Effects And Mechanisms Study Of Arsenic Trioxide Tracheal Instillation On Lipopolysaccharide-induced Pneumonia In Rats

BackgroundWith the rapid development of social economy and urbanization in China,air pollution,especially urban air pollution,is becoming more and more serious.In recent years,the haze in large cities has become the most important environmental factors that seriously threaten the health of the residents.It is an important environmental and health problem that the government and the public need to pay close attention to.A large number of studies have approved that fine particulate matter(PM2.5)is the main component of haze and has potential health impact.Epidemiological studies have shown that air pollutants,especially PM2.5,are associated with increased residents' mortality,leading to acute exacerbations of heart and lung diseases.Meanwhile,studies have also shown that PM2.5 can cause inflammation in human lungs,and there may be a correlation between inflammation and pollutant components.The composition of PM2.5 is complex,carrying a large number of biological,chemical and physical substances.Studies found many kinds of microorganisms that can cause infectious pneumonia can be detected on PM2.5,including bacteria and viruses.Heavy metal pollution is serious in cities,especially in industrialized cities.Heavy metals are easily attached to PM2.5.They can easily accumulate in the human body but are difficult to degrade.Further analysis of PM2.5 indicates that arsenic(As)is one of its important components.The current research is mainly focused on the distribution,occurrence,and source identification of the single component in the PM2.5,however,the biological toxicities and pathogenic mechanisms of PM2.5 components on healthy residents and pneumonia patients need further study.The analysis of the PM2.5 revealed that arsenic(As)oxide is one of the heavy metal compounds on the PM2.5 in industrial cities.At the same time,As oxide is also a classic toxic.It is not clear whether As oxide is one of the contributors to health hazards of haze.Therefore,the toxic effects of arsenic trioxide(ATO)on lungs were studied by means of tracheal instillation to elucidate the health hazards of haze and its mechanism.Epidemiological studies have shown that residents with underlying lung diseases such as pneumonia,bronchitis and chronic obstructive pulmonary disease are vulnerable to haze.Lipopolysaccharide(LPS)is a component of the cell wall of gram-negative bacteria and is used in the construction of an animal model of pneumonia.In this study,LPS-pretreated rats were used to construct a pneumonia model.Through ATO exposure,the toxicity of ATO to pneumonia rats was observed,and elucidated the health hazards of haze on residents with underlying diseases.ObjectTo study the effects and mechanisms of arsenic trioxide(ATO)on the lung injury and the air blood barrier function in healthy rats and pneumonia rats.Method1.Toxic and dose determination: in nature,As has four valence states of As+5,As+3,As,and As-3,while atmospheric inorganic As only exists as two oxides of As+3 and As+5.The average concentrations of As+3 and As+5 in the atmosphere are 5.1±2.7 ng/m3 and 67±35 ng/m3,respectively accounting for 2.2%–74.6% and 12%–100% of total atmospheric arsenic.Toxicological studies have shown that the biological toxicity of As was determined by its chemical valency.The biological toxicity of As+3 is 35-60 times that of As+5,and As+3 is easier to enter cells.Therefore,we selected As+3,which had a relatively high biological toxicity,that is,ATO,as a toxicant,to study its pulmonary toxicity.The standard limit of air As concentration in China's ambient air quality standards is 6 ng/m3.However,the analysis of PM2.5 in China found that the average concentration of As fluctuates greatly and can reach 920 ng/m3 under heavy pollution weather,exceeding the standard limit of 153 times.Based on the above actual conditions,we set a low-dose exposure group(LD group)of 60 ng/m3 to simulate the level of arsenic expourse under mild air pollution conditions,and with a 10-fold increase in concentration,that is,600 ng/m3 and 6000 ng/m3 exposure concentrations for the medium dose(MD)and high dose(HD)groups,respectively,were used to simulate acute As exposure levels in moderate and very severe air pollution.Considering the tidal volume of rats,we determined that the doses of ATO in the LD,MD,and HD groups were 0.16,1.60,and 16.00 ?g/kg,respectively.2.Establishment of pneumonia rat model: with reference to Jiao Guangyu et al.,an animal model of pneumonia was constructed with an airway exposure of 0.20 mg/kg LPS.4h after LPS exposure can cause pulmonary interstitial edema,alveolar inflammatory cell infiltration,leakage of albumin and red blood cell,and these injuries were not completely recovered after 48 h.Study the effects of ATO on the development of lung injury in pneumonia rats and its mechanism.3.ATO exposed healthy rats and groups: the male Wistar rats were randomly divided into CON group and LD,MD,HD ATO exposure groups.Each group was exposed to normal saline and ATO of 0.16,1.6,and 16.00 ?g/kg by tracheal instillation,respectively.Samples were collected at 6,12,24,48,and 72 hours after exposure.4.ATO exposed pneumonia rat models and groups: LPS tracheal instillation was used to establish a pneumonia rat model.The male Wistar rats were randomly divided into control group(the CON group),ATO exposure group(16.0?g/kg ATO,the ATO group),the LPS pneumonia model group(0.2mg/kg,LPS,the LPS group)and the ATO exposed LPS pneumonia group(16.0?g/kg ATO +0.2mg/kg LPS,the ATO+LPS group).12 h after exposure,blood and lung tissue samples from each group were collected for testing and analysis.5.Sample detection methods: use automatic blood cell analyzer for blood cell count;ELISA kit for detection of serum inflammatory factor concentration;weighing method,coomassie brilliant blue method for detecting lung water content and lung lavage fluid protein content;the pathological changes of lung pathology were observed under microscope,ultrastructural changes of cells were observed by transmission electron microscopy,changes of air-blood barrier permeability were detected by Evans blue and lanthanum nitrate tracer;the expression of JNK1/2 and p38 protein in the inflammatory pathway was detected by Western Blot,and the expression of Claudin-3,Claudin-4,and Claudin-18 protein and cell localization were observed by laser confocal microscopy.6.Statistical analysis: SPSS 18.0 statistical software was used for analysis.The results were expressed as mean ± standard deviation((?)±s).One-way analysis of variance was used for comparison between groups,and SNK(Student-Newman-Keuls)method was used for comparison between groups.Pearson analysis was used for multivariate correlation analysis.P<0.05 was considered statistically significant.Result1.Effects of different doses of ATO expourse on lung in healthy ratsCompared with the CON group,the WBC count in LD group decreased significantly in 6h and 12 h,while the 6h,12 h and 24 h in the MD and HD groups decreased significantly.The TNF-a in the expourse groups increased significantly in 48 h and only HD group was still significantly higher at 72 h.The IL-6 and IL-10 in the LD and MD groups increased significantly in the 24 h,and the HD group increased significantly in 12 h,72h and 12 h,24h and 72 h.The lung water content in the LD and MD groups increased in 24 h,the HD group increased at 5 points,the concentration of prote in the BALF in the LD group increased in 24 h and 72 h,the MD group began to increase at 12 h and the HD group began to increase at 6h,and did not recover at 72 h.HE staining showed that exposure to MD and HD of ATO could cause pulmonary interstitial thickening,alveolar edema,alveolar structure destruction and inflammatory cell infiltration,especially in the HD group at 12 h,24h and 48 h.Protein detection showed that after 12 h and 24 h ATO exposure resulted in increased phosphorylation of JNK1/2 and p38 proteins.2.Effects of ATO exposure on lung tissue structure in pneumonia ratsHE staining showed that the pulmonary interstitial thickening,alveolar membrane edema and inflammatory cell infiltration were observed in both ATO and LPS groups,and lung injury was more significant in ATO+LPS group,the damage of alveolar structure could be observed.Observation of the ultrastructure of the cells by scanning electron microscopy revealed that the ATO group and LPS group showed vacuolization of the AT-I and AT-II cells,nuclear pyknosis,and collapse of the mitochondria and lamellar bodies of the AT-II cells.In the ATO+LPS group,the matrix of cells was vacuolized,the mitochondria and lamellar bodies of AT-II cells collapsed,and even the nuclear lysis was observed.The results showed that ATO exposure may increase lung injury in pneumonia rats,suggesting that there may be a common mechanism for ATO and LPS-induced lung injury.3.Effects of ATO exposure on serum inflammatory factors and MAPK pathway in pneumonia ratsCompared with the CON group,the ATO group,the LPS group and the ATO+LPS group all showed a significant decrease in WBC,and the greatest reduction was observed in the ATO+LPS group.There was no obvious change in TNF-? after exposure to ATO,but it was significantly increased in the LPS and ATO+LPS groups,however,the ATO+LPS group was significantly higher than the ATO and LPS groups.The ATO group,LPS group and ATO+LPS group all showed increased in IL-6,IL-10,lung water content,and BALF protein content,and were highest in the ATO+LPS group.The detection of protein expression showed that the phosphorylation levels of JNK1/2 and p38 proteins increased in ATO group,LPS group and ATO+LPS group.However,the phosphorylation level of JNK1/2 in ATO+LPS group was not significantly different from that in ATO group and LPS group,but the phosphorylation level of p38 in ATO+LPS group was significantly higher than that in ATO group and LPS group.It is suggested that ATO aggravates the degree of lung injury induced by LPS in pneumonia rats,and MAPK signaling pathway may play a regulatory role in this process.4.Effects of ATO exposure on the permeability of lung air-blood barrier in healthy rats and pneumonia ratsCompared with the CON group,there was no significant change in Evans blue exudation in the lungs of the ATO group,but the lung Evans blue concentration in the LPS and ATO+LPS groups was significantly higher,and the concentration in the ATO+LPS group was higher than that in the ATO or LPS groups.In the ATO group,lanthanum nitrate appeared in the pulmonary vascular cavity and interspace of alveolar cell;in LPS group,lanthanum nitrate was limited in the pulmonary intravascular cavity;while the lanthanum nitrate in the ATO+LPS group was distributed in the pulmonary vascular cavity and interspace of alveolar cell,and was also distributed in the alveolar cells.These results indicate that ATO exposure can increase the permeability of pulmonary air-blood barrier in healthy and pneumonia rats,and the increase of pulmonary air-blood barrier permeability in pneumonia rats is more obvious.5.Effects of ATO exposure on the expression of Claudin proteins in healthy rats and pneumonic ratsCompared with the CON group,the expression of Claudin-4 and Claudin-18 proteins in the ATO group and LPS group decreased significantly,and the expression of Claudin-3 protein increased significantly,while,in ATO+LPS group,the expression of Claudin-4 and Claudin-18 proteins was further reducedand and the expression of Claudin-3 protein was further increased.It is suggested that the abnormal expression of Claudin-3,Claudin-4 and Claudin-18 protein may be one of the reasons that the increase of air-blood barrier permeability of healthy and pneumonia rats caused by ATO exposure.Conclusion1.The arsenic exposure in the LD group did not cause significant damage to the lungs of the rats.As exposure in the MD group began to show lung injury,and As exposure in the HD group resulted in significant lung injury,showing pulmonary edema,pulmonary interstitial thickening.It is suggested that inhalation of As in the case of haze has no obvious effect on human lung injury.Only in the extreme heavy haze,a large amount of As entering the body may cause lung injury to healthy people.2.ATO exposure can aggravate lung injury in pneumonia rats,aggravate the alveolar cell membrane edema,aggravate organelle damage,and increase inflammatory response.MAPK pathway may be a regulatory pathway for ATO to aggravate lung inflammation in pneumonia rats.It is suggested that acute As exposure may play a role in increasing the incidence of respiratory-related diseases in residents with underlying respiratory diseases under heavy haze.3.ATO can increase the permeability of lung air-blood barrier in healthy rats,and can lead to higher permeability of air-blood barrier in LPS induced pneumonia rats.The abnormal expression of Claudin family may be the reason for the increased permeability.It is suggested that,under heavy haze,the destruction of the air-blood barrier caused by acute As exposure may be the reason for the increased incidence of respiratory-related diseases in healthy residents,especially pneumonia patients.4.PM2.5 is a compound containing a large number of chemical components.Acute exposure to high-dose arsenic can aggravate lung injury in healthy rats,especially in pneumonia rats.We speculate that other chemical components of PM2.5,especially in industrial cities,may also be the cause of haze respiratory toxicity and need further studies.