Effect of fluoride exposure on endoplasmic reticulum-mitochondrial calcium transfer and apoptosis in primary nerve cells

doi:10.12307/2025.929

Abstract

Abstract: BACKGROUND: Previous studies have found that neuronal damage caused by continuous excessive fluoride exposure is related to Ca2+ overload, but the mechanism of Ca2+ flow conversion between intracellular calcium stores and cell apoptosis damage is still unclear.
OBJECTIVE: To investigate the effect of fluoride exposure on Ca2+ transport channel proteins and apoptosis levels in the mitochondria-associated endoplasmic reticulum membrane of primary cultured neural cells.
METHODS: Primary nerve cells of neonatal SD rats were cultured in vitro and identified by immunofluorescence staining with neuronal nucleus-specific antibody up to day 7. The nerve cells were divided into control group (containing 0 mmol/L sodium fluoride), low fluoride group (containing 0.5 mmol/L sodium fluoride), and high fluoride group (containing 1 mmol/L sodium fluoride). The cell morphological changes were observed by light microscope 24 hours after fluorine exposure. The expression levels of apoptosis-related protein BAX/BCL-2 and calcium transfer-related pathways VDAC1, GRP 75, and IP3R were detected using western blot assay. The expression levels of VDAC1, GRP 75, and IP3R mRNA were detected by RT-PCR. Ca2+ levels were detected by Rhood-2AM Ca2+ probe. Mitochondrial membrane potential detection kit was used to detect the change in mitochondrial membrane potential. The level of apoptosis was determined by flow cytometry and TUNEL staining.
RESULTS AND CONCLUSION: (1) The purity of neurons cultured on day 7 had been determined to be over 90%, with few impurities, good growth status, and tight cell network connections, meeting the requirements of subsequent experiments. (2) Compared with the control group, growth of neural cell clusters in the low-fluoride group and the high-fluoride group increased; the processes were broken; the cell body was rounded, and the connection network between cells was destroyed. Compared with the low-fluoride group, the cell damage changes in the high-fluoride group were more obvious. (3) Compared with the control group, the protein expressions of VDAC1, GRP75, and IP3R were increased in the low-fluoride group and the high-fluoride group (P < 0.05), and the ratio of apoptosis-related protein BAX/BCL-2 was increased (P < 0.05). Compared with the control group, the expression of VDAC1 and GRP75 mRNA in the low-fluoride group was significantly increased (P < 0.05); the expression levels of VDAC1, GRP75, and IP3R mRNA in the high-fluoride group were significantly increased (P < 0.01). (4) The level of cell apoptosis increased significantly after fluoride exposure, and the high-fluoride group was significantly higher than the control and low-fluoride groups (P < 0.01). (5) After fluoride exposure, the concentration of mitochondrial Ca2+ in nerve cells increased significantly (P < 0.05), the mitochondrial membrane potential decreased (P < 0.01), and the degree of damage in the high-fluoride group was more obvious (P < 0.05). The results show that fluoride exposure impairs the morphological structure of primary neural cells, resulting in upregulation of Ca2+ transfer pathway protein expression between the endoplasmic reticulum and mitochondria, mitochondrial Ca2+ overload, mitochondrial damage, and increased levels of apoptosis.

Key words: fluoride exposure, primary nerve cell, Ca2+ transfer, apoptosis, mitochondrial membrane potential, mitochondrial damage, mitochondria-associated endoplasmic reticulum membrane, calcium transport channel

CLC Number:

Lu Yongheng, Zhu Shuang, Zhao Feiyan, Ai Fujun, Liu Yanjie, Dong Yangting, Guan Zhizhong, Wei Na. Effect of fluoride exposure on endoplasmic reticulum-mitochondrial calcium transfer and apoptosis in primary nerve cells[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(1): 111-119.

Figures/Tables 2

2.1 原代神经细胞的形态如图1所示，培养至第7天的神经细胞状态良好，体积较大、胞体圆润，细胞质丰富，光晕明显，细胞呈轻度聚集生长，细胞突起丰富，细胞之间相互连接形成紧密网络。 2.2 原代神经细胞的鉴定结果使用NeuN进行免疫荧光染色鉴定，NeuN是一种神经元特异性核蛋白，主要表达于成熟的神经细胞核中，表达量与神经细胞数量呈正相关，可用于定量分析神经细胞数目变化(图2所示，蓝色：DAPI，红色：NeuN)。随机拍取6个200倍视野，使用Image J计算神经细胞的纯度为(95.00±2.31)%。 2.3 氟处理后神经细胞形态变化如图3所示，氟暴露处理24 h后，于光学显微镜下观察神经细胞形态变化，可见对照组神经细胞呈轻度聚集生长，状态良好，胞体饱满，圆润，突起丰富，突起之间的连接较为紧密；与对照组比较，氟暴露组神经细胞胞体出现肿胀、圆缩，细胞之间聚集生长程度增加，细胞突起变短或缺失，细胞间突起连接减少；与低氟组比较，高氟组细胞形态变化更明显，甚至部分细胞出现死亡，细胞碎片增加。 2.4 染氟处理后线粒体Ca2+浓度变化如图4所示，经线粒体Ca2+指示剂Rhod-2-AM处理后，荧光显微镜下可见Ca2+广泛分布于神经细胞胞质及突起内，且随氟暴露浓度增加，细胞内Ca2+荧光染色逐渐增强，高氟组线粒体Ca2+浓度明显高于对照组和低氟组(P < 0.05)。 2.5 染氟处理后线粒体膜电位变化线粒体膜电位检测是一种以JC-1为荧光探针，检测细胞、组织等膜电位变化的实验方法，可用于早期的细胞凋亡检测，当线粒体膜电位较高时，JC-1可以在线粒体的基质中形成聚合物(J-aggregates)，产生红色荧光，当线粒体膜电位较低时，JC-1不能聚集在线粒体的基质中，以单体(monomer)形式存在，产生绿色荧光。通过JC-1从红色荧光到绿色荧光的转变可以很容易地检测到细胞膜电位的下降。如图5所示，氟暴露导致原代神经细胞线粒体膜电位降低，且高氟组神经细胞膜电位下降最为明显。"

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