脊髓磷酸化胞外信号调节蛋白激酶在大鼠切口痛痛觉过敏中的作用

目的 研究切口痛模型大鼠脊髓磷酸化胞外信号调节蛋白激酶(p-ERK)表达的变化及鞘内注射ERK上游激酶MEK的抑制剂U0126对其机械性痛觉阈值的影响.方法 雄性SD大鼠32只按随机数字表法分为假手术组、模型组、DMSO组、U0126组,每组8只,前2组大鼠鞘内注射生理盐水20μL;后2组大鼠分别鞘内注射DMSO、U0126 10μL后用生理盐水10μL冲管.注药10min后除假手术组外,后3组大鼠均制备右后足趾部切口痛模型.分别于制作模型前、模型后2、24、48 h应用YLS-3E电子压痛仪测定各组大鼠右足的机械性痛觉阈值.另取SD大鼠24只.分组方法 和处理同上,每组6只.每组分别在模型后2h、24h选择3只应用免疫组化染色检测大鼠脊髓背角p-ERK的表达.结果模型组、DMSO组大鼠模型后2、24、48 h及U0126组大鼠模型后2、24 h有足机械性痛觉阈值均低于模型前,差异有统计学意义(P<0.05);DMSO组和模型组之间比较大鼠机械性痛觉阈值差异无统计学意义(P>0.05);与同一时间点DMSO组和模型组比较,U0126组大鼠模型后2、24、48 h右足机械性痛觉阈值均增高,差异有统计学意义(P<0.05).免疫组化染色结果发现.与假手术组比较模型组和DMSO组模型后2、24 h患侧脊髓背角P-ERK免疫阳性细胞增多;与模型组和DMSO组同一时间比较,U0126组患侧脊髓背角p-ERK阳性细胞减少,差异均有统计学意义(P相似文献   

脊髓磷酸化胞外信号调节蛋白激酶在大鼠切口痛痛觉过敏中的作用

Objective To investigate the changes of phosphorylated extracellular signal-regulated kinase (p-ERK) expression in the immunoreactive cells of the spinal dorsal hom after plantar incision,and explore the effects of intrathecal administration of MEK inhibitor U0126 on physiological pain threshold in rat models with incisional pain.Methods Thirty-two male SD rats were equally randomized into control group (C group),incisional pain group (I group),intrathecal U0126 group (Ugroup) and intrathecal DMSO group (D group).Twenty-μL physiological saline was injected into the rats of the C group and I group,respectively.Ten-μL DMSO and U0126 were injected into the rats in the U group and D group,respectively.Rat models with incisionai pain were induced in the other 3 groups except the C group.Mechanical hyperalgesia were evaluated by paw-pressure before and 2,24 h and 2 d after the inducement.Another 24 rats were treated as the above method and equally divided into 4 groups; the numbers of p-ERK immunoreactive cells in the dorsal horn were quantified to determine the ERK activation 2 and 24 after the model inducement.Results The paw-pressure threshold in I group and D group 2 and 24 h,and 2 d after the incision,and that in U group 2 and 24 h after the incision were significantly decreased as compared with that in C group (P<0.05); that between I group and D group showed no significant difference (P>0.05); that in the U group was obviously higher than that in the I group and D group at the same time points (P<0.05).Significantly increased numbers of p-ERK immunoreactive cells in the I group and D group were observed as compared with those in the C group (P<0.05); those in the U group was obviously decreased as compared with those in the I group and D group at the same time points (P<0.05).Conclusion Plantar incision-induced mechanical hyperalgesia can be prevented by intrathecal injection of U0126 through decreasing the expression of p-ERK positive cells,indicating that ERK pathway in the spinal dorsal horn involves in the incision-induced mechanical hyperalgesia in rats.     

脊髓磷酸化胞外信号调节蛋白激酶在大鼠切口痛痛觉过敏中的作用

Objective To investigate the changes of phosphorylated extracellular signal-regulated kinase (p-ERK) expression in the immunoreactive cells of the spinal dorsal hom after plantar incision,and explore the effects of intrathecal administration of MEK inhibitor U0126 on physiological pain threshold in rat models with incisional pain.Methods Thirty-two male SD rats were equally randomized into control group (C group),incisional pain group (I group),intrathecal U0126 group (Ugroup) and intrathecal DMSO group (D group).Twenty-μL physiological saline was injected into the rats of the C group and I group,respectively.Ten-μL DMSO and U0126 were injected into the rats in the U group and D group,respectively.Rat models with incisionai pain were induced in the other 3 groups except the C group.Mechanical hyperalgesia were evaluated by paw-pressure before and 2,24 h and 2 d after the inducement.Another 24 rats were treated as the above method and equally divided into 4 groups; the numbers of p-ERK immunoreactive cells in the dorsal horn were quantified to determine the ERK activation 2 and 24 after the model inducement.Results The paw-pressure threshold in I group and D group 2 and 24 h,and 2 d after the incision,and that in U group 2 and 24 h after the incision were significantly decreased as compared with that in C group (P<0.05); that between I group and D group showed no significant difference (P>0.05); that in the U group was obviously higher than that in the I group and D group at the same time points (P<0.05).Significantly increased numbers of p-ERK immunoreactive cells in the I group and D group were observed as compared with those in the C group (P<0.05); those in the U group was obviously decreased as compared with those in the I group and D group at the same time points (P<0.05).Conclusion Plantar incision-induced mechanical hyperalgesia can be prevented by intrathecal injection of U0126 through decreasing the expression of p-ERK positive cells,indicating that ERK pathway in the spinal dorsal horn involves in the incision-induced mechanical hyperalgesia in rats.     

电针干预自发性高血压大鼠主动脉丝裂原活化蛋白激酶磷酸酶1及磷酸化细胞外信号调节激酶1/2蛋白的表达

背景：近年来大量临床研究表明针刺风池、太冲、曲池等穴位能有效降低血压,可用于高血压,但对其治疗的分子机制尚未阐明。 目的：观察针刺大鼠风池、太冲、曲池等穴位对丝裂原活化蛋白激酶信号转导调控系统的影响,从而探讨针刺治疗高血压的分子机制。 方法：选取8月龄自发性高血压雄性Wistar大鼠14只,随机分为针刺组和模型组,每组7只;另选取同月龄正常血压雄性Wistar-Kyoto大鼠7只作为对照组。对针刺组大鼠采用电针针刺双侧风池、曲池和三阴交穴,毫针刺太溪和太冲穴。3周后采用RT-PCR方法检测各组大鼠主动脉组织丝裂原活化蛋白激酶磷酸酶1 mRAN的表达,Western blot方法检测丝裂原活化蛋白激酶磷酸酶1、磷酸化细胞外信号调节激酶1/2蛋白表达。 结果与结论：与对照组比较,模型组主动脉组织磷酸化细胞外信号调节激酶1/2蛋白表达水平升高,丝裂原活化蛋白激酶磷酸酶1 mRNA及其蛋白表达水平降低(P < 0.01);与模型组比较,针刺组主动脉组织磷酸化细胞外信号调节激酶1/2蛋白表达水平降低,丝裂原活化蛋白激酶磷酸酶1 mRNA及其蛋白表达水平升高(P < 0.05)。提示针刺治疗自发性高血压大鼠可能是通过调控丝裂原活化蛋白激酶信号转导途径,增强磷酸化细胞外信号调节激酶1/2蛋白表达,降低丝裂原活化蛋白激酶磷酸酶1蛋白表达,从而改善血管重塑,降低血压。     

p38丝裂原活化蛋白激酶与缺血性脑损伤

丝裂原活化蛋白激酶(mitogen activated protein kinase．MAPK)超家族广泛分布于细胞浆内。是一族含有丝氨酸／苏氨酸残基的蛋白激酶，是将细胞外刺激信号传递到细胞核．引起细胞生物学反应的重要信号传导系统。目前MAPK超家族在哺乳动物中至少发现4种亚家族，分别为细胞外信号调节激酶(extracelluar signal regulated protein kinase，     

马来酸桂哌齐特对脑缺血大鼠模型胞外信号调节激酶表达的影响

目的观察马来酸桂哌齐特预处理对大鼠脑缺血半暗带区胞外信号调节激酶(ERK1/2)磷酸化表达的影响,探讨其对脑缺血的保护作用及可能的作用机制。方法采用改良线栓法建立大鼠永久性大脑中动脉阻塞(pMCAO)模型。SD大鼠随机分为1马来酸桂哌齐特组(pMCAO模型大鼠,n=57。尾静脉注射马来酸桂哌齐特3.0 mg·kg-1,×5d);2对照组(pMCAO模型大鼠,n=57。尾静脉注射生理盐水0.5 mL,×5 d);3假手术组(不插线栓,n=50。尾静脉注射生理盐水0.5 mL,×5 d);4正常组(n=3,不做任何处理)。应用TTC染色法测定梗死体积,蛋白印迹法和免疫组化法检测不同时间点(术后3、6、24、48和72 h)缺血半暗带区ERK蛋白的表达。结果马来酸桂哌齐特组与对照组比较,梗死体积减少17.91%(P=0.001)。马来酸桂哌齐特组缺血半暗带pERK1/2表达于3 h开始增加,24 h达高峰[为正常的(5.75±0.70)倍]后逐渐降低,72 h仍为正常的(2.89±0.51)倍,各时间点与正常组比较,均差异有显著统计学意义(P0.01)。6、24和48 h 3个时间点,马来酸桂哌齐特组缺血半暗带内pERK1/2表达较对照组增加(P0.05)。pERK的免疫组化检测示马来酸桂哌齐特预处理能上调缺血半暗带区内ERK磷酸化表达(P0.05)。结论马来酸桂哌齐特预处理可减少脑梗死体积,并能上调缺血半暗带区的ERK磷酸化表达;参与MAPK信号通路、上调缺血半暗带区内ERK磷酸化的表达,可能是其保护缺血性脑损伤的机制之一。     

实验性自身免疫性脑脊髓炎小鼠神经损害与脑组织中丝裂原活化蛋白激酶表达的关系

目的观察实验性自身免疫性脑脊髓炎(EAE)模型小鼠脑组织中丝裂原活化蛋白激酶(MAPKs)表达变化及其与神经损害的关系。方法 C57BL/6小鼠随机分为:EAE组(n=12),采用髓鞘少突胶质细胞糖蛋白35-55多肽(MOG35-55)制备成抗原乳剂免疫小鼠;对照组(n=10),用生理盐水处理小鼠。每日观察两组小鼠的行为学变化,并进行神经功能障碍评分。于高峰期处死小鼠,冰冻处理脑与脊髓,行苏木精-伊红染色观察脊髓组织的炎症细胞浸润,LFB染色观察脊髓组织的髓鞘脱失,蛋白印迹法检测小鼠脑组织中MAPKs表达。分析EAE小鼠神经功能障碍改变与中枢神经组织MAPKs表达量的相关性。结果 EAE组与对照组比较:日均神经行为学评分增加(P0.01);脊髓炎症细胞浸润增多(P0.001),髓鞘脱失增多(P0.001)。P-ERK(42)、P-ERK(44)、P-JNK(54)表达量均增多(P0.01、P0.05、P0.05)。神经功能障碍与P-ERK(42)、P-ERK(44)、P-JNK(54)表达呈正相关。结论 EAE高峰期神经损伤程度与中枢神经组织中的P-ERK(42)、P-ERK(44)、P-JNK(54)表达增加相平行,提示MOG35-55诱导的EAE中枢神经损伤可能与MAPKs所激活的信号通路有关。     

三七总皂甙通过细胞外信号调节蛋白激酶1/2信号通路促进大鼠

背景：三七总皂甙对骨髓基质细胞向神经样细胞分化的效应及相关信号通路目前尚不明确。 目的：探讨细胞外信号调节蛋白激酶1/2信号通路对三七总皂甙调节大鼠骨髓基质细胞向神经细胞增殖、分化的作用。 设计、时间及地点：细胞学体外观察,于2008-05/10在汕头大学医学院分子生物学实验室完成。 材料：4~6周龄雄性SD大鼠9只,由汕头大学医学院实验动物中心提供。三七总皂甙为广西梧州制药集团股份有限公司产品,细胞外信号调节蛋白激酶的抑制剂PD98059为Cell Signaling Tech 公司产品。 方法：全骨髓法体外分离纯化大鼠骨髓基质细胞,取传至第3代细胞进行诱导分化,设立3组：单纯诱导组向培养液中加入10 μg/L碱性成纤维细胞生长因子,预诱导24 h后全量换为正式诱导液(含10 μg/L碱性成纤维细胞生长因子、2% DMSO、200 μmol/L丁羟回醚的α-MEM培养基),每24 h半量换诱导液1次;三七总皂甙组向预诱导液和正式诱导液内加入终浓度100 mg/L三七总皂甙;PD98059组在三七总皂甙组培养液基础上加入10 μmol/L PD98059。 主要观察指标：细胞形态学观察,MTT法检测细胞增殖情况,免疫组织化学方法鉴定细胞Nestin的表达。 结果：诱导6 h后,少数细胞呈锥形,细胞突起增多伸长,类似神经元,继续诱导培养后细胞突起增多,突起相互连接成网状。与单纯诱导组、PD98059组比较,诱导6,12,24 h三七总皂甙组细胞均明显增殖(P < 0.05),且随诱导时间延长呈递增趋势(P < 0.05);单纯诱导组、PD98059组间比较无明显差异(P > 0.05)。与单纯诱导组比较,诱导24 h后三七总皂甙组Nestin阳性率明显升高(P < 0.05),PD98059组Nestin阳性率明显降低(P < 0.05)。 结论：三七总皂甙可以促进骨髓基质细胞的神经分化和分化后增殖,细胞外信号调节蛋白激酶1/2的特异性抑制剂PD98059能够拮抗三七总皂甙促增殖分化作用,提示细胞外信号调节蛋白激酶1/2信号通路是三七总皂甙促进骨髓基质细胞向神经细胞分化和增殖的重要环节。     

隐源性难治性颞叶癫疒间患者脑内丝裂原活化蛋白激酶激酶激酶5基因表达的研究

目的 检测丝裂原活化蛋白激酶激酶激酶5(MAPKKK5)mRNA及其编码蛋白产物在隐源性难治性颞叶癫疒间(TLE)患者脑内的表达,从分子水平探讨难治性TLE可能的发病机制.方法 取10例隐源性难治性TLE患者手术切除的颞叶组织,10例脑外伤患者(病例对照组)相应部位颞叶组织,利用RT-PCR与Western blot方法检测两组患者MAPKKK5 mRNA (MAPKKK5 mRNA/β-actin)与MAPKKK5蛋白(MAPKKK5/β-actin)的表达.结果 难治性TLE患者颞叶组织MAPKKK5 mRNA (1.001±0.321) 和MAPKKK5蛋白(0.359±0.299)的表达水平均上调,与病例对照组的(0.648±0.157)和(0.137±0.084)比较差异有统计学意义(均P<0.05).结论 MAPKKK5 基因表达上调可能参与难治性TLE的发病机制.     

Jiawei Wendan decoction affects mitogen-activated protein kinase signal pathway in the hippocampus of depression rats

A previous study from our group showed that Jiawei Wendan decoction inhibits protein expression of interleukin-1β, 2, and 6, as well as plasma neuropeptide Y, P substance and somatostatin in the hippocampus of depression rat models. The present study analyzed the influence of Jiawei Wendan decoction on the mitogen-activated protein kinase signal transduction pathway in the hippocampus. Results demonstrated that Jiawei Wendan decoction effectively upregulated expression of small molecular G proteins, extracellular regulated kinase 1/2, and activated ribosomal S6 kinase protein in the rat hippocampus. In addition, Jiawei Wendan decoction exhibits antidepressant effects similar to fluoxetine. The underlying mechanisms were shown to be dependent on increased mitogen-activated protein kinase signal transduction pathway activity.     

电针可影响脑缺血再灌注大鼠抗氧化应激MAPK信号的转导

针刺脑缺血再灌流模型大鼠 “百会”和“大椎”穴后,可见脑皮质及纹状体组织ERK表达增加,血清谷胱甘肽还原酶、谷胱甘肽过氧化物酶活性与血清谷胱甘肽含量上调,脑缺血再灌流大鼠神经行为学评分改善,应用MAPK阻滞剂PD98059可拮抗上述作用。说明电针能逆转脑缺血再灌流所造成自由基连锁反应及氧应激损伤,产生脑神经元的保护作用,MAPK信号通路可能是电针抗氧应激及氧化还原敏感信号的重要转导途径之一。     

淋巴细胞特异性酪氨酸蛋白激酶基因多态性与阿尔茨海默病的相关性研究

目的探讨淋巴细胞特并性酪氨酸蛋白激酶（LCK）基因多态性、载脂蛋白E（apoE）基因多态性和阿尔茨海默病（AD）的相关性。方法用TaqMan—PCR法检测单核苷酸多态性（SNP）,对380例日本人AD患者[包括327例迟发型AD（LOAD）与53例早发型AD（EOAD）]和380例非痴呆对照纽中。观察LCK基因及apoE基因的多态性分布,并分析其与AD的相关性。结果（1）LCK基因＋6424A/G多态位点的G／G基因型息AD风险为非G／G型的1．41倍（95％CI=1．06～1．87）,患LOAD风险为1．37倍（95％CI=1．02～1．85）;（2）apoEε4基因携带者患AD风险为非apoEε4基因携带者的5．11倍（95％CI=3．63～7．19）;（3）排除apoE基因型对AD风险的影响后,G／G基因型患AD风险为非G／G型的1．66倍（95％CI=1．16～2．38）,患LOAD风险为1．64倍（95％CI=1．12～2．40）,同时风险等位基因G与AD（P〈0．05）和LOAD（P〈0．05）具有相关性。结论LCK基因＋6424A／G多态位点与AD风险的增加呈正性相关性,apoEε4增加了AD的发病风险,LCK是独立于APOE基因的又一新的风险基因。     

Non-photic phase shifting of the circadian clock: role of the extracellular signal-responsive kinases I/II/mitogen-activated protein kinase pathway

The master circadian clock, located in the suprachiasmatic nucleus (SCN), is synchronized to the external world primarily through exposure to light. A second class of stimuli based on arousal or activity can also reset the hamster circadian clock in a manner distinct from light. The mechanism underlying these non-photic phase shifts is unknown, although suppression of canonical clock genes and immediate early genes has been implicated. Recently, suppression of one of the mitogen-activated protein kinases (MAPK), namely extracellular signal-responsive kinases I/II (ERK), has been implicated in phase shifts to dark pulses, a stimulus with both photic and non-photic components. We investigated the involvement of the ERK/MAPK pathway in phase shifts in response to 3 h of sleep deprivation initiated at mid-day. About three-quarters of animals subjected to this procedure demonstrated large phase advances of about 3 h. Those that shifted exhibited a significant decrease in phosphorylated ERK (p-ERK) in the SCN. Those animals that were perfused during the sleep deprivation also exhibited immunoreactivity for p-ERK in a distinct portion of the ventrolateral SCN. Finally, injections of U0126 to the SCN to prevent phosphorylation of ERK significantly decreased levels of p-ERK but did not produce phase shifts. These data demonstrate that a purely non-photic manipulation is able to alter the activity of the MAPK pathway in the SCN, with downregulation in the SCN shell and activation in a portion of the SCN core.     

Regulation of the phosphoinositide-3 kinase and mitogen-activated protein kinase signaling pathways by progesterone and its reduced metabolites in the rat brain

Several growth factors, such as vascular endothelial growth factor, brain-derived neurotrophic factor, and insulin-like growth factor-I are involved in the actions of progesterone in the central nervous system. Previous studies in neuronal and glial cultures have shown that progesterone may regulate growth factor signaling, increasing the phosphorylation of extracellular-signal regulated kinase (ERK) and the phosphorylation of Akt, components of the mitogen-activated protein kinase (MAPK) and the phosphoinositide-3 kinase (PI3K) signaling pathways, respectively. In this study, we have evaluated whether progesterone and its reduced metabolites, dihydroprogesterone and tetrahydroprogesterone, regulate PI3K and MAPK signaling in the brain of ovariectomized rats in vivo. Significant increases in the phosphorylation of ERK, in the expression of the catalytic (p110) and the regulatory (p85) subunits of PI3K and in the phosphorylation of Akt were observed in the hypothalamus, the hippocampus, and the cerebellum 24 hr after progesterone administration. Progesterone metabolites partially mimicked the effect of progesterone and had a stronger effect on MAPK and PI3K signaling in the hypothalamus than in the other brain regions. These findings suggest that progesterone regulates MAPK and PI3K signaling pathways in the central nervous system in vivo by direct hormonal actions and by mechanisms involving progesterone metabolites.     

Weighted gene co-expression network analysis identifies specific modules and hub genes related to subsyndromal symptomatic depression

Abstract

Objectives: The identification of the potential molecule targets for subsyndromal symptomatic depression (SSD) is critical for improving the effective clinical treatment on the mental illness. In the current study, we mined the genome-wide expression profiling and investigated the novel biological pathways associated with SSD.

Methods: Expression of differentially expressed genes (DEGs) were analysed with microarrays of blood tissue cohort of eight SSD patients and eight healthy subjects. The gene co-expression is calculated by WGCNA, an R package software. The function of the genes was annotated by gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.

Results: We identified 11 modules from the 9,427 DEGs. Three co-expression modules (blue, cyan and red) showed striking correlation with the phenotypic trait between SSD and healthy controls. Gene ontology and KEGG pathway analysis demonstrated that the function of these three modules was enriched with the pathway of inflammatory response and type II diabetes mellitus. Finally, three hub genes, NT5DC1, SGSM2 and MYCBP, were identified from the blue module as significant genes.

Conclusions: This first blood gene expression study in SSD observed distinct patterns between cases and controls which may provide novel insight into understanding the molecular mechanisms of SSD.     

5-羟色胺2A受体基因多态性与抑郁症的关联

目的：探讨中国汉族人群难治性抑郁症患者与5-羟色胺2A(5-HT2A)受体基因的T102C多态性之间的关系。方法：抽取79例难治性抑郁症患者作研究，以102名正常人作对照。应用聚合酶链式反应(PCR)扩增技术及限制性片段长度多态性(RFLP)分别测定所有研究对象的5-HT2A受体基因的基因型和等位基因。结果：5-HT2A受体基因的3种基因型(A1／A1，A1／A2和A2／A2)在难治性抑郁症组的分布分别为31．6％、54．4％和13．9％，在对照组分别为29．4％、45．1％和25．5％，两组间差异无显著性。结论：5-HB。受体基因的T102C多态性与难治性抑郁症之间无显著关联。     

5-羟色胺2C受体基因多态性与抑郁症的关联

目的：探讨5-羟色胺2C（5-HT2C）受体基因-759C/T多态性与抑郁症发病机制之间的关系。方法：50例抑郁症患者作为研究组,30名正常者作为对照组,应用聚合酶链式反应（PCR）扩增技术及限制性片段长度多态性（RFLP）分别测定所有受试者的5-HT2C受体基因的基因型和等位基因,由于5-HT2C基因位于X性染色体,故对女性个体行基因鉴定,对男性患者只行半合子型鉴定。结果：与对照组相比,抑郁症组男性半合子型T、C分布频率分别为31.8%和68.2%,对照组为41.7%和58.3%,两组比较差异无显著性（χ^2=2.11,P〉0.05）。抑郁症组女性基因型TT、CC分布频率分别为46.4%和53.6%,对照组为16.7%和83.3%,无T、C杂合子基因型出现,两组比较差异有显著性（χ^2=20.4,P〈0.001）。抑郁症组T、C等位基因分布频率分别为42.3%和57.7%,对照组为22.9%和77.1%,两组比较差异有显著性（χ^2=8.72,P〈0.01）。结论：5-HT2C受体基因启动子区-759C/T单核苷酸置换多态性与女性伴自杀行为抑郁症的发病机制可能存在相关性,而男性患者的发病可能与此无关。