Resveratrol evokes neuroprotective effects and improves foot stance following kainate-induced excitotoxic damage to the mouse spinal cord.

Excitotoxicity, characterized by over-activation of glutamate receptors, is a major contributor to spinal cord injury (SCI) pathophysiology, resulting in neuronal death and loss of locomotor function. In our previous in vitro studies, we showed that excitotoxicity induced by the glutamate analogue kainate (KA) leads to a significant reduction in the number of neurons, providing a model for SCI. Our current objective was to assess the neuroprotective role of resveratrol (RESV), a natural polyphenol, following KA-induced SCI. In vivo excitotoxicity was induced by intraspinal injection of KA immediately followed by RESV administration to Balb/C adult male mice. In neonatal mouse spinal cord preparations, excitotoxicity was transiently induced by bath-applied KA, either with or without RESV. KA administration resulted in a significant deterioration in hindlimb motor coordination and balance during locomotion, which was partially reverted by RESV. Additionally, RESV preserved neurons in both dorsal and ventral regions. Sirtuin 2 (SIRT2) immunoreactive signal was increased by RESV, while the selective SIRT1 inhibitor 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide (EX-527) attenuated RESV neuroprotective effects. These findings suggest that RESV attenuation of excitotoxic-induced neuronal loss and locomotor deficits is mediated, at least in part, through the activation of SIRT1, potentially involving SIRT2 as well. Indeed, our results highlight the potential use of RESV to enhance neuroprotective strategies for SCI.

Isobavachalcone, a natural sirtuin 2 inhibitor, exhibits anti-triple-negative breast cancer efficacy in vitro and in vivo.

Triple-negative breast cancer (TNBC) is the most aggressive and lethal clinical subtype and lacks effective targeted therapies at present. Isobavachalcone (IBC), the main active component of Psoralea corylifolia L., has potential anticancer effects. Herein, we identified IBC as a natural sirtuin 2 (SIRT2) inhibitor and characterized the potential mechanisms underlying the inhibition of TNBC. Molecular dynamics analysis, enzyme activity assay, and cellular thermal shift assay were performed to evaluate the combination of IBC and SIRT2. The therapeutic effects, mechanism, and safety of IBC were analyzed in vitro and in vivo using cellular and xenograft models. IBC effectively inhibited SIRT2 enzyme activity with an IC50 value of 0.84 ± 0.22 µM by forming hydrogen bonds with VAL233 and ALA135 within its catalytic domain. In the cellular environment, IBC bound to and stabilized SIRT2, consequently inhibiting cellular proliferation and migration, and inducing apoptosis and cell cycle arrest by disrupting the SIRT2/α-tubulin interaction and inhibiting the downstream Snail/MMP and STAT3/c-Myc pathways. In the in vivo model, 30 mg/kg IBC markedly inhibited tumor growth by targeting the SIRT2/α-tubulin interaction. Furthermore, IBC exerted its effects by inducing apoptosis in tumor tissues and was well-tolerated. IBC alleviated TNBC by targeting SIRT2 and triggering the reactive oxygen species ROS/ß-catenin/CDK2 axis. It is a promising natural lead compound for future development of SIRT2-targeting drugs.

Deacetylase sirtuin 2 negatively regulates myeloid-derived suppressor cell functions in allograft rejection.

Although myeloid-derived suppressor cells (MDSCs) are critical for allograft survival, their regulatory mechanism remains unclear. Herein, our results showed that metabolism sensor sirtuin 2 (SIRT2) negatively regulates the functions of MDSCs in inducing allogeneic skin graft rejection. Genetic deletion of SIRT2 in myeloid cells (Sirt2Δmye) increased the number of CD11b+Gr1+ MDSCs in bone marrow, spleens, draining lymph nodes, and allografts, inhibited the production of proinflammatory cytokine tumor necrosis factor ɑ, enhanced the production of anti-inflammatory cytokine interleukin 10, and potentiated the suppressive activation of MDSCs in prolonging allograft skin survival. C-X-C motif chemokine receptor 2 is critical for mediating the recruitment and cytokine production of MDSCs induced by SIRT2. Mechanistically, Sirt2Δmye enhanced NAD+ levels, succinate dehydrogenase subunit A (SDHA) activities, and oxidative phosphorylation (OXPHOS) levels in MDSCs after transplantation. Pharmacologically blocking nicotinamide phosphoribosyltransferase effectively reverses the production of cytokines and suppressive activities of MDSC induced by Sirt2Δmye. Blocking OXPHOS with knockdown of SDHA or pharmacological blocking of SDHA significantly restores Sirt2Δmye-mediated stronger MDSC suppressive activity and inflammatory factor productions. Thus, our findings identify a previously unrecognized interplay between NAD+ and SDH-mediated OXPHOS metabolic pathways in regulating MDSC functions induced by the metabolic sensor SIRT2 in allogeneic transplantation.

Ginsenoside Rh4 inhibits the malignant progression of multiple myeloma and induces ferroptosis by regulating SIRT2.

Multiple myeloma (MM) has a high mortality rate, and the exploration of therapeutic drugs for MM with low side effects is a hot topic at the moment. Ginsenoside Rh4 has been shown to inhibit tumour growth in many cancers. However, the role of ginsenoside Rh4 in MM and its reaction mechanism have not been reported so far. After the treatment with different concentrations of ginsenoside Rh4, the proliferation of NCI-H929 cells was detected by Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine staining. The cell apoptosis and cycle arrest were detected by flow cytometry and western blot. The thiobarbituric acid-reactive substances (TBARS) production was assessed with TBARS assay, whereas Fe2+ fluorescence assay was used for the measurement of Fe2+ level. The production of reactive oxygen species was evaluated with dichloro-dihydro-fluorescein diacetate staining, and western blot was applied for the estimation of ferroptosis-related proteins. The potential targets of ginsenoside Rh4 were predicted by molecular docking technology and verified by western blot. The transfection efficacy of overexpression-SIRT2 was examined with quantitative reverse transcription polymerase chain reaction and western blot. To figure out the detailed reaction mechanism between ginsenoside Rh4 and SIRT2 in MM, rescue experiments were conducted. We found that ginsenoside Rh4 inhibited cell proliferation, induced cell apoptosis, promoted cycle arrest and facilitated ferroptosis in MM. Moreover, ginsenoside Rh4 inhibited SIRT2 expression in MM cells. The overexpression of SIRT2 reversed the effects of ginsenoside Rh4 on cell proliferation, cell apoptosis, cycle arrest and ferroptosis in MM. Overall, ginsenoside Rh4 inhibited the malignant progression of MM and induced ferroptosis by regulating SIRT2.

Effects of curcumin and melatonin treatment in the cerebral cortex of adult rats.

The study investigated the effect of exogenous melatonin and (or) curcumin treatment on the cerebral cortex of adult rats. In this context, malondialdehyde (MDA), nitric oxide (NO), glutathione (GSH), superoxide dismutase (SOD), nuclear factor E2-related factor 2 (Nrf2) and SIRT2 protein expression were examined. A total of 30 Wistar albino rats involved in the study were randomly divided into five groups. Over 30 days, the control groups received phosphate-buffered saline or dimethyl sulfoxide injections, and the treatment groups received melatonin, curcumin, or a combination of melatonin and curcumin injections. In the cerebral cortex homogenates, the MDA, GSH, and sum of NO were respectively determined by the thiobarbituric acid, modified Ellman and Griess test methods. The SOD and Nrf2 levels were examined using the ELISA method and SIRT2 protein expression using the Western blot technique. The study found that both melatonin and curcumin treatments significantly reduced lipid peroxidation and SIRT2 protein expression levels (p < 0.05) and increased the Nrf2 level in the cytoplasm (p < 0.05). The study revealed that curcumin and melatonin treatments reduced MDA and SIRT2 protein expression level and increased intracellular Nrf2, GSH, and SOD in the cortex tissue. We also found that the combined melatonin and curcumin treatment produced no synergistic effect.

A novel circ_0018553 protects against angiotensin-induced cardiac hypertrophy in cardiomyocytes by modulating the miR-4731/SIRT2 signaling pathway.

Due to the complicated pathophysiology of cardiac hypertrophy, there are no effective therapies for the treatment of pathological cardiac hypertrophy. Accumulating evidence has demonstrated that circRNAs participate in the pathophysiology of cardiac hypertrophy. In this study, we investigated the regulatory mechanisms of the novel circ_0018553 in angiotensin II (Ang II)-induced cardiac hypertrophy. Circ_0018553 was enriched in endothelial progenitor cell (EPC)-derived exosomes, and circ_0018553 expression was downregulated in a cellular model of Ang II-induced cardiac hypertrophy. Silencing circ_0018553 promoted cardiac hypertrophy in the Ang II-induced cardiac hypertrophy cellular model, while overexpression of circ_0018553 significantly attenuated Ang II-induced cardiac hypertrophy in cardiomyocytes. Moreover, mechanistic studies revealed that circ_0018553 acted as a sponge for miR-4731 and that miR-4731 repressed sirtuin 2 (SIRT2) expression by targeting the 3'UTR of SIRT2. MiR-4731 overexpression promoted cardiac hypertrophy in the Ang II-induced cardiac hypertrophy cellular model, while inhibition of miR-4731 significantly attenuated Ang II-induced cardiac hypertrophy in cardiomyocytes. The rescue experiments showed that miR-4731 overexpression attenuated the protective effects of circ_0018553 overexpression on the cardiac hypertrophy induced by Ang II; SIRT2 silencing also attenuated the protective effects of miR-4731 inhibition on the Ang II-induced cardiac hypertrophy. In conclusion, our results indicated that EPC-derived exosomal circ_0018553 protected against Ang II-induced cardiac hypertrophy by modulating the miR-4731/SIRT2 signaling pathway.

Glyphosate decreases bovine oocyte quality by inducing oxidative stress and apoptosis.

Glyphosate is a universal herbicide with genital toxicity, but the effect of glyphosate on oocytes has not been reported. This study aimed to evaluate the effect of glyphosate (0, 10, 20, 50 and 100 mM) on bovine oocyte in vitro maturation. We showed that 50 mM glyphosate adversely affects the development of bovine oocytes. Exposure of oocytes to 50 mM glyphosate caused an abnormal reduction in oxidative (redox) levels compared with that in the control group, with a significantly higher reactive oxide species level (P < 0.05) and significantly lower glutathione (GSH) expression (P < 0.05). Additionally, the mRNA levels of antioxidant genes (SOD1, SOD2, SIRT2, SIRT3) and the mitochondrial membrane potential (MMP) were significantly reduced (P < 0.05). Furthermore, treatment with 50 mM glyphosate-induced apoptosis, and the mRNA levels of the apoptotic genes Caspase-3 and Caspase-4 were significantly higher than those in the control group (P < 0.05); however, the mRNA level of BAX was significantly higher than that in the control group (P < 0.01). Additionally, the mRNA levels of the anti-apoptotic genes Survivin and BCL-XL were significantly lower than those in the control group (P < 0.05), and oocyte quality was adversely affected. Together, our results confirmed that glyphosate impairs the quality of oocytes by promoting abnormal oocyte redox levels and apoptosis.

SIRT2 Affects Cell Proliferation and Apoptosis by Suppressing the Level of Autophagy in Renal Podocytes.

Purpose: Despite the discovery of many important molecules in diabetic nephropathy, there has been very limited progress in the management of diabetic kidney diseases and the design of new drugs. To fill this gap, the present study explored the expression of SIRT2 in high-glucose murine kidney foot cells and its impact on cell biological functions. Methods: Expression levels of SIRT2 in the MPC-5 of murine kidney foot cells after high and normal glucose treatment or in cells targeted with siRNA were detected using qRT-PCR. Cellular proliferation and programmed cell death were analyzed via the CCK8 assay and flow cell technique, separately. Levels of autophagy markers were measured by western blotting, and chloroquine treatment was applied to the cells to observe the effect of SIRT2 on cell proliferation and apoptosis after treatment. Results: The expression level of SIRT2 was remarkably upregulated in the high-GLU group in contrast to the low-GLU group. The cell proliferation and autophagy levels were significantly reduced, and apoptosis was remarkably reinforced in the high-GLU group in contrast to the normal GLU group. However, knocking down the expression level of SIRT2 caused an increase in cell proliferation and cell autophagy levels and significantly weakened apoptosis. Chloroquine influenced cell proliferation and apoptosis in cells targeted with SIRT2 siRNA. Conclusion: SIRT2 expression was upregulated in hyperglycaemic murine kidney foot cells, and knocking down the expression level of SIRT2 affected the biological function of the cells. We found that SIRT2 may modulate cell proliferation and apoptosis by regulating cell autophagy.

Soluble RAGE attenuates myocardial I/R injuries via FoxO3-Bnip3 pathway.

Soluble receptor for advanced glycation end-products (sRAGE) was reported to inhibit cardiac apoptosis through the mitochondrial pathway during myocardial ischemia/reperfusion (I/R) injury. Meanwhile, the proapoptotic protein Bcl2 and adenovirus E1B 19-kDa-interacting protein 3 (Bnip3) was reported to mediate mitochondrial depolarization and be activated by the Forkhead box protein O3 (FoxO3a). Therefore, it is supposed that FoxO3a-Bnip3 pathway might be involved in the inhibiting effects of sRAGE on mitochondrial apoptosis during I/R. I/R surgery or glucose deprivation/reoxygenation was adopted to explore mitochondrial depolarization, apoptosis and related signaling pathways in mice hearts and cultured cardiomyocytes. The results showed that overexpression of sRAGE in cardiomyocytes dramatically improved cardiac function and reduced infarct areas in I/R treated mice. sRAGE inhibited mitochondrial depolarization and cardiac apoptosis during I/R, which correlated with reduced expression of Bnip3, Sirt2, phosphorylation of Akt and FoxO3a which translocated into nucleus in cultured cardiomyocytes. Either Sirt2 or FoxO3a silencing enhanced the inhibiting effects of sRAGE on mitochondrial depolarization induced by I/R in cultured cardiomyocytes. Meanwhile, overexpression or silencing of FoxO3a affected the inhibiting effects of sRAGE on Bnip3 and cleaved caspase-3 in cultured cardiomyocytes. Therefore, it is suggested that sRAGE inhibited I/R injuries via reducing mitochondrial apoptosis through the FoxO3a-Bnip3 pathway.

M1 macrophage-derived exosomes impair beta cell insulin secretion via miR-212-5p by targeting SIRT2 and inhibiting Akt/GSK-3ß/ß-catenin pathway in mice.

AIMS/HYPOTHESIS: Macrophage levels are elevated in pancreatic islets, and the resulting inflammatory response is a major contributor to beta cell failure during obesity and type 2 diabetes mellitus. Previous studies by us and others have reported that exosomes released by macrophages play important roles in mediating cell-to-cell communication, and represent a class of inflammatory factors involved in the inflammatory process associated with type 2 diabetes mellitus. However, to date, no reports have demonstrated the effect of macrophage-derived exosomes on beta cells, and little is known regarding their underlying mechanisms in beta cell injury. Thus, we aimed to study the impact of macrophage-derived exosomes on islet beta cell injury in vitro and in vivo. METHODS: The phenotypic profiles of islet-resident macrophages were analysed in C57BL/6J mice fed a high-fat diet (HFD). Exosomes were collected from the medium of cultured bone marrow-derived macrophages (BMDMs) and from isolated islet-resident macrophages of HFD-fed mice (HFD-Exos). The role of exosomes secreted by inflammatory M1 phenotype BMDMs (M1-Exos) and HFD-Exos on beta cell function was assessed. An miRNA microarray and quantitative real-time PCR (qPCR) were conducted to test the level of M1-Exos-derived miR-212-5p in beta cells. Then, miR-212-5p was overexpressed or inhibited in M1-Exos or beta cells to determine its molecular and functional impact. RESULTS: M1-polarised macrophages were enriched in the islets of obese mice. M1 macrophages and islet-resident macrophages of HFD-fed mice impaired beta cell insulin secretion in an exosome-dependent manner. miR-212-5p was notably upregulated in M1-Exos and HFD-Exos. Enhancing the expression of miR-212-5p impaired beta cell insulin secretion. Blocking miR-212-5p elicited a significant improvement in M1-Exos-mediated beta cell insulin secretion during injury. Mechanistically, M1-Exos mediated an intercellular transfer of the miR-212-5p, targeting the sirtuin 2 gene and regulating the Akt/GSK-3ß/ß-catenin pathway in recipient beta cells to restrict insulin secretion. CONCLUSIONS/INTERPRETATION: A novel exosome-modulated mechanism was delineated for macrophage-beta cell crosstalk that drove beta cell dysfunction and should be explored for its therapeutic utility.

Acetylation of muscle creatine kinase negatively impacts high-energy phosphotransfer in heart failure.

A hallmark of impaired myocardial energetics in failing hearts is the downregulation of the creatine kinase (CK) system. In heart failure patients and animal models, myocardial phosphocreatine content and the flux of the CK reaction are negatively correlated with the outcome of heart failure. While decreased CK activity is highly reproducible in failing hearts, the underlying mechanisms remains elusive. Here, we report an inverse relationship between the activity and acetylation of CK muscle form (CKM) in human and mouse failing hearts. Hyperacetylation of recombinant CKM disrupted MM homodimer formation and reduced enzymatic activity, which could be reversed by sirtuin 2 treatment. Mass spectrometry analysis identified multiple lysine residues on the MM dimer interface, which were hyperacetylated in the failing hearts. Molecular modeling of CK MM homodimer suggested that hyperacetylation prevented dimer formation through interfering salt bridges within and between the 2 monomers. Deacetylation by sirtuin 2 reduced acetylation of the critical lysine residues, improved dimer formation, and restored CKM activity from failing heart tissue. These findings reveal a potentially novel mechanism in the regulation of CK activity and provide a potential target for improving high-energy phosphoryl transfer in heart failure.

Overexpression of SIRT2 Alleviates Neuropathic Pain and Neuroinflammation Through Deacetylation of Transcription Factor Nuclear Factor-Kappa B.

Sirtuin 2 (SIRT2), a member of the mammalian sirtuin family, plays an important role in the pathogenesis of various neurological diseases. However, whether SIRT2 is involved in the regulation of neuropathic pain remains unclear. In this study, we aimed to investigate the potential role of SIRT2 in regulating neuropathic pain in a rat model induced by chronic constriction injury (CCI). We found that SIRT2 was downregulated in the dorsal root ganglion (DRG) in CCI rats. Intrathecal injection of a recombinant adenovirus expressing SIRT2 markedly alleviated mechanical allodynia and thermal hyperalgesia in CCI rats. This also inhibited the expression of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6 in the DRG of CCI rats. Moreover, our results showed that overexpression of SIRT2 inhibited the acetylation of the nuclear factor-kappa B (NF-κB) p65 protein in the DRG of CCI rats. Additionally, treatment with a SIRT2 specific inhibitor significantly aggravated neuropathic pain and attenuated the inhibitory effect of SIRT2 overexpression on neuropathic pain development. Taken together, these results suggest that overexpression of SIRT2 alleviates neuropathic pain associated with inhibition of NF-κB signaling and neuroinflammation. Therefore, SIRT2 may serve as a potential therapeutic target for treatment of neuropathic pain.

PEP-1-SIRT2-induced matrix metalloproteinase-1 and -13 modulates type II collagen expression via ERK signaling in rabbit articular chondrocytes.

Matrix metalloproteinases (MMPs) are critical for the degradation of the extracellular matrix (ECM), which includes cartilage-specific collagen types I, II and XI. We previously found that PEP-1-sirtuin (SIRT)2 could induce dedifferentiation of articular chondrocytes; however, the underlying mechanisms remains unclear. We addressed this in the present study by examining the association between PEP-1-SIRT2 and the expression of MMP-1 and MMP-13 and type II collagen in rabbit articular chondrocytes. We found that PEP-1-SIRT2 increased MMP-1 and -13 expression in a dose- and time-dependent manner, as determined by western blotting. A similar trend in MMP-1 and -13 levels was observed in cultures during expansion to four passages. Pharmacological inhibition of MMP-1 and -13 blocked the PEP-1-SIRT2-induced decrease in type II collagen level. Phosphorylation of extracellular regulated kinase (ERK) was increased by PEP-1-SIRT2; however, treatment with the mitogen-activated protein kinase inhibitor PD98059 suppressed PEP-1-SIRT2-induced MMP-1 and -13 expression and dedifferentiation while restoring type II collagen expression in passage 2 cells. These results suggest that PEP-1-SIRT2 promotes MMP-induced dedifferentiation via ERK signaling in articular chondrocytes.

tRNA synthetase counteracts c-Myc to develop functional vasculature.

Recent studies suggested an essential role for seryl-tRNA synthetase (SerRS) in vascular development. This role is specific to SerRS among all tRNA synthetases and is independent of its well-known aminoacylation function in protein synthesis. A unique nucleus-directing domain, added at the invertebrate-to-vertebrate transition, confers this novel non-translational activity of SerRS. Previous studies showed that SerRS, in some unknown way, controls VEGFA expression to prevent vascular over-expansion. Using in vitro, cell and animal experiments, we show here that SerRS intervenes by antagonizing c-Myc, the major transcription factor promoting VEGFA expression, through a tandem mechanism. First, by direct head-to-head competition, nuclear-localized SerRS blocks c-Myc from binding to the VEGFA promoter. Second, DNA-bound SerRS recruits the SIRT2 histone deacetylase to erase prior c-Myc-promoted histone acetylation. Thus, vertebrate SerRS and c-Myc is a pair of 'Yin-Yang' transcriptional regulator for proper development of a functional vasculature. Our results also discover an anti-angiogenic activity for SIRT2.DOI: http://dx.doi.org/10.7554/eLife.02349.001.