创伤后应激障碍样行为异常大鼠海马钙依赖性反应紊乱

目的探讨情感行为异常大鼠海马钙离子及其相关的钙依赖性反应,进一步认识创伤后应激障碍(PTSD)样行为异常的神经生物学基础。方法将240只雄性Wistar大鼠随机分组为海马阈下电刺激组(SE,n=96)、海马电极埋植对照组(CE,n=96)和正常对照组(NC,n=48),采用频率25Hz、波宽1ms、串长10s、串隔7min、强度100μA的恒流、单脉冲电流,反复刺激大鼠海马以建立PTSD样行为异常动物模型;采用神经生化、流式细胞仪、荧光标记术及Westernblotting等方法,定量观测了实验大鼠海马Na+-K+-ATP酶与Ca2+-ATP酶活性,细胞内游离钙离子含量与钙调素(CaM)相对活性平均通道荧光,以及海马组织总CaM表达的动态变化规律。结果电刺激停止后12h阈下刺激大鼠海马细胞线粒体Na+-K+-ATP酶活性即明显下降犤(0.56±0.17)mmol/(kg·s),F=4.438,P<0.05犦,48h仍显著低于NC组犤(0.61±0.17)mmol/(kg·s),P=0.026犦;24h线粒体Ca2+-ATP酶活性亦明显降低犤(0.53±0.14)mmol/(kg·s),F=4.999,P<0.05犦,72h仍显著低于NC组犤(0.61±0.17)mmol/(kg·s),P=0.027犦。海马细胞内游离钙离子含量于电刺激停止后12～48h明显高于两对照组(F=16.355,P<0.01),72h仍显著高于NC组犤(290±70)nmol/L,P=0.03犦,游离CaM平均通道荧光则同步降低(F=10.655,P<0.05),而海马组     

氯丙嗪对大鼠牙齿矿化抑制作用的实验研究

目的：探讨氯丙嗪(chlorpromazine,CPZ)对大鼠切牙硬组织矿化的影响。方法;采用酶联反应仪及高压液相色谱仪（HPLC）检测CPZ对培养不同时间牙胚ALP及Ca^2 的含量,利用显微放射照相技术观察CPZ对大鼠硬组织矿化的情况。结果：培养0-7d牙胚Ca^2 含量及ALP逐渐上升,培养7-10d的牙胚中,实验组Ca^2 含量及ALP活性的上升幅度显著减弱;CPZ剂量依赖性地抑制大鼠牙本质形成,并且对血浆中Ca、P无明显影响。结论：CPZ对大鼠牙齿矿化过程具有抑制作用。该作用是通过成牙本质细胞的钙调节系统实现的。     

硬组织替代材料对成骨细胞内游离钙及钙调素水平的影响

目的;研究羟基聚磷酸钙钠（ＨＰＡ）、羟基磷灰石（ＨＡ）、生物玻璃陶瓷（ＢＧＣ）和纯钛（Ｔｉ）４种硬组织替代材料对成骨细胞的细胞内游离钙（〖Ｃａ＾２＋〗ｉ）浓度、钙调素（ＣａＭ）含量以及细胞膜ＡＴＰ酶活性的影响,探讨这４处材料的生物相容性。方法：选用ＳＤ乳鼠颅顶骨皮骨细胞进行体外培养,把ＨＰＡ、ＨＡ、ＢＧＣ和Ｔｉ粉分别制成材料浸提液,将培养成活的成骨细胞接种于材料浸提液中培养,３ｄ后测定成骨细胞内（     

Ca2+/calmodulin‐dependent protein kinase II in spinal dorsal horn contributes to the pain hypersensitivity induced by γ‐aminobutyric acid type a receptor inhibition

The fast inhibitory synaptic transmission mediated by the γ‐aminobutyric acid type A receptor (GABA_AR) within spinal dorsal horn exerts a gating control over the synaptic conveyance of nociceptive information from the periphery to higher brain regions. Although a large body of evidence has demonstrated that the impairment of GABAergic inhibition alone is sufficient to elicit pain hypersensitivity in intact animals, the underlying mechanisms remain to be characterized. The present study shows that Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) is an important signaling protein downstream of reduced GABAergic inhibition. We found that pharmacological removal of inhibition by intrathecal application of the GABA_AR antagonist bicuculline significantly enhanced the autophosphorylation of CaMKII at Thr286 in spinal dorsal horn of mice. In addition to increased CaMKII activity, bicuculline also promoted CaMKII interaction with N‐methyl‐D‐aspartate (NMDA)‐subtype glutamate receptors and induced the translocation of CaMKII from cytosolic compartments to the synaptosomal membrane fraction. Immunoblotting analysis revealed that the phosphorylation levels of NMDA receptor NR2B subunit at Ser1303 and of AMPA‐subtype glutamate receptor GluR1 subunit at Ser831, two important CaMKII phosphorylation sites, were substantially enhanced after bicuculline application. Behavioral tests illustrated that intrathecal administration of the CaMKII inhibitor KN‐93, NMDA receptor antagonist D‐APV, or AMPA receptor antagonist GYKI 52466 effectively ameliorated the mechanical allodynia evoked by bicuculline. These data thus indicate that CaMKII signaling is critical for the reduced inhibition to evoke spinal sensitization. © 2013 Wiley Periodicals, Inc.     

脑血栓形成急性期患者血小板静息及激活状态胞浆游离钙离子浓度钙调素含量的实验研究

目的探讨血小板静息状态胞浆游离钙离子浓度([Ca2 ]i)、激活状态胞浆游离钙离子浓度([Ca2 ]ic)、钙调素含量 (CaM)在脑血栓形成(CT)急性期的变化及其作用。方法应用Fura-2荧光标记示踪法及酶联免疫法(ELISA)测定了31例CT急性期患者血小板[Ca2 ]i、[Ca2 ]ic浓度和CaM含量。结果脑血栓形成急性期患者血小板[Ca2 ]i、[Ca2 ]ic、CaM含量分别为(152．25 ±30．57)nmol／L,(200．36±33．62)nmol／L,(441．67±209．31)fg／102个血小板;正常对照组含量分别为(111．31±21．85)nmol／L, (158．43±22．44)nmol／L,(301．88 ±218．10)fg／102个血小板,脑血栓形成急性期患者血小板[Ca2 ]i、[Ca2 ]ic、CaM含量均明显高于正常对照(P<0．01,P<0．05)。结论脑血栓形成急性期患者,应用钙拮抗剂降低血小板胞浆钙离子浓度,可抑制血小板功能。     

Inhibition of calcium/calmodulin‐dependent protein kinase kinase (CaMKK) exacerbates impairment of endothelial cell and blood–brain barrier after stroke

Brain microvascular endothelial cells play an essential role in maintaining blood–brain barrier (BBB) integrity, and disruption of the BBB aggravates the ischemic injury. CaMKK (α and β) is a major kinase activated by elevated intracellular calcium. Previously, we demonstrated that inhibition of CaMKK exacerbated outcomes, conversely, overexpression reduced brain injury after stroke in mice. Interestingly, CaMKK has been shown to activate a key endothelial protector, sirtuin 1 (SIRT1). We hypothesized that CaMKK protects brain endothelial cells via SIRT1 activation after stroke. In this study, Oxygen‐Glucose Deprivation (OGD) was performed in human brain microvascular endothelial cells. Stroke was induced by middle cerebral artery occlusion (MCAO) in male mice. Knockdown of CaMKK β using siRNA increased cell death following OGD. Inhibition of CaMKK β by STO‐609 significantly and selectively down‐regulated levels of phosphorylated SIRT1 after OGD. Changes in the downstream targets of SIRT1 were observed following STO‐609 treatment. The effect of STO‐609 on cell viability after OGD was absent, when SIRT1 was concurrently inhibited. We also demonstrated that STO‐609 increased endothelial expression of the pro‐inflammatory proteins ICAM‐1 and VCAM‐1 and inhibition of CaMKK exacerbated OGD‐induced leukocyte‐endothelial adhesion. Finally, intracerebroventricular injection of STO‐609 exacerbated endothelial apoptosis and reduced BBB integrity after 24‐hr reperfusion following MCAO in vivo. Collectively, these results demonstrated that CaMKK inhibition reduced endothelial cell viability, exacerbated inflammatory responses and aggravated BBB impairment after ischemia. CaMKK activation may attenuate ischemic brain injury via protection of the microvascular system and a reduction in the infiltration of pro‐inflammatory factors.     

晶状体主要内源性蛋白MIP的突变体致先天性白内障的发生机制

目的　研究晶状体主要内源性蛋白（ｍａｊｏｒｉｎｔｒｉｎｓｉｃｐｒｏｔｅｉｎ,ＭＩＰ）Ｒ２３３Ｋ突变体的产生是否影响ＭＩＰ与钙调蛋白的相互作用,继而阐述此突变致先天性白内障的发病机制。方法　构建连有ＥＧＦＰ的ＭＩＰ野生型及Ｒ２３３Ｋ突变体表达质粒,分别转染Ｈｅｌａ细胞系,确定Ｒ２３３Ｋ突变体的亚细胞定位。用免疫共沉淀技术来检测Ｒ２３３Ｋ突变体与钙调蛋白的相互作用。合成野生型及突变型ＭＩＰ羧基末端多肽链,用荧光素报告基因技术来验证这些多肽链的功能。结果　在ＥＧＦＰ的显影下,野生型及Ｒ２３３Ｋ突变体均定位于细胞膜上,ｐ．Ｒ２３３Ｋ突变未影响ＭＩＰ的亚细胞定位及其在细胞内的蛋白表达量。而免疫共沉淀实验显示,与野生型ＭＩＰ相比,位于羧基末端的Ｒ２３３Ｋ突变体与钙调蛋白的相互作用明显减弱。荧光素报告基因实验证实,突变后的ＭＩＰ与钙调蛋白的绑定亲和力明显下降。结论　Ｒ２３３Ｋ突变体影响了ＭＩＰ与钙调蛋白的相互作用,降低了ＭＩＰ羧基末端与钙调蛋白的绑定亲和力,继而影响了晶状体纤维细胞的水通透性,导致了先天性白内障的发生。     

Ca2+–Calmodulin–Calcineurin Signaling Modulates α-Synuclein Transmission

Background

The intercellular transmission of pathogenic proteins plays a crucial role in the progression of neurodegenerative diseases. Previous research has shown that the neuronal uptake of such proteins is activity-dependent; however, the detailed mechanisms underlying activity-dependent α-synuclein transmission in Parkinson's disease remain unclear.

Objective

To examine whether α-synuclein transmission is affected by Ca²⁺–calmodulin–calcineurin signaling in cultured cells and mouse models of Parkinson's disease.

Methods

Mouse primary hippocampal neurons were used to examine the effects of the modulation of Ca²⁺–calmodulin–calcineurin signaling on the neuronal uptake of α-synuclein preformed fibrils. The effects of modulating Ca²⁺–calmodulin–calcineurin signaling on the development of α-synuclein pathology were examined using a mouse model injected with α-synuclein preformed fibrils.

Results

Modulation of Ca²⁺–calmodulin–calcineurin signaling by inhibiting voltage-gated Ca²⁺ channels, calmodulin, and calcineurin blocked the neuronal uptake of α-synuclein preformed fibrils via macropinocytosis. Different subtypes of voltage-gated Ca²⁺ channel differentially contributed to the neuronal uptake of α-synuclein preformed fibrils. In wild-type mice inoculated with α-synuclein preformed fibrils, we found that inhibiting calcineurin ameliorated the development of α-synuclein pathology.

Conclusion

Our data suggest that Ca²⁺–calmodulin–calcineurin signaling modulates α-synuclein transmission and has potential as a therapeutic target for Parkinson's disease. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.     

基于串联质谱标签定量蛋白质组学的痹祺胶囊治疗类风湿性关节炎的作用机制

目的 利用串联质谱标签（tandem mass tags,TMT）定量蛋白质组学技术研究痹祺胶囊治疗类风湿性关节炎（rheumatoid arthritis,RA）的作用机制。方法 建立II型胶原蛋白诱导的RA大鼠模型，并给予痹祺胶囊进行干预，取对照组（C）、模型组（M）、痹祺胶囊0.4 g/kg给药组（BQ）的左膝关节组织，采用TMT定量蛋白质组学技术分析鉴定各组大鼠膝关节组织中的蛋白，以表达差异倍数≥1.2或差异倍数≤0.83且P<0.05筛选M vs C、BQ vs M、BQ vs C差异表达蛋白，并分析经痹祺胶囊干预后有回调趋势的差异表达蛋白。最后通过GeneCards、OMIN数据库检索RA疾病靶点，与回调蛋白进行交集分析得到与RA疾病相关的回调蛋白，对其进行基因本体（gene ontology,GO）功能和京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes,KEGG）通路富集分析，明晰痹祺胶囊对RA的作用机制。结果 经痹祺胶囊干预后有121个差异蛋白具有回调趋势，其中38个与RA疾病相关，如β2整合素（integr...