Hydrogen sulfide inhibits homocysteine-induced endoplasmic reticulum stress and neuronal apoptosis in rat hippocampus via upregulation of the BDNF-TrkB pathway.

AIM
Homocysteine (Hcy) can elicit neuronal cell death, and hyperhomocysteinemia is a strong independent risk factor for Alzheimer's disease. The aim of this study was to examine the effects of hydrogen sulfide (H2S) on Hcy-induced endoplasmic reticulum (ER) stress and neuronal apoptosis in rat hippocampus.


METHODS
Adult male SD rats were intracerebroventricularly (icv) injected with Hcy (0.6 μmol/d) for 7 d. Before Hcy injection, the rats were treated with NaHS (30 or 100 μmol·kg(-1)·d(-1), ip) and/or k252a (1 μg/d, icv) for 2 d. The apoptotic neurons were detected in hippocampal coronal slices with TUNEL staining. The expression of glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), cleaved caspase-12, and BDNF in the hippocampus were examined using Western blotting assays. The generation of H2S in the hippocampus was measured with the NNDPD method.


RESULTS
Hcy markedly inhibited the production of endogenous H2S and increased apoptotic neurons in the hippocampus. Furthermore, Hcy induced ER stress responses in the hippocampus, as indicated by the upregulation of GRP78, CHOP, and cleaved caspase-12. Treatment with the H2S donor NaHS increased the endogenous H2S production and BDNF expression in a dose-dependent manner, and significantly reduced Hcy-induced neuronal apoptosis and ER stress responses in the hippocampus. Treatment with k252a, a specific inhibitor of TrkB (the receptor of BDNF), abolished the protective effects of NaHS against Hcy-induced ER stress in the hippocampus.


CONCLUSION
H2S attenuates ER stress and neuronal apoptosis in the hippocampus of Hcy-treated rats via upregulating the BDNF-TrkB pathway.

Emerging evidence indicates that endoplasmic reticulum (ER) stress plays a pivotal role in the development or pathology of AD, which is characterized by an abnormal formation of inclusion bodies and aggregation of misfolded proteins [15][16][17] . The ER is a sophisticated luminal network for the synthesis, www.nature.com/aps Wei HJ et al Acta Pharmacologica Sinica npg maturation, folding, and transportation of proteins, which are required for cell survival and normal cellular functions [18,19] . Disturbance of protein folding under various physiological or pathological conditions, such as alteration of cellular redox, deprivation of glucose, aberration of Ca 2+ regulation, and viral infections, creates the ER stress [20] . Wei et al reported that exposure to Hcy of the rat embryonic heart-derived cell line H9c2 can increase the expression of ER stress response genes, such as glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), and cleaved caspase-12 [21] , suggesting that ER stress is involved in the mechanisms of Hcy-induced cellular dysfunction [8,[22][23][24][25] . Therefore, it is logical to speculate that inhibiting Hcy-induced ER stress may be a novel therapeutic strategy to prevent and ameliorate AD progression. Hydrogen sulfide (H 2 S), the third gaseous mediator, has recently been recognized as an important endogenous neuromodulator and neuroprotectant [26][27][28][29] . The most recent in vitro studies by our group have demonstrated that Hcy-associated neurotoxicity to PC12 cells is due to reduced endogenous generation of H 2 S [30] and that supplementation of sodium hydrosulfide (NaHS), the H 2 S-donor, ameliorates the Hcy elicited-neurotoxicity in PC12 cells [31] . However, the mechanisms underlying the protective effect of H 2 S against Hcy neurotoxicity are not completely understood. Recent studies have demonstrated that H 2 S inhibits ER stress in apolipoprotein E knockout mice [32] and in doxorubicin-treated H9c2 cells [33] . In addition, H 2 S has been shown to attenuate 6-hydroxydopamine-induced ER stress in SH-SY5Y cell line [34] . Furthermore, H 2 S could antagonize cardiomyocytic ER stress in Hcyinduced cardiomyocytic injury [21] .
The impact of H 2 S on ER stress involved in the neurotoxicity of Hcy is not yet known. Given the importance of Hcy in the pathogenesis of AD and the elevated ER stress response to Hcy, it is imperative to understand if the Hcy-induced ER stress status of neurocytes is regulated by H 2 S and to investigate the underlying mechanisms. Brain-derived neurotrophic factor (BDNF), a neurotrophic factor acting on the central nervous system, prevents ordinary types of neuronal cell death induced by various stimulants. Increasing evidence has demonstrated that prevention of ER stress contributes to BDNF-mediated neuroprotection [35][36][37] . Therefore, the present study was undertaken to examine whether H 2 S could regulate Hcy-induced neuronal ER stress in a rat model of Hcy neurotoxicity in vivo. Furthermore, the involvement of the BDNFtyrosine protein kinase B (TrkB) pathway in H 2 S-attenuated ER stress induced by Hcy was also investigated. In this study, we demonstrated for the first time the protective effects of H 2 S against Hcy-induced ER stress in the hippocampus of rats and that the underlying mechanism involves the upregulation of the BDNF-TrkB pathway.

Materials and methods
Reagents NaHS, Hcy, and k252a (an inhibitor of the BDNF receptor TrkB) were purchased from Sigma-Aldrich (St Louis, MO, USA). The in situ apoptosis detection kit was supplied by Roche Diagnostics (Indianapolis, IN, USA). Specific monoclonal anti-BDNF, anti-GRP78, and anti-CHOP antibodies were purchased from Epitomics Inc (Burlingame, CA, USA). Specific monoclonal anti-caspase-12 antibody was obtained from Sigma-Aldrich. RPMI-1640 medium, horse serum, and fetal bovine serum (FBS) were supplied by Gibco BRL (Grand Island, NY, USA).
Cell culture PC12 cells, originally derived from a transplantable rat pheochromocytoma, were supplied by Sun Yat-sen University Experimental Animal Center (Guangzhou, China) and maintained on tissue culture plastic in RPMI-1640 medium supplemented with 10% heat-inactivated horse serum and 5% FBS at 37 °C under an atmosphere of 5% CO 2 and 95% air. The culture medium was changed three times per week.

Animals
Adult male Sprague-Dawley rats (250-280 g), obtained from the SJA Lab Animal Center of Changsha (Changsha, China), were housed individually in a temperature-and humiditycontrolled room and kept on a 12-h light/12-h dark cycle with free access to food and water. The rats were handled (2-3 min per rat per day) for one week to be accustomed to the experimenter. Experiments were conducted in accordance with the National Institute of Health's Guide for the Care and Use of Laboratory Animals and were approved by the Animal Use and Protection Committee of University of South China. All efforts were made to minimize animal's suffering.

Drugs and treatments
Hcy and NaHS were dissolved in 0.9% nonpyrogenic NaCl and the solution was filtered through a 0.2-μm PTFE filter (Sigma-Aldrich). K252a was dissolved in sterilized artificial cerebrospinal fluid/dimethyl sulfoxide (ACSF/DMSO; volum ratio, 1:1) (Sigma-Aldrich). Hcy and k252a were injected intracerebroventricularly (icv), and NaHS was injected intraperitoneally (ip). Rats were divided into four treatment groups: 1) a control group, in which subjects were treated with saline (ip) and ACSF/DMSO (icv) every day for 9 d; 2) an Hcy-treated alone group, in which subjects were treated with 0.6 μmol/d Hcy (icv) for 7 d; 3) a co-treated with Hcy and NaHS group, in which subjects were pretreated with 30 or 100 μmol·kg -1 ·d -1 NaHS (ip) for 2 d and then co-treated with 0.6 μmol/d Hcy (icv) for 7 d; and 4) a k252a-inhibition group, in which subjects were pretreated with 30 or 100 μmol/kg NaHS (ip) and 1 μg/d k252a (icv) for 2 d and then co-treated with 0.6 μmol Hcy (icv) per day for 7 d. The intraperitoneal treatments were administered in a volume of 1 mL/kg body weight. The intracerebroventricular treatments were administered in a volume of 2.5 μL.

Intracerebroventricular injection
The rats were anesthetized with sodium pentobarbital (45 mg/kg, ip) and placed into a stereotaxic apparatus for operation. Saline, ACSF/DMSO, Hcy (0.6 μmol), or k252a (1 μg) www.chinaphar.com Wei HJ et al Acta Pharmacologica Sinica npg with a volume of 2.5 μL was injected into the bilateral ventricle at the following coordinates: anterior/posterior -1.4 mm, medial/lateral 1.8 mm, dorsal/ventral -3.0 mm from the bregma, respectively, with an injection rate of 0.5 μL/min under the control of a micropump. To ensure that the entire injection had been delivered, the injection cannula was allowed to remain in place for an additional minute before being removed.
TdT-mediated dUTP nick-end labeling (TUNEL) staining After anesthesia, rats were fixed by perfusing 200-300 mL of Zamboni's fixative (4% paraformaldehyde in 0.1 mol/L phosphate buffer, pH=7.4) through the left ventricle of the heart. The hippocampus was removed and post-fixed in the perfusion fixative for 2 h at room temperature and then immersed in a 20% sucrose solution until the tissue sank. The hippocampus was coronally sectioned with a cryostat at a thickness of 10 μm.
To detect the apoptotic cell death in the hippocampus, we processed the sections of the hippocampus for TUNEL staining using an in situ apoptosis detection kit (Roche Diagnostics). Briefly, sections were digested with 15 µg/mL proteinase K for 15 min at room temperature and then washed with phosphate buffered saline (PBS). Endogenous peroxidase activity was quenched with 3% H 2 O 2 for 5 min at room temperature. After being washed with PBS, the sections were immersed in terminal deoxynucleotidyl transferase (TdT) labeling buffer, covered with TdT buffer containing TdT, 1 mmol/L Mn 2+ , and biotinylated dNTP, and incubated in a humid atmosphere at 37 °C for 90 min before being washed with PBS. The sections were then incubated with streptavidin-horseradish peroxidase for 30 min. The sections were rinsed with PBS, and immersed in diaminobenzidine solution. The slides were counterstained for 1 min with 1% methyl green. The total number of TUNELpositive cells on each section was counted under a microscope. The data are presented as the number of positive neurons/mm 2 .

Statistical analysis
Data are expressed as the mean±SEM. The significance of inter-group differences was evaluated by one-way analysis of variance (ANOVA), followed by a Newman-Keuls test. Differences were considered significant at two-tailed P<0.05.

H 2 S attenuates Hcy-induced neuronal apoptosis in the hippocampal CA1 region
We first detected the apoptotic cells in the hippocampus slices by TUNEL staining to confirm the protection of H 2 S against Hcy-induced neurotoxicity in vivo. As shown in Figure 1, after 7-d treatment with Hcy (0.6 μmol/d, icv), significant amounts of TUNEL-positive neurons appeared in the hippocampal CA1 region in the rats. However, the TUNEL-positive neurons in the hippocampal CA1 region were markedly decreased in the rats pretreated with NaHS (100 μmol·kg -1 ·d -1 for 2 d, ip) and then co-treated with Hcy (0.6 μmol/d for 7 d, icv).

H 2 S upregulates BDNF expression in hippocampus of rats
To explore if the upregulation of BDNF is involved in the mechanism underlying the protective effect of H 2 S against Hcy-elicited neurotoxicity, we investigated the effect of H 2 S on BDNF expression in the hippocampus of rats. BDNF expression was markedly downregulated in the hippocampus of rats  (Figure 2A). In addition, the expression of BDNF was upregulated in the hippocampus of rats treated with NaHS (30 and 100 μmol·kg -1 ·d -1 for 9 d, ip) ( Figure 2B).
H 2 S decreases Hcy-induced upregulation of GRP78 expression in the hippocampus of rats, and this effect is abolished by blocking the BDNF-TrkB pathway To investigate whether Hcy induces ER stress in the hippocampus of rats and whether H 2 S inhibits Hcy-induced ER stress, we first sought to explore the expression of GRP78, an important marker for ER stress, in the hippocampus of rats. As shown in Figure 3A, Hcy (0.6 μmol/d for 7 d, icv) significantly increased GRP78 expression in the hippocampus of rats. However, pretreatment with NaHS (30 and 100 μmol·kg -1 ·d -1 for 2 d, ip) before the co-treatment with Hcy for 7 d clearly decreased the upregulated expression of GRP78 induced by Hcy. These findings indicate the effects of Hcy on ER stress in the hippocampus and the inhibitory effect of H 2 S on Hcyinduced ER stress.
To determine the involvement of the BDNF-TrkB pathway in H 2 S neuroprotection against Hcy-induced ER stress, we investigated the effect of k252a on the inhibitory role of H 2 S in Hcy-upregulated GRP78 expression. As shown in Figure 3B, treatment with k252a (1 μg/d for 9 d, icv) dramatically curbed the preventive effects of NaHS (100 μmol·kg -1 ·d -1 for 9 d, ip) on the expression of GRP78 in the hippocampus of rats treated with Hcy (0.6 μmol/d for 7 d, icv), indicating that the protective effect of H 2 S against Hcy-induced ER stress is mediated by stimulation of the BDNF-TrkB pathway.

S represses Hcy-induced upregulation of CHOP expression in hippocampus of rats, and this effect is blocked by inhibiting the BDNF-TrkB pathway
To further ascertain whether exposure to Hcy induces ER stress, we proceeded to monitor the CHOP protein level in the hippocampus of rats. CHOP expression in the hippocampus of rats treated with Hcy (0.6 μmol/d for 7 d, icv) was clearly increased. However, upregulated CHOP expression was attenuated in the hippocampus of rats by pretreating with NaHS (30 and 100 μmol·kg -1 ·d -1 for 2 d, ip) before co-treatment with Hcy (0.6 μmol/d for 7 d, icv) ( Figure 4A). These data also indicated the protective action of H 2 S on Hcy-induced ER stress.
We also explored the effect of k252a on the suppressive role of H 2 S in Hcy-upregulated CHOP expression. Western blotting analysis revealed that administration of k252a (1 μg/d, icv, for 9 d) prevented NaHS (100 μmol·kg -1 ·d -1 for 9 d, ip) from suppressing the expression of CHOP in the hippocampus of rats treated with Hcy (0.6 μmol/d for 7 d, icv) ( Figure 4B). This suggests an important role of the BDNF-TrkB pathway in mediating the neuroprotective effect of H 2 S against ER stress induced by Hcy.  Cleaved caspase-12 participates in ER stress-induced apoptosis. Therefore, we further investigated the expression of cleaved caspase-12 in the hippocampus of rats. As shown in Figure 5A, Hcy (0.6 μmol/d for 7 d, icv) significantly upregulated the expression of cleaved caspase-12 in the hippocampus of rats. However, pretreatment with NaHS (30 and 100 μmol·kg -1 ·d -1 for 2 d, ip) before co-treatment with Hcy for 7 d clearly suppressed the upregulation of cleaved caspase-12 induced by Hcy. These data suggested that H 2 S treatment produces a protective role in ER stress-induced apoptosis. We further investigated the effect of k252a on the inhibitory role of H 2 S in Hcy-upregulated expression of cleaved caspase-12. As shown in Figure 5B, administration of the inhibitor of the BDNF receptor k252a (1 μg/d for 9 d, icv) prevented NaHS (100 μmol·kg -1 ·d -1 for 9 d, ip) from inhibiting the upregulated expression of cleaved caspase-12 in the hippocampus of rats treated with Hcy (0.6 μmol/d for 7 d, icv). This suggests that the BDNF-TrkB pathway mediates the neuroprotective effect of H 2 S against Hcy-induced ER stress.

H 2 S prevents Hcy-induced ER stress in PC12 cells
To further confirm the protective effect of H 2 S against Hcyinduced ER stress, we investigated the effects of H 2 S on Hcyinduced ER stress in PC12 cells. As illustrated in Figure 6, pretreatment with NaHS (200 μmol/L) for 30 min significantly attenuated the increases in the expression levels of GRP78, CHOP, and cleaved caspase-12 in PC12 cells induced by treatment with 5 mmol/L of Hcy for 24 h. These data confirm the protective effect of H 2 S against Hcy-induced ER stress in vitro.

Hcy inhibits endogenous hippocampal H 2 S production, which is reversed by NaHS administration
To know the feedback effect of ER stress on H 2 S production in the hippocampus, we explored the level of hippocampal endogenous H 2 S production in the rats treated with Hcy and the restorative effect of NaHS administration. As shown in Figure 7, the generation of H 2 S in the hippocampus of rats treated with Hcy (0.6 μmol/d for 7 d, icv) was markedly inhibited. However, the generation of H 2 S in the hippocampus of rats was rescued by pretreating with NaHS (30 and 100 μmol·kg -1 ·d -1 for 2 d, ip) before co-treatment with Hcy (0.6 μmol/d for 7 d, icv).

Discussion
H 2 S is an emerging novel endogenous neuroprotectant. We have previously demonstrated the protective effect of H 2 S against the neurotoxicity of Hcy. The present study was designed to investigate the role of H 2 S in regulating Hcyinduced neuronal ER stress and the underlying mechanisms. We showed that H 2 S upregulates the expression of BDNF in the hippocampus of rats and that intracerebroventricular injection of Hcy leads to an increase in the expression of various ER stress-associated proteins, including GRP78, CHOP, and cleaved caspase-12, in the hippocampus of rats. Notably, we found that H 2 S was able to downregulate the elevated ER stress markers. Furthermore, blocking of the BDNF-TrkB pathway by the inhibitor of TrkB, k252a, reverses the protective role of H 2 S in Hcy-induced ER stress. Collectively, these findings suggest that H 2 S could attenuate neuronal ER stress in Hcy-induced neurotoxicity by upregulating the pathway of BDNF-TrkB.
Hcy is an independent risk factor for AD [10][11][12][13][14] . ER stress is implicated in the development or pathology of AD [15][16][17] .  Although it is not clear if Hcy triggers neuronal ER stress, ER stress has been proposed to explain the pathogenic effects of Hcy in cardiovascular disease [21] , insulin resistance of adipose tissue [38] , apoptosis of osteoblastic cells [39] , type 2 diabetes mellitus [40] , and hepatic steatosis [41] . ER stress may be a common pathway of the injury of tissues and cells induced by Hcy. In the present work, we examined the effects of Hcy on the expression of protein markers of ER stress, such as GRP78, CHOP, and cleaved caspase-12, in the hippocampus of rats. We found that expressions of GRP78, CHOP, and cleaved caspase-12 in the hippocampus of rats were all upregulated by intracerebroventricular injection of Hcy. This indicated that Hcy-induced, severe ER stress was present in the hippocampus, suggesting that ER stress is a contributory factor in Hcyinduced neurotoxicity.
Interestingly, H 2 S and Hcy are metabolites of methionine [42] , but they exert entirely opposite effects on the viability of neurocytes. Hcy induces accumulation of reactive oxygen species (ROS) and stimulates neurotoxicity [43,44] , whereas H 2 S scavenges ROS and protects neurons against oxidative stress [45][46][47][48] . Furthermore, both elevation of Hcy and decrease of H 2 S are detected in the brains of AD patients [49] . Therefore, we have previously explored if H 2 S directly antagonizes the toxicity of Hcy to neuronal cells, and our previous data revealed that H 2 S could attenuate the neurotoxicity of Hcy [31] . It has been reported that H 2 S serves as a protective gaseous signaling molecule in the nervous system by preserving mitochondrial function [50,51] . The ER is another important subcellular organelle critical for protein folding and the formation of disulfide bonds [52] . Investigating if H 2 S can affect the ER function is important for elucidating the mechanisms underlying the protective effect of H 2 S against Hcy-induced neurotoxicity.
In the present study, we demonstrated that administration of NaHS alleviated the expression of GRP78, CHOP, and cleaved caspase-12 in the hippocampus of rats treated intracerebroventricularly with Hcy. The fact that H 2 S suppresses Figure 6. NaHS suppresses Hcy-induced ER stress in PC12 cells. After pretreatment with NaHS (200 μmol/L) for 30 min, PC12 cells were exposed to Hcy (5 mmol/L) for 24 h. The levels of GRP78 (A), CHOP (B), and cleaved caspase-12 (C) expression in PC12 cells were detected by Western blotting using anti-GRP78, -CHOP, and -cleaved caspase-12 antibody, respectively. In all blots, staining for β-actin was used as a loading control. Values are presented as the mean±SEM (n=3). c P<0.01 vs control group. f P<0.01 vs Hcy-treated alone group. Figure 7. Hcy inhibits hippocampal endogenous H 2 S generation, which is rescued by the administration of NaHS. Rats were pretreated with NaHS (30 and 100 μmol·kg -1 ·d -1 for 2 d, ip) and then co-treated with Hcy (0.6 μmol/d for 7 d, icv). The hippocampus of rats was homogenized and the generation of H 2 S in the hippocampus was measured by the NNDPD method as described in the Materials and Methods section. Values are expressed as the mean±SEM (n=3-5). c P<0.01 vs control group. f P<0.01 vs Hcy-treated alone group.
www.nature.com/aps Wei HJ et al Acta Pharmacologica Sinica npg the expression of GRP78, CHOP, and cleaved caspase-12 is consistent with its effect on the toxicity of Hcy in cardiomyocytes [21] . Moreover, the protective effects of H 2 S against Hcyinduced ER stress were further confirmed in PC12 cells. Our results indicated that H 2 S counteracts Hcy-induced ER stress, contributing to the role of H 2 S in protecting against Hcyinduced neurotoxicity. We further examined the possible signaling mechanisms for the protective effect of H 2 S against Hcy-induced ER stress. BDNF, a member of the neurotrophin family, regulates the development, maintenance, plasticity, and function of the nervous system [53,54] . In vitro and in vivo studies indicate that BDNF functions as a neuroprotective agent and rescues neurons from various insults [55,56] . Increasing evidence has shown that BDNF plays an important role in counteracting ER stress [35][36][37] , suggesting that the suppression of ER stress may contribute to BDNF-mediated neuroprotection. The action of BDNF is mediated by its binding to the TrkB receptor. In the present study, we investigated the contribution of the BDNF-TrkB pathway to the inhibitory role of H 2 S in Hcy-induced ER stress. We found that NaHS upregulated the expression of BDNF in the hippocampus of rats. Furthermore, our present data demonstrated that blocking the BDNF-TrkB pathway by the inhibitor of TrkB reversed the inhibitory effect of H 2 S on the expression of GRP78, CHOP, and cleaved caspase-12 in the hippocampus of rats treated with Hcy. Our results suggest that H 2 S exerts its protective effects against Hcy-induced ER stress by upregulation of the BDNF-TrkB pathway.
In conclusion, the present observations identify the beneficial role of H 2 S in protecting the hippocampus against Hcyinduced ER stress. The neuroprotective effect of H 2 S involves the regulation of the BDNF-TrkB pathway. The findings of this work provide novel insights into the mechanisms of H 2 Smediated protection against Hcy neurotoxicity. As Hcy is an independent risk factor for AD [10][11][12][13][14] and ER stress is a crucial process in the pathogenesis of AD [15][16][17] , our findings suggest that H 2 S has potential therapeutic value in the treatment of neurodegenerative diseases such as AD.