Hydrogen Sulfide Inhibits Homocysteine-Induced Neuronal Senescence by Up-Regulation of SIRT1

Homocysteine (Hcy) accelerates neuronal senescence and induces age-related neurodegenerative diseases. Silence signal regulating factor 1 (SIRT1) prolongs lifespan and takes neuroprotective effects. We have previously demonstrated that hydrogen sulfide (H2S) prevents Hcy-induced apoptosis of neuronal cells and has neuroprotective effect. In the present work, we aimed to investigate whether H2S protects HT22 cells against Hcy-induced neuronal senescence and whether SIRT1 mediates this role of H2S. We found that Hcy induced cellular senescence in HT22 cells, as determined by β-galactosidase staining, expressions of P16INK4a, P21CIPL, and trypan blue Staining, which are the markers of cellular senescence. However, sodium hydrosulfide (NaHS, the donor of H2S) significantly reversed Hcy-induced cellular senescence. Interestingly, NaHS not only up-regulated the expression of SIRT1 in HT22 cells but also reversed Hcy-downregulated the expression of SIRT1 in HT22 cells. Furthermore, we found that pretreatment with Sirtinol (an inhibitor of SIRT1) markedly reversed the protection of NaHS against Hcy-induced HT22 cells senescence and apoptosis. Our findings illustrated that H2S protects HT22 cells against Hcy-induced senescence by up-regulating SIRT1.


Introduction
Homocysteine (Hcy) is a sulfur-containing nonprotein amino acid derive from methionine metabolism [1]. Hcy promotes neuronal degeneration and thus it contributes to age-related neurodegenerative diseases, such as dementia, Alzheimer's disease (AD), Parkinson's disease(PD), and stroke [2,3]. The emerging evidence suggests that a potentially important contributor to aging and age-related neurodegenerative diseases is cellular senescence [4], a process that imposes permanent proliferative arrest on cells responding to various stressors [5]. Therapeutic strategies that safely interfere with the detrimental effects of cellular senescence are gaining significant attention [4,6]. Interestingly, accumulating evidence showed that cellular senescence is causally implicated in Hcy-generated age-related diseases [7][8][9]. Thus, finding a new strategy to antagonize Hcyinduced neuronal senescence has important value in the prevention and treatment of age-related neurodegenerative diseases.
Hydrogen sulfide (H2S) is a novel gaseous molecule with an extremely unpleasant odor. In the brain, H 2 S is an important neuroprotective agent and has therapeutic potential in neurodegenerative diseases of aging, such as AD and PD [10,11]. Recently, H 2 S was identifiedas a new approach to Ivyspring International Publisher prolong lifespan [12]. H 2 S inhibits the production of mitochondrial ROS [13], decreases oxidative stress [14,15], and repairs DNA damage [16] to protect cells from senescence. Previous studies revealedthat H 2 S protects endothelial cells against nicotinamide- [17] and H 2 O 2 - [18] induced cellular senescence. Our previous study indicated that H 2 S prevents Hcy-induced neurotoxicity [19,20]. In this study, we explored whether ameliorating Hcy-induced neuronal senescence contributes to the protection of H 2 S against Hcy-induced neurotoxicity.
Silent mating type information regulator 2 homolog 1 (SIRT1) is a NAD+-dependent deacetylase of lysine residue of the target protein. SIRT1 extends lifespan [21] and improves cell tolerance to inhibit environmental stress [22,23]. Recently, it has shown that H2S extends lifespan by activation of SIRT1 [12]. Furthermore, Our previous data demonstrated that H 2 S up-regulates the expression of SIRT1 in PC12 cells [20]. Thus, we further investigated whether SIRT1 mediates the inhibitory role of H 2 S in Hcy-induced neuronal senescence.
The present studies demonstrated the ability of H 2 S to inhibit Hcy-induced senescence in HT-22 cells and identified the mediatory role of SIRT1 in the protective action of H 2 S from Hcy-induced neuronal senescence. It provides novel strategy to prevent Hcy-induced neurotoxicity via inhibition of senescence-associated neuronal aging. Thus, our research identifies SIRT-1 as a potent therapeutic target and H2S as a potent treatment for Hcy-related neurodegenerative diseases.

Cell viability assay
Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay. HT22 Cells of logarithmic phase growth were seeded into a 96-well plate at a density of 1 × 10 4 cells/well. The cells were treated with 2.5, 5.0, 10.0 mM Hcy, or/and 100, 200, 400 μmol/L NaHS and routinely incubated for 24 hours, 48 hours, and 72 hours. After treatment, viable cells were stained with 5μL of CCK-8 solution (CCK-8, Sigma, USA) and incubated at 37°C for 4 h. Absorbance was measured at 450 nm by a microplate reader (ELX-800, BIO-TEK, USA). The experiment was performed three times.

Trypan blue Staining
The cells were grew at concentration of 1 × 10 4 cells/ml in 24 well-plates to 60-70% confluence by incubation for 24 h, then treated with 2.5mM, 5.0mM, 10mM Hcy or and 100, 200, 400 μmol/L NaHS for 24 h, 48 h, 72 h. Floating and adhering cells were collected, washed once with PBS (pH 7.4), centrifuged, suspended in 10% DMEM and stained with 0.4% trypan blue (sigma, St. Louis, MO, USA) at room temperature. About 10 μl of sample was loaded on hemocytometer chamber and numbers of blue cells and non-blue cells were counted under a light microscope (Mike audi, China). The results were expressed as percentage of the control.

Western blotting
Cells of logarithmic phase growth were plated in 50ml culture bottle. After incubated with DMEM for 24h, the cells were treated with Hcy or/ and NaHS at different concentrations for 48 h. Then the treated cells were resuspended in 100 uM cell lysis buffers and PMSF incubation on ice for 30 min. The supernatant was collected after centrifuged at 12,000 rpm for 10 min at 4˚C. The protein concentrations were detected by BCA method (Solarbio, Beijing, China). Equal amounts of total protein extracts were electrophoresed through 10% or 12% SDS-PAGE gel, then transferred to polyvinylidene difluoride membranes (Solarbio, China). After blocked with TBST (50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.1% Tween-20) containing 5% BSA (Sigma, USA) for 2 h, the membranes were incubated with primary antibodies including monoclonal antibody for P16 INK4a , P21 CIPL , and SIRT1(dilution, 1:1,000) at 4˚C overnight. After washed with TBST for 5 min 5 times, the membranes were incubated with secondary antibodies including Goat anti-rabbit (Proteintech, USA) (dilution, 1: 5,000) at room temperature for 2 h. After washed for three times with TBST for 3 times, the membranes were visualized with Western Blotting Chemiluminescence Reagent (Solarbio, Beijing, China), followed by apposition of the membranes with autoradiographic films (Kodak, China). The expression of β-actin for each sample was used as a control.

Flow cytometry analysis
HT22 cells of the log phase were grew in 6-well plates and treated with Hcy or NaHS for 48h. Cells were stained 50 μg/ml propidium iodide (PI) (Roche, Mannheim, Germany) and 10 μg/ml RNase A (Sigma, St. Louis, MO, USA). Percentages of cells existing within the various phases (G0/G1, S, G2/M) of the cell cycle were calculated by the mean fluorescence intensity.

Statistical analysis
Experiments were repeated at least three times. Values were expressed as mean ± standard error of the mean (S.E.M.). All data were analyzed by SPSS version 21.0. Data were evaluated for statistical significance with using one-way analysis of variance (ANOVA) followed by LSD post hoc tests. P < 0.05 was considered to indicate a statistically significant difference.

Hcy induced the cellular senescence in HT22 cells
We first explored whether Hcy induces cellular senescence in HT22 cells. After treatment with Hcy (2.5, 5, 10 mM) for 48 h, the percentage of senescenceassociated beta-galactosidase (SA-β-Gal)positive cells was increased (Fig. 1A), the expressions of P16 INK4a and P21 CIPL were upregulated (Fig. 1B), and the cell density was decreased (Fig. 1C) in HT22 cells, which indicated that Hcy induces the cellular senescence in HT22 cells.

H 2 S prevented Hcy-induced cellular senescence in HT22 cells
Next, we explored the effect of H 2 S on Hcy-induced cellular senescence in HT22 cells. HT22 cells were pretreated with NaHS (100, 200, and 400 μM) for 30 min and then cotreated with 5 mM Hcy for 48 h. We found that pretreatment of NaHS (100, 200, or 400 mM) significantly decreased the percentage of SA-β-galpositive cells ( Fig. 2A) and the expressions of P16 INK4a and P21 CIPL (Fig. 2B), while increased the cell density ( Fig. 2C) in Hcy-treated HT22 cells. These results demonstrated that H 2 S prevented Hcyinduced cellular senescence in HT22 cells.

NaHS upregulated the expression of SIRT1 in HT22 cells
To explore the mediatory role of SIRT1 in the protection of H2S against Hcy-induced cellular senescence in HT22 cells, we first investigated the expression of SIRT1 in different treated HT22 cells. After the expression of SIRT1 in HT22 cells was markedly down-regulated by treatment with Hcy (2.5, 5.0, 10.0 mM) for 48 h (Fig. 3A), while was up-regulated by treatment with NaHS (100, 200, and 400 μM) alone for 48 h (Fig. 3B). Furthermore, preteatment with NaHS (100, 200, and 400 μM) restored the expression of SIRT1 in Hcy-treated HT22 cells (Fig. 3C). These results suggest that NaHS not only upregulated the expression of SIRT1 in HT22 cells but also reversed the down-regulation of SIRT1 in Hcy-treated HT22 cells.

Sirtinol reversed the protection of NaHS against Hcy-induced cellular senescence in HT22 cells
To further confirm whether SIRT1 mediates the protection of NaHS against Hcy-induced cellular senescence in HT22 cells, we explored whether sirtinol, a specific inhibitor of SIRT1, reversed this protective role of NaHS. We found that pretreatment with sirtinol (15 μM, for 30 min) increased the percentage of SA-β-gal-positive cells (Fig. 4A) as well as the expressions of P16 INK4a , P21 CIPL (Fig. 4B), while decreased the cell density (Fig. 4C) in HT22 cells cotreated with 5 mM Hcy and 400 μM NaHS for 48 h. These findings verified that sirtinol reverses the protection of NaHS against Hcy-induced cellular senescence in HT22 cells.

Sirtinol reversed the protective effect of H 2 S on Hcy-indcued neurotoxicity to HT22 cells
We have previously demonstrated that H 2 S inhibits Hcy-induced neurotoxicity. Therefore, we next investigated whether blockage of SIRT1 reverses the protective effect of H 2 S against Hcy-induced neurotoxicity to HT22 cells. As shown in Fig. 5A, pretreatment of NaHS (100, 200, or 400 μM) significantly increased the cell viability of HT22 cells exposed to 5 mM of Hcy for 48 h, which indicated that NaHS inhibites Hcy-induced cytotoxicity to HT22 cells. However, pretreatment with sirtinol (15 μM) markedly decreased the cell viability of HT22 cells cotreated with Hcy (5 mM) and NaHS (400 μM) (Fig.  5B), which indiccated that sirtinol reverses the protection of NaHS against Hcy-induced cytotoxicity to HT22 cells. In addition, pretreatment of NaHS (400 μM, for 30 min) significantly decreased the apoptosis in HT22 cells treated with 5 mM of Hcy for 48 h, while pretreatment with sirtinol (15 μM, for 30 min) increased the apoptosis in HT22 cells cotreated with Hcy and NaHS (Fig. 5C), which indiccated that sirtinol reverses the protection of NaHS against Hcy-induced apoptosis to HT22 cells. These results demonstrated that inhibition of SIRT1 reverses the protection of H2S against Hcy-induced neurotoxicity in HT22 cells.

Discussion
Our previous studies have demonstrated that H 2 S has a protective effect against Hcy-evoked neurotoxicity [20,[24][25][26]. Considering the cellular senescence is prominent in the neurotoxicity of Hcy [3,[27][28][29], the present work was designed to explore whether the protection of H2S against the neurotoxicity of Hcy is associated with regulating neuronal senescence.
The main findings of the present work are the following: (i) H 2 S suppressed Hcy-induced cellular senescence in HT22; (ii) H 2 S up-regulated the expression of SIRT1 in Hcy-exposed HT22 cells. (iii) Inhibition of SIRT1 reversed the protective effect of H 2 S against Hcy-induced senescence and neurotoxicity in HT22 cells. Together, we demonstrated that the protection of H 2 S against Hcy-induced neurotoxicity involves inhibition of neuronal senescence through upregulating SIRT1 signaling.
Cellular senescence, a process that imposes permanent proliferative arrest on cells in response to various stressors, has emerged as a potentially important contributor to aging and age-related disease, and it represents an attractive target for therapeutic exploitation [4]. Hcy is an independent risk factor for neurological and cardiovascular disease [30,31]. The level of Hcy in bodies increases with age [32]. Therefore, understanding whether Hcy induces neuronal senescence is necessary for a greater understanding of the neurotoxicity of Hcy, which may be part of the mechanisms leading to age-relate neurological diseases [33,34]. SA-β-Gal staining remains as the most widely used biomarker for cellular senescence [35,36]. P21 CIPL is a cyclin-dependent kinase inhibitor, with regulating cell cycle progression at G1 and S phase [37]. P16 INK4a plays a critical role on controlling cellular senescence [38]. The present work demonstrated that the percentage of SA-β-Gal positive cells and the expressions of age-related markers P16 INK4a as well as P21 CIPL were significantly increased in Hcy-treated HT22 cells. Furthermore, we found that Hcy led to cell growth arrest. These data concluded that Hcy induces cellular senescence in HT22 cells. Therefore, modulation of neuronal senescence might represent a novel therapeutic strategy to overcome Hcy-induced neurotoxicity.  H 2 S, a well-known regulator of inflammation [39], ER stress [40] and cytotoxicity [41], is recognized as a new approach to prolong lifespan [12]. H 2 S has been described to prevent endothelial cell senescence [18,42] and delays aging [43]. Furthermore, we have previously found that H 2 S inhibits formaldehydeinduced cellular senescence [44] and protects against Hcy-induced neurotoxicity [25]. Therefore, it is imperative to assess the effect of H 2 S on Hcy-induced cellular senescence. In the present work, we found that H 2 S increased the growth curve and decreased the percentage of SA-β-Gal positive cells as well as the expressions of P16 INK4a and P21 CIPL in Hcy-treated HT22 cells. Therefore, we for the first demonstrated that H 2 S prevents Hcy-induced cellular senescence in HT22 cells. It has been confirmed that H 2 S prevents the process of senescence in the endothelium [42], kidney [45,46], vascular [47], heart [48] and brain [43] of mice. Furthermore, increasing the content of endogenous H 2 S by proper diet extends the life span of the aged mice [49,50]. These previous findings offered a reasonable explanation for the results obtained in the present study. Therefore, the regulation of neuronal senescence offers insights into the protection of H2S against the neurotoxicity of Hcy. In our previous work, we used PC12 cells as a cell model to explore the protection of H2S against Hcy-induced endoplasmic reticulum (ER) stress [20]. This previous work focused on the protective role of H 2 S in Parkinson's disease. Thus, we used PC12 cells to explore the protection of H 2 S against Hcy-induced ER stress because PC12 cells are dopaminergic neurons and are commonly used as the cell model of PD. In the present study, the purpose is to explore the effect of H 2 S on Hcy-induced impairment in learning and memory. Learning and memory are closely related to hippocampus [51]. Thus, HT22 cells were used in the present work because they are hippocampal neurons [52]. Hcy may be also induced ER stress in HT22 cells. In our future, we will explore the role of ER stress in Hcy-induced neuronal senescence in HT22 cells.
The present study also investigated the possible underlying mechanism for the protective role of H2S in Hcy-induced senescence. Sirtuin was regarded as the lifespan-extending gene in the past years [53]. SIRT1, an important member of sirtuin family, also takes an important role in delaying cellular senescence and extending longevity [29,54]. Numerous studies have demonstrated that SIRT1 ameliorate neurodegenerative disease, such as AD [55], PD [56], subarachnoid hemorrhage [57], which reveal that SIRT1 delays senescence in the brain. Our previous study demonstrated that SIRT1 mediates the protective effect of H 2 S on Hcy-induced neurotoxicity [20]. Therefore, we speculated whether SIRT1 mediates the protection of H 2 S against Hcy-induced senescence in HT22 cells. How might Hcy mediate the blockade of SIRT1? Previous study suggests that Hcy increases ROS generation by activating protein kinase C-β (PKCβ), which in turn improves SIRT1 degradation through a proteasome-dependent mechanism [58]. In line with this finding, our present work demonstrated that Hcy downregulated the expression of SIRT1 in HT22 cells. Notably, we found that H2S not only increased the expressions of SIRT1 in HT22 cells, but also reversed Hcy-reduced the expressions of SIRT1 in HT22 cells. These results implied that the upregulation of SIRT1 contributed to the protective effect of H2S on Hcy-induced senescence. To further confirm whether the SIRT1 mediates the protection of H 2 S against Hcy-induced senescence, we explored whether the blockage of SIRT1 abolishes this protection of H 2 S. We utilized Sirtinol as the inhibitor of SIRT1, which get a broaden acceptance [59]. Our results showed that inhibited SIRT1 by Sirtinol abolished the protection of H 2 S against Hcy-induced increase in the percentage of SA-β-Gal positive cells, up-regulations of P16 INK4a and P21 CIP1 , and the arrest of cell growth. Taken together, these results indicated that SIRT1 mediates H 2 S-exerted protection against Hcy-induced senescence in HT-22 cells. Simultaneously, the blockage of SIRT1 also eliminated the protection of H 2 S against Hcy-induced neurotoxicity, which further demonstrated that the involvement of reduced cellular senescence in the H 2 S-exerted protection against the neurotoxicity of Hcy. The possible molecular mechanism of H 2 S action on SIRT1 is unknown. A recent study has described that H 2 S increases intracellular NAD+ levels that are known to fuel SIRT1 activity [60] and that SIRT1 increases phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), which may be related to the regulation of anti-apoptosis and stress response [61,62]. Although the precise molecular mechanisms of H2S action on SIRT1 remain to be determined, the most promising candidates are NAD+ or the pathway of PKCβ-ROS. Future works will be required to precisely define whether NAD+ and ERK1/2 implicate in the mechanisms underlying the regulatory role of H2S in the action of SIRT1.

Conclusion
In this study, we demonstrated that treatment with NaHS, a donor of H 2 S, attenuated Hcy-induced cellular senescence and upregulated SIRT1 expression in Hcy-exposed HT22 cells. Furthermore, we showed the reversing role of inhibited SIRT1 in the protection of NaHS against Hcy-induced cellular senescence and neurotoxicity. These data suggested that H 2 S inhibits Hcy-induced cellular senescence by upregulation of SIRT1. Our results provide important insights into the molecular mechanism underlying H 2 Sexerted protection against Hcy-induced neurotoxicity and provide a basis for investigating H 2 S as a therapeutic approach for Hcy-related neurodegenerative disease.