Electroacupuncture Mediates Fat Metabolism and Autophagy via a Sirt3-Dependent Mechanism in Mice Fed High-Fat Diet.

This study investigates the therapeutic potential of electroacupuncture (EA) on obesity, focusing on its influence on autophagy and energy metabolism, utilizing a high-fat diet (HFD)-induced mouse model. Treatment with EA significantly reduces body weight, fat deposition, and lipid accumulation in HFD-fed mice. Additionally, EA effectively ameliorates metabolic imbalances, reducing blood glucose levels and plasma markers of liver function. At the molecular level, EA enhances the expression of thermogenesis-associated genes in brown adipose tissue and decreases p53 expression, suggesting a decrease in apoptosis. Autophagy in white adipose tissue is inhibited by EA, as demonstrated by the suppression of key autophagy-related proteins. Further experiments highlight the critical role of Sirtuin 3 (Sirt3) in EA's anti-obesity effects. Sirt3 supplementation combined with EA results in reduced body weight, fat deposition, and lipid accumulation, along with modulations in key metabolic indicators. Moreover, EA's modulatory effect on uncoupling protein 1 (Ucp1), Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc-1α), and p53 is found to be Sirt3 dependent. In conclusion, EA exerts beneficial effects against obesity through Sirt3-dependent modulation of autophagy and energy metabolism, indicating a potential therapeutic approach for obesity and related metabolic disorders.

Auranofin attenuates Schistosoma mansoni egg-induced liver granuloma and fibrosis in mice.

Schistosomiasis is a serious tropical disease. Despite extensive research into the etiology of liver fibrosis, effective therapeutic options remain limited. This study aims to assess the effectiveness of auranofin in treating hepatic granuloma and fibrogenesis produced by Schistosoma (S.) mansoni eggs. Auranofin is a gold complex that contains thioglucose tetraacetate and triethylphosphine. Eighty BALB/c male mice were divided into four groups (n=20/group): negative control (GI), positive control (GII), and early (GIII) and late (GIV) treatment groups with oral auranofin according to beginning of treatment 4th week and 6th week post-infection. Mice were infected subcutaneously in a dose of 60±10 cercariae/mouse. Worm counts, egg loads, and oogram patterns were determined. Biochemical, histological, and immunostaining of interleukin-1ß (IL-1ß), Sirtuin 3 (SIRT3), and smooth muscle actin (SMA) were assessed. GIII showed a significant decrease in the total S. mansoni worm burden and ova/gram in liver tissue (with reduction percent of 63.07% and 78.26%, respectively). Schistosomal oogram patterns, immature and mature ova, also showed a significant decrease. The reduction in granuloma number and size was 40.63% and 48.66%, respectively, in GIII, whereas in GIV, the reduction percent was 76.63% and 67.08%. In addition, the degree of fibrosis was significantly diminished in both treated groups. GIV showed significant reduction in IL-1ß and SMA expression and increase in SIRT3 expression. These findings reveal how auranofin suppresses the development of liver fibrosis. Therefore, it is crucial to take another look at auranofin as a prospective medication for the treatment of S. mansoni egg-induced hepatic granuloma and consequent fibrosis.

Salvianolic Acid B Inhibits Oxidative Stress in Glomerular Mesangial Cells Alleviating Diabetic Nephropathy by Regulating SIRT3/FOXO1 Signaling.

INTRODUCTION: Oxidative stress is pivotal in advancing diabetic nephropathy (DN). Salvianolic acid B (SAB), derived from Radix Salviae miltiorrhizae, exhibits renoprotective effects. However, the mechanisms underlying its action in DN are not fully elucidated. This study explores SAB's protective effect on DN, focusing on its antioxidative properties in glomerular mesangial cells. METHODS: The renoprotective effects of various SAB dosages on DN rats were assessed by evaluating kidney tissue pathological alterations through hematoxylin and eosin, periodic acid-Schiff, Masson, TUNEL staining, and kidney function through biochemical detection. Cell counting kit-8 and lactate dehydrogenase cytotoxicity assays were utilized to evaluate the viability of high glucose (HG)-induced HBZY-1 cells treated with various SAB dosages. Oxidative stress and inflammation levels were measured using enzyme-linked immunosorbent assay kits. The Sirtuin 3 (SIRT3)/Forkhead box transcription factor O1 (FOXO1) pathway was examined through Western blot and immunohistochemistry. RESULTS: SAB mitigated kidney histopathological alterations and function and cell apoptosis in DN rats at various dosages. It enhanced the activity of glutathione peroxidase and superoxide dismutase while decreasing reactive oxygen species and malondialdehyde levels both in vivo and in vitro. SAB also suppressed the levels of pro-inflammatory cytokines (IL-1ß, IL-6, MCP-1, and TNF-α) and the expression of collagen IV and fibronectin in HG-induced HBZY-1 cells. Furthermore, SAB activated the SIRT3/FOXO1 signaling pathway. CONCLUSION: Our findings suggest that SAB may alleviate oxidative stress in DN both in vivo and in vitro, potentially through the activation of the SIRT3/FOXO1-mediated signaling pathway. This study provides initial insights into the possible antioxidative and renoprotective effects of SAB, indicating its potential utility as a therapeutic agent for DN.

PACAP-Sirtuin3 alleviates cognitive impairment through autophagy in Alzheimer's disease.

BACKGROUND: Autophagy is vital in the pathogenesis of neurodegeneration. Thus far, no studies have specifically investigated the relationship between pituitary adenylate cyclase-activating polypeptide (PACAP) and autophagy, particularly in the context of Alzheimer's disease (AD). This study used in vitro and in vivo models, along with clinical samples, to explore interactions between PACAP and autophagy in AD. METHODS: AD model mice were administered 6 µl of 0.1 mg/ml PACAP liquid intranasally for 4 weeks, then subjected to behavioral analyses to assess the benefits of PACAP treatment. The underlying mechanisms of PACAP-induced effects were investigated by methods including real-time quantitative polymerase chain reaction, RNA sequencing, immunofluorescence, and western blotting. Exosomes were extracted from human serum and subjected to enzyme-linked immunosorbent assays to examine autophagy pathways. The clinical and therapeutic implications of PACAP and autophagy were extensively investigated throughout the experiment. RESULTS: Impaired autophagy was a critical step in amyloid ß (Aß) and Tau deposition; PACAP enhanced autophagy and attenuated cognitive impairment. RNA sequencing revealed three pathways that may be involved in AD progression: PI3K-AKT, mTOR, and AMPK. In vivo and in vitro studies showed that sirtuin3 knockdown diminished the ability of PACAP to restore normal autophagy function, resulting in phagocytosis dysregulation and the accumulation of pTau, Tau, and Aß. Additionally, the autophagic biomarker MAP1LC3 demonstrated a positive association with PACAP in human serum. CONCLUSIONS: PACAP reverses AD-induced cognitive impairment through autophagy, using sirtuin3 as a key mediator. MAP1LC3 has a positive relationship with PACAP in humans. These findings provide insights regarding potential uses of intranasal PACAP and sirtuin3 agonists in AD treatment. TRIAL REGISTRATION: NCT04320368.

Retuning Mitochondrial Apoptosis/Mitophagy Balance via SIRT3-Energized and Microenvironment-Modulated Hydrogel Microspheres to Impede Osteoarthritis.

Full-range therapeutic regimens for osteoarthritis (OA) should consider organs (joints)-tissues (cartilage)-cells (chondrocytes)-organelles cascade, of which the subcellular mitochondria dominate eukaryotic cells' fate, and thus causally influence OA progression. However, the dynamic regulation of mitochondrial rise and demise in impaired chondrocytes and the exact role of mitochondrial metronome sirtuins 3 (SIRT3) is not clarified. Herein, chondrocytes are treated with SIRT3 natural agonist dihydromyricetin (DMY) or chemical antagonist 3-TYP, respectively, to demonstrate the positive action of SIRT3 on preserving cartilage extracellular matrix (ECM). Molecular mechanical investigations disclose that SIRT3-induced chondroprotection depended on the repression of mitochondrial apoptosis (mtApoptosis) and the activation of mitophagy. Inspired by the high-level matrix proteinases and reactive oxygen species (ROS) in the OA environment, by anchoring gelatin methacrylate (GelMA) and benzenediboronic acid (PBA) to hyaluronic acid methacrylate (HAMA) with microfluidic technology, a dual-responsive hydrogel microsphere laden with DMY is tactfully fabricated and named as DMY@HAMA-GelMA-PBA (DMY@HGP). In vivo injection of DMY@HGP ameliorated cartilage abrasion and subchondral bone sclerosis, as well as promoted motor function recovery in post-traumatic OA (PTOA) model via recouping endogenous mtApoptosis and mitophagy balance. Overall, this study unveils a novel mitochondrial dynamic-oriented strategy, holding great promise for the precision treatment of OA.

Melatonin attenuates lung ischemia-reperfusion injury through SIRT3 signaling-dependent mitophagy in type 2 diabetic rats.

Background: Lung ischemia-reperfusion injury (LIRI) remains the major cause of primary lung dysfunction after lung transplantation. Diabetes mellitus (DM) is an independent risk factor for morbidity and mortality following lung transplantation. Mitochondrial dysfunction is recognized as a key mediator in the pathogenesis of diabetic LIRI. Melatonin has been reported to be a safe and potent preserving mitochondrial function agent. This study aimed at investigating the potential therapeutic effect and mechanisms of melatonin on diabetic LIRI. Methods: High-fat-diet-fed streptozotocin-induced type 2 diabetic rats were exposed to melatonin, with or without administration of the SIRT3 short hairpin ribonucleic acid (shRNA) plasmid following a surgical model of ischemia-reperfusion injury of the lung. Lung function, inflammation, oxidative stress, cell apoptosis, and mitochondrial function were examined. Results: The SIRT3 signaling and mitophagy were suppressed following diabetic LIRI. Treatment with melatonin markedly induced mitophagy and restored SIRT3 expression. Melatonin treatment also attenuated subsequent diabetic LIRI by improving lung functional recovery, suppressing inflammation, decreasing oxidative damage, diminishing cell apoptosis, and preserving mitochondrial function. However, either administration of SIRT3 shRNA or an autophagy antagonist 3-methyladenine (3-MA) suppressing mitophagy, and compromised the protective action of melatonin. Conclusion: Data indicated that melatonin attenuates diabetic LIRI through activation of SIRT3 signaling-mediated mitophagy.

SIRT3 Activator Honokiol Inhibits Th17 Cell Differentiation and Alleviates Colitis.

BACKGROUND: Honokiol (HKL), a natural extract of the bark of the magnolia tree and an activator of the mitochondrial protein sirtuin-3 (SIRT3), has been proposed to possess anti-inflammatory effects. This study investigated the inhibitory effects of HKL on T helper (Th) 17 cell differentiation in colitis. METHODS: Serum and biopsies from 20 participants with ulcerative colitis (UC) and 18 healthy volunteers were collected for the test of serum cytokines, flow cytometry analysis (FACS), and relative messenger RNA (mRNA) levels of T cell subsets, as well as the expression of SIRT3 and phosphorylated signal transducer and activator of transcription/retinoic acid-related orphan nuclear receptor γt (p-STAT3/RORγt) signal pathway in colon tissues. In vitro, naïve clusters of differentiation (CD) 4 + T cells isolated from the mouse spleen differentiated to subsets including Th1, Th2, Th17, and regulatory T (Treg) cells. Peripheral blood monocytes (PBMCs) from healthy volunteers were induced to the polarization of Th17 cells. After HKL treatment, changes in T cell subsets, related cytokines, and transcription factors were measured. The dextran sulfate sodium (DSS)-induced colitis and interleukin (IL)-10-deficient mice were intraperitoneally injected with HKL. These experiments were conducted to study the effect of HKL on the development, cytokines, and expression of signaling pathway proteins in colitis. RESULTS: Patients with UC had higher serum IL-17 and a higher proportion of Th17 differentiation in blood compared with healthy participants; while IL-10 level and the proportion of Treg cells were lower. Higher relative mRNA levels of RORγt and a lower SIRT3 expression in colon tissues were observed. In vitro, HKL had little effect on the differentiation of naïve CD4+ T cells to Th1, Th2, or Treg cells, but it downregulated IL-17 levels and the Th17 cell ratio in CD4+ T cells from the mouse spleen and human PBMCs under Th17 polarization. Even with a STAT3 activator, HKL still significantly inhibited IL-17 levels. In DSS-induced colitis mice and IL-10 deficient mice treated with HKL, the length of the colon, weight loss, disease activity index, and histopathological scores were improved, IL-17 and IL-21 levels, and the proportion of Th17 cells were decreased. Sirtuin-3 expression was increased, whereas STAT3 phosphorylation and RORγt expression were inhibited in the colon tissue of mice after HKL treatment. CONCLUSIONS: Our study demonstrated that HKL could partially protect against colitis by regulating Th17 differentiation through activating SIRT3, leading to inhibition of the STAT3/RORγt signaling pathway. These results provide new insights into the protective effects of HKL against colitis and may facilitate the research of new drugs for inflammatory bowel disease.

[Cigarette Smoke Induces Gefitinib Resistance in NSCLC Cells 
via ROS/Sirt3/SOD2 Pathway].

BACKGROUND: Epidermal growth factor receptor (EGFR) gene mutations are the most common driver mutations in non-small cell lung cancer (NSCLC). To prolong the survival of the patients, EGFR tyrosine kinase inhibitors (TKIs) resistance in NSCLC is a major challenge that needs to be addressed urgently, and this study focuses on investigating the mechanism of cigarette smoke (CS) induced Gefitinib resistance in NSCLC. METHODS: PC-9 and A549 cells were cultured in vitro and treated with 1 µmol/L Gefitinib for 4 h and 10% cigarette smoke extract (CSE) for 48 h. Western blot was used to detect Sirtuin 3 (Sirt3) and superoxide dismutase 2 (SOD2) protein expressions; DCFH-DA probe was used to detect intracellular reactive oxygen species (ROS); CCK-8 kit was used to detect cell activity, and EdU was used to detect cell proliferation ability. Sirt3 overexpression plasmid (OV-Sirt3) was transfected in PC-9 and A549 cells and treated with 1 µmol/L Gefitinib for 4 h and 10% CSE for 48 h after N-acetylcysteine (NAC) action. The expressions of Sirt3 and SOD2 were detected by Western blot; the ROS level in the cells was detected by DCFH-DA probe, and the cell activity was detected by CCK-8. RESULTS: CSE induced an increase in the 50% inhibitory concentration (IC50) of both PC-9 and A549 cells to Gefitinib (P<0.01) and enhanced the proliferation of PC-9 and A549 cells, suggesting that CS induced Gefitinib resistance in NSCLC. ROS was involved in CSE-induced Gefitinib resistance (P<0.05). CSE induced low expressions of Sirt3 and SOD2 (P<0.01), and Sirt3/SOD2 was associated with poor prognosis in lung cancer patients (P<0.05). OV-Sirt3 in PC-9 and A549 cells reversed CSE-induced Gefitinib resistance (P<0.05) and significantly reduced ROS production. NAC reversed CSE-induced Gefitinib resistance in PC-9 and A549 cells (P<0.05). CONCLUSIONS: The ROS/Sirt3/SOD2 pathway is involved in CS-induced Gefitinib resistance in NSCLC.

Baicalein attenuates bleomycin-induced lung fibroblast senescence and lung fibrosis through restoration of Sirt3 expression.

CONTEXT: Fibroblast senescence was reported to contribute to the pathological development of idiopathic pulmonary fibrosis (IPF), and baicalein is reported to attenuate IPF. OBJECTIVE: This study explores whether baicalein attenuates lung fibrosis by regulating lung fibroblast senescence. MATERIALS AND METHODS: Institute of Cancer Research (ICR) mice were randomly assigned to control, bleomycin (BLM), baicalein and BLM + baicalein groups. Lung fibrosis was established by a single intratracheal dose of BLM (3 mg/kg). The baicalein group received baicalein orally (100 mg/kg/day). Sirtuin 3 (Sirt3) siRNA (50 µg) was injected through the tail vein once a week for 2 weeks to explore its effect on the anti-pulmonary fibrosis of baicalein. RESULTS: BLM-treated mice exhibited obvious lung fibrosis and fibroblast senescence by showing increased levels of collagen deposition (27.29% vs. 4.14%), hydroxyproline (208.05 vs. 40.16 ng/mg), collagen I (25.18 vs. 9.15 µg/mg), p53, p21, p16, MCP-1, PAI-1, TNF-α, MMP-10 and MMP-12 in lung tissues, which were attenuated by baicalein. Baicalein also mitigated BLM-mediated activation of TGF-ß1/Smad signalling pathway. Baicalein restored the BLM-induced downregulation of Sirt3 expression in lung tissues and silencing of Sirt3 abolished the inhibitory role of baicalein against BLM-induced lung fibrosis, fibroblast senescence and activation of TGF-ß1/Smad signalling pathway. CONCLUSIONS: Baicalein preserved the BLM-induced downregulation of lung Sirt3 expression, and thus the suppression of TGF-ß1/Smad signalling pathway and lung fibrosis, which might provide an experimental basis for treatment of IPF.

Role of Estrogen-Related Receptor γ and PGC-1α/SIRT3 Pathway in Early Brain Injury After Subarachnoid Hemorrhage.

Estrogen-related receptors (ERRs) were shown to play an important role in the regulation of free radical-mediated pathology. This study aimed to investigate the neuroprotective effect of ERRγ activation against early brain injury (EBI) after subarachnoid hemorrhage (SAH) and the potential underlying mechanisms. In a rat model of SAH, the time course of ERRs and SIRT3 and the effects of ERRγ activation were investigated. ERRγ agonist DY131, selective inhibitor GSK5182, or SIRT3 selective inhibitor 3-TYP were administered intracerebroventricularly (icv) in the rat model of SAH. The use of 3-TYP was for validating SIRT3 as the downstream signaling of ERRγ activation. Post-SAH assessments included SAH grade, neurological score, Western blot, Nissl staining, and immunofluorescence staining in rats. In an vitro study, the ERRγ agonist DY131 and ERRγ siRNA were administered to primary cortical neurons stimulated by Hb, after which cell viability and neuronal deaths were accessed. Lastly, the brain ERRγ levels and neuronal death were accessed in SAH patients. We found that brain ERRγ expressions were significantly increased, but the expression of SIRT3 dramatically decreased after SAH in rats. In the brains of SAH rats, ERRγ was expressed primarily in neurons, astrocytes, and microglia. The activation of ERRγ with DY131 significantly improved the short-term and long-term neurological deficits, accompanied by reductions in oxidative stress and neuronal apoptosis at 24 h after SAH in rats. DY131 treatment significantly increased the expressions of PGC-1α, SIRT3, and Bcl-2 while downregulating the expressions of 4-HNE and Bax. ERRγ antagonist GSK5182 and SIRT3 inhibitor 3-TYP abolished the neuroprotective effects of ERRγ activation in the SAH rats. An in vitro study showed that Hb stimulation significantly increased intracellular oxidative stress in primary cortical neurons, and DY131 reduced such elevations. Primary cortical neurons transfected with the ERRγ siRNA exhibited notable apoptosis and abolished the protective effect of DY131. The examination of SAH patients' brain samples revealed increases in ERRγ expressions and neuronal apoptosis marker CC3. We concluded that ERRγ activation with DY131 ameliorated oxidative stress and neuronal apoptosis after the experimental SAH. The effects were, at least in part, through the ERRγ/PGC-1α/SIRT3 signaling pathway. ERRγ may serve as a novel therapeutic target to ameliorate EBI after SAH.

Cigarette Smoke Induces Gefitinib Resistance in NSCLC Cells via ROS/Sirt3/SOD2 Pathway / 中国肺癌杂志

BACKGROUND@#Epidermal growth factor receptor (EGFR) gene mutations are the most common driver mutations in non-small cell lung cancer (NSCLC). To prolong the survival of the patients, EGFR tyrosine kinase inhibitors (TKIs) resistance in NSCLC is a major challenge that needs to be addressed urgently, and this study focuses on investigating the mechanism of cigarette smoke (CS) induced Gefitinib resistance in NSCLC.@*METHODS@#PC-9 and A549 cells were cultured in vitro and treated with 1 µmol/L Gefitinib for 4 h and 10% cigarette smoke extract (CSE) for 48 h. Western blot was used to detect Sirtuin 3 (Sirt3) and superoxide dismutase 2 (SOD2) protein expressions; DCFH-DA probe was used to detect intracellular reactive oxygen species (ROS); CCK-8 kit was used to detect cell activity, and EdU was used to detect cell proliferation ability. Sirt3 overexpression plasmid (OV-Sirt3) was transfected in PC-9 and A549 cells and treated with 1 µmol/L Gefitinib for 4 h and 10% CSE for 48 h after N-acetylcysteine (NAC) action. The expressions of Sirt3 and SOD2 were detected by Western blot; the ROS level in the cells was detected by DCFH-DA probe, and the cell activity was detected by CCK-8.@*RESULTS@#CSE induced an increase in the 50% inhibitory concentration (IC50) of both PC-9 and A549 cells to Gefitinib (P<0.01) and enhanced the proliferation of PC-9 and A549 cells, suggesting that CS induced Gefitinib resistance in NSCLC. ROS was involved in CSE-induced Gefitinib resistance (P<0.05). CSE induced low expressions of Sirt3 and SOD2 (P<0.01), and Sirt3/SOD2 was associated with poor prognosis in lung cancer patients (P<0.05). OV-Sirt3 in PC-9 and A549 cells reversed CSE-induced Gefitinib resistance (P<0.05) and significantly reduced ROS production. NAC reversed CSE-induced Gefitinib resistance in PC-9 and A549 cells (P<0.05).@*CONCLUSIONS@#The ROS/Sirt3/SOD2 pathway is involved in CS-induced Gefitinib resistance in NSCLC.

Therapeutic effect of Sirtuin 3 on ameliorating nonalcoholic fatty liver disease: The role of the ERK-CREB pathway and Bnip3-mediated mitophagy.

Increased mitochondrial damage is related to the progression of a diet-induced nonalcoholic fatty liver disease. The aim of our study is to investigate the role of Sirtuin 3 (Sirt3) in treating nonalcoholic fatty liver disease with a focus on mitophagy and the ERK-CREB pathway. Our data indicated that Sirt3 was downregulated in liver tissue in response to chronic HFD treatment. Interestingly, re-introduction of Sirt3 protected hepatic function, attenuated liver fibrosis, alleviated the inflammatory response, and prevented hepatocyte apoptosis. Molecular investigations demonstrated that lipotoxicity was associated with an increase in mitochondrial apoptosis as evidenced by reduced mitochondrial potential, augmented ROS production, increased cyt-c leakage into the nucleus, and activated caspase-9 apoptotic signalling. Additionally, Sirt3 overexpression protected hepatocytes against mitochondrial apoptosis via promoting Bnip3-required mitophagy. Functional studies showed that Sirt3 reversed Bnip3 expression and mitophagy activity via the ERK-CREB signalling pathway. Blockade of the ERK-CREB axis repressed the promotive effects of Sirt3 on Bnip3 activation and mitophagy augmentation, finally negating the anti-apoptotic influences of Sirt3 on hepatocytes in the setting of high-fat-stress. Collectively, our data show that high-fat-mediated liver damage is associated with Sirt3 downregulation, which is followed by ERK-CREB pathway inactivation and Bnip3-mediated inhibition of mitophagy, causing hepatocytes to undergo mitochondria-dependent cell death. Based on this, strategies for enhancing Sirt3 activity and activating the ERK-CREB-Bnip3-mitophagy pathways could be used to treat nonalcoholic fatty liver disease.

Emerging beneficial roles of sirtuins in heart failure.

Sirtuins are a highly conserved family of histone/protein deacetylases whose activity can prolong the lifespan of model organisms such as yeast, worms and flies. In mammalian cells, seven sirtuins (SIRT1-7) modulate distinct metabolic and stress-response pathways, SIRT1 and SIRT3 having been most extensively investigated in the cardiovascular system. SIRT1 and SIRT3 are mainly located in the nuclei and mitochondria, respectively. They participate in biological functions related to development of heart failure, including regulation of energy production, oxidative stress, intracellular signaling, angiogenesis, autophagy and cell death/survival. Emerging evidence indicates that the two sirtuins play protective roles in failing hearts. Here, we summarize current knowledge of sirtuin functions in the heart and discuss its translation into therapy for heart failure.