The Role And Mechanism Of Cuproptosis In Lung Injury Caused By SARS-CoV-2 Infection

Background:Coronavirus disease 2019（COVID-19）is a highly contagious and serious disease caused by the severe acute respiratory syndrome coronavirus 2（SARS-Co V-2）.It exhibits diverse clinical manifestations,rapid progression,and a poor prognosis in severe and critical cases.As of January 7,2024,it has been responsible for more than7 million deaths worldwide.Recently,the role of metal elements in the pathogenesis of COVID-19 has garnered significant attention.Among these metals,iron,copper,zinc,selenium,and other metal ions have been particularly scrutinized.Zinc is regarded as an important predictor of severe COVID-19 outcomes,and ferroptosis has been reported to play a role in myocardial injury.As an essential trace element in the human body,copper serve as critical catalytic cofactors in various biological processes.Similar to iron,excessive accumulation of copper can trigger"cuproptosis"in cells.Our study had revealed that the serum concentrations of copper in COVID-19 patients is significantly elevated compared to healthy controls.Furthermore,bioinformatics analysis has demonstrated that cuproptosis-related genes influence the development of COVID-19.Based on these findings,we hypothesize that cuproptosis may play a role in the pathogenic process leading to lung injury caused by SARS-Co V-2 infection.Therefore,investigating the levels of serum copper in different types of COVID-19patients and their correlation with disease severity,as well as elucidating the mechanistic role of copper in the pathogenic process of COVID-19,will provide crucial cytological and molecular insights for subsequent clinical prognosis assessments and the development of anti-COVID drugs.Objective:The aim of this study was to investigate the serum copper level in COVID-19patients and clarify the correlation between copper and COVID-19 through statistical analysis.Additionally,we aimed to explore the relationship between SARS-Co V-2spike protein-induced alveolar epithelial cell injury and cuproptosis.This research will provide a theoretical basis for the development of precision therapeutic targets for COVID-19.Methods:1.Discussion on serum concentrations of copper in patients with different types of COVID-19（1）To collect clinical data of confirmed COVID-19 cases who were hospitalized from December 2022 to February 2023 in the Department of Respiratory and Critical Care Medicine at the Second Hospital of Jilin University.（2）The levels of cytokines（IL-1β,IL-6,IL-18,IFN-γ,TNF-α,TGF-β1）as well as copper,ceruloplasmin（CP）,and FDX1,a key regulator of cuproptosis,were measured in serum samples obtained from both patients and healthy controls.2.To investigate the effect of copper on alveolar epithelial cells（1）A549 cells were stimulated by copper ionophore alone or in combination with a variety of metal ions,and cell survival was detected by CCK8.（2）Cisplatin or Elesclomol-Cu Cl₂ stimulated A549 cells,the protein expression of Caspase 3 and Cleaved-Caspase 3 was detected by Western blot and AO/EB staining was used to check positive cells.After pretreatment with multiple inhibitors of cell death mechanism,the cells were stimulated with Elesclomol-Cu Cl₂,cisplatin and ML162,and the cell survival was detected by CCK8.（3）After Elesclomol-Cu Cl₂ stimulates A549 cells,the intracellular copper concentration was detected by colorimetric method.Western blot was used to detect the expression levels of Lipoy-DLAT,Lipoy-DLST,FDX1,DLAT,DLST,HSP70 and SLC31A1.（4）After Elesclomol-Cu Cl₂ stimulated A549 cells,AGEs and intracellular ATP content were detected by ELISA,mitochondrial morphological changes were observed by transmission electron microscopy,cell senescence was observed byβ-galactosidase staining,and the expression level of mitochondrial oxidative phosphorylation complex（OXPHOS）and COX IV were detected by Western blot.3.To explore the relationship between alveolar epithelial cell injury induced by the SARS-Co V-2 spike protein and cupoptosis（1）A549 cells were stimulated with Spike protein with different concentration gradients,and the intracellular copper concentration was detected by colorimetric method,the m RNA expression levels of copper transporter SLC31A1,ATP7A and ATP7B were detected by q RT-PCR,the expression of CP and FDX1 was detected by ELISA,the expression of SLC31A1 and FDX1 was detected by Western blot,and the level of cytokines was detected by ELISA.（2）A549 cells were stimulated with 0 ng/m L and 25 ng/m L Spike protein or pretreated with copper chelator（TTM）and then given 25 ng/m L Spike protein,colorimetric method to detect intracellular copper concentration,ELISA to detect CP and FDX1 concentrations,q RT-PCR to detect cuproptosis related genes and P65m RNA expression levels,and Western blot to the expression of cuproposis-related proteins,AGEs and intracellular ATP content were detected by ELISA,mitochondrial morphology changes were observed by transmission electron microscopy,cell senescence was observed byβ-galactosidase staining,and OXPHOS complex and COX IV expression levels were detected by Western blot.ELISA was used to detect the levels of cytokines.（3）Macrophages were stimulated with Spike Protein,and the culture supernatant was retained.Subsequently,either the macrophage supernatant or TTM pretreatment was applied,followed by the administration of the macrophage supernatant to stimulate A549 cells,and the intracellular copper concentration was detected by colorimetric method,CP and FDX1 concentrations were detected by ELISA,cuproptosis-related genes and P65 m RNA expression levels were detected by q RT-PCR,and the expression level of cuproptosis-related protein was detected by Western blot,the content of AGEs and intracellular ATP was detected by ELISA,the morphological changes of mitochondria were observed by transmission electron microscopy,the senescence of cells was observed byβ-galactosidase staining,and the expression level of OXPHOS complex and COX IV was detected by Western blot.ELISA was used to detect the levels of cytokines.4.To explore the relationship between NF-κB signaling pathway and SARS-Co V-2spike protein-induced copper death in alveolar epithelial cellsA549 cells were stimulated by Spike protein or NF-κB pathway inhibitor pretreatment,and the expression level of phosphorylated protein related to NF-κB pathway was detected by Western blot,the level of cytokines was detected by ELISA,the intracellular copper concentration was detected by colorimetric method,the content of CP and FDX1 was detected by ELISA,and the gene related to cuproposis and P65m RNA were detected by q RT-PCR,AGEs and intracellular ATP content were detected by ELISA,mitochondrial morphology was observed by transmission electron microscopy,and cell senescence was observed byβ-galactosidase staining.Results:1.Discussion on serum concentrations of copper in patients with different types of COVID-19（1）A total of 44 patients were enrolled in this study.They were categorized into a severe group and a non-severe group based on the severity of their condition.Specifically,32 patients were assigned to the severe group,12 to the non-severe group,and 10 healthy individuals served as controls.Notably,the patients in the severe group exhibited a significantly higher age compared to those in the non-severe group,with a higher proportion of patients aged over 65 years（P<0.05）.Additionally,the severe group demonstrated a significantly faster heart rate and a lower oxygenation index（P<0.05）.When comparing the severe group with the non-severe group,there were significant increases in the levels of white blood cell count,neutrophil percentage,neutrophil count,lactate dehydrogenase,high-sensitivity troponin,D-dimer,fibrinogen degradation products,procalcitonin,hypersensitive C-reactive protein,C-reactive protein,IL-6,and ferritin.Conversely,the levels of lymphocyte percentage,total lymphocyte count,CD3⁺T lymphocyte count,CD4⁺T lymphocyte count,CD8⁺T lymphocyte count,albumin,and 25-hydroxyvitamin D were significantly decreased.（2）The serum copper content of COVID-19 patients was significantly higher than that of the healthy control group,and the severe group had a higher level than the non-severe group（P<0.05）.Additionally,the serum levels of cytokines such as IL-1β,IL-6,IL-18,TNF-α,IFN-γ,TGF-β1,and ceruloplasmin（CP）in COVID-19 patients were elevated,while the level of FDX1,a key regulator of cuproptosis,was lower compared to the healthy control group.Specifically,the levels of IL-1β,IL-6,IL-18,TNF-α,IFN-γ,TGF-β1,and CP in the severe group were significantly higher than those in the non-severe group,positively correlating with the severity of the disease.Conversely,the level of FDX1 in the severe group was lower than that in the non-severe group,indicating a negative correlation with disease severity（P<0.05）.（3）Through the analysis of ROC curves of various risk factors,the results showed that the area under the curve of oxygenation index was the largest（AUC 0.995）,followed by FDX1（AUC 0.977）,and the AUC for copper and CP were 0.843 and 0.903,respectively.The results showed that FDX1,copper,CP and oxygenation index had predictive value for severe COVID-19（P<0.05）.2.To investigate the effect of copper on alveolar epithelial cells（1）Compared with other metal ions,only copper ions and copper ionophores were found to significantly reduce cell survival.Following pulse stimulation of A549 cells with the 1:1 concentration of Elesclomol and Cu Cl₂,the viability of these cells decreased notably as the duration of cell culture increased.（2）The positive rate of AO/EB staining and the protein expression of Cleaved-Caspase 3/Caspase 3 increased（P<0.05）after cisplatin stimulation of cells,but the above manifestations were not found in Elesclomol-Cu Cl₂-stimulated cells.（3）Elesclomol-Cu Cl₂ up-regulated the intracellular copper concentration,and led to the down-regulation of the expression levels of Lipoy-DLAT,Lipoy-DLST,DLAT,DLST,and FDX1,and the up-regulation of the expression levels of heat shock protein70（HSP70）and copper transporter SLC31A1（P<0.05）.（4）Elesclomol-Cu Cl₂ induces mitochondrial dysfunction in cells,which is manifested by mitochondrial morphological damage under transmission electron microscopy,increased expression of senescence-relatedβ-galactosidase,decreased intracellular ATP content,and down-regulated expression levels of mitochondrial OXPHOS complex（CI,CII,CIII,CIV,CV）and COX IV（P<0.05）.3.To explore the relationship between alveolar epithelial cell injury induced by the SARS-Co V-2 spike protein and cupoptosis（1）After A549 cells were stimulated with different concentration gradients of Spike protein,it was observed that the intracellular copper concentration increased,which was positively correlated with the increase of Spike protein concentration.The increase of SLC31A1 m RNA,CP and cytokines（IL-1β,IL-6,IL-18,TNF-α,INF-γ,TGF-β1）were positively correlated with the increase of Spike protein concentration and the decrease of ATP7A m RNA,ATP7B m RNA,FDX1 concentration and protein were negatively correlated with the increase of Spike protein concentration（P<0.05）.（2）After A549 cells were stimulated with Spike protein,it was observed that the concentration of the intracellular copper and CP increased,the concentration of FDX1decreased,the m RNA expression levels of cuproposis-related genes FDX1,DLD,DLAT,and NDUFS8 decreased,and the m RNA expression levels of DLST,MTF1,GLS,and P65 increased,and the cuproposis-related proteins Lipoy-DLAT and Lipoy-DLST,FDX1,DLAT,DLST protein was down-regulated,the expression level of HSP70 and SLC31A1 protein was up-regulated,the concentration of AGEs increased,the intracellular ATP content decreased,the mitochondria were significantly swollen,the lumen was expanded,the number of cristae decreased or even disappeared,and vacuolization was observed,the expression of aging-relatedβ-galactosidase was increased,and the expression of mitochondrial OXPHOS complex（CI,CII,CIII,CIV,CV）and COX IV were down-regulated.The expression of cytokines（IL-1β,IL-6,IL-18,TNF-α,INF-γ,TGF-β1）was up-regulated（P<0.05）.However,the above changes were alleviated by the pre-application of TTM treatment（P<0.05）.（3）Stimulation with macrophage supernatant produces comparable results to those observed with Spike protein stimulation,and these effects are also alleviated by TTM（P<0.05）.4.To explore the relationship between NF-κB signaling pathway and SARS-Co V-2spike protein-induced copper death in alveolar epithelial cellsSpike protein up-regulates the expression levels of phosphorylated proteins p-P65,p-IκBαand inflammatory cytokines（IL-1β,IL-6,TNF-α）associated with the NF-κB pathway.Compared with the Spike protein group,the expression levels of NF-κB pathway-related phosphorylated proteins and cytokines in the cells of the BAY11-7082+Spike protein group were down-regulated,and the m RNA expression of P65was decreased,the concentration of the intracellular copper and CP decreased,the concentration of FDX1 increased,the m RNA expressions of FDX1,DLD,DLAT,and NDUFS8 were up-regulated,and the m RNA expressions of DLST,MTF1,GLS decreased,and Mitochondrial morphological damage was alleviated,aging-relatedβ-galactosidase expression decreased,AGEs concentration decreased,and intracellular ATP content increased（P<0.05）.Conclusions:1.In this study,it was found that the increase of serum copper and ceruloplasmin in COVID-19 patients was positively correlated with the severity of the disease,and the decrease in the level of FDX1 was negatively correlated with the severity of the disease.2.Serum copper,ceruloplasmin,and FDX1 may be independent risk factors for predicting severe COVID-19 and have diagnostic value.3.SARS-Co V-2 spike protein may induce the accumulation of intracellular copper in alveolar epithelial cells,leading to cuproptosis,and the copper chelator TTM can alleviate the cell damage caused by the spike protein.NF-κB signaling pathway may be involved in the process of spike protein-induced cuproptosis in alveolar epithelial cells.