In daily life, La can enter organism via contact or the food chain, especially in areas rich in REEs. A large number of population surveys have shown that it is difficult to excrete La through metabolism after it enters the body. When La accumulates in the nervous system, it exerts a certain toxic effect. Therefore, La is often used to explore the role of REEs on the central nervous system [4, 5, 15]. According to reports, when La is ingested by growing offspring rats through drinking water, it causes growth retardation [15]. In this experiment, the weight of the offspring rats from the La exposure groups and the weight of the offspring rats from the control group were significantly different, demonstrating that LaCl3 affects the normal growth and development of offspring rats.
Hippocampal Nissl bodies are special nerve cell structures that are widely distributed in neurons. Their main function is to synthesize proteins and renew some cell components. The large size and large number of Nissl bodies can reflect the ability of nerve cells to synthesize proteins. In the central nervous system, lanthanum can accumulate in the hippocampus by passing through the blood–brain barrier, thereby causing damage to nerve cells [16]. In this study, in offspring from the control group, hippocampal pyramidal cells in the hippocampal CA1, CA3, and DG regions were arranged neatly, and the cytoplasm contained a large number of Nissl bodies. However, in the offspring rats exposed to lanthanum, Nissl body number in the CA1, CA3 and DG regions of the hippocampus declined to varying degrees, the number of hippocampal neurons was reduced, and the cell arrangement was disordered. This indicates that the accumulation of LaCl3 in offspring has a toxic effect on the hippocampal cells during the growth and development period.
The ultrastructure of the hippocampal CA1 area of rat offspring was observed by electron microscopy. We found that compared with the lanthanum groups, there were more mitochondria near the nucleus in the control group, and the mitochondrial matrix electron density was higher. and the mitochondria had more and longer ridges. As the dose of lanthanum increased, the number of mitochondria near the nucleus decreased, the electron density of the mitochondrial matrix decreased, the mitochondria appeared swelled, and mitochondrial ridges decreased in number and became shorter. Mitochondrial vacuolation was observed in the hippocampal neurons of some offspring in the 1.0% LaCl3 group. Our data indicate that LaCl3 causes substantial damage to hippocampal nerve cells. The higher the dose of LaCl3 is, the more severe the damage to organelles in hippocampal neurons.
The Morris water maze experiment involves directional navigation and spatial probe tests. These two behavioural tests are commonly used to test and evaluate the learning and memory of offspring rats. Here, offspring rats from the control group showed clear goals and found the hidden platform in the two tests, but as the dose of LaCl3 gradually increased, the swimming trajectories of the offspring rats became more irregular. The offspring rats in the 1.0% LaCl3 group showed irregular swimming trajectories or swam in circles. These results indicate that lanthanum-exposed rat offspring exhibit significantly impaired memory and learning after impairment of hippocampal nerve cell development.
Necrosis caused by changes in the TNFR-RIPK1/RIPK3 pathway is an important mechanism underlying nervous system damage. Necrosis, as a programmed cell death, is initiated by TNF-α. TNF-α outside the cell binds to TNFR1 on the cell membrane, activates TNFR1, and signals to TNF-related proteins (TNFR-associated death domain, TRADD) and RIPK1 with the same type of death domain in the cell through the death domain. Furthermore, TRADD also recruits TNF receptor-associated factor 2 (TNFR-associated factor 2, TRAF2), intracellular apoptosis protein inhibitor 1/2 (cIAP1/2), and ubiquitinase to form complex I [17, 18]. In complex I, RIPK1 relies on its three domains, namely, the intermediate domain (ID), the amino-terminal kinase domain (KD) and the carboxyterminal death domain (DD) [19], to regulate cell survival. The occurrence of necroptosis mainly depends on the KD domain of RIPK1[20]. When RIPK1 dissociates from complex I and the ubiquitination of RIPK1 is blocked, RIPK1 dissociates from complex I and forms complex II together with FADD and Caspase8. If Caspase-8 activity is not suppressed at this time, Caspase-mediated apoptosis occurs [21, 22]. In contrast, if Caspase-8 activity is suppressed at this time, the Caspase signalling pathway is inhibited, and RIPK1 in complex II interacts with the RIP homotypic interaction motif (RHIM) domain of RIPK3 through the homotypic domain in the C-terminus to form a RIPK1-RIPK3 complex [23]. The formation and phosphorylation of this complex are key and specific steps in necroptosis [24]. Phosphorylated RIPK3 in the complex recruits mixed lineage kinase domain protein (MLKL). When MLKL is recruited to necrotic cells, RIPK3 phosphorylates the 357th threonine residue and the 358th serine residue of MLKL. The N-terminal death effect domain 4HBD of MLKL is exposed, and oligomers are formed from monomers [25–27] and transferred from the cytoplasm to the cell membrane, causing the cells to swell and ultimately rupture and die, leading to leakage of cell contents and completing the necroptosis process [16, 28].
TNFR1 is the initial receptor protein of the signalling pathway, and tumour necrosis factor receptor is of great significance to the survival of nerve cells [17, 18, 29]. Studies have shown that necroptosis of hippocampal neurons can affect the learning and memory of rat offspring and is the cause of certain neurodegenerative diseases [2]. According to the results, t TNFR1 level in the hippocampi of 49-day-old offspring increased as the dose of lanthanum increased, indicating that La promoted this pathway to a certain extent.
RIPK1 is a key protein that inhibits learning and memory resulting from necroptosis of hippocampal nerve cells and determines the mode of cell death. Relevant experiments have shown that the RIPK1 inhibitor Nce-1 can alleviate pathological changes in a major cerebral artery occlusion model of stroke [30]. Zhang et al. proved that phosphorylated RIPK1 participates in TNF-α-induced necroptosis [31]. Therefore, the level of P-RIPK1 can also indicate the occurrence of necroptosis. Here, P-RIPK1 level in the hippocampi of offspring rats from the 0.5% and 1.0% lanthanum-exposed groups was markedly higher than that in offspring rats from the control group. It is speculated that there is a basis for necroptosis of hippocampal cells of lanthanum-exposed rat offspring.
RIPK3 is a key protein involved in RIPK1-related reactions and in necroptosis. Studies have shown that rats in which RIPK3 is inhibited have the ability to resist necroptosis [32]. The P-RIPK3 complex forms after binding to RIPK1, and phosphorylation of this complex leads to the recruitment and phosphorylation of the MLKL protein, leading to necroptosis of hippocampal nerve cells. In this experiment, as the dose of lanthanum increased, the content of P-RIPK3 increased. The P-RIPK3 level in the hippocampus in the 1.0% LaCl3 group was upregulated in comparison to the control group, which indicated that there was a key protein complex for necroptosis formed through the TNFR-RIPK1/RIPK3 signalling pathway in the hippocampi of the offspring.
The MLKL protein is downstream of RIPK1/RIPK3, and necroptosis is ultimately caused by MLKL-related pathways [32]. P-MLKL is considered a marker of TNF-driven necrosis [33]. In this experiment, the P-MLKL content in the hippocampus in the lanthanum-exposed groups, especially in the 0.5% LaCl3 and 1.0%LaCl3 groups, was markedly increased (P < 0.05). A higher level of P-MLKL indicates a higher degree of necrosis, which further suggests that neuronal necrosis in the hippocampi of the offspring was increased, in turn leading to abnormal learning and memory functions.
Based on previous studies and the results of this experiment, LaCl3 has a significant effect on rat offspring from birth to 49 days. The higher the dose of lanthanum was, the slower the growth and development of the rat offspring. In the hippocampal CA1, CA3, and DG regions of offspring rats, there were fewer Nissl bodies, abnormal arrangement of nerve cells, damage to mitochondria that could be observed under an electron microscope, mitochondrial swelling, a reduction in the number of mitochondrial ridges, and more severe neurodevelopmental damage. The greater the impairment of learning and memory was, the greater the levels of necroptosis-related factors TNFR1, P-RIPK1, P-RIPK3 and P-MLKL in the hippocampus was. Based on the above results, it can be speculated that lanthanum causes hippocampal nerve cell damage in offspring rats and that this effect is related to the TNFR-RIPK1/RIPK3 necroptosis signaling pathway.