Effects of Chronic Fluoride Poisoning on Calcium Signaling Pathways in the Hippocampus of Rats
Posted: Mon Oct 12, 2020 7:12 am
Zhang J - "Effects of Chronic Fluoride Poisoning on Calcium Signaling Pathways in the Hippocampus of Rats" Master's Thesis (2010), Zhejiang Normal University, Jinhua, Zhejiang Province, China.
https://www.zhangqiaokeyan.com/academic ... 76499.html
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Summary: An appropriate amount of fluoride is described as necessary for maintaining normal physiological activities, but fluoride is also described as a cumulative protoplasmic poison. Long-term excessive fluoride intake can damage bone organs (skeletal fluorosis, dental fluorosis) and non-skeletal organs (including blood and nervous systems). Nerve tissue is described as highly sensitive to fluoride, and the hippocampus is described as a central nervous system target site where neurons may undergo plastic changes due to fluoride. Mechanistic explanations proposed for fluoride neurotoxicity include G-protein signal transduction and oxidative stress. A commonly cited concept is that neurotoxin-related nervous system injury is closely related to Ca2+ homeostasis imbalance within G-protein signalling, potentially involving calcium-regulated NF-κB expression in oxidative stress sensitive neurons. This study used a chronic drinking-water fluorosis animal model, assessed dental fluorosis, blood fluoride, hippocampal neuronal morphology, synaptosomal Ca2+ concentration, and expression of Ca2+ signaling and apoptosis-related proteins (CaMKⅡα, c-fos, NF-κB p65, Bcl-2, Bax) in the hippocampal CA3 region.
Research method: A total of 152 newly weaned male SD rats were randomly divided into four groups: the control group (drinking tap water, with water fluoride content less than 0.5 mg/L), the low fluoride group, the medium fluoride group, and the high fluoride group (drinking water containing sodium fluoride at 15 mg/L, 30 mg/L, and 60 mg/L). Each group used the prepared solution as the only source of drinking water, and the rats had free access to food and water. The feeding time was 18 months. At the initial stage of fluoride exposure (3 months of age), the rats were tested for dental fluorosis and blood fluoride. The rats were then decapitated in two batches at the middle of fluoride exposure (9 months of age) and at the end of fluoride exposure (18 months of age). The hippocampus was examined by HE staining to detect pathological changes in the CA3 area. The fluorescent indicator Fura-2/AM was used to detect changes in Ca2+ concentration in hippocampal synapses in rats with chronic fluorosis. Immunohistochemistry was used to detect changes in the expression of CaMKⅡα, c-fos, NF-κB p65, Bcl-2, and Bax proteins in the hippocampal CA3 area.
Results: Excessive fluoride intake caused dental fluorosis in rats. With increasing fluoride concentration, the symptoms of dental fluorosis in rats increased, and the incisors were obviously deformed and defective, with chalky horizontal stripes. The blood fluoride concentration in each fluoride group was significantly or extremely significantly increased compared with the control group (P < 0.05 or P < 0.01), indicating that the animal fluorosis model was successfully replicated. HE staining results showed that chronic fluorosis damaged the morphological structure of rat nerve cells. Compared with the control group, hippocampal nerve cells in the fluoride groups were sparsely distributed, cell outlines disappeared, and vacuoles appeared. These changes became more significant with increasing fluoride exposure time and fluoride concentration. Results for synaptosomal Ca2+ concentration showed that, with increasing age in rats, synaptosomal Ca2+ concentration in the control group tended to increase, and fluoride treatment aggravated Ca2+ overload. At the mid-stage of fluoride exposure, compared with the control group, synaptosomal Ca2+ concentration in the medium fluoride group and the high fluoride group increased significantly or extremely significantly (P < 0.05 or P < 0.01). At the end of fluoride exposure, compared with the control group, synaptosomal Ca2+ concentration in the medium fluoride group and the high fluoride group increased significantly (P < 0.01). Immunohistochemistry results showed that, with increasing age in rats, the protein expression of CaMKⅡα, c-fos, and NF-κB p65 in the control group and in each fluoride-exposed group increased to different degrees, and increasing fluoride exposure time and fluoride dose made these changes more pronounced. At the mid-stage of fluoride exposure, compared with the control group, CaMKⅡα expression in the medium and high fluoride groups increased significantly (P < 0.05), c-fos expression increased significantly or extremely significantly (P < 0.05 or P < 0.01), and although there was no significant difference in NF-κB p65 expression, it tended to increase with increasing fluoride dose. At the end of fluoride exposure, compared with the control group, CaMKⅡα and c-fos expression in the medium fluoride group and the high fluoride group increased significantly (P < 0.01), and NF-κB p65 expression in the fluoride groups increased significantly (P < 0.05). Immunohistochemistry results also showed that, with increasing age, the expression levels of Bcl-2 and Bax proteins in the control group and in each fluoride-exposed group declined to different degrees, and increasing fluoride exposure time and fluoride dose made these declines more pronounced. At the mid-stage of fluoride exposure, compared with the control group, Bcl-2 expression in the medium and high fluoride groups decreased significantly or extremely significantly (P < 0.05 or P < 0.01), and Bax expression decreased significantly (P < 0.01). At the end of fluoride exposure, compared with the control group, Bcl-2 expression in the medium fluoride group and the high fluoride group decreased significantly or extremely significantly (P < 0.05 or P < 0.01), and Bax expression in the low fluoride group, the medium fluoride group, and the high fluoride group decreased significantly or extremely significantly (P < 0.05, P < 0.01, P < 0.01). At the same time, at the mid-stage of fluoride exposure, the ratio of Bax to Bcl-2 (Bax/Bcl-2) tended to increase with fluoride exposure time and concentration. At the end of fluoride exposure, compared with the control group, the Bax/Bcl-2 ratio in the medium fluoride group increased significantly (P < 0.05), suggesting that nerve cells were in an apoptotic state.
Conclusion: In summary, chronic fluoride exposure has toxic effects on the central nervous system. Fluoride can cause changes in the morphology and structure of hippocampal nerve cells, resulting in hippocampal synaptosomal Ca2+ overload, which in turn leads to abnormal protein expression of CaMKⅡα, c-fos, NF-κB p65, Bcl-2, and Bax in the calcium ion signaling pathway, and finally makes nerve cells prone to apoptosis. During aging, degenerative changes in nerve cells can also lead to apoptosis of rat nerve cells, and fluoride treatment exacerbates this change. This study further clarified the molecular mechanism of fluoride neurotoxicity through the calcium ion signaling pathway and provided a theoretical basis for deeper exploration of the molecular biological mechanism by which chronic fluorosis affects the central nervous system. In addition, NF-κB expression has a sensitive dose effect relationship with fluoride exposure time and concentration, and it may be a target molecule related to fluoroneurotoxicity. This provides a certain theoretical basis for the development of biomarkers of fluoroneurotoxicity.
《浙江师范大学》 2010年收藏 | 手机打开
慢性氟中毒对大鼠脑海马钙离子信号通路的影响
张璟
【摘要】： 适量氟是维持机体正常生理活动的基础,但氟是一种蓄积性原生质毒物,机体长期过量摄入氟可引起氟骨症、氟斑牙等骨相器官的损害以及血液、神经系统等非骨相器官的损害。神经组织对氟的敏感性很高,海马作为中枢神经系统中氟中毒的靶部位之一,其神经细胞易受氟的影响而发生可塑性变化。近年来,通过对氟神经毒性作用机制的研究,人们主要提出了G蛋白信号转导理论与氧化应激理论。 目前普遍认为,神经毒物引起的神经系统损伤与G蛋白信号转导通路中Ca2+稳态失调有密切关系。其机制可能与神经细胞内对氧化应激敏感且受钙离子调控的核转录因子(nuclear factor kappa-B, NF-κB)表达等有关。因此,本实验通过复制饮水型慢性氟中毒动物模型,以观察和检测氟中毒引起氟斑牙、血氟水平异常及海马神经细胞形态结构改变为基础,首次在氟的神经毒性研究中将钙离子信号通路与氧化应激理论结合,检测氟中毒对大鼠海马突触体内Ca2+浓度以及钙离子信号通路中CaMKⅡ、c-fos、NF-κB、Bcl-2及Bax蛋白表达的影响。 研究方法：152只初断乳雄性SD大鼠随机分为四组,即对照组(饮用自来水,水氟含量低于0.5mg/L),低氟组、中氟组、高氟组(饮水含氟化钠分别为15mg/L、30mg/L、60mg/L)。各组以配制的溶液作为饮水唯一来源,自由摄食、饮水,饲养时间为18个月。染氟初期(3月龄)对大鼠进行氟斑牙和血氟检测,染氟中期(9月龄)和染氟结束(18月龄)分两批断头处死大鼠,HE染色检测海马CA3区病理学改变,荧光指示剂Fura-2/AM检测慢性氟中毒大鼠海马突触体内Ca2+浓度的变化,免疫组化法检测海马CA3区CaMKⅡα、c-fos、NF-κBρ65、Bcl-2及Bax蛋白表达的改变。 1、过量氟摄入会导致大鼠氟斑牙发生,随染氟浓度升高,大鼠氟斑牙症状加剧,切齿明显变形、缺损并出现白垩状横纹；各染氟组血氟浓度与对照组相比显著或极显著升高(P0.05或P0.01)。表明动物氟中毒模型复制成功。 2、HE染色结果显示,慢性氟中毒可损伤大鼠神经细胞形态结构。与对照组相比,染氟组大鼠海马神经细胞分布稀疏,细胞轮廓消失,并出现空泡。且这种变化随染氟时间和染氟浓度的递增而更为显著。 3、突触体Ca2+浓度检测结果显示,随大鼠月龄增长,对照组突触体Ca2+浓度有升高的趋势,而染氟处理加剧了Ca2+的超载。染氟中期：与对照组相比,中氟组、高氟组突触体Ca2+浓度显著或极显著升高(P0.05或P0.01)；染氟结束：与对照组相比,中氟组、高氟组突触体Ca2+浓度极显著升高(P0.01)。 4、IHC结果显示,随大鼠月龄增长,对照组及各染氟组CaMKⅡα、c-fos、NF-κBρ65蛋白表达均有不同水平的上升,且染氟时间和染氟剂量的递增使这种变化更为显著。染氟中期：与对照组相比,中氟组、高氟组CaMKⅡα表达显著升高(P0.05),c-fos表达显著或极显著升高(P0.05或P0.01),NF-κBρ65表达虽无显著性差异,但随染氟剂量升高其表达有上升的趋势；染氟结束：与对照组相比,中氟组、高氟组CaMKⅡαc-fos表达极显著升高(P0.01),中氟组NF-κBρ65表达显著升高(P0.05)。 5、IHC结果显示,随大鼠月龄增长,对照组及各染氟组Bcl-2和Bax蛋白表达水平均有不同水平的下降,且染氟时间和染氟剂量的递增使这种变化更为显著。染氟中期：与对照组相比,中氟组、高氟组Bcl-2表达显著或极显著下降(P0.05或P0.01),Bax表达极显著下降(P0.01)；染氟结束：与对照组相比,中氟组、高氟组Bcl-2表达显著或极显著下降(P0.05或P0.01),低氟组、中氟组、高氟组Bax表达显著或极显著下降(P0.05,P0.01,P0.01)。同时,染氟中期：Bax与Bcl-2比值(Bax/Bcl-2)随染氟时间和染氟浓度的递增有升高的趋势；染氟结束：与对照组相比,中氟组、高氟组Bax/Bcl-2比值显著上升(P0.05),提示神经细胞处于凋亡状态。 综合上述,慢性氟暴露对中枢神经系统有毒性作用。氟可导致海马神经细胞形态结构发生改变,引起海马突触体Ca2+超载,继而导致钙离子信号通路中CaMKⅡα、c-fos、NF-κBρ65、Bcl-2及Bax的蛋白表达异常,最终诱导神经细胞趋于凋亡。衰老过程中,神经细胞的退行性改变也可导致大鼠神经细胞凋亡,而染氟处理加剧了这种变化。本研究进一步阐明了氟神经毒性对钙离子信号通路影响的分子机理,为深入探讨慢性氟中毒对中枢神经系统毒性作用的分子生物学机制提供了一定的理论基础。此外,NF-κB的表达与染氟时间、浓度有敏感的量效关系,可能是氟神经毒性相关靶分子,这为开展氟神经毒性的生物标志物研究提供一定的理论依据。
https://www.zhangqiaokeyan.com/academic ... 76499.html
https://xueshu.baidu.com/ndscholar/brow ... =xueshu_se
Summary: An appropriate amount of fluoride is described as necessary for maintaining normal physiological activities, but fluoride is also described as a cumulative protoplasmic poison. Long-term excessive fluoride intake can damage bone organs (skeletal fluorosis, dental fluorosis) and non-skeletal organs (including blood and nervous systems). Nerve tissue is described as highly sensitive to fluoride, and the hippocampus is described as a central nervous system target site where neurons may undergo plastic changes due to fluoride. Mechanistic explanations proposed for fluoride neurotoxicity include G-protein signal transduction and oxidative stress. A commonly cited concept is that neurotoxin-related nervous system injury is closely related to Ca2+ homeostasis imbalance within G-protein signalling, potentially involving calcium-regulated NF-κB expression in oxidative stress sensitive neurons. This study used a chronic drinking-water fluorosis animal model, assessed dental fluorosis, blood fluoride, hippocampal neuronal morphology, synaptosomal Ca2+ concentration, and expression of Ca2+ signaling and apoptosis-related proteins (CaMKⅡα, c-fos, NF-κB p65, Bcl-2, Bax) in the hippocampal CA3 region.
Research method: A total of 152 newly weaned male SD rats were randomly divided into four groups: the control group (drinking tap water, with water fluoride content less than 0.5 mg/L), the low fluoride group, the medium fluoride group, and the high fluoride group (drinking water containing sodium fluoride at 15 mg/L, 30 mg/L, and 60 mg/L). Each group used the prepared solution as the only source of drinking water, and the rats had free access to food and water. The feeding time was 18 months. At the initial stage of fluoride exposure (3 months of age), the rats were tested for dental fluorosis and blood fluoride. The rats were then decapitated in two batches at the middle of fluoride exposure (9 months of age) and at the end of fluoride exposure (18 months of age). The hippocampus was examined by HE staining to detect pathological changes in the CA3 area. The fluorescent indicator Fura-2/AM was used to detect changes in Ca2+ concentration in hippocampal synapses in rats with chronic fluorosis. Immunohistochemistry was used to detect changes in the expression of CaMKⅡα, c-fos, NF-κB p65, Bcl-2, and Bax proteins in the hippocampal CA3 area.
Results: Excessive fluoride intake caused dental fluorosis in rats. With increasing fluoride concentration, the symptoms of dental fluorosis in rats increased, and the incisors were obviously deformed and defective, with chalky horizontal stripes. The blood fluoride concentration in each fluoride group was significantly or extremely significantly increased compared with the control group (P < 0.05 or P < 0.01), indicating that the animal fluorosis model was successfully replicated. HE staining results showed that chronic fluorosis damaged the morphological structure of rat nerve cells. Compared with the control group, hippocampal nerve cells in the fluoride groups were sparsely distributed, cell outlines disappeared, and vacuoles appeared. These changes became more significant with increasing fluoride exposure time and fluoride concentration. Results for synaptosomal Ca2+ concentration showed that, with increasing age in rats, synaptosomal Ca2+ concentration in the control group tended to increase, and fluoride treatment aggravated Ca2+ overload. At the mid-stage of fluoride exposure, compared with the control group, synaptosomal Ca2+ concentration in the medium fluoride group and the high fluoride group increased significantly or extremely significantly (P < 0.05 or P < 0.01). At the end of fluoride exposure, compared with the control group, synaptosomal Ca2+ concentration in the medium fluoride group and the high fluoride group increased significantly (P < 0.01). Immunohistochemistry results showed that, with increasing age in rats, the protein expression of CaMKⅡα, c-fos, and NF-κB p65 in the control group and in each fluoride-exposed group increased to different degrees, and increasing fluoride exposure time and fluoride dose made these changes more pronounced. At the mid-stage of fluoride exposure, compared with the control group, CaMKⅡα expression in the medium and high fluoride groups increased significantly (P < 0.05), c-fos expression increased significantly or extremely significantly (P < 0.05 or P < 0.01), and although there was no significant difference in NF-κB p65 expression, it tended to increase with increasing fluoride dose. At the end of fluoride exposure, compared with the control group, CaMKⅡα and c-fos expression in the medium fluoride group and the high fluoride group increased significantly (P < 0.01), and NF-κB p65 expression in the fluoride groups increased significantly (P < 0.05). Immunohistochemistry results also showed that, with increasing age, the expression levels of Bcl-2 and Bax proteins in the control group and in each fluoride-exposed group declined to different degrees, and increasing fluoride exposure time and fluoride dose made these declines more pronounced. At the mid-stage of fluoride exposure, compared with the control group, Bcl-2 expression in the medium and high fluoride groups decreased significantly or extremely significantly (P < 0.05 or P < 0.01), and Bax expression decreased significantly (P < 0.01). At the end of fluoride exposure, compared with the control group, Bcl-2 expression in the medium fluoride group and the high fluoride group decreased significantly or extremely significantly (P < 0.05 or P < 0.01), and Bax expression in the low fluoride group, the medium fluoride group, and the high fluoride group decreased significantly or extremely significantly (P < 0.05, P < 0.01, P < 0.01). At the same time, at the mid-stage of fluoride exposure, the ratio of Bax to Bcl-2 (Bax/Bcl-2) tended to increase with fluoride exposure time and concentration. At the end of fluoride exposure, compared with the control group, the Bax/Bcl-2 ratio in the medium fluoride group increased significantly (P < 0.05), suggesting that nerve cells were in an apoptotic state.
Conclusion: In summary, chronic fluoride exposure has toxic effects on the central nervous system. Fluoride can cause changes in the morphology and structure of hippocampal nerve cells, resulting in hippocampal synaptosomal Ca2+ overload, which in turn leads to abnormal protein expression of CaMKⅡα, c-fos, NF-κB p65, Bcl-2, and Bax in the calcium ion signaling pathway, and finally makes nerve cells prone to apoptosis. During aging, degenerative changes in nerve cells can also lead to apoptosis of rat nerve cells, and fluoride treatment exacerbates this change. This study further clarified the molecular mechanism of fluoride neurotoxicity through the calcium ion signaling pathway and provided a theoretical basis for deeper exploration of the molecular biological mechanism by which chronic fluorosis affects the central nervous system. In addition, NF-κB expression has a sensitive dose effect relationship with fluoride exposure time and concentration, and it may be a target molecule related to fluoroneurotoxicity. This provides a certain theoretical basis for the development of biomarkers of fluoroneurotoxicity.
《浙江师范大学》 2010年收藏 | 手机打开
慢性氟中毒对大鼠脑海马钙离子信号通路的影响
张璟
【摘要】： 适量氟是维持机体正常生理活动的基础,但氟是一种蓄积性原生质毒物,机体长期过量摄入氟可引起氟骨症、氟斑牙等骨相器官的损害以及血液、神经系统等非骨相器官的损害。神经组织对氟的敏感性很高,海马作为中枢神经系统中氟中毒的靶部位之一,其神经细胞易受氟的影响而发生可塑性变化。近年来,通过对氟神经毒性作用机制的研究,人们主要提出了G蛋白信号转导理论与氧化应激理论。 目前普遍认为,神经毒物引起的神经系统损伤与G蛋白信号转导通路中Ca2+稳态失调有密切关系。其机制可能与神经细胞内对氧化应激敏感且受钙离子调控的核转录因子(nuclear factor kappa-B, NF-κB)表达等有关。因此,本实验通过复制饮水型慢性氟中毒动物模型,以观察和检测氟中毒引起氟斑牙、血氟水平异常及海马神经细胞形态结构改变为基础,首次在氟的神经毒性研究中将钙离子信号通路与氧化应激理论结合,检测氟中毒对大鼠海马突触体内Ca2+浓度以及钙离子信号通路中CaMKⅡ、c-fos、NF-κB、Bcl-2及Bax蛋白表达的影响。 研究方法：152只初断乳雄性SD大鼠随机分为四组,即对照组(饮用自来水,水氟含量低于0.5mg/L),低氟组、中氟组、高氟组(饮水含氟化钠分别为15mg/L、30mg/L、60mg/L)。各组以配制的溶液作为饮水唯一来源,自由摄食、饮水,饲养时间为18个月。染氟初期(3月龄)对大鼠进行氟斑牙和血氟检测,染氟中期(9月龄)和染氟结束(18月龄)分两批断头处死大鼠,HE染色检测海马CA3区病理学改变,荧光指示剂Fura-2/AM检测慢性氟中毒大鼠海马突触体内Ca2+浓度的变化,免疫组化法检测海马CA3区CaMKⅡα、c-fos、NF-κBρ65、Bcl-2及Bax蛋白表达的改变。 1、过量氟摄入会导致大鼠氟斑牙发生,随染氟浓度升高,大鼠氟斑牙症状加剧,切齿明显变形、缺损并出现白垩状横纹；各染氟组血氟浓度与对照组相比显著或极显著升高(P0.05或P0.01)。表明动物氟中毒模型复制成功。 2、HE染色结果显示,慢性氟中毒可损伤大鼠神经细胞形态结构。与对照组相比,染氟组大鼠海马神经细胞分布稀疏,细胞轮廓消失,并出现空泡。且这种变化随染氟时间和染氟浓度的递增而更为显著。 3、突触体Ca2+浓度检测结果显示,随大鼠月龄增长,对照组突触体Ca2+浓度有升高的趋势,而染氟处理加剧了Ca2+的超载。染氟中期：与对照组相比,中氟组、高氟组突触体Ca2+浓度显著或极显著升高(P0.05或P0.01)；染氟结束：与对照组相比,中氟组、高氟组突触体Ca2+浓度极显著升高(P0.01)。 4、IHC结果显示,随大鼠月龄增长,对照组及各染氟组CaMKⅡα、c-fos、NF-κBρ65蛋白表达均有不同水平的上升,且染氟时间和染氟剂量的递增使这种变化更为显著。染氟中期：与对照组相比,中氟组、高氟组CaMKⅡα表达显著升高(P0.05),c-fos表达显著或极显著升高(P0.05或P0.01),NF-κBρ65表达虽无显著性差异,但随染氟剂量升高其表达有上升的趋势；染氟结束：与对照组相比,中氟组、高氟组CaMKⅡαc-fos表达极显著升高(P0.01),中氟组NF-κBρ65表达显著升高(P0.05)。 5、IHC结果显示,随大鼠月龄增长,对照组及各染氟组Bcl-2和Bax蛋白表达水平均有不同水平的下降,且染氟时间和染氟剂量的递增使这种变化更为显著。染氟中期：与对照组相比,中氟组、高氟组Bcl-2表达显著或极显著下降(P0.05或P0.01),Bax表达极显著下降(P0.01)；染氟结束：与对照组相比,中氟组、高氟组Bcl-2表达显著或极显著下降(P0.05或P0.01),低氟组、中氟组、高氟组Bax表达显著或极显著下降(P0.05,P0.01,P0.01)。同时,染氟中期：Bax与Bcl-2比值(Bax/Bcl-2)随染氟时间和染氟浓度的递增有升高的趋势；染氟结束：与对照组相比,中氟组、高氟组Bax/Bcl-2比值显著上升(P0.05),提示神经细胞处于凋亡状态。 综合上述,慢性氟暴露对中枢神经系统有毒性作用。氟可导致海马神经细胞形态结构发生改变,引起海马突触体Ca2+超载,继而导致钙离子信号通路中CaMKⅡα、c-fos、NF-κBρ65、Bcl-2及Bax的蛋白表达异常,最终诱导神经细胞趋于凋亡。衰老过程中,神经细胞的退行性改变也可导致大鼠神经细胞凋亡,而染氟处理加剧了这种变化。本研究进一步阐明了氟神经毒性对钙离子信号通路影响的分子机理,为深入探讨慢性氟中毒对中枢神经系统毒性作用的分子生物学机制提供了一定的理论基础。此外,NF-κB的表达与染氟时间、浓度有敏感的量效关系,可能是氟神经毒性相关靶分子,这为开展氟神经毒性的生物标志物研究提供一定的理论依据。