AMPK信号通路在大鼠慢性糖皮质激素应激中的作用及机制

发布时间：2018-03-22 05:15

本文选题：地塞米松　切入点：星形胶质细胞　出处：《华中科技大学》2013年博士论文　论文类型：学位论文

【摘要】：第一部分糖皮质激素对培养大鼠皮层神经细胞AMPK信号通路的影响及机制目的：腺苷酸活化蛋白激酶（AMP-activated protein kinase，AMPK）是机体重要的能量调节因子，在中枢神经系统有广泛分布。抑郁模型动物下丘脑AMPK磷酸化水平是降低的，而抑郁症患者和动物模型均存在糖皮质激素高表达。糖皮质激素作用是否会下调AMPK磷酸化？尚未见相关研究报道。本实验旨在通过给予大鼠和原代培养皮层细胞糖皮质激素暴露，研究该过程中AMPK的变化及其机制。方法：皮下注射连续21d给予大鼠皮质酮40mg/kg/d。分别给予原代培养皮层神经元，小胶质细胞和星形胶质细胞1μM地塞米松2h，48h或72h作用。通过Western Blot方法检测蛋白水平的变化。结果：慢性糖皮质激素暴露降低大鼠海马及前额皮层磷酸化AMPK（pAMPK）水平。给予地塞米松作用2h，48h，72h均不影响神经元和小胶质细胞的pAMPK水平。给予地塞米松作用2h，48h和72h降低星形胶质细胞pAMPK水平。给予皮质酮作用2h，48h和72h也降低星形胶质细胞pAMPK水平。给予星形胶质细胞地塞米松2h，48h和72h升高p(Thr32)FOXO3a水平。血清和糖皮质激素诱导蛋白激酶1（serum-and glucocorticoid-inducible kinase1，SGK1）抑制剂GSK650394可抑制地塞米松导致的星形胶质细胞pAMPK降低和p(Thr32)FOXO3a升高。但糖皮质激素受体（glucocorticoid receptor，GR）拮抗剂RU38486不能阻断地塞米松导致的星形胶质细胞pAMPK降低。而且给予GSK650394可逆转地塞米松导致的p（T32）FOXO3a升高和pAMPK下降。结论：地塞米松通过激活SGK1抑制FOXO3a转录活性，减少LKB1表达，选择性地抑制星形胶质细胞的pAMPK水平。第二部分AMPK对培养大鼠星形胶质细胞糖皮质激素受体表达的影响及机制目的：有研究表明慢性糖皮质激素暴露可降低动物大脑GR表达。给予糖皮质激素可抑制星形胶质细胞pAMPK。有报道，外周AMPK可以调节GR功能，但未见中枢神经系统的相关研究报道。本实验旨在研究地塞米松作用于原代培养皮层星形胶质细胞该过程中，AMPK信号通路介导糖皮质激素诱导的糖皮质激素受体表达下调中的作用及机制。方法：给予原代培养皮层星形胶质细胞1μM地塞米松作用2h，48h或72h；通过Western Blot方法检测各蛋白水平的变化。结果：给予地塞米松2h，48h或72h不影响神经元GR表达。给予地塞米松48h或72h降低小胶质细胞GR和iNOS表达。给予地塞米松48h或72h可降低星形胶质细胞GR，脑源性神经营养因子（Brain-Derived Neurotrophic Factor，BDNF）表达，增加诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）表达。给予皮质酮48h或72h可降低星形胶质细胞GR，，BDNF表达。在星形胶质细胞，AMPK抑制剂compound C，Ara-A48h和72h均减少GR和BDNF的表达；给予AMPK激动剂AICAR，阿司匹林和二甲双胍可逆转地塞米松导致的GR和BDNF的表达下降；SGK1抑制剂GSK650394也可阻断或逆转地塞米松导致的GR和BDNF的表达下降。GR阻断剂RU38486不能阻断地塞米松的作用；AMPK激动剂AICAR，阿司匹林和二甲双胍均可逆转地塞米松导致的GR和BDNF表达变化；地塞米松暴露可以导致FOXO3a抑制性位点Thr32磷酸化水平升高；地塞米松暴露可以导致组蛋白去乙酰化酶5（Histone deacetylases5，HDAC5）位点S295和S498磷酸化水平升高，而且AICAR可以逆转该变化； HDAC5抑制剂SAHA和SB939均可以阻断和逆转地塞米松导致的GR和BDNF下降；SGK1抑制剂可以阻断和逆转地塞米松导致GR和BDNF变化。结论：地塞米松通过抑制AMPK对HDAC5的磷酸化，增加HDAC5活性，从而降低星形胶质细胞GR的表达。第三部分AMPK激动剂二甲双胍对慢性糖皮质激素暴露所致大鼠抑郁样行为的作用及机制目的：AMPK激动剂阿司匹林，白藜芦醇有一定的抗抑郁作用。很多抗糖尿病药物也可缓解抑郁症状。但是，上述作用机制并不明确。抗糖尿病药物二甲双胍是AMPK激动剂，少见其抗抑郁报道。本实验旨在通过慢性糖皮质激素暴露大鼠，探讨二甲双胍对慢性糖皮质激素暴露导致大鼠抑郁样行为及GR表达下降的影响和作用机制。方法：给予大鼠连续21d皮下注射皮质酮40mg/kg/d，第15d开始，同时给予300mg/kg/d二甲双胍或氟西汀10mg/kg/d连续14d；采用强迫游泳评估大鼠抑郁样行为；采用高架十字迷宫评估大鼠焦虑样行为；采用高尔基染色方法检测树突棘密度；采用Western Blot方法检测大鼠脑区蛋白水平的变化。结果：慢性皮质酮暴露大鼠出现抑郁样行为增加和体重增长下降，焦虑样行为无变化；海马和前额皮层的树突棘密度降低；海马和前额皮层pAMPK，GR，BDNF表达减少。给予14d二甲双胍或氟西汀，除对体重增长减少无作用外，均可以逆转慢性皮质酮暴露导致的上述其他变化。结论：二甲双胍可改善慢性皮质酮暴露导致的大鼠抑郁样行为，海马和前额皮层树突棘密度降低及pAMPK，GR和BDNF表达减少。该作用可能是通过激活AMPK介导GR表达实现的。
[Abstract]:The effect and mechanism of glucocorticoid on AMPK signaling pathway in cultured rat cortical neurons
Objective: adenosine monophosphate activated protein kinase (AMP-activated protein, kinase, AMPK) is the most important energy regulator, is widely distributed in the central nervous system. The animal model of depression in the phosphorylation level of AMPK is lower, and the depression patients and animal models have high expression in glucocorticoid. Whether glucocorticoid action will cut AMPK phosphorylation? Has not yet been reported. This study aims to give and primary culture of rat cortical cells of glucocorticoid exposure, changes and mechanism of AMPK in the process.
Methods: after subcutaneous injection of continuous 21d, corticosterone 40mg/kg/d. was given to primary cultured cortical neurons, microglia and astrocytes 1, M dexamethasone 2h, 48h or 72h. The protein level was detected by Western Blot.
Results: chronic glucocorticoid exposure decreased rat hippocampus and prefrontal cortex of phosphorylated AMPK (pAMPK) level. Given dexamethasone 2h, 48h, 72h had no effect on neurons and microglia pAMPK level. Dexamethasone 2h, 48h and 72h decreased astrocyte pAMPK level. 48h and 2H give the effect of corticosterone. 72h also decreased pAMPK of astrocytes. Astrocytes level given dexamethasone 2h, 48h and 72h increased P (Thr32) FOXO3a. The level of serum and glucocorticoid inducible kinase 1 (Serum-and glucocorticoid-inducible, kinase1, SGK1) astrocytes pAMPK inhibitor GSK650394 inhibited dexamethasone resulted in a decrease in FOXO3a and P (Thr32) but increased. The glucocorticoid receptor (glucocorticoid, receptor, GR) antagonist RU38486 blocked astrocytes pAMPK and dexamethasone resulted in a decrease in GSK650394. The increase of P (T32) FOXO3a and pAMPK decreased by reversing dexamethasone.
Conclusion: Dexamethasone inhibits the transcriptional activity of FOXO3a by activating SGK1 and reduces the expression of LKB1, and selectively inhibits the pAMPK level of astrocytes.
The effect of second part AMPK on the expression of glucocorticoid receptor in cultured rat astrocytes and its mechanism
Objective: studies have shown that chronic glucocorticoid exposure can reduce the animal's brain. The GR expression of glucocorticoid can inhibit astrocytes pAMPK. reported that peripheral AMPK can regulate the function of GR, but there is no report on the central nervous system. The purpose of this experiment was to study effects of DEX on primary cultured cortical astrocytes of the in the process, AMPK signaling pathway mediated by glucocorticoid receptor glucocorticoid induced expression effect and mechanism of cut in.
Methods: the primary cultured cortical astrocytes were treated with 1 M dexamethasone for 2h, 48h or 72h, and the changes of protein levels were detected by Western Blot.
Results: Dexamethasone 2h, 48h or 72h did not affect the expression of GR neurons. Dexamethasone 48h or 72h decreased expression of GR in microglia and iNOS. Dexamethasone 48h or 72h can reduce the GR of astrocytes, brain-derived neurotrophic factor (Brain-Derived Neurotrophic, Factor, BDNF) increased expression of inducible nitric oxide synthase (iNOS inducible nitric oxide synthase, 48h). The expression given corticosterone or 72h can decrease GR in astrocytes. The expression of BDNF in astrocytes, AMPK inhibitor compound C, Ara-A48h and 72h decreased the expression of GR and BDNF; AMPK agonist AICAR, decreased expression of aspirin and metformin can reverse the dexamethasone induced GR and BDNF GSK650394; expression of SGK1 inhibitors can also lead to block or reverse the decline of BDNF GR and dexamethasone.GR antagonist RU38486 did not block the effect of dexamethasone; AMP K agonist AICAR, aspirin and metformin can lead to changes in the expression of GR and BDNF reversed dexamethasone; dexamethasone exposure can cause FOXO3a inhibition of Thr32 phosphorylation levels; dexamethasone exposure can lead to histone deacetylase 5 (Histone, deacetylases5, HDAC5) increased S498 phosphorylation site of S295 and AICAR, and can reverse the change;
HDAC5 inhibitors SAHA and SB939 can block and reverse the decrease of GR and BDNF induced by dexamethasone, and SGK1 inhibitors can block and reverse the change of GR and BDNF caused by dexamethasone.
Conclusion: dexamethasone reduces the phosphorylation of AMPK to HDAC5 and increases the activity of HDAC5, thus reducing the expression of GR in astrocytes.
The effect and mechanism of the third AMPK agonist, metformin, on the depressive behavior of rats induced by chronic glucocorticoid exposure
Objective: AMPK agonist aspirin, resveratrol has certain antidepressant effect. Many anti diabetes drugs also can alleviate the symptoms of depression. However, the mechanism is not clear. The anti diabetic drug metformin is a AMPK agonist, the rare antidepressant reported. This experiment aimed at exposing rats by chronic glucocorticoid, explore metformin chronic glucocorticoid exposure leads to depression like behavior of rats and the expression of GR decreased the effect and mechanism of action.
Methods: rats were given continuous subcutaneous injection of 21d 40mg/kg/d 15d, corticosterone, 300mg/kg/d given metformin or fluoxetine 10mg/kg/d continuous 14d; evaluation by forced swimming depression like behaviors in rats; assess the anxiety like behavior of rats using the elevated plus maze; using Golgi staining method to detect the density of dendritic spines; to detect the changes of protein levels in the rat brain using Western Blot method.
Results: chronic corticosterone exposure rats showed depression like behavior and weight gain decreased growth, no change in anxiety like behavior; the density of dendritic spines in hippocampus and prefrontal cortex in the hippocampus and prefrontal cortex decreased; pAMPK, GR, BDNF expression decreased. 14d given metformin or fluoxetine, in addition to reduce the growth of body weight had no effect, can reverse the chronic the other changes in exposure to corticosterone.
Conclusion: metformin can improve depressive behavior induced by chronic corticosterone exposure in rats, decrease in dendritic spine density and decrease in expression of pAMPK, GR and BDNF in hippocampus and prefrontal cortex, which may be mediated by activation of AMPK mediated GR expression.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R749.4

【共引文献】