透過您的圖書館登入
IP:54.159.186.146
  • 學位論文

探討新生期dexamethasone處理對大鼠海馬迴中動情素受體之表現量及長期增益效應之影響

Elucidate the effects of neonatal dexamethasone treatment on hippocampal estrogen receptor expression and long-term potentiation of rats

指導教授 : 呂國棟
若您是本文的作者,可授權文章由華藝線上圖書館中協助推廣。

摘要


地塞米松(dexamethasone, DEX)是一種人工合成的糖皮質固醇(synthetic glucocorticoids),數十年來被廣泛用作抗發炎藥物 (anti-inflammatory drugs)。同時也用來治療及預防出生體重過低 (extreme low birth weight, ELBW)早產兒的慢性肺部疾病 (chronic respiratory disease)。許多研究指出,新生兒時期的地塞米松治療 (neonatal dexamethasone treatment, NDT)可能對早產兒的情緒 (emotional)和認知 (cognitive)功能產生長期不良的作用。 我們先前的研究指出NDT於雄鼠與雌鼠身上,均會造成長期的不良影響,包含類憂鬱行為(depression-like behavior)的增加,以及雌性大鼠海馬迴 (hippocampus)中α型雌激素受體 (estrogen receptor alpha, ERα)和G蛋白雌激素受體 (G-protein coupled estrogen receptor, GPER so-called GPR30)的表現量下降,且可能為NDT對雌鼠造成長期不良影響的重要原因。我們本研究中驗證是哪一種雌激素受體,為造成NDT青少年雌鼠長期不良反應的主因。 本實驗中使用新生的Wistar大鼠,從出生後第一天到第三天 (postnatal day 1~3, PND 1-3),給予劑量遞減式 (tapering)的DEX皮下注射,自PND 1-3其劑量依序為0.5、0.3和0.1 mg/kg。 於六周齡時,利用開放空間試驗(open field test, OFT)評估NDT大鼠自發性運動功能(spontaneous activity),另外透過強迫游泳試驗 (forced swimming test, FST),評測NDT大鼠的類憂鬱行為。 此外,準備平行組(parallel group)的NDT大鼠,於相同周齡時進行即時聚合酶鏈鎖反應 (real-time polymerase chain reaction, qPCR)評估海馬迴中Erα, Erβ, Gper, Grin1, Grin2a, Grin2b等基因表現。並利用西方墨點法(western blotting)評估mitogen activated protein kinase (MAPK) 的蛋白質表現量及其磷酸化水平(phosphorylation level)。 亦進行了離體胞外電生理記錄 (in vitro extracellular recording),測量海馬迴以高頻刺激誘導的長期增益作用(high frequency stimulation-induced long-term potentiation, HFS-LTP),以評估其海馬迴的神經可塑性(neuroplasticity)。最後利用強迫游泳試驗,以評估投予ERα致效劑(agonist),是否對NDT大鼠具有抗憂鬱(anti-depressant)的功能。 實驗結果顯示,NDT動物青春期前的體重顯著降低,這證明DEX的有效投藥。 OFT的結果顯示NDT動物的自發運動功能未受影響。FST的結果顯示NDT會導致類憂鬱行為的增加。雌性青少年大鼠NDT組的不掙扎的時間(immobility)與對照組相比有顯著增加。而雄性青少年NDT大鼠,則必須先給予高台曝露(platform exposure)之急性壓力處理後,其不掙扎的時間才有顯著增加。qPCR結果顯示,雌性青少年NDT大鼠海馬迴中Erα的表現顯著降低,而Erβ 和 Gper表現量則無明顯差異。NDT雄性青少年大鼠海馬迴中Erα, Erβ 及 Gper的表現量,與控制組間相較,皆無呈現顯著差異。雌性青少年NDT大鼠海馬迴中Grin1, Grin2a 及 Grin2b表現量皆沒有呈現差異。電生理實驗結果顯示NDT母鼠的海馬迴HFS-LTP有顯著下降的現象,而輸入和輸出曲線比值(input/output curve ratio, I/O curve)結果顯示,NDT老鼠與對照組相比有顯著下降的現象。而在配對脈衝促進 (pair-pulse facilitation, PPF) 實驗結果中並無呈現顯著差異。透過表面灌流 (suprafusion) ERα致效劑丙吡唑三醇 (propyl pyrazole triol, PPT)後,NDT雌性大鼠的海馬迴HFS-LTP恢復至正常範圍。而預先投予ERα的拮抗劑methyl-piperidino-pyrazole (MPP),可以阻擋PPT對HFS-LTP的恢復效果;而NDT組經皮下注射PPT後,其不掙扎的時間明顯減少,與對照組相比無顯著差異,顯示其具有降低類憂鬱行為的效果。最後我們利用GPER的致效劑同時為ERα和ERβ的抑制劑氟維司群(fulvestrant)進行驗證,投予fulvestrant無法使NDT雌性大鼠的海馬迴HFS-LTP恢復至正常範圍。 西方墨點法結果顯示,NDT並未影響雌性青少年大鼠海馬迴中MAPK 的蛋白質表現量及磷酸化水平。 綜合上述實驗結果,我們推論NDT青少年雌性大鼠的不良反應,較可能是藉由影響ERα的表現而造成。實驗結果可供後續臨床研究參考,例如投予PPT治療NDT所造成的長期不良反應。

並列摘要


Dexamethasone (DEX) is a synthetic glucocorticoid and frequently used as an anti-inflammation agent for decades. It is also applied neonatally as a therapeutic agent to prevent and cure chronic lung diseases in extremely low birth weight (ELBW) prematurely born infants. However, accumulated results pointed out that neonatal dexamethasone treatment (NDT) may have long-term deleterious effects on emotional and cognitive function. Our previous results demonstrated that NDT might cause depression-like behavior and alter the hippocampal estrogen receptors (ERs) gene expression, including the alpha type estrogen receptor (ERα) and G-protein coupled estrogen receptor (GPER, so-called GPR30). The change of hippocampal ERs gene expression is responsible for the long-term adverse effect of NDT on female NDT rats. We decided to determine which type of ERs participated in the NDT adverse effects on juvenile female rats. Briefly, newborn Wistar rats were subjected to subcutaneous tapering-dose injections of DEX (0.5, 0.3, and 0.1 mg/kg, respectively) from postnatal days one to three (PND 1-3). Animals were then subjected to an open field test (OFT) and forced swimming test (FST) for evaluating spontaneous motor function and depression-like behavior at the age of six weeks old. Additional NDT group animals were prepared and subjected to real-time PCR (qPCR) for evaluating the hippocampal Erα, Erβ, Gper, Grin1, Grin2a, Grin2b gene expression. The western blot was also applied to determine the protein expression and the phosphorylation level of the mitogen-activated protein kinase (MAPK). Furthermore, we used in vitro extracellular recording of the hippocampal high-frequency stimulation-induced long-term potentiation (HFS-LTP) to assess neuroplasticity in the hippocampus. Finally, the NDT animals were administered with ERα agonist then using the forced swimming test to evaluate its anti-depressive function. Results showed a significant somatic growth impairment of the NDT animals, which proven a successful drug administration. The OFT results showed that NDT did not affect the spontaneous movement of animals. The FST results revealed that the immobility time in the NDT group of juvenile female rats was significantly increased compared with the control group. However, juvenile male rats must be given acute stress (flap-top exposure). The immobility time in the NDT group was significantly increased compared with the control group. Our qPCR results showed that NDT juvenile female rats significantly reduced Erα in the hippocampus. Still, there was no difference between Erβ and Gper. There was no difference in Erα, Erβ and Gper expression in the hippocampus of NDT juvenile male rats. There was no significant difference in the Grin1, Grin2a, and Grin2 expression in the hippocampus of NDT juvenile female rats. The results of in vitro extracellular recording showed that the hippocampal HFS-LTP of the NDT juvenile female rats had a significant decrease, and the input/output curve ratio (I/O curve) results showed that the NDT rats had a significant decrease compared with the control group. In the pair-pulse facilitation (PPF) experiment, the results showed no significant difference. Through superfusion (suprafusion) ERα agonist propyl pyrazole triol (PPT), the hippocampal HFS-LTP of NDT juvenile female rats restored to the normal range. Besides, the ERα antagonist methyl-piperidino-pyrazole (MPP) could block the restoration effect of PPT in the hippocampal HFS-LTP. Finally, we used GPER agonist as both ERα and ERβ antagonist fulvestrant, which could not restore the hippocampal HFS-LTP. Western blot results showed no difference in the hippocampal MAPK protein expression and phosphorylation level of NDT female rats. After subcutaneous injection of PPT, the percentage time of immobility of NDT female rats was reduced. There was no difference in the percentage time of immobility between the NDT combined PPT injection group (NDT+PPT) compared with control group animals. In summary, we conclude that the adverse effect on NDT juvenile female rats could be ascribed to the function of ERα. Our results will be helpful for subsequent clinical administration as a reference, such as the administration of PPT as a pharmacological treatment to reduce adverse reactions caused by NDT.

參考文獻


Reference
Adams, M.M., Fink, S.E., Janssen, W.G., Shah, R.A. & Morrison, J.H. (2004) Estrogen modulates synaptic N-methyl-D-aspartate receptor subunit distribution in the aged hippocampus. J Comp Neurol, 474, 419-426.
Agorastos, A., Pervanidou, P., Chrousos, G.P. & Kolaitis, G. (2018) Early life stress and trauma: developmental neuroendocrine aspects of prolonged stress system dysregulation. Hormones (Athens), 17, 507-520.
Ajj, H., Chesnel, A., Pinel, S., Plenat, F., Flament, S. & Dumond, H. (2013) An alkylphenol mix promotes seminoma derived cell proliferation through an ERalpha36-mediated mechanism. PLoS One, 8, e61758.
Alkalay, A.L., Klein, A.H., Nagel, R.A. & Pomerance, J.J. (1996) Evaluation of hypothalamic-pituitary-adrenal axis in premature infants treated with dexamethasone. Am J Perinatol, 13, 473-477.

延伸閱讀