Effects Of PM2.5 On Airway Mucus Secretion In Rats And Hydrogen Intervention

Objective: In recent years,more and more attention has been paid to the effect of fine particulate matter（PM2.5）on health.Acting as the gateway for air pollutants coming into the body and the direct affected target organ,the respiratory system is the first to be harmed by PM2.5.Many researches have revealed many impacts of PM2.5 on respiratory system.The mucus secreting from airway epithelial cells plays an important role in innate immune system of the respiratory system.But over-production of mucus is associated with many chronic airway inflammatory diseases.However,there is still a lack of research about how PM2.5 affecting the airway mucus hypersecretion.At the same time,little progress has been made on how to prevent and reduce the harm of PM2.5.Hydrogen（H₂）is known as a flammable gas.But H₂ has been reported to selectively reduce hydroxyl radical and the most cytotoxicity of ROS.There are many studies showing that inflammation and oxidative stress play an important role in the pathogenesis of PM2.5.However,no reports have shown that H₂ can reduce the effect of PM2.5 induced airway mucus hypersecretion.In this study,Concentrated Ambient Particulate Exposure Facility was used,and the rats were exposed to high concentrations of PM2.5 by inhalation.The effect of PM2.5 on airway mucus secretion in rats and related mechanisms were investigated.In addition,H₂ was used to see if it could mitigate the harm caused by PM2.5.Methods:1 Animals and exposure regimen: Male Wistar rats were divided randomly into five groups with seven animals each: fresh air control group（FA）,PM2.5 exposed group（PM）,hydrogen treatment group（PM+H）,oxygen treatment group（PM+O）and hydrogen control group（H）.Rats in PMgroup were exposed to concentrated ambient particles（CAP_s）,for 5consecutive hours/day,5days/week for 25 days,with the CAP_s concentration about 1500 μg/m3;For PM+H group,5 hours CAP_s followed by 2 hours hydrogen oxygen mixed gas inhalation treatment,containing 66% H₂;For PM+O group,5 hours CAP_s followed by 2 hours nitrogen oxygen mixture（33% O2）inhalation to exclude the effect of O2;Rats in the FA or H group were both exposed to filtered fresh air instead of CAP_s,but H group treated 2 hours hydrogen oxygen mixture inhalation each time after the air exposure.2 Measurement indexes: Measurement of lung mechanics and Pulmonary function was performed after the last exposure.Then rats were sacrificed,and bronchoalveolar lavage fluid（BALF）were collected for the analysis of proinflammatory factors（TNF-a,IL-8,IL-2,IL-1β,IL-10,MMP-12）and oxidative stress indicators（SOD,8-iso-PG）.At the same time,the right caudal lobe was removed,fixed in formalin solution,stained by HE stain,PAS stain and immunohistochemistry,for observing the change of morphological and mucus secretion;the right cranial lobe were stored at-80℃ until MDA levels in lung tissue measured for oxidative stress indicator;the right middle lobe were stored at-80℃ until processed for isolation of total RNA for analyzing MUC5 AC mRNA levels by real-time PCR.3 Statistical analysis: SPSS version 21.0 was applied for all statistical analyses.The data were expressed as the mean±SD.Comparisons among groups were tested by One-Way ANOVA analysis and Student-Newman-Keuls（SNK）test,Kruskal-Wallis H Test was used if the results were homogeneity of variance or non-normal distribution.For all tests performed,P<0.05 was considered statistically significant.Results:1 Forced vital capacity（FVC）and Peak expiratory flow（PEF）were significantly decreased with PM2.5 exposures,while total respiratory system elastance（Ers）and total respiratory system resistance（Rrs）were significantly augmented in the PM and PM+O group compared with the FA（P<0.01）.Andthese changes were significantly abrogated by hydrogen inhalation.2 PAS staining showed that PM2.5 exposure significantly increased PAS-positive rate as compared with that in the FA（P<0.01）.As expected,hydrogen significantly decreased PM-induced positive staining rate as compared with that in the PM and PM+O group（P<0.01）.At the same time,semi-quantitative analyses of IHC demonstrated that the percentage of bronchial epithelium muc5ac-positive area significantly increased in the PM2.5 exposure group as compared with that in the FA（P<0.01）,and this increase was significantly abrogated by hydrogen（P<0.01）.Meanwhile,the level of muc5 ac mRNA increased significantly in the rat lungs of PM2.5exposure group as compared with that in the control group（P<0.01）,Hydrogen significantly decreased PM2.5-induced muc5 ac mRNA level（P<0.01）.3 Compared with the FA group,the levels of pro-inflammatory mediators including TNF-a,IL-8,IL-2,IL-1β and IL10 was significantly higher in the PM and PM+O group（P<0.01）.And Hydrogen treatment significantly attenuated PM2.5-induced changes in above mentioned indicators（P<0.01,IL8 P<0.05）.4 The content of MDA,SOD and 8-iso-PG in the lung from the PM and PM+O group was significantly higher than that from the FA（P<0.01）.The oxidative stress indicators level of the PM+H group decreased significantly as compared with the PM and PM+O（P<0.01）.HE staining showed that PM2.5could induced rat lung pathological damage and inflammatory responses.The inflammatory scores of lungs from the the PM and PM+O group were significantly higher than that from the FA（P<0.01）,but the change induced by PM2.5 was effectively abrogated by hydrogen（P<0.01）.Conclusions:1 These findings indicated that subacute high concentration PM2.5inhalation exposure could induce airway mucus hypersecretion in healthy rats.This was associated with PM2.5 induced airway inflammation and oxidative stress.2 This was the first report to demonstrate that Hydrogen treatment couldattenuate PM2.5-induced airway mucus hypersecretion.To a certain extent,Hydrogen reduced the other harm of respiratory system caused by PM2.5exposure.Therefore,H₂ inhalation was expected to be applied to prevent and control of PM2.5 caused harm to the human body.