Mechanism Of Cadmium Induced Pyroptosis By Regulating PERK In SH-SY5Y Cells

Background:Cadmium（Cd）is one of the most toxic heavy metals of environmental pollutants and is a major risk factor for neurological disease,including neurodegenerative diseases.Therefore,it is a significant strategy to explore the specific mechanism of Cd-induced nerve cell damage for prevention and treatment of nervous system diseases.Pyroptosis is a newly discovered form of inflammatory programmed cell death,which can induce strong inflammatory reaction and death.NLRP3inflammasome has been proved to activate caspase-1 and induced pyroptosis.It is reported that TXNIP can directly combined with NLRP3 inflammasome to promote its activation.And endoplasmic reticulum（ER）is the major target organelle of Cd-induced toxicity.As a transmembrane protein on the ER,PERK can sense stress-related information transmitted by the endoplasmic reticulum,and can promote the expression of TXNIP.So,we speculate that Cd may regulate the expression of TXNIP through the PERK protein and activate the NLRP3 inflammasome,and finally induce the cell pyroptosis and damage.The production of reactive oxygen species（ROS）is a crucial mechanism of Cd-induced toxicity,and it is also an early trigger of Cd activated ER stress.N-acetylcysteine（NAC）,as a common active oxygen scavenger,it has not been reported whether NAC can regulate endoplasmic reticulum stress and inhibit pyroptosis by downregulating the level of ROS in SH-SY5Y cells.Consequently,we further study the specific molecular mechanism of Cd-induced nerve cells pyroptosis and the effect of NAC on pyroptosis.It has potential application value for the prevention and treatment of inflammation-related neurological diseases induced by Cd exposure.Objective:The study is aim to explore whether Cd can cause cell pyroptosis and the role of endoplasmic reticulum stress in this process.And we also testify the protective effect of NAC on Cd-induced nerve cell pyroptosis.Methods:In this study,the human neuroblastoma cell SH-SY5Y was applied to establish an in vitro model.First,CCK-8 and flow cytometry were used to detected the cell viability and pyroptosis,and determine the treatment concentration of CdCl₂ based on the above results.After that,the cells were divided into the control group and the CdCl₂treatment group,then the reactive oxygen species kit was used to detect the level of intracellular ROS.And Western Blotting and RT-q PCR were used to detect the expression of PERK,TXNIP,NLRP3,Caspase-1,IL-1βand IL-18.Secondly,the experiment was divided into four groups:control group,CdCl₂ group,GSK2606414group and GSK2606414+CdCl₂ group,and the above methods were used to detect changes in cell viability,pyroptotic and the expression of pyroptosis related molecules.Finally,the experiment was divided into four groups:control group,CdCl₂ group,NAC group and NAC+CdCl₂ group.Then the cell viability,the level of intracellular ROS,the changes of pyroptotic and the expression of pyroptosis related molecules were detected.Results:1.After treatment with CdCl₂,SH-SY5Y cells were significantly damaged which was characterized by decreased cell viability.And the result of flow cytometry showed that the percentage of cell pyroptosis gradually increased with elevation of Cd dose.The level of intracellular ROS raised in the CdCl₂group.Western blotting and real-time PCR showed that the level of PERK,TXNIP,NLRP3,caspase-1,IL-1βand IL-18 were up-regulated after cells were treated with 10μM CdCl₂.2.After pretreatment of SH-SY5Y cells with PERK inhibitor,GSK2606414,the cell viability in the GSK2606414+CdCl₂ group improved compared with the CdCl₂ group;the proportion of caspase-1 and PI double-positive cells in the GSK2606414+CdCl₂group is modestly decreased;the activation of NLRP3 and the expression level of TXNIP,caspase-1,IL-1βand IL-18 were decreased.3.Compared with the CdCl₂ group,the viability of cells in the NAC and CdCl₂co-treatment group was enhanced,the level of ROS production and the percent of double-positive cells stained with Caspase-1 and PI were all reduced;the phosphorylation of PERK and the level of TXNIP,NLRP3,caspase-1,IL-1βand IL-18were also downregulated in the NAC+CdCl₂ group.Conclusion:Cadmium exposure can cause pyroptosis of SH-SY5Y cells through PERK/TXNIP/NLRP3 signaling pathway and NAC can significantly alleviate the pyroptosis induced by Cd.