SIRT1 Serum Concentrations in Lipodystrophic Syndromes.

Lipodystrophies (LDs) are rare, complex disorders of the adipose tissue characterized by selective fat loss, altered adipokine profile and metabolic impairment. Sirtuins (SIRTs) are class III NAD+-dependent histone deacetylases linked to fat metabolism. SIRT1 plays a critical role in metabolic health by deacetylating target proteins in tissue types including liver, muscle, and adipose. Circulating SIRT1 levels have been found to be reduced in obesity and increased in anorexia nervosa and patients experiencing weight loss. We evaluated circulating SIRT1 levels in relation to fat levels in 32 lipodystrophic patients affected by congenital or acquired LDs compared to non-LD subjects (24 with anorexia nervosa, 22 normal weight, and 24 with obesity). SIRT1 serum levels were higher in LDs than normal weight subjects (mean ± SEM 4.18 ± 0.48 vs. 2.59 ± 0.20 ng/mL) and subjects with obesity (1.7 ± 0.39 ng/mL), whereas they were close to those measured in anorexia nervosa (3.44 ± 0.46 ng/mL). Our findings show that within the LD group, there was no relationship between SIRT1 levels and the amount of body fat. The mechanisms responsible for secretion and regulation of SIRT1 in LD deserve further investigation.

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.

Excess glucose alone depress young mesenchymal stromal/stem cell osteogenesis and mitochondria activity within hours/days via NAD+/SIRT1 axis.

BACKGROUND: The impact of global overconsumption of simple sugars on bone health, which peaks in adolescence/early adulthood and correlates with osteoporosis (OP) and fracture risk decades, is unclear. Mesenchymal stromal/stem cells (MSCs) are the progenitors of osteoblasts/bone-forming cells, and known to decrease their osteogenic differentiation capacity with age. Alarmingly, while there is correlative evidence that adolescents consuming greatest amounts of simple sugars have the lowest bone mass, there is no mechanistic understanding on the causality of this correlation. METHODS: Bioinformatics analyses for energetics pathways involved during MSC differentiation using human cell information was performed. In vitro dissection of normal versus high glucose (HG) conditions on osteo-/adipo-lineage commitment and mitochondrial function was assessed using multi-sources of non-senescent human and murine MSCs; for in vivo validation, young mice was fed normal or HG-added water with subsequent analyses of bone marrow CD45- MSCs. RESULTS: Bioinformatics analyses revealed mitochondrial and glucose-related metabolic pathways as integral to MSC osteo-/adipo-lineage commitment. Functionally, in vitro HG alone without differentiation induction decreased both MSC mitochondrial activity and osteogenesis while enhancing adipogenesis by 8 h' time due to depletion of nicotinamide adenine dinucleotide (NAD+), a vital mitochondrial co-enzyme and co-factor to Sirtuin (SIRT) 1, a longevity gene also involved in osteogenesis. In vivo, HG intake in young mice depleted MSC NAD+, with oral NAD+ precursor supplementation rapidly reversing both mitochondrial decline and osteo-/adipo-commitment in a SIRT1-dependent fashion within 1 ~ 5 days. CONCLUSIONS: We found a surprisingly rapid impact of excessive glucose, a single dietary factor, on MSC SIRT1 function and osteogenesis in youthful settings, and the crucial role of NAD+-a single molecule-on both MSC mitochondrial function and lineage commitment. These findings have strong implications on future global OP and disability risks in light of current worldwide overconsumption of simple sugars.

Salidroside Ameliorates Lung Injury Induced by PM 2.5by Regulating SIRT1-PGC-1α in Mice.

Objective: This study aimed to clarify the intervention effect of salidroside (SAL) on lung injury caused by PM 2.5 in mice and illuminate the function of SIRT1-PGC-1ɑ axis. Methods: Specific pathogen-free (SPF) grade male C57BL/6 mice were randomly assigned to the following groups: control group, SAL group, PM 2.5 group, SAL+PM 2.5 group. On the first day, SAL was given by gavage, and on the second day, PM 2.5 suspension was given by intratracheal instillation. The whole experiment consist of a total of 10 cycles, lasting 20 days. At the end of treatment, blood samples and lung tissues were collected and analyzed. Observation of pathological changes in lung tissue using inverted microscopy and transmission electron microscopy. The expression of inflammatory, antioxidants, apoptosis, and SIRT1-PGC-1ɑ proteins were detected by Western blotting. Results: Exposure to PM 2.5 leads to obvious morphological and pathologica changes in the lung of mice. PM 2.5 caused a decline in levels of antioxidant-related enzymes and protein expressions of HO-1, Nrf2, SOD2, SIRT1 and PGC-1ɑ, and an increase in the protein expressions of IL-6, IL-1ß, Bax, caspase-9 and cleaved caspase-3. However, SAL reversed the aforementioned changes caused by PM 2.5 by activating the SIRT1-PGC-1α pathway. Conclusion: SAL can activate SIRT1-PGC-1ɑ to ameliorate PM 2.5-induced lung injury.

[Exercise promotes irisin expression to ameliorate renal injury in type 2 diabetic rats].

OBJECTIVE: To investigate the role of irisin in exercise-induced improvement of renal function in type 2 diabetic rats. METHODS: Forty male SD rats aged 4-6 weeks were randomized into normal control group, type 2 diabetes mellitus model group, diabetic exercise (DE) group and diabetic irisin (DI) group (n=8). The rats in DE group were trained with treadmill running for 8 weeks, and those in DI group were given scheduled irisin injections for 8 weeks. After the treatments, blood biochemical parameters of the rats were examined, and renal histopathology was observed with HE, Masson and PAS staining. Western blotting was used to detect the protein expression levels in the rats'kidneys. RESULTS: The diabetic rats showed significantly increased levels of fasting insulin, total cholesterol, triglyceride, serum creatinine and blood urea nitrogen with lowered serum irisin level (all P < 0.05). Compared with those in DM group, total cholesterol, triglyceride, serum creatinine and blood urea nitrogen levels were decreased and serum irisin levels were increased in both DE and DI groups (all P < 0.05). The rats in DM group showed obvious structural disorders and collagen fiber deposition in the kidneys, which were significantly improved in DE group and DI group. Both regular exercises and irisin injections significantly ameliorated the reduction of FNDC5, LC3-II/I, Atg7, Beclin-1, p-AMPK, AMPK and SIRT1 protein expressions and lowered of p62 protein expression in the kidneys of the diabetic rats (all P < 0.05). CONCLUSION: Both exercise and exogenous irisin treatment improve nephropathy in type 2 diabetic rats possibly due to irisin-mediated activation of the AMPK/SIRT1 pathway in the kidneys to promote renal autophagy.

SIRT1 maintains bone homeostasis by regulating osteoblast glycolysis through GOT1.

The silent information regulator T1 (SIRT1) is linked to longevity and is a crucial mediator of osteoblast function. We investigated the direct role of Sirt1 during bone modeling and remodeling stages in vivo using Tamoxifen-inducible osteoblast-specific Sirt1 conditional knockout (cKO) mice. cKO mice exhibited lower trabecular and cortical bone mass in the distal femur. These phenotypes were coupled with lower bone formation and bone resorption. Metabolomics analysis revealed that the metabolites involved in glycolysis were significantly decreased in cKO mice. Further analysis of the quantitative acetylome revealed 11 proteins with upregulated acetylation levels in both the femur and calvaria of cKO mice. Cross-analysis identified four proteins with the same upregulated lysine acetylation site in both the femur and calvaria of cKO mice. A combined analysis of the metabolome and acetylome, as well as immunoprecipitation, gene knockout, and site-mutation experiments, revealed that Sirt1 deletion inhibited glycolysis by directly binding to and increasing the acetylation level of Glutamine oxaloacetic transaminase 1 (GOT1). In conclusion, our study suggested that Sirt1 played a crucial role in regulating osteoblast metabolism to maintain bone homeostasis through its deacetylase activity on GOT1. These findings provided a novel insight into the potential targeting of osteoblast metabolism for the treatment of bone-related diseases.

Punicalagin attenuates isoproterenol-induced myocardial infarction through nuclear factor erythroid 2-related factor 2/silent information regulator transcript-1-mediated inhibition of inflammation and cardiac stress markers in experimental animal models.

Myocardial infarction (MI) is a significant global health issue and the leading cause of death. Myocardial infarction (MI) is characterized by events such as damage to heart cells and stress generated by inflammation. Punicalagin (PCN), a naturally occurring bioactive compound found in pomegranates, exhibits a diverse array of pharmacological effects against many disorders. This study aimed to assess the preventive impact of PCN, with its potential anti-inflammatory and antioxidant properties, on myocardial injury caused by isoproterenol (ISO) in rats and elucidate the possible underlying mechanisms. Experimental rats were randomly categorized into four groups: control group (fed a regular diet for 15 days), PCN group (orally administered PCN at 50 mg/kg body weight (b.w.) for 15 days), ISO group (subcutaneously administered ISO (85 mg/kg b.w.) on days 14 and 15 to induce MI), and PCN+ISO group (orally preadministered PCN (50 mg/kg b.w.) for 15 days and administered ISO (85 mg/kg b.w.) on days 14 and 15). The rat cardiac tissue was then investigated for cardiac marker, oxidative stress marker, and inflammatory marker expression levels. PCN prevented ISO-induced myocardial injury, suppressing the levels of creatine kinase-myocardial band, C-reactive protein, homocysteine, cardiac troponin T, and cardiac troponin I in the rats. Moreover, PCN treatment reversed (P<0.01) the ISO-induced increase in blood pressure, attenuated lipid peroxidation markers, and depleted both enzymatic and nonenzymatic markers in the rats. Additionally, PCN inhibited (P<0.01) ISO-induced overexpression of oxidative stress markers (p-38, p-c-Jun N-terminal kinase, and p-extracellular signal-regulated kinase 1), inflammatory markers (nuclear factor-kappa B, tumor necrosis factor-alpha, and interleukin-6), and matrix metalloproteinases and decreased the levels (P<0.01) of apoptosis proteins in the rats. Nuclear factor erythroid 2-related factor 2/silent information regulator transcript-1 (Nrf2/Sirt1) is a major cellular defense protein that regulates and scavenges oxidative toxic substances through apoptosis. Therefore, overexpression of Nrf2/Sirt1 to inhibit inflammation and oxidative stress is considered a novel target for preventing MI. PCN also significantly enhanced the expression of Nrf2/Sirt1 in ISO-induced rats. Histopathological analyses of cardiac tissue revealed that PCN treatment exhibited a protective effect on the heart tissue, mitigating damage. These findings show that by activating the Nrf2/Sirt1 pathway, PCN regulates oxidative stress, inflammation, and apoptosis, hence providing protection against ISO-induced myocardial ischemia.

Mechanism of ameliorating cerebral ischemia/reperfusion injury by antioxidant inhibition of autophagy based on network pharmacology and experimental verification.

Cerebral ischemia-reperfusion injury (CIRI) is one of the most difficult challenges in cerebrovascular disease research. It is primarily caused by excessive autophagy induced by oxidative stress. Previously, a novel compound X5 was found, and the excellent antioxidant activity of it was verified in this study. Moreover, network pharmacological analysis suggested that compound X5 was closely associated with autophagy and the mTOR pathway. In vitro, X5 could significantly inhibit the expression of autophagy proteins Beclin-1 and LC3-ß, which are induced by H2O2, and promote the expression of SIRT1. In vivo, compound X5 significantly reduced the infarct size and improved the neurological function scores in the middle cerebral artery occlusion (MCAO) model of rats. In conclusion, ROS-induced autophagy is closely related to mTOR, SIRT1 and others, and X5 holds promise as a candidate for the treatment of CIRI.

BMAL1 alleviates myocardial damage in sepsis by activating SIRT1 signaling and promoting mitochondrial autophagy.

BACKGROUND: Brain and muscle arnt-like protein-1 (BMAL1) deficiency is associated with myocardial dysfunction and suppressed sirtuin 1 (SIRT1). However, whether BMAL1 promotes mitophagy via SIRT1 to alleviate myocardial injury in sepsis remains unknown. METHODS: An in vitro myocardial injury model was established using lipopolysaccharide (LPS)-treated H9C2 cells. Knockdown or overexpression of genes was performed using plasmid transfection. Gene and protein expression was assessed by qRT-PCR and Western blot, respectively. Cell proliferation was evaluated using cell counting kit-8, and cellular apoptosis and reactive oxygen species (ROS) levels were analyzed using flow cytometry. An in vivo myocardial injury model of sepsis was established by cecal ligation and puncture in rats. Myocardial function was characterized by analyzing the damage-associated proteins, inflammatory factors, ejection fraction, and fraction shortening. RESULTS: sgBMAL1 significantly decreased BMAL1 levels and remarkably increased the sensitivity of H9C2 cells to LPS stimulation, consequently enhancing LPS-induced apoptosis, inflammation, and ROS levels. These effects were further attenuated by BMAL1 overexpression. BMAL1 knockdown inhibited the expression of SIRT1 and mitophagy-associated proteins. SIRT1 overexpression reversed the enhancement of shBMAL1 on cell proliferation and inflammation. In the rat model of sepsis, BMAL1 overexpression decreased the myocardial injury-associated proteins to recover the myocardial function and suppressed inflammatory activities by promoting mitophagy via SIRT1. CONCLUSION: BMAL1 enhances mitophagy dependent on SIRT1, thereby alleviating myocardial injury in sepsis.

Effect of polydimethylsiloxane surface morphology on osteogenic differentiation of mesenchymal stem cells through SIRT1 signalling pathway.

Constructing surface topography with a certain roughness is a widely used, non-toxic, cost-effective and effective method for improving the microenvironment of cells, promoting the proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs), and promoting the osseointegration of grafts and further improving their biocompatibility under clinical environmental conditions. SIRT1 plays an important regulatory role in the osteogenic differentiation of bone marrow-derived MSCs (BM-MSCs). However, it remains unknown whether SIRT1 plays an important regulatory role in the osteogenic differentiation of BM-MSCs with regard to surface morphology. Polydimethylsiloxane (PDMS) with different surface morphologies were prepared using different grits of sandpaper. The value for BMSCs added on different surfaces was detected by cell proliferation assays. RT-qPCR and Western blotting were performed to detect SIRT1 activation and osteogenic differentiation of MSCs. Osteogenesis of MSCs was detected by alkaline phosphatase (ALP) and alizarin red S staining. SIRT1 inhibition experiments were performed to investigate the role of SIRT1 in the osteogenic differentiation of MSCs induced by surface morphology. We found that BM-MSCs have better value and osteogenic differentiation ability on a surface with roughness of PDMS-1000M. SIRT1 showed higher gene and protein expression on a PDMS-1000M surface with a roughness of 13.741 ± 1.388 µm. The promotion of the osteogenic differentiation of MSCs on the PDMS-1000M surface was significantly decreased after inhibiting SIRT1 expression. Our study demonstrated that a surface morphology with certain roughness can activate the SIRT1 pathway of MSCs and promote the osteogenic differentiation of BMSCs via the SIRT1 pathway.

SIRT1 alleviates Cd nephrotoxicity through NF-κB/p65 deacetylation-mediated pyroptosis in rat renal tubular epithelial cells.

Cadmium (Cd) is a widely distributed environmental pollutant, primarily causing nephrotoxicity through renal proximal tubular cell impairment. Pyroptosis is an inflammation-related nucleotide-binding oligomerization segment-like receptor family 3 (NLRP3)-dependent pathway for programmed cell death. We previously reported that inappropriate inflammation caused by Cd is a major contributor to kidney injury. Therefore, research on Cd-induced inflammatory response and pyroptosis may clarify the mechanisms underlying Cd-induced nephrotoxicity. In this study, we observed that Cd-induced nephrotoxicity is associated with NLRP3 inflammasome activation, leading to an increase in proinflammatory cytokine expression and secretion, as well as pyroptosis-related gene upregulation, both in primary rat proximal tubular (rPT) cells and kidney tissue from Cd-treated rats. In vitro, these effects were significantly abrogated through siRNA-based Nlrp3 silencing; thus, Cd may trigger pyroptosis through an NLRP3 inflammasome-dependent pathway. Moreover, Cd exposure considerably elevated reactive oxygen species (ROS) content. N-acetyl-l-cysteine, an ROS scavenger, mitigated Cd-induced NLRP3 inflammasome activation and subsequent pyroptosis. Mechanistically, Cd hindered the expression and deacetylase activity of SIRT1, eventually leading to a decline in SIRT1-p65 interactions, followed by an elevation in acetylated p65 levels. The administration of resveratrol (a SIRT1 agonist) or overexpression of Sirt1 counteracted Cd-induced RELA/p65/NLRP3 pathway activation considerably, leading to pyroptosis. This is the first study to reveal significant contributions of SIRT1-triggered p65 deacetylation to pyroptosis and its protective effects against Cd-induced chronic kidney injury. Our results may aid in developing potential therapeutic strategies for preventing Cd-induced pyroptosis through SIRT1-mediated p65 deacetylation.

Nicotinamide Mononucleotide improves oocyte maturation of mice with type 1 diabetes.

BACKGROUND: The number of patients with type 1 diabetes rises rapidly around the world in recent years. Maternal diabetes has a detrimental effect on reproductive outcomes due to decreased oocyte quality. However, the strategies to improve the oocyte quality and artificial reproductive technology (ART) efficiency of infertile females suffering from diabetes have not been fully studied. In this study, we aimed to examine the effects of nicotinamide mononucleotide (NMN) on oocyte maturation of mouse with type 1 diabetes mouse and explore the underlying mechanisms of NMN's effect. METHODS: Streptozotocin (STZ) was used to establish the mouse models with type 1 diabetes. The successful establishment of the models was confirmed by the results of body weight test, fasting blood glucose test and haematoxylin and eosin (H&E) staining. The in vitro maturation (IVM) rate of oocytes from diabetic mice was examined. Immunofluorescence staining (IF) was performed to examine the reactive oxygen species (ROS) level, spindle/chromosome structure, mitochondrial function, actin dynamics, DNA damage and histone modification of oocytes, which are potential factors affecting the oocyte quality. The quantitative reverse transcription PCR (RT-qPCR) was used to detect the mRNA levels of Sod1, Opa1, Mfn2, Drp1, Sirt1 and Sirt3 in oocytes. RESULTS: The NMN supplementation increased the oocyte maturation rate of the mice with diabetes. Furthermore, NMN supplementation improved the oocyte quality by rescuing the actin dynamics, reversing meiotic defects, improving the mitochondrial function, reducing ROS level, suppressing DNA damage and restoring changes in histone modifications of oocytes collected from the mice with diabetes. CONCLUSION: NMN could improve the maturation rate and quality of oocytes in STZ-induced diabetic mice, which provides a significant clue for the treatment of infertility of the patients with diabetes.

Esculin suppresses the PERK-eIF2α-CHOP pathway by enhancing SIRT1 expression in oxidative stress-induced rat chondrocytes, mitigating osteoarthritis progression in a rat model.

OBJECTIVE: Osteoarthritis (OA) is a degenerative disease characterized by the gradual degeneration of chondrocytes, involving endoplasmic reticulum (ER) stress. Esculin is a natural compound with antioxidant, anti-inflammatory and anti-tumor properties. However, its impact on ER stress in OA therapy has not been thoroughly investigated. We aim to determine the efficiency of Esculin in OA treatment and its underlying mechanism. METHODS: We utilized the tert-butyl hydroperoxide (TBHP) to establish OA model in chondrocytes. The expression of SIRT1, PERK/eIF2α pathway-related proteins, apoptosis-associated proteins and ER stress-related proteins were detected by Western blot and Real-time PCR. The apoptosis was evaluated by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. X-ray imaging, Hematoxylin & Eosin staining, Safranin O staining and immunohistochemistry were used to assess the pharmacological effects of Esculin in the anterior cruciate ligament transection (ACLT) rat OA model. RESULTS: Esculin downregulated the expression of PERK/eIF2α pathway-related proteins, apoptosis-associated proteins and ER stress-related proteins, while upregulated the expression of SIRT1 and Bcl2 in the TBHP-induced OA model in vitro. It was coincident with the results of TUNEL staining and flow cytometry. We further confirmed the protective effect of Esculin in the rat ACLT-related model. CONCLUSION: Our results suggest the potential therapeutic value of Esculin on osteoarthritis. It probably inhibits the PERK-eIF2α-ATF4-CHOP pathway by upregulating SIRT1, thereby mitigating endoplasmic reticulum stress and protecting chondrocytes from apoptosis.

miRNA-142-3p aggravates hydrogen peroxide-induced human umbilical vein endothelial cell premature senescence by targeting SIRT1.

Vascular endothelial cell premature senescence plays an important part in stroke. Many microRNAs (miRNAs) are known to be involved in the pathological process of vascular endothelial cell premature senescence. The present study aimed to investigate the mechanism of hydrogen peroxide (H2O2)-induced premature senescence in human umbilical vein endothelial cells (HUVECs) and effect of miR-142-3p on hydrogen peroxide (H2O2)-induced premature senescence. HUVECs were exposed to H2O2 to establish a model premature senescence in endothelial cells. CCK-8 assay was performed to detect cell viability. Senescence-associated ß-galactosidase staining assay and senescence-related proteins p16 and p21 were used to detect changes in the degree of cell senescence. RT-qPCR and Western blot were conducted to measure mRNA and protein levels, respectively. The scratch wound-healing assay, transwell assay, and EdU assay were performed to evaluate the ability of migration and proliferation, respectively. miRNA-142-3p and silencing information regulator 2 related enzyme 1 (SIRT1) binding was verified using Targetscan software and a dual-luciferase assay. We found that miRNA-142-3p is abnormally up-regulated in HUVECs treated with H2O2. Functionally, miRNA-142-3p inhibition may mitigate the degree of HUVEC senescence and improve HUVEC migration and proliferation. Mechanistically, SIRT1 was validated to be targeted by miRNA-142-3p in HUVECs. Moreover, SIRT1 inhibition reversed the effects of miRNA-142-3p inhibition on senescent HUVECs exposed to H2O2. To our knowledge, this is the first study to show that miRNA-142-3p ameliorates H2O2-induced HUVECs premature senescence by targeting SIRT1 and may shed light on the role of the miR-142-3p/SIRT1 axis in stroke treatment.

Apoptotic Effect of Isoimpertorin via Inhibition of c-Myc and SIRT1 Signaling Axis.

Though Isoimperatorin from Angelicae dahuricae is known to have antiviral, antidiabetic, anti-inflammatory and antitumor effects, its underlying antitumor mechanism remains elusive so far. Hence, the apoptotic mechanism of Isoimperatorin was explored in hepatocellular carcinomas (HCCs). In this study, Isoimperatorin inhibited the viability of Huh7 and Hep3B HCCs and increased the subG1 apoptotic portion and also abrogated the expression of pro-poly-ADP ribose polymerase (pro-PARP) and pro-caspase 3 in Huh7 and Hep3B cells. Also, Isoimperatorin abrogated the expression of cyclin D1, cyclin E1, CDK2, CDK4, CDK6 and increased p21 as G1 phase arrest-related proteins in Huh7 and Hep3B cells. Interestingly, Isoimperatorin reduced the expression and binding of c-Myc and Sirtuin 1 (SIRT1) by Immunoprecipitation (IP), with a binding score of 0.884 in Huh7 cells. Furthermore, Isoimperatorin suppressed the overexpression of c-Myc by the proteasome inhibitor MG132 and also disturbed cycloheximide-treated c-Myc stability in Huh7 cells. Overall, these findings support the novel evidence that the pivotal role of c-Myc and SIRT1 is critically involved in Isoimperatorin-induced apoptosis in HCCs as potent molecular targets in liver cancer therapy.

ADNP dysregulates methylation and mitochondrial gene expression in the cerebellum of a Helsmoortel-Van der Aa syndrome autopsy case.

BACKGROUND: Helsmoortel-Van der Aa syndrome is a neurodevelopmental disorder in which patients present with autism, intellectual disability, and frequent extra-neurological features such as feeding and gastrointestinal problems, visual impairments, and cardiac abnormalities. All patients exhibit heterozygous de novo nonsense or frameshift stop mutations in the Activity-Dependent Neuroprotective Protein (ADNP) gene, accounting for a prevalence of 0.2% of all autism cases worldwide. ADNP fulfills an essential chromatin remodeling function during brain development. In this study, we investigated the cerebellum of a died 6-year-old male patient with the c.1676dupA/p.His559Glnfs*3 ADNP mutation. RESULTS: The clinical presentation of the patient was representative of the Helsmoortel-Van der Aa syndrome. During his lifespan, he underwent two liver transplantations after which the child died because of multiple organ failure. An autopsy was performed, and various tissue samples were taken for further analysis. We performed a molecular characterization of the cerebellum, a brain region involved in motor coordination, known for its highest ADNP expression and compared it to an age-matched control subject. Importantly, epigenome-wide analysis of the ADNP cerebellum identified CpG methylation differences and expression of multiple pathways causing neurodevelopmental delay. Interestingly, transcription factor motif enrichment analysis of differentially methylated genes showed that the ADNP binding motif was the most significantly enriched. RNA sequencing of the autopsy brain further identified downregulation of the WNT signaling pathway and autophagy defects as possible causes of neurodevelopmental delay. Ultimately, label-free quantification mass spectrometry identified differentially expressed proteins involved in mitochondrial stress and sirtuin signaling pathways amongst others. Protein-protein interaction analysis further revealed a network including chromatin remodelers (ADNP, SMARCC2, HDAC2 and YY1), autophagy-related proteins (LAMP1, BECN1 and LC3) as well as a key histone deacetylating enzyme SIRT1, involved in mitochondrial energy metabolism. The protein interaction of ADNP with SIRT1 was further biochemically validated through the microtubule-end binding proteins EB1/EB3 by direct co-immunoprecipitation in mouse cerebellum, suggesting important mito-epigenetic crosstalk between chromatin remodeling and mitochondrial energy metabolism linked to autophagy stress responses. This is further supported by mitochondrial activity assays and stainings in patient-derived fibroblasts which suggest mitochondrial dysfunctions in the ADNP deficient human brain. CONCLUSION: This study forms the baseline clinical and molecular characterization of an ADNP autopsy cerebellum, providing novel insights in the disease mechanisms of the Helsmoortel-Van der Aa syndrome. By combining multi-omic and biochemical approaches, we identified a novel SIRT1-EB1/EB3-ADNP protein complex which may contribute to autophagic flux alterations and impaired mitochondrial metabolism in the Helsmoortel-Van der Aa syndrome and holds promise as a new therapeutic target.

Geneticin ameliorates pulmonary fibrosis by attenuating the TGF-ß/Smad via modulating AMPK/SIRT1 signaling.

AIM: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive condition with unknown aetiology that causes the lung parenchyma to scar incessantly, lowering the quality of life and hastening death. In this investigation, we studied the anti-fibrotic activity of Geneticin (a derivative of gentamycin) using in vitro and in vivo models. MAIN METHODS: The TGF-ß-mediated differentiation model was adopted to investigate (fibrotic marker's levels/expression) the anti-fibrotic activity of geneticin (GNC) in in-vitro scenarios (LL29 and DHLF cells). In vivo, the bleomycin (BLM)-induced pulmonary fibrosis model was employed by administering BLM intratracheally. Post 14 days of BLM administration, animals were treated with geneticin (6.25, 12.5, and 25 mg·kg-1) for another 14 days, and their therapeutic effect was investigated using a spectrum of techniques. KEY FINDINGS: RTqPCR and western-blot results revealed that geneticin treatment significantly attenuated the TGF-ß/BLM mediated fibrotic cascade of markers in both in-vitro and in-vivo models respectively. Further, the BLM-induced pulmonary fibrosis model revealed, that geneticin dose-dependently reduced the BLM-induced inflammatory cell infiltrations, and thickness of the alveoli walls, improved the structural distortion of the lung, and aided in improving the survival rate of the rats. Picrosirus and Masson's trichrome staining indicated that geneticin therapy reduced collagen deposition and, as a result, lung functional characteristics were improved as assessed by flexivent. Mechanistic studies have shown that geneticin reduced fibrosis by attenuating the TGF-ß/Smad through modulating the AMPK/SIRT1 signaling. SIGNIFICANCE: These findings suggest that geneticin may be a promising therapeutic agent for the treatment of pulmonary fibrosis in clinical settings.

Nephroprotective effect of Ginsenoside Rg1 in lipopolysaccharide-induced sepsis in mice through the SIRT1/NF-κB signaling.

INTRODUCTION: During sepsis, the kidney is one of the most vulnerable organs. Sepsis-associated acute kidney injury (S-AKI) is hallmarked by renal inflammation, apoptosis, and oxidative injury. Ginsenoside Rg1 (Rg1) is a natural product that possesses abundant pharmacological actions and protects against many sepsis-related diseases. Nevertheless, its role and related mechanism in S-AKI remain to be determined. MATERIALS AND METHODS: S-AKI was induced using lipopolysaccharide (LPS, 10 mg/kg) via a single intraperitoneal injection. Rg1 (200 mg/kg) was intraperitoneally administered for 3 consecutive days before LPS treatment. For histopathological examination, murine kidney tissues were stained with hematoxylin and eosin. Tubular injury score was calculated to evaluate kidney injury. Serum creatinine and BUN levels were measured for assessing renal dysfunction. The levels and activities of oxidative stress markers (MDA, 4-HNE, PC, GSH, SOD, and CAT) in renal tissue were measured by corresponding kits. Renal cell apoptosis was detected by TUNEL staining. The protein levels of apoptosis-related markers (Bcl-2, Bax, and Cleaved caspase-3), proinflammatory factors, SIRT1, IκBα, p-NF-κB p65, and NF-κB p65 in kidneys were determined using western blotting. Immunofluorescence staining was employed to assess p-NF-κB p65 expression in renal tissues. RESULTS: LPS-induced injury of kidneys and renal dysfunction in mice were ameliorated by Rg1. Rg1 also impeded LPS-evoked renal cell apoptosis in kidneys. Moreover, Rg1 attenuated LPS-triggered inflammation and oxidative stress in kidneys by inhibiting proinflammatory cytokine release, enhancing antioxidant levels and activities, and reducing lipid peroxidation. However, all these protective effects of Rg1 in LPS-induced AKI mice were reversed by EX527, an inhibitor of sirtuin 1 (SIRT1). Mechanistically, Rg1 upregulated SIRT1 protein expression, increased SIRT1 activity, and inactivated NF-κB signaling in the kidney of LPS-induced AKI mice, which was also reversed by EX527. CONCLUSIONS: Rg1 ameliorates LPS-induced kidney injury and suppresses renal inflammation, apoptosis, and oxidative stress in mice via regulating the SIRT1/NF-κB signaling.

Pyrroloquinoline quinone promotes human mesenchymal stem cell-derived mitochondria to improve premature ovarian insufficiency in mice through the SIRT1/ATM/p53 pathway.

BACKGROUND: DNA damage and oxidative stress induced by chemotherapy are important factors in the onset of premature ovarian insufficiency (POI). Studies have shown that mitochondria derived from mesenchymal stem cells (MSC-Mito) are beneficial for age-related diseases, but their efficacy alone is limited. Pyrroloquinoline quinone (PQQ) is a potent antioxidant with significant antiaging and fertility enhancement effects. This study aimed to investigate the therapeutic effect of MSC-Mito in combination with PQQ on POI and the underlying mechanisms involved. METHODS: A POI animal model was established in C57BL/6J mice by cyclophosphamide and busulfan. The effects of MSC-Mito and PQQ administration on the estrous cycle, ovarian pathological damage, sex hormone secretion, and oxidative stress in mice were evaluated using methods such as vaginal smears and ELISAs. Western blotting and immunohistochemistry were used to assess the expression of SIRT1, PGC-1α, and ATM/p53 pathway proteins in ovarian tissues. A cell model was constructed using KGN cells treated with phosphoramide mustard to investigate DNA damage and apoptosis through comet assays and flow cytometry. SIRT1 siRNA was transfected into KGN cells to further explore the role of the SIRT1/ATM/p53 pathway in combination therapy with MSC-Mito and PQQ for POI. RESULTS: The combined treatment of MSC-Mito and PQQ significantly restored ovarian function and antioxidant capacity in mice with POI. This treatment also reduced the loss of follicles at various stages, improving the disrupted estrous cycle. In vitro experiments demonstrated that PQQ facilitated the proliferation of MitoTracker-labelled MSC-Mito, synergistically restoring mitochondrial function and inhibiting oxidative stress in combination with MSC-Mito. Both in vivo and in vitro, the combination of MSC-Mito and PQQ increased mitochondrial biogenesis mediated by SIRT1 and PGC-1α while inhibiting the activation of ATM and p53, consequently reducing DNA damage-mediated cell apoptosis. Furthermore, pretreatment of KGN cells with SIRT1 siRNA reversed nearly all the aforementioned changes induced by the combined treatment. CONCLUSIONS: Our research findings indicate that PQQ facilitates MSC-Mito proliferation and, in combination with MSC-Mito, ameliorates chemotherapy-induced POI through the SIRT1/ATM/p53 signaling pathway.

Water-soluble 4-(dimethylaminomethyl)heliomycin exerts greater antitumor effects than parental heliomycin by targeting the tNOX-SIRT1 axis and apoptosis in oral cancer cells.

The antibiotic heliomycin (resistomycin), which is generated from Streptomyces resistomycificus, has multiple activities, including anticancer effects. Heliomycin was first described in the 1960s, but its clinical applications have been hindered by extremely low solubility. A series of 4-aminomethyl derivatives of heliomycin were synthesized to increase water solubility; studies showed that they had anti-proliferative effects, but the drug targets remained unknown. In this study, we conducted cellular thermal shift assays (CETSA) and molecular docking simulations to identify and validate that heliomycin and its water-soluble derivative, 4-(dimethylaminomethyl)heliomycin (designated compound 4-dmH) engaged and targeted with sirtuin-1 (SIRT1) in p53-functional SAS and p53-mutated HSC-3 oral cancer cells. We further addressed the cellular outcome of SIRT1 inhibition by these compounds and found that, in addition to SIRT1, the water-soluble 4-dmH preferentially targeted a tumor-associated NADH oxidase (tNOX, ENOX2). The direct binding of 4-dmH to tNOX decreased the oxidation of NADH to NAD+ which diminished NAD+-dependent SIRT1 deacetylase activity, ultimately inducing apoptosis and significant cytotoxicity in both cell types, as opposed to the parental heliomycin-induced autophagy. We also observed that tNOX and SIRT1 were both upregulated in tumor tissues of oral cancer patients compared to adjacent normal tissues, suggesting their clinical relevance. Finally, the better therapeutic efficacy of 4-dmH was confirmed in tumor-bearing mice, which showed greater tNOX and SIRT1 downregulation and tumor volume reduction when treated with 4-dmH compared to heliomycin. Taken together, our in vitro and in vivo findings suggest that the multifaceted properties of water-soluble 4-dmH enable it to offer superior antitumor value compared to parental heliomycin, and indicated that it functions through targeting the tNOX-NAD+-SIRT1 axis to induce apoptosis in oral cancer cells.