Effects Of Lanthanum On Autophagy And Apoptosis Of Primary Neurons In Rats And Their Correlation

Objective:Rare earth elements?REEs?include lanthanide elements in the periodic table and samarium?Sc?and yttrium?Y?which have properties similar to those of 15lanthanide elements.REEs have long been reported to be toxic to the brain and central nervous system.La is a representative element of LREE,which can be highly accumulated in the brain and has chemical activity.It is currently the most commonly used representative substance for studying the neurotoxicity caused by REEs.Studies have shown that La can damage the blood-brain barrier and the ultrastructure of neurons and synapses,cause neuronal calcium homeostasis,cause neurotransmitter uptake and release disorders,induce excessive activation of glutamate receptors,and memory Relevant genes and protein expression are suppressed,neurobehavioral abnormalities,etc.,have exact central nervous system toxicity,but the related mechanisms have not been fully elucidated.Autophagy is an evolutionarily conserved mechanism for cell self-repair and survival,and it is widespread in eukaryotes.Apoptosis is an autonomous and orderly cell death process,also known as type I programmed cell death.It plays an important role in biological evolution,maintaining internal environment stability,and the development of multiple system organs.Under physiological conditions,moderate autophagy and apoptosis are beneficial to the body,but under pathological conditions,excessive or insufficient autophagy and apoptosis can cause damage to related tissues and organs and abnormal functions.The interaction between apoptosis and autophagy is crucial to cell fate,and the two can be interdependent and transform each other.Beclin1 is the first gene found in mammals to regulate cell autophagy.By combining with type III PI3K/VPS34 to form a complex,it recruits other autophagic regulatory proteins in the cytoplasm to form protein complexes and promote autophagosome membrane Formation.The Bcl-2family is a key regulator of apoptosis.Studies have found that Beclin-1 and anti-apoptotic proteins Bcl-2 and Bcl-xl both contain BH3 domains,so Bcl-2 or Bcl-xl can bind to Beclin-1 through this domain to form a dimer.This prevents the latter from binding to type III PI3K/VPS34 and inhibits autophagy.Caspase-3 and caspase-6 are important apoptotic effector proteins,and Beclin1 is one of its substrates.Caspase-3/6 cleavage Beclin 1 can generate two main fragments Beclin1-N and Beclin1-C.Beclin1-C can be localized to mitochondria and lead to the release of apoptotic factors such as Cyto c,and promote the occurrence of apoptosis.In addition,pro-apoptotic single BH3 proteins,including BAD,BID,BNIP3,NOXA,PUMA,etc.,can competitively bind Bcl-2/Bcl-xl,on the one hand,depolymerize Beclin 1 and Bcl-2/Bcl-xl to It plays a role in promoting autophagy,and at the same time replaces and releases the pro-apoptotic protein BAX/BAK from Bcl-2 or Bcl-xl,which is beneficial to the occurrence of apoptosis.Neurons are the key structural and functional units of the central nervous system,and are the main performers of learning and memory.Neurons belong to special differentiated cells after mitosis.They cannot divide and proliferate excess or harmful substances like other cells.Therefore,the self-stabilizing mechanism is particularly important for maintaining their normal structure and function.This study used in vitro experiments to observe the effects of lanthanum chloride on primary cultured rat cortical neurons Bcl-2,Bcl-xl,Beclin1,caspase 3,caspase 6,BAD,BID,Effects of mRNA transcription and protein expression levels of NOXA,PUMA,BNIP3,analysis of protein expression changes of Beclin1-C and mitochondria and Cyto c in cytoplasm,and measurement of neuronal apoptosis rate by flow cytometry,observation with transmission electron microscope Changes in the ultrastructure of neurons in order to clarify the effects of lanthanum chloride on neuron apoptosis and autophagy and explore the relationship between the two,and provide new laboratory data for further revealing the neurotoxic mechanism of lanthanum.Methods:Several adult Wistar rats provided by the Experimental Animal Center of China Medical University weighed 290±10g.Female and male rats were mated in cages at a ratio of 2:1.Female mice were housed separately after confirming pregnancy.The cerebral cortex was harvested within 24 h after birth of the pups,and primary neurons were cultured.Immunofluorescence method was used to identify the purity of neurons.The cck-8 method was used to determine the cell survival rate and determine the concentration and duration of exposure.Real-time quantitative PCR was used to determine the mRNA transcription levels of neurons Bcl-xl,Bcl-2,Beclin1,caspase3,caspase6,BID,BAD,NOXA,PUMA,and BNIP3.Western blot was used to determine the Bcl-xl,Bcl-2,Beclin1,caspase3,caspase6,BAD,BID,PUMA,NOXA,BNIP3,Beclin1-C,Cyto c in mitochondria and cytoplasm expression of protein in neurons.Flow cytometry was used to determine the apoptosis rate of neurons.The ultrastructure of neurons was observed by transmission electron microscope.Results:1.The rate of NSE-positive cells in cultured cells is over 90%.The cultured cells are identified as neurons,which can be used as an in vitro model for subsequent experiments.2.With the increase of LaCl₃ treatment concentration,the survival rate of rat cortical neurons decreased significantly.According to the analysis results of cck-8,in the subsequent experiments,0.025 mmol/L LaCl₃,0.05 mmol/L LaCl₃,and 0.1mmol/L LaCl₃ were used as the treatment concentration,and the treatment time was24 h.3.LaCl₃ can up-regulate Beclin1 mRNA and protein expression levels of neurons;down-regulate Bcl-2,Bcl-xl mRNA and protein expression levels.4.LaCl₃ can up-regulate the expression of caspase 3,caspase 6 mRNA and protein,increase the expression level of Beclin1-C fragment and cytoplasmic Cyto c,down-regulate the expression level of mitochondrial Cyto c,and significantly increase the neuronal apoptosis rate.5.LaCl₃ can up-regulate the expression levels of BAD,BID,BNIP3,PUMA,NOXAmRNA and protein in neuronal cells.6.LaCl₃ treatment can damage the ultrastructure of neurons,which shows that the nuclear membrane gap is enlarged and chromatin is concentrated around the nuclear membrane;multiple vacuoles and autophagosomes appear in the cytoplasm;the number of organelles is reduced,mitochondria are swollen,Sudden destruction,vacuole-like changes.Conclusions:1.LaCl₃ can not only increase the apoptosis of rat cortical neurons,but also promote the autophagy of neurons.2.LaCl₃ inhibits the expression of Bcl-2,Bcl-xl,up-regulates the expression of caspase 3 and caspase 6,increases the production of C-terminal fragments of Beclin 1 and promotes the release of Cyto c in mitochondria,and up-regulates the expression of BAD,BID,BNIP3,NOXA,PUMA to prevent Various mechanisms such as the binding of Bcl-2,Bcl-xl and pro-apoptotic proteins play a role in promoting neuronal apoptosis.3.LaCl₃ releases Beclin1 by increasing the competitive combination of BAD,BID,BNIP3,NOXA,PUMA and Bcl-2,Bcl-xl by up-regulating the expression of Beclin1,which is conducive to the role of this autophagy-related protein,leading to enhanced neuronal autophagy.4.There is a close relationship between the neuronal apoptosis and autophagy mechanism caused by LaCl₃.The autophagy-related protein Beclin1 is an important meeting point among them.Multiple members of the Bcl-2 family participate in it and play an important role.