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  本文選題：地塞米松　切入點：星形膠質細胞　出處：《華中科技大學》2013年博士論文　論文類型：學位論文

【摘要】：第一部分糖皮質激素對培養(yǎng)大鼠皮層神經細胞AMPK信號通路的影響及機制 目的：腺苷酸活化蛋白激酶（AMP-activated protein kinase，AMPK）是機體重要的能量調節(jié)因子，在中樞神經系統(tǒng)有廣泛分布。抑郁模型動物下丘腦AMPK磷酸化水平是降低的，而抑郁癥患者和動物模型均存在糖皮質激素高表達。糖皮質激素作用是否會下調AMPK磷酸化？尚未見相關研究報道。本實驗旨在通過給予大鼠和原代培養(yǎng)皮層細胞糖皮質激素暴露，研究該過程中AMPK的變化及其機制。 方法：皮下注射連續(xù)21d給予大鼠皮質酮40mg/kg/d。分別給予原代培養(yǎng)皮層神經元，小膠質細胞和星形膠質細胞1μM地塞米松2h，48h或72h作用。通過Western Blot方法檢測蛋白水平的變化。 結果：慢性糖皮質激素暴露降低大鼠海馬及前額皮層磷酸化AMPK（pAMPK）水平。給予地塞米松作用2h，48h，72h均不影響神經元和小膠質細胞的pAMPK水平。給予地塞米松作用2h，48h和72h降低星形膠質細胞pAMPK水平。給予皮質酮作用2h，48h和72h也降低星形膠質細胞pAMPK水平。給予星形膠質細胞地塞米松2h，48h和72h升高p(Thr32)FOXO3a水平。血清和糖皮質激素誘導蛋白激酶1（serum-and glucocorticoid-inducible kinase1，SGK1）抑制劑GSK650394可抑制地塞米松導致的星形膠質細胞pAMPK降低和p(Thr32)FOXO3a升高。但糖皮質激素受體（glucocorticoid receptor，GR）拮抗劑RU38486不能阻斷地塞米松導致的星形膠質細胞pAMPK降低。而且給予GSK650394可逆轉地塞米松導致的p（T32）FOXO3a升高和pAMPK下降。 結論：地塞米松通過激活SGK1抑制FOXO3a轉錄活性，減少LKB1表達，選擇性地抑制星形膠質細胞的pAMPK水平。 第二部分AMPK對培養(yǎng)大鼠星形膠質細胞糖皮質激素受體表達的影響及機制 目的：有研究表明慢性糖皮質激素暴露可降低動物大腦GR表達。給予糖皮質激素可抑制星形膠質細胞pAMPK。有報道，外周AMPK可以調節(jié)GR功能，但未見中樞神經系統(tǒng)的相關研究報道。本實驗旨在研究地塞米松作用于原代培養(yǎng)皮層星形膠質細胞該過程中，AMPK信號通路介導糖皮質激素誘導的糖皮質激素受體表達下調中的作用及機制。 方法：給予原代培養(yǎng)皮層星形膠質細胞1μM地塞米松作用2h，48h或72h；通過Western Blot方法檢測各蛋白水平的變化。 結果：給予地塞米松2h，48h或72h不影響神經元GR表達。給予地塞米松48h或72h降低小膠質細胞GR和iNOS表達。給予地塞米松48h或72h可降低星形膠質細胞GR，腦源性神經營養(yǎng)因子（Brain-Derived Neurotrophic Factor，BDNF）表達，增加誘導型一氧化氮合酶（inducible nitric oxide synthase，iNOS）表達。給予皮質酮48h或72h可降低星形膠質細胞GR，，BDNF表達。在星形膠質細胞，AMPK抑制劑compound C，Ara-A48h和72h均減少GR和BDNF的表達；給予AMPK激動劑AICAR，阿司匹林和二甲雙胍可逆轉地塞米松導致的GR和BDNF的表達下降；SGK1抑制劑GSK650394也可阻斷或逆轉地塞米松導致的GR和BDNF的表達下降。GR阻斷劑RU38486不能阻斷地塞米松的作用；AMPK激動劑AICAR，阿司匹林和二甲雙胍均可逆轉地塞米松導致的GR和BDNF表達變化；地塞米松暴露可以導致FOXO3a抑制性位點Thr32磷酸化水平升高；地塞米松暴露可以導致組蛋白去乙�；�5（Histone deacetylases5，HDAC5）位點S295和S498磷酸化水平升高，而且AICAR可以逆轉該變化； HDAC5抑制劑SAHA和SB939均可以阻斷和逆轉地塞米松導致的GR和BDNF下降；SGK1抑制劑可以阻斷和逆轉地塞米松導致GR和BDNF變化。 結論：地塞米松通過抑制AMPK對HDAC5的磷酸化，增加HDAC5活性，從而降低星形膠質細胞GR的表達。 第三部分AMPK激動劑二甲雙胍對慢性糖皮質激素暴露所致大鼠抑郁樣行為的作用及機制 目的：AMPK激動劑阿司匹林，白藜蘆醇有一定的抗抑郁作用。很多抗糖尿病藥物也可緩解抑郁癥狀。但是，上述作用機制并不明確�？固悄虿∷幬锒纂p胍是AMPK激動劑，少見其抗抑郁報道。本實驗旨在通過慢性糖皮質激素暴露大鼠，探討二甲雙胍對慢性糖皮質激素暴露導致大鼠抑郁樣行為及GR表達下降的影響和作用機制。 方法：給予大鼠連續(xù)21d皮下注射皮質酮40mg/kg/d，第15d開始，同時給予300mg/kg/d二甲雙胍或氟西汀10mg/kg/d連續(xù)14d；采用強迫游泳評估大鼠抑郁樣行為；采用高架十字迷宮評估大鼠焦慮樣行為；采用高爾基染色方法檢測樹突棘密度；采用Western Blot方法檢測大鼠腦區(qū)蛋白水平的變化。 結果：慢性皮質酮暴露大鼠出現(xiàn)抑郁樣行為增加和體重增長下降，焦慮樣行為無變化；海馬和前額皮層的樹突棘密度降低；海馬和前額皮層pAMPK，GR，BDNF表達減少。給予14d二甲雙胍或氟西汀，除對體重增長減少無作用外，均可以逆轉慢性皮質酮暴露導致的上述其他變化。 結論：二甲雙胍可改善慢性皮質酮暴露導致的大鼠抑郁樣行為，海馬和前額皮層樹突棘密度降低及pAMPK，GR和BDNF表達減少。該作用可能是通過激活AMPK介導GR表達實現(xiàn)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.

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R749.4

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