| Objective: Realgar is a traditional Chinese medicine containing metalloid arsenic and has a wide range of clinical applications.Some classic recipes such as Angong Niuhuang Pills,Bezoar Detoxification Tablets,etc.contain it.However,due to the influence of the traditional concept of non-toxic side effects of traditional Chinese medicines,drug-induced arsenic poisoning caused by the abuse of realgar or its compound preparations has been reported from time to time.Epidemiological and animal experiments have shown that long-term arsenic exposure can cause not only skin damage,digestive damage,and cancer,but also central nervous system damage in severe cases.Therefore,the effects on human health caused by arsenic exposure and accumulation effects caused by realgar into drugs have attracted widespread public attention.The brain is one of the target organs of arsenic toxicity,so it is of important theoretical and practical significance to explore the mechanism of toxicity of realgar in the central nervous system and discover the sensitive molecular targets for early changes in toxicity,which is of great theoretical and practical significance for taking effective measures to prevent the occurrence of pharmaceutical-induced arsenic poisoning and guiding the rational clinical use of drugs.As an important self-regulation mechanism,autophagy participates in various biological functions such as growth and development,cell differentiation,and resistance to pathogens.Disturbances in the autophagy process of nerve cells can also lead to the accumulation of protein aggregates,disrupting cellular homeostasis,and producing neurotoxicity.The classical autophagy process can be divided into 3 stages: autophagy induction stage,autophagosome formation stage and autophagic lysosomal degradation stage,and damage at any stage will lead to cell homeostasis imbalance.Studies have shown that when autophagy induces enhancement,a large amount of p62 in neurons is recruited to autophagosomes and subsequently degraded in lysosomes,while when p62 cannot be degraded,it can induce apoptosis of nerve cells by activating the caspase-8inflammatory cascade and increasing the lysis of caspase-9 after accumulation in autophagosomes.The above suggests that realgar may lead to p62 accumulation by activating the autophagy-inducing phase or inhibiting the autophagy degradation phase,resulting in neurotoxicity.The JNK/Vps34 complex pathway plays an important role in regulating autophagy-induced non-m TOR dependent pathways.When cytokines,stress,inflammatory factors and other factors activate the JNK/c-Jun signaling pathway,it will promote the formation of Beclin1-Vps34 core complex and induce autophagy,microtubule-associated protein LC3 hydrolysis to LC3 I,and then the cytoplasmic form of LC3 I is converted to LC3 II,p62 is recruited and bound to the autophagosome membrane to participate in autophagosome formation,and finally degraded in lysosomes.After autophagy is overactivated,efficient degradation of autophagy is essential for maintaining cell survival,and the activity of lysosomal membrane proteins,lysosomal catheteolyases,and the maintenance of the acidic environment of lysosomes will directly affect the degradation function.At present,it is not clear whether realgar can activate autophagy and promote p62 aggregation through JNK-mediated Beclin1-Vps34 complex pathway.In addition,previous studies have shown that realgar can activate nuclear factor erythroid 2-related factor 2(NRF2)in cortex,a key transcription factor involved in anti-apoptotic and antioxidant defense.It is worth noting that NRF2 can directly induce p62 transcription and promote the expression of p62 protein,and p62 can compete with NRF2 to bind KEAP1,promoting NRF2 activation,thereby forming a p62-NRF2 feedback loop.However,it is not clear whether the change of p62-NRF2 feedback loop promotes the accumulation of p62 induced by realgar.Mitochondria are highly dynamic organelles that play important roles in biological processes such as energy metabolism,apoptosis,autophagy,and cellular aging.Mitochondrial dysfunction is also strongly associated with nerve damage.The results of the previous experiments of the research group showed that after the exposure of realgar,the morphology and structure of mitochondria in cortical neurons were significantly damaged.but the role mitochondria play in realgar-induced central nervous system toxicity has not been studied.Mitochondria regulate mitochondrial morphology,number,distribution,and function through continuous fusion and division,and mitochondrial disruption due to reduced fusion and/or increased division triggers mitochondrial apoptosis pathways.There is growing evidence that DRP1,a key protein that regulates mitochondrial division,is recruited to the mitochondrial membrane in large numbers in neurological,liver,and cancer diseases to mediate mitochondrial overdivision,thereby promoting apoptosis and activating mitochondrial autophagy.However,on the one hand,there is still a lot of exploration about which receptor proteins DRP1 is recruited;On the other hand,as a double-edged sword for maintaining cellular homeostasis,the role and mechanism of mitophagy in promoting apoptosis by disrupting mitochondrial dynamics and its mechanism have also been elucidated.To sum up,this study uses a combination of animal and cell experiments to explore the mechanism of central nervous system toxicity induced by realgar from the perspective of “interaction dialogue between autophagy flow and p62-NRF2 feedback loop mediated p62 accumulation”,and “UBXD8-DRP1 mediated mitochondrion division and PINK1-Parkin regulated mitochondrial autophagy interaction dialogue”,in order to provide research basis and new experimental data for the study of realgar toxicity mechanism.Methods: 1.Construction of rat models exposed to different doses of realgar 303-week-old,female Sprague Dawley rats were selected and divided into control group,0.3 g/kg and 0.9 g/kg realgar groups according to the random number table method.After8 weeks of realgar exposure,new object recognition test and open field test were used to evaluate the changes of cognitive ability,emotional state and motor ability of rats;liquid chromatography-atomic fluorescence spectroscopy(LC-AFS)was used to detect urine,Various forms and contents of arsenic in blood and cerebral cortex;use molecular biology techniques such as transmission electron microscopy,immunofluorescence,western blot,q PCR,Tunel staining,etc.to study the damage to the central nervous system after realgar exposure.2.Construction of JNK inhibitor,autophagy inhibitor and autophagy activator intervention rat model after realgar exposure.3-week-old,female Sprague Dawley rats were selected and divided into: control group,0.9 g/kg realgar,according to the random number table method group,JNK inhibitor intervention group,autophagy inhibitor intervention group,autophagy activator intervention group,JNK inhibitor group,autophagy inhibitor group,autophagy activator group.After the animals were treated for 8 weeks,molecular biology techniques such as western blotting and q PCR were used to study the molecular mechanism of realgar-induced neuronal apoptosis by disrupting autophagy.3.Use DMA to treat SH-SY5 Y cells to establish an in vitro realgar exposure model.SH-SY5 Y cells were treated with different concentrations of DMA(0,2.5,5,10 m M)for different time or 24 h.Cell viability was detected by MTS method;apoptosis rate was detected by flow cytometry and Tunel staining method;autophagosome formation was observed by transmission electron microscope;molecular biology techniques such as western blotting and q PCR were used to detect autophagy-related proteins,The expression levels of NRF2 signaling pathway related proteins and m RNA;the hydrolysis activities of cathepsin B and cathepsin D were detected by kit method;the toxic effect of DMA on SH-SY5 Y cells was studied.4.The SH-SY5 Y cell line was selected and treated with DMA.The small molecule compound SP600125 was used to inhibit the JNK signaling pathway,the specific activator rapamycin(Rapamycin)to activate the induction of autophagy,and the small molecule compound 3-MA to inhibit the induction of autophagy.,small RNAs interfered with the expression of p62 and NRF2,and molecular biology techniques such as western blotting and q PCR were used to detect autophagy-related proteins and NRF2 signaling pathway-related proteins and m RNA expression levels;to explore the way DMA interferes with autophagy and the p62-NRF2 feedback loop The molecular mechanism of inducing apoptosis of SH-SY5 Y cells provides more convincing results and new ideas for further elucidating the mechanism of realgar-induced neurotoxicity.Results: 1.Levels of morphological arsenic in the cerebral cortex,blood,and urine of rats after exposure to realgar.LC-AFS results showed that i As,DMA and MMA were detected in urine after exposure to realgar,but only DMA was detected in blood,and arsenic accumulated in brain tissue was mainly present in the form of DMA,suggesting that the end product of realgar’s metabolism in the brain is DMA.2.Effects of realgar on neurobehavioral studies in rats.The results of the novel thing recognition experiment showed that in the test stage,compared with the control group,the rats in the 0.3 g/kg and 0.9 g/kg exposure groups did not show a significant preference for new things and old things,indicating that realgar caused cognitive impairment in rats.Compared with the control group,the number of times rats entered the central area,the time spent in the central area,the exercise distance and speed of the rats in the realgar exposure group decreased significantly,and there was a significant dose-effect relationship,indicating that the rats induced anxiety-like behavior and damaged exercise ability.3.Effects of androgens on neuronal ultrastructure and apoptosis.Realgar can induce damage to the ultrastructure(mitochondria,endoplasmic reticulum,and Golgi)of rat cortical neurons and apoptosis.4.Realgar perturbates autophagy flow homeostasis,mediates p62 and promotes apoptosis.The results showed that realgar induced homeostatic imbalance in autophagy,which was manifested as increased expression of LC3II/LC3 I,increased autophagosomes,significantly increased expression of p62 protein,and reduced apoptosis after silencing p62.5.Realgar activates the autophagy-induced process through the JNK/Vps34 complex pathway to promote p62 aggregation.The results showed that the JNK signaling pathway and autophagy-induced the expression of Beclin1 and Vps34 could be activated after exposure to realgar,and the binding ability of Beclin-Vps34 complex could be enhanced,and after inhibiting the JNK signaling pathway,the expression of autophagy-related proteins Beclin1,Vps34 and LC3 II proteins decreased,the binding capacity of Beclin1-Vps34 complex was also significantly reduced,and the expression of p62 protein was not significantly changed.6.Realgar hinders the degradation process of autophagy by inhibiting lysosomal hydrolase activity,resulting in p62 accumulation.The results showed that after the exposure of realgar,lysosomal hydrolase B and D activities decreased significantly,and after DMA treatment,the fusion process of autophagosomes and lysosomes was observed in SH-SY5 Y cells without being affected,but the acidic environment was reduced.7.Realgar promotes the accumulation of p62 by activating the p62-NRF2 feedback loop.The results showed that the level of ROS in cortical neurons and the expression level of NRF2 protein increased significantly after exposure of realgar,and the results of in vitro experiments showed that the binding ability of p62-NRF2 decreased after DMA treatment,and the activation of NRF2 was inhibited after silencing p62,which indicated that NRF2 was activated in a p62-dependent manner,which enhanced the p62-NRF2 feedback loop and contributed to the accumulation of p62 induced by autophagic flow disorder.8.Effects of realgar on total arsenic levels in blood and cerebral cortex.The results showed that the total arsenic content in the blood was significantly increased,but not dose-dependent,and the total arsenic content in the cortex was significantly and dose-dependent.9.Realgar induces mitochondrial dysfunction in the cerebral cortex of rats and triggers mitochondrial apoptosis pathways.The results showed that after exposure to realgar,the mitochondrial electronic respiratory chain complex ETC decreased,ATP decreased,and the expression levels of Cyt-c and caspase-3 proteins increased significantly.10.Realgar inhibits mitochondrial fusion and regulates mitochondrial division through the UBXD8-DRP1 pathway,promoting apoptosis.The results showed that the expression of fusion proteins OPA1 and MFN1 was downregulated after exposure of androgen,the expression of division protein DRP1 and mitochondrial membrane protein UBXD8 was upregulated,and after silencing UBXD8,the colocalization of UBXD8 and DRP1 was reduced,mitochondrial division was reduced,and the expression level of mitochondrial apoptosis protein decreased significantly after mitochondrial division was inhibited by Mdivi-1.11.Mitochondrial division is involved inrealgar-induced mitophagy activation.The results showed that the expression of PINK1,Parkin,LC3II/LC3 I and p62 proteins were significantly increased after the exposure of realgar,and the colocalization of PINK1 and LC3 decreased after the use of Mdivi-1 to inhibit mitochondrial division,and the expression of PINK1 and Parkin proteins decreased significantly.12.PINK1-Parkin-mediated mitophagy regulates arsenic-induced mitochondrial division and apoptosis.The results showed that after silencing PINK1 inhibited autophagy induction,mitochondrial division was inhibited and apoptosis was alleviated.Conclusion: 1.Realgar exposure induced apoptosis of nerve cells led to cognitive impairment,mood changes,and decreased exercise capacity in rats.2.Realgar activated the autophagy induction stage and recruited p62 in large quantities through the JNK/Vps34 complex pathway,while directly inhibiting lysosomal function rather than autophagic lysosomal fusion process,hindering p62 degradation,and then promoting p62 accumulation and apoptosis of nerve cells.3.The enhancement of p62-NRF2 feedback loop has a promoting effect on p62 accumulation.4.Realgar inhibits mitochondrial fusion and promotes mitochondrial excessive division through the UBXD8-DRP1 pathway,leading to mitochondrial dynamics imbalance,mitochondrial dysfunction,inducing apoptosis,while activating the PINK1/Parkin pathway to regulate mitochondrial autophagy. |
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