The Pathological Characteristics And Preliminary Mechanism Of Mouse Lung Injury Induced By N-nTiO₂

BackgroundNitrogen-doped nano-titanium dioxide（N-nTiO₂）is a novel nano-material doped with non-metallic nitrogen in nano-titanium dioxide（nTiO₂）lattice.N-nTiO₂has more efficient visible light catalytic activity than other types of nTiO₂,so it has a wide range of indoor application prospects.A large number of studies have reported a variety of toxic effects of different types of nTiO₂.However,the biological effects of N-nTiO₂ are still lacking,and the adverse effects and mechanism of N-nTiO₂ exposure on the body are still unclear.Our previous study found that subacute exposure to N-nTiO₂ through the respiratory tract can damage lung function and cause pathological changes such as pulmonary congestion,edema,and alveolar septal thickening in rats.This result suggests that N-nTiO₂ may accumulate in the lungs and cause damage to the respiratory system under repeated respiratory exposure.Pulmonary fibrosis is a kind of interstitial lung disease with poor prognosis,mainly manifested as lung inflammation,alveolar structure destruction and collagen fiber deposition.Prior research has shown that nano carbon black particles,nTiO₂and nano zinc oxide particles can induce pulmonary fibrosis in mice through the long-term respiratory tract exposure.These nano-particles can cause pulmonary fibrosis through inflammation,oxidative stress,etc.The mechanism of nano-particles induce pulmonary fibrosis has not been fully elucidated.Alveolar macrophage（AM）is considered to be the main target cell of inhaled particles.Recent studies have shown that AM injury plays a key role in the development of pulmonary fibrosis caused by particles.Oxidative stress,autophagy disorder and other pathways may play an important role in AM injury caused by particles.The main exposure route of N-nTiO₂ is usually the respiratory tract.In order to explore the pathological characteristics and mechanism of lung injury caused by repeated exposure to N-nTiO₂ through the respiratory tract,this study intends to establish repeated mouse exposures model by improving the intratracheal instillation method and cell exposures model of N-nTiO₂.In addition,this study preliminarily explored the role and mechanism of AM in N-nTiO₂-induced lung injury in mice.Methods1.The establishment of respiratory exposure animal model of N-nTiO₂To establish the animal model of respiratory exposure to N-nTiO₂,it is necessary to perform repeated intratracheal instillation in experimental animals.The current non-exposed intratracheal instillation method in mice have the problems of low reliability,complex operation and low efficiency.In order to reduce the operation time,standardize the operation process,and reduce the interference of tracheal injury and irritation caused by the operation on the results,this study designed and manufactured a non-exposed intratracheal instillation device.The device is improved in terms of identifying the tracheal opening in mice and judging the success of tracheal intubation.The reliability and accuracy of the device were verified by intratracheal instilling ink solution and PM_2.5 suspension in the mice.After characterizing N-nTiO₂,the N-nTiO₂respiratory exposures mouse model was established by non-exposure intratracheal instillation.Forty-eight SPF male C57BL/6J mice were randomly divided into 4 groups:control group（PBS）and low,medium and high dose exposure groups（5,10,20μg/μl N-nTiO₂）,with instillation volume of 35μl.intratracheal instillation was performed every 5 days for the first 5 weeks,and no treatment was performed for the last 5weeks.The body weight of mice was recorded during the entire experiment period.2.The pathological characteristics of mouse lung injury induced by N-nTiO₂Whole body plethysmography（WBP）was used for non-invasive pulmonary function detection,and micro CT was used for lung imaging examination.Gross lung injury was observed,and pathological injury of lung tissue was observed by hematoxylin-eosin staining（HE）,MASSON staining and immunohistochemical staining.And the content of hydroxyproline（HYP）in lung tissue was detected.TUNEL staining and Western Blot（WB）were used to detect the apoptosis of lung tissue.3.The Preliminary mechanism of mouse lung injury induced by N-nTiO₂Bronchoalveolar lavage fluid（BALF）smear,transmission electron microscope（TEM）and energy spectrum analysis were used to observe the deposition of N-nTiO₂ in lung and its localization in alveolar macrophages.The mouse alveolar macrophage cell line（MH-S cell）was used to construct N-nTiO₂ exposure model in vitro.After exposure of N-nTiO₂,the cell activity and apoptosis of MH-S cells were detected,and the phagocytosis of N-nTiO₂ were observed.The effects of N-nTiO₂ exposure on apoptosis and autophagy dysfunction of MH-S cell were detected by fluorescence probe and Western Blot（WB）,it was also tested in mouse.Results1.The establishment of respiratory exposure animal model of N-nTiO₂The design and assembly of non-exposed intratracheal instillation device for mice were completed.After verification,a single operator can quickly and accurately complete the mouse intratracheal instillation operation,and the device can accurately and quickly identify the location of the mouse tracheal opening and judge whether the tracheal intubation is successful.The black ink blot was found only in the lung,but not in the gastrointestinal tract.And the acute lung injury model constructed by instillation of PM_2.5 suspension was effective.The instillation experiment showed that the method has high reliability and accuracy.A mouse model of repeated exposure to N-nTiO₂ through respiratory tract was successfully established by this non-exposed intratracheal instillation method.2.The pathological characteristics of mouse lung injury induced by N-nTiO₂Non-invasive pulmonary function test showed that the high-dose exposure group had decreased lung function compared with the control group.Micro CT showed that the lung marking in the exposure group was enhanced compared with that in the control group,and the diffuse increased density shadow was visible.Significant damage was observed in the mouse lungs in the exposure group.HE staining showed that the normal alveolar structure disappeared,inflammatory cell infiltration and lung consolidation occurred in the exposure group,and particles were deposited in the injured sites of the lungs.In the exposure group,MASSON staining and immunohistochemistry showed that the collagen fiber deposition and hydroxyproline content increased in the lungs,the fibrosis-related marker Fibronectin protein expression was increased.And the EMT-related marker Vimentin protein expression was increased and E-cadherin protein expression was decreased.N-nTiO₂-induced pulmonary fibrosis occurred in exposure group mice.TUNEL staining and WB results showed that the apoptosis level of lung tissue increased in the exposed group.3.The Preliminary mechanism of mouse lung injury induced by N-nTiO₂AM that ingest particles was observed in the BALF smear in the exposure group,and TEM showed that spindle-shaped fine particles were engulfed by AM.Ti element was found in exposure group lung tissue by EDX.N-nTiO₂exposures induced the proliferation activity of MH-S cells decreased and apoptosis increased in a dose-dependent manner.Bright-field microscopy and TEM showed that N-nTiO₂ was engulfed by MH-S cells,and spindle-shaped fine particles were encapsulated in autophagosomes.The number of autophagolysosomes in MH-S cells increased after N-nTiO₂ exposures.WB results showed that N-nTiO₂ exposures increased the expression of apoptosis-related protein BAX and autophagy-related protein LC3BII and p62 in MH-S cells,leading to apoptosis and autophagy dysfunction in AM.WB results also showed that the autophagy-related proteins LC3B-II and p62 were increased in exposure group mice.Conclusions1.The non-exposed intratracheal instillation method proposed in this study has high reliability and accuracy,and it can be used in the study of repeated intratracheal instillation exposure in mouse.2.Repeated exposures to N-nTiO₂ through the respiratory tract can cause lung tissue injury and lung function impairment in mice,N-nTiO₂ can be deposited in lung tissue,which will cause damage to normal alveolar structure,thickening of alveolar septum,and increase deposition of collagen fibers in lung tissue.3.Alveolar macrophages may be the main target cells of N-nTiO₂,the apoptosis and autophagy dysfunction may play an important role in mouse lung injury induced by N-nTiO₂.