内源性Ghrelin受体在MPTP诱导的小鼠黑质多巴胺能神经元损伤中的作用及机制研究
发布时间：2018-10-23 19:05
【摘要】：帕金森病(Parkinson’s disease,PD)是主要的中枢神经系统退行性疾病之一,其主要病理学特征是中脑黑质多巴胺(dopamine,DA)能神经元选择性死亡,以及残存的神经元内出现α-突触核蛋白(α-synuclein,α-Syn)的异常聚集,形成Lewy小体(Lewy bodies,LBs)。由于释放到纹状体(striatum,Str)的DA减少,造成基底神经节的直接通路和间接通路失衡,引起静止性震颤、肌僵直、运动迟缓和姿势反射障碍等临床表现。到目前为止,PD的病因尚未明确,遗传、环境、老龄化、氧化应激,炎症等因素均参与了PD黑质DA能神经元变性死亡的过程。Ghrelin作为一种内源性脑肠肽,是生长激素促分泌素受体(growth hormone secretagogue receptor,GHS-R)的唯一内源性配体,GHS-R主要有1a和1b两种亚型,其中1a亚型是其主要的功能性受体。在中枢神经系统,GHS-R1a主要分布在下丘脑、垂体、海马、中脑、黑质等部位,具有调节生长激素分泌,食物摄入、能量代谢及神经保护作用。本课题组前期研究发现,Ghrelin可以通过激活其受体GHS-R1a介导的细胞内信号转导过程,使黑质DA能神经元的兴奋性增加,从而导致纹状体的DA释放量增加和代谢率增高。Ghrelin还可以通过其受体激活的细胞内信号转导途径,通过抗氧化、抗炎和抗凋亡机制拮抗神经毒素1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)诱导的黑质DA能神经元的损伤。并且在Ghrelin存在的条件下,外源性过表达GHS-R1a可以显著拮抗1-甲基-4-苯基吡啶阳离子(1-methyl-4-phenylpyridinium ion,MPP+)诱导的黑质DA能神经元的损伤。以上结果提示,在PD中,Ghrelin能够通过与其受体GHS-R1a结合发挥神经保护作用。同时,也有研究指出,在没有天然配体Ghrelin存在的情况下,GHS-R1a也能够与多种G蛋白偶联受体发生二聚化形成异源二聚体,如多巴胺1型受体(dopamine type 1 receptor,D1R),多巴胺2型受体(dopamine type 2 receptor,D2R),黑皮质素3型受体(melanocortin-3 receptor,MC3R),血清素2C型受体(serotonin type 2C receptor,5-HT2C)和大麻素1型受体(cannabinoid type 1 receptor,CB1)等,进而通过激活细胞内信号转导过程发挥重要的生理功能。但是内源性GHS-R1a在PD中黑质DA能神经元损伤中的作用尚未见任何报道。为了探讨内源性GHS-R1a在PD中黑质DA能神经元损伤中的作用及其机制,本实验拟在Ghrelin受体敲除的小鼠(Ghsr+/-小鼠)上,给予MPTP腹腔注射,观察Ghrelin受体敲除对MPTP所致的小鼠黑质DA能神经元损伤的影响。实验分为四组,WT-NS组、WT-MPTP组、Ghsr+/--NS组以及Ghsr+/--MPTP组,分别给予腹腔注射MPTP或者NS 5 w,应用高效液相色谱技术(high-performance liquid chromatography,HPLC)观察Str区DA含量的变化;应用免疫荧光技术,观察中脑黑质酪氨酸羟化酶(tyrosine hydroxylase,TH)阳性细胞和小胶质细胞数量变化;应用Western blots技术检测黑质区TH、超氧化物歧化酶1(superoxide dismutase 1,SOD1)、白细胞介素-6(interleukin-6,IL-6)的蛋白表达变化以及Bcl-2/Bax比值的变化,以期阐明内源性GHS-R1a在PD中黑质DA能神经元损伤中的作用。结果如下:1.MPTP注射1 w、3 w和5 w后,WT小鼠黑质区TH阳性细胞数量分别减少了16%、34%和48%,TH蛋白表达水平分别降低了26%、51%和44%,差别具有统计学意义(P0.05)。而Ghsr+/-小鼠,MPTP注射1 w、3 w和5 w后,黑质区TH阳性细胞数量分别减少了40%、50%和58%,TH蛋白表达水平分别降低了42%、61%和74%,与对照组相比,差别具有统计学意义(P0.05)。与WT-MPTP组小鼠相比,Ghsr+/--MPTP组小鼠黑质区TH阳性细胞的数量分别减少了33%、26%和30%,TH蛋白表达水平分别降低了21%、40%和59%,差别具有统计学意义(P0.05)。2.MPTP注射5 w后,WT小鼠Str内DA含量由13.81±0.27 ng/mg降低至6.65±0.67ng/mg,与对照组相比,降低了51%,差别具有高度统计学意义(P0.01)。而Ghsr+/-小鼠,MPTP注射5 w后,Str内DA含量由11.67±2.25 ng/mg降低至3.81±0.49ng/mg,与对照组相比,降低了67%,与WT-MPTP组小鼠相比,降低了42%,差别具有高度统计学意义(P0.01)。3.MPTP注射5 w后,WT小鼠黑质区小胶质细胞数量增加了66%,与对照组相比,差别具有高度统计学意义(P0.001)。而Ghsr+/-小鼠,MPTP注射3w后,黑质区小胶质细胞数量即增加了60%,注射5 w后,增加了107%,与对照组相比,差别具有高度统计学意义(P0.001)。与WT-MPTP组小鼠相比,Ghsr+/--MPTP组小鼠黑质小胶质细胞数量在MPTP注射5 w后,增加了31%,差别具有高度统计学意义(P0.001)。4.在MPTP注射5 w后,WT-MPTP小鼠黑质IL-6蛋白表达量上升了33%,而Ghsr+/--MPTP小鼠黑质IL-6蛋白表达量上升了65%,差别具有统计学意义(P0.05)。与WT-MPTP小鼠对比,Ghsr+/--MPTP小鼠黑质区IL-6蛋白表达量增加了23%,差别具有统计学意义(P0.05)。5.Western blots检测结果发现,在MPTP注射3 w和5 w后,WT小鼠黑质区SOD1蛋白表达量分别降低了29%和36%,而Ghsr+/-小鼠,在MPTP注射3 w和5 w后,黑质区SOD1蛋白表达量分别降低42%和63%,差别具有统计学意义(P0.05)。与WT-MPTP小鼠对比,Ghsr+/--MPTP小鼠黑质SOD1蛋白表达量在MPTP注射5 w后减少了42%。6.WT小鼠在给药5 w后,黑质Bcl-2/Bax比值降低了30%,差别具有高度统计学意义(P0.001),而Ghsr+/-小鼠在给药3 w后,黑质Bcl-2/Bax比值就降低了33%,5 w后降低了50%,差别具有统计学意义(P0.05)。与WT-MPTP小鼠对比,Ghsr+/--MPTP小鼠黑质Bcl-2/Bax比值在MPTP注射5 w后降低34%,差别具有统计学意义(P0.05)。综上所述,内源性GHS-R1a的缺失能够明显增强MPTP的神经毒性作用,在MPTP注射的早期,即出现中脑黑质DA能神经元的损伤,表现为TH阳性细胞数量的减少和TH蛋白表达水平的降低,其机制可能与Ghrelin受体敲除后,MPTP引起的小胶质细胞的激活,导致炎性反应增强,抗氧化应激能力减弱促进细胞凋亡所致。本研究表明,内源性GHS-R1a在黑质DA能神经元上具有重要的生理学意义,它的缺失会使黑质DA能神经元更容易受到神经毒素MPTP所致的损伤。
[Abstract]:Parkinson's disease (PD) is one of the main degenerative diseases of the central nervous system. The main pathological features of Parkinson's disease (PD) are the selective death of dopaminergic neurons in mesencephalic mesencephalic dopaminergic neurons, and the presence of NPY-syncretin in the remaining neurons. The abnormal aggregation of n-Syn forms a Lewy small body (LBs). As a result of the reduction of DA released to striatum (Str), the direct pathway and indirect pathway imbalance in basal ganglia lead to the clinical manifestations of stationary tremor, muscle stiffness, exercise delay, and postural reflex disorder. So far, the etiology of PD is not clear, genetic, environment, aging, oxidative stress, inflammation and other factors are involved in the degeneration and death of PD/ DA neurons. Ghrelin, which is the only endogenous ligand of growth hormone secretin receptor (GHS-R), is the only endogenous ligand of GHHS-R, and there are two subtypes of GHS-R, among which 1a is its main functional receptor. In central nervous system, GHS-R1a is mainly distributed in hypothalamus, pituitary, hippocampus, midbrain, cerebral cortex and so on. It has effects of regulating growth hormone secretion, food intake, energy metabolism and neuroprotection. We found that Ghrelin can increase the excitability of dopaminergic neurons by activating the intracellular signal transduction pathway mediated by GHS-R1a, which leads to an increase in DA release and an increase in the amount of dopamine in the striatum. Ghrelin can also antagonize the neuronal damage induced by 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyl-1, 2, 3, 6-tetrahydro-1 (MPTP) by anti-oxidation, anti-inflammatory and anti-apoptotic mechanisms through intracellular signal transduction pathways activated by its receptor. Moreover, under the presence of Ghrelin, exogenous overexpression of GHS-R1a can significantly antagonize the damage of the dopaminergic neurons induced by 1-methyl-4-phenylcarbyl cation (MPP +). These results suggest that in PD, Ghrelin can play a neuroprotective role in combination with its receptor GHS-R1a. At the same time, it is also pointed out that in the absence of a natural ligand Ghrelin, GHS-R1a can also dimerize with a variety of G protein coupled receptors to form heterogenous dimers, such as dopamine type 1 receptor (D1R), dopamine type 2 receptor (D2R), Melanocortin-3 receptor (MC3R), serotonin 2C receptor (5-HT2C) and cannabinoid type 1 receptor (CCR5), etc., can play an important role in activating intracellular signal transduction. However, the role of endogenous GHS-R1a in dopaminergic neuronal damage in PD has not been reported. In order to investigate the role and mechanism of endogenous GHS-R1a in the neuronal damage induced by PDDA in PD, the effect of Ghrelin receptor knockout on the neuronal damage induced by MPTP was observed in the mouse (Ghsr +/-mouse) knockout mice (Ghsr +/-mice). The experiment was divided into four groups: WT-NS group, WT-MPTP group, Ghsr +/--NS group and Gsr +/-MPTP group. MPTP or NS 5w was injected into the abdominal cavity respectively, and the change of DA content in Str region was observed by high-performance liquid chromatography (HPLC). The changes of TH, superoxide dismutase 1 (SOD1), interleukin-6 (IL-6) and the ratio of Bcl-2/ Bax were detected by Western blots technique. The aim of this study was to elucidate the role of endogenous GHS-R1a in dopaminergic neuron injury in PD. Results The number of TH positive cells decreased by 16%, 34% and 48% in WT mice after 1 w, 3 w and 5 w, respectively. The expression level of TH protein decreased by 26%, 51% and 44%, respectively (P0.05). In Gsr +/-mice, the number of TH positive cells decreased by 40%, 50% and 58%, respectively, and the level of TH protein expression decreased by 42%, 61% and 74%, respectively, compared with the control group (P <0.05). Compared with WT-MPTP group, the number of TH positive cells decreased by 33%, 26% and 30%, respectively, and the level of TH protein expression decreased by 21%, 40% and 59%, respectively. In WT mice, the content of DA in Str decreased from 13.81 ng/ mg to 6. 65% 0. 67ng/ mg. Compared with the control group, the content of DA decreased by 51%, and the difference was highly significant (P0.01). In Gsr +/-mice, after 5 w injection of MPTP, the content of DA in Str decreased from 11.67 to 2.25ng/ mg to 3.81 0.49ng/ mg. Compared with the control group, the content of DA decreased by 67%. Compared with WT-MPTP group, the content of DA decreased by 42%, and the difference was highly significant (P0.01). The number of microglial cells increased by 66% in WT mice, and the difference was highly significant compared with the control group (P0.001). In Gsr +/-mice, after 3w injection of MPTP, the number of microglial cells increased by 60% and the injection 5w increased by 107%. Compared with the control group, the difference was highly significant (P0.001). Compared with WT-MPTP group, the number of microglial cells in Gsr +/-MPTP group increased 31% after MPTP injection, and the difference was highly significant (P0.001). After 5 w injection of MPTP, the expression of IL-6 protein in WT-MPTP mice increased by 33%, while the expression of IL-6 protein in Gsr +/-MPTP mice increased by 65%, and the difference was statistically significant (P0.05). Compared with WT-MPTP mice, the expression of IL-6 protein increased by 23% in Gsr +/-MPTP mice, and the difference was statistically significant (P0.05). After MPTP injection of 3w and 5w, the expression of SOD1 protein decreased by 42% and 63%, respectively, and the difference was statistically significant (P0.05). Compared with WT-MPTP mice, the expression of SOD1 protein in Gsr +/-MPTP mice decreased by 42% after 5 w injection of MPTP. 5w decreased by 50% and the difference was statistically significant (P0.05). Compared with WT-MPTP mice, the ratio of Bcl-2/ Bax in Gsr +/-MPTP mice decreased by 34% after 5 w injection of MPTP, and the difference was statistically significant (P0.05). In conclusion, the deletion of endogenous GHS-R1a can significantly enhance the neurotoxicity of MPTP. In the early stage of MPTP injection, the damage of DA energy neurons in midbrain can be reduced, the decrease of TH positive cells and the decrease of TH protein expression level, and the mechanism may be related to the knockout of Ghrelin receptor. MPTP's activation of microglial cells leads to an enhanced inflammatory response, a decrease in oxidative stress, and the promotion of apoptosis. The present study shows that endogenous GHS-R1a plays an important physiological role in the dopaminergic neurons, and the deletion of endogenous GHS-R1a can make the dopaminergic neurons more susceptible to the damage caused by the neurotoxin MPTP.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.5
本文编号：2290205
[Abstract]:Parkinson's disease (PD) is one of the main degenerative diseases of the central nervous system. The main pathological features of Parkinson's disease (PD) are the selective death of dopaminergic neurons in mesencephalic mesencephalic dopaminergic neurons, and the presence of NPY-syncretin in the remaining neurons. The abnormal aggregation of n-Syn forms a Lewy small body (LBs). As a result of the reduction of DA released to striatum (Str), the direct pathway and indirect pathway imbalance in basal ganglia lead to the clinical manifestations of stationary tremor, muscle stiffness, exercise delay, and postural reflex disorder. So far, the etiology of PD is not clear, genetic, environment, aging, oxidative stress, inflammation and other factors are involved in the degeneration and death of PD/ DA neurons. Ghrelin, which is the only endogenous ligand of growth hormone secretin receptor (GHS-R), is the only endogenous ligand of GHHS-R, and there are two subtypes of GHS-R, among which 1a is its main functional receptor. In central nervous system, GHS-R1a is mainly distributed in hypothalamus, pituitary, hippocampus, midbrain, cerebral cortex and so on. It has effects of regulating growth hormone secretion, food intake, energy metabolism and neuroprotection. We found that Ghrelin can increase the excitability of dopaminergic neurons by activating the intracellular signal transduction pathway mediated by GHS-R1a, which leads to an increase in DA release and an increase in the amount of dopamine in the striatum. Ghrelin can also antagonize the neuronal damage induced by 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyl-1, 2, 3, 6-tetrahydro-1 (MPTP) by anti-oxidation, anti-inflammatory and anti-apoptotic mechanisms through intracellular signal transduction pathways activated by its receptor. Moreover, under the presence of Ghrelin, exogenous overexpression of GHS-R1a can significantly antagonize the damage of the dopaminergic neurons induced by 1-methyl-4-phenylcarbyl cation (MPP +). These results suggest that in PD, Ghrelin can play a neuroprotective role in combination with its receptor GHS-R1a. At the same time, it is also pointed out that in the absence of a natural ligand Ghrelin, GHS-R1a can also dimerize with a variety of G protein coupled receptors to form heterogenous dimers, such as dopamine type 1 receptor (D1R), dopamine type 2 receptor (D2R), Melanocortin-3 receptor (MC3R), serotonin 2C receptor (5-HT2C) and cannabinoid type 1 receptor (CCR5), etc., can play an important role in activating intracellular signal transduction. However, the role of endogenous GHS-R1a in dopaminergic neuronal damage in PD has not been reported. In order to investigate the role and mechanism of endogenous GHS-R1a in the neuronal damage induced by PDDA in PD, the effect of Ghrelin receptor knockout on the neuronal damage induced by MPTP was observed in the mouse (Ghsr +/-mouse) knockout mice (Ghsr +/-mice). The experiment was divided into four groups: WT-NS group, WT-MPTP group, Ghsr +/--NS group and Gsr +/-MPTP group. MPTP or NS 5w was injected into the abdominal cavity respectively, and the change of DA content in Str region was observed by high-performance liquid chromatography (HPLC). The changes of TH, superoxide dismutase 1 (SOD1), interleukin-6 (IL-6) and the ratio of Bcl-2/ Bax were detected by Western blots technique. The aim of this study was to elucidate the role of endogenous GHS-R1a in dopaminergic neuron injury in PD. Results The number of TH positive cells decreased by 16%, 34% and 48% in WT mice after 1 w, 3 w and 5 w, respectively. The expression level of TH protein decreased by 26%, 51% and 44%, respectively (P0.05). In Gsr +/-mice, the number of TH positive cells decreased by 40%, 50% and 58%, respectively, and the level of TH protein expression decreased by 42%, 61% and 74%, respectively, compared with the control group (P <0.05). Compared with WT-MPTP group, the number of TH positive cells decreased by 33%, 26% and 30%, respectively, and the level of TH protein expression decreased by 21%, 40% and 59%, respectively. In WT mice, the content of DA in Str decreased from 13.81 ng/ mg to 6. 65% 0. 67ng/ mg. Compared with the control group, the content of DA decreased by 51%, and the difference was highly significant (P0.01). In Gsr +/-mice, after 5 w injection of MPTP, the content of DA in Str decreased from 11.67 to 2.25ng/ mg to 3.81 0.49ng/ mg. Compared with the control group, the content of DA decreased by 67%. Compared with WT-MPTP group, the content of DA decreased by 42%, and the difference was highly significant (P0.01). The number of microglial cells increased by 66% in WT mice, and the difference was highly significant compared with the control group (P0.001). In Gsr +/-mice, after 3w injection of MPTP, the number of microglial cells increased by 60% and the injection 5w increased by 107%. Compared with the control group, the difference was highly significant (P0.001). Compared with WT-MPTP group, the number of microglial cells in Gsr +/-MPTP group increased 31% after MPTP injection, and the difference was highly significant (P0.001). After 5 w injection of MPTP, the expression of IL-6 protein in WT-MPTP mice increased by 33%, while the expression of IL-6 protein in Gsr +/-MPTP mice increased by 65%, and the difference was statistically significant (P0.05). Compared with WT-MPTP mice, the expression of IL-6 protein increased by 23% in Gsr +/-MPTP mice, and the difference was statistically significant (P0.05). After MPTP injection of 3w and 5w, the expression of SOD1 protein decreased by 42% and 63%, respectively, and the difference was statistically significant (P0.05). Compared with WT-MPTP mice, the expression of SOD1 protein in Gsr +/-MPTP mice decreased by 42% after 5 w injection of MPTP. 5w decreased by 50% and the difference was statistically significant (P0.05). Compared with WT-MPTP mice, the ratio of Bcl-2/ Bax in Gsr +/-MPTP mice decreased by 34% after 5 w injection of MPTP, and the difference was statistically significant (P0.05). In conclusion, the deletion of endogenous GHS-R1a can significantly enhance the neurotoxicity of MPTP. In the early stage of MPTP injection, the damage of DA energy neurons in midbrain can be reduced, the decrease of TH positive cells and the decrease of TH protein expression level, and the mechanism may be related to the knockout of Ghrelin receptor. MPTP's activation of microglial cells leads to an enhanced inflammatory response, a decrease in oxidative stress, and the promotion of apoptosis. The present study shows that endogenous GHS-R1a plays an important physiological role in the dopaminergic neurons, and the deletion of endogenous GHS-R1a can make the dopaminergic neurons more susceptible to the damage caused by the neurotoxin MPTP.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.5
【参考文献】
相关期刊论文 前1条
1 Sang Ryong Kim;;Inhibition of microglial activation and induction of neurotrophic factors by flavonoids:a potential therapeutic strategy against Parkinson's disease[J];Neural Regeneration Research;2015年03期
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