cAMP/PKA/CREB信号通路调控组织器官细胞纤维化及中医药干预作用的研究进展

环磷酸腺苷（cAMP）通过活化cAMP依赖的蛋白激酶A（PKA）,使cAMP反元件结合蛋白（CREB）磷酸化,从而调节基因转录,广泛参与神经系统的学习记忆过程,而近年来研究显示cAMP/PKA/CREB信号通路参与组织器官细胞的纤维化过程。机体损伤后在其组织器官细胞修复过程中,细胞外基质异常增生谓之纤维化,纤维化可使肝、肺、肾及心等脏器组织功能下降。中医药在治疗纤维化等慢性复杂疾病过程中有独特的优势,而调节cAMP/PKA/CREB信号通路是其防治组织器官细胞纤维化的机制之一。     

五味子乙素对慢性应激抑郁大鼠海马BDNF/TrkB/CREB通路的影响

目的 研究五味子乙素对慢性应激抑郁大鼠海马脑源性神经营养因子（BDNF）/酪氨酸激酶B（TrkB）/环磷腺苷效应元件结合蛋白（CREB）信号通路的影响。方法 40只SD大鼠随机选择10只作为对照组,其余大鼠采用慢性不可预知温和应激（chronic unpredictable mild stress,CUMS）结合孤养制备抑郁症模型,造模结束后随机分为3组：模型组、盐酸氟西汀（3 mg·kg^-1）组、五味子乙素（5 mg·kg^-1）组,每天ig给药1次,连续8周。分别于造模前、造模后及给药后进行旷场、悬尾、强迫游泳行为学实验;通过苏木素-伊红（HE）染色观察大鼠海马形态学改变;免疫组织化学染色（IHC）法观察大鼠海马BDNF蛋白表达;实时荧光定量PCR（qRT-PCR）法检测大鼠海马BDNF、TrkB、CREB mRNA相对表达量;Westernblotting检测大鼠海马BDNF、TrkB、CREB蛋白相对表达量。结果 与对照组比较,模型组大鼠旷场实验水平、垂直得分显著降低（P＜0.05）,悬尾不动时间和强迫游泳漂浮时间显著增加（P＜0.05）;HE染色结果显示海马神经元结构损伤,IHC结果显示海马BDNF表达明显降低;海马BDNF、TrkB、CREB mRNA及蛋白相对表达显著降低（P＜0.05）。与模型组比较,盐酸氟西汀及五味子乙素组大鼠水平、垂直得分显著增加（P＜0.05）,不动时间和漂浮时间显著减少（P＜0.05）;海马神经元结构明显复原,海马组织中BDNF染色明显增加;BDNF、TrkB、CREB mRNA和蛋白相对表达量显著增加（P＜0.05）。结论 五味子乙素可以改善慢性应激抑郁大鼠抑郁样行为、海马区神经元数量及形态,其机制可能与上调BDNF/TrkB/CREB信号通路有关。     

奥拉西坦/丁基苯酞对新生大鼠缺氧缺血性脑病的相关研究

目的：研究联合应用奥拉西坦（ORS）和丁基苯酞（NBP）在新生大鼠缺氧缺血性脑病中的早期治疗作用。方法：出生7d大鼠共分为4组，每组25只。（1）空白组：分离左侧颈总动脉后在动脉下置线，不结扎亦不低氧。（2）模型组：制备大鼠缺血缺氧性脑病模型立即腹腔注射生理盐水0．1mL／只。（3）对照组：建模后立即腹腔注射ORS100mg／kg。（4）实验组：造模成功后立即腹腔注射0RS100mg／kg，30min后腹腔注射丁基苯酞10mg／kg。所有动物于注射药物后12h、24h、3d、7d后断头处死取大脑组织，并对脑组织中含水量、SOD含量进行检测，通过免疫组化及westernblot方法检测水通道蛋白-4（AQP-4）和环磷酸腺苷反应元件结合蛋白（CREB）的变化情况。结果：与模型组比较，实验组脑组织含水量明显低于时照组，但SOD结果显示高于对照组（P〈0．05）；通过免疫组化及westernblot分析与模型组比较，可见AQP-4表达在对照组和实验组均有降低，但实验组低于对照组接近空白组，CREB蛋白在实验组中呈高表达接近空白组（P〈0．05）。结论：大脑缺氧缺血后应用ORS／NBP可上调CREB蛋白并下调AQP-4，时新生大鼠缺氧缺血性脑病具有早期治疗作用。     

加味四逆散对皮质酮和谷氨酸致损伤的PCI2细胞中cAMP反应元件结合蛋白及其磷酸化的影响

目的研究中药复方加味四逆散（JWSNS）对皮质酮和谷氨酸致损伤的PCI2细胞中cAMP反应元件结合蛋白（CREB）及其磷酸化的影响。方法以200μmol·L^-1Cort和50μmol·L^-1Glu损伤的PC12细胞作为体外药理学研究模型,制备JWSNS含药血清,采用免疫组织化学法和Western blot法检测PC12细胞损伤模型中CREB和磷酸化CREB蛋白的表达。结果200μmol·L^-1 Cort和50Ixmol·L^-1 Glu可导致PCI2细胞中CREB和p-CREB表达下降（CREB阳性细胞率分别为17．1％±4．2％和20．8％±5．7％,p—CREB表达量分别为0．587±0．123和0．515±0．157,P＜0．05或P＜0．01）,10％JWSNS含药血清能升高CREB和P—CREB的表达（CREB阳性细胞率分别为62．6％±11．7％和79．5％±7．6％,p-CREB表达量分别为1．298±0．112和1．269±0．128,P＜0．05或P＜0．01）。结论10％JWSNS含药血清能通过调节cAMP—CREB信号通路发挥其神经保护和抗抑郁的作用。     

miR-134调控CREB/BDNF通路参与脑卒中后抑郁的作用机制研究#br#

摘要：目的　探讨微小RNA（miR）-134调节环腺苷酸应答元件结合蛋白（CREB）/脑源性神经营养因子（BDNF）通路参与脑卒中后抑郁（PSD）的作用机制。方法　32只SD大鼠按照随机数字表法分为假手术组、模型组、antagomir NC组、miR-134 antagomir组,每组8只;除假手术组外,其余各组行大脑中动脉阻塞（MCAO）术,然后采用慢性不可预见温和应激（CUMS）诱导建立PSD模型,假手术组除不插入线栓外其他操作相同。MCAO术后CUMS前antagomir NC组、miR-134 antagomir组分别于大脑双侧海马区注射5 μL antagomir NC、miR-134 antagomir（1 μmol/L）,5 d注射1次,连续5次。假手术组、模型组注射等体积生理盐水。CUMS应激前,应激第7、14、21天对大鼠称体质量,动物敞箱实验评价大鼠运动行为改变、蔗糖偏爱实验评价大鼠快感缺乏行为改变。实验结束后处死大鼠,分离海马CA1区,实时荧光定量PCR（qPCR）检测miR-134、CREB、BDNF mRNA表达,免疫组化染色检测CREB、BDNF蛋白表达情况。双荧光素酶报告基因实验鉴定CREB与miR-134的靶向关系。结果　与假手术组相比,模型组大鼠体质量、水平和垂直活动得分、糖水饮用比例降低（P＜0.05）;与模型组、antagomir NC组相比,miR-134 antagomir组大鼠体质量、水平和垂直活动得分、糖水饮用比例升高（P＜0.05）,随着应激时间的延长,该效应逐渐明显。与假手术组相比,模型组海马CA1区miR-134水平升高（P＜0.05）,CREB、BDNF mRNA表达和阳性细胞数减少（P＜0.05）;与模型组、antagomir NC组相比,miR-134 antagomir组海马CA1区miR-134水平降低（P＜0.05）,CREB、BDNF mRNA表达和阳性细胞数增多（P＜0.05）。与miR-134 mimic NC+CREB-WT相比,miR-134 mimic+CREB-WT共转染细胞荧光素酶相对活性下降（P＜0.05）,证明CREB序列上存在miR-134特异结合位点。结论　降低miR-134可促进CREB/BDNF通路蛋白的表达,进而改善抑郁状态,实现对PSD大鼠的保护。     

CREB与学习记忆及情绪关系

目的研究环单磷酸腺苷反应元件结合蛋白(CREB)与学习记忆和情绪的关系。方法通过查阅国内外CREB相关文献及近年来对CREB与学习记忆和情绪关系所做的临床和动物实验分析。结果相关实验结果显示:在学习及情绪障碍患者和动物模型的神经系统内发现CREB含量减少。结论脑组织中CREB含量的减少可导致学习记忆下降和各种情绪障碍。     

栝楼桂枝颗粒对神经元兴奋性毒性损伤后钙信号通路的影响

目的 研究栝楼桂枝颗粒对N-甲基-D-天冬氨酸（N-methyl-D-aspartic acid,NMDA）诱导神经元兴奋性毒性损伤后钙信号通路的影响,并探讨其可能的机制。方法 建立NMDA诱导神经元兴奋性毒性损伤模型,栝楼桂枝颗粒干预后,采用MTT、LDH法检测神经元活性,免疫荧光染色法检测神经元特异性指标MAP-2蛋白的表达。Real-time PCR法检测神经元中CaMKⅡ、CREB mRNA的表达,Western blot法检测p-CREB、p-CaMKⅡ、CaMKⅡ、CREB及CaM蛋白的表达。结果 与NMDA组比较,栝楼桂枝颗粒（200,300 μg·mL^-1）组可显著提高细胞活力,降低LDH浓度（P<0.05或P<0.01）,明显升高CREB、CaMKⅡ mRNA水平（P<0.05或P<0.01）,明显升高MAP-2、p-CREB、p-CaMKⅡ及CREB水平（P<0.01）,而显著降低CaM水平（P<0.01）。结论 栝楼桂枝颗粒对NMDA诱导的神经元兴奋性毒性损伤具有保护作用,此作用可能与促进CREB、CaMKⅡ的磷酸化,抑制CaM的表达有关。     

cAMP反应元件结合蛋白与神经退行性疾病

cAMP反应元件结合蛋白(cAMP response elem ent b ind-ing prote in,CREB)是位于细胞核内的转录因子。CREB的活性受到信号通路中许多因子的调控。CREB活化后与真核生物靶基因CRE序列结合并调节其转录,发挥多种生物学效应。在中枢神经系统,CREB调节着神经细胞生长发育,参与神经细胞突触可塑性、长时程记忆的形成过程。CREB参与阿尔采末病、血管性痴呆、亨廷顿舞蹈病及H IV-相关痴呆等神经退行性疾病的病理生理机制研究也取得了进展,成为以CREB作为靶点控制神经退行性疾病病程的理论基础。     

高血氨对大鼠脑内NMDAR1 CREB表达的影响

目的探讨高血氨大鼠学习记忆能力改变的分子机制。方法雄性SD大鼠40只,随机分为2组,每组20只：正常对照组（A组）,高血氨模型组（B组）,Morris水迷宫观察动物学习记忆的变化,大鼠处死后,检测血氨,原位杂交检测N-甲基-D-天门冬氨酸受体1（NMDAR1）基因的表达,实时荧光定量聚合酶链反应（RTqPCR）检测大鼠脑组织环磷酸腺苷（cAMP）应答元件结合蛋白（CREB）基因的表达。结果与正常对照组比较,高血氨模型组大鼠血氨水平明显升高,其平均逃避潜伏期、游泳总距离均延长;并且大脑皮层、海马NMDAR1mRNA表达均下降,CREB基因表达亦均下降。结论高血氨模型大鼠学习记忆功能下降可能由于脑组织NMDAR1、CREB等基因表达下调所致。     

酒精依赖大鼠皮质CREB变化及氟西汀对其影响

目的　研究酒依赖大鼠皮质cAMP反应元件结合蛋白 (CREB)、磷酸化CREB( p CREB)蛋白表达的变化 ,并观察氟西汀对其影响。方法　给大鼠自由饮含低浓度乙醇 (体积分数为 6% )的水溶液 2 8d。采用免疫组织化学方法检测大鼠皮质CREB、p CREB蛋白表达。结果　撤除酒精后大鼠前额皮质、梨状皮质的p CREB蛋白表达均降低 ,其中以戒断 2 4h时降低最明显 ,与对照组比较两部位分别降低5 7 2 8%、5 5 84%。氟西汀 ( 10mg·kg-1,ip)能减轻饮酒大鼠戒断症状 ,增加大鼠戒断 2 4h时前额皮质、梨状皮质 p CREB水平 ,与饮酒组戒断 2 4h比较两部位分别增加2 92 5 8%、12 8 44 %。结论　CREB可能是介导酒精依赖的受体后信号转导物质之一 ,氟西汀对酒精戒断症状的改善与其拮抗酒精戒断诱导的 p CREB降低有关     

Neuropeptide Y Y1 receptor knockdown can modify glutathione peroxidase and c-AMP response element-binding protein in phenylpropanolamine-treated rats

It has been reported that antioxidative enzymes, neuropeptide Y (NPY), and c-AMP response element-binding protein (CREB) are involved in regulating phenylpropanolamine (PPA)-mediated appetite suppression. Here, we investigated whether Y1 receptor (Y1R) might be involved in this regulation. Rats were daily treated with PPA for 4 days. Changes in the contents of NPY, Y1R, glutathione peroxidase (GP), and CREB were assessed and compared. Results showed that Y1R, GP, and CREB increased, with a maximal increase about 100, 200, and 150 %, respectively, on Day 2. By contrast, NPY decreased with a biggest reduction about 48 % on Day 2 and the pattern of expression during PPA treatment was opposite to those of Y1R, GP, and CREB. Central knockdown (using antisense) or inhibition (using antagonist) of Y1R expression modulated the anorectic response of PPA and the reciprocal regulation between NPY and GP (or CREB), revealing an essential role of Y1R in regulating NPY, GP, and CREB. These results suggest that Y1R participates in the reciprocal regulation of NPY, GP, and CREB in the hypothalamus during PPA treatment in conscious rats. The present results may aid the therapeutic research of PPA and related antiobesity drugs.     

cAMP/PKA信号通路在肾上腺肿瘤中的作用

目的：探讨cAMP/PKA信号通路在肾上腺肿瘤中的作用。方法将人肾上腺肿瘤H295R细胞随机分为对照组、实验组A及实验组B,对照组不进行处理,实验组A加入cAMP衍生物db-cAMP,实验组B加入db-cAMP及cAMP/PKA信号通路抑制剂H-89,分别检测细胞PKA活性;应用Western Blot方法检测cAMP/PKA信号通路下游CREB蛋白的磷酸化水平;CCK8实验检测细胞72 h的增殖水平;糖皮质激素分泌实验检测3组H295R的激素分泌能力。结果实验组A的PKA活性水平、CREB蛋白的磷酸化水平、细胞增殖水平、糖皮质激素分泌水平均较对照组和实验组B显著升高(P<0．05);而实验组B与对照组比较差异无统计学意义(P<0．05)。结论 cAMP/PKA信号通路的异常活化能够促进肾上腺肿瘤的形成与功能的发挥。     

The induction of orphan nuclear receptor Nur77 expression by n-butylenephthalide as pharmaceuticals on hepatocellular carcinoma cell therapy

N-butylidenephthalide (BP), isolated from the chloroform extract of Angelica sinensis, has been examined for its antitumor effects on glioblastoma multiforme brain tumors; however, little is known about its antitumor effects on hepatocellular carcinoma cells. Two hepatocellular carcinoma cell lines, HepG2 and J5, were treated with either N-butylidenephthalide or a vehicle, and cell viability and apoptosis were evaluated. Apoptosis-related mRNA and proteins expressed, including orphan receptor family Nurr1, NOR-1, and Nur77, were evaluated as well as the effect of N-butylidenephthalide in an in vivo xenograft model. N-butylidenephthalide caused growth inhibition of both the cell lines at 25 microg/ml. Furthermore, N-butylidenephthalide-induced apoptosis seems to be related to Nur77 translocation from nucleus to cytosol, which leads to cytochrome c release and caspase-3-dependent apoptosis. N-butylidenephthalide-related tumor apoptosis was associated with phosphatidylinositol 3-kinase/protein kinase B (AKT)/glycogen synthase kinase-3beta rather than the mitogen-activated protein kinase or protein kinase C pathway. Blockade of AKT activation enhanced proliferation inhibition and the induction of phosphor-Bcl-2 and Nur77 proteins. Besides, the increasing apoptosis by BP via transfection wild-type cAMP-response element-binding protein (CREB) into tumor cell was suppressed by dominant phosphorylation site mutation of CREB. This finding suggested CREB pathway was also partly involved in tumor apoptosis caused by BP. Administration of N-butylidenephthalide showed similar antitumoral effects in both HepG2 and J5 xenograft tumors. N-Butylidenephthalide induced apoptosis in hepatocellular carcinoma cells, both in vitro and in vivo, suggesting a potential clinical use of this compound for improving the prognosis of hepatocellular carcinoma cells.     

羟甲芬太尼立体异构体对培养的海马神经元cAMP-反应元件结合蛋白磷酸化的影响

AIM: To define the effects and signal pathways of ohmefentanyl stereoisomers [(-)-cis-(3R,4S,2'R) OMF (F9202),( )-cis-(3R,4S,2'S) OMF (F9204), and (-)-cis-(3S,4S,2'R) OMF (F9203)] on the phosphorylation of cAMP-re-sponse element binding protein (CREB) in cultured rat hippocampal neurons. METHODS: The effects of the three OMF stereoisomers and morphine (Mor) on cAMP accumulation and CREB phosphorylation were monitored by radioimmunoassay and Western blot analysis, respectively. RESULTS: The three OMF stereoisomers and Mor could all partially inhibit forskolin-stimulated (25μmol/L, 15min) cAMP accumulation in a dose-dependent manner and this effect could be reversed by naloxone. F9202, F9204, and Mor could significantly increase CREB phosphorylation from 2.88 to 3.59 folds over control levels after 30-min exposure. This effect was reversed by naloxone,but F9203 failed to increase CREB phosphorylation. KN-62 and staurosporine significantly blocked the opioidsinduced CREB phosphorylation, while H-89 and PD 98059 had no effect on the actions. CONCLUSION: Mor,F9202, and F9204, which could induce psychological dependence affected via the μ-opioid receptor, stimulated intracellular signal pathways involving Ca^2 /calmodulin-dependent protein kinases (CCDPK) and protein kinase C(PKC) pathways, which in turn initiated CREB phosphorylation. F9203, which could not induce dependence, had no effect on CREB phosphorylation in hippocampal neurons. The increased CREB phosphorylation in hippocampal neurons may play a role in opioids dependence.