| Hydrogen sulfide (H2S) was known to be a toxic gas and an environmental hazard for many decades. However, it is now recognized that H2S may serve as a gaseous mediator, which is endogenously produced to influence biological functions in mamalian. Together with nitric oxide and carbon monoxide, they form the group of mediators that has been termed the'gasotransmitters'. The past decade has seen an exponential growth of scientific interest in the physiological and pathological significance of H2S especially with respect to its role in the central nervous system (CNS) and the cardiovascular system. In the CNS, H2S facilitates long-term potentiation (LTP) and regulates intracellular calcium concentration and pH level in brain cells. Intriguingly, H2S produces anti-oxidant, anti-inflammatory and anti-apoptotic effects that may have relevance to neurodegenerative disorders. Abnormal generation and metabolism of H2S have been reported in the pathogenesis of ischemic stroke, Alzheimer's disease (AD), and recurrent febrile seizure. Exogenously applied H2S is demonstrated to have value for the treatment of febrile seizure. However, whether endogenous H2S is involved in the progress of PD and the therapeutic effects of H2S on PD are still unclear so far. Therefore, more attention should be focus on its neuroprotective effects and the underlying cellular and molecular mechanisms in neurodegeneration.Parkinson's disease (PD), the second most popular neurodegenerative disorder, is characterized by selective degeneration of dopamine neurons in the substantia nigra and aggregation of Lewy bodies (LBs) in neuron. The incidence rate of the disease increases along with aging, and more than 2% of the population aged over 65 years are attacked by the disease. In current, the first chosen for clinical treatment of PD is administration of L-DOPA, which alleviates the symptoms only and can not retard dopaminergic neuron degeneration. Although various hypotheses, including genetic factors, mitochondrial dysfunction, oxidative stress, excitotoxicity, neuroinflammation and apoptosis had been proposed to be involved in the pathogenesis of PD, the exact mechanisms governing dopaminergic neuron loss remain unclear. The predominant obstacle to developing neuroprotective therapies is a limited understanding of the key molecular events that provoke neurodegeneration.Therefore, the most important concern should be focused on the investigation of effective targets for PD treatment in future study. Previous studies reported that the level of H2S is decreased in hippocampus of AD patients, suggesting H2S be involved in the progress of AD. Our previous investigations also demonstrated that exogenous H2S inhibited LPS stimulated microglial activation and rotenone induced mitochondrial dysfunction. Moreover, both microglial inflammation and mitochondrial apoptosis are triggers for the development of PD. All of these studies reveal that H2S has potential influence in neurodegeneration. So far, however, there is no direct evidences support the hypothesis that H2S exerts neuroprotective effect in Parkinson's disease.Base on these findings, the aim of present studies is to investigate the role of H2S in PD model. We first established 6-OHDA lesioned PD rat model to investigate the endogenous H2S level in injured striatum and the neuroprotective effect of exogenous H2S on neural injury in PD rat model. Secondly, we explored the regulatory effect of H2S on glutamate uptake function in primary cultured astrocytes in vitro. Finally, Kir6.2 knockout mice were used to establish PD mouse model by subcutaneous injection of MPTP. We observed the protective effects of H2S on neurotransmitter and pathological alterations in PD mice and demonstrated the potential molecular targets for H2S. The results suggested that H2S may serve as a neuroprotectant in neurotoxin-induced neurodegeneration via multiple mechanisms and therefore has potential therapeutic value for treatment of PD.AIM: The present study was designed to examine the endogenous H2S level in injured striatum of PD rats and investigate the therapeutic effect of H2S on behavioral symptom and loss of TH neuron in PD model.METHODS: Unilateral injection of 6-OHDA in striatum (AP: +3.0 mm; ML: +1.0 mm; DV: -4.5 mm) was performed to establish rat PD model. Apomorphine was subcutaneously injected to induce contralateral rotations, which were recorded with a video camera at weekly intervals, considering as the behavioral symptom of PD model. NaHS (1.68 mg/kg and 5.6 mg/kg) was systemic administrated in successful PD rats for 3 weeks to examine the therapeutic effect of H2S. Endogenous H2S level was measured with zinc acetate trapping method. Immunohistochemistry was performed to detect the staining of tyrosine hydroxylase (TH) in substantia nigra and striatum. Western blotting confirmed the expression of TH in SN and striatum. The product of lipid peroxidation, malondialdehyde (MDA), was measured by commercial assay kit. SH-SY5Y cells were cultured to carry out cell fractionation. The membrane and cytosolic fractions were probed with antibody against gp91, p47 and ERK1/2.RESULTS: 1) Apomorphine induced marked contralateral rotations in the rats accepted unilateral injection of 6-OHDA, suggesting that the animal PD model was established successfully. The endogenous H2S level was significantly reduced in the lesioned SN of PD rat, indicating that H2S be involved in the development of PD. 2) Systemic administration of NaHS (1.68 and 5.6 mg/kg, i.p.) for 3 weeks dramatically alleviated the progression of movement dysfunction and attenuated the loss of dopaminergic neurons in the SN and striatum. Western blotting confirmed that NaHS reversed the downregulation of TH expression in SN and striatum. 3) NaHS inhibited the elevated MDA level in injured striatum of PD rat. Furthermore, NaHS specifically suppressed 6-OHDA evoked NADPH oxidase activation by inhibiting upregulation of gp91 in cytomembrane and trafficking of p47. CONCLUSION:1. Reduction of endogenous H2S level is involved in the development of PD rat model.2. H2S has potential therapeutic value for behavioral symptom and loss of dopaminergic neurons in 6-OHDA lesioned rats.3. H2S exerts anti-oxidative role via inhibition of NADPH oxidase activation.AIM: The present study was designed to investigate the effect of hydrogen sulfide (H2S), a novel neuromodulator, on hydrogen peroxide (H2O2)-induced glutamate uptake impairment and cellular injuries in primary cultured rat cortical astrocytes.METHODS: Primary cultured SD rat astrocytes were used to establish H2O2 injured cellular model. MTT assay was employed to examine cell viability and LDH measurement was applied to detect cellular injury. GSH and ROS level were measured to reflect the anti-oxidative effect of H2S in astrocytes. Experiment of [3H] labeled glutamate uptake was performed to investigate the effect of H2S on H2O2 injured astrocytic glutamate transporter. Western blotting was used to analysis the phosphorylation of ERK1/2, expression of total ERK1/2, trafficking of GLT-1 from cytoplasma to cytomembrane.RESULTS: 1) H2O2 (200μM) significantly decreased astrocytic cell viability and stimulated massive LDH release. NaHS attenuated H2O2 induced decline of cell survival in a concentration dependent manner and reversed H2O2 decreased intracellular GSH production. The protective effect of H2S could be abolished by PDC, which is specific inhibitor for glutamate uptake. NaHS also attenuated ROS accumulation and ATP deficiency induced by H2O2. 2) CBS inhibitor, AOAA, aggravated H2O2 induced reduction of cell viability, increase of LDH release and cleaved PARP expression, indicating that endogenous H2S exerts anti-oxidative effect. 3) NaHS reversed H2O2 impaired [3H]-glutamate uptake function, and enhanced the trafficking of GLT-1 from cytoplasma to cytomembrane. PD98059, an ERK1/2 inhibitor, exerted similar effect on GLT-1 trafficking. Moreover, NaHS suppressed H2O2 evoked phosphorylation of ERK1/2, promoting GLT-1 transport via inhibition of MAPK signal pathway. CONCLUSION:1. H2S has protective effect on oxidative stress-induced astrocyte impairment via enhancing glutamate uptake function.2. H2S may promote glutamate uptake activity via decreasing ROS generation, enhancing ATP production and suppressing ERK1/2 activation.Part III The neuroprotective effects of hydrogen sulfide in MPTP mouse model of Parkinson's diseaseAIM: To investigate the role and the mechanism of H2S on MPTP-induced degeneration of dopaminergic neurons in MPTP/p PD model using Kir6.2 or UCP2 deficiency mice.METHODS: Wild type, Kir6.2-/- and UCP2-/- mice were treated with MPTP (20 mg·kg-1 s.c.) and probenecid (250 mg·kg-1 i.p.) daily for 5 days. NaHS (5.6 mg·kg-1·day-1, i.p.) was administered to mice 3 days before the first injection with MPTP and last for 8 days totally. BrdU (50 mg·kg-1 i.p, every 2 h, total 4 times) was injected one day before sacrifice. Mice were killed 3.5 days after the final injection of MPTP. Immunohistochemistry was performed to examine tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), macrophage-1 antigen (Mac-1) and 5-bromodeoxyuridine (BrdU) expression. The total numbers of TH, GFAP and Mac-1 positive cells in the SNc and BrdU-positive cells in the subventricular zone (SVZ) and subgranular zone (SGZ) were obtained stereologically using the optical fractionator method. HPLC with electrochemical detectionwas used to measure striatal levels of different neurotransmitters, including DA, DOPAC, HVA, 5-HT, 5-HIAA, glutamate and GABA. Mesencephalic primary neuron was cultured to detect the neurotoxicity induced by MPP+. The levels of GRP78, CHOP, caspase12, LC3 and NF-κB were determined by Western blotting.RESULTS: 1) There was no significant difference (p>0.05) in MPTP/p induced impairment of neral pathology and neurotransmitters between Kir6.2+/+ and Kir6.2-/- mice. 2) Pretreatment with NaHS (5.6 mg/kg) decreased mortality induced by MPTP/p and attenuated loss of TH neuron, activation of astrocytes and microglia in SNc and inhibition of cell proliferation in SGZ of both Kir6.2+/+ and Kir6.2-/- mice (p<0.05). However, NaHS had no effect on decreased dopamine level in striatum of PD mouse (p>0.05). 3) Pretreatment with NaHS protected primary mesencephalic neurons against MPP+-induced cytotoxicity in both Kir6.2+/+ and Kir6.2-/- neurons. The mechanisms for the effect of NaHS are involved in suppressing MPP+ induced upregulation of GRP78, CHOP, caspase12 and LC3 expression. NaHS also inhibited activation of NF-kB pathway by reducing p65 transported into nucleus. 4) UCP2 knockout abolished the neuroprotective effects of H2S on MPP+-induced damage of TH neurons in both in vivo and in vitro study, suggesting that UCP2 may be the target for H2S.CONCLUSION:1. H2S alleviates the loss of TH neuron, the activation of astrocytes and microglia and attenuates the inhibition of cell proliferation in MPTP/p mouse model, suggesting that H2S may exert neuroprotective effects on neurodegeneration.2. The neuroprotective effect of H2S is independent on Kir6.2/K-ATP channel in MPTP/p mouse model. However, UCP2 located in mitochondrial membrane may be the molecular target for H2S.3. H2S inhibits endoplasmic reticulum stress and downstream pathway, exerting neuroprotective effects. In summary, the major contributions of the present study lie in:1. Endogenous H2S decline participates in the initiation and progress of PD. The level of endogenous H2S decreases significantly in striatum of neurotoxin induced PD animal model. This work proposes the direct evidences that H2S is involved in the development of PD.2. Exogenous H2S has neuroprotective effects in neurotoxin induced PD model. H2S alleviates the behavioral symptom and attenuates the loss of TH neuron, the activation of astrocytes and microglia and the inhibition of cell proliferation in PD animal model. We demonstrate for the first time that H2S may serve as a neuroprotectant to treat and prevent neurotoxin-induced neurodegeneration and therefore has potential therapeutic value for treatment of PD.3. The neuroprotective effect of H2S is independent on K-ATP channel. Previous studies reported that Kir6.2/K-ATP channel that locates in neuron mediates the neuroprotective effects of H2S in in-vitro research. It's found in this investigation that Kir6.2 knockout can not abolish the protective effect of H2S on neural damage in both in-vivo and in-vitro study, indicating the neuroprotective effect of H2S is independent on K-ATP channel. However, UCP2 knockout abolishes H2S protecting dopaminergic neurons against MPP+-induced injury, suggesting that UCP2 located in mitochondrial membrane may be the molecular target for H2S. |
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