【作者】 袁世英；

【导师】 陈建国；

【作者基本信息】 华中科技大学 ， 药理学， 2013， 博士

【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。更多还原

【Abstract】 Part Ⅰ Effects of glucocorticoids on AMPK signaling pathway incultured rat cortical cellAims: AMP-activated protein kinase (AMPK) is an important energy metabolismmodulating factor, which is also widely distributed in the CNS. The phosphorylationlevel of AMPK in hypothalamus of depression-model animals is lower than thecontrol ones, while glucocorticoid (GC) levels are increased in both depressionpatients and animal models. Is the decreased phosphorylation of AMPK induced bythe chronic surge of GC? There is still no report on this question. Our recent researchfocused on the GC induced AMPK phosphorylation changes and its mediatingmechanisms in vivo and in vitro.Methods: Administration of Corticosterone on rat at the dosage of40mg/kg/d for21days through s.c. injection. Primary cultures of cerebral cortical neurons, microgliaand astrocytes were treated with1μM dexamethasone for2h,48h or72h. TheAMPK signal modifications were determined with Western blot.Results: The AMPK phosphorylation levels in rat hippocampus and prefrontal cortex(PFC) declined in the group treated with chronically administered corticosterone.Dexamethasone treatments for2h,48h and72h had no effects on thephosphorylation of AMPK in primarily cultured neurons and microglia, while in theastrocytes, the AMPK phosphorylation levels were decreased significantly.Dexamethasone treatments could also increase the inhibitory phosphorylation level ofFOXO3a on the Thr32site. All the phosphorylation level changes could be preventedwith a serum-and glucocorticoid-inducible kinase1(SGK1) inhibitor GSK650394. While GSK650394could attenuate the effects of dexamethasone on astrocytes,RU38486, a glucocorticoid receptor (GR) inhibitor, did not influence thedexamethasone effects.Conclusion: Dexamethasone inhibits the transcriptional activity of FOXO3a viaSGK1, leading to the reduced phosphorylation level and activity of AMPK inastrocytes. Part Ⅱ Effects of AMPK on the expression of glucocorticoidreceptor in cultured cortical astrocyteAims: It has been reported that chronic administration of GC on rat can reduce theglucocorticoids receptor (GR) expression in the brain, and that GC also inhibits theactivity of AMPK in astrocytes. Investigations have shown that AMPK modulates thefunction of GR, but related study in CNS is still lacking. Our recent research aimed atthe mechanism via which AMPK regulates GR expression in primarily culturedcortical astrocyte exposed to dexamethasone.Methods: After treatments with dexamethasone for2h,48h or72h, the indicationswere detected by Western blot.Results: Exposure to dexamethasone for2h,48h and72h did not influence the GRlevel in cortical neuron, while had inhibitory effects on the GR and iNOS expressionin primarily cultured microglia. On the primary culture of cortical astrocyte, the sametreatment could drastically reduce the expression level of GR and BDNF, andstimulate the expression of iNOS. Interestingly, administration of AMPK inhibitors,compound C and Ara-A, could imitate the dexamethasone-induced alterations, while AMPK activators, AICAR, aspirin and metformin, showed inverting effects ondexamethasone-induced changes in cortical astrocyte. SGK1inhibitor GSK65394alsofeatured preventive and reversing effect on the dexamethasone effect in astrocytewhile the GR inhibitor RU38486failed to. Dexamethasone exposure drasticallyincreased the inhibitory phosphorylation of FOXO3a and decreased the AMPKphosphorylation sites on histone deacetylases5(HDAC5) Ser259and Ser498, whichcould both be restored with AICAR. In addition, HDAC5inhibitors SAHA andSB939, and SGK1specific inhibitor GSK, could attenuate and invert thedexamethasone-induced GR and BDNF decrease in cortical astrocytes.Conclusion: Dexamethasone reduces the expressions of GR and BDNF in astrocytethrough APMK and HDAC5pathway. Part Ⅲ Effects of AMPK agonist metformin ondepression-behaviors induced by chronic glucocorticoid exposureAims: AMPK activators, such as aspirin and resveratrol, play certain antidepressanteffects, whose underlying mechanisms are not clear yet. Many anti-diabetics emergeantidepressant effect in the treatment of diabetes. The anti-diabetics metformin is anAMPK activator, however there is no report about its antidepressant studies inAnimals. In this study, we aim to identify the effects of metformin administration onchanges of behaviors and the expression of GR in hippocampus and prefrontal cortexinduced by chronic corticosterone exposure in rats.Methods: Administration of300mg/kg metformin or10mg/kg fluoxetine daily for14d after21-d repeated40mg/kg/d corticosterone-injections in rats. Identified depression-like behaviors of rats by forced swimming test; Identified anxiety-likebehaviors of rats by elevated plus maze test. To identify the density of dendritic spinesGolgi staining was used. Western Blot was used to detect the expression of proteins inhippocampus and prefrontal cortex.Results: Compared with vehicle group, depression-like behaviors was increased andbody weight gain was slowed in corticosterone exposed groups, furthermore, dendriticspine density and expression of pAMPK, GR and BDNF in hippocampus andprefrontal cortex were all decreased in21-day repeated corticosterone exposed rats,but anxiety-like behaviors was not affected. Excitingly, metformin or fluoxetineadministration attenuated the changes above induced by repeated corticosteroneinjection, except for the weight gain loss.Conclusion: Metformin can improve alterations in depression-like behaviors anddecline of GR, BDNF expression in hippocampus and prefrontal cortex induced bychronic corticosterone exposure in rats.更多还原