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.     

氟对大鼠骨组织3-磷酸肌醇激酶和蛋白激酶B1表达的影响

目的 观察慢性氟中毒对大鼠骨组织中3-磷酸肌醇激酶(PI3K)、蛋白激酶B1(Akt1)蛋白和mRNA表达的影响,探讨PI3K/Akt信号通路在氟骨症发病机制中的作用.方法 将36只SD大鼠按性别和体质量随机分为3组:对照组、低氟组、高氟组,每组12只.对照组自由饮用自来水(含氟量<0.5 mg/L),低、高氟组大鼠分别饮用氟化钠(NaF)配制的含氟量为5.0、50.0 mg/L的自来水.实验6个月后处死大鼠,收集血清,用酶联免疫吸附测定(ELISA)法检测骨钙素(BGP)、组织蛋白酶K(Cath-K).取大鼠股骨下段,用免疫组织化学方法和实时荧光定量PCR法检测骨组织中PI3K、Akt1蛋白和mRNA的表达.结果 各组大鼠血清BGP、Cath-K 水平比较,差异有统计学意义(F值分别为73.45、39.36,P均<0.05).与对照组[(0.15±0.03)μg/L、(18.32±2.27)pmol/L]比较,低、高氟组血清BGP[(1.99±0.62)、(2.38±0.16)μg/L]、Cath-K[(89.07±19.66)、(110.16±9.81)pmol/L]明显升高(P均<0.05),且高氟组明显高于低氟组(P均<0.05).各组大鼠骨组织PI3K、Akt1蛋白和mRNA表达水平比较,差异有统计学意义(F值分别为178.16、118.08,38.81、52.31,P均<0.05).与对照组(181.55±4.24、188.46±2.18,3.84±1.69、4.33±0.89)比较,低、高氟组大鼠骨组织PI3K(171.66±2.85、154.12±4.15,11.31±4.18、20.54±6.68)、Akt1蛋白和mRNA表达(177.47±3.16、156.42±3.18.12.52±3.13、19.43±5.36)明显增高(P均<0.05),且高氟组明显高于低氟组(P均<0.05).结论 血清BGP、Cath-K可作为慢性氟中毒骨病变的代谢指标.氟可导致大鼠骨组织中PI3K、Akt1蛋白和mRNA表达水平增高,PI3K/Akt 信号通路可能参与了氟引起的骨骼损伤机制.

Abstract：

Objective To observe the expression of phosphoinositide 3-kinase(PI3K) and protein kinase B1 (Akt1) in PI3K/Akt signaling pathway in rat bones with fluorosis, and to reveal the mechanisms of the skeletal fluorosis. Methods Thirty-six SD rats were randomly divided into 3 groups (control group, low-dose fluorosis group, high-dose fluorosis group) and 12 rats were in each group according to body weight. The rats were fed with different concentrations of fluoride (NaF) to establish fluorosis models. Controls were fed with tap water( < 0.5 mg/L), experimental animals in low- or high-dose groups were fed with water containing NaF 5.0,50.0 mg/L, respectively. Rats were sacrificed after 6 months of treating with fluoride and the serum was kept for testing the bone metabolic markers of none gla protein(BGP) and cathepsin K(Cath-K) by enzyme-linked immunosorbent assay(ELISA), the proteins and mRNA levels of PI3K and Akt1 in rat bones were detected by immunohistochemistry and real time PCR, respectively. Results Each group of serum BGP and Cath-k were compared, the difference was statistically significant(F = 73.45,39.36, all P < 0.05). The contents of BGP[(1.99 ± 0.62), (2.38 ± 0.16)μg/L] and Cath-K [(89.07 ± 19.66), (110.16 ± 9.81)pmol/L] in the low-and high-dose fluorosis groups were higher than those in the control group[(0.15 ± 0.03)μg/L,( 18.32 ± 2.27)pmol/L], and the high fluorosis group was obviously higher than the low fluorosis group (all P < 0.05). Each group of serum PI3K and Akt1 protein and mRNA were compared, the difference was statistically significant(F- 178.16,118.08,38.81,52.31, all P< 0.05). Compared to the control group (181.55 ± 4.24,188.46 ± 2.18,3.84 ± 1.69,4.33 ± 0.89), the protein and mRNA expressions of PI3K(171.66 ± 2.85,154.12 ± 4.15,11.31 ± 4.18,20.54 ± 6.68), Akt1(177.47 ± 3.16,156.42 ± 3.18,12.52 ± 3.13,19.43 ± 5.36) were higher in the low- and high-dose fluorosis groups (all P < 0.05), and the high fluorosis group was obviously higher than the low fluorosis group (all P < 0.05). Conclusions BGP and Cath-K contents could be used as bone metabolic indices in the endemic fluorosis disease. Fluoride can increase the expression of PI3K and Akt1 mRNA and protein in bone tissue of fluorosis rats, and PI3K/Akt1 signaling pathway may be involved in the pathogenesis of bone injury caused by fluoride.     

慢性氟中毒对大鼠大脑皮质神经细胞活性氧水平和线粒体融合的影响

目的 观察慢性氟中毒对大鼠大脑皮质神经细胞活性氧(ROS)水平和线粒体融合的影响,并分析二者间的关系.方法 选择SD大鼠120只,按性别和体质量随机分为3组:对照组、低氟组、高氟组,每组40只.对照组大鼠自由饮用自来水(含氟量<0.5 mg/L);低、高氟组分别饮用氟化钠(NaF)配制的含氟量为10.0、50.0 mg/L的自来水.分别于3、6个月时处死大鼠,采集大脑组织制作冰冻切片,采用荧光测定法检测皮质神经细胞ROS水平和线粒体形态变化.结果 3、6个月时各组大鼠大脑皮质神经细胞ROS荧光计数和Ⅱ型线粒体计数水平比较,差异有统计学意义(F值分别为3.07、3.06,3.05、3.07,P均<0.05).3个月时,与对照组(10.43±5.98、4.12±3.86)比较,高氟组大鼠大脑皮质神经细胞ROS荧光计数(25.48±6.09)和Ⅱ型线粒体计数(20.47±6.09)明显升高(P均<0.05),而低氟组(11.67±3.49、6.68±3.48)未见明显改变(P均>0.05);6个月时,与对照组(25.26±6.41、20.26±6.41)比较,低、高氟组大鼠大脑皮质神经细胞ROS荧光计数和Ⅱ型线粒体计数(63.02±8.15、65.60±7.40,49.33±8.61、53.10±6.95)均明显增高(P均<0.05).ROS荧光计数与Ⅱ型线粒体计数间呈明显正相关(r值分别为0.93、0.81,P均<0.05).结论 摄入过量的氟导致大鼠大脑皮质神经细胞氧化应激水平升高,线粒体融合功能障碍,这些改变与染氟时间和剂量密切相关.线粒体异常改变的机制可能与慢性氟中毒引起的氧化应激水平升高有关.

Abstract：

Objective To investigate the changes of reactive oxygen species(ROS) level and mitochondria fission-fusion-balance in cortical neurons of rats with chronic fluorosis and reveal the correlation between these two factors. Methods One hundred and twenty rats were randomly divided into 3 groups(control group, low-dose fluorosis group, high-dose fluorosis group) and 40 rats were in each group according to body weight and the experiments were carried out for 3 months or 6 months. The rats were fed with different concentrations of fluoride (NaF) to establish fluorosis models. Controls were fed with tap water( < 0.5 mg/L), experimental animals in low- or high-dose group were fed with water containing NaF 10.0,50.0 mg/L, respectively. The level of ROS and the morphology in mitochondria fission-fusion balance in neurons of the cortex of rat brains prepared with cortical frozen sections were detected with ROS fluorescent probe and MitoTracker RED probe, respectively. Results Significant differences of the level of ROS and the numbers of abnormal mitochondria in morphology in the cortical neurons were found between 3 groups at the experiment period of 3 month and 6 month(F= 3.07,3.06,3.05,3.07, all P < 0.05). As compared with control group(10.43 ± 5.98,4.12 ± 3.86) at the experiment period of 3 month, the level of ROS and the numbers of abnormal mitochondria in morphology in the cortical neurons were obviously increased in high-dose fluorosis group(25.48 ± 6.09,20.47 ± 6.09, all P < 0.05), whereas no significant changes were found in low-dose fluorosis group(11.67 ± 3.49,6.68 ± 3.48, all P> 0.05). Furthermore, the increases in both ROS level and abnormal numbers of mitochondria were significant observed in the cortical neurons of low-dose fluorosis group (63.02 ± 8.15, 49.33 ± 8.61) and high-dose fluorosis group(65.60 ± 7.40,53.10 ± 6.95) as compared with the control group (25.26 ± 6.41,20.26 ± 6.41) at the experimental period of 6 month (all P < 0.05). The abnormal numbers of mitochondria correlated with ROS level(r = 0.93,0.81, all P < 0.05). Conclusions Taking excessive amount of fluoride results in high level of oxidative stress and impaired the balance of mitochondrial fission-fusion,which is dependent on the feeding times and doses of fluoride. The mechanism of the mitochondrial abnormalities might be associated with the high level of oxidative stress induced by chronic fluorosis.     

慢性氟中毒大鼠脑组织中c-Jun氨基末端激酶表达水平观察

Objective To investigate the expression of c-Jun-N-terminal kinase(JNK) in rat brains with chronic fluorosis and try to reveal the molecular mechanism for the neural impairment induced by the disease.Methods The rats were randomly divided into 3 groups, normal control group(drinking water containing less than 0.5 mg/L of sodium fluoride, NaF), lower fluoride exposed group(drinking water containing 5 mg/L NaF) and higher fluoride exposed group(drinking water containing 50 mg/L NaF), 24 in every group. The rats were examined at the sixth month after feeding. The concentration of fluorine in urine and blood was detected by F-ion selective electrode. The expression of JNK in brains was investigated by using Western blotting and immunohitochemistry staining, and analyze the correlation between activating of JNK and the concentration of fluorine in blood. Results The increased concentration of fluorine in urine(control: 0.92 ± 0.30, lower fluoride exposed group: 2.56 ± 0.91,higher fluoride exposed group: 5.73 ± 3.14, P ＜ 0.05) were observed when 6 months after the beginning of the experiment, and the amount of fluorine in blood was also higher in rats with fluorosis(control: 0.12 ± 0.07, lower fluoride exposed group: 0.36 ± 0.14, higher fluoride exposed group: 0.50 ± 0.18, P ＜ 0.05). The expression of phospho-JNK at protein levels were higher in the brains of rats with fluorosis than that of controls (control: 1.00 ± 0.37, lower fluoride exposed group: 1.20 ± 0.28, higher fluoride exposed group: 1.74 ± 0.69, P ＜ 0.05), whereas no change of total-JNK was found(F = 0.046, P ＞ 0.05). Furthermore, the expression of phospho-JNK in the parietal cortex(119.3 ± 14.1), occipital cortex(112.7 ± 5.4), hippocampus CA3(100.6 ± 8.9), dorsal thalamus (117.8 ± 10.4) and olivary nucleus( 112.6 ± 5.9) of rats in higher fluoride exposed group were higher than that in control( 104.1 ± 8.9,106.6 ± 9.6,106.6 ± 9.7,108.9 ± 6.4,100.3 ± 8.4, all P ＜ 0.05) and lower fluoride exposed group(96.7 ± 17.1,102.5 ± 8.3,106.4 ± 6.5,110.2 ± 9.3,102.4 ± 4.7,102.5 ± 9.8, all P＜ 0.05). The positive stained neurons of total-JNK also distributed in the same brain regions of rats, but no difference was detected between the rats with fluorosis and controls(all P ＞ 0.05). The increased level of phospho-JNK was positively correlated with the fluoride contents in blood of the rats with fluorosis (r = 0.677). Conclusions The expression of phospho-JNK in brains of rats with fluorosis was significantly increased with a correlation to fluoride content in blood, which might be connected to the mechanism of neural impairment induced by chronic fluorosis.     

硒对氟致大鼠肾脏损伤保护作用的实验观察

目的 观察硒对氟致大鼠肾脏损伤的保护作用,探讨硒的最佳作用剂量及作用靶点.方法 断乳SD雄性大鼠80只,按体质量随机分8组,每组10只.对照组饮用自来水;染氟组饮用50 mg/L的氟化钠溶液;低、中、高硒组分别饮用0.375、0.750、1.500 mg/L的亚硒酸钠溶液;氟+低、中、高硒组分别饮用50 mg/L的氟化钠和0.375、0.750、1.500 mg/L的亚硒酸钠两两组合的溶液.染毒6个月后,测大鼠肾脏组织的氧化水平和核因子κB(NF-κB)的表达量.结果 染氟组大鼠体质量[(695.95±55.89)g]低于对照组[(782.69±56.12)g,P＜0.01],染氟组谷胱甘肽过氧化物酶(GSH-Px)活性[(55.86±5.09)U/mgprot]与对照组[(68.66±4.52)U/mgprot]比较,差异无统计学意义(P＞0.05),但有降低的趋势.染氟组大鼠总抗氧化能力(T-AOC)水平[(7.54±1.35)U/mgprot]低于对照组[(9.03±0.37)U/mgprot,P＜0.05],染氟组丙二醛(MDA)水平[(3.86±0.31)nmol/mgprot]高于对照组[(3.14±0.32)nmol/mgprot,P＜0.05].氟+高硒组GSH-Px活性[(74.99±8.41)U/mgprot]高于染氟组[(55.86±5.09)U/mgprot,P＜0.05],MDA水平[(3.17±0.20)nmol/mgprot]低于染氟组[(3.86±0.31)nmol/mgprot,P＜0.05].染氟组、高硒组和氟+低硒组的NF-κB的表达水平(0.360±0.015,0.367±0.007,0.376±0.006)高于对照组(0.312±0.022,P均＜0.05),氟+高硒组(0.312±0.005)低于染氟组(0.360±0.015,P＜0.05).结论 1.500 mg/L硒是本实验条件下硒对慢性氟中毒致大鼠肾脏损伤的最佳保护作用剂量,NF-κB可能是硒拮抗氟中毒的药物靶点.

Abstract：

Objective To explore the protective effect of selenium, an antioxidant, on fluoride-induced renal injury in rats and find out the optimal level of selenium against fluoride toxicity and its valid molecular target.Methods All 80 male weanling SD rats were randomly divided into 8 groups by body weight as follows: normal control group(drinking tap water), fluoride exposed group (drinking water containing 50 mg/L of NaF), low, middle,high selenium exposed groups(drinking water containing 0.375, 0.750, 1.500 mg/L of Na2SeO3) and low, middle,high Se-fluoride groups (drinking water containing both 50 mg/L NaF and three doses of Na2SeO3 as abovementioned, respectively). After 6 months, the rats were killed then the oxidation level and nuclear factor κB(NF-κB)expression level in kidney were measured. Results The weight of the fluoride exposed group[(695.95 ± 55.89 )g]was significantly deceased than the controls[(782.69 ± 56.12)g, P ＜ 0.01]. Glutathione peroxidase(GSH-Px)activity of fluoride exposed group[(55.86 ± 5.09)U/mgprot] was not significantly different but decreased. Tatal antioxidant capacity (T-AOC) activity in fluoride exposed group [(7.54 ± 1.35)U/mgprot] significantly decreased than the controls[(9.03 ± 0.37 )U/mgprot, P ＜ 0.05]. In addition, a significant increase of malondialdehyde ( MDA )in fluoride exposed group[(3.86 ± 0.31 )mnol/mgprot, P ＜ 0.05] was observed than the controls[(3.14 ± 0.32)nmol/mgprot, P ＜ 0.05]. GSH-Px activity of high Se-fluoride group[(74.99 ± 8.41 )U/mgprot] was significantly higher than the fluoride exposed group[(55.86 ± 5.09)U/mgprot, P ＜ 0.05] and its MDA level[(3.17 ± 0.20)nmol/mgprot] was lower than the fluoride exposed group[(3.86 ± 0.31 ) nmol/mgprot, P ＜ 0.05]. NF-κB expression levels of fluoride group, high selenium group and low Se-fluoride group(0.360 ± 0.015,0.367 ± 0.007,0.376 ± 0.006,respecyively) were obviously increased compared with the controls(0.312 ± 0.022, P ＜ 0.05); it was significantly lower in high Se-fluoride group(0.312 ± 0.005) than in fluoride exposed group(0.360 ± 0.015, P ＜ 0.05). Conclusions Na2SeO3 of 1.5 mg/L is the optimal dose against chronic fluorosis on kidney injury under this experimental condition.NF-κB is likely to be a target molecule of the selenium as an antagonist on fluorosis.     

燃煤污染型氟中毒大鼠学习记忆能力及胆碱酯酶活性改变

Objective To observe the influence of coal burning fluorosis on learning and memory ability in rats and reveal its possible mechanisms. Methods Healthy 48 SD rats were divided into control, low-fluoride and high-fluoride group. All rats in fluoride exposed groups were fed with the eom polluted by drying processes with burning coal containing high level of fluoride obtained from the endemic fluorosis area to produce the animal model of fluorosis. The experiment period were 3,6 mouths, respectively. The ability of leaning and memory was measured by Morris test and cholinesterase activity detected by photometric method at 3 or 6 month after experiment, respectively. Results Fluoride contents signifieantlly influenced the escape latency, the numbers of crossing the platforms and the time of staying the platforms(the value of F was 29.29,6.47,6.50, respectively, P<0.01).In addition, the numbers of crossing the platforms and the time of staying the platforms were influenced by the exposed time(the value of F was 16.11,45.59, P<0.01). Furthermore, the fluoride contents and the exposed time had an interaction between the numbers of crossing the platforms and the time of staying the platforms (the value of F was 4.67,5.68, P<0.05 or<0.01). Three months after the experiment, the mean values of escape latency [(14.71± 4.85)s] of rats in highly fluoride exposed group were significantly prolonged as compared with controls [(9.28±4.22)s]; 6 month after the experiment, the mean values of escape latency[(12.42±8.03)s, (17.48± 8.05)s] of rats in both groups exposed to fluoride were significantly prolonged as compared to controls [(7.04± 3.29)s, P<0.05]. The decreased numbers of crossing the platforms[(1.62±0.87)number] and the declined time of staying the platforms[(16.70±5.02)s] were found in the rats exposed to high fluoride as compared to controls [(3.53±1.67 )number, (23.33±5.35)s, P<0.05]. The fluoride contents obviously influenced the activities of acetylcholinesterase and butylcolinesterase (the value of F was 12.83,13.27, P<0.01). On the other hand, the times of breeding also influnced the activities of butylcolinesterase (the value of F was 16.26, P<0.01). In 3 months of the experiment, the activities of butylcolinesterase [(0.55±0.12)kU/g] in low fluoride exposed group were significantly decreased in comparison with controls[(0.73±0.10)kU/g, P<0.05]. The activities of acetylcholinesterase[(0.62±0.42)kU/g] and butylcolioesterase[(0.58±0.10)kU/g] in high fluoride group were significantly decreased as compared to eontrois[(1.41±0.52), (0.73±0.10)kU/g, P<0.05]. The correlation analysis showed that there was a negative correlation between the cholinesterase and the escape latency(r=-0.68, P< 0.01), and a positive correlation between the cholinesterase and the time of staying the platforms(r=0.57, P< 0.01). Conclusions The ability of learning and memory in rats with coal buring fluorosis was decreased, which might be connected to the decreased activity of cholinesterase in a dose-effect correlation.     

Correlation between expression of peroxisome proliferator-activated receptor γ and oxidative stress in brains of rats with chronic fluorosis

正Objective To detect the expression of peroxisome proliferator-activated receptorγ(PPARγ) in the brains of rats with chronic fluorosis and elucidate the relationship between PPARγand oxidative stress in chronic fluorosis.Methods According to body weight (100-120 g),sixty healthy SD rats were divided into control group(less than 0. 5 mg/L fluoride in drinking water), low fluoride group (5. 0 mg/L fluoride in drinking water,prepared by NaF), and high fluoride group (50. 0 mg/L fluoride in drinking water) via the random number table     

慢性氟中毒大鼠软骨的损害及COLIXA3蛋白、表达的实验研究

目的 探讨不同程度的氟中毒是否影响染氟大鼠软骨组织中COLIXA3蛋白的表达.方法 选用3～4周龄健康雄性Wistar大鼠40只,按体质量随机分为5组,每组8只,分笼饲养,期间分别自由饮用含氟化钠0(对照)、25、50、100、150mg/L的蒸馏水,饲喂6月后建立氟中毒大鼠模型.应用光学显微镜分析实验大鼠骨组织的病理形态学变化过程.并采用免疫组织化学技术检测大鼠股骨干骺端COLIXA3蛋白的表达情况.结果 大鼠骨组织HE染色显示,各染氟组股骨干骺端出现不同程度软骨骨化,骨密度增加,具有硬化性氟骨症病变.对照组软骨组织未见明显异常.大鼠软骨细胞COLIXA3免疫组织化学染色结果为阳性,胞浆内可见有棕黄色颗粒,25、50、100 mg/L组在软骨组织中COLIXA3蛋白表达(23.3±4.5、41.2±5.6、26.4±7.5)增强.其中50、100 mg/L组表达与对照组(6.1±3.5)相比明显增加,组间比较差异有统计学意义(P均<0.05).150 mg/L组COLIXA3蛋白(13.3±4.2)较前面3组表达减弱,仍高于对照组,组问比较差异无统计学意义(P>0.05).结论 动物模型中,大鼠病理学为单纯性骨硬化表现.低剂量氟促进,高剂量抑制大鼠软骨细胞的增生.随着染氟时间的延长,外环境中氟浓度过高时,对软骨细胞就表现为氟离子的直接毒性作用.氟化物影响染氟大鼠软骨组织中COLIXA3蛋白的表达,低剂量氟可以促进COLIXA3蛋白的表达,随剂量增加氟的促进作用减弱.

Abstract：

Objective To explore whether different degrees of fluorosis influence the expression of cartilage COLIXA3 protein in fluorosis model rats. Methods Forty male Wistar rats 3 to 4 weeks old were randomly divided into 5 groups according to body mass, and these rats were fed with distilled water containing sodium fluoride(NaF) of 0(control), 25, 50, 100 and 150 mg/L for 6 months, respectively, in order to establish the animal model of drinking water type fluorosis. Pathomorphologieal changes of the osseous tissues of rats were analyzed under light microscope and transmission electron microscope, and the expression of COLIXA3 protein of femur metaphysis was examined by immunohistochemistry. Results HE staining showed different degrees of femoral metaphyseal ossification of cartilage in each experimental group, bone density increased, with sclerotic lesions of skeletal fluorosis. The control group showed no abnormal cartilage. Electron microscopy showed that the experimental groups with varying degrees of cartilage cell swelling, cell matrix fades, 50 mg/L group .showed hyperplasia, and 100,150 mg/L groups were observed with organelles decreased, part of the disintegration of the cartilage cell lacunae, lmmunohistochemical staining of rat chondrocytes COLIXA3 was positive, cytoplasm with brown granules, cartilage COLIXA3 protein expression(23.3 ± 4.5, 41.2 ± 5.6, 26.4 ~ 7.5) in the 25, 50 and 100 mg/L groups enhanced. Compared to the control group (6.1 ± 3.5), the expression of 50 and 100 mg/L groups was significantly increased, and the differences were statistically significant(all P < 0.05). The expression(13.3 ± 4.2)of COLIXA3 protein in 150 mg/L group was decreased compared with the previous three, but is still higher than that of control, and the difference was not statistically significant(P > 0.05). Conclusions There has pathological changes of sclerosing skeletal fluorosis in animal model. Low-dose fluoride promotes while high-dose inhibits cartilage cell proliferation. When fluorine concentration in external environment is too high and with extended exposure to fluoride, direct toxic effects of fluoride on cartilage cells is observed. Fluorine affects and promotes the expression of COLIXA3 protein in cartilage. Low-dose fluoride can promote COLIXA3 protein expression, as the dose increases (over 100 mg/L), the effect decreases.     

氟对雄性大鼠睾丸组织金属元素水平的影响

目的 探讨慢性氟中毒对大鼠睾丸组织金属元素水平的影响,为氟的生殖毒性研究提供一定实验依据.方法 健康雄性Wistar大鼠32只,体质量150～180 g,按体质量随机分为4组,生理盐水(对照)组、低、中、高氟组(100、200、300 mg-kg-1·d-1NaF),每组8只,灌胃染毒90 d,每天称体质量.染氟结束次日,颈椎脱位法处死大鼠,摘取睾丸组织,原子吸收分光光度计测定睾丸组织中会属元素钙(Ca)、铁(Fe)、锌(Zn)、铜(Cu)和镁(Mg)水平.结果 染氟第30天大鼠体质量组间比较,差异有统计学意义(F=3.884,P<0.05),其中低、中氟组[(235.00±14.56)、(235.44±24.99)g]高于高氟组[(206.00±18.16)g,P均<0.05];第0、60、90天大鼠体质量组间比较,差异无统计学意义(F值分别为0.501、0.578、1.893,P均>0.05).4组大鼠睾丸组织Ca、Zn和Mg组间比较,差异有统计学意义(F值分别为6.630、6.844、5.333,P均<0.05),其中元素Ca低氟组[(56.15±4.21)mg/kg]较对照组[(77.57±6.66)mg/kg]降低,元素Zn低、中、高氟组[(4.80±0.55)、(4.56±0.33)、(5.46±0.79)mg/kg]较对照组[(7.16±0.28)mg/kg]降低,元素Mg高氟组[(32.44±1.53)mg/kg]较对照组[(42.54±8.07)mg/kg]降低(P均<0.05);4组大鼠睾丸组织Fe和Cu组间比较,差异无统计学意义(F值分别为1.324、0.207,P均>0.05).结论 慢性氟中毒可通过影响大鼠睾丸组织金属元素水平损害大鼠生殖系统.

Abstract：

Objective To probe into the effects of fluoride on metal elements in the testis tissue of male rats, and provide experimental basis to further research for reproductive toxicity of fluoride. Methods Thirty-two healthy male Wistar rats, weighting 150 - 180 g, were randomly divided into 4 groups, normal sodium(control) by intragastrie administration for 90 days, and body weight was observed daily. After the last intragastric administration, all rats were killed by cervical dislocation. The contents of calcium(Ca), ferri(Fe), zincum(Zn),cuprum(Cu ) and magnesium(Mg) in the testis tissue were measured by atomic absorption speetrophotometry.Results After 30 days exposure, the difference of body weight between groups was statistically significant(F=3.884, P < 0.05). The body weight in low- and medium-dose groups[(235.00 :t: 14.56), (235.44 ± 24.99)g] were significant increased than high-dose group[(206.00 ± 18.16)g, all P < 0.05]. There was no significant difference of body weight between the groups at 0, 60 and 90 days(F = 0.501, 0.578, 1.893, all P > 0.05). The difference of Ca, Zn and Mg levels among four groups was statistically significant(F = 6.630, 6.844, 5.333, all P < 0.05). The content of Ca of the low-dose group[(56.15 + 4.21 )mg/kg] decreased than that of the control group[(77.57 ± 6.66)mg/kg, P < 0.05];the content of Zn of the low-, medium- and high-dose groups[(4.80 ± 0.55), (4.56 ± 0.33),(5.46 ± 0.79 )mg/kg] deceased than that of the control group [(7.16 ± 0.28 )mg/kg, all P < 0.05];the content of Mg of the high-dose group [(32.44 ± 1.53 ) mg/kg] decreased than that of the control group [(42.54 ± 8.07 ) mg/kg,all P < 0.05]. The difference of testis Fe and Cu between four groups was not statistically significant(F = 1.324,0.207, all P > 0.05). Conclusion Chronic fluorosis can affect the levels of metal elements in rat testis and damage the reproductive system.     

氟中毒对体外培养破骨细胞数量及骨吸收功能的影响

目的 观察氟中毒对体外培养破骨细胞数量及骨吸收功能的影响,探讨其作用机制.方法 机械分离法作用于新生SD大鼠四肢长骨,于TC199培养液(含10%胎牛血清)中获得破骨细胞和骨髓基质细胞.将破骨细胞接种于96孔培养板和象牙片培养,而骨髓基质细胞接种于6孔培养板培养,分别于2 h后换液并染氟(氟化钠),对照组、低氟组、中氟组、高氟组染氟剂量分别为0、2.5×10-5、5.0×10-5、10.0×10-5mol/L.培养2、5d后对培养板中破骨细胞进行抗酒石酸酸性磷酸酶(TRAP)染色,光镜下计数破骨细胞数量;培养5 d后象牙片经1%甲苯胺蓝染色,光镜下分析破骨细胞骨吸收陷窝面积.骨髓基质细胞染氟作用8 h后提取总RNA,实时荧光定量PCR法检测细胞核因子κβ受体活化因子配体(RANKL)和骨保护素(0PG)mRNA表达水平.结果 ①体外培养2 d时,对照组、低氟组、中氟组、高氟组破骨细胞数量分别为(337.5 4-70.5)、(447.5 ±43.4)、(472.9±34.8)、(475.3±24-3)个/孔,各染氟组明显高于对照组(P均<0.05);体外培养5 d时,对照组、低氟组、中氟组、高氟组破骨细胞数量分别为(92.5±22.1)、(123.0±26.4)、(135.5 ±22.2)、(136.9 ±23.0)个/孔,各染氟组明显高于对照组(P均<0.05).②体外培养5 d时,对照组、低氟组、中氟组、高氟组破骨细胞骨吸收陷窝面积分别为(0.088±0.030)、(0.100 ±0.018)、(0.152±0.015)、(0.242±0.031)mm2/片,中氟组和高氟组明显高于对照组(P均<0.05).③对照组、低氟组、中氟组、高氟组骨髓基质细胞RANKL/OPG mRNA表达比值分别为100.00±56.02、144.95±97.21、223.25 ±184.48、193.98 ±137.93,中氟组和高氟组明显高于对照组(P均<0.05).结论 氟中毒可引起体外培养破骨细胞数量增多,促进其细胞分化及骨吸收活性,该作用可能与其上调 RANKL/OPC,mRNA表达比值有关.

Abstract：

Objective To determine the effects of fluoride on osteoclasts's quantity and bone resorption function in vitro and its mechanisms. Methods The osteoclasts and bone marrow stromal cells(BMSCs) isolated from long bone of new born rats were cultured respectively in TC199 medium (containing 10% fetal bovine serum) with fluoride. The osteoclasts were inoculated in 96-well culture plate and ivory slice, BMSCs were inoculated in 6- well culture plate, respectively, medium were changed after 2 hours incubation. They were divided into control group, low-dose fluoride, medium-dose fluoride and high-dose fluoride groups, the doses of sodium fluoride were 0,2.5 × 10-5,5.0 × 10-5,10.0 × 10-5 mol/L, respectively. Tartrate-resistant acid phosphatase(TRAP) staining positive cells were counted under light microscope after TRAP staining on the 2nd and the 5th day and the pit formed in ivory slices were measured by histomorphometry after staining with toludine blue. The expression of receptor activator of NK-κβ ligand(RANKL) and osteoprotegerin(OPC) was detected by real-time fluorescence quantitative (337.5 ± 70.5), (447.5 ± 43.4), (472.9 ± 34.8), (475.3 ± 24.3)/well in the control group, the low-dose, mediumdose and high-dose fluoride groups, respectively. The differences were statistically significant between these groups and the control group (all P < 0.05). After in vitro culture for 5 days, the numbers of osteoclasts were (92.5 ± 22.1), (123.0 ± 26.4), (135.5 ± 22.2), (136.9 ± 23.0) per well in the control group, the low-dose, medium-dose and high-dose fluoride groups, respectively. The differences were statistically significant between these groups and the (0.088 ± 0.030), (0.100 ± 0.018), (0.152 ± 0.015), (0.242 ± 0.031 )mm2 per piece in the control group, the lowdose, medium-dose and high-dose fluoride groups, respectively. The values of medium-dose and high-dose fluoride BMSCs in the control group, the low-dose, medium-dose and high-dose fluoride groups were 100.00 ± 56.02, 144.95 ± 97.21,223.25 ± 184.48,193.98 ± 137.93, respectively. The values of medium-dose and high-dose fluoride groups were significantly higher than that of control group (all P < 0.05). Conclusions Fluoride can cause increase in the number of osteoclasts in vitro and promote their cell differentiation and bone resorption activity, which may be related to increased expression ratio of RANKL/OPG mRNA in BMSCs.     

氟中毒大鼠骨组织中内质网应激实验研究

目的 观察内质网应激在氟中毒大鼠骨组织中的变化,探索内质网应激在氟骨症发病机制中的可能作用.方法 48只Wistar大鼠,按体质量分成4组,每组12只.对照组和低钙组分别饲以常食饲料(含钙量为0.790%)和自制低钙饲料(含钙量为0.063%),饮用自来水(含NaF＜1 mg/L);高氟组和低钙高氟组分别饲以常食饲料和自制低钙饲料,饮用加氟(NaF,221 mg/L)自来水.实验期间动物自由进食、进水,每周测体质量1次.实验期3个月.生化方法检测大鼠血清氧化应激酶、尿酸(URIC)和碱性磷酸酶(ALP)活性.抽提大鼠一侧股骨骨干的总RNA,利用RT-PCR技术分析内质网应激相关基因BIP、Xbp1、CHOP和PDI的表达水平.结果 低钙高氟组血清丙二醛(MDA)水平高于对照组[(14.74±3.11)μmol/L比(10.15±1.96)μmol/L,P＜0.05];高氟组血清谷胱甘肽过氧化物酶(GPx)的活性高于对照组[(3.87±0.41)×103 U/L比(2.85±0.55)×103U/L,P＜0.05];高氟组和低钙高氟组的尿酸(URIC)分别低于对照组和低钙组[(73.95±9.52)μmol/L比(110.43±25.48)μmol/L,(54.32±22.09)μmol/L比(101.71±17.01)μmol/L/L,P＜0.05].低钙高氟组大鼠的ALP活性高于对照组[(24.77±4.57)×103 U/L比(12.91±3.97)×103 U/L,P＜0.01)].低钙组和低钙高氟组BIP/GAPDH的表达高于对照组(1.38±0.24、1.35±0.12比1.14±0.06,P＜0.05).低钙高氟组的Xbp1/GAPDH的表达高于对照组和低钙组(1.48±0.20比1.02±0.25、1.07±0.25,P＜0.01);低钙高氟组的CHOP/GAPDH的表达高于对照组(0.84±0.18比0.52±0.07,P＜0.05).结论 氟中毒大鼠机体内氧化应激态和骨形成有明显的增强,并伴有骨组织细胞的内质网应激.说明内质网应激与氧化应激很可能都参与了氟骨症的发病机制.     

骨桥蛋白在低钙和燃煤污染型氟中毒大鼠肾组织中的表达

目的 观察骨桥蛋白(OPN)在低钙和氟中毒大鼠肾组织中的表达,探讨OPN与氟中毒肾损害的关系.方法 1月龄Wistar大鼠48只,雌雄各半,体质量80～100 g.按2×2析因设计将大鼠按质量随机分为4组:对照组、高氟组、低钙组、低钙高氟组,每组12只,雌雄各半.采用合成饲料喂养,高氟组和低钙高氟组饲料中的玉米来自燃煤污染型氟中毒病区,含氟量为100 mg/kg,对照组和低钙组饲料中的玉米来自于非病区,含氟量为5 mg/kg.喂养16周后处死大鼠,观察各组大鼠牙齿变化,计算大鼠氟斑牙检出率.取大鼠肾脏,采用RT-PCR技术及免疫组化技术检测大鼠肾脏OPN蛋白和OPN mRNA表达.结果 对照组和低钙组牙齿生长良好,高氟组、低钙高氟组大鼠均出现明显的氟斑牙,氟斑牙检出率为100%.免疫组化结果表明,OPN主要定位于肾组织的肾小管上皮细胞中.对照组和低钙组肾组织OPN阳性细胞淡染、散在分布,高氟组与低钙高氟组OPN阳性细胞着色较深,广泛分布在肾小管上皮细胞中.OPN蛋白表达显示,高氟组(168.64±13.21)和低钙高氟组(169.26±8.92)高于对照组(145.78±10.26,P均<0.01)和低钙组(149.60±16.84,P均<0.01);OPN mRNA表达显示,高氟组(1.89±0.37)和低钙高氟组(1.94±0.22)高于对照组(1.32±0.26,P均<0.05)和低钙组(1.30±0.18,P均<0.05).高氟影响OPN蛋白和OPN mRNA表达(F=13.821、4.24,P均<0.05),低钙不影响OPN蛋白和OPN mRNA表达(F=2.164、0.58,P均>0.05),但高氟和低钙联合作用时,二者对OPN蛋白和OPN mRNA表达有交互作用(F=6.257、4.32,P均<0.05).结论 过量氟可促使大鼠肾组织的OPN表达增加,提示OPN与氟中毒肾损害程度密切相关,可考虑作为一种氟中毒肾损害的标志,并且,高氟与低钙同时存在时,大鼠肾损害会进一步加重,提示低钙是氟化物损害肾脏的重要环节.     

投氟不同时间大鼠血清氧化应激和碱性磷酸酶活性的变化研究

目的 观察过量氟处理的大鼠在不同时间内氧化应激态与碱性磷酸酶(ALP)活性的变化.方法 24只Wistar大鼠,按体质量随机分成对照组、高氟组,每组12只.对照组大鼠饮用自来水(氟化钠<1 mg/L),高氟组大鼠饮用自来水中加入剂量为221 ms/L的氟化钠.实验期间动物自由进食、饮水,每周测体质量1次.实验时间分别为l、4、 8、 12周.通过生化方法检测大鼠血清丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GPx)、尿酸和ALP活性.结果 投氟与时间两因素对ALP活性影响有交互作用(F=4.690,P<0.05).投氟1周与12周大鼠血清的ALP活性[(19.29±3.69)、(15.72±0.79)kU/L]较对照组[(14.08±1.99)、(12.91±3.97)kU/L]明显升高(P均<0.05);投氟1周和4周大鼠血清MDA[(13.37±4.38)、(11.82±2.08)μmol/L]较对照组[(8.75±3.24)、(7.42±2.62)μmol/L]明显增高(P均<0.05);高氟组SOD、GPx与对照组比较,差异无统计学意义(P均>0.05);投氟1、4周大鼠血清尿酸[(89.53±13.21)、(88.47±19.78)μmol/L]较对照组[(77.79±11.43)、(65.42±13.42)μmol/L]明显增高(P均<0.05),而投氟8、12周大鼠尿酸[(67.21±9.44)、(73.95±9.52)μmol/L]低于对照组[(77.79±11.43)、(65.42±13.42)μmol/L,P均<0.05].结论 投与过量氟大鼠的ALP活性变化具有一定的时间依赖性;而投氟刺激大鼠氧化应激水平增强,这种刺激与投氟时间没有明显关联.     

燃煤污染型氟中毒大鼠学习记忆能力及胆碱酯酶活性改变

目的 观察燃煤污染型氟中毒大鼠学习记忆能力的变化,探讨其发生机制.方法 健康SD大鼠48只,按染氟剂量分为对照、低氟、高氟3组.低氟组、高氟组大鼠以燃煤型地方病氟中毒重病区燃煤烘烤的当地玉米为主要饲料,复制氟中毒大鼠模型,实验期为3、6个月,各8只大鼠.实验结束时用Morris水迷宫方法检测大鼠行为学变化,并用比色法测定脑组织乙酰胆碱酯酶和丁酰胆碱酯酶活性.结果 染氟剂量对逃避潜伏期时间、第7天穿过平台次数、逗留平台象限时间有明显影响(F值分别为29.29、6.47、6.50,P均<0.01):染氟时间对第7天穿过平台次数和逗留平台象限时间有明显影响(F值分别为16.11、45.59,P均<0.01);染氟剂量和时间对第7天穿过平台次数和逗留平台象限时间有交互作用(F值分别为4.67、5.68,P<0.05或<0.01).3个月时,高氟组大鼠逃避潜伏期时间[(14.71±4.85)s]较对照组[(9.28±4.22)s]明显增加(P<0.05);6个月时,低氟组、高氟组大鼠逃避潜伏期时间[(12.42±8.03)、(17.48±8.05)s]较对照组[(7.04±3.29)s]延长(P均<0.05),高氟组第7天穿过平台次数[(1.62±0.87)次]和逗留平台象限时间[(16.70±5.02)s]较对照组[(3.53±1.67)次、(23.33±5.35)s]降低(P均<0.05).染氟剂量对乙酰胆碱酯酶与丁酰胆碱酯酶活性均有明显影响(F值分别为12.83、13.27,P均<0.01);染氟时间对丁酰胆碱酯酶活性有明显影响(F=16.26,P<0.01).3个月时,低氟组丁酰胆碱酯酶活性[(0.55±0.12)kU/g]明显低于对照组[(0.73±0.10)kU/g,P<0.05],高氟组乙酰胆碱酯酶[(0.62±0.42)kU/g]和丁酰胆碱酯酶活性[(0.58±0.10)kU/g]均明显低于对照组[(1.41±0.52)、(0.73±0.10)kU/g,P均<0.05].胆碱酯酶活性与逃避潜伏期时间呈负相关(r=-0.68,P<0.01),胆碱酯酶活性与逗留平台象限时间呈正相关(r=0.57,P<0.01).结论 燃煤污染型慢性氟中毒大鼠学习记忆能力减退,可能与脑组织胆碱酯酶活性下降有关,二者间有量效关系.     

氟中毒对仔鼠行为学及脑尼古丁受体的影响

目的 探讨燃煤型氟中毒对仔鼠行为学及脑尼古丁受体的影响及其发生机制。方法SD大鼠随机分为2组,即对照组、高氟组。高氟组以氟病区燃煤烘烤的玉米为主要饲料,来复制氟中毒动物模型,饲养6个月后雌雄合笼,取30d龄仔鼠,用Morris水迷宫方法检测仔鼠行为学变化;比色法测定脑胆碱酯酶活性;蛋白印迹方法测定尼古丁受体蛋白水平;实时荧光定量PCR方法测定尼古丁受体mRNA水平。结果与对照组比较,高氟组仔鼠学习记忆能力、脑胆碱酯酶活性均显著降低,尼古丁受体α3、α4亚单位蛋白和mRNA水平平均降低[（30％及55％）,（8％及14％）;t=3．22,4．34,3．19,2．51],α7亚单位蛋白水平下降（48％,t=6．23）,mRNA水平升高（9％,t=-3.88）。α4、α7亚单位蛋白水平与仔鼠学习记忆能力呈显著相关。结论燃煤型氟中毒仔鼠学习记忆能力降低,可能与尼古丁受体蛋白含量降低及脑胆碱酯酶活性下降有关。     

燃煤污染型氟中毒大鼠肾组织中PURA基因及其蛋白的表达

目的 探讨PURA基因及其蛋白在燃煤污染型氟中毒大鼠肾组织中的表达情况.方法 SD大鼠36只,体质量80-100 g.将大鼠按体质量随机分对照组、加氟组、高氟组,每组12只,雌雄各半.对照组、加氟组和高氟组大鼠分别饲以含氟量为1.5、25.0、60.0 ms/ks的饲料.4个月后,采用RT-PCR技术及免疫组化技术检测大鼠肾组织PURA基因及其蛋白表达水平.结果 加氟组和高氟组大鼠肾组织PIJRA mRNA[(2.74±1.06)、(4.29±2.11)]及蛋白表达[(28 827.91±4801.94)、(61 146.96±4997.55)]均高于对照组[(1.13±0.87)、(7131.95±1524.54),P均<0.05)],高氟组大鼠肾组织PURA mRNA及蛋白表达高于低氟组(P均<0.05).结论 高氟可以导致大鼠肾组织PURA mRNA及蛋白表达水平增强.     

燃煤型氟中毒对大鼠中脑黑质神经元的影响

目的观察燃煤型氟中毒大鼠中脑黑质神经元的形态学变化,为地方性氟中毒引起脑损伤的发病机制提供实验依据。方法取SPF级SD大鼠90只,随机分为正常对照组、低氟组（3.3 mg/kg）和高氟组（106 mg/kg）,每组30只,雌雄各半。除对照组食用正常饲料外,其他各组均食用不同配方饲料,复制氟中毒大鼠模型。6个月后采用比色法测定各组大鼠黑质胆碱酯酶活性,然后取中脑黑质进行尼氏染色,TUNEL法细胞凋亡染色及TH免疫组化染色,光镜观察3组大鼠黑质神经元的形态变化,并测量TH阳性反应产物的平均光密度。结果随着染毒剂量的增加主要有：尿氟含量逐渐增多（P〈0.05）;学习记忆能力与胆碱酯酶活性逐渐下降（P〈0.05）;黑质细胞凋亡数量增多（P〈0.05）,TH阳性神经元减少（P〈0.05）。结论燃煤型氟中毒大鼠随染毒剂量的增加中脑黑质TH阳性神经元减少,细胞凋亡数量增多,这些变化可能是氟的神经毒性作用之一。     

慢性氟中毒大鼠脑组织中c-Jun氨基末端激酶表达水平观察

目的 观察慢性氟中毒大鼠脑组织中c-Jun氨基末端激酶(JNK)信号转导激酶表达变化,进一步揭示慢性氟中毒神经损伤的分子机制.方法 SD大鼠随机分为3组:对照组、低氟组、高氟组,每组24只,饮用水含氟量分别为＜0.5和5.0、50.0 mg/L,实验期为6个月.用氟离子选择电极法测定大鼠尿氟及血氟,用Western blotting和免疫组织化学方法检测脑组织中JNK信号转导激酶的表达和分布,并分析血氟与活化的JNK激酶的相关关系.结果低氟组和高氟组大鼠尿氟[(2.56±0.91)、(5.73±3.14)mg/L]和血氟[(0.36±0.14)、(0.50±0.18)mg/L]均较对照组[(0.92±0.30)、(0.12±0.07)mg/L]升高(P均＜0.05).高氟组(1.74±0.69)脑组织phospho-JNK表达高于对照组(1.00±0.37)和低氟组(1.20±0.28,P均＜0.05);total-JNK蛋白表达水平3组间比较,差异无统计学意义(F=0.046,P＞0.05).phospho-JNK、total-JNK阳性表达神经元主要集中在皮质、海马和背侧丘脑,其中高氟组大鼠phospho-JNK在顶叶皮质(119.3±14.1)、枕叶皮质(112.7±5.4)、海马CA3区(100.6±8.9)、背侧丘脑(117.8±10.4)及橄榄核(112.6±5.9)中阳性表达较对照组(104.1±8.9、106.6±9.6、106.6±9.7、108.9±6.4、100.3±8.4)和低氟组(96.7±17.1、102.5±8.3、106.4±6.5、110.2±9.3、102.4±4.7、102.5±9.8)明显增高(P均＜0.05),而total-JNK在各组大鼠脑组织中阳性表达分布未见明显改变(P均＞0.05).相关分析结果发现,随大鼠血氟升高,脑组织中phospho-JNK表达呈增高趋势,二者存在正相关关系(r=0.677).结论慢性氟中毒导致脑组织中磷酸化JNK表达改变,并与机体中氟蓄积量存在相关关系,这些改变可能与慢性氟中毒导致的神经损伤有关系.