【作者】 鲁明；

【导师】 胡刚；

【作者基本信息】 南京医科大学 ， 药理学， 2011， 博士

【摘要】 硫化氢（H₂S）是一种内源性气体信号分子,参与哺乳动物众多生物学功能的调节。因此,H₂S与一氧化氮和一氧化碳一起被称作“气体递质”。近十年来,人们对于H₂S在中枢神经系统（central nervous system, CNS）和心血管系统中作用的研究日益深入,发现H₂S作为脑内一种新的神经调质,促进海马长时程增强效应,调节细胞内钙离子稳态和pH水平,调节多种重要的生理功能。已证实H₂S代谢异常参与缺血性脑卒中、阿尔茨海默症（AD）、亨廷顿舞蹈病（HD）和反复发作的热性惊厥等中枢神经系统疾病的发生发展。研究也已阐明H₂S参与脑内氧化应激、神经炎症和细胞凋亡的调节,可能与帕金森病（Parkinson’s disease, PD）等神经退行性疾病的病理机制相关,但至今尚未见确切报道。PD是一种严重危害中老年人健康、以黑质-纹状体多巴胺（DA）能神经元进行性丢失为主要病理特征、发病率仅次于AD的第二大类神经退行性疾病。在我国65岁以上人群中PD的发病率约2%,占世界PD患者总数的40%以上,且有逐年增高的趋势。预期未来10年内我国罹患PD的人数将占全球患者总数的60%以上。半个多世纪以来,左旋多巴替代疗法一直是临床治疗PD的主要手段。然而,左旋多巴仅能缓解症状,并不能阻止PD的病理进程,且长期使用还可导致许多严重的不良反应。针对DA能神经元损伤的病理机制,尽管已提出兴奋性毒性、线粒体功能障碍、氧化应激、炎症等学说,但导致DA能神经元损伤的确切机制目前仍不清楚,这已经成为制约研发理想治疗药物和临床治疗学突破的瓶颈。显然,加强PD的病因学研究,阐明PD中多巴胺能神经元进行性变性、坏死的病理机制,探索神经保护的新策略,显得十分紧迫和必要。研究已表明AD患者相关脑区内H₂S产生和代谢异常,提示H₂S可能参与了AD的发生、发展,其机制涉及H₂S抗氧化、抗凋亡及抗炎和促炎的双重作用。本实验室前期研究发现,应用外源性H₂S可抑制LPS诱导的小胶质细胞活化和鱼藤酮诱导的神经元线粒体功能障碍。而小胶质细胞活化介导的神经炎症和线粒体功能障碍激活的神经元凋亡是PD发生的重要病理机制。尽管研究提示H₂S可能与PD相关,但目前尚未获得直接的支持证据。本文工作首先研究内源性H₂S与6-OHDA诱导的PD大鼠模型的病理相关性,研究给予外源性H₂S对PD模型大鼠行为学和病理学损伤的影响及其作用机制。第二部分工作应用原代培养的大鼠星形胶质细胞建立氧化损伤细胞模型,研究H₂S对星形胶质细胞损伤的保护作用,阐明H₂S对谷氨酸摄取功能的影响及机制。第三部分工作应用野生型（wild-type,WT）小鼠、Kir6.2敲除（Kir6.2 knockout,Kir6.2-/-）小鼠和线粒体解耦联蛋白2敲除(UCP2 knockout,UCP2^-/-)小鼠,建立亚急性MPTP/p PD小鼠模型,研究H₂S对PD小鼠病理学和神经递质水平的影响,阐明H₂S对PD发挥神经保护作用的靶点和可能的分子机制。本文工作的研究结果为H₂S参与PD病程的发生、发展提供直接的支持证据,并阐明外源性H₂S在PD神经损伤中发挥多靶点的保护作用,为PD的临床治疗学提供新的思路和策略。目的:研究、阐明H₂S与PD的相关性及外源性H₂S对PD模型大鼠的神经保护作用。方法:建立单侧纹状体立体定位注射6-OHDA诱导的PD大鼠模型,通过皮下注射阿扑吗啡诱导动物对侧旋转行为。筛选出成功的PD大鼠给予NaHS（1.68 mg/kg和5.6 mg/kg,i.p.）治疗3周,动态观测行为学症状的变化。给药结束后,处死大鼠、灌注取脑并收集新鲜脑组织。醋酸锌法测定相关脑区H₂S浓度;免疫组织化学染色观察中脑黑质和纹状体TH神经元损伤;应用Western-blotting法检测上述脑区酪氨酸羟化酶表达;应用MDA试剂盒测定中脑脂质过氧化产物MDA含量。培养SH-SY5Y神经元细胞株,分离纯化胞浆蛋白和胞膜蛋白,应用Western-blotting法检测NADPH氧化酶胞膜亚基gp91的表达及胞浆亚基p47从胞浆向胞膜转运情况。结果:1)单侧纹状体内注射6-OHDA四周后,阿扑吗啡诱导的PD模型大鼠出现明显的健侧旋转行为,表明模型建立成功;相较于伪手术组大鼠,6-OHDA模型大鼠患侧纹状体内H₂S浓度显著降低（P<0.05）,健侧亦有下降趋势,但无显著性差异（P>0.05）,提示脑内内源性H₂S与PD的发生、发展密切相关。2)NaHS治疗3周显著抑制模型大鼠对侧旋转症状进行性加重,且显著改善6-OHDA诱导的患侧黑质致密部和纹状体内多巴胺能神经元减少;同时,Western blotting分析发现NaHS逆转上述脑区酪氨酸羟化酶（TH）表达的下调。3)NaHS显著减轻6-OHDA引起的纹状体内MDA合成增加,提示H₂S对PD模型大鼠的神经保护作用与其抗氧化应激损伤相关;整体和离体研究均证实NaHS显著抑制NADPH氧化酶膜亚基gp91表达上调和胞浆亚基p47的转运,且该作用依赖抑制ERK1/2磷酸化。结论:1、脑内H₂S水平与6-OHDA诱导的PD大鼠模型的发生、发展相关。2、外源应用H₂S能够改善模型动物行为学和病理学损伤,发挥神经保护作用。3、H₂S神经保护作用的机制涉及抑制NADPH氧化酶活化,减轻氧化应激损伤。目的:研究H₂S对原代培养的SD大鼠星形胶质细胞损伤的保护作用及其对谷氨酸摄取功能的影响,阐明H₂S对PD的神经损伤发挥保护作用的细胞与分子机制。方法:分离、培养SD大鼠脑内星形胶质细胞,给予内源性氧化物质H₂O₂,建立损伤模型,研究H₂S对H₂O₂诱导的星形胶质细胞损伤作用的调节。MTT法和LDH检测研究H₂O₂和H₂S对星形胶质细胞活力的影响。GSH和ROS水平测定观察H₂S的抗氧化应激作用。应用同位素标记的谷氨酸摄取实验检测H₂S对星形胶质细胞谷氨酸再摄取功能的影响。应用Western-blotting法观察星形胶质细胞谷氨酸转运体GLT-1在细胞内的转运（trafficking）状态以及调节其转运的MAPK信号通路的活化情况。结果:1)H₂S供体NaHS呈浓度依赖性地减轻H₂O₂（200μM）引起的星形胶质细胞活力下降和LDH释放增加;NaHS（100μM）预处理15分钟逆转H₂O₂引起的细胞内抗氧化物质还原型谷胱甘肽（GSH）的合成减少。上述作用均可被特异性谷氨酸摄取抑制剂PDC所取消;同时,NaHS改善H₂O₂引起的胞内ROS蓄积和ATP生成减少。2)H₂S合成酶CBS抑制剂AOAA加重H₂O₂所致星形胶质细胞活力下降、LDH释放增加以及裂解型PARP蛋白表达上调,提示内源性H₂S参与细胞内抗氧化损伤作用。3)NaHS预处理逆转H₂O₂损伤的星形胶质细胞[3H]谷氨酸摄取功能,且促进谷氨酸转运体GLT-1从胞浆向胞膜转运。同时发现ERK1/2磷酸化抑制剂PD98059具有相似的作用。NaHS显著抑制H₂O₂激活的ERK1/2磷酸化,表明H₂S通过MAPK信号通路影响GLT-1的转运。结论:1、离体研究发现H₂S增强SD大鼠星形胶质细胞谷氨酸摄取功能,促进胞内抗氧化物质GSH合成,可能是H₂S对氧化应激损伤发挥保护作用的主要机制之一。2、硫化氢抑制谷氨酸转运体氧化失活,促进其从胞浆向胞膜转运,并保障主动摄取所需的能量,从而调节星形胶质细胞谷氨酸摄取功能。目的:应用野生型（wild-type,WT）、Kir6.2敲除(Kir6.2 knockout,Kir6.^2-/-)和UCP2敲除(UCP2 knockout,UCP^2-/-)小鼠,建立亚急性MPTP/p PD小鼠模型,从整体、细胞及分子水平研究、阐明H₂S对PD的神经保护作用及其机制。方法:应用MPTP(20 mg·kg^-1)皮下注射,间隔1小时腹腔注射丙磺舒(250 mg·kg^-1),连续给药5天,制备亚急性PD小鼠模型。给药结束后2天进行5-溴脱氧尿核苷(5-bromodeoxyuridine,BrdU,50 mg·kg^-1,i.p. every 2 h,共4次)标记。NaHS(5.6 mg·kg^-1·day^-1, i.p.)在MPTP首次给药前3天应用,连续给药8天。造模结束后3.5天处死动物,收集标本。应用免疫组织化学结合体视学计数、ImageJ软件分析黑质致密部（substantia nigra pars compacta, SNpc）DA能神经元损伤;同时检测黑质致密部星形胶质细胞和小胶质细胞增殖活化;室管膜下层（subventricular zone, SVZ）和颗粒细胞下层（subgranular zone, SGZ）神经再生情况;应用高效液相色谱法（HPLC）检测纹状体脑区单胺类及氨基酸类神经递质及其代谢产物水平的变化。离体培养小鼠中脑神经元,应用免疫细胞化学染色检测MPP+所致神经元数目及突起长度损伤;应用Western-blotting分析内质网应激启动的GRP78、CHOP和Caspase12及溶酶体自噬标志物LC3的表达。结果:1)在亚急性MPTP/p PD模型中,Kir6.^2+/+和Kir6.^2-/-两种基因型小鼠中脑TH神经元损伤,胶质细胞活化,神经再生及纹状体单胺类和氨基酸类递质水平变化均无显著性差异（p>0.05）。2)NaHS预处理提高模型中Kir6.^2+/+和Kir6.^2-/-两种基因型小鼠的存活率,且显著改善两种基因型小鼠黑质致密部TH神经元减少、星形胶质细胞和小胶质细胞的增殖活化;减轻SGZ区神经干细胞增殖的抑制（p<0.05）;但对纹状体DA及其代谢产物水平改变无显著影响（p>0.05）。3)NaHS改善MPP+诱导的两种基因型中脑TH神经元数目和平均突起长度减少。其机制涉及NaHS抑制MPP+引起的内质网应激中蛋白伴侣分子GRP78、转录因子CHOP、效应分子Caspase12和溶酶体自噬标志物LC3的表达上调,并抑制下游NF-κB信号通路的激活。4)线粒体内膜解耦联蛋白2（uncoupling protein 2,UCP2）敲除取消NaHS对亚急性MPTP模型小鼠SNc区TH神经元的保护作用,同时取消NaHS改善MPP+导致离体培养的中脑TH神经元数目和平均突起长度减少,LDH释放增加的作用,提示H₂S的神经保护作用依赖UCP2。结论:1、H₂S对MPTP/p PD模型小鼠的神经损伤具有确切的保护作用。2、H₂S的神经保护作用不依赖Kir6.2/K-ATP通道,线粒体内膜上的UCP2可能是H₂S的作用靶点。3、H₂S抑制ROS引起的内质网应激及下游凋亡通路,发挥神经保护作用。综上所述,本文工作的主要创新之处在于:1、阐明内源性H₂S与PD的发生、发展密切相关本文研究发现神经毒素诱导的PD模型大鼠相关脑区内H₂S水平显著降低,提示脑内H₂S水平降低参与了PD的发生发展过程,为H₂S与PD的相关性提供了直接的实验证据。2、发现外源性H₂S对PD的神经损伤具有保护作用通过在体和离体研究,发现应用外源性H₂S对PD模型动物出现的行为学症状、TH神经元丢失、星形胶质细胞功能障碍、小胶质细胞增殖活化和神经干细胞增殖抑制均有显著的改善作用,在学术界首次报道H₂S作为一种神经保护剂对PD神经损伤具有确切的保护作用,为临床防治PD提供了新的思路和策略。3、揭示H₂S对PD模型动物的神经保护作用不依赖K-ATP通道本文研究发现敲除表达于神经元的K-ATP通道孔道形成亚基Kir6.2,不能取消H₂S对PD模型小鼠神经损伤的保护作用,表明H₂S对PD小鼠的神经保护作用不依赖K-ATP通道。而线粒体UCP2敲除则能取消H₂S对中脑TH神经元的保护作用,提示H₂S的作用靶点可能是位于K-ATP通道上游的UCP2。该发现不仅揭示了H₂S保护作用的机制,也为PD的神经保护和研发理想治疗药物提供了有益的靶标。更多还原

【Abstract】 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 UCP^2-/- 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.更多还原