天麻粉预防痴呆小鼠模型发病及其对抗氧化作用的影响

目的:研究长期灌胃天麻粉是否可以改善APP/PS1双转基因阿尔茨海默病(AD)模型小鼠学习记忆能力以及延缓AD进程,这些作用是否与调控Kelch样环氧氯丙胺相关蛋白1(Keap1)-核转录因子E2相关因子2(Nrf2)/血红素加氧酶-1(HO-1)抗氧化应激通路有关,进一步探讨天麻粉的神经保护作用机制以及对AD预防和治疗的作用。方法:APP/PS1双转基因小鼠模型,将实验小鼠分为5组,正常组,正常给药组,模型组,天麻粉预防组,天麻粉治疗组。正常给药组,天麻粉预防组在小鼠8周龄时每日灌胃同等剂量的天麻粉(1.5 g·kg^-1);正常组、模型组同时期给予等量的生理盐水,直至24周龄时行Morris水迷宫检测小鼠学习和记忆能力,天麻粉治疗组待小鼠22周龄时,连续2周每天灌胃同等剂量天麻粉(1.5 g·kg^-1),灌胃至24周龄时行Morris水迷宫检测小鼠穿越平台次数、逃避潜伏期及平台停留时间;提取小鼠海马组织RNA,实时荧光定量PCR(Real-time PCR)检测Nrf2,HO-1,Keap1的mRNA水平;蛋白免疫印迹法(Western blot)检测小鼠海马组织中Nrf2,HO-1,Keap1蛋白的表达水平。结果:与正常组比较,水迷宫检测结果显示,模型组小鼠的学习记忆能力显著降低(P<0.01);与模型组比较,天麻粉预防组、天麻粉治疗组小鼠学习记忆能力显著提高(P<0.01);与正常组比较,模型组小鼠海马组织中Nrf2,HO-1的mRNA及蛋白水平均有不同程度的降低(P<0.05,P<0.01),与模型组比较,天麻粉预防组小鼠海马组织中Nrf2,HO-1的mRNA及蛋白水平均有不同程度的增高(P<0.05,P<0.01),天麻粉治疗组Nrf2,HO-1 mRNA水平显著增高(P<0.01),Nrf2,HO-1蛋白表达水平变化差异无统计学意义,各组间Keap1的mRNA及蛋白水平变化无统计学差异。结论:Morris水迷宫及其他结果显示天麻粉能够改善APP/PS1小鼠的学习和记忆能力,且其可能通过调控Keap1-Nrf2/HO-1通路,增强下游抗氧化基因的表达发挥抗氧化功能,进而改善APP/PS1小鼠的学习记忆能力。     

慢性氟中毒大鼠大脑皮质神经细胞线粒体融合分裂基因转录水平观察

目的 观察慢性氟中毒大鼠大脑皮质神经细胞线粒体融合分裂基因mRNA含量和活性氧(ROS)水平,探讨其在氟中毒线粒体损伤中的作用.方法 选取SD大鼠120只,按体重随机分为3组:对照组、低氟组、高氟组,每组40只.在饮水中加入不同剂量的氟化钠,含氟量分别为＜0.5、10、50 mg/L,饲养3、6个月.采用线粒体ROS荧光探针法检测大鼠大脑皮质神经细胞线粒体ROS水平,实时荧光定量PCR法检测线粒体融合基因Mfn1及分裂基因Fis1、Drp1转录水平.结果 3个月时,与对照组[10.43±5.98、(3.4±0.6)×103、(8.8±1.4)×10、(1.2±0.2)×102]比较,低氟组大鼠大脑皮质神经细胞线粒体ROS水平(11.67±3.49)未见明显改变(P＞ 0.05),线粒体融合分裂基因Mfn1、Drp1、Fis1 mRNA表达水平未见明显改变[(3.1±0.3)×104、(6.7±2.7)×10、(5.0±0.9)×10,P均＞0.05];而高氟组大鼠大脑皮质神经细胞线粒体ROS水平(25.48±6.09)显著升高(P＜0.05),线粒体融合分裂基因Mfn1、Drp1、Fis1 mRNA表达水平[(1.0±0.2)×1011、(3.0±1.6)×103、(8.9±3.6)×102]明显升高(P均＜ 0.05).6个月时,与对照组[25.26±6.41、(3.0±0.8)×109、(5.1±0.8)×103、(2.8±0.7)×102]比较,低、高氟组大鼠大脑皮质神经细胞线粒体ROS水平(63.02±8.15、65.60±7.40)明显升高,线粒体融合基因Mfn1mRNA表达水平[(3.0±0.4)×106、(4.0±0.9)×104]显著降低,分裂基因Fis1、Drp1 mRNA表达水平[(2.0±0.8)×106、(4.0±0.6)×105,(3.8±1.3)×103、(1.3±0.2)×103]明显升高(P均＜0.05).结论 摄入过量的氟导致大鼠大脑皮质神经细胞ROS水平升高,线粒体融合基因转录降低、分裂基因转录升高,这种改变与染氟时间和剂量呈正相关.线粒体融合分裂基因转录水平改变可能与慢性氟中毒引起的线粒体氧化应激升高有关.     

慢性氟中毒性肝损伤及其发病机制

地方性氟中毒是一种在特定地理环境中发生的生物地球化学性疾病,除了引起氟斑牙和氟骨症外,还引起机体多个器官和组织发生病理损害[1-3].     

贵州苗族、布依族、侗族人群线粒体DNA 9 bp序列缺失频率分析

目的 调查贵州苗族、布依族、侗族人群线粒体DNA细胞色素氧化酶Ⅱ和t RNALys基因间小非编码Ⅴ区串联重复序列9 bp的缺失频率.方法 采用聚合酶链反应-聚丙烯酰胺凝胶方法及DNA序列分析法对105名苗族、97名布依族、102名侗族的男性个体线粒体DNA的9 bp缺失多态频率进行分析.结果 在304名男性中仅发现标准型和短型(即9 bp缺失)两种多态,缺失频率为23.0％(70/304).其中苗族缺失频率为28.6％(30/105)、布依族为26.8％(26/97)、侗族为13.7％(14/102).三者之间的差异具有统计学意义(P＜0.05).结论 贵州苗族、布依族、侗族人群线粒体DNA 9 bp缺失基因频率均较高,三者之间差异有统计学意义.     

贵州省地方性氟中毒地区人群GSTP1基因Ile105Val位点多态性分析

Objective To investigate plasma glutathione S-transferase(GSTs) activity and GSTP1 gene Ile105Val polymorphism in Bijie City, Guizhou Province, a coal-burning fluorosis endemic area. Methods One hundred and sixty villagers from Yachi Twon using non-improved cooking stoves were selected as the non-intervened group in Bijie City, Guizhou Province where coal-burning fluorosis was prevailing; 153 villagers as the intervented group were chosen from Changchun Twon, where cooking stoves were improved; 151 villagers were served as the control group from Baiyunshan Twon, Changshun County without endemic fluorosis. The activity of GSTs was tested by colorimetric analysis with spectrophotometer. The genotype of the GSTP1 gene Ile105Val polymorphism, presenting as either homozygous wild-type (AA), or heterozygous mutation type (AG), or homozygous mutation type (GG), was detected through the PCR-RFLP procedure. Results The activity of GSTs in plasma of non-intervened group [(12.44±4.97) kU/L]was significantly lower than that of intervened group (P < 0.05), and that of intervened group[(20.78±6.20)kU/L]was significantly lower than that of control group[(24.30±6.27)kU/L, P< 0.05]. The difference of the enzyme activity of three groups were statistically significant (F = 51.71, P < 0.05), but this enzyme activity did not vary significantly in each sex of each grnup(P > 0.05). Compared intervened group [AA:67.3%(103/153), AG:29.4%(45/153),GG:3.3%(5/153)]and non-intervened group[AA:66.9%(107/160), AG:30%(48/160), GG:3.1%(5/160)]with control group[AA:74.8%(113/151), AG:25.2%(38/151), GG:0 (0/151)], the Ile105Val polymorphism site of GSTP1 gene had significant difference(χ2= 6.04,6.07, both P< 0.05), but not significant between intervened and non-intervened groups(χ2 = 0.02, P>0.05). Conclusions Fluorosis can decrease the activity of GSTs and introduce the GSTP1 gene Ile105Val polymorphism, intervention with the fluorine intake will improve the effect of fluoride on the body.     

MAPKs阻断剂U0126对神经细胞α7尼古丁受体表达的影响

目的：研究老年性痴呆发病中α7尼古丁受体（nAChR）与ERK MAPK信号转导通路之间的关系。方法：用MEK阻断剂U0126阻断SH-SY5Y细胞ERK蛋白的活化及表达,实时荧光定量PCR（Real-time PCR）及Western blotting方法测定α7 nAChR mRNA及蛋白表达水平。结果：细胞经U0126处理后p-MEK和p-ERK1/2的水平明显降低,提示MEK和ERK 1/2的磷酸化被阻断,同时U0126处理组α7 nAChR mRNA及蛋白的表达量降低。结论：U0126能抑制SH-SY5Y细胞ERK MAPK信号转导通路,ERK MAPK信号转导通路的活化可能与神经细胞α7尼古丁受体的正常分泌及胆碱能信号传递密切相关。     

SH-SY5Y神经细胞中α3尼古丁受体基因表达沉默对β分泌酶蛋白表达的影响

目的 研究α3神经型尼古丁受体(nAChR)基因沉默对β分泌酶(BACE)蛋白表达的影响.方法 稳定转染α3 nAChR siRNA表达质粒及空质粒的SH-SY5Y神经细胞与正常对照组细胞用Real time-PCR和Western印迹法检测α3 nAChR mRNA表达和蛋白水平表达,以评价RNA的干扰效率;用蛋白质印迹方法 分别检测细胞中β分泌酶两个亚型(BACE1和BACE2)蛋白表达水平的变化.结果 稳定转染α3 nAChR siRNA表达质粒的细胞克隆株,与空质粒和正常对照组相比BACE1蛋白表达水平增加,BACE2蛋白表达水平减少.结论 α3 nAChR可以通过增加BACE2蛋白表达水平及减少BACE1蛋白表达水平,影响Aβ生成从而减少Aβ的神经毒性作用,发挥神经保护作用.     

慢性氟中毒对大鼠脑组织Phospho-Elk-1表达的影响

目的 观察慢性氟中毒大鼠脑组织中细胞外调节蛋白激酶(ERK1/2)信号转导通路下游作用底物三元复合物因子Phospho-Elk-1的表达和分布,探讨慢性氟中毒所致学习记忆损害的发生机制.方法 SD 大鼠72只,体质量100～120 g,按体质量随机分为3组,每组24只,雌雄各半.对照组饮用自来水(含氟量<0.5 mg/L),低氟组和高氟组饮用加入氟化钠的自来水(P质量浓度分别为5.0、50.0 mg/L).6个月后,称取大鼠体质量,观察氟斑牙发生情况,用氟离子选择电极法检测大鼠尿氟及骨氟;用Morris水迷宫方法的定向航行实验检测大鼠学习能力,空间探索实验检测大鼠记忆能力;用免疫组织化学方法检测大鼠脑组织中Phospho-Elk-1在蛋白水平的表达和分布.结果 低氟组和高氟组大鼠体质量[(449.2±77.1)、(312.8 ±89.7)g]较对照组[(635.5±76.2)g]显著下降(P均<0.05),出现不同程度氟斑牙(x2=7.83,P<0.05),尿氟[(2.56±0.91)、(5.73±3.14)mg/L]及骨氟[(709.2±37.4)、(1306.3 ±102.4)mg/kg]较对照组[(0.92±0.30)mg/L、(348.5 ±89.2)mg/kg]明显升高(P均<0.05).低氟组和高氟组大鼠逃避潜伏期[(7.4±4.1)、(12.2±5.7)s]较对照组[(4.8±2.7)s]明显延长(P均<0.05),第1次穿越平台区时同[(4.18±1.10)、(5.89±0.56)s]较对照组[(1.17±0.75)s]显著延长(P均<0.05),均以高氟组尤为明显(P均<0.05).低氟组和高氟组大鼠海马CA1区(167.4±8.3、163.2±9.4)、CA2区(175.7±5.0、183.3±4.2)、CA3区(165.2±11.6、162.9±4.4)、CA4 区(168.7±6.9、169.5±5.3)、齿状回(185.2 ±4.0、193.1±6.1)及尾壳核(181.4±3.8、179.8±5.5)神经细胞Phospho-Elk-1表达水平较对照组(142.4±8.1、144.9±8.4、143.6±5.8、116.8±9.1、140.2±7.8、163.1±13.1)显著增加(P均<0.05).结论 慢性氟中毒可引起大鼠脑组织海马及尾壳核区域Pbospho-Elk-1表达水平升高,这种改变可能与大鼠学习记忆能力下降机制有一定关系.

Abstract：

Objective To investigate the expression and distribution of the downstream substrate of extracellular regulated protein kinase(ERK1/2) pathway, ternary complex factor phospho-Elk-1, in rat brains with chronic fluorosis, and reveal the mechanism of the impaired learning and memory ability caused by chronic fluorosis. Methods Seventy-two SD rats, weighing 100 - 120 g, were randomly divided into 3 groups, 24 in each group (half male and half female). The rats in control group were fed with tap water (fluoride < 0.5 mg/L); low- and high-dose fluoride groups were fed with tap water with different concentrations of NaF(5.0,50.0 mg/L F-, respectively). After 6 months, body weight was weighed, dental fluorosis was determined by observation and urinary fluoride and bone fluoride were detected by fluorine ion-selective electrode; the learning ability of rats was measured by navigation test of Morris water maze, and memory ability by spatial probe test in Morris water maze; the expression and distribution of phospho-Elk-1 in different brain regions were detected by immunohistochemistry method. Results In low- and high-fluoride groups, the body weight of rat[(449.2 ± 77.1), (312.8 ± 89.7)g] was significantly decreased than that of control [(635.5 ± 76.2 )g, all P< 0.05], the varying degrees of dental fluorosis were observed(x2 = 7.83, P<0.05), urinary fluoride[(2.56 ±0.91),(5.73 ±3.14)mg/L] and bone fluoride[(709.2 ± 37.4) ,(1306.3 ± 102.4) mg/kg] were significantly higher than those in controls[(0.92 ± 0.30)mg/L,(348.5 ± 89.2)mg/kg, all P< 0.05]. The escape latency of low- and high-fluoride groups[ (7.4 ± 4.1), (12.2 ± 5.7)s] was longer than that of control [(4.8 ± 2.7 )s, all P < 0.05] and the escape latency in high-fluoride group was significantly longer than that in other groups (all P < 0.05); in spatial probe test, the time of first crossing platform was longer in rats with fluorosis [(4.18 ± 1.10),(5.89 ± 0.56)s] as compared to control[(1.17 ± 0.75)s, all P< 0.05]. Expressions of phospho-Elk-1 in the hippocampus CA1(167.4 ± 8.3,163.2 ± 9.4), CA2(175.7 ± 5.0,183.3 ± 4.2), CA3(165.2 ± 11.6,162.9 ± 4.4), CA4(168.7± 6.9,169.5 ±5.3), fascia dentate (185.2 ±4.0,193.1 ±6.1) and caudate putamen( 181.4 ± 3.8, 179.8 ± 5.5) in low- and high-fluoride groups were higher than those of controls(142.4 ± 8.1,144.9 ± 8.4,143.6 ± 5.8, 116.8 ± 9.1,140.2 ± 7.8,163.1 ± 13.1, all P< 0.05). Conclusion Chronic fluorosis can cause increased expression of phospho-Elk-1 in the hippocampus and caudate putamen region of rat brains, which might be related to the mechanisms of decreased learning and memory ability of rats overexposed to fluoride.     

β-淀粉样肽对人SH-SY5Y细胞突触素、发动蛋白Ⅰ及衔接蛋白180表达的影响

目的 探讨β-淀粉样肽(Aβ)对人SH-SY5Y神经母细胞瘤细胞突触素(Syn)、发动蛋白Ⅰ (Dyn Ⅰ)及衔接蛋白180(AP180)表达的影响。 方法 分别应用0.01、0.1、1、2及5μmol/LAβ1-42处理SH-SY5Y细胞,对照组细胞不加任何处理。MTT法检测细胞增殖情况,Western blotting 检测0.5、1μmol/L Aβ1-42处理组及对照组细胞Syn、DynⅠ和AP180蛋白表达,RT-PCR检测1 μmol/L Aβ1-42处理组及对照组细胞Syn、DynⅠ和AP180 mRNA表达。 结果 0.1 μmol/L Aβ1-42处理细胞即可出现细胞增殖率下降,并呈剂量依赖性负性相关关系,与对照组比较差异均有统计学意义(P＜0.05); 0.5 μmol/L Aβ1-42处理细胞可见DynⅠ蛋白表达降低,与对照组比较差异有统计学意义(P＜0.05);1 μmol/L Aβ1-42处理细胞可见Syn、DynⅠ蛋白及mRNA表达均降低,与对照组比较差异均有统计学意义(P＜0.05);但未见AP180蛋白及mRNA表达水平发生改变。 结论 Aβ1-42可引起人SH-SY5Y细胞Syn和DynⅠ表达降低,该改变可能与AD发病中学习记忆能力减退有关。     

不同聚集形式β-淀粉样肽对人SH-SY5Y细胞发动蛋白Ⅰ和突触素表达的影响

目的探讨不同聚集形式的β-淀粉样肽(Aβ)对人神经母细胞瘤细胞(SH-SY5Y细胞)发动蛋白Ⅰ(DynⅠ)和突触素(Syn)表达的影响。方法制备不同聚集形式的Aβ1～42处理SH-SY5Y细胞,采用蛋白质印迹法检测细胞中DynⅠ及Syn蛋白表达水平;采用免疫组织化学染色方法检测10例阿尔茨海默病(AD)患者及8例老年同龄对照者尸解后脑海马结构中β-淀粉样肽寡聚体(AβOs)表达水平。结果 Aβ单体或Aβ纤丝体处理SH-SY5Y细胞未见DynⅠ及Syn蛋白表达水平显著变化,AβOs处理细胞可见DynⅠ及Syn蛋白表达水平降低(P<0.05);AD患者海马及内嗅区皮质神经元内AβOs的表达显著高于老龄对照者(P<0.05)。结论细胞内表达的AβOs可能是引起AD患者脑中网格蛋白内吞调节蛋白———DynⅠ表达下降及突触丧失的主要Aβ神经毒性形式,该改变可能与AD发病机制中学习记忆能力减退有关。