Sirtuins (SIRT) exhibit deacetylation or ADP-ribosyltransferase activity and regulate a wide range of cellular processes in the nucleus, mitochondria, and cytoplasm. The role of the only sirtuin that resides in the cytoplasm, SIRT2, in the development of ischemic injury and cardiac hypertrophy is not known. In this paper, we show that the hearts of mice with deletion of
Purpose: Various sources of radiation including radiofrequency, electromagnetic radiation (EMR), low- dose X-radiation, low-level microwave radiation and ionizing radiation (IR) are indispensable parts of modern life. In the current review, we discussed the adaptive responses of biological systems to radiation with a focus on the impacts of radiation-induced oxidative stress (RIOS) and its molecular downstream signaling pathways.Materials and methods: A comprehensive search was conducted in Web of Sciences, PubMed, Scopus, Google Scholar, Embase, and Cochrane Library. Keywords included Mesh terms of "radiation," "electromagnetic radiation," "adaptive immunity," "oxidative stress," and "immune checkpoints." Manuscripts published up until December 2019 were included.Results: RIOS induces various molecular adaptors connected with adaptive responses in radiation exposed cells. One of these adaptors includes p53 which promotes various cellular signaling pathways. RIOS also activates the intrinsic apoptotic pathway by depolarization of the mitochondrial membrane potential and activating the caspase apoptotic cascade. RIOS is also involved in radiation-induced proliferative responses through interaction with mitogen-activated protein kinases (MAPks) including p38 MAPK, ERK, and c-Jun N-terminal kinase (JNK). Protein kinase B (Akt)/phosphoinositide 3-kinase (PI3K) signaling pathway has also been reported to be involved in RIOS-induced proliferative responses. Furthermore, RIOS promotes genetic instability by introducing DNA structural and epigenetic alterations, as well as attenuating DNA repair mechanisms. Inflammatory transcription factors including macrophage migration inhibitory factor (MIF), nuclear factor κB (NF-κB), and signal transducer and activator of transcription-3 (STAT-3) paly major role in RIOS-induced inflammation.Conclusion: In conclusion, RIOS considerably contributes to radiation induced adaptive responses. Other possible molecular adaptors modulating RIOS-induced responses are yet to be divulged in future studies.
Abstract Traumatic brain injury (TBI) is one of the most concerning health issues in which the normal brain function may be disrupted as a result of a blow, bump, or jolt to the head. Loss of consciousness, amnesia, focal neurological defects, alteration in mental state, and destructive diseases of the nervous system such as cognitive impairment, Parkinson's, and Alzheimer's disease. Parkinson's disease is a chronic progressive neurodegenerative disorder, characterized by the early loss of striatal dopaminergic neurons. TBI is a major risk factor for Parkinson's disease. Existing therapeutic approaches have not been often effective, indicating the necessity of discovering more efficient therapeutic targets. The mammalian target of rapamycin (mTOR) signaling pathway responds to different environmental cues to modulate a large number of cellular processes such as cell proliferation, survival, protein synthesis, autophagy, and cell metabolism. Moreover, mTOR has been reported to affect the regeneration of the injured nerves throughout the central nervous system (CNS). In this context, recent evaluations have revealed that mTOR inhibitors could be potential targets to defeat a group of neurological disorders, and thus, a number of clinical trials are investigating their efficacy in treating dementia, autism, epilepsy, stroke, and brain injury, as irritating neurological defects. The current review describes the interplay between mTOR signaling and major CNS‐related disorders (esp. neurodegenerative diseases), as well as the mTOR signaling–TBI relationship. It also aims to discuss the promising therapeutic capacities of mTOR inhibitors during the TBI.
Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Sirtuins (SIRT) exhibit deacetylation or ADP-ribosyltransferase activity and regulate a wide range of cellular processes in the nucleus, mitochondria, and cytoplasm. The role of the only sirtuin that resides in the cytoplasm, SIRT2, in the development of ischemic injury and cardiac hypertrophy is not known. In this paper, we show that the hearts of mice with deletion of Sirt2 (Sirt2-/-) display improved cardiac function after ischemia-reperfusion (I/R) and pressure overload (PO), suggesting that SIRT2 exerts maladaptive effects in the heart in response to stress. Similar results were obtained in mice with cardiomyocyte-specific Sirt2 deletion. Mechanistic studies suggest that SIRT2 modulates cellular levels and activity of nuclear factor (erythroid-derived 2)-like 2 (NRF2), which results in reduced expression of antioxidant proteins. Deletion of Nrf2 in the hearts of Sirt2-/- mice reversed protection after PO. Finally, treatment of mouse hearts with a specific SIRT2 inhibitor reduced cardiac size and attenuates cardiac hypertrophy in response to PO. These data indicate that SIRT2 has detrimental effects in the heart and plays a role in cardiac response to injury and the progression of cardiac hypertrophy, which makes this protein a unique member of the SIRT family. Additionally, our studies provide a novel approach for treatment of cardiac hypertrophy and injury by targeting SIRT2 pharmacologically, providing a novel avenue for the treatment of these disorders. Editor's evaluation In this manuscript, the authors examine the role of Sirt2 on cardiac hypertrophy by using 2 in-vivo models- systemic KO of Sirt2 and cardiac specific KO of Sirt 2. They have shown that Sirt2 is important for development of heart failure and cardiac hypertrophy. Mechanistically, the authors show that Sirt2 regulates NRF2 and that deletion of Sirt2 is protective through stabilization and increased nuclear translocation of NRF2. https://doi.org/10.7554/eLife.85571.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Sirtuin (SIRT) family of proteins comprise class III of histone deacetylases. SIRTs require NAD+ to carry out their enzymatic reaction, and have been implicated in a wide range of cellular processes including aging, apoptosis, response to stress and inflammation, control of energy efficiency, circadian clocks, and mitochondrial biogenesis (Baur et al., 2012; Preyat and Leo, 2013). In mammals, seven sirtuins (SIRT1-7) have been identified, which are categorized according to their subcellular localization to the nucleus (SIRT1, -6, and -7), mitochondria (SIRT3, -4, and -5), and cytoplasm (SIRT2). SIRT1–3 have a robust deacetylation activity, while SIRT4 is reported to display ADP-ribosyltransferase activity. SIRT5 may function as a protein desuccinylase and demalonylase, and SIRT6 and SIRT7 display weak deacetylase activity (Du et al., 2011; Haigis et al., 2006; Michishita et al., 2005; Pan et al., 2011). A number of SIRTs have been studied in the heart (for review, please see Matsushima and Sadoshima, 2015). The effects of SIRT1 in the heart are complex. Sirt1 deletion protects against pressure overload (PO)-induced cardiac hypertrophy (Oka et al., 2011; Sundaresan et al., 2011); however, low-level overexpression of SIRT1 in the heart attenuates age-associated cardiac hypertrophy, fibrosis, and cardiac dysfunction, while high-level overexpression of SIRT1 increases these pathological effects (Alcendor et al., 2007). In the setting of ischemia-reperfusion (I/R), SIRT1 exerts protective effects: Sirt1 knockout (KO) in the heart increases I/R-induced injury, while its overexpression protects against I/R-induced injury (Hsu et al., 2010). Thus, it appears that the effects of SIRT1 on cardiac response to stress are dependent on its expression levels as well as the context of injury. SIRT3 has also been studied in the heart and shown to protect against both cardiac hypertrophy and I/R injury (Porter et al., 2014; Sundaresan et al., 2009), while SIRT6 KO mice exhibit cardiac hypertrophy (Sundaresan et al., 2012). Recent studies have assessed the role of SIRT2 in the heart. One study showed that deletion of Sirt2 reduces AMPK activation and increases age-related and angiotensin II-mediated cardiac hypertrophy (Tang et al., 2017), while another showed that advanced glycation end products and its receptor promote diabetic cardiomyopathy through suppression of SIRT2, however, KO mice were not used for these studies (Yuan et al., 2015). Another study showed that Sirt2 deficiency increases nuclear localization of NFATc2 and its transcription activity, and that NFAT inhibition rescues the cardiac dysfunction in mice with Sirt2 deletion (Sarikhani et al., 2018). Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a transcription factor that activates a number of cytoprotective genes, including antioxidative enzymes (Tufekci et al., 2011). Under normal conditions, NRF2 resides in the cytoplasm, and is degraded primarily through its interaction with Keap1 (kelch-like ECH-associated protein 1), which also serves as a bridge between NRF2 and cullin 3-ubiquitination complex (Tufekci et al., 2011). Under oxidative stress, NRF2 escapes degradation, translocates into the nucleus, and binds to antioxidant response elements in the promoter of a number of genes (Kaspar et al., 2009). NRF2 acetylation is decreased with SIRT1 overexpression (Kawai et al., 2011); however, an association between SIRT1 and NRF2 has not been demonstrated, and the functional consequences of NRF2 deacetylation have not been studied. NRF2 KO mice developed cardiac hypertrophy and heart failure (HF) after trans-aortic constriction (TAC) (Li et al., 2009), indicating the NRF2 is protective against cardiac stress. We recently showed that SIRT2 mediates NRF2 deacetylation in the liver cells and its translocation in the nucleus to regulate antioxidant genes (Yang et al., 2017). In this paper, we show that SIRT2 plays a detrimental role in the heart in response to injury, in contrast to a previously published report (Tang et al., 2017). Mechanistically, deletion of Sirt2 is protective through stabilization and increased nuclear translocation of NRF2, leading to increased expression of antioxidant genes. Finally and most importantly, we show that pharmacological inhibition of SIRT2 protects the heart against the development of cardiac hypertrophy, opening potential treatment for this disorder. Results SIRT2 is expressed in the heart and its levels are elevated in HF We first showed that SIRT2 is expressed in the heart (Figure 1—figure supplement 1A) and in H9c2 cardiomyoblasts (Figure 1—figure supplement 1B) at relatively high levels. We also found that SIRT2 expression was higher in the hearts of mice 4 weeks after TAC compared to sham (Figure 1A), while the levels of other sirtuin family members with major deacetylation activity that have been studied in the heart (i.e., SIRT1, SIRT3, and SIRT6) were not different. Additionally, we noted a significant increase in the levels of SIRT2 in the explanted hearts from end-stage HF patients with dilated cardiomyopathy (Figure 1B). We also assessed the levels of SIRT2 in explanted hearts from patients with ischemic cardiomyopathy and showed that SIRT2 is increased in these hearts (Figure 1C). These results indicate that SIRT2 levels are increased in HF and ischemic injury. Figure 1 with 1 supplement see all Download asset Open asset SIRT2 is upregulated in heart failure (HF). (A) SIRT1, SIRT2, SIRT3, and SIRT6 in mouse hearts after trans-aortic constriction (TAC). (B) SIRT2 in human hearts from healthy patients and patients with dilated cardiomyopathy. (C) SIRT2 protein levels in the hearts of control individual and patients with ischemic heart failure. *p<0.05 by Student's t-test. Data presented as mean ± SEM. Figure 1—source data 1 SIRT1, -2, -3, and -6 after sham and trans-aortic constriction (TAC) surgery as shown in Figure 1A. https://cdn.elifesciences.org/articles/85571/elife-85571-fig1-data1-v2.csv Download elife-85571-fig1-data1-v2.csv Figure 1—source data 2 SIRT2 in non-failing and failing human hearts as shown in Figure 1B. https://cdn.elifesciences.org/articles/85571/elife-85571-fig1-data2-v2.csv Download elife-85571-fig1-data2-v2.csv Figure 1—source data 3 SIRT2 in non-failing and ischemic human hearts as shown in Figure 1C. https://cdn.elifesciences.org/articles/85571/elife-85571-fig1-data3-v2.csv Download elife-85571-fig1-data3-v2.csv Figure 1—source data 4 Full gels for Figure 1A–C. https://cdn.elifesciences.org/articles/85571/elife-85571-fig1-data4-v2.pptx Download elife-85571-fig1-data4-v2.pptx Figure 1—source data 5 Full gels for Figure 1A–C unedited. https://cdn.elifesciences.org/articles/85571/elife-85571-fig1-data5-v2.pptx Download elife-85571-fig1-data5-v2.pptx Figure 1—source data 6 Full gels for Figure 1A–C unedited. https://cdn.elifesciences.org/articles/85571/elife-85571-fig1-data6-v2.zip Download elife-85571-fig1-data6-v2.zip Sirt2 deficiency preserves cardiac function in response to PO and I/R injury We first used mice with global deletion of Sirt2 KO (Sirt2-/-) for our studies. We assessed whether Sirt2 deletion affects the levels of other sirtuin family members in the heart. Sirt2-/- hearts displayed no change in other sirtuin family members at the mRNA level, and no change in protein levels of SIRT1, SIRT3, or SIRT6 (sirtuins with major deacetylation activity) was detected (Figure 2—figure supplement 1). We then assessed whether Sirt2 deletion protects against PO. Sirt2-/- mice displayed normal cardiovascular parameters at baseline and no overt phenotype. However, in response to TAC, Sirt2-/- mice displayed improved cardiac function than littermate controls, as assessed by fractional shortening (FS) and ejection fraction (EF) (Figure 2A and B). Additionally, Sirt2-/- mice displayed evidence of less cardiac hypertrophy, as evidenced by lower interventricular septal (IVS) thickness on echocardiography (Figure 2C), and reduced cardiac size and heart weight to body weight ratio on gross examination (Figure 2D and E). Histological examination of the hearts also showed smaller cardiomyocytes in Sirt2-/- hearts after PO, as assessed by H&E staining (Figure 2F and G). These data indicate that deletion of Sirt2 results in protection of the heart against PO with improved cardiac function and less cardiac hypertrophy. Figure 2 with 1 supplement see all Download asset Open asset Sirt2 deficiency protects the heart against cardiac dysfunction after trans-aortic constriction (TAC). Sirt2-/- and wild-type (WT) littermates were subjected to TAC and ejection fraction (EF) (A), fractional shortening (FS) (B), and interventricular septal thickness during diastole (C) were assessed 4 weeks later (N=6–9). (D–F) Representative hearts (D), HW/BW (E) (N=3–5), H&E staining, (F) and the summary of cross-sectional area of cardiomyocytes (G) in WT and Sirt2-/- hearts (N=20 cardiomyocytes), *p<0.05 by one-way ANOVA and post hoc Tukey analysis (A, B, C, and E) and unpaired Student's t-test (G). Bars represent group mean. Figure 2—source data 1 Ejection fraction (EF) in wild-type (WT) and Sirt2-/- mice after sham or trans-aortic constriction (TAC) as shown in Figure 2A. https://cdn.elifesciences.org/articles/85571/elife-85571-fig2-data1-v2.csv Download elife-85571-fig2-data1-v2.csv Figure 2—source data 2 Fractional shortening (FS) in wild-type (WT) and Sirt2-/- mice after sham or trans-aortic constriction (TAC) as shown in Figure 2B. https://cdn.elifesciences.org/articles/85571/elife-85571-fig2-data2-v2.csv Download elife-85571-fig2-data2-v2.csv Figure 2—source data 3 Interventricular septal (IVS) thickness diastole in wild-type (WT) and Sirt2-/- mice after sham or trans-aortic constriction (TAC) as shown in Figure 2C. https://cdn.elifesciences.org/articles/85571/elife-85571-fig2-data3-v2.csv Download elife-85571-fig2-data3-v2.csv Figure 2—source data 4 HW/BW in wild-type (WT) and Sirt2-/- mice after sham or trans-aortic constriction (TAC) as shown in Figure 2E. https://cdn.elifesciences.org/articles/85571/elife-85571-fig2-data4-v2.csv Download elife-85571-fig2-data4-v2.csv Figure 2—source data 5 CSA in wild-type (WT) and Sirt2-/- hearts as shown in Figure 2G. https://cdn.elifesciences.org/articles/85571/elife-85571-fig2-data5-v2.csv Download elife-85571-fig2-data5-v2.csv To better assess the role of SIRT2 in the development of HF and ischemic damage, we then studied the effects of Sirt2 deletion in the heart on the response to I/R. We subjected Sirt2-/- and their littermate wild-type (WT) controls to I/R and cardiac function was assessed after 7 and 21 days. At both time points, EF and FS were significantly higher in Sirt2-/- mice compared to controls (Figure 3A and B). Time course of cardiac assessment showed that while FS was comparable between WT and Sirt2-/- on day 3, it quickly deteriorated in WT mice, consistent with transition into HF, while Sirt2-/- mice maintained their cardiac function (Figure 3C). To further support these findings, we assessed the effects of Sirt2 modulation on cell death in response to H2O2 in neonatal rat cardiomyocytes (NRCMs) treated with control or Sirt2 siRNA by measuring propidium iodide (PI) positive cells. We found that cells with Sirt2 knockdown (KD) displayed improved cell viability in response to H2O2 (Figure 3D and E). Overall, these results indicate that SIRT2 exerts detrimental effects in the heart in response to PO and I/R, and that its deletion leads to protective effects. Figure 3 Download asset Open asset Hearts from Sirt2-/- mice are protected against ischemia-reperfusion (I/R) injury. Ejection fraction (EF) and fractional shortening (FS) in wild-type (WT) and Sirt2-/- mice 7 (A) and 21 days (B) after I/R (N=4–5). (C) Time course of FS in Sirt2-/- mice after I/R injury (N=4–5). (D, E) Cell death assessed by propidium iodide (PI), in neonatal rat cardiomyocyte (NRCM) treated with control or Sirt2 siRNA and with 500 µM of H2O2. *p<0.05 by ANOVA for all panels expect for panel C, where Student's t-test was used for comparison between the two time points. Bars represent mean (A, B), and data presented as mean ± SEM. Figure 3—source data 1 Ejection fraction (EF) and fractional shortening (FS) in wild-type (WT) and Sirt2-/- mice after ischemia-reperfusion (I/R) as shown in Figure 3A. https://cdn.elifesciences.org/articles/85571/elife-85571-fig3-data1-v2.csv Download elife-85571-fig3-data1-v2.csv Figure 3—source data 2 Ejection fraction (EF) and fractional shortening (FS) in wild-type (WT) and Sirt2-/- mice after ischemia-reperfusion (I/R) as shown in Figure 3B. https://cdn.elifesciences.org/articles/85571/elife-85571-fig3-data2-v2.csv Download elife-85571-fig3-data2-v2.csv Figure 3—source data 3 Time course of fractional shortening (FS) in wild-type (WT) and Sirt2-/- mice after ischemia-reperfusion (I/R) as shown in Figure 3C. https://cdn.elifesciences.org/articles/85571/elife-85571-fig3-data3-v2.csv Download elife-85571-fig3-data3-v2.csv Figure 3—source data 4 Propidium iodide (PI) positive cells as shown in Figure 3E. https://cdn.elifesciences.org/articles/85571/elife-85571-fig3-data4-v2.csv Download elife-85571-fig3-data4-v2.csv The experiments in Figures 2 and 3 were conducted in mice with global deletion of Sirt2. To confirm a role for SIRT2 in cardiomyocyte response to injury, we then generated cardiac-specific Sirt2 KO mice (cs-Sirt2-/-) by crossing Sirt2 floxed mice with αMHC-Cre mice. We confirmed lack of SIRT2 expression and no change in SIRT1 and SIRT3 in the hearts of cs-Sirt2-/- mice (Figure 4—figure supplement 1). The cs-Sirt2-/- mice were then subjected to TAC with littermate Cre negative mice as control, and cardiac functions (EF and FS) were assessed at 1 and 2 weeks after injury. At both time points, cs-Sirt2-/- mice displayed improved cardiac function compared to WT controls (Figure 4A and B). Consistent with these data, HF markers in the heart, including Nppa and Nppb, were significantly lower in cs-Sirt2-/- mice after TAC (Figure 4C and D). To determine whether these effects are gender specific, we also preformed TAC in female Sirt2f/f and cs-Sirt2-/- mice and demonstrated that they are also protected against TAC at 7 and 14 days (Figure 4—figure supplement 2). Finally, the hearts of cs-Sirt2-/- mice are also protected against I/R, as their function was significantly higher (Figure 4E) and degree of ischemic damage was lower (Figure 4F) than Sirt2f/f mice 14 days after I/R. Figure 4 with 2 supplements see all Download asset Open asset cs-Sirt2-/- hearts are protected against trans-aortic constriction (TAC) and ischemia-reperfusion (I/R). Ejection fraction (EF) and fractional shortening (FS) in Sirt2f/f and cs-Sirt2-/- mice 7 (A) and 14 days (B) after TAC (N=5–9). (C,D) mRNA levels of Anf (C) and Bnp (D) in the hearts of Sirt2f/f and cs-Sirt2-/- mice 4 weeks after TAC (N=7–8). (E) EF and FS in Sirt2f/f and cs-Sirt2-/- mice 7 and 14 days after I/R (N=4). (F) Necrotic area (representing the degree of ischemic damage) in Sirt2f/f and cs-Sirt2-/- mice 14 days after MI. *p<0.05 by ANOVA for panels A and B, and Student's t-test was used for panels C and D. Data are presented as mean ± SEM. Figure 4—source data 1 Ejection fraction (EF) and fractional shortening (FS) in Sirt2f/f and cs-Sirt2-/- mice 7 days after ischemia-reperfusion (I/R) as shown in Figure 4A. https://cdn.elifesciences.org/articles/85571/elife-85571-fig4-data1-v2.csv Download elife-85571-fig4-data1-v2.csv Figure 4—source data 2 Ejection fraction (EF) and fractional shortening (FS) in Sirt2f/f and cs-Sirt2-/- mice 14 days after ischemia-reperfusion (I/R) as shown in Figure 4B. https://cdn.elifesciences.org/articles/85571/elife-85571-fig4-data2-v2.csv Download elife-85571-fig4-data2-v2.csv Figure 4—source data 3 Nppa mRNA in Sirt2f/f and cs-Sirt2-/- hearts as shown in Figure 4C. https://cdn.elifesciences.org/articles/85571/elife-85571-fig4-data3-v2.csv Download elife-85571-fig4-data3-v2.csv Figure 4—source data 4 Nppb mRNA in Sirt2f/f and cs-Sirt2-/- hearts as shown in Figure 4D. https://cdn.elifesciences.org/articles/85571/elife-85571-fig4-data4-v2.csv Download elife-85571-fig4-data4-v2.csv Figure 4—source data 5 Echo parameters in Sirt2f/f and cs-Sirt2-/- hearts as shown in Figure 4E. https://cdn.elifesciences.org/articles/85571/elife-85571-fig4-data5-v2.xlsx Download elife-85571-fig4-data5-v2.xlsx SIRT2 deacetylates NRF2 resulting in decreased transcriptional activity in the heart We previously showed that SIRT2 deacetylates NRF2 protein in the liver and alters iron release from hepatocytes (Yang et al., 2017). Since deacetylation of NRF2 leads to protein destabilization and NRF2 regulates the expression of many antioxidant genes, we hypothesized that the mechanism for the protective effects of Sirt2 deletion in response to stress is through decreased NRF2 deacetylation and degradation, resulting in increased expression of antioxidant proteins. To test this hypothesis, we first assessed whether there is physical interaction between SIRT2 and NRF2 in the heart. Co-immunoprecipitation (IP) experiments showed that SIRT2 interacts with NRF2 in the heart of WT mice (Figure 5A). Figure 5 with 3 supplements see all Download asset Open asset SIRT2 interacts with nuclear factor (erythroid-derived 2)-like 2 (NRF2) and regulates its activity in the heart. (A) Co-immunoprecipitation (IP) of SIRT2 and NRF2 in extracts of hearts from wild-type (WT) mice. (B) Endogenous NRF2 acetylation levels in the hearts of WT and Sirt2-/- mice at the baseline. Acetylated proteins were IPed by anti-acetyl antibody followed by immunoblotting with anti-NRF2 antibody. (C) NRF2 protein levels in neonatal rat cardiomyocytes (NRCMs) treated with Sirt2 siRNA. (D) NRF2 protein levels in H9c2 cells treated with control or Sirt2 siRNA and harvested at different time points after treatment with 100 µg/ml of CHX. (E) NRF2 protein levels in the nucleus in NRCMs treated with control or Sirt2 siRNA. (F–H) mRNA levels of NRF2 target genes in pentose phosphate pathway (F), quinone and glutathione-based detoxification (G), thioredoxin production (H) in H9c2 cells overexpressing empty vector (white bars) or SIRT2 (gray bars). *p<0.05 by Student's t-test. Figure 5—source data 1 mRNA with overexpression of EV or SIRT2 as shown in Figure 5F. https://cdn.elifesciences.org/articles/85571/elife-85571-fig5-data1-v2.csv Download elife-85571-fig5-data1-v2.csv Figure 5—source data 2 mRNA with overexpression of EV or SIRT2 as shown in Figure 5G. https://cdn.elifesciences.org/articles/85571/elife-85571-fig5-data2-v2.csv Download elife-85571-fig5-data2-v2.csv Figure 5—source data 3 mRNA with overexpression of EV or SIRT2 as shown in Figure 5H. https://cdn.elifesciences.org/articles/85571/elife-85571-fig5-data3-v2.csv Download elife-85571-fig5-data3-v2.csv Figure 5—source data 4 Uncropped gels for Figure 5. https://cdn.elifesciences.org/articles/85571/elife-85571-fig5-data4-v2.pptx Download elife-85571-fig5-data4-v2.pptx Figure 5—source data 5 Uncropped gels for Figure 5 unedited. https://cdn.elifesciences.org/articles/85571/elife-85571-fig5-data5-v2.pptx Download elife-85571-fig5-data5-v2.pptx Figure 5—source data 6 Uncropped gels for Figure 5 unedited. https://cdn.elifesciences.org/articles/85571/elife-85571-fig5-data6-v2.zip Download elife-85571-fig5-data6-v2.zip We then measured acetylation levels of NRF2 in WT and Sirt2-/- hearts, and showed that NRF2 acetylation levels are increased within Sirt2-/- hearts (Figure 5B). We then assessed whether deacetylation of NRF2 alters its levels in the cardiac cells, as shown before in the liver (Yang et al., 2017). Treatment of NRCMs with Sirt2 siRNA resulted in increased NRF2 protein levels compared with control siRNA, indicating that SIRT2 leads to a reduction in the levels of NRF2 protein (Figure 5C). Since NRF2 protein levels are higher in Sirt2-/- hearts, we next assessed whether SIRT2 alters the stability of NRF2 protein. NRF2 levels were significantly lower starting at 60 min after treatment with the protein synthesis inhibitor cycloheximide (CHX), leading to almost complete degradation at 120 min in cells treated with control siRNA. However, we noted no change in NRF2 protein levels in cells treated with Sirt2 siRNA (Figure 5D). These data indicate that SIRT2 binds to NRF2, and its deacetylation leads to the instability and degradation of NRF2. NRF2 is a transcription factor and upon activation, translocates into the nucleus to exert its transcriptional activity (Hybertson et al., 2011). Thus, we measured nuclear level of NRF2 and found it to be increased in NRCMs with Sirt2 KD (Figure 5E). Since the increase in nuclear levels of NRF2 suggests possibly higher transcriptional activity of the protein, we next assessed the effects of Sirt2 modulation on NRF2 transcriptional activity in H9c2 cells treated with lentivirus expressing either control or SIRT2 lentivirus. Consistent with its increased nuclear levels, SIRT2 overexpression in H9c2 cells resulted in lower levels of known NRF2 target genes (Figure 5F–H). However, the mRNA levels of non-NRF2 targeted antioxidant genes were not affected by SIRT2 overexpression (Figure 5—figure supplement 1). We confirmed the data in mouse HL-1 atrial cell line and showed an increase in NRF2 protein with Sirt2 KD and a decrease in NRF2 target proteins with overexpression of SIRT2 (Figure 5—figure supplement 2). Since our data suggest a role for SIRT2 in the regulation of NRF2-mediated expression of antioxidant genes, we next assessed whether SIRT2 has an effect on reactive oxygen species (ROS) production. NRCMs treated with Sirt2 siRNA displayed less ROS levels after treatment with H2O2 (Figure 5—figure supplement 3), further supporting a role for SIRT2 in regulating oxidative state of cardiomyocytes. Sirt2/Nrf2 double KO mice display more cardiac damage after I/R compared to Sirt2-/- mice Our results thus far demonstrate that NRF2 is a target of SIRT2 and that SIRT2 regulates NRF2 acetylation and protein levels. To determine whether the protective effects of SIRT2 are mediated through NRF2, we generated Sirt2/Nrf2 double KO mice and subjected the mice to I/R injury. The Sirt2/Nrf2 double KO mice displayed reduced EF and FS compared to Sirt2-/- mice (Figure 6A and B), indicating that deletion of Nrf2 reverses the protective effects of SIRT2. Figure 6 Download asset Open asset Nrf2 deletion and SIRT2 inhibitors protected against cardiac damage and cardiac hypertrophy. Ejection fraction (EF) (A) and fractional shortening (FS) (B) in wild-type (WT), Sirt2-/-, and Sirt2-/-/Nrf2-/- double knockout (KO) mice 28 days after ischemia-reperfusion (I/R) (N=4–5). (C) Protocol for treatment of mice with SIRT2 inhibitor, AGK2. (D) Echo images of hearts from WT mice treated with either vehicle or AGK2. (E–J) EF (E), FS (F), left ventricular diameter during diastole (LVDd) (G), left ventricular diameter during systole (LVDs) (H), IVSd (I), and posterior wall thickness during diastole (PWTd) (J) in WT mice treated with AGK after trans-aortic constriction (TAC) according to the protocol in panel C (N=6–10). *p<0.05 by ANOVA for panels A–B or Student's t-test for panels E–J. Figure 6—source data 1 Ejection fraction (EF) in wild-type (WT), Sirt2-/-, and Sirt2-/-/Nrf2-/- mice after ischemia-reperfusion (I/R) as shown in Figure 6A. https://cdn.elifesciences.org/articles/85571/elife-85571-fig6-data1-v2.csv Download elife-85571-fig6-data1-v2.csv Figure 6—source data 2 Fractional shortening (FS) in wild-type (WT), Sirt2-/-, and Sirt2-/-/Nrf2-/- mice after ischemia-reperfusion (I/R) as shown in Figure 6B. https://cdn.elifesciences.org/articles/85571/elife-85571-fig6-data2-v2.csv Download elife-85571-fig6-data2-v2.csv Figure 6—source data 3 Ejection fraction (EF) with AGK2 as shown in Figure 6E. https://cdn.elifesciences.org/articles/85571/elife-85571-fig6-data3-v2.csv Download elife-85571-fig6-data3-v2.csv Figure 6—source data 4 Fractional shortening (FS) with AGK2 as shown in Figure 6F. https://cdn.elifesciences.org/articles/85571/elife-85571-fig6-data4-v2.csv Download elife-85571-fig6-data4-v2.csv Figure 6—source data 5 Left ventricular diameter during diastole (LVDd) with AGK2 as shown in Figure 6G. https://cdn.elifesciences.org/articles/85571/elife-85571-fig6-data5-v2.csv Download elife-85571-fig6-data5-v2.csv Figure 6—source data 6 LVDs with AGK2 as shown in Figure 6H. https://cdn.elifesciences.org/articles/85571/elife-85571-fig6-data6-v2.csv Download elife-85571-fig6-data6-v2.csv Figure 6—source data 7 IVSd with AGK2 as shown in Figure 6I. https://cdn.elifesciences.org/articles/85571/elife-85571-fig6-data7-v2.csv Download elife-85571-fig6-data7-v2.csv Figure 6—source data 8 Posterior wall thickness during diastole (PWTd) with AGK2 as shown in Figure 6J. https://cdn.elifesciences.org/articles/85571/elife-85571-fig6-data8-v2.csv Download elife-85571-fig6-data8-v2.csv Pharmacological inhibition of SIRT2 protects the heart against ischemic damage Since a reduction in SIRT2 levels led to protection against the development of HF and cardiac hypertrophy, we next studied whether pharmacological inhibition of SIRT2 also exerts protective effects in the heart in response to PO. For these studies, we used AGK2, a selective SIRT2 inhibitor (He et al., 2012; Outeiro et al., 2007; Petrilli et al., 2013). Eight-week-old C57B6 mice were underwent TAC and 1 day later, they were randomized to treatment with 40 mg/kg of AGK2 or vehicle intraperitoneally twice a week for 4 weeks. At the conclusion of the study, their cardiac function and heart chamber size were assessed using echocardiography (Figure 6C). Treatment with AGK2 did not change the systolic function of the heart, as assessed by EF, FS (Figure 6D–F). However, measures of cardiac size, as assessed by left ventricular (LV) diameter during diastole and systole (LVDd and LVDs, respectively), were increased, while measures of LV wall diameter, as assessed by IVSd and posterior wall thickness during diastole (PWTd), were reduced (Figure 6G–J). These results indicate that pharmacological inhibition of SIRT2 can protect the heart against cardiac hypertrophy and improve cardiac remodeling in response to PO. Discussion Sirtuins play a major role in post-translational modification of proteins, and their deletion have been shown to lead to a number of physiological changes and pathological conditions (Baur et al., 2012; Watroba and Szukiewicz, 2021; Zhao et al., 2020). Although multiple sirtuins have been investigated in the context of cardiovascular diseases (Tang et al., 2017; Yuan et al., 2015; Sarikhani et al., 2018), it is not known whether SIRT2 has a role in protection against HF and cardiac hypertrophy. In this paper, we used genetic models to show that SIRT2 has detrimental effects in the heart in the setting of cardiac insults and demonstrate that the deleterious effects of SIRT2 is through increased NRF2 deacetylation and its degradation and eventual reduction in the levels of antioxidant genes. We also show that deletion of Nrf2 reverses the protective effects of Sirt2 deletion. Finally, we provide a clinical significance for our findings and show that
Background : Gastric cancer (GC) is one of the most common cancers worldwide and in Iran. Conventional therapies are surgery and chemotherapy. Current studies are evaluating natural compounds in inhibiting growth of cancer cell. In this study isolated peptide melittin with 26 amino acids from bee venom and its impact on the viability and proliferation of gastric cancer cells was investigated. Methods : At first melittin was purified from honeybee venom using a reversed-phase high performance liquid chromatography (RP- HPLC) and C18 column. In order to investigate whether melittin, a 26 amino acids peptide which is the main components of honeybee venom, inhibits proliferation of human gastric adenocarcinoma cell line (AGS cells), MTT ((3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide) assay was performed. Hemolytic assay carried out in order to confirm the biologic activity of the isolated melittin. AGS cells were plated in a 96-well plate and treated with serially diluted concentrations of melittin for 6 and 12 hours. The mortality of the cells was measured via MTT assay at 540 nm. Results : The obtained chromatogram from RP-HPLC showed that melittin comprises 50% of the studied bee venom. SDS-PAGE analysis of melittin fraction confirmed purity of isolated melittin. Hemolytic activity assay indicates that isolated melittin shows a strong hemolytic activity (HD50=0.5). MTT assay showed that melittin strongly inhibits proliferation of gastric cancer cells at concentrations more than 2µg/ml. This inhibitory effect is dependent to melittin concentration and incubation time. Conclusion : This study provides evidence that melittin inhibits proliferation of the gastric cancer cells. Results showed that isolated melittin from honey bee venom have cytotoxic effect on AGS cell line with a trend of increasing cytotoxicity with increasing concentration and incubation time.
It has been previously reported that melittin, the main ingredient of honey bee venom, has anticancer properties. However, there appears to be no earlier study focusing on the isolation of melittin from Iranian honey bee venom (Apis mellifera meda), and evaluation of its effect on cancerous cells.We isolated melittin using reversed-phase high performance liquid chromatography, and its potential toxicity on gastric cancer AGS cells was determined with an MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay. Furthermore, to ascertain whether melittin induces apoptosis or necrosis in these cells, morphological evaluation, DNA fragmentation assay, propidium podide and annexin-V-FITC dual staining, and flow cytometric analysis were also conducted.The results of our study suggested that melittin inhibited the proliferation of AGS cells in a dose and time-dependent trend. All of the above four distinct assays indicated that melittin induces necrosis in AGS cells at concentrations of ≥ 1 μg/mL.The present study indicated that melittin has an anticancer effect on gastric cancer AGS cells and stimulates necrotic cell death in these cells.
Hemiscorpius lepturus scorpionism poses one of the most dangerous health problems in many parts of the world. The common therapy consists of using antivenom antibody fragments derived from a polyclonal immune response raised in horses. However, this immunotherapy creates serious side effects, including anaphylactic shock sometimes even leading to death. Thus, many efforts have been made to introduce new replacement therapeutics that cause less adverse reactions. One of the most attractive approaches to replacing the available therapy is offered by single-domain antibody fragments, or nanobodies (Nbs). We immunized dromedaries with H. lepturus toxin and identified a functional recombinant Nb (referred to as F7Nb) against heminecrolysin (HNc), the major known hemolytic and dermonecrotic fraction of H. lepturus venom. This Nb was retrieved from the immune library by phage display selection. The in vitro neutralization tests indicated that 17.5 nmol of the F7Nb can inhibit 45% of the hemolytic activity of 1 EC100 (7.5 μg/ml) of HNc. The in vivo neutralization tests demonstrated that F7Nb had good antihemolytic and antidermonecrotic effects against HNc in all tested mice. Surprisingly, F7Nb (8.75 nmol) neutralized 1 LD100 of HNc (10 μg) via an intracerebroventricular route or 1 LD100 (80 μg) via a subcutaneous route. All of the control mice died. Hence, this Nb is a potential leading novel candidate for treating H. lepturus scorpionism in the near future.—Yardehnavi, N., Behdani, M., Pooshang Bagheri, K., Mahmoodzadeh, A., Khanahmad, H., Shahbazzadeh, D., Habibi-Anbouhi, M., Hassanzadeh Ghassabeh, G., Muyldermans, S. A camelid antibody candidate for development of a therapeutic agent against Hemiscorpius lepturus envenomation. FASEB J. 28, 4004-4014 (2014). www.fasebj.org
Background: Human Papillomavirus (HPV) is the main biological agent causing Sexually Transmitted Diseases (STDs), including precancerous lesions and several types of prevalent cancers. To date, numerous types of vaccines are designed to prevent high-risk HPV. However, their prophylactic effect is not the same and does not clear previous infections. Therefore, there is an urgent need for developing therapeutic vaccines that trigger cell-mediated immune responses for the treatment of HPV. The HPV16 E6 and E7 proteins are ideal targets for vaccine therapy against HPV. Fusion protein vaccines, which include both immunogenic interest protein and an adjuvant for augmenting the immunogenicity effects, are theoretically capable of guaranteeing the power of the immune system against HPV. Methods: A vaccine construct, including HPV16 E6/E7 proteins along with a heat shock protein GP96 (E6/E7-NTGP96 construct), was designed using in silico methods. By the aid of the SWISS-- MODEL server, the optimal 3D model of the designed vaccine was selected, and then the physicochemical and molecular parameters were performed using bioinformatics tools. Docking studies were done to evaluate the binding interaction of the vaccine. Allergenicity, immunogenicity, B, and T cell epitopes of the designed construct were predicted. Results: Immunological and structural computational results illustrated that our designed construct is potentially proper for stimulation of cellular and humoral immune responses against HPV. Conclusion: Computational studies showed that the E6/E7-NTGP96 construct is a promising candidate vaccine that needs further in vitro and in vivo evaluations.
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