內(nèi)源性Ghrelin受體在MPTP誘導(dǎo)的小鼠黑質(zhì)多巴胺能神經(jīng)元損傷中的作用及機(jī)制研究
發(fā)布時(shí)間：2018-10-23 19:05
【摘要】：帕金森病(Parkinson’s disease,PD)是主要的中樞神經(jīng)系統(tǒng)退行性疾病之一,其主要病理學(xué)特征是中腦黑質(zhì)多巴胺(dopamine,DA)能神經(jīng)元選擇性死亡,以及殘存的神經(jīng)元內(nèi)出現(xiàn)α-突觸核蛋白(α-synuclein,α-Syn)的異常聚集,形成Lewy小體(Lewy bodies,LBs)。由于釋放到紋狀體(striatum,Str)的DA減少,造成基底神經(jīng)節(jié)的直接通路和間接通路失衡,引起靜止性震顫、肌僵直、運(yùn)動(dòng)遲緩和姿勢(shì)反射障礙等臨床表現(xiàn)。到目前為止,PD的病因尚未明確,遺傳、環(huán)境、老齡化、氧化應(yīng)激,炎癥等因素均參與了PD黑質(zhì)DA能神經(jīng)元變性死亡的過(guò)程。Ghrelin作為一種內(nèi)源性腦腸肽,是生長(zhǎng)激素促分泌素受體(growth hormone secretagogue receptor,GHS-R)的唯一內(nèi)源性配體,GHS-R主要有1a和1b兩種亞型,其中1a亞型是其主要的功能性受體。在中樞神經(jīng)系統(tǒng),GHS-R1a主要分布在下丘腦、垂體、海馬、中腦、黑質(zhì)等部位,具有調(diào)節(jié)生長(zhǎng)激素分泌,食物攝入、能量代謝及神經(jīng)保護(hù)作用。本課題組前期研究發(fā)現(xiàn),Ghrelin可以通過(guò)激活其受體GHS-R1a介導(dǎo)的細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)過(guò)程,使黑質(zhì)DA能神經(jīng)元的興奮性增加,從而導(dǎo)致紋狀體的DA釋放量增加和代謝率增高。Ghrelin還可以通過(guò)其受體激活的細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)途徑,通過(guò)抗氧化、抗炎和抗凋亡機(jī)制拮抗神經(jīng)毒素1-甲基-4-苯基-1,2,3,6-四氫吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)誘導(dǎo)的黑質(zhì)DA能神經(jīng)元的損傷。并且在Ghrelin存在的條件下,外源性過(guò)表達(dá)GHS-R1a可以顯著拮抗1-甲基-4-苯基吡啶陽(yáng)離子(1-methyl-4-phenylpyridinium ion,MPP+)誘導(dǎo)的黑質(zhì)DA能神經(jīng)元的損傷。以上結(jié)果提示,在PD中,Ghrelin能夠通過(guò)與其受體GHS-R1a結(jié)合發(fā)揮神經(jīng)保護(hù)作用。同時(shí),也有研究指出,在沒(méi)有天然配體Ghrelin存在的情況下,GHS-R1a也能夠與多種G蛋白偶聯(lián)受體發(fā)生二聚化形成異源二聚體,如多巴胺1型受體(dopamine type 1 receptor,D1R),多巴胺2型受體(dopamine type 2 receptor,D2R),黑皮質(zhì)素3型受體(melanocortin-3 receptor,MC3R),血清素2C型受體(serotonin type 2C receptor,5-HT2C)和大麻素1型受體(cannabinoid type 1 receptor,CB1)等,進(jìn)而通過(guò)激活細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)過(guò)程發(fā)揮重要的生理功能。但是內(nèi)源性GHS-R1a在PD中黑質(zhì)DA能神經(jīng)元損傷中的作用尚未見(jiàn)任何報(bào)道。為了探討內(nèi)源性GHS-R1a在PD中黑質(zhì)DA能神經(jīng)元損傷中的作用及其機(jī)制,本實(shí)驗(yàn)擬在Ghrelin受體敲除的小鼠(Ghsr+/-小鼠)上,給予MPTP腹腔注射,觀察Ghrelin受體敲除對(duì)MPTP所致的小鼠黑質(zhì)DA能神經(jīng)元損傷的影響。實(shí)驗(yàn)分為四組,WT-NS組、WT-MPTP組、Ghsr+/--NS組以及Ghsr+/--MPTP組,分別給予腹腔注射MPTP或者NS 5 w,應(yīng)用高效液相色譜技術(shù)(high-performance liquid chromatography,HPLC)觀察Str區(qū)DA含量的變化;應(yīng)用免疫熒光技術(shù),觀察中腦黑質(zhì)酪氨酸羥化酶(tyrosine hydroxylase,TH)陽(yáng)性細(xì)胞和小膠質(zhì)細(xì)胞數(shù)量變化;應(yīng)用Western blots技術(shù)檢測(cè)黑質(zhì)區(qū)TH、超氧化物歧化酶1(superoxide dismutase 1,SOD1)、白細(xì)胞介素-6(interleukin-6,IL-6)的蛋白表達(dá)變化以及Bcl-2/Bax比值的變化,以期闡明內(nèi)源性GHS-R1a在PD中黑質(zhì)DA能神經(jīng)元損傷中的作用。結(jié)果如下:1.MPTP注射1 w、3 w和5 w后,WT小鼠黑質(zhì)區(qū)TH陽(yáng)性細(xì)胞數(shù)量分別減少了16%、34%和48%,TH蛋白表達(dá)水平分別降低了26%、51%和44%,差別具有統(tǒng)計(jì)學(xué)意義(P0.05)。而Ghsr+/-小鼠,MPTP注射1 w、3 w和5 w后,黑質(zhì)區(qū)TH陽(yáng)性細(xì)胞數(shù)量分別減少了40%、50%和58%,TH蛋白表達(dá)水平分別降低了42%、61%和74%,與對(duì)照組相比,差別具有統(tǒng)計(jì)學(xué)意義(P0.05)。與WT-MPTP組小鼠相比,Ghsr+/--MPTP組小鼠黑質(zhì)區(qū)TH陽(yáng)性細(xì)胞的數(shù)量分別減少了33%、26%和30%,TH蛋白表達(dá)水平分別降低了21%、40%和59%,差別具有統(tǒng)計(jì)學(xué)意義(P0.05)。2.MPTP注射5 w后,WT小鼠Str內(nèi)DA含量由13.81±0.27 ng/mg降低至6.65±0.67ng/mg,與對(duì)照組相比,降低了51%,差別具有高度統(tǒng)計(jì)學(xué)意義(P0.01)。而Ghsr+/-小鼠,MPTP注射5 w后,Str內(nèi)DA含量由11.67±2.25 ng/mg降低至3.81±0.49ng/mg,與對(duì)照組相比,降低了67%,與WT-MPTP組小鼠相比,降低了42%,差別具有高度統(tǒng)計(jì)學(xué)意義(P0.01)。3.MPTP注射5 w后,WT小鼠黑質(zhì)區(qū)小膠質(zhì)細(xì)胞數(shù)量增加了66%,與對(duì)照組相比,差別具有高度統(tǒng)計(jì)學(xué)意義(P0.001)。而Ghsr+/-小鼠,MPTP注射3w后,黑質(zhì)區(qū)小膠質(zhì)細(xì)胞數(shù)量即增加了60%,注射5 w后,增加了107%,與對(duì)照組相比,差別具有高度統(tǒng)計(jì)學(xué)意義(P0.001)。與WT-MPTP組小鼠相比,Ghsr+/--MPTP組小鼠黑質(zhì)小膠質(zhì)細(xì)胞數(shù)量在MPTP注射5 w后,增加了31%,差別具有高度統(tǒng)計(jì)學(xué)意義(P0.001)。4.在MPTP注射5 w后,WT-MPTP小鼠黑質(zhì)IL-6蛋白表達(dá)量上升了33%,而Ghsr+/--MPTP小鼠黑質(zhì)IL-6蛋白表達(dá)量上升了65%,差別具有統(tǒng)計(jì)學(xué)意義(P0.05)。與WT-MPTP小鼠對(duì)比,Ghsr+/--MPTP小鼠黑質(zhì)區(qū)IL-6蛋白表達(dá)量增加了23%,差別具有統(tǒng)計(jì)學(xué)意義(P0.05)。5.Western blots檢測(cè)結(jié)果發(fā)現(xiàn),在MPTP注射3 w和5 w后,WT小鼠黑質(zhì)區(qū)SOD1蛋白表達(dá)量分別降低了29%和36%,而Ghsr+/-小鼠,在MPTP注射3 w和5 w后,黑質(zhì)區(qū)SOD1蛋白表達(dá)量分別降低42%和63%,差別具有統(tǒng)計(jì)學(xué)意義(P0.05)。與WT-MPTP小鼠對(duì)比,Ghsr+/--MPTP小鼠黑質(zhì)SOD1蛋白表達(dá)量在MPTP注射5 w后減少了42%。6.WT小鼠在給藥5 w后,黑質(zhì)Bcl-2/Bax比值降低了30%,差別具有高度統(tǒng)計(jì)學(xué)意義(P0.001),而Ghsr+/-小鼠在給藥3 w后,黑質(zhì)Bcl-2/Bax比值就降低了33%,5 w后降低了50%,差別具有統(tǒng)計(jì)學(xué)意義(P0.05)。與WT-MPTP小鼠對(duì)比,Ghsr+/--MPTP小鼠黑質(zhì)Bcl-2/Bax比值在MPTP注射5 w后降低34%,差別具有統(tǒng)計(jì)學(xué)意義(P0.05)。綜上所述,內(nèi)源性GHS-R1a的缺失能夠明顯增強(qiáng)MPTP的神經(jīng)毒性作用,在MPTP注射的早期,即出現(xiàn)中腦黑質(zhì)DA能神經(jīng)元的損傷,表現(xiàn)為TH陽(yáng)性細(xì)胞數(shù)量的減少和TH蛋白表達(dá)水平的降低,其機(jī)制可能與Ghrelin受體敲除后,MPTP引起的小膠質(zhì)細(xì)胞的激活,導(dǎo)致炎性反應(yīng)增強(qiáng),抗氧化應(yīng)激能力減弱促進(jìn)細(xì)胞凋亡所致。本研究表明,內(nèi)源性GHS-R1a在黑質(zhì)DA能神經(jīng)元上具有重要的生理學(xué)意義,它的缺失會(huì)使黑質(zhì)DA能神經(jīng)元更容易受到神經(jīng)毒素MPTP所致的損傷。
[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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R742.5
本文編號(hào)：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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R742.5
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
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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