| Objective A rat alveolar proteinosis model was established by non-exposure tracheal perfusion nano-indium tin oxide exposure method.The success of the model was verified by pathology.The structural damage,oxidative stress and expression level and change rule of inflammatory factors of rat lung tissue caused by nano-indium tin oxide were studied from two aspects of different doses and different exposure time of single dose.The inflammatory effect and pathogenesis caused by nano-indium tin oxide were preliminarily discussed,so as to find out the key points of nano-indium tin oxide poisoning.Methods Specific pathogen Free(SPF)male Sprague-Dawley rats at 6weeks of age were randomly divided into two groups according to their body weight.There were 40 rats in the dose group which were randomly divided into 4 groups: normal saline control group,low-dose exposure group(1.2mg/kg),medium-dose exposure group(3mg/kg)and highdose exposure group(6mg/kg).Each group had 10 rats,and were exposed twice a week with an interval of 3 days for 12 weeks.In the time group,120 rats were randomly divided into 3 days,7 days,14 days,28 days,56 days and 84 days of 6mg/kg nano-indium tin oxide lung irrigation group.The exposure group at each time was respectively provided with the corresponding time control group and the same amount of normal saline lung irrigation,with 10 rats in each group.Twice a week,with an interval of three days,the weight changes of the rats were recorded weekly,and their mental state,diet and drinking water were observed.After the exposure,chloral hydrate(5ml/kg)was injected intraperitoneally to dissect after anesthesia.Blood was taken from the abdominal aorta,serum was taken centrifugally,right and left lower lung lobes were ligated,lung lavage fluid was reserved,and lung lobes that were not lavaged were removed.We detected the serum indium content by inductively coupled plasma mass spectrometry(ICP-MS).The ultrastructural changes and ion distribution of lung tissue were observed by transmission electron microscopy(FETEM).The lung tissue was embedded in paraffin,sliced and fixed on glass slides.Hematoxylin-eosin HE staining was used to observe the pathological changes of lung tissue and PAS staining was used to detect alveolar proteinosis.The contents of SP-A,SP-D,KL-6 and HO-1 in lung tissues and the contents of GM-CSF,TNF-α,IL-6 in serum were determined by enzyme-linked immunosorbent assay.The content of LDH in alveolar lavage fluid was determined by microenzymatic labeling method;the content of T-SOD in alveolar lavage fluid was determined by hydroxylamine method;the content of MDA in alveolar lavage fluid was determined by thiobarbituric acid;the content of T-AOC in lung tissue was determined by FRAP.Immunohistochemistry was used to determine the expression levels of TNF-α and IL-6 beta-protein in lung tissues,and the Western Blot was used to detect the expression level of cytokines.Results 1 During the exposure period,the mental state of rats in each group was not significantly abnormal,the diet and water intake were in good conditions.There was no significant difference in the average body weight of rats in each group(P>0.05).The pulmonary coefficient of rats increased with the raise of the dose of infection.The pulmonary coefficient of each infected group increased significantly compared with that of the control group(P <0.05).Compared with the low-dose group,the pulmonary coefficient of rats in the high-dose group also increased significantly(P <0.05).The pulmonary coefficient of rats in the infected group was significantly higher than that in the control group after 14 days of exposure(P<0.05).2 The pathological results showed that the toxic lung tissues in each dose group were damaged to different degrees,and it could be seen that there were uniform,eosinophilic,non-structured fine granular substances in the alveolar cavity with macrophage proliferation and foam macrophage proliferation;That eosinophilic deposits of fine granular material without structure appeared in the alveolar cavity of the infected group from day 14,and alveolar macrophages increased and were mostly foamy.3 The PAS results showed that there were granular eosinophilic and foamy macrophages in the alveoli and pink floc deposits in the alveolar cavity of the dose group,which increased with the increase of the dose;After 28 days of infection,PAS stained positive cells were found in all infected groups,which increased with the increase of infection time.4 Compared with the control group,the serum indium content in each dose infected group was significantly increased(P<0.05)(100～1000 times),and increased with the increase of the infected dose.The serum indium content in the time infected group was significantly increased(P<0.05),and increased with the increase of the time of infection.5The serum levels of TNF-ɑ,IL-1β and IL-6 in the exposed groups were decreased to different degrees compared with those in the control group(P<0.05).Western Blot results showed that TNF-ɑ and IL-1β in lung tissues of the exposed groups were decreased compared with those of the control group.Immunohistochemical results showed that the expression of TNF-ɑ and IL-1β was consistent with that of Western Blot.The time group results showed an upward to downward trend in TNF-ɑ,IL-1β,and IL-6 levels,with a turning point between 14 and 28 days of exposure.Compared with the control group,SP-A,SP-D and KL-6 in the lung tissues of the exposed groups were all increased(P<0.05).SPA was significantly increased on days 14,28,56,and 84(P<0.05),SP-D was significantly increased on days 28,56,and 84(P<0.05),and KL-6 was significantly increased on days56 and 84(P<0.05).The levels of GM-CSF in the serum of the infected group were significantly lower than those of the control group(P<0.05).The results of the time group showed that GM-CSF in serum was significantly decreased at 56 and 84 days compared with the control group(P<0.05).Compared with the control group,the contents of T-SOD and MDA in the lavage fluid of the exposed groups were significantly increased(P<0.05),and the LDH content in the alveolar lavage fluid of the exposed groups was significantly increased(P<0.05).The content of T-SOD,MDA and LDH in the lavage solution all showed an increasing trend,and MDA content in each infected group was significantly higher than that in the control group after 7 days of infection(P<0.05).After 28 days of TAOC poisoning,all infected groups were lower than their control groups(P<0.05).Conclusions The deposition of ITO NPs in the lungs of rats increases the content of serum indium,which leads to the accumulation of a large number of proteins in the alveoli and causes lung injury.ITO NPs induced alveolar proteinosis in rats may be related to the accumulation of ITO NPs and the destruction of rat alveolar macrophage,which can clear the metabolism of surfactant and lead to the accumulation of a large number of alveolar surfactant.ITO NPs can cause oxidative stress,inflammatory response and imbalance of oxidative antioxidants in rat lung tissues,which may be one of the mechanisms of alveolar proteinosis caused by ITO NPs.Figure20;Table19;Reference 97... |
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