SIRT1 Deficiency Research Articles

SIRT1 mediates the inflammatory response of macrophages and regulates the TIMP3/ADAM17 pathway in atherosclerosis

ObjectiveMacrophage polarization and the resulting phenotype have versatile roles in atherosclerosis. The study aims to decipher the role of SIRT1 in regulating macrophage phenotypes and atherosclerosis development. MethodsTwo mouse lines of SIRT1△Mac/ApoE−/− and SIRT1fl/fl/ApoE−/− were fed with high-fat diet to generate atherosclerotic lesion. Mouse peritoneal macrophages were isolated and transfected with SIRT1-overexpressing vector or vector-null. ResultsThe SIRT1△Mac/ApoE−/− mice exhibited greater atherosclerotic lesions, stronger immunofluorescence staining for M1-like macrophage marker, iNOS, and weaker immunofluorescence staining for M2-like macrophage marker, Arginase-1, than the SIRT1fl/fl/ApoE−/− littermates. The gene expressions of M1 markers (IL-1β, IL-6, and iNOS) were increased and those of M2 markers (IL-10 and Arg-1) decreased in both aortic roots and peritoneal macrophages from SIRT1△Mac/ApoE−/− mice, whereas SIRT1 overexpression rectified the changes in M1/M2 expression. A declined expression of TIMP3 with an increased expression of ADAM17 was noted in SIRT1△Mac/ApoE−/− macrophages, whereas SIRT1 overexpression rescued TIMP3 expression and inhibited ADAM17 expression. ConclusionOur data suggest that SIRT1 deficiency may promote macrophage M1 polarization and regulate the TIMP3/ADAM17 pathway thus favoring atherosclerosis development, indicating an anti-atherosclerotic role of macrophage SIRT1.

Melatonin Prevents Thioacetamide-Induced Gut Leakiness and Liver Fibrosis Through the Gut-Liver Axis via Modulating Sirt1-Related Deacetylation of Gut Junctional Complex and Hepatic Proteins.

Intestinal barrier dysfunction with high serum endotoxin is common in patients with liver fibrosis, but the mechanisms underlying liver fibrosis remain unclear. Melatonin is a well-recognized antioxidant and an anti-inflammatory agent that benefits multiple organs. However, the beneficial effects of melatonin on gut leakiness-associated liver fibrosis have not been systemically studied. Here, we investigated the protective mechanisms of melatonin against thioacetamide (TAA)-induced gut barrier dysfunction and hepatic fibrosis by focusing on posttranslational protein modifications through the gut-liver axis. Our results showed that gut leakiness markers, including decreased gut tight/adherens junction proteins (TJ/AJs) with increased intestinal deformation, apoptosis, and serum endotoxin, were observed early at 1 week after TAA exposure. Liver injury, apoptosis, and fibrosis were prominent at 2 and 4 weeks. Mechanistically, we found that gut TJ/AJs were hyper-acetylated, followed by ubiquitin-dependent proteolysis, leading to their degradation and gut leakiness. Gut dysbiosis, hepatic protein hyper-acetylation, and SIRT1 downregulation were also observed. Consistently, intestinal Sirt1 deficiency greatly enhanced protein hyper-acetylation, gut leakiness, endotoxemia, and liver fibrosis. Pretreatment with melatonin prevented or improved all these changes in both the gut and liver. Furthermore, melatonin blunted protein acetylation and injury in TAA-exposed T84 human intestinal and AML12 mouse liver cells. Overall, this study demonstrated novel mechanisms by which melatonin prevents gut leakiness and liver fibrosis through the gut-liver axis by attenuating the acetylation of intestinal and hepatic proteins. Thus, melatonin consumption can become a potentially safe supplement for liver fibrosis patients by preventing protein hyper-acetylation and gut leakiness.

Hyperglycemia-induced Sirt3 downregulation increases microglial aerobic glycolysis and inflammation in diabetic neuropathic pain pathogenesis.

Hyperglycemia-induced neuroinflammation significantly contributes to diabetic neuropathic pain (DNP), but the underlying mechanisms remain unclear. To investigate the role of Sirt3, a mitochondrial deacetylase, in hyperglycemia-induced neuroinflammation and DNP and to explore potential therapeutic interventions. Here, we found that Sirt3 was downregulated in spinal dorsal horn (SDH) of diabetic mice by RNA-sequencing, which was further confirmed at the mRNA and protein level. Sirt3 deficiency exacerbated hyperglycemia-induced neuroinflammation and DNP by enhancing microglial aerobic glycolysis invivo and invitro. Overexpression of Sirt3 in microglia alleviated inflammation by reducing aerobic glycolysis. Mechanistically, high-glucose stimulation activated Akt, which phosphorylates and inactivates FoxO1. The inactivation of FoxO1 diminished the transcription of Sirt3. Besides that, we also found that hyperglycemia induced Sirt3 degradation via the mitophagy-lysosomal pathway. Blocking Akt activation by GSK69093 or metformin rescued the degradation of Sirt3 protein and transcription inhibition of Sirt3 mRNA, which substantially diminished hyperglycemia-induced inflammation. Metformin invivo treatment alleviated neuroinflammation and diabetic neuropathic pain by rescuing hyperglycemia-induced Sirt3 downregulation. Hyperglycemia induces metabolic reprogramming and inflammatory activation in microglia through the regulation of Sirt3 transcription and degradation. This novel mechanism identifies Sirt3 as a potential drug target for treating DNP.

Klotho enhances diastolic function in aged hearts through Sirt1-mediated pathways.

Aging leads to a progressive decline in cardiac function, increasing the risk of heart failure with preserved ejection fraction (HFpEF). This study elucidates the impact of α-Klotho, an anti-aging hormone, on cardiac diastolic dysfunction and explore its downstream mechanisms. Aged wild-type and heterozygous Klotho-deficient mice received daily injection of soluble α-Klotho (sKL) for 10weeks, followed by a comprehensive assessment of heart function by echocardiography, intracardiac pressure catheter, exercise tolerance, and cardiac pathology. Our findings show that klotho deficiency accentuated cardiac hypertrophy, diastolic dysfunction, and exercise intolerance, while sKL treatment ameliorates these abnormalities and improves cardiac capillary densities. Downstream of klotho, we focused on the Sirtuin1 (Sirt1) signaling pathway to elucidate the potential underlying mechanism by which Klotho improves diastolic function. We found that decreased Klotho levels were linked with Sirt1 deficiency, whereas sKL treatment restored Sirt1 expression in aged hearts and mitigated the DNA damage response pathway activation. Through tandem mass tag proteomics and unbiased acetylomics analysis, we identified 220 significantly hyperacetylated lysine sites in critical cardiac proteins of aged hearts. We found that sKL supplementation attenuated age-dependent DNA damage and cardiac diastolic dysfunction. In contrast, Klotho deficiency significantly increased hyperacetylation of several crucial cardiac contractile proteins, potentially impairing ventricular relaxation and diastolic function, thus predisposing to HFpEF. These results suggest the potential benefit of sKL supplementation as a promising therapeutic strategy for combating HFpEF in aging.

P7C3 suppresses astrocytic senescence to protect dopaminergic neurons: Implication in the mouse model of Parkinson's disease.

Astrocytic senescence is inextricably linked to aging and neurodegenerative disorders, including Parkinson's disease (PD). P7C3 is a small, neuroprotective aminopropyl carbazole compound that exhibits anti-inflammatory properties. However, the effects of P7C3 on astrocytic senescence in PD remain to be elucidated. An invitro, long culture-induced, replicative senescence cell model and a 1-methyl-4-phenylpyridinium (MPP+)/rotenone-induced premature senescence cell model were used to investigate the effects of P7C3 on astrocytic senescence. An invivo, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse PD model was used to study the role of P7C3 in astrocytic senescence. Immunoblotting, real-time quantitative RT-PCR (qPCR), immunofluorescence, subcellular fractionation assays, and immunohistochemistry were utilized to confirm the effects of P7C3 on astrocytic senescence and elucidate its underlying mechanisms. This study determined that P7C3 suppressed the senescence-associated secretory phenotype (SASP) in both cell models, as demonstrated by the reduction in the critical senescence marker p16 and proinflammatory factors (IL-6, IL-1β, CXCL10, and MMP9) and increased laminB1 levels, implying that P7C3 inhibited replicative astrocytic senescence and MPP+/rotenone-induced premature astrocytic senescence, Most importantly, we demonstrated that P7C3 prevented the death of dopamine (DA) neurons and reduced the behavioral deficits in the MPTP-induced mouse model of PD, which is accompanied by a decrease in senescent astrocytes in the substantia nigra compacta (SNc). Mechanistically, P7C3 promoted Nrf2/Sirt3-mediated mitophagy and reduced mitochondrial reactive oxygen species (mitoROS) generation, which contributed to the suppression of astrocytic senescence. Furthermore, Sirt3 deficiency obviously abolished the inhibitory effects of P7C3 on astrocytic senescence. This study revealed that P7C3 inhibited astrocytic senescence via increased Nrf2/Sirt3-mediated mitophagy and suppression of mitoROS, which further protected against DA neuronal loss. These observations provide a prospective theoretical basis for P7C3 in the treatment of age-associated neurodegenerative diseases, such as PD.

Sirt1 Deficiency Promotes Age-Related AF Through Enhancing Atrial Necroptosis by Activation of RIPK1 Acetylation.

Aging is one of the most potent risk determinants for the onset of atrial fibrillation (AF). Sirts (sirtuins) have been implicated in the pathogenesis of cardiovascular disease, and their expression declines with aging. However, whether Sirts involved in age-related AF and its underlying mechanisms remain unknown. The present study aims to explore the role of Sirts in age-related AF and delineate the underlying molecular mechanisms. Sirt1 levels in the atria of both elderly individuals and aging rats were evaluated using quantitative real-time polymerase chain reaction and Western blot analysis. Mice were engineered to specifically knockout Sirt1 in the atria and right ventricle (Sirt1mef2c/mef2c). Various techniques, such as echocardiography, atrial electrophysiology, and protein acetylation modification omics were employed. Additionally, coimmunoprecipitation was utilized to substantiate the interaction between Sirt1 and RIPK1 (receptor-interacting protein kinase 1). We discerned that among the diverse subtypes of sirtuin proteins, only Sirt1 expression was significantly diminished in the atria of elderly people and aged rats. The Sirt1mef2c/mef2c mice exhibited an enlarged atrial diameter and heightened vulnerability to AF. Acetylated proteomics and cell experiments identified that Sirt1 deficiency activated atrial necroptosis through increasing RIPK1 acetylation and subsequent pseudokinase MLKL (mixed lineage kinase domain-like protein) phosphorylation. Consistently, necroptotic inhibitor necrosulfonamide mitigated atrial necroptosis and diminished both the atrial diameter and AF susceptibility of Sirt1mef2c/mef2c mice. Resveratrol prevented age-related AF in rats by activating atrial Sirt1 and inhibiting necroptosis. Our findings first demonstrated that Sirt1 exerts significant efficacy in countering age-related AF by impeding atrial necroptosis through regulation of RIPK1 acetylation, highlighting that the activation of Sirt1 or the inhibition of necroptosis could potentially serve as a therapeutic strategy for age-related AF.

Abstract 6272: Loss of SIRT6 diverts TGF-β signaling to promote liver cancer

Abstract Background: In the US, ~35% of individuals on the waitlist for a liver transplant who had hepatocellular cancer (HCC) also had metabolic dysfunction-associated steatohepatitis (MASH). Yet, key signaling mechanisms controlling cellular transformation in this obesity-driven HCC remain only partially understood. SIRT6 is NAD+-dependent protein deacetylases that limits the activity of the lipogenic transcription factors SREBP1/2. Reduced SIRT6 is associated with human fatty liver and cancer. However, SIRT6 also limits liver fibrosis by inhibiting TGF-β signaling through the deacetylation of SMAD2 and SMAD3. We found that Smad3 driven TGF-β signaling is modulated by βII-Spectrin (encoded by SPTBN1), and that a hot mutation in HCC- D1089Y encompasses the SPTBN1-SMAD3 binding site (Gastro 2018; 154:195-210). In stressed hepatocytes, βII-Spectrin promotes lipogenesis through interactions between caspase-cleaved forms of both SREBP1 and βII-Spectrin (Sci Transl Med 2021;13:624). Liver-specific knockout of βII-Spectrin (LSKO) blocks fatty liver and HCC. Here we explored the extent of βII-spectrin-D1089Y regulation of SIRT6 expression through SMAD3, and the combined roles of SIRT6 and SPTBN1 in human HCC. Methods: We generated a SPTBN1-D1089Y mutant mouse and examined SIRT6 in the SPTBN1-D1089Y mutant mouse liver tissues and MEFs. TGF-β/Smad3 pathway activity was analyzed in SPTBN1-D1089Y mutant MEFs via western blot and RNA-seq. SIRT6 expression was examined in mouse MAFLD/MASH (GSE156059). Structure-based molecular docking simulations using three-dimensional structural coordinates of human SIRT6 (ID: Q8N6T7) and β2SP (ID: Q01082) from the AlphaFold database, followed by interactions between SIRT6 and SPTBN1. Results: SIRT6 levels are decreased in SPTBN1-D1089Y mutant liver cancer cell line and mouse liver tissues. TGF-β/SMAD3 pathway activity was altered in SPTBN1-D1089Y mutant MEF cells with decreased phospho-SMAD3 levels. The structural model predicted specific interactions between human SPTBN1 and SIRT6. Overexpression of Flag-SIRT6 reduced βII-Spectrin acetylation in HepG2 cells. TGF-β induced SMAD3, βII-Spectrin coimmunoprecipitation with SIRT6. SIRT6 knockdown increases nSREBP1, SCD1 (encoded by a nSREBP1 target gene), and βII-Spectrin in HepG2 cells. SIRT6 RNA expression is significantly decreased in liver macrophages from mice with NAFLD/NASH. Conclusions: Our data indicated a SMAD dependent as well as a SMAD3 independent role of SPTBN1 in regulating SIRT6. βII-spectrin-D1089Y together with SIRT6 deficiency may increase the susceptibility to MASH/HCC via regulating the crosstalk between macrophage and hepatocytes or regulating MASH and HCC. Citation Format: Kazufumi Ohshiro, Xiyan Xiang, Xiaochun Yang, Krishanu Bhowmick, Mohammad I. Hassan, Taj Mohammad, Lopa Mishra. Loss of SIRT6 diverts TGF-β signaling to promote liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6272.

Sirt1 alleviates osteoarthritis via promoting FoxO1 nucleo-cytoplasm shuttling to facilitate autophagy

This study aims to investigate the role and underlying mechanisms of Sirt1 in the pathophysiological process of OA. Safranine O and HE staining were utilized to identify pathological changes in the cartilage tissue. Immunohistochemistry was employed to evaluate the expression levels of proteins. IL-1β treatment and TamCartSirt1flox/flox mice were utilized to induce OA model both in vitro and in vivo. Key autophagy-related transcription factors, autophagy-related genes, and chondrocyte extracellular matrix (ECM) breakdown enzyme markers were examined using multi assays. Immunofluorescence staining revealed subcellular localization and gene expression patterns. ChIP assay and Co-immunoprecipitation assay were conducted to investigate the interactions between FoxO1 and the promoter regions of Atg7 and Sirt1. Our results demonstrate that Sirt1 deficiency exhibited inhibitory effects on ECM synthesis and autophagy, as well as exacerbated angiogenesis. Moreover, Atg7, Foxo1, and Sirt1 could form a protein complex. Sirt1 was observed to facilitate nuclear translocation of FoxO1, enhancing its transcriptional activity. Furthermore, FoxO1 was found to bind to the promoter regions of Atg7 and Sirt1, potentially regulating their expression. This study provides valuable insights into the involvement of Sirt1-Atg7-FoxO1 loop in OA, opening new avenues for targeted therapeutic interventions aiming to mitigate cartilage degradation and restore joint function.

Lack of the Histone Deacetylase SIRT1 Leads to Protection against Endoplasmic Reticulum Stress through the Upregulation of Heat Shock Proteins.

Histone deacetylase SIRT1 represses gene expression through the deacetylation of histones and transcription factors and is involved in the protective cell response to stress and aging. However, upon endoplasmic reticulum (ER) stress, SIRT1 impairs the IRE1α branch of the unfolded protein response (UPR) through the inhibition of the transcriptional activity of XBP-1 and SIRT1 deficiency is beneficial under these conditions. We hypothesized that SIRT1 deficiency may unlock the blockade of transcription factors unrelated to the UPR promoting the synthesis of chaperones and improving the stability of immature proteins or triggering the clearance of unfolded proteins. SIRT1+/+ and SIRT1-/- fibroblasts were exposed to the ER stress inducer tunicamycin and cell survival and expression of heat shock proteins were analyzed 24 h after the treatment. We observed that SIRT1 loss significantly reduced cell sensitivity to ER stress and showed that SIRT1-/- but not SIRT1+/+ cells constitutively expressed high levels of phospho-STAT3 and heat shock proteins. Hsp70 silencing in SIRT1-/- cells abolished the resistance to ER stress. Furthermore, accumulation of ubiquitinated proteins was lower in SIRT1-/- than in SIRT1+/+ cells. Our data showed that SIRT1 deficiency enabled chaperones upregulation and boosted the proteasome activity, two processes that are beneficial for coping with ER stress.

Sirtuin 3 regulates astrocyte activation by reducing Notch1 signaling after status epilepticus.

Sirtuin3 (Sirt3) is a nicotinamide adenine dinucleotide enzyme that contributes to aging, cancer, and neurodegenerative diseases. Recent studies have reported that Sirt3 exerts anti-inflammatory effects in several neuropathophysiological disorders. As epilepsy is a common neurological disease, in the present study, we investigated the role of Sirt3 in astrocyte activation and inflammatory processes after epileptic seizures. We found the elevated expression of Sirt3 within reactive astrocytes as well as in the surrounding cells in the hippocampus of patients with temporal lobe epilepsy and a mouse model of pilocarpine-induced status epilepticus (SE). The upregulation of Sirt3 by treatment with adjudin, a potential Sirt3 activator, alleviated SE-induced astrocyte activation; whereas, Sirt3 deficiency exacerbated astrocyte activation in the hippocampus after SE. In addition, our results showed that Sirt3 upregulation attenuated the activation of Notch1 signaling, nuclear factor kappa B (NF-κB) activity, and the production of interleukin-1β (IL1β) in the hippocampus after SE. By contrast, Sirt3 deficiency enhanced the activity of Notch1/NF-κB signaling and the production of IL1β. These findings suggest that Sirt3 regulates astrocyte activation by affecting the Notch1/NF-κB signaling pathway, which contributes to the inflammatory response after SE. Therefore, therapies targeting Sirt3 may be a worthy direction for limiting inflammatory responses following epileptic brain injury.

Sirt6 ablation in the liver causes fatty liver that increases cancer risk by upregulating Serpina12

Non-alcoholic fatty liver disease is a chronic liver abnormality that exhibits high variability and can lead to liver cancer in advanced stages. Hepatic ablation of SIRT6 results in fatty liver disease, yet the potential mechanism of SIRT6 deficiency, particularly in relation to downstream mediators for NAFLD, remains elusive. Here we identify Serpina12 as a key gene regulated by Sirt6 that plays a crucial function in energy homeostasis. Specifically, Sirt6 suppresses Serpina12 expression through histone deacetylation at its promoter region, after which the transcription factor, Cebpα, binds to and regulates its expression. Sirt6 deficiency results in an increased expression of Serpina12 in hepatocytes, which enhances insulin signaling and promotes lipid accumulation. Importantly, CRISPR-Cas9 mediated Serpina12 knockout in the liver ameliorated fatty liver disease caused by Sirt6 ablation. Finally, we demonstrate that Sirt6 functions as a tumor suppressor in the liver, and consequently, deletion of Sirt6 in the liver leads to not only the spontaneous development of tumors but also enhanced tumorigenesis in response to DEN treatment or under conditions of obesity.

Inhibition of Sirt3 activates the cGAS-STING pathway to aggravate hepatocyte damage in hepatic ischemia–reperfusion injury mice

Hepatic ischemia–reperfusion injury (IRI) typically manifests during subtotal hepatectomy and inflicts substantial damage to liver function in the perioperative period. Although the central role of cGAS-STING-mediated immune inflammation in hepatocyte damage during hepatic IRI is acknowledged, the precise regulatory mechanisms remain elusive. The current study aims to elucidate how Sirt3 inhibition activates the cGAS-STING pathway and exacerbates hepatocyte damage in hepatic IRI. We established both in vivo and in vitro models by creating hepatic IRI mice model and subjecting AML-12 hepatocyte cell lines to oxygen-glucose deprivation/reperfusion (OGD/R). Hepatic IRI compromised liver and mitochondrial function while elevating cytosolic mitochondrial DNA (mtDNA) levels in hepatocytes. Additionally, both in vivo hepatic IRI and in vitro OGD/R induced increased phosphorylation and activation of cGAS, STING, and IRF3, accompanied by heightened levels of pro-inflammatory factors, including TNF-α, IL-1β, and type I interferon (IFN-β). Importantly, knockdown of cGAS or STING through siRNA effectively attenuated hepatic IRI-induced inflammation and ameliorated liver function in both experimental settings, underscoring the dynamic involvement of the cGAS-STING pathway in hepatic IRI-induced inflammation. Furthermore, we observed a significant reduction in Sirt3 expression following hepatic IRI, both in vivo and in vitro. Then we generated Sirt3-deficient mice and applied Sirt3 knockdown in AML-12 hepatocytes. Notably, Sirt3 deficiency led to increased phosphorylation and activation of cGAS, STING, and IRF3, coupled with elevated TNF-α, IL-1β, and IFN-β levels in both in vivo and in vitro conditions. Moreover, upon silencing various downstream targets of Sirt3, such as transcription factors Sp1, Pu1, and p65, we observed that specifically knocking down p65 in AML-12 hepatocytes reduced cGAS mRNA levels. Co-immunoprecipitation assays confirmed a direct interaction between Sirt3 and p65. The absence of Sirt3 significantly increased nuclear translocation of p65 in mice, whereas Sirt3 knockdown in AML-12 hepatocytes heightened nuclear translocation of p65. ChIP-PCR assays demonstrated that Sirt3 deficiency notably enhanced the binding of p65 to two cGAS promoters, ultimately promoting cGAS transcription. Collectively, our results underscored that inhibition of Sirt3 activates the cGAS-STING pathway to aggravate hepatocyte damage by increasing cytosolic mtDNA and promoting nuclear translocation of p65 to promote cGAS transcription in hepatic IRI. These findings hold promise for potential therapeutic interventions in hepatic IRI by targeting the Sirt3-cGAS-STING axis, offering new avenues for the development of clinical strategies to mitigate liver damage during the perioperative period.

Particulate matter-induced metabolic recoding of epigenetics in macrophages drives pathogenesis of chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a group of illnesses associated with unresolved inflammation in response to toxic environmental stimuli. Persistent exposure to PM is a major risk factor for COPD, but the underlying mechanism remains unclear. Using our established mouse model of PM-induced COPD, we find that repeated PM exposure provokes macrophage-centered chronic inflammation and COPD development. Mechanistically, chronic PM exposure induces transcriptional downregulation of HAAO, KMO, KYNU, and QPRT in macrophages, which are the enzymes of de novo NAD+ synthesis pathway (kynurenine pathway; KP), via elevated chromatin binding of the CCCTC-binding factor (CTCF) near the transcriptional regulatory regions of the enzymes. Subsequent reduction of NAD+ and SIRT1 function increases histone acetylation, resulting in elevated expression of pro-inflammatory genes in PM-exposed macrophages. Activation of SIRT1 by nutraceutical resveratrol mitigated PM-induced chronic inflammation and COPD development. In agreement, increased levels of histone acetylation and decreased expression of KP enzymes were observed in pulmonary macrophages of COPD patients. We newly provide an evidence that dysregulated NAD+ metabolism and consecutive SIRT1 deficiency significantly contribute to the pathological activation of macrophages during PM-mediated COPD pathogenesis. Additionally, targeting PM-induced intertwined metabolic and epigenetic reprogramming in macrophages is an effective strategy for COPD treatment.

Pterostilbene exerts cytotoxicity on activated hepatic stellate cells by inhibiting excessive proliferation through the crosstalk of Sirt1 and STAT3 pathways.

Pterostilbene (PTE), a natural analogue of resveratrol, abundantly exists in blueberries and grapes and has several beneficial potentials against oxidative stress, inflammation, and cancer. In current study, we investigated the effects of PTE on hepatic fibrosis in vitro and in vivo. Activation of hepatic stellate cells (HSCs) is an initiating event in the initiation of hepatic fibrosis. MTT assay revealed that PTE (3.125-12.5 μM) displayed cytotoxicity on activated HSCs, no cytotoxicity on AML-12 and quiescent HSCs. PTE significantly inhibited the expressions of α-SMA, collagen Ⅰ and TIMP-1/MMP13 ratio; suppressed inflammatory cascade activation to reduce inflammatory cytokines release, such as Caspase-1, IL-1β and IL-6. PTE activated Sirt1 and decreased STAT3 phosphorylation, functioning as SRT1720 and Niclosamide. Sirt1 deficiency significantly elevated p-STAT3 expression, while STAT3 deficiency resulted in Sirt1 increasing and inhibited fibrosis and inflammatory cytokines expressions. In mice with hepatic fibrosis induced by thioacetamide (TAA), PTE significantly decreased ALT and AST activities, reduced fibrosis markers, STAT3 phosphorylation and activated Sirt1 expression. PTE showed cytotoxicity on activated HSCs to ameliorate hepatic fibrosis via regulating fibrogenesis, energy metabolism and inflammation and targeting the crosstalk of Sirt1 and STAT3. In conclusion, PTE could be potentially beneficial as a natural plant metabolite in preventing and treating hepatic fibrosis.

Sirt3 deficiency promotes endothelial dysfunction and aggravates renal injury.

Sirtuin 3 (SIRT3), the main deacetylase of mitochondria, modulates the acetylation levels of substrates governing metabolism and oxidative stress. In the kidney, we showed that SIRT3 affects the proper functioning of high energy-demanding cells, such as tubular cells and podocytes. Less is known about the role of SIRT3 in regulating endothelial cell function and its impact on the progression of kidney disease. Here, we found that whole body Sirt3-deficient mice exhibited reduced renal capillary density, reflecting endothelial dysfunction, and VEGFA expression compared to wild-type mice. This was paralleled by activation of hypoxia signaling, upregulation of HIF-1α and Angiopietin-2, and oxidative stress increase. These alterations did not result in kidney disease. However, when Sirt3-deficient mice were exposed to the nephrotoxic stimulus Adriamycin (ADR) they developed aggravated endothelial rarefaction, altered VEGFA signaling, and higher oxidative stress compared to wild-type mice receiving ADR. As a result, ADR-treated Sirt3-deficient mice experienced a more severe injury with exacerbated albuminuria, podocyte loss and fibrotic lesions. These data suggest that SIRT3 is a crucial regulator of renal vascular homeostasis and its dysregulation is a predisposing factor for kidney disease. By extension, our findings indicate SIRT3 as a pharmacologic target in progressive renal disease whose treatments are still imperfect.

Insufficient SIRT1 in macrophages promotes oxidative stress and inflammation during scarring.

Macrophage is a critical regulator in wound healing and scar formation, and SIRT1 is related to macrophage activation and polarization, while the specific mechanism is still unclear. To explore the specific effects of SIRT1 in scarring, we established a skin incision mouse model and LPS-induced inflammation cell model. The expression of SIRT1 in tissue and macrophage was detected, and the level of SIRT1 was changed to observe the downstream effects. LPS-induced macrophages with or without SIRT1 deficiency were used for TMT-based quantitative proteomic analysis. SIRT1 was suppressed in scar while increased in macrophages of scar tissue. And macrophages were proven to be necessary for wound healing. In the early stage of wound healing, knockout of SIRT1 in macrophage could greatly strengthen inflammation and finally promote scarring. NADH-related activities and oxidoreductase activities were differentially expressed in TMT-based quantitative proteomic analysis. We confirmed that ROS production and NOX2 level were elevated after LPS stimulation while the Nrf2 pathway and the downstream proteins, such as Nqo-1 and HO-1, were suppressed. In contrast, the suppression of SIRT1 strengthened this trend. The NF-κB pathway was remarkably activated compared with the control group. Insufficient increase of SIRT1 in macrophage leads to over activated oxidative stress and activates NF-κB pathways, which then promotes inflammation in wound healing and scarring. Further increasing SIRT1 in macrophages could be a promising method to alleviate scarring. KEY MESSAGES: SIRT1 was suppressed in scar while increased in macrophages of scar tissue. Inhibition of SIRT1 in macrophage leads to further activated oxidative stress. SIRT1 is negatively related to oxidative stress in macrophage. The elevation of SIRT1 in macrophage is insufficient during scarring.

NAMPT/SIRT1 Expression Levels in White Blood Cells Differentiate the Different Rheumatoid Arthritis Subsets: An Inspiration from Traditional Chinese Medicine.

Rheumatoid arthritis (RA) patients are prone to developing different metabolic complications. Traditional Chinese Medicine attributes this uncertainty to varied syndrome types. We retrospectively analyzed some serological indicators of active RA patients and healthy individuals. Randomly selected RA patients were divided into three groups according to NAMPT and SIRT1 expression levels in white blood cells (WBCs). Their disease severity and metabolic status were compared. Representative blood samples were subjected to a UPLC-MS/MS-based metabolomics analysis. Different human WBCs were treated with oleic acid and palmitic acid in vitro. The results indicated that blood glucose and lipid levels were decreased in RA patients, but their decrease was not in accordance with disease severity. Nutrients in the patients highly expressing SIRT1 were well preserved, with the lowest levels of RF and β-CTX and the highest levels of adiponectin and resistin. Most of them exhibited cold symptoms. When SIRT1 deficiency was obvious, lipid depletion became evident, irrespective of expression levels of NAMPT. Simultaneous high-expression of SIRT1 and NAMPT coincided with the increase in production of lactic acid and the prevalence of hot symptoms. Despite the low levels of IL-6, joint injuries were severe. The corresponding WBCs were especially sensitive to fatty acids anti-inflammatory treatments. The levels of CCL27, CCL11, CCL5, AKP, CRP and ESR were similar among all the groups. NAMPT overexpression is a risk factor for joint injuries and nutrient depletion in RA. Supplementation with lipids would exert beneficial effects on these RA patients. Its aftermath would cause even severe inflammation. Contrarily, SIRT1 up-regulation restrains inflammation and lipid depletion.

Cartilage-specific Sirt6 deficiency represses IGF-1 and enhances osteoarthritis severity in mice

ObjectivesPrior studies noted that chondrocyte SIRT6 activity is repressed in older chondrocytes rendering cells susceptible to catabolic signalling events implicated in osteoarthritis (OA). This study aimed to define the effect...

SIRT6 deficiency in endothelial cells exacerbates oxidative stress by enhancing HIF1α accumulation and H3K9 acetylation at the Ero1α promoter.

SIRT6, an important NAD+ -dependent protein, protects endothelial cells from inflammatory and oxidative stress injuries. However, the role of SIRT6 in cardiac microvascular endothelial cells (CMECs) under ischemia-reperfusion injury (IRI) remains unclear. The HUVECs model of oxygen-glucose deprivation/reperfusion (OGD/R) was established to simulate the endothelial IRI in vitro. Endoplasmic reticulum oxidase 1 alpha (Ero1α) mRNA and protein levels in SIRT6-overexpressing or SIRT6-knockdown cells were measured by qPCR and Western blotting. The levels of H2 O2 and mitochondrial reactive oxygen species (ROS) were detected to evaluate the status of oxidative stress. The effects of SIRT6 deficiency and Ero1α knockdown on cellular endoplasmic reticulum stress (ERS), inflammation, apoptosis and barrier function were detected by a series of molecular biological experiments and functional experiments in vitro. Chromatin immunoprecipitation, Western blotting, qPCR, and site-specific mutation experiments were used to examine the underlying molecular mechanisms. Furthermore, endothelial cell-specific Sirt6 knockout (ecSirt6-/- ) mice were subjected to cardiac ischemia-reperfusion surgery to investigate the effects of SIRT6 in CMECs in vivo. The expression of Ero1α was significantly upregulated in SIRT6-knockdown endothelial cells, and high Ero1α expression correlated with the accumulation of H2 O2 and mitochondrial ROS. In addition, SIRT6 deficiency increased ERS, inflammation, apoptosis and endothelial permeability, and these effects could be significantly attenuated by Ero1α knockdown. The deacetylase catalytic activity of SIRT6 was important in regulating Ero1α expression and these biological processes. Mechanistically, SIRT6 inhibited the enrichment of HIF1α and p300 at the Ero1α promoter through deacetylating H3K9, thereby antagonizing HIF1α/p300-mediated Ero1α expression. Compared with SIRT6-wild-type (SIRT6-WT) cells, cells expressing the SIRT6-H133Y-mutant and SIRT6-R65A-mutant exhibited increased Ero1α expression. Furthermore, ecSirt6-/- mice subjected to ischemia-reperfusion surgery exhibited increased Ero1α expression and ERS in CMECs and worsened injuries to microvascular barrier function and cardiac function. Our results revealed an epigenetic mechanism associated with SIRT6 and Ero1α expression and highlighted the therapeutic potential of targeting the SIRT6-HIF1α/p300-Ero1α axis.

Sirt3 improves monosodium urate crystal-induced inflammation by suppressing Acod1 expression

BackgroundPrevious studies have revealed that Sirt3 deficiency is associated with several inflammatory responses. The purpose of this study is to investigate the role and potential molecular mechanisms of Sirt3 in the inflammation induced by monosodium urate (MSU) crystals.MethodsThe Sirt3 expression level in the peripheral blood mononuclear cells (PBMCs) of patients with gout was measured. Function and molecular mechanism of Sirt3 in MSU crystal-induced inflammation were investigated in bone marrow-derived macrophages (BMDMs), C57BL/6 mouse, and Sirt3−/− mouse.ResultsSirt3 expression was decreased in the PBMCs of patients with gout. Sirt3 agonist (Viniferin) inhibited the acetylation levels of mitochondrial proteins including the SOD2 protein. RNA sequencing, bio-informatics analysis, RT-PCR, and Western blot demonstrated that Sirt3 could suppress the expression of Acod1 (Irg1), which plays an important role in gout. In BMDMs treated with palmitic acid (C16:0) plus MSU crystals, Acod1 knockdown repressed mitochondrial reactive oxygen species (mtROS) over-production, macrophage migration, and mitochondrial fragmentation, and Acod1 improved AMPK activity. The over-expression of Acod1 did not significantly affect the level of itaconic acid, but greatly decreased the levels of some important intermediate metabolites of the tricarboxylic acid (TCA) cycle. These data indicate that Acod1 exerts a pro-inflammatory role in MSU crystal-induced inflammation and is independent of the metabolic level of itaconic acid. Sirt3 deficiency exacerbates inflammatory response induced by MSU crystals in vitro and in vivo.ConclusionThe current study has shown that Sirt3 can alleviate the MSU crystal-induced inflammation by inhibiting the expression of Acod1.