RESUMEN
Objective:To study the effects of fluoride on apoptosis and oxidative stress levels of spinal cord nerve cells in rats.Methods:A total of 54 6-week-old Sprague-Dawley female rats, weighing 150 - 200 g, were selected and fed for 1 week. They were divided into a control group [given deionized water containing 0 mg/L sodium fluoride (NaF)], a low fluoride group (given deionized water containing 50 mg/L NaF), and a high fluoride group (given deionized water containing 100 mg/L NaF) using a random number table method, with 18 rats in each group. All groups received standard feed. After 4, 8, and 12 weeks of fluoride exposure, six rats were selected from each group to observe the occurrence of dental fluorosis, and the motor function of hind limbs in rats was evaluated based on the Basso-Beattie-Bresnahan (BBB) score. Then the rats were anesthetized with 5% chloral hydrate via intraperitoneal injection and euthanized by cardiac puncture. Spinal cord tissue of the rats was collected to detect the activities of oxidative stress factors such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), as well as the contents of malondialdehyde (MDA) and catalase (CAT). After 12 weeks of fluoride exposure, morphologic changes in rat spinal cord neurons were observed using Nissl staining, and apoptosis of spinal cord nerve cells was detected using the TdT mediated dUTP nick end labeling (TUNEL) cell apoptosis detection kit. The Western blotting was used to detect the expression of B-lymphoblastoma-2 (Bcl-2) gene related X protein (Bax), Bcl-2 promoter (Bad), and Bcl-2 protein in rat spinal cord tissue; immunofluorescence staining was used to observe the expression of Bax and Bcl-2 protein in spinal cord neurons.Results:After 12 weeks of fluoride exposure, rats in both the low fluoride and high fluoride groups developed varying degrees of dental fluorosis; the differences of BBB scores of rats in the control, low fluoride, and high fluoride groups were statistically significant ( F = 14.09, P < 0.001). The differences of SOD [(124.04 ± 4.87), (96.66 ± 15.01), (91.12 ± 15.87) U/mg prot] and GSH-Px activitives [(561.92 ± 59.65), (456.83 ± 29.51), (385.07 ± 74.87) U/mg prot], MDA [(9.96 ± 1.50), (16.64 ± 2.05), (20.80 ± 3.37) nmol/mg prot] and CAT contents [(8.97 ± 1.05), (6.39 ± 0.97), (6.42 ± 0.83) nmol/mg prot] among the control, low fluoride, and high fluoride groups were statistically significant ( F = 11.17, 14.19, 30.12, 14.52, P < 0.05). Among them, the SOD, GSH-Px activities, and CAT content in the low fluoride and high fluoride groups were lower than those in the control group, while the MDA content was higher than that in the control group ( P < 0.05). The GSH-Px activity in the high fluoride group was lower than that in the low fluoride group, and MDA content was higher than that in the low fluoride group ( P < 0.05). The intact neuronal structures and clear visible nuclei were seen, and Nissl bodies were uniformly stained in the spinal cord neurons of the control group rats, with more numbers, and no apoptotic cells were observed; the staining of Nissl bodies in the spinal cord neurons of rats was uneven in the low fluoride and high fluoride groups, with fewer numbers, and more apoptotic cells. There were statistically significant differences in the apoptosis rate of spinal cord nerve cells and the expression levels of Bax, Bad, and Bcl-2 protein in the spinal cord tissues of rats in the control, low fluoride, and high fluoride groups ( F = 272.81, 35.53, 17.57, 92.50, P < 0.05). The results of immunofluorescence staining showed that there were statistically significant differences in the fluorescent intensity of Bax and Bcl-2 proteins in the spinal cord neurons of rats in the control, low fluoride, and high fluoride groups ( F = 12.67, 22.14, P < 0.05). Conclusion:Chronic fluorosis induces a decrease in antioxidant enzyme activity, an increase in lipid peroxidation levels, and an increase in neuronal apoptosis in the spinal cord of rats.
RESUMEN
Objective:To explore the changes of mitophagy- and apoptosis-related protein expressions after spinal cord injury in rats and its related mechanism.Methods:Ninety-six healthy female SD rats were divided into sham surgery group ( n=48) and spinal cord injury group ( n=48) according to the random number table. Each group was divided into six time points of 1, 3, 7, 14, 21 and 28 days with 8 rats at each time point. In the sham surgery group, the T 8-9 spinous processes and vertebral plates were removed without damage to the spinal cord; in the spinal cord injury group, the spinal cord injury model was established using Allen′s method. At each post-injury time point in two groups, BBB score was used to evaluate the motor function of the rats; HE staining was used to observe the histopathological changes of the spinal cord; immunofluorescence was used to observe the co-localized positive cells of the voltage-dependent anion channel protein 1 (VDAC1) with microtubule-associated protein 1 light chain 3 (LC3) II, E3 ubiquitin ligase (Parkin), and polyubiquitin-binding protein (p62); Western blotting was used to observe expressions of the mitochondrial LC3 II/LC3 I, cytoplasmic Parkin, mitochondrial Parkin, cytoplasmic p62, mitochondrial p62, cytoplasmic apoptotic proteins (Bax), mitochondrial Bax, cytoplasmic cytochrome C (Cyt C), and mitochondrial Cyt C. Results:(1) Compared with the sham surgery group [(21.00±0.00)points at all time points], the BBB scores in the spinal cord injury group were (0.94±0.50)points, (1.69±0.70)points, (4.13±0.99)points, (11.81±1.03)points, (15.06±1.12)points and (18.38±0.83)points at 1, 3, 7, 14, 21 and 28 days after injury, respectively ( P<0.01). (2) Compared with the sham surgery group, the structure in the spinal cord injury group was significantly disrupted at 1 and 3 days after injury, when a large number of hemorrhagic foci, inflammatory cell infiltration, neuronal swelling, and cavity formation were observed. At 7 and 14 days after injury, the number of hemorrhagic foci was markedly reduced compared with the earlier period, when swollen neuronal cytosols, inflammatory cell infiltration and a large number of cavities were found. At 21 and 28 days after injury, a large number of cells were seen to be involved in the repair and the arrangement of cells was disorganized. (3) Compared with the sham surgery group, the number of co-localized positive cells of VDAC1 with LC3 II, Parkin and p62 separately in the spinal cord injury group, markedly increased at 1 and 3 days after surgery, and gradually decreased after that. (4) In the sham surgery group and at 1, 3, 7, 14, 21 and 28 days after injury in the spinal cord injury group, the mitochondrial LC3 II/LC3 I expression levels were 0.56±0.05, 1.00±0.05, 1.19±0.11, 0.86±0.05, 0.80±0.08, 0.66±0.13 and 0.51±0.11, respectively; the cytoplasmic Parkin expression levels were 0.80±0.13, 0.47±0.08, 0.29±0.06, 0.57±0.07, 0.70±0.05, 0.97±0.09 and 0.88±0.12, respectively; the mitochondrial Parkin expression levels were 0.67±0.09, 1.07±0.18, 1.27±0.15, 0.82±0.12, 0.59±0.09, 0.53±0.13 and 0.57±0.14, respectively; the cytoplasmic p62 expression levels were 1.25±0.08, 1.04±0.04, 0.94±0.05, 1.09±0.05, 1.19±0.06, 1.20±0.04 and 1.27±0.05, respectively; the mitochondrial p62 expression levels were 0.61±0.06, 0.88±0.07, 1.09±0.09, 0.98±0.07, 0.70±0.08, 0.68±0.08 and 0.60±0.09, respectively; the cytoplasmic Bax expression levels were 0.92±0.08, 0.67±0.07, 0.36±0.08, 0.48±0.08, 0.69±0.06, 0.88±0.11 and 0.94±0.08, respectively; the mitochondrial Bax expression levels were 0.57±0.04, 0.74±0.04, 0.91±0.05, 0.76±0.05, 0.63±0.08, 0.61±0.05 and 0.57±0.05, respectively; the cytoplasmic Cyt C expression levels were 0.28±0.05, 0.81±0.07, 1.12±0.08, 0.64±0.07, 0.67±0.13, 0.60±0.11 and 0.37±0.06, respectively; and the mitochondrial Cyt C expression levels were 1.02±0.07, 0.91±0.14, 0.37±0.07, 0.73±0.06, 0.91±0.11, 0.95±0.13 and 1.10±0.15, respectively. Compared with the sham surgery group, in the spinal cord injury group mitochondrial LC3II/LC3I had the highest expression level at 3 days after injury; cytoplasmic Parkin had the lowest expression level at 3 days after injury; mitochondrial Parkin had the highest expression level at 3 days after injury; cytoplasmic p62 had the lowest expression level at 3 days after injury; mitochondrial p62 had the highest expression level at 3 days after injury; cytoplasmic Bax had the lowest expression level at 3 days after injury; mitochondrial Bax had the highest expression level at 3 days after injury; cytoplasmic Cyt C had the highest expression level at 3 days after injury; and mitochondrial Cyt C had the lowest expression level at 3 days after injury. Conclusion:Mitochondrial autophagy and apoptosis are enhanced after spinal cord injury in rats, and the potential mechanism may be associated with the transfer of Parkin and P62 from the cytoplasm to the damaged mitochondria for enhanced mitophagy and the release of a large amount of Cyt C from the mitochondria caused by the transfer of Bax from the cytoplasm to the damaged mitochondria for enhanced apoptosis.
RESUMEN
Fluorosis is an endemic disease caused by prolonged exposure to excessive fluoride and is considered a serious public health problem in many countries. In recent years, the damage of chronic fluorosis to the central nervous system has attracted extensive attention from scholars at home and abroad. The mechanisms of neurotoxicity caused by fluorosis include oxidative stress, inflammatory reaction, autophagy, neurotransmitters and related enzymes, changes in neural signaling pathways, abnormal neuronal energy metabolism of neurons, cell apoptosis, etc., causing permanent damage to human brain structure, impaired learning ability, memory dysfunction and behavioral problems. This article reviews the effects of fluorosis on the nervous system and related mechanisms, and provides a reliable basis for prevention and treatment of fluorosis.