Research On The Mechanisms Of PC12 Cells Autophagic Death Induced By Zinc Oxide Nanoparticles

Nanoparticles are particles with at least one dimension less than 100 nm in a three-dimensional space.With the vigorous development of science and technology,nanoparticles have become more and more widely used in production,life and other aspects.The wide application of nanoparticles has led to an increase in the risk of their exposure.However,due to the small particle size and high surface activity of nanoparticles,when they enter the human body through respiration,skin contact,etc.,they can pass through various barriers and be distributed in different tissues and organs,thereby inducing body damage.Therefore,we must pay attention to the potential harm that nanoparticles may cause.In recent years,nanotoxicology has become a major research hotspot in the field of environmental science.Zinc oxide nanoparticles（ZnO NPs）are one of the most widely used metal oxide nanoparticles and are often used in the production of sunscreens,tooth fillers,food additives,semiconductors and other materials.Therefore,the biosafety assessment of ZnO NPs is particularly important.At present,studies have shown that ZnO NPs could pass through the blood-brain barrier,placental barrier,olfactory bulb or taste nerve pathway and entered the central nervous system.However,there are few studies on the potential neurotoxic effects of ZnO NPs and the specific mechanisms.Therefore,there is an urgent need to carry out related work.PartⅠ:The rat adrenal pheochromocytoma（PC12 cells）is a neuron-like cell line that can simulate the occurrence of a variety of neurodegenerative diseases.Therefore,we selected PC12 cells as in vitro cell model to explore the neurotoxic effects of ZnO NPs and the specific mechanisms.The successful uptake of ZnO NPs by PC12 cells is the primary factor in determining its toxic effects.Therefore,we first analyzed the uptake of ZnO NPs by PC12 cells.Electron microscopy results showed that after ZnO NPs were exposed to PC12 cells for 2 hours,the presence of ZnO NPs could be observed in the cells.Further study found that the side scatter angle and the total zinc element content of PC12 cells were increased in a concentration dependent manner after ZnO NPs exposure.These results all indicated that ZnO NPs could be taken up by PC12cells.In order to detect the ion release capabilities of ZnO NPs in PC12 cells,we used a Zn²⁺-specific fluorescence probe Fluor ^TMZn-520 to detect the change of Zn²⁺concentration in the cells after ZnO NPs exposure.The results showed after exposure to ZnO NPs,the free Zn²⁺level in the cells increased in a time-dependent manner.Further detection Zn²⁺subcellular positioning found that Zn²⁺co-localized with lysosomes.Therefore,ZnO NPs would release Zn²⁺in the lysosome after entering the cells.Next,we analyzed the toxic effect of ZnO NPs on PC12 cells by PI staining.The results showed that the percentage of PI-positive cells increased significantly when exposed to ZnO NPs,indicating that ZnO NPs could induce PC12 cells death.It was found by DCFH-DA staining that ZnO NPs exposure would increase the level of ROS in PC12cells,and when the ROS scavenger NAC was used to remove excess ROS,the toxicity of ZnO NPs to PC12 cells was alleviated,indicating that ZnO NPs induced an oxidative cell death.Further studies have found that there was an obvious positive correlation between the Zn²⁺level in cells and the toxicity of ZnO NPs to PC12 cells,that is,the higher the concentration of Zn²⁺released by ZnO NPs,the stronger the toxicity to PC12cells.Chelating excess Zn²⁺in cells with Zn²⁺chelating agent TPEN could effectively alleviate the death of PC12 cells induced by ZnO NPs,indicating that the toxic effect of ZnO NPs on PC12 cells was at least partially dependent on the release of Zn²⁺.Finally,by detecting the level of ROS after chelating Zn²⁺,it was found that the regulation of Zn²⁺on the toxicity of ZnO NPs was achieved by regulating the level of ROS,because the pretreatment of Zn²⁺chelating agent TPEN could significantly inhibit the increase of ROS levels in PC12 cells induced by ZnO NPs.In addition,TPEN and NAC could alleviate ZnO NPs-induced PC12 cells death to the same extent,this phenomenon could also illustrate the regulation of Zn²⁺to ROS.In summary,the experiments in this chapter demonstrated that after ZnO NPs entered PC12 cells,they could release Zn²⁺through lysosomes,and Zn²⁺would further induce the production of a large amount of ROS,which caused oxidative stress to the cells and ultimately caused cells death.PartⅡ:In this part,we first explored how ZnO NPs induced PC12 cells death.The classic cell death methods mainly include apoptosis,necrosis,autophagy and ferroptosis.We judged the methods of ZnO NPs-induced PC12 cells death by detecting the effects of these inhibitors of death methods on the toxicity of ZnO NPs.Both PI staining and CCK-8 results showed that apoptosis,necrosis and ferroptosis played only a small role in the process of ZnO NPs-induced PC12 cells death,and autophagy was the main cause of PC12 cells death.Further analysis by electron microscopy and MDC staining method showed that after ZnO NPs exposure,autophagosomes did accumulate in PC12 cells.The time-dependent up-regulation of autophagy-related proteins LC3B-Ⅱand Beclin1 levels detected by western blot assay also confirmed the above view.By monitoring the expression level of the autophagy degradation substrate P62,it was found that the autophagosomes induced by ZnO NPs could not be degraded normally.Therefore,ZnO NPs not only induced the formation of autophagosomes but also blocked the degradation of autophagosomes.Next,we explored the specific mechanism of autophagosome accumulation induced by ZnO NPs.Akt/m TOR and AMPK are the main signal pathways known to regulate the formation of autophagy.However,western blot results showed that although the phosphorylation level of m TOR decreased after 1h of ZnO NPs exposure,over time,it was recovered,and the phosphorylation level of AMPK has not changed significantly,indicating that Akt/m TOR and AMPK did not play an important role in ZnO NPs-induced autophagy in PC12 cells.MAPKs is another signal pathway that is known to participate in autophagy formation,including JNK,P38and ERK.We found that JNK and ERK were obviously activated after ZnO NPs exposure,which prompted us that JNK and ERK may be the key molecules that regulated autophagy in PC12 cells.Then,we investigated the effects of JNK and ERK inhibitors on the toxicity of ZnO NPs by PI staining and CCK-8,and found that the JNK inhibitor SP600125 significantly alleviated the toxicity of ZnO NPs to PC12 cells,while ERK inhibitor PD98059 aggravated the toxicity of ZnO NPs,indicating that only JNK promoted PC12 cells death induced by ZnO NPs,and the activation of ERK was related to cell survival.Further studies have found that when JNK inhibitor SP600125 inhibited the activation of JNK,the production of autophagosomes in PC12 cells induced by ZnO NPs was also inhibited,which could be explained by western blot assay,GFP-LC3plasmid transfection and MDC dyeing experiments.Therefore,we concluded that ZnO NPs induced autophagy by activating JNK.Western blot experiments showed that under the exposure of ZnO NPs,the phosphorylation level of Bcl-2 in PC12 cells increased in a time-dependent manner,and the co-exposure of the JNK inhibitor SP600125 and ZnO NPs could reduce the phosphorylation level of Bcl-2,which suggested that Bcl-2phosphorylation may be the key to JNK-mediated autophagy.Further through immunoprecipitation experiments,it was found that compared with the control group,ZnO NPs exposure inhibited the interaction between Bcl-2 and Beclin1 in PC12 cells,and pretreatment with JNK inhibitor SP600125 could reverse this phenomenon.Based on these results,we concluded that JNK was first activated under the action of ZnO NPs,the activated JNK led to the phosphorylation of Bcl-2,phosphorylation of Bcl-2 further dissociated the Bcl-2/Beclin1 complex,which led to the occurrence of autophagy.Next,we explored the reasons why ZnO NPs induced autophagic flow blockade.Through the detection of lysosome size,number,p H and permeability we found that ZnO NPs did not cause damage to lysosome function.Further western blot experiments found that the exposure of ZnO NPs would significantly down-regulated the expression of LAMP-2,indicating that the abnormal expression of LAMP-2 may be the reason why autophagosomes in PC12 cells could not be degraded normally.In addition,we also found that the blocking of autophagic flux was also related to the activation of JNK.Finally,we studied the relationship between Zn²⁺and autophagy,and found that chelating excess Zn²⁺in cells with Zn²⁺chelating agent TPEN could effectively reduce the level of autophagosomes in PC12 cells,indicating that Zn²⁺promoted the occurrence of autophagy.Further western blot experiments showed that the ROS scavenger NAC could inhibit the activation of JNK and the accumulation of autophagosomes,indicating that ROS promoted the activation of JNK and then induced the occurrence of autophagy.And ROS was produced by Zn²⁺.Based on these,it could be concluded that ZnO NPs released Zn²⁺,generated ROS,activated JNK,induced accumulation of autophagosomes and ultimately caused PC12 cells death.In addition,we also found that the pretreatment of autophagy inhibitors 3-MA and SP600125 could reduce the concentration of Zn²⁺in cells,indicating that autophagy could in turn promote the release of Zn²⁺.In summary,the experiments in this chapter demonstrated that the toxic mechanisms of ZnO NPs to PC12 cells involved the Zn²⁺-ROS-JNK-autophagy positive feedback regulatory loop.