A large body of research has demonstrated that ACR can cause significant neurotoxicity in both occupational exposure in humans and animal experiments (Murray, et al. 2019;Park 2021), including symptoms such as gait abnormalities, muscle weakness, and ataxia, with clear evidence from in vivo and in vitro studies (Pennisi, et al. 2013;Zhang, et al. 2017). In this study, rats exposed to ACR exhibited symptoms such as limb weakness, gait abnormalities, increased hindlimb extension, hindlimb weakness, and difficulty in movement. After 28 days of exposure, the symptoms in the high-dose group (18 mg/kg ACR) were more pronounced than those in the low-dose group (6 mg/kg ACR), indicating cumulative neurotoxicity in the rats following ACR exposure. In addition, the results of hindlimb clasping test after 28 days of ACR exposure showed that the hindlimb clasping effect of rats was more obvious with the increase of exposure concentration. Above results suggest that ACR can produce significant neurotoxicity in rats, which is consistent with previously reported studies.
Studies have shown that ACR can cause damage to the neurogenesis of the hippocampus, induce neuronal loss and glial cell proliferation in rat hippocampus, and induce synaptic damage in the hippocampus (Park, et al. 2010;Yan, et al. 2022). In this study, HE staining showed that after ACR exposure, the distribution of rat hippocampal nerve cells became loose, the nuclei were condensed, and the neurons underwent vacuolar degeneration. Transmission electron microscopy observation showed that after ACR exposure, different degrees of cell boundary blurring were observed, the chromatin in the nucleus decreased, the volume of mitochondria swelled and the cristae decreased, the mitochondria became vacuolar in appearance, and the degree of damage increased with the increase of ACR exposure time and dose. This indicates that ACR can cause damage to the hippocampus of rats and induce neurotoxicity.
Brain-derived neurotrophic factor (BDNF) is one of the most abundant, widely distributed, and extensively studied neurotrophic factors in the neurotrophic factor family, playing a crucial role in the growth and development of neurons and synapses, and brain plasticity (Bazzari and Bazzari 2022;Wang, et al. 2022). Current research suggests that BDNF can act as a key neuroprotective factor in neurodegenerative diseases (Lu, et al. 2013), such as Parkinson's disease (Palasz, et al. 2020), Alzheimer's disease (Amidfar, et al. 2020), and Huntington's chorea (Zhou, et al. 2021). Synapsin I (Syn1) is a major peripheral protein regulating the release of mature neuronal synaptic vesicles on the surface of synaptic vesicle cytoplasm (Park, et al. 2021;Corradi, et al. 2008). It is primarily expressed in the central and peripheral nervous systems and is associated with early neuronal development, synaptic growth and development, and regulation of neurotransmitter release. Changes in Syn1 lead to alterations in synaptic homeostasis, which have been shown to cause neuronal developmental disorders, and the regulation of Syn1 expression has been shown to promote neuronal dendrite growth (Yan, et al. 2022;Hedegaard, et al. 2013;Parenti, et al. 2022). The results of this study showed that as the exposure concentration increased, the protein expression levels of BDNF and Syn1 in the hippocampal tissue of SD rats were significantly reduced. In vitro experiments, the expression results of BDNF and Syn1 were the same as in vivo experiments. This indicates that ACR affects neuronal development and function in the rat hippocampus and affects synaptic function in the hippocampus, resulting in neurotoxicity.
At present, there are many mechanisms underlying the neurotoxicity caused by ACR, among which neuro-oxidative damage caused by ACR has received much attention from researchers. Studies have shown that antioxidant parameters such as SOD, MDA, and CAT play important roles in ACR-induced oxidative damage (Bicer, et al. 2022;Elsawy, et al. 2021). Nrf2, as a key transcription factor regulating oxidative stress, plays an important role in inducing the body's antioxidant response. Heme Oxygenase-1 (HO-1), as a downstream gene of Nrf2, is positively regulated by Nrf2 signaling and can play an important role in cell protection by countering various forms of cell death (Loboda, et al. 2016;Ryter 2021). Our study results showed that as the concentration of ACR increased, the expression of Nrf2 in rat hippocampal tissue also increased. RT-qPCR results showed that the expression of Nrf2 and HO-1 in rat hippocampal tissue also increased with increasing concentration of ACR. The results of in vitro experiments were consistent with those of in vivo experiments. Our study suggests that ACR can cause oxidative damage in rat hippocampal tissue and activate the Nrf2 pathway to counteract the oxidative damage caused by ACR.
Our research group previously found through serum proteomic analysis of ACR occupational population that the glycolytic enzyme PGK1 was significantly elevated in the serum of ACR occupational population compared with that of non ACR exposed population. To confirm whether PGK1 plays a role in ACR-induced neuronal damage, we conducted in vivo and in vitro experiments. The results of in vivo studies showed that the mRNA and protein expression levels of PGK1 in the rat hippocampal tissue increased with the increasing concentration of ACR exposure. Meanwhile, the immunohistochemistry and immunofluorescence results showed that the expression of PGK1 also significantly increased with the increasing concentration of ACR exposure. The results of in vitro studies also showed that the mRNA and protein expression levels of PGK1 increased with the increasing concentration of ACR exposure. These results suggest that PGK1 may play a role in ACR-induced neuronal damage.
PGK1 mutations may affect the peripheral and central nervous system(Echaniz-Laguna, et al. 2019). Changes in PGK1 can affect nerve cells by stimulating glycolysis or acting on other pathways. Studies have shown that enhanced PGK1 protects cell damage by promoting glycolysis and increasing ATP levels. Reducing PGK1 expression can reduce oxidative damage by activating oxidative stress signaling pathways(Cai, et al. 2019;Xu, et al. 2022). A study has shown that inhibition of PGK1 protects nerve cells from damage caused by neurotoxins, In order to investigate the specific role of PGK1 in ACR-induced nerve damage, we used PGK1 siRNA in vitro experiments. The results of the in vitro experiments showed that the cell survival rate of the PGK1 siRNA transfection group was higher than that of the experimental group at the same exposure concentration (1.25 mM and 2.5 mM). Electron microscopy results showed that at the same exposure concentration (2.5 mM), the cell nucleus of the PGK1 siRNA transfection group was not squeezed, mitochondria swelled but did not exhibit a large number of vacuoles, and there were no giant mitochondria. Compared with the experimental group, the overall state of the cells was improved. Western blot results showed that at the same exposure concentration (2.5 mM), compared with the experimental group, the protein expression of BDNF and Syn1 increased in the PGK1 siRNA transfection group, while the protein expression of Nrf2 decreased. This suggests that inhibiting the expression of PGK1 can improve ACR-induced nerve damage and may play a protective role in ACR-induced cellular oxidative damage.