Progression Of Zinc Oxide Nanoparticles-induced Acute Lung Inflammation Of Mice And The Protective Mechanism Of NRF2

Objective:In recent years,the development of nanomaterials has brought great conveniences to human life.Zinc oxide nanoparticles(Zn ONPs),as one of the most common nanomaterials,are widely used in various fields.However,toxicity researchs of Zn ONPs have lagged far behind the application studies.Evidences have shown that Zn ONPs can lead to acute lung inflammation in healthy volunteers at concentrations below the occupational exposure limit(5 mg/m~3).Therefore,it is crucial to investigate the toxic effects and mechanisms and find potential prevention strategies to protect people exposure to Zn ONPs.Exposure to Zn ONPs can lead to lung inflammation and oxidative stress,but the relationship is still unclear.In vivo experimental models of respiratory exposure mostly take chronic or subacute route,however,acute and multi-temporal study is unsubstantiated.The first part of this study selected multiple time points to observe the temporal changes of pulmonary toxicological effects after Zn ONPs exposure in mice.The small particle size and large surface area of Zn ONPs make particles easy to entry into cells,fuse with lysosomes,and trigger lysosomal membrane permeabilization(LMP).Severe LMP can further lead to programmed cell death.To investigate the toxicological mechanism of Zn ONPs,in the second part we used the mouse alveolar type II epithelial cell line(MLE12)to investigate whether Zn ONPs could cause cell death via LMP.Nuclear factor erythroid2-related factor 2(NRF2)plays an important role in exogenous-induced injury by transcriptionally activating a series of antioxidant enzymes to exhibit the body’s antioxidant defense response.To investigate the role of NRF2 in Zn ONPs-induced lung injury in mice and its mechanism,we observed the NRF2 signals of lung tissues and used Nrf2 knockdown MLE12 cells to observe the effects of LMP pathways and cell death.In the third part,we used the NRF2 agonist—CDDO-Im to intervene and explore whether it could reduce Zn ONPs-induced lung inflammation.The three parts of the paper systematically sort out the changes of pulmonary toxicological effects of Zn ONPs exposure,the mechanisms of toxicological effects and the potential intervention targets,providing new ideas for the prevention and treatment of hazards arising from human exposure to Zn ONPs.Methods:1.Wild type C57BL/6J mice(8-12 weeks,Female,n=11)were randomly divided into 9 groups according to body weight.The groups were:control group(Cont),10 min group after Zn ONPs exposure(10 min),30 min group(30 min),1 h group(1 h),3 h group(3 h),6 h group(6 h),1 d group(1 d),3 d group(3 d),and 7 d group(7 d).The mice were anesthetized by intraperitoneal injection of 50 mg/kg sodium pentobarbital,followed by oropharyngeal aspiration of 50μL phosphate buffer solution(PBS,containing 5%fetal bovine serum,FBS)or 20μg Zn ONPs in PBS,and the relevant indexes were measured at different time points.Lung zinc levels were detected by inductively coupled plasma mass spectrometry(ICP MS);The levels of oxidative stress of lung tissues were reflected by malondialdehyde(MDA)assay and total antioxidant capacity(T-AOC)assay;the levels of lactic dehydrogenase(LDH)and HMGB1 in BALF supernatant were measured to reflect lung epithelial cell injury;lung inflammatory response were reflected by inflammatory cell count in BALF,H&E staining and immunohistochemical staining;the changes of NRF2 signaling pathway in lung tissues were detected by western blotting.2.Using normal MLE12 cells,control cells of MLE12(Scramble)and Nrf2-knock down(Nrf2-KD)cells,treated with Zn ONPs at different concentrations and times,respectively.Cell viability was detected by CCK8 assay;changes of lysosome-associated protein levels were measured by western blotting;CTSB enzyme released by lysosomes was examined by enzymatic activity assays;LMP levels were observed by Lysotracker and Lysosensor fluorescence staining;the intracellular H2DCFDA probe fluorescence intensity was detected by flow cytometry to reflect cellular ROS levels;the relationship between LMP and ROS was investigated by CTSB inhibitor(CTSB(-))and ROS inhibitor(N-Acetyl-L-cysteine,NAC),respectively.3.Wild type C57BL/6J mice(8-12 weeks,Female,n=10)were randomly divided into 4 groups according to body weight.The groups were:control group(Cont),CDDO-Im intervention group(CDDO-Im),Zn ONPs treatment group(Zn ONPs)and CDDO-Im intervention group plus Zn ONPs treatment group(CDDO-Im+Zn ONPs).Mice were injected intraperitoneally with corn oil or CDDO-Im(2 mg/kg)at-1 d and 1 d,and oropharyngeally inhaled with PBS(5%FBS)or 20μg Zn ONPs at 0 d and observed at 3 d.The changes of NRF2 signaling pathway in lung tissues were detected by western blotting;the m RNA expression of NRF2 downstream genes and inflammatory factors were detected by RT-q PCR;lung inflammatory responses were reflected by inflammatory BALF cell counts,H&E staining and immunohistochemical staining.The above indicators were applied to observe whether CDDO-Im intervention could reduce the lung inflammation of mice caused by Zn ONPs.Results:1.Mice oropharynx inhalation of Zn ONPs resulted in a significant increase in lung zinc levels after 10 min and 30 min,and were largely excreted from the lung after 6 h,suggesting that the subsequent injury was a secondary effect rather than due to Zn ONPs itself.Lung tissue underwent oxidative stress characterized by elevated MDA and decreased T-AOC levels at 6 h,followed by elevated LDH and HMGB1 levels in BALF at12 h–1 d,representing pulmonary epithelial cell death,and an acute lung inflammatory response dominated by neutrophils infiltration at 3 d.At 7 d,macrophages became the dominant cells in BALF indicating a repair phase of inflammation at this time.Lung histopathology showed time-dependent alveolar wall thickening and inflammatory cells infiltration.The NRF2 signaling pathway was upregulated during Zn ONPs exposure,suggesting that the lung tissue may initiated an antioxidant defense system.2.Zn ONPs treatment caused LMP and cell death in MLE12 cells,as evidenced by increased levels of CALAPIN1 protein,mature forms of CTSB and CTSD protein,and CTSB activity released from lysosomes;decreased fluorescence intensity of Lysotracker and Lysosensor.Zn ONPs also lead to cell death by increasing intracellular ROS levels.Application of CTSB inhibitors and NAC revealed a mutual promotion between LMP and ROS due to Zn ONPs treatment.In Nrf2-KD cells,LMP,ROS levels and cell death after Zn ONPs treatment were more significant compared to Scramble cells,showing the protective effect of NRF2 on Zn ONPs-induced cell death.3.Intervention with the NRF2 agonist,CDDO-Im,was found to increase lung antioxidant levels and effectively reduce BALF inflammatory cell numbers,lung inflammatory factor levels and histopathological abnormalities of mice exposure to Zn ONPs.Conclusions:1.Mice oropharyngeal aspiration to Zn ONPs led to oxidative stress,lung epithelial cell injury and inflammation,while NRF2 signaling pathways were upregulated during the exposure.2.LMP and ROS increasing were the cause of Zn ONPs-induced cell death,and LMP and ROS can promote each other.Nrf2-KD further increased LMP and ROS levels and thus promoted cell death.3.Intervention with CDDO-Im could protect against Zn ONPs-induced acute lung inflammation in mice by increasing the lung antioxidant capacity and reducing inflammatory factors.