), promoted apoptotic cell death and ER stress flowing into autophagy (p < 0.001) in HCT 116 and HT-29 cells. These cellular events were accompanied by sirtuin 3 (SIRT3) upregulation (p < 0.01), as also supported by SIRT3 transient silencing experiments resulting in the attenuation of the effects of iSGLT2 on the cellular metabolic/energetic alterations and the induction of programmed cell death. The identification and validation of dipeptidyl peptidase 4 (DPP4) as potential common target of SGLT2 and SIRT3 were also assessed. CONCLUSIONS: These results deepened knowledge on the iSGLT2 contribution in limiting CRC tumorigenesis unveiling the SGLT2/SIRT3 axis in the cytotoxic mechanisms.

Exosomes regulate SIRT3-related autophagy by delivering miR-421 to regulate macrophage polarization and participate in OSA-related NAFLD.

PURPOSE: To analyze the role of and mechanism underlying obstructive sleep apnea (OSA)-derived exosomes in inducing non-alcoholic fatty liver (NAFLD). METHODS: The role of OSA-derived exosomes was analyzed in inducing hepatocyte fat accumulation in mice models both in vivo and in vitro. RESULTS: OSA-derived exosomes caused fat accumulation and macrophage activation in the liver tissue. These exosomes promoted fat accumulation; steatosis was more noticeable in the presence of macrophages. Macrophages could internalize OSA-derived exosomes, which promoted macrophage polarization to the M1 type. Moreover, it inhibited sirtuin-3 (SIRT3)/AMP-activated protein kinase (AMPK) and autophagy and promoted the activation of nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasomes. The use of 3-methyladenine (3-MA) to inhibit autophagy blocked NLRP3 inflammasome activation and inhibited the M1 polarization of macrophages. miR-421 targeting inhibited SIRT3 protein expression in the macrophages. miR-421 was significantly increased in OSA-derived exosomes. Additionally, miR-421 levels were increased in OSA + NAFLD mice- and patient-derived exosomes. In the liver tissues of OSA and OSA + NAFLD mice, miR-421 displayed similar co-localization with the macrophages. Intermittent hypoxia-induced hepatocytes deliver miR-421 to the macrophages via exosomes to inhibit SIRT3, thereby participating in macrophage M1 polarization. After OSA and NAFLD modeling in miR-421-/- mice, liver steatosis and M1 polarization were significantly reduced. Additionally, in the case of miR-421 knockout, the inhibitory effects of OSA-derived exosomes on SIRT3 and autophagy were significantly alleviated. Furthermore, their effects on liver steatosis and macrophage M1 polarization were significantly reduced. CONCLUSIONS: OSA promotes the delivery of miR-421 from the hepatocytes to macrophages. Additionally, it promotes M1 polarization by regulating the SIRT3/AMPK-autophagy pathway, thereby causing NAFLD.

2-APQC, a small-molecule activator of Sirtuin-3 (SIRT3), alleviates myocardial hypertrophy and fibrosis by regulating mitochondrial homeostasis.

Sirtuin 3 (SIRT3) is well known as a conserved nicotinamide adenine dinucleotide+ (NAD+)-dependent deacetylase located in the mitochondria that may regulate oxidative stress, catabolism and ATP production. Accumulating evidence has recently revealed that SIRT3 plays its critical roles in cardiac fibrosis, myocardial fibrosis and even heart failure (HF), through its deacetylation modifications. Accordingly, discovery of SIRT3 activators and elucidating their underlying mechanisms of HF should be urgently needed. Herein, we identified a new small-molecule activator of SIRT3 (named 2-APQC) by the structure-based drug designing strategy. 2-APQC was shown to alleviate isoproterenol (ISO)-induced cardiac hypertrophy and myocardial fibrosis in vitro and in vivo rat models. Importantly, in SIRT3 knockout mice, 2-APQC could not relieve HF, suggesting that 2-APQC is dependent on SIRT3 for its protective role. Mechanically, 2-APQC was found to inhibit the mammalian target of rapamycin (mTOR)-p70 ribosomal protein S6 kinase (p70S6K), c-jun N-terminal kinase (JNK) and transforming growth factor-ß (TGF-ß)/ small mother against decapentaplegic 3 (Smad3) pathways to improve ISO-induced cardiac hypertrophy and myocardial fibrosis. Based upon RNA-seq analyses, we demonstrated that SIRT3-pyrroline-5-carboxylate reductase 1 (PYCR1) axis was closely assoiated with HF. By activating PYCR1, 2-APQC was shown to enhance mitochondrial proline metabolism, inhibited reactive oxygen species (ROS)-p38 mitogen activated protein kinase (p38MAPK) pathway and thereby protecting against ISO-induced mitochondrialoxidative damage. Moreover, activation of SIRT3 by 2-APQC could facilitate AMP-activated protein kinase (AMPK)-Parkin axis to inhibit ISO-induced necrosis. Together, our results demonstrate that 2-APQC is a targeted SIRT3 activator that alleviates myocardial hypertrophy and fibrosis by regulating mitochondrial homeostasis, which may provide a new clue on exploiting a promising drug candidate for the future HF therapeutics.

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.

SIRT1 restores mitochondrial structure and function in rats by activating SIRT3 after cerebral ischemia/reperfusion injury.

Mitochondrial dysfunction contributes to cerebral ischemia-reperfusion (CI/R) injury, which can be ameliorated by Sirtuin-3 (SIRT3). Under stress conditions, the SIRT3-promoted mitochondrial functional recovery depends on both its activity and expression. However, the approach to enhance SIRT3 activity after CI/R injury remains unelucidated. In this study, Sprague-Dawley (SD) rats were intracranially injected with either adeno-associated viral Sirtuin-1 (AAV-SIRT1) or AAV-sh_SIRT1 before undergoing transient middle cerebral artery occlusion (tMCAO). Primary cortical neurons were cultured and transfected with lentiviral SIRT1 (LV-SIRT1) and LV-sh_SIRT1 respectively before oxygen-glucose deprivation/reoxygenation (OGD/R). Afterwards, rats and neurons were respectively treated with a selective SIRT3 inhibitor, 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP). The expression, function, and related mechanism of SIRT1 were investigated by Western Blot, flow cytometry, immunofluorescence staining, etc. After CI/R injury, SIRT1 expression decreased in vivo and in vitro. The simulation and immune-analyses reported strong interaction between SIRT1 and SIRT3 in the cerebral mitochondria before and after CI/R. SIRT1 overexpression enhanced SIRT3 activity by increasing the deacetylation of SIRT3, which ameliorated CI/R-induced cerebral infarction, neuronal apoptosis, oxidative stress, neurological and motor dysfunction, and mitochondrial respiratory chain dysfunction, promoted mitochondrial biogenesis, and retained mitochondrial integrity and mitochondrial morphology. Meanwhile, SIRT1 overexpression alleviated OGD/R-induced neuronal death and mitochondrial bioenergetic deficits. These effects were reversed by AAV-sh_SIRT1 and the neuroprotective effects of SIRT1 were partially offset by 3-TYP. These results suggest that SIRT1 restores the structure and function of mitochondria by activating SIRT3, offering neuroprotection against CI/R injury, which signifies a potential approach for the clinical management of cerebral ischemia.

Colorectal cancer cells with stably expressed SIRT3 demonstrate proliferating retardation by Wnt/ß-catenin cascade inactivation.

Colorectal cancer (CRC) is a typical and lethal digestive system malignancy. In this study, we investigated the effect of sirtuin 3 (SIRT3) expression, a fidelity mitochondrial protein, on the proliferation of CRC cells and the mechanisms involved. Using the University of Alabama at Birmingham Cancer Data Analysis Portal database and the Clinical Proteomic Tumour Analysis Consortium database, we discovered that low expression of SIRT3 in CRC was a negative factor for survival prognosis (P < .05). Meanwhile, SIRT3 expression was correlated with distant metastasis and tumour, node, metastasis stage of CRC patients (P < .05). Subsequently, we observed that CRC cells with stable SIRT3 expression exhibited a significant decrease in proliferative capacities both in vitro and in vivo, compared to their counterparts (P < .05). Further investigation using western blot, immunoprecipitation and TOPflash/FOPflash assay showed the mechanism of growth retardation of these cells was highly associated with the degradation of ß-catenin in cytosol, and the localization of ß-catenin/α-catenin complex in the nucleus. In conclusion, our findings suggest that the inhibition of CRC cell proliferation by SIRT3 is closely associated with the inactivation of the Wnt/ß-catenin signalling pathway.