丁基苯酞对大鼠脑缺血再灌注损伤后Bcl-2表达的影响

[目的]探讨丁基苯酞(NBP)对大鼠脑缺血再灌注损伤后Bcl-2蛋白和mRNA表达的影响。[方法]利用大脑中动脉线栓法将♂SD大鼠建立局灶性缺血再灌注模型,随机分为假手术组(不阻断大脑中动脉)、脑缺血再灌注组(I/R组)和NBP治疗组(NBP组),每组20只大鼠;脑缺血2h后开始再灌注。NBP组每天2次灌胃NBP,每次25mg/kg,I/R组及假手术组灌胃相同剂量食用植物油。再灌注72h后行神经功能缺损评分,然后断头取脑,TTC染色观察脑梗死体积,免疫组织化学(SP)方法检测梗死核心区、梗死周围区、海马区Bcl-2蛋白的表达,原位杂交方法(POD法)检测Bcl-2mRNA的表达。[结果]NBP组较I/R组大鼠脑梗死体积小(P﹤0.05),神经功能缺损改善(P﹤0.05);梗死核心区NBP组和I/R组Bcl-2蛋白和mRNA表达差异无统计学意义(P﹥0.05),梗死周围区和海马区NBP组Bcl-2蛋白和mRNA表达均高于I/R组和假手术组(P﹤0.05)。[结论]NBP减少脑缺血再灌注后大鼠脑梗死体积,改善神经功能,促进大鼠脑梗死周围区和海马区Bcl-2蛋白和mRNA的表达,可能为NBP抗凋亡和保护缺血脑组织的机制之一。     

Glutathione conjugation and bacterial mutagenicity of racemic and enantiomerically pure cis-and trans-methyl epoxycinnamates

This paper describes the ability of racemic, and enantiomerically pure cis-and trans-methyl epoxycinnamates (methyl 3-phenyl-2,3-epoxy-propanoates) to undergo glutathione conjugation and subsequent excretion as mercapturic acid and on the mutagenicities of these epoxy esters in the Ames assay. In incubation mixtures containing rat liver cytosol (9,000 g), the decrease of glutathione due to the epoxy esters occurred enzymatically. The highest glutathione depletion was found for the cis-epoxy cinnamic esters. Adult male rats administered a single i.p. dose of racemic trans- and cis-epoxy cinnamates (0.7 mmol/kg, n = 4) excreted thioethers in urine. Higher urinary thioether excretion was found after the cis-epoxy ester dosing. The structures of the thioether metabolites isolated from the urinary extracts were identified by TLC and confirmed by synthesis and mass spectrometry (FAB⁺). The thioethers appeared to be hydroxy mercapturic acids. The N-alkylating potential of the racemic epoxy esters was determined using 4-(p-nitrobenzyl)pyridine (=NBP). The trans-epoxy ester appeared to react much better with NBP than the cis-compound. Mutagenic effects of racemic trans-epoxy cinnamate as well as the enantiomerically pure trans-epoxy cinnamates were observed in the Ames test with S.typhimurium strains TA1535, TA1537, TA1538 and TA100 without metabolic activation. No mutagenic responses were detected using any of the epoxy cinnamates with S9 activation. By comparing the mutagenicity and the enzymatically catalyzed glutathione conjugation it follows that the activity of the respective enantiomeric methyl cinnamates goes in the opposite order. Glutathione conjugation plays a protective role in the detoxication in living organism of the potentially toxic methyl epoxy cinnamates.     

Reactions of the trifunctional nitrogen mustard tris(2-chloroethyl)-amine (HN3) with human erythrocyte membranes in vitro.

Covalent labeling of membrane proteins and cytoplasmic components in washed human erythrocytes by the alkylating antitumor agent HN3 has been studied in vitro. Using ¹⁴C-labeled HN3 and SDS-polyacrylamide-gel-electrophoresis for separation of the reaction products the following results have been obtained: (1) The membrane protein spectrin (bands 1,2) disappeared from the SDS-polyacrylamide-gel-electrophoresis pattern. Formation of proteins with higher molecular weight suggested cross-linking of spectrin to itself and to other proteins from membrane and cytoplasm. (2) The carbohydrate containing protein glycophorin (PASI) and the protein in band 7 were labeled by the compound. Other membrane proteins reacted to a lower extent. (3) The alkylating agent penetrated the membrane and reacted with hemoglobin in the cytoplasm. The formation of globin dimers was detected.     

丁基苯酞抗大鼠大脑皮质神经元氧糖剥夺/复氧损伤及其机制

本研究探讨丁基苯酞抗大鼠大脑皮质神经元氧糖剥夺/复氧损伤及其机制。原代培养大鼠大脑皮质神经元，建立氧糖剥夺/复氧模型(OGD/R)，采用MTT法、酶学检查、免疫组化、RT-PCR等观察丁基苯酞(各浓度组)的保护作用及其机制。在氧糖剥夺4 h/复氧8 h时丁基苯酞各浓度组可增加神经元的细胞活力和减少神经元LDH(乳酸脱氢酶)的释放，可显著降低神经元表达iNOS mRNA(诱生型一氧化氮合酶)和NF-κB(核因子κB) p65蛋白(增加)。不同剂量丁基苯酞(100、 10、 1和0.1 μmol·L^-1)在增加细胞活力、减少LDH释放及降低神经元表达iNOS mRNA等方面，高浓度的作用强于低浓度，且丁基苯酞100 μmol·L^-1组与吡咯烷二硫代氨基甲酸酯(pyrrolidine dithiocarbamate，PDTC) 100 μmol·L^-1组差异显著。在OGD 4 h/R 8 h时丁基苯酞可能抑制iNOSmRNA的表达及NF-κB的活化，从而有效保护氧糖剥夺/复氧中损伤的大脑皮质神经元。     

氨丁苯酞对血小板聚集功能的影响

目的　观察氨丁苯酞 (GZ 0 2 ) ,丁苯酞 (NBP)的结构衍生物对血小板聚集功能的影响。方法　参照Tamura法 ,观察GZ 0 2对局部脑缺血大鼠血小板聚集率的影响 ,并用比蚀法测定了GZ 0 2体内和体外给药对AA、ADP和胶原诱导的血小板聚集率的影响。结果　GZ 0 2能明显降低局部脑缺血大鼠血小板聚集率的异常增高 ,体内体外条件下均能明显抑制AA诱导的血小板聚集。结论　实验证明GE 0 2具有抑制血小板聚集功能的作用     

丁苯酞对血管性痴呆小鼠认知功能的影响及Nrf2/SIRT3信号通路的调节作用

目的探讨丁苯酞对血管性痴呆(vascular dementia,VD)小鼠认知功能及Nrf2/SIRT3信号通路中的调节作用。方法将36只野生型小鼠(Nrf2^+/+)按照随机数字表法分为假手术组(Sham组)、模型组(Nrf2^+/+VD组)、丁苯酞治疗组(Nrf2^+/+NBP组),每组12只。将24只Nrf2基因敲除小鼠(Nrf2^-/-)按照随机数字表法分为Nrf2^-/-模型组(Nrf2^-/-VD组)和Nrf2^-/-治疗组(Nrf2^-/-NBP组),每组12只。采用反复三次结扎小鼠双侧颈总动脉的方法建立脑缺血再灌注致认知功能障碍的小鼠模型;应用Morris水迷宫实验检测小鼠的认知功能,HE染色观察海马CA1区神经元形态结构的变化,免疫组化分析小鼠海马CA1区caspase-3、caspase-9的阳性表达,Western blot检测小鼠海马Nrf2、p62、LC3、SIRT3的蛋白表达。结果(1)Morris水迷宫实验中:与VD组小鼠相比,Sham组与Nrf2^+/+NBP组小鼠第5天逃避潜伏期明显缩短[(20.69±8.91)s,(7.58±9.47)s,(8.41±12.20)s;q=3.58,5.07,均P<0.05],目标象限停留时间百分比明显增加[(16.80±3.27)%,(25.25±5.51)%,(24.18±6.46)%;q=3.36,4.43,均P<0.05];与VD组比较,Nrf2^-/-VD组小鼠第5天逃避潜伏期明显延长[(33.71±9.05)s],目标象限停留时间百分比明显降低[(10.84±3.26)%],均差异有统计学意义(q=3.56,3.58;均P<0.05)。与Nrf2^-/-VD组比较,Nrf2^-/-NBP组小鼠逃避潜伏期、目标象限停留时间百分比均差异无统计学意义(均P>0.05)。(2)病理学结果显示:与VD组小鼠比较,Sham组与Nrf2^+/+NBP组小鼠海马CA1区锥体神经元形态结构损伤更轻,Nrf2^-/-VD组的损伤更为严重,且经NBP治疗后神经元形态改善并不明显。(3)凋亡蛋白的表达:与VD组相比,Sham组与Nrf2^+/+NBP组小鼠海马CA1区caspase-3、caspase-9表达均降低(t=3.48,2.95,3.46,2.93,均P<0.01),而Nrf2^-/-VD组的表达均增加(t=-2.99,-3.77,均P<0.01);同Nrf2^-/-VD组小鼠比较,Nrf2^-/-NBP组小鼠海马CA1区caspase-3、caspase-9的表达差异无统计学意义(均P>0.05)。(4)Western blot结果显示:与VD组相比,Nrf2^+/+NBP组小鼠海马Nrf2、SIRT3、p62蛋白表达增加,LC3Ⅱ/Ⅰ比值降低(t=-3.24,-4.04,-4.03,3.62,均P<0.01);Sham组Nrf2表达、LC3Ⅱ/Ⅰ比值降低,SIRT3、p62表达增加(t=3.44,4.72,-3.92,-4.19,均P<0.01);Nrf2^-/-VD组Nrf2、SIRT3、p62蛋白表达降低,LC3Ⅱ/Ⅰ比值升高(t=9.14,4.20,4.30,-3.78,均P<0.01);同Nrf2^-/-NBP比较,Nrf2^-/-VD组Nrf2、SIRT3、p62蛋白表达降低,LC3Ⅱ/Ⅰ比值升高(t=2.40,3.24,1.21,-1.16,均P<0.01);Nrf2^+/+NBP组Nrf2、SIRT3、p62蛋白表达升高,LC3Ⅱ/Ⅰ比值降低(t=-3.29,-5.00,-7.12,6.24,均P<0.01)。结论丁苯酞可以减轻VD小鼠海马区的凋亡损伤,改善反复缺血再灌注损伤所致的认知功能障碍,其机制可能与调控Nrf2/SIRT3通路、抑制海马区神经细胞凋亡、自噬有关。     

丁苯酞对SOD1~(G93A)转基因鼠脊髓Ferritin表达的影响

目的探讨丁苯酞对SOD1G 93A转基因鼠的神经保护作用及其可能的机制。方法选取SOD1G 93A阳性小鼠48只,随机分为丁苯酞组(发病早期及终末期)和安慰剂组(发病早期及终末期),每组12只,观察SOD1G 93A转基因鼠的发病时间和生存期,采用免疫组化和蛋白印迹的方法观察SOD1G 93A转基因鼠腰髓铁蛋白(Ferritin)表达。结果丁苯酞组的发病时间为118.0±2.5d,与安慰剂组的109.5±2.2d相比较,P<0.05;生存时间为138.6±2.3d,与安慰剂组的128.1±2.8d相比较,P<0.05;免疫组化显示:发病初期,安慰剂组Ferritin阳性细胞显示胞浆和胞膜周围均匀着色,丁苯酞组Ferritin仅胞膜周围着色,胞核和胞浆着色不明显;终末期,丁苯酞组与安慰剂组相比,二者的Ferritin在胞核和胞浆均有着色;蛋白印迹显示:发病初期,Ferritin在腰髓表达量的相对值,丁苯酞组为0.81±0.23,与安慰剂组的1.57±0.43相比较,P<0.05;终末期,丁苯酞组为1.45±0.45,与安慰剂组的1.73±0.36相比较,P>0.05。结论丁苯酞延迟ALS小鼠的发病时间并延长其生存期,丁苯酞延迟小鼠发病可能与调节铁代谢有关。     

The astrocyte network in the ventral nerve cord neuropil of the Drosophila third-instar larva

Understanding neuronal function at the local and circuit level requires understanding astrocyte function. We have provided a detailed analysis of astrocyte morphology and territory in the Drosophila third-instar ventral nerve cord where there already exists considerable understanding of the neuronal network. Astrocyte shape varies more than previously reported; many have bilaterally symmetrical partners, many have a high percentage of their arborization in adjacent segments, and many have branches that follow structural features. Taken together, our data are consistent with, but not fully explained by, a model of a developmental growth process dominated by competitive or repulsive interactions between astrocytes. Our data suggest that the model should also include cell-autonomous aspects, as well as the use of structural features for growth. Variation in location of arborization territory for identified astrocytes was great enough that a standardized scheme of neuropil division among the six astrocytes that populate each hemi-segment is not possible at the third instar. The arborizations of the astrocytes can extend across neuronal functional domains. The ventral astrocyte in particular, whose territory can extend well into the proprioceptive region of the neuropil, has no obvious branching pattern that correlates with domains of particular sensory modalities, suggesting that the astrocyte would respond to neuronal activity in any of the sensory modalities, perhaps integrating across them. This study sets the stage for future studies that will generate a robust, functionally oriented connectome that includes both partners in neuronal circuits—the neurons and the glial cells, providing the foundation necessary for studies to elucidate neuron–glia interactions in this neuropil.     

丁苯酞对脑缺血再灌注大鼠HSP70及TLR4的影响研究

目的：研究丁苯酞预处理对脑缺血再灌注大鼠细胞凋亡及HSP70及TLR4表达的影响。方法：将144只实验大鼠随机分成：假手术组（ Sham组）、缺血再灌注组（ IR组）、给予NBP干预的缺血再灌注组（ NBP组）各48只,各组再根据缺血2 h后再灌注的时间6 h、12 h、24 h和48 h分为4个小组,每小组各12只。通过Longa改良线栓法制备大鼠缺血再灌注模型,NBP组待大鼠缺血再灌注模型建立后立即予NBP灌胃,IR及Sham组作为对照组,予等量的生理盐水灌胃。比较不同组大鼠神经功能缺损评分、细胞凋亡数、HSP70及TLR4表达的差异。结果：再灌注时间为12 h、24 h和48 h时,NBP组的神经功能缺损评分均低于IR组（P均＜0.05）;在任意灌注时间段内,IR、NBP组的凋亡细胞数均显著高于Sham组（P均＜0.01）,NBP组的细胞凋亡数均显著低于IR组（P均＜0.01）;在任意灌注时间段内,NBP、IR组的HSP70和TLR4阳性细胞数均显著高于Sham组（ P均＜0.01）;与IR组相比, NBP组的HSP70和TLR4的表达量显著降低（ P均＜0.01）。结论：NBP可以通过抑制HSP70和TLR4的表达量,改善细胞的凋亡数和机体炎症反应,从而起到保护神经功能的作用。     

脑红蛋白在脑梗死大鼠表达及丁苯酞干预效应

目的探讨脑红蛋白在脑梗死大鼠中的表达和丁苯酞干预在氧化应激损伤中作用。方法将90只雄性SD大鼠随机分为假手术组、模型组、治疗组。每组再分为3个亚组:1 d组、3 d组和7 d组,每亚组10只。模型组和治疗组采用线栓法制作大鼠大脑中动脉闭塞模型(MCAO),假手术组只分离不结扎。治疗组在动物苏醒后以丁苯酞植物油灌胃,假手术组和模型组同法给予等量植物油灌胃。每只大鼠在术后3h和处死前行神经功能评分,应用RT-PCR和免疫组化检测脑组织脑红蛋白(NGB)表达,化学比色法检测超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量。结果 (1)神经功能评分,模型组和治疗组术后3 h评分差异无统计学意义(P>0.05),各时间点处死前评分差异均有统计学意义(P<0.05)。(2)NGB mRNA和免疫组化表达随时间延长逐渐降低,各时间点模型组较假手术组、治疗组较模型组高,差异均有统计学意义(P<0.05)。(3)SOD活性和MDA含量随时间延长逐渐减少。SOD活性假手术组较治疗组、治疗组较模型组高,差异均有统计学意义(P<0.05);MDA含量假手术组较治疗组、治疗组较模型组低,差异均有统计学意义(P<0.05)。结论丁苯酞促进脑缺血大鼠脑红蛋白表达,减少氧化应激损伤。