基于表观遗传学及PKA信号通路探讨针刺对BDNF蛋白及基因表达的影响

发布时间：2018-05-17 01:25

  本文选题：表观遗传学 + 慢性应激　； 参考：《北京中医药大学》2016年博士论文

【摘要】：目的本研究采用慢性应激大鼠模型,从表观遗传学(DNA甲基化和组蛋白乙酰化)和细胞信号通路(PKA信号通路)的角度,探讨针刺对慢性应激模型(CUMS)大鼠行为学和脑源性神经营养因子(BDNF)蛋白及基因表达的影响,为阐明针刺的抗抑郁作用机制提供一定的依据。方法1：40只雄性SD大鼠,体重(220～240)g,随机分为空白对照组(空白组)、模型组、模型+氟西汀组(氟西汀组)、模型+针刺组(针刺组)。除空白组,其余大鼠均接受28 d慢性温和不可预知刺激,氟西汀组在应激前30 min灌胃给药(1.8mg/kg/d),针刺组在应激前30 min针刺“百会”和“印堂”穴,每天一次。在实验第0d、7d、14d、21d、 28 d分别进行旷场、糖水实验,称取体重。实验结束后取海马组织和血清。ELISA法检测血清BDNF蛋白,RT-PCR法检测海马BDNF mRNA, Western-blot法检测海马BDNF、乙酰化组蛋白H3K9 (acH3K9)、脱乙酰化酶2(HDAC2)蛋白,甲基化PCR法检测海马BDNF基因启动子I甲基化。2：60只雄性SD大鼠,体重(220～240)g,随机分为空白对照组(空白组)、模型组、模型+氟西汀组(氟西汀组)、模型+针刺组(针刺组)、模型+氟西汀+H89组(氟西汀+H89组)、模型+针刺+H89组(针刺+H89组)。除空白组,其余大鼠均接受21d慢性温和不可预知刺激。针刺组和氟西汀组的造模和治疗干预方法同上。氟西汀+H89组和针刺+H89组在造模前1周进行侧脑室套管埋置手术,在应激前60 min给予侧脑室注射H89溶液(10μm,5μL),隔天一次。两个组在应激前30 min分别进行灌胃和针刺,每天一次。所有大鼠在实验第0d和第21d进行旷场、糖水实验,称取体重。实验结束后取海马组织。Western-blot法检测海马中蛋白激酶A (PKA)、 cAMP反应元件结合蛋白(CREB)和磷酸化CREB (p-CREB)的蛋白含量。结果1.针刺和氟西汀对模型大鼠行为学的影响实验前,各组大鼠行为学检测均无统计学差异(P0.05)。造模后,与空白组相比,模型组的运动次数、糖水偏好百分比和体质量明显降低(P0.01,P0.01,P0.01)。与模型组相比,针刺和氟西汀均能提高模型大鼠的运动次数(P0.05,P0.05)、糖水偏好百分比(P0.01,P0.01)和体质量(P0.05,P0.01),但针刺与氟西汀对各项行为学的作用趋势不同。2.针刺和氟西汀对血清、海马中BDNF蛋白和海马BDNF mRNA的影响与空白组相比,模型组大鼠血清中和海马中BDNF蛋白含量均显著降低(P0.01,P0.01),海马中BDNF mRNA也明显降低(P0.01)；与模型组相比,氟西汀能显著增加血清BDNF蛋白表达(P0.01)和海马BDNF mRNA表达(P0.01),针刺能显著增加血清和海马BDNF蛋白表达(P0.01,P0.01)和海马BDNF mRNA表达(P0.01),但针刺增加血清BDNF蛋白和海马BDNF mRNA作用不如氟西汀(P0.05,P0.01)。3.血清BDNF蛋白、海马BDNF蛋白与行为学的相关性分析血清BDNF蛋白表达与海马BDNF蛋白表达存在正相关性(P0.05)。旷场运动次数与血清BDNF蛋白存在正相关性(P0.05),但糖水偏好百分比与血清BDNF蛋白相关性不显著(P0.05)；海马BDNF蛋白与行为学(旷场运动次数、糖水偏好百分比)均存在正相关性(P0.05,P0.01)。4.海马BDNF蛋白、海马BDNF mRNA与行为学的相关性分析海马BDNF蛋白与海马BDNF mRNA相关性不显著(P0.05)。但旷场运动次数和糖水偏好百分比与海马BDNF mRNA均有显著正相关性(P0.000,P0.005)。5.针刺和氟西汀对海马BDNF DNA启动子I甲基化的影响各组大鼠海马BDNF DNA启动子I甲基化差异均没有统计学意义(B 0.05)。6.针刺和氟西汀对海马acH3K9和HDAC2蛋白的影响与空白组相比,模型组海马内acH3K9蛋白含量明显降低(P0.05)；与模型组相比,氟西汀组和针刺组的acH3K9蛋白含量均有上升趋势,但是没有统计学意义(P0.05,P0.05)。氟西汀组与针刺组相比差异也不显著(P0.05)。与空白组相比,模型组海马内HDAC2蛋白含量显著升高(P0.01)；与模型组相比,氟西汀和针刺均可降低海马内HDAC2的蛋白表达(P0.01)。针刺组和氟西汀组之间的差异不显著(P0.05)。7.海马acH3K9蛋白、HDAC2蛋白与BDNF mRNA的相关性分析海马acH3K9蛋白表达与海马HDAC2蛋白表达有显著的负相关性(P0.005)。海马BDNF mRNA表达与海马acH3K9蛋白表达有一定的正相关性(P0.05),与海马HDAC2蛋白表达有显著的负相关性(P0.000)。8.海马acH3K9蛋白、HDAC2蛋白与行为学的相关性分析旷场运动次数与海马acH3K9蛋白表达有一定正相关性(P0.05),与海马HDAC2蛋白表达有显著的负相关性(P0.000)。糖水偏好百分比与海马acH3K9蛋白表达没有显著相关性(P0.05),与海马HDAC2蛋白表达有显著的负相关性(P0.000)。9.针刺和氟西汀对海马PKA-CREB信号通路的影响PKA特异性阻断剂H89能阻断氟西汀和针刺对模型大鼠行为学(水平穿越格数、竖立次数、糖水消耗量)的改善作用,且对氟西汀治疗作用的阻断更加显著。各组海马内CREB蛋白表达差异不显著(P0.05)。与空白组相比,模型组海马内PKA-α和p-CREB蛋白表达明显降低(P0.01,P0.01)。与模型组相比,氟西汀组的PKA-α蛋白显著增高(P0.05),p-CREB蛋白表达有上升趋势,但是没有统计学意义(P0.05),针刺组的PKA-α和p-CREB蛋白表达均显著增高(P0.01,P0.05),氟西汀+H89组和针刺+H89组的PKA-α蛋白表达也增高(P0.05,P0.05)。与氟西汀组相比,针刺组的PKA-α和p-CREB蛋白表达均无显著差异(P0.05,P0.05),氟西汀+H89组的PKA-α蛋白表达无显著差异(P0.05),但p-CREB蛋白表达显著下降(P0.01)。与针刺组相比,针剌+H89组的PKA蛋白表达也无显著差异(P0.05),但针刺+H89组的p-CREB蛋白含量显著降低(P0.01)。结论1.慢性应激可以降低大鼠的自主探究行为,抑制奖赏系统和抑制体重增长。针刺能改善慢性应激诱导的抑郁样行为。2.慢性应激诱导的糖水偏好百分比和旷场运动次数下降与海马BDNF蛋白和BDNF mRNA含量降低有关,针刺可以通过提高海马BDNF蛋白和BDNF mRNA表达改善模型大鼠的抑郁样行为。3.慢性应激诱导的大鼠抑郁样行为及海马BDNF mRNA降低与海马内HDAC2蛋白含量增加有关,与组蛋白H3K9乙酰化(acH3K9)含量降低。针刺可以通过下调海马HDAC2蛋白表达,促进组蛋白H3K9乙酰化,增加BDNF mRNA表达,缓解模型大鼠的抑郁样行为。4.慢性应激诱导的大鼠抑郁样行为与海马内PKA信号通路受到抑制有关,针刺可通过激活海马内PKA信号通路发挥抗抑郁作用。5.海马内BDNF、 acH3K9、 HDAC2及PKA信号通路在抑郁症的病理生理学中均起重要作用,本研究证实针刺可以通过调节上述指标缓解模型大鼠的抑郁样行为,为阐明针刺的抗抑郁作用机制提供了科学依据。
[Abstract]:Objective to explore the effects of acupuncture on behavior and brain derived neurotrophic factor (BDNF) protein and gene expression in chronic stress model (CUMS) rats from the angle of epigenetics (DNA methylation and histone acetylation) and cell signaling pathway (PKA signaling pathway), and to elucidate the antidepressant effect of acupuncture in order to clarify the antidepressant effect of acupuncture. Methods at 1:40 male SD rats, weight (220~240) g, were randomly divided into blank control group (blank group), model group, model + fluoxetine group (fluoxetine group), model + acupuncture group (acupuncture group). Except for blank group, the other rats were treated with 28 d chronic mild unpredictable stimulation, and fluoxetine group was given 30 min before stress. Drug (1.8mg/kg/d), acupuncture group at 30 min before stress "Baihui" and "printing hall" point, once a day. In the experiment, 0d, 7d, 14d, 21d, 28 d respectively to carry out the open field, sugar water experiment, weighed. After the experiment, the hippocampus and serum.ELISA method were taken to detect serum BDNF protein, RT-PCR method was used to detect hippocampus BDNF mRNA, hippocampus detection method was used for detection hippocampus BDNF, acetylation histone H3K9 (acH3K9), deacetylase 2 (HDAC2) protein, methylation PCR method to detect the I methylation of BDNF gene promoter in the hippocampus of the male SD rats and weight (220~240) g, randomly divided into blank control group (blank group), model group, model + fluoxetine group (fluoxetine group), model + acupuncture group (acupuncture group), model + fluoxetine +H89 Group (group of fluoxetine +H89), model + acupuncture +H89 group (group of acupuncture +H89). Except for blank group, the rest rats were treated with 21d chronic mild unpredictable stimulation. The model and treatment intervention of the acupuncture group and fluoxetine group were same. The lateral ventricle burial operation was performed in the group of fluoxetine +H89 and the acupuncture group +H89 at 1 weeks before the model, and the side brain was given to the side brain before stress. Intravorum injection of H89 solution (10 mu m, 5 u L) one time every other day. Two groups were gavage and acupuncture at 30 min before stress, once a day. All rats were in the open field of experimental 0d and 21d, sugar water experiment, and weighed. After the experiment, the hippocampus tissue.Western-blot method was used to detect the protein kinase A (PKA), cAMP reaction element binding protein (CR). EB and phosphorylated CREB (p-CREB) protein content. Results 1. acupuncture and fluoxetine had no statistical difference (P0.05) before the experiment on behavior of model rats (P0.05). Compared with the blank group, the number of movement times, the percentage of sugar water preference and body mass decreased significantly (P0.01, P0.01, P0.01). And the model was compared with the model group. Compared with the group, acupuncture and fluoxetine could improve the number of exercise times (P0.05, P0.05), P0.01, P0.01 and body mass (P0.05, P0.01) in the model rats, but the effects of acupuncture and fluoxetine on various behaviourology were different from that of.2. acupuncture and fluoxetine on serum, BDNF protein in hippocampus and BDNF mRNA of hippocampus in the blank group. In the model group, the content of BDNF protein in the serum and hippocampus decreased significantly (P0.01, P0.01), and the BDNF mRNA in the hippocampus decreased significantly (P0.01). Compared with the model group, fluoxetine could significantly increase the expression of serum BDNF protein (P0.01) and the expression of BDNF mRNA in the hippocampus (P0.01). The needle spines could significantly increase the expression of the protein and the sea hippocampal BDNF protein. The expression of horse BDNF mRNA (P0.01), but the increase of serum BDNF protein and hippocampal BDNF mRNA effect is not as good as the.3. serum BDNF protein of fluoxetine (P0.05, P0.01), the correlation between the BDNF protein of the hippocampus and the behaviourology, the positive correlation between the expression of the serum BDNF protein and the expression of hippocampal protein is positive. P0.05, but there was no significant correlation between the percentage of sugar water preference and serum BDNF protein (P0.05), and there was a positive correlation between hippocampal BDNF protein and behavior (the number of open field movements, the percentage of sugar water preference) (P0.05, P0.01).4. hippocampal BDNF protein, and the correlation analysis of hippocampal BDNF mRNA and Study on the relationship between hippocampal BDNF protein and hippocampal BDNF No significant (P0.05). But there was a significant positive correlation between the frequency of open field movement and the percentage of sugar water preference and hippocampal BDNF mRNA (P0.000, P0.005).5. acupuncture and the effect of fluoxetine on the I methylation of the promoter of BDNF DNA in the hippocampus. There was no significant difference in the difference of I methylation of BDNF DNA promoter in hippocampus of rats (0.05) Compared with the blank group, the content of acH3K9 protein in the hippocampus of the model group was significantly lower than that in the blank group (P0.05). Compared with the model group, the content of acH3K9 protein in the fluoxetine group and the acupuncture group increased, but there was no statistical significance (P0.05, P0.05). The difference between the fluoxetine group and the acupuncture group was not significant (P0.05) and the blank group (P0.05, P0.05). The cH3K9 protein content was significantly lower than that in the model group (P0.05, P0.05). Compared with the model group, the content of HDAC2 protein in the hippocampus increased significantly (P0.01). Compared with the model group, fluoxetine and acupuncture could reduce the protein expression of HDAC2 in the hippocampus (P0.01). The difference between the acupuncture group and the fluoxetine group was not significant (P0.05).7. hippocampal acH3K9 protein, the correlation of HDAC2 egg white and BDNF mRNA was analyzed, and the expression of acH3K9 protein in the hippocampus and the sea were in the sea. The expression of horse HDAC2 protein has significant negative correlation (P0.005). There is a positive correlation between the expression of hippocampal BDNF mRNA and the expression of acH3K9 protein in the hippocampus (P0.05), and there is a significant negative correlation with the expression of HDAC2 protein in the hippocampus (P0.000).8. hippocampal acH3K9 protein, the correlation of HDAC2 protein and behavioral studies analysis of the frequency of open field and the expression of the hippocampal acH3K9 protein There was a positive correlation (P0.05), and there was a significant negative correlation with the expression of HDAC2 protein in the hippocampus (P0.000). There was no significant correlation between the percentage of sugar water preference and the expression of acH3K9 protein in the hippocampus (P0.05). There was a significant negative correlation with the expression of HDAC2 protein in the hippocampus (P0.000).9. acupuncture and fluoxetine's effect on the PKA specific resistance of the hippocampus PKA-CREB signaling pathway. H89 can block the improvement of fluoxetine and acupuncture on the behavior of model rats (horizontal crossing number, vertical number, sugar water consumption), and the blocking of fluoxetine treatment is more significant. There is no significant difference in the expression of CREB protein in hippocampus (P0.05). Compared with the blank group, the expression of PKA- alpha and p-CREB protein in the hippocampus of the model group is obvious. Lower (P0.01, P0.01). Compared with the model group, the PKA- alpha protein of the fluoxetine group increased significantly (P0.05), and the expression of p-CREB protein increased, but there was no statistical significance (P0.05). The expression of PKA- alpha and p-CREB protein in the acupuncture group increased significantly (P0.01, P0.05), and the expression of the protein expression in the fluoxetine +H89 group and the needle +H89 group increased. Compared with the fluoxetine group, there was no significant difference in the expression of PKA- alpha and p-CREB protein in the acupuncture group (P0.05, P0.05), and there was no significant difference in the expression of PKA- alpha protein in the fluoxetine +H89 group (P0.05), but the expression of p-CREB protein decreased significantly (P0.01). Compared with the acupuncture group, there was no significant difference in the expression of PKA protein in the needle +H89 group (P0.05). The white content was significantly decreased (P0.01). Conclusion 1. chronic stress can reduce the behavior of autonomic exploration, inhibit reward system and inhibit the growth of body weight. Acupuncture can improve the depression like behavior induced by chronic stress, the percentage of sugar water preference and the decline of the number of open field exercise times induced by chronic stress induced by.2. and the decrease of the content of BDNF protein and BDNF mRNA in the hippocampus are reduced. Acupuncture can improve the depressive behavior of the model rats by improving the expression of BDNF protein and BDNF mRNA in the hippocampus and the depression like behavior induced by.3. chronic stress in rats and the decrease of BDNF mRNA in the hippocampus is related to the increase of the content of HDAC2 protein in the hippocampus, and the content of the histone H3K9 acetylation (acH3K9) decreases. The acupuncture can reduce the hippocampus HDAC2 protein table. Da, promote histone H3K9 acetylation, increase BDNF mRNA expression, alleviate the depressive behavior of model rats,.4. chronic stress induced depression like behavior in rats is related to the inhibition of PKA signaling pathway in the hippocampus. Acupuncture can play antidepressant in hippocampus BDNF, acH3K9, HDAC2, and PKA signaling pathway by activating the PKA signaling pathway in the hippocampus. It plays an important role in the pathophysiology of depression. This study confirms that acupuncture can relieve the depressive behavior of rats by regulating these indexes and provide a scientific basis for clarifying the mechanism of antidepressant action of acupuncture.
【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】：2016
【分类号】：R245
，

本文编号：1899294