Effects And Mechanisms Of Lung Injury Induced By Nitrogen-droped Nano-titanium Dioxide

Objective:As a novel nanomaterial,nitrogen-doped nano titanium dioxide（N-nTiO₂）exerts a photocatalytic action in which an electron-hole pair formed on the surface under natural light irradiation undergoes a redox reaction with water and oxygen in the air to generate a superoxide anion（·O₂^-）and a hydroxyl group.Free radicals（·OH-）further decompose the harmful organics in the environment and in the air,making them harmless carbon dioxide（CO₂）and water（H₂O）,thereby serving the purpose of disinfection,sterilization and purification of air.This subject intends to study the damage of the N-nTiO₂ to the lung and its possible toxic mechanism.Methods:Forty-two Sprague-Dawley rats were divided into 6 groups of 7 rats randomly,including the control group,and five dose groups（13.64mg/kg,27.27mg/kg,40.91mg/kg,54.55mg/kg,and 68.18mg/kg body weight）of N-nTiO₂.The rats were exposed by intratracheal instillation every 3 days for 28 days,and were then killed by heart bloodletting.Weekly weekends were weighed and recorded,and marked as 0th,1st,2nd,3rd,and 4th week weights of rats.24 hours before the first instillation and 24 hours after the last instillation,the lung function was measured by BUXCO spirometer and related index data such as respiratory rate（f）,minute ventilation（MV）,tidal volume（TV）,inspiratory peak（PIF）,expiratory peak（PEF）,and enhanced respiratory patency（Pehn）;after the final lung function data collection was completed,rats were anesthetized with 10%chloral hydrate and full lung tissue taken for weighing and calculating the ratio of lung to body.Collected 10 to 15 ml of bronchoalveolar lavage fluid,usually flush the left lung 3 times,inject 5 ml of preheated normal saline each time,and store at-20C.Use a multi-functional microplate reader to measure the total protein（TP）content in bronchoalveolar lavage fluid,as well as the activity of lactate dehydrogenase（LDH）and alkaline phosphatase（AKP）.After the non-irrigated right lung tissue was fixed in 10%neutral formalin for72 hours,histopathological sections of the lung were prepared and observed.At the same time,another lung tissue was fixed in 0.25%glutaraldehyde after 24 hours.Transmission electron microscope was used to observe the distribution of sodium and rice materials and the number of autophagosomes in lung tissue.The lung homogenates was collected and a multi-functional microplate reader was used to measure the activities of superoxide（SOD）,glutathione（GSH）,catalase（CAT）and the malondialdehyde（MDA）content.Western Blot was used to detect the expression of autophagy-related proteins LC3 and Beclin1.Results:The body weight of the rats showed a gradually increasing trend during the experiment,in which the weight gain of the dose group increased slowly compared with the blank control group,Compared with the control group at week 0,there was a significant decrease in body weight at doses of 13.64mg/kg and above N-nTiO₂exposed at week 1,week 2,week 3,and week 4（P<0.05）.The results of the ratio of lung to body increased above 27.27mg/kg N-nTiO₂ exposed group compared with control group（P<0.05）.Pulmonary function test results showed that compared with the control group,there was no significant change at f and Pehn in each exposed group,and both TV and PIF increased,but only increased in the 68.18 mg/kg N-nTiO₂exposed group was significant（P<0.05）.MV and PEF were significantly increased in54.55 and 68.18 mg/kg N-nTiO₂ exposed groups（P<0.05）.The results of enzyme activity in alveolar lavage showed that LDH was significantly higher in the27.27mg/kg and above N-nTiO₂ exposure group than in the blank control group（P<0.05）.AKP was significantly increased in the 54.55,68.18mg/kg N-nTiO₂exposure group（P<0.05）.TP was only significantly increased in the 40.91mg/kg N-nTiO₂ exposed group（P<0.05）.Histological sections of the lung showed that the lung tissue damage was aggravated as the dose increased.The main manifestations were cell proliferation,hyperemia,inflammatory cell infiltration,pulmonary obstruction,atelectasis and consolidation of lung.Ultramicroscopic results of lung tissue showed that the low-dose N-nTiO₂ group showed the deposition of nanoparticles,the macrophages in the middle-dose group increased and the concentration of alveolar cells became smaller,the autophagy bodies aggregated in the high dose group,the autophagy-related proteins LC3 and Beclin1 were detected at the same time.The results showed that the expression of LC3 and Beclin1 were Increased.Antioxidant enzyme activity results showed that compared with the control group,SOD was significantly decreased in the exposed group of 54.55mg/kg and68.18mg/kg N-nTiO₂（P<0.05）.GSH was significantly decreased in the 13.64mg/kg and above N-nTiO₂ exposed group（P<0.05）.MDA content and CAT were significantly increased in the 13.64mg/kg and above N-nTiO₂ exposed group（P<0.05）.Conclusion:After tracheal instillation of N-nTiO₂,the lung pathology of rats and the lung function were induced.The possible mechanism is that N-nTiO₂ activates the body’s oxidative stress（OS）through the generated ROS,thereby increasing the lipid peroxidation level and decreasing the antioxidant enzyme activity.Autophagy increased the toxicity of the experimental animals by increasing the dose of OS,and some eventually evolved into lung consolidation.Therefore,personal protection should be strengthened in actual work to avoid exposure to N-nTiO₂ at high concentration for a long time.