Effect Of Lanthanum On Autophagy Of Central Nervous System Neurons In Rats And Its Related Mechanisms

Objective:The rare earth element?REE?is a general term for the special elements in 17of the periodic table,including 15 lanthanides and scandium?Sc?and yttrium?Y?which are similar in electronic structure and chemical properties.Lanthanum,as a representative of light rare earth elements,has unique physicochemical properties of rare earth elements,and is often used as a representative substance for studying the biological effects of REEs.Studies have shown that REEs can enter the body and accumulate in tissues and organs,causing damage and dysfunction of the body.Epidemiological surveys in China have shown that children with high rare earths area have significantly lower IQ and memory than children with low rare earths area.Fan found that the rare earth content in the blood of children aged 7-10 years in the rare earth mining area was1.73 times that of the children in the control area,and the IQ score was related to the distance between the home and the rare earth mining area.Injecting lanthanum chloride?LaCl₃?into the center of the forebrain hemisphere of day-old chicks can inhibit the recovery of memory.Oral intake of LaCl₃ can cause abnormal neurobehaviness in mice.Feng found that LaCl₃ exposure can hinder the development of the central nervous system,interfere with the distribution of trace elements in the brain and neurotransmitter levels,and impair learning and memory.Our previous studies showed that LaCl₃ can affect the transcription and expression of memory-related proteins by inhibiting cAMP/PKA/CREB signaling pathway,[Ca²⁺]_i/CaM/CaMKIV/CREB signaling pathway and NF-?B signaling pathway.It can also disturb the Glu-Gln cycle,abnormally increase glutamate,D-serine levels,over-activate hippocampal NMDA receptors,induce excitotoxicity,and cause excessive apoptosis of nerve cells via mitochondrial pathway.Despite this,the mechanism of neurotoxicity of sputum has not yet been fully elucidated and further research is needed.Autophagy is a lysosomal-dependent degradation pathway widely distributed in eukaryotic cells,which can eliminate abnormal proteins and damaged organelles present in the cytoplasm and provide energy and raw materials for cell repair and reconstruction.Cells face self-balancing and survival mechanisms of harmful stress.However,excessive enhancement or inhibition of autophagy levels can have an adverse effect on the body.Autophagy is regulated by several signaling pathways centered on mTORC1.The most representative ones are type I PI3K/AKT signaling pathway and AMPK signaling pathway.Type I PI3K/AKT is the main negative regulatory signal of autophagy.During autophagy,type I PI3K phosphorylates the substrate PIP2 to generate PIP3,and PIP3 acts as a second messenger to bind to the PH domain-containing signaling proteins Akt and PDK1,promoting the threonine Thr308 and serine Ser473 of Akt.The site is activated by phosphorylation,and the activated Akt indirectly activates mTORC1 located downstream thereof and inhibits autophagy by inhibiting the activity of TSC1 and 2.The AMPK signaling pathway is a positive regulatory signal for autophagy.In contrast to the action of AKT,AMPK initiates autophagy by activating the TSC 1/2 complex to inhibit the activation of Rheb and mTORC1.Previous studies have shown that LaCl₃ exposure can significantly increase the reactive oxygen species?ROS?in rat hippocampus and primary cultured neurons and primary cultured astrocytes,and a recent study showed that excessive production of ROS by LaCl₃ exposure can affect autophagy-related genes atg4,BECN1?atg6?and map1lc3b?atg8?by activating c-Jun N-terminal kinase?JNK?signaling pathway in nuclear and cytoplasmic phosphorylation of c-Jun and FoxO1/3 respectively,suggesting that LaCl₃may affect its normal physiological function by affecting the level of autophagy in rat hippocampal neurons.Based on previous research results,this study used a combination of in vivo and in vitro,using neurobehavioral and molecular biology techniques to change the level of autophagy from La and related regulatory signaling pathway AKT/mTOR,The research on the change of activation state of AMPK provides new clues and evidence for revealing the mechanism of neurotoxicity of La.Methods:In the in vivo experiment,96 healthy adult Wistar rats?48 males,240±10 g in weight;48 females,weighing 220±10 g?were housed in the animal room for 5 days to acclimate to the surrounding environment.Female rats were randomly divided into four groups?12 in each group?,and then females and males were mated in a 1:1 ratio,and the next day,the females were found to be the 0th day of pregnancy.During the gestation period?3 weeks?and lactation?3 weeks?,each group of rats was exposed to an aqueous solution containing 0,0.25%,0.5%,1.0%LaCl₃,respectively.After weaning,each group of rats took water to take the corresponding concentration of LaCl₃ aqueous solution,and ended at 1 month after weaning,and conducted behavioral tests and determination of various indicators.The Morris water maze test was used to train and test the spatial learning and memory ability of rats.Realtime PCR and Western Blot were used to detect LC3B,p62,Beclin1,Bcl-2,p-Bcl-2 and Lamp2a in hippocampal neurons of rats.mRNA transcription and protein expression levels of type III PI3K,AKT,p-AKT,mTOR,p-mTOR,AMPK,p-AMPK,TFEB.Intracellular autophagosomes were detected by transmission electron microscopy.In vitro experiments were performed on primary cultures of brain neurons using pups within 24 hours of birth.Neurons were identified using neuron-specific enolase.The expression levels and colocalization of LC3B and p62 in neurons were detected by immunofluorescence.Detection of LC3B,p62,Beclin1,Bcl-2,p-Bcl-2,Lamp2a,III PI3K,AKT,p-AKT,mTOR,p-mTOR,AMPK in primary cultured neurons by Realtime PCR and Western Blot mRNA transcription and protein expression levels of p-AMPK and TFEB.After the intervention of the autophagy inhibitors wortmannin and bafilomycin A1,the autophagy-related protein index was again determined to avoid false positive results.Finally,the autophagosomes in primary cultured neurons were detected by transmission electron microscopy.Verify again that La can lead to an abnormal increase in autophagy.Results:1.The effect of LaCl₃ on learning and memory ability of offspring rats.In the five training days,the learning ability of the La group was significantly weaker than that of the control group.In the place navigation test,as the dose of La-expose increased,the escape latency and swimming distance of the platform found by the pups increased gradually.The swimming trajectory showed that the path of the La group was chaotic,and there was no clear purpose,and the swimming distance increased.In the spatial probe test,the number of entrances to the target quadrant,the residence time in the target quadrant and the swimming distance in the target quadrant decreased with the increase of the dose of La.The trajectory heat map showed that the swimming trajectory of the La group was disordered.There is no clear exploration strategy,and even rarely enter the target quadrant.2.The effect of LaCl3 on the level of autophagy and the expression of AKT/mTOR and AMPK in autophagy-related signaling pathways.With the increase of La exposure dose,the AKT/mTOR signaling pathway was inhibited,the AMPK signaling pathway was activated,and the level of autophagy was significantly increased.3.The effect of LaCl₃ on the ultrastructure of hippocampal neurons in rats.The cell membrane of hippocampal neurons in the control group was smooth and intact,the cell body was large and round,and the organelles in the cytoplasm were abundant and clearly visible.There were very rich and well-structured endoplasmic reticulum,Golgi and mitochondria.The nucleus is clear and the nucleolus nuclear membrane is smooth and intact.There is no double-layer membrane vacuole-like structure in the cytoplasm.In the1.0%LaCl₃ group,the structure of neurons changed,the shape of the cell body was irregular,the endoplasmic reticulum and Golgi were visible in the cytoplasm,the mitochondria were significantly reduced,and a large number of bilayer membrane vacuoles-like structures appeared in the cytoplasm.,in line with the structural features of autophagosomes.The shape of the nuclear membrane changed,and the nucleolus could not be observed,indicating that LaCl₃ has a strong damage effect on hippocampal neurons.4.The effect of LaCl₃ on the ultrastructure of primary cultured neurons.The cell membrane of the control group was smooth and intact,the cell body was large and round,the nucleus was large and intact,and a small amount of vacuolar-like structure was observed in the cytoplasm.The neurons in the neurons after Wortmannin inhibition are large and round,and a small number of organelles are visible in the cytoplasm,and there are no other abnormal structures.After inhibition of autophagy with Baf-A1,a large number of bilayer membrane vacuolar-like structures containing contents are present in neurons,which should be autophagosomes that cannot be fused.A large number of mitochondria appear in the cytoplasm,the nucleus is small,and the edge of the nuclear membrane blurry.After exposure to sputum,a large number of bilayer membrane vacuolar-like structures appeared in the neurons,and there were many inclusions in the vacuoles,which were characterized by autophagosomes,indicating that sputum can induce autophagy in primary cultured neurons.After the addition of the autophagy upstream inhibitor Wortmannin,there were almost no abnormal structures in the neurons and no autophagosomes were present,indicating that the inhibitor inhibited autophagy from the upstream.The autophagosome fusion inhibitor Baf-A1 was added at the same time as the sputum exposure.The results showed that a large number of bilayer membrane vacuolar-like structures appeared in the neurons added to Baf-A1,and the number was more than that of the exposed neurons alone.After inhibition of autophagosome fusion,autophagosomes are retained in the cells and accumulated in large amounts.Conclusion:1.LaCl₃ can cause spatial learning and memory impairment in offspring rats.2.LaCl₃ can inhibit the AKT/mTOR signaling pathway in progeny rats,activate AMPK-related signaling pathway and TFEB,resulting in elevated levels of autophagy.3.LaCl₃ can cause changes in the ultrastructure of neural cells in the offspring,and the number of autophagosomes in the cytoplasm increases significantly.4.LaCl₃ can inhibit the AKT/mTOR signaling pathway of primary rat neurons,activate AMPK and TFEB signaling pathways,and promote phosphorylation of Bcl-2,resulting in abnormally elevated autophagy levels.5.Autophagy inhibitor Wortmannin and Baf-A1 intervention experiments and transmission electron microscopy results can rule out the interference of false positives,to determine the ability of La to promote autophagy.6.The abnormal enhancement of autophagy induced by La may be one of the important mechanisms leading to the damage and death of this type of cells.