Study On The Senescence And Ferroptosis Of Human Bronchial Epithelial Cells Induced By Paraquat

Cellular senescence refers to a relatively stable state in which cells in a state of continuous division irreversibly lose the ability to proliferation,triggered by DNA damage or abnormal gene expression.Senescent cells gradually accumulate over time and play a pathophysiological role in the loss of function and regeneration capacity of tissues and organs.Paraquat(PQ;1,1 ’-dimethyl-4,4’-bipyridine-dichloride(1,1’-dimethyl-4,4’dipyridine-dichloride)is a highly effective non-selective herbicide commonly used in agricultural production.It can cause acute poisoning through oral administration,skin contact,and respiratory inhalation.After PQ poisoning,the concentration is the highest in the lung.Because the lung is the main affected tissue of PQ poisoning,the main cause of death from paraquat poisoning is respiratory insufficiency and respiratory failure.However,there is no direct effective drug for paraquat poisoning,and the mechanism of paraquat poisoning is not fully understood.This study found that PQinduced lung injury is related to lung cell senescence and ferroptosis induced by paraquat.In this study,we first determined the effect of paraquat-induced senescence in human bronchial epithelial Beas-2B cells and investigated the underlying mechanism.Firstly,we used MTT assay to detect the cytotoxicity of PQ on human bronchial epithelial cells Beas-2B at 24h,36h,48h and 72h.The results showed that PQ had strong cytotoxicity on Beas-2B cells.PQ treatment inhibited the proliferation activity of Beas-2B cells in a time-and dose-dependent manner.Next,to investigate the senescence effect of PQ,we used β-galactosidase(SA-β-gal)staining to quantify the degree of PQ-induced senescence and found that the content of senescent cells gradually increased with the gradual increase of PQ concentration.Previous studies have shown that persistently activated DNA damage response signals are the main cause of cellular senescence.Next,we used single cell gel electrophoresis to assess the degree and integrity of DNA damage in Beas-2B cells treated with PQ,and the results showed that PQ caused DNA damage in a dose-dependent manner.Thus,these results suggest that PQ causes senescence and DNA damage in Beas-2B cells.Subsequently,we used flow cytometry to detect the effect of PQ on cell cycle,and the results showed that Beas-2B cells showed obvious typical S-phase arrest after PQ treatment in a dosedependent manner.In addition,we analyzed the changes in the expression of S-phase related proteins cdc2 and cyclinE in Beas-2B cells treated with PQ by Western blotting.The results showed that PQ could significantly reduce the protein expression of cdc2 and cyclinE in Beas-2B cells.To further explore the mechanism of PQ-induced senescence in Beas-2B cells,we used Western Blot to analyze the changes in the expression of related proteins in PQ-treated Beas-2B cells.The results showed that the expression of p53,p21 and Rb was up-regulated,and subsequently,We used RT-PCR to assess changes in the expression of relevant mrnas at the RNA level.The results of RT-PCR were consistent with those of western blot.As an inhibitor of p53,PTF-α can specifically inhibit the transcriptional activity of p53,we pretreated Beas-2B cells with PTF-α and then treated with PQ to detect whether the proportion of senescent cells changed.The results of SA-β-gal staining showed that the percentage of SA-β-gal in senescent cells in the PTF-α treatment group was significantly lower than that in the non-PTF-α group.Western Blot results showed that the expressions of p53,p21 and Rb were down-regulated after PTF-a pretreatment compared with PQ alone.These results suggest that PQ may induce Beas-2B cell senescence by activating the transcriptional activity of p53.Ferroptosis is a new type of iron-dependent cell death caused by lipid peroxidation,which is closely related to the maintenance of living homeostasis and the occurrence of diseases.Ferroptosis is associated with oxidative stress.The DNA damage observed in PQ-induced human bronchial epithelial cell senescence suggests that ferroptosis may occur simultaneously during cell senescence.Based on this,we conducted an experimental study on PQ-induced ferroptosis in human bronchial epithelial Beas-2B cells and the related mechanisms.Firstly,the effect of ferroptosis inhibitor Fer-1 on the viability of human bronchial epithelial Beas-2B cells and the effect of Fer-1 pretreatment on PQ-induced cell damage were detected by MTT assay.The results showed that a concentration of 10μM or less had little effect on cell viability,and inhibition of ferroptosis significantly improved cell damage.Next,we pretreated Beas-2B cells with Fer-1 for 30min followed by PQ treatment.Based on the characteristics of ferroptosis of cells,we further examined the ROS content,and the results of flow cytometry showed that ROS content increased after PQ treatment and decreased after pretreatment with Fer-1 compared with PQ alone.GSH,MDA and SOD are important indicators of ferroptosis.We used the related kits,and the results showed that the GSH and SOD activities were decreased and the MDA level was up-regulated after PQ treatment in a dose-dependent manner.After adding Fer-1 pretreatment,the MDA level was decreased and the GSH and SOD activities were increased compared with PQ alone.To further explore the mechanism of PQ-induced ferroptosis in Beas-2B cells,the expression of ferroptosis-related proteins was detected by immunoblotting.The results showed that the expression of ferroptosis-related proteins GPX4,SLC7A11,and FTH1 was down-regulated and P53 expression was upregulated in a dose-dependent manner after PQ treatment.After preconditioning with Fer-1,the expression of GPX4,SLC7A11,and FTH1 was up-regulated and P53 expression was down-regulated compared with PQ alone.Subsequently,we used RTPCR to assess the changes in the expression of relevant mrnas at the RNA level.The results of RT-PCR were consistent with those of western blot.Subsequently,in order to verify whether the regulation is mediated by activating the transcriptional activity of p53,Beas-2B cells were pretreated with PTF-α and then treated with PQ to detect the related changes.The results showed that PTF-αpretreatment decreased the levels of ROS and MDA,increased the activities of SOD and GSH,up-regulated the expression of GPX4,SLC7A11 and FTH1,and downregulated the expression of P53 compared with PQ alone.The results of RT-PCR were consistent with those of western blot.PTF-α,a specific inhibitor of p53,effectively attenuated PQ-induced ferroptosis in Beas-2B cells.Based on the above experimental results,we inferred that PQ may induce ferroptosis in Beas-2B cells by activating the transcriptional activity of p53.In conclusion,PQ-induced senescence and ferroptosis in human bronchial epithelial Beas-2B cells are related to the activation of p53.The results of this study provide new data for the mechanism of PQ-induced lung injury,and provide a new perspective for the prevention of PQ-induced lung injury.