基于JNK信号通路探讨针刀干预对帕金森病大鼠机制的研究

发布时间：2018-05-30 17:26

本文选题：帕金森病 + JNK信号通路　；参考：《北京中医药大学》2017年博士论文

【摘要】：目的:观察针刀干预对帕金森病模型大鼠黑质神经炎症、神经细胞凋亡及相关因子的影响,并探讨JNK信号通路在针刀干预帕金森病中的作用机制。方法:雄性SD大鼠72只,分为空白组、假手术组、模型组、药物组、电针组和针刀组。采用单侧纹状体双靶点注射6-OHDA建立PD模型,通过阿朴吗啡(APO)诱发行为学检测验证模型。空白组不做任何处理;假手术组与模型组的注射方法和部位相同,仅注射等量的含0.2%维生素C的生理盐水;造模成功后,药物组美多芭片50mg/kg灌胃,电针组进行针刺治疗,针刀组给予针刀干预。治疗四周后各组取材检测,检测指标包括:(1)行为学检测:记录大鼠60min内旋转圈数;(2)HE染色观察黑质神经细胞形态学;(3)免疫荧光双标法检测黑质COX-2/TH,p-c-Jun/TH的表达;(4)Westernblot检测黑质JNK,P-JNK,p-c-Jun,COX-2及TH表达;(5)免疫组织化学检测黑质TH神经细胞;(6)Real-TimePCR检测黑质DATmRNA含量;(7)TUNEL染色检测凋亡细胞(8)免疫荧光双标法检测黑质Caspase-3/TH的表达(9)Westernblot检测黑质Caspase-3,Bcl-2,Beclin-1 的表达结果:(1)行为学检测:模型组出现旋转行为且治疗前后旋转圈数无差异(P0.05);药物组、电针组、针刀组干预后大鼠旋转圈数明显减少,活动增多,干预前后有明显差异(P0.01),与模型组相比差异显著(P0.01)。(2)形态学检测:①HE染色:不同干预方法均可使HE染色黑质神经细胞数目增多,形态结构趋于正常,药物组黑质神经细胞排列有序,数量与模型组比较增多,细胞变性程度减轻,胶质细胞增生不明显;电针组黑质神经细胞数量与模型组比较增多较为明显,细胞变性程度明显减轻;针刀组黑质神经细胞数目与模型组比较明显增多,排列较密集,细胞变性减轻。②黑质TH的免疫组织化学染色:正常组和假手术组的黑质均可见密集的TH免疫反应阳性细胞,呈棕褐色,胞体较大,胞体主要为圆形或卵圆形,少数为多边形。模型组黑质可见少量、稀疏的TH免疫反应阳性神经元,胞体缩小,轮廓不清晰;药物组、电针组及针刀组TH免疫反应阳性神经元数量增多较为明显,胞体轮廓清晰可见,染色强度上亦有增加。③免疫荧光双标记黑质COX-2/TH,p-c-Jun/TH,Caspase-3/TH检测结果:正常组和假手术组的黑质均可见密集的TH免疫反应阳性细胞,呈绿色,胞体较大,胞体主要为圆形或卵圆形,同时,COX-2,p-c-Jun,Caspase-3/TH,呈红色荧光,在数量上和染色强度上表达较弱;模型组黑质可见少量、稀疏的TH免疫反应阳性神经元,呈绿色,细胞肿胀变性,或皱缩,数量较少,染色强度减低,同时,COX-2,p-c-Jun,Caspase-3,呈红色荧光,在数量上和染色强度上表达增加;药物组、电针组及针刀组TH免疫反应阳性神经元呈绿色,排列较有序,数量增多,胞体变性程度减轻,染色强度上亦有增加;同时,COX-2,p-c-Jun,Caspase-3/TH,呈红色荧光,较模型组,在数量上减少,染色强度减低。与电针组和药物组比较,针刀组TH神经元数目更多,胞体变性程度更轻;针刀组COX-2和p-c-Jun在数量上较电针组少,但比药物组多;药物组Caspase-3在数量上比电针组和针刀组少。(3)Western blot 检测结果:①与空白组比较,模型组大鼠黑质内JNK含量无明显差异(P0.05);P-JNK,P-JNK/JNK,p-c-Jun表达升高(P0.05),COX-2表达显著升高(P0.01);与模型组比较,药物组、电针组、针刀组大鼠黑质内JNK含量无明显差异(P0.05);与模型组比较,P-JNK和p-c-Jun含量在药物组和电针组中有差异(P0.05),针刀组具有显著差异(P0.01),与模型组比,P-JNK/JNK在药物组,电针组和针刀组中有差异(P0.05),COX-2含量电针组具有差异(P0.05),在药物组和针刀组中有显著差异(P0.01);②与空白组比较,模型组大鼠黑质内TH表达显著降低(P0.01)。与模型组比较,药物组、电针组大鼠黑质内TH表达均升高(P0.05),针刀组大鼠黑质内TH表达显著升高(P0.01);③与空白组比较,模型组大鼠黑质内Caspase-3表达升高(P0.05),Beclin-1表达显著升高(P0.01),Bcl-2表达显著下降(P0.01);与模型组比较,Caspase-3含量在药物组和电针组中降低(P0.05),针刀组显著降低(P0.01);Beclin-1含量在电针组表达降低(P0.05),在药物组和针刀组中表达显著降低(P0.01);Bcl-2在三组中表达均升高(P0.05)。(4)Real time-PCR检测结果:与空白组比较,模型组大鼠黑质内DAT表达显著降低(P0.01);与模型组比较,药物组大鼠黑质内DAT表达显著升高(P0.01),电针组和针刀组大鼠黑质内DAT表达均升高(P0.05)。(5)TUNEL染色:与空白组比较,模型组大鼠神经元凋亡数显著增加(P0.01);与模型组比较,药物组、电针组及针刀针组大鼠神经元凋亡数显著降低(P0.01);结论:(1)针刀干预能有效减轻PD大鼠黑质神经元炎症反应,在JNK通路上,其可能通过降低COX-2/p-c-Jun的活性,降低JNK磷酸化水平,提高TH的抗氧化活性而发挥对神经元细胞保护作用。(2)针刀干预可使TH神经元形态改善、数量增多以及TH阳性细胞的表达增多,提高DAT的表达,从而发挥对神经元的保护作用。(3)针刀干预能有效的减轻PD大鼠黑质神经元的凋亡,其可能通过降低Caspase-3的促凋亡活性,激活Bcl-2的抗凋亡活性,抑制自噬基因Beclin-1活性,提高TH的抗氧化活性而发挥对神经元细胞保护作用。(4)对JNK信号的通路的抑制可能是针刀发挥作用的机制之一。
[Abstract]:Objective: To observe the effect of needle knife intervention on the inflammation of substantia nigra, nerve cell apoptosis and related factors in Parkinson's disease model rats, and to explore the mechanism of JNK signaling pathway in the intervention of needle knife in Parkinson's disease. Methods: 72 male SD rats were divided into blank group, sham operation group, model group, drug group, electroacupuncture group and needle knife group. The PD model was established by injection of 6-OHDA, and the model was tested by apomorphine (APO). The blank group did not do any treatment. The injection method and location of the sham operation group and the model group were the same, only the same amount of normal saline containing 0.2% vitamin C was injected; after the work was made, the drug group was treated with 50mg/kg gavage, and the electroacupuncture group was carried out. Acupuncture treatment, needle knife group was given needle intervention. After four weeks of treatment, the test indexes included: (1) behavioral test: record the number of rotations in 60min in rats; (2) HE staining to observe the morphology of substantia nigra neurons; (3) double immunofluorescence double labeling method for the detection of substantia nigra COX-2/TH, p-c-Jun/TH expression; (4) Westernblot detection of substantia nigra JNK, P-JNK, p-c-Jun COX-2 and TH expression; (5) immunohistochemical detection of substantia nigra TH nerve cells; (6) Real-TimePCR detection of substantia nigra DATmRNA content; (7) TUNEL staining detection of apoptotic cells (8) immunofluorescence double labeling method for the detection of substantia nigra Caspase-3/TH (9) Westernblot detection of substantia nigra Caspase-3, Bcl-2, Beclin-1 expression: (1) behavioral test: model group appearance There was no difference in rotation circle before and after treatment (P0.05). The number of rotating rings in the drug group, the electroacupuncture group and the needle knife group were significantly reduced, the activity increased, and there were significant differences (P0.01) before and after intervention (P0.01). (2) morphological examination: (1) HE staining: the number of different intervention methods can cause the number of HE dyed black matter nerve cells. In the drug group, the number of substantia nigra neurons in the drug group was orderly, the number and the model group increased, the degree of cell degeneration was reduced, and the proliferation of glial cells was not obvious; the number of black matter neurons in the electroacupuncture group was more obvious than the model group, and the degree of cell degeneration was obviously reduced; the number and model of the black matter nerve cells in the needle knife group were the number and model. The type group was more obvious, the arrangement was more dense and the cell degeneration was reduced. (2) the immunohistochemical staining of the substantia nigra TH: the substantia nigra of the normal group and the sham operation group were all dense TH immunoreactive positive cells, brown and large, and the cell body was mainly round or oval, and the few were polygons. The model group had a small amount of substantia nigra and sparsely TH free The number of immunoreactive neurons in the drug group, the electroacupuncture group and the needle knife group increased more obviously, the body contour was clearly visible and the staining intensity increased. (3) the results of immunofluorescence double labelled melanin COX-2/TH, p-c-Jun/TH, Caspase-3/TH detection: the black of the normal group and the sham operation group. The cytoplasm of TH immunoreactive positive cells was green, the cell body was large, and the cell body was mainly round or oval, and COX-2, p-c-Jun, Caspase-3/TH were red fluorescence, and the expression was weak in quantity and intensity. The model group had a few and sparse TH immunoreactive neurons, green, swollen and denatured cells, or COX-2, p-c-Jun, Caspase-3, with red fluorescence, increased in quantity and intensity. The TH immunoreactive neurons in the drug group, the electroacupuncture group and the needle knife group were green, arranged in a more orderly manner, increased in number, reduced the degree of body degeneration, and increased in dyeing strength; meanwhile, COX-2, p-c-J UN, Caspase-3/TH, red fluorescence, compared with model group, decreased in quantity, and the intensity of dyeing decreased. Compared with the electroacupuncture group and the drug group, the number of TH neurons in the needle knife group was more and the degree of cell degeneration was lighter; the number of COX-2 and p-c-Jun in the needle knife group was less than the electroacupuncture group, but more than the drug group; the quantity of Caspase-3 in the drug group was more than the electroacupuncture group and the needle knife group. (3) Western blot detection results: (1) compared with the blank group, there was no significant difference in the content of JNK in the substantia nigra of the model group (P0.05); P-JNK, P-JNK/JNK, p-c-Jun expression increased (P0.05), and the COX-2 expression increased significantly (P0.01). Compared with the model group, there was no significant difference in the JNK content of the substantia nigra in the drug group, electroacupuncture group and the needle knife group (P0.05); compared with the model group, The contents of P-JNK and p-c-Jun were different in the drug group and the electroacupuncture group (P0.05), and there were significant differences in the needle knife group (P0.01). Compared with the model group, the P-JNK/JNK was different in the drug group, the electroacupuncture group and the needle knife group (P0.05), the COX-2 content in the electroacupuncture group was different (P0.05), and there was a significant difference between the drug group and the needle knife group (P0.01); (2) compared with the blank group, the model was compared with the blank group. The expression of TH in the substantia nigra of the rats was significantly decreased (P0.01). Compared with the model group, the expression of TH in the substantia nigra of the rats in the drug group and the electroacupuncture group increased (P0.05), and the TH expression in the substantia nigra of the Acupotomy rats increased significantly (P0.01). (3) the expression of Caspase-3 in the substantia nigra of the model group increased (P0.05), and the Beclin-1 expression increased significantly (P0.01), Bcl-2 expression was obvious. Decrease (P0.01). Compared with the model group, the content of Caspase-3 decreased (P0.05) in the drug group and the electroacupuncture group (P0.01), the expression of Beclin-1 decreased (P0.05) in the electroacupuncture group (P0.05), and the expression in the drug group and the needle knife group was significantly decreased (P0.01); Bcl-2 in the three groups increased (P0.05). (4) Real time-PCR detection results: and blank Compared with the model group, the expression of DAT in the substantia nigra was significantly decreased (P0.01). Compared with the model group, the expression of DAT in the substantia nigra of the drug group increased significantly (P0.01), and the expression of DAT in the substantia nigra of the electroacupuncture group and the Acupotomy group increased (P0.05). (5) TUNEL staining: compared with the blank group, the neuron apoptosis increased significantly (P0.01) in the model group (P0.01); compared with the model group, the rat model group was compared with the model group. Compared with the drug group, electroacupuncture group and needle knife group, the neuron apoptosis was significantly decreased (P0.01). Conclusion: (1) acupuncture intervention can effectively reduce the inflammatory response of the substantia nigra neurons in PD rats. On the JNK pathway, it may reduce the activity of COX-2/p-c-Jun, reduce the level of JNK phosphorylation and improve the antioxidant activity of TH to protect the neuron cells. (2) acupuncture intervention can improve the morphology of TH neurons, increase the number of TH positive cells, increase the expression of DAT, and improve the protective effect of the neurons. (3) acupuncture intervention can effectively reduce the apoptosis of the neurons in the substantia nigra of PD rats. It may pass the apoptotic activity of Caspase-3 and activate the anti apoptosis of Bcl-2. Activity, inhibit the activity of autophagy gene Beclin-1, improve the antioxidant activity of TH and play a protective role on neuron cell. (4) inhibition of JNK signaling pathway may be one of the mechanisms of acupotomy.
【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R245

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