MtROS-mediated Downregulation Of SIRT1 Induces Cellular Senescence In Cadmium Exposure-induced Chronic Obstructive Pulmonary Disease

Objective: This study aimed to investigate whether cellular senescence caused by downregulation of SIRT1 is involved in cadmium(Cd)exposure-induced chronic obstructive pulmonary disease(COPD).Method: 1.First,an animal model of COPD induced by cadmium exposure was established.C57BL/6J mice purchased from Vital River were randomly divided into two groups: Control(Ctrl)and Cd groups.The Cd group was inhaled Cd Cl2(0.1 mg/m L)every other day for 10 weeks.Mice in the ctrl group are exposed to normal saline in the same way.After 10 weeks,mice were tested for lung function,the remaining mice were collected after sacrifice.H&E staining was used to assess lung injury in mice and to perform pathological scoring.2.We conducted mouse experiments and human pulmonary epithelial cells(BEAS-2B cells)experiments to investigate the effects of Cd exposure on telomere damage and cellular senescence.In vivo experiments,frozen sections were prepared,and SA-β-gal staining was performed to evaluate cellular senescence in the lungs of mice.Western blot assay was used to detect the expression of lamin B1 and the telomere protective proteins TRF2 and TIN2.In vitro experiments,BEAS-2B cells were divided into 2 groups: Ctrl group and Cd group.Cells from the Cd group were maintained in an environment exposed to Cd(2 μM).SA-β-gal staining was performed after 5 weeks to observe the level of cellular senescence.The proteins were collected after 3 and 5 weeks of Cd exposure in order to detect the expression of lamin B1,TRF2 and TIN2.3.We conducted mouse experiments and BEAS-2B cells experiments to investigate the effects of Cd exposure on the expression of SIRT1.In vivo experiments,lung tissue protein was extracted to detect the expression of SIRT1.In vitro experiments,cells were harvested to detect SIRT1 expression after 0,6,12 and 24 hours of treatment with Cd(2 μM).4.We conducted BEAS-2B cells experiments to investigate the effect of SIRT1 on the level of TRF2 acetylation.After co-culture of BEAS-2B cells with Cd(2 μM)for 24 h,cells were harvested.Colocalization of SIRT1 and TRF2 was detected by immunofluorescence,and the interaction between SIRT1 and TRF2 was detected by co-immunoprecipitation.Changes in TRF2 acetylation levels were then examined.In cells exposed to Cd for 24 h,the level of TRF2 acetylation in BEAS-2B cells was determined by co-immunoprecipitation.5.We conducted mouse experiments and BEAS-2B cells experiments to study the effect of Cd exposure on mitochondrial stress.Expression of mitochondrial stress marker HSP70 and CLPP were detected in in vivo and in vitro experiments.Mito SOXTM Red was detected and JC-1 staining were then performed.6.We conducted mouse experiments to investigate the effects of mitochondrial stress on Cd exposure-induced telomere damage,cellular senescence and COPD.All C57BL/6J mice were randomly divided into 4 groups: Ctrl,Mito-TEMPO(MT),Cd and Cd+MT.In the Cd and Cd+MT groups,mice were inhaled Cd Cl2(0.1 mg/m L)every other day for 10 weeks.In the MT and Cd+MT groups,MT(1 mg/kg)was given by intraperitoneal injection 1 h before Cd exposure.After 10 weeks,lung function was determined in the mice and lung tissue was collected.H&E staining was used to assess lung injury in mice.SA-β-gal staining was used to detect cellular senescence in mouse lungs.Western blot was used to detect the expression of HSP70,CLPP,SIRT1,lamin B1,TRF2 and TIN2 in the lungs of mice.Results: 1.After chronic Cd exposure,the lung weight and lung coefficient of mice were significantly increased.Normal alveolar structures were damaged,inflammatory infiltrates were pronounced,airway walls were thicken,and lung function was significantly decreased.2.In mouse lungs and lung epithelial cells,the secretion of the cellular senescence marker SA-β-gal was increased and Lamin B1 was decreased after Cd exposure.And telomere damage marker TRF2 and TIN2 was significantly downregulated.3.Furthermore,the expression of SIRT1 was downregulated after Cd exposure in mouse lungs and lung epithelial cells.4.Mechanistically,the interaction between SIRT1 and TRF2 was weakened after Cd exposure in BEAS-2B cells.An interaction between SIRT1 and TRF2 was observed in BEAS-2B cells,and the interaction was attenuated after Cd exposure.In addition,the level of TRF2 acetylation was significantly increased in BEAS-2B cells after Cd exposure.5.In Cd-exposed cells,mitochondrial stress markers HSP70 and CLPP were upregulated,mitochondrial reactive oxygen species were increased and mitochondrial membrane potential was decreased.6.In vivo,MT,a targeted mitochondrial scavenger,alleviated Cd-induced mitochondrial stress,downregulation of SIRT1 and downregulation of telomere damage markers and cellular senescence markers.At the same time,MT pretreatment significantly attenuated the damage to alveolar structure,inflammatory cell infiltration,thickening of the small airway wall and decline in pulmonary function induced by Cd exposure.Conclusion: mtROS-mediated downregulation of SIRT1 induces cellular senescence in cadmium exposure-induced chronic obstructive pulmonary disease.