The Effects And Mechanisms Of Short-Term Exposure To PM_2.5on Bleomycin-induced Pulmonary Fibrosis In Rats

Objective: Interstitial lung diseases is characterized by accumulation ofvarious inflammatory cells, thickened alveolar septum with collagendeposition and reduced lung compliance, leading to impaired gas exchange,without known cause. But growing evidence suggests that severalenvironmental agents may have a causal role in selected disorders ofinterstitial lung diseases. Idiopathic pulmonary fibrosis is defined as a morecommon form of interstitial lung diseases. As component of ambientenvironmental air pollutants, PM_2.5show greater deposition in the deep lungupon inhalation. Evidence suggests PM_2.5had stronger associations withpulmonary diseases, such as COPD and asthma. In this study, we investigatedthe effects of PM_2.5on pulmonary inflammatory and fibrotic changes inducedby intratracheal bleomycin （BLM） in mice. Mice were intratracheallyadministered either PM_2.5, BLM or BLM plus PM_2.5. We assessed fibroticchanges by the content of hydroxyproline （HYP） and histopathology of lung.To elucidate how PM_2.5contributes to the development of BLM-inducedfibrosis, we collected bronchoalveolar lavage fluid （BALF） and detected thetotal and differential cell counts and concentrations of proinflammatorycytokine interleukin-6（IL-6）、TNF-α and fibrogenic mediator transforminggrowth factor-β1（TGF-β1）. Western blot was performed to detect the proteinlevels of TLR2and TLR4in lung of rats.Methods:96healthy Sprague-Dawley （SD） female rats of clean grade,were divided into four groups randomly.①c ontrol group（group N）;②PM_2.5group （group PM）;③BLM group （group B）;④PM_2.5plus BLM group （groupPM+B）；24in each group，each group were randomly into four groups by fourtime periods （the3thday, the7thday, the14thday and the21thday）. The controlgroup were injected of normal saline （1.5ml/1kg） into trachea, group PM received3.75mg/kg of PM_2.5suspended in saline （1.5ml/kg）, group B wereinduced by intratracheal injection of Bleomycin （3.5mg/kg）, group PM plus Breceived PM_2.5（3.75mg/kg） combined with BLM （3.5/kg） in saline （1.5ml/kg）.The rats were sacrificed by deep anesthesia on days3,7,14and21afteradministration. The lower lobes of right lungs were cut into sections andstained with hematoxylin-eosin （H-E） or Masson’s trichrome to estimate thedegree of alveolitis and pulmonary fibrosis by the method of Szapiel into4grades. Bronchoalveolar lavage fluid （BALF） was collected by cannulatingthe trachea and lavaging the left lungs. The levels of IL-6and TNF-α on days3and7and activated transforming growth factor-β1（TGF-β1） on days3,7,14and21in BALF were detected by the method of enzyme linkedimmunosorbent assay （ELISA）. Western blot was performed to detect theprotein levels of TLR2and TLR4in middle lobe of right lung on day3. Themiddle lobes of right lung were made into tissue homogenate which were usedfor measuring the contents of HYP in pulmonary tissue on day21. Statisthicswork was done with SPSS13.0statistical software.Results:1Body weight changes: On the same time, we confirmed that the miceinstilled with PM_2.5alone did not exhibit any significant difference in bodyweight compared with the control group （P>0.05）. In the groups that receivedBLM or PM_2.5plus BLM, body weight of rats was significantly decreasedcompared with the control group （P<0.05）. Moreover, in the groups thatreceived PM_2.5plus BLM, body weight of rats was significantly decreasedcompared with that in the group B （P<0.05）. In the groups that received BLMor PM_2.5plus BLM, body weight of rats was significantly decreased on day7compared with that on the other days （P<0.05）.2The results of pulmonary pathology: On the same time point, group B,group PM+B exhibited significant alveolar inflammatory and fibrosis changescompared with group N and group PM （P<0.05）, the degree of alveolarinflammation in group PM+B is more serious than that in group B on day3and7（P<0.05）. Group PM+B exhibited significant fibrosis changes with group B on day14（P<0.05）. Group PM exhibited significant nflammatorychanges compared with group N on day3（P<0.05）, There was no significantdifference in the degree of fibrosis changes between group N and group PMon days3,7,14and21（P>0.05）.3The total cell count of BALF and cell classification: In comparisonwith the control group, the mice administered BLM or PM_2.5plus BLMshowed significant increases in the numbers of total cells, percentage ofneutrophils at any time point （P<0.05）. The total cell numbers in BALF weresignificantly increased in group PM+B as compared with that in group B atany time point （P<0.05）. Intratracheal PM_2.5alone could induce significantincrease in the numbers of total cells, percentage of neutrophils comparedwith the control group on the3thday in BALF （P<0.05）. Intratracheal PM_2.5plus BLM could induce significant increase in the numbers of total cells ondays7and14, percentage of neutrophils on day7compared with that on theother days in BALF （P<0.05）. Intratracheal BLM could induce significantincrease in the numbers of total cells on days7, percentage of neutrophils ondays7,14and21compared with that on the other days in BALF （P<0.05）.4The changes of concentration of IL-6, TNF-α and TGF-β1in BALF:We measured the levels of TNF-α and IL-6in BAL fluid as potent acuteproinflammatory mediators. The concentration of IL-6and TNF-α in BALfluid were significantly elevated in Group B and group PM+B compared withthat in group N and group PM on days3and7（P<0.05）. Compared with thatof group B, the concentrations of IL-6and TNF-α were significantly increasedin group PM+B （P<0.05）.There was significant difference in the concentrationof TNF-α and IL-6between group N and group PM on day3（P<0.05）.Additionally, to evaluate the effect of PM_2.5on BLM-induced upregulation ofthe fibrogenic mediators, the levels of TGF-β1in BAL fluid were measuredon days3,7,14and21. The concentration of TGF-β1were significantlyelevated in group B and group PM+B compared with that in group N andgroup PM at any time point （P<0.05）. The concentration of TGF-β1weresignificantly elevated in group PM+B compared with that in group B on days 3,7and14（P<0.05）. There was no significant difference in the level ofTGF-β1between the control and group PM at any time point （P>0.05）. Theconcentration of TNF-α and IL-6were significantly elevated in group PM+Band group B on day7compared with that on day3（P<0.05）. Theconcentration of TGF-β1were significantly elevated in group B on days14and21compared with that on the other days （P<0.05）, the concentration ofTGF-β1were significantly elevated in group PM+B on day14compared withthat on the other days （P<0.05）.5The contents of hydroxyproline （HYP） in pulmonary tissue: To evaluatethe development of pulmonary fibrosis, the middle lobes of right lung fromthe mice on day21were analyzed for the contents of HYP. The contents ofHYP were significantly elevated in pulmonary tissue in group PM+B andgroup B compared with that in control group （P<0.05）. The contents of HYPin pulmonary tissue in the group PM+B was significantly higher than that inthe group B （P<0.05）. There were no statistical significance between groupB and group N （P＞0.05）.6The protein levels of TLR2and TLR4in lung: Western blot wasperformed to detect the protein levels of TLR2and TLR4in middle lobe ofright lung on day3. Injection of PM_2.5, BLM or PM_2.5plus BLM intotratracheal caused significant increase in the protein levels of TLR2and TLR4in the lungs compared with injectiong of saline in the control group （P<0.05）.The protein levels of TLR2in the lungs in group B and in group PM+B wassignificantly higher than that in group PM. The protein levels of TLR2in thelungs in group PM+B was significantly greater than that in group B （P<0.05）.The protein levels of TLR4in the lungs in group PM+B was significantlyhigher than those of group B （P<0.05）.Conclusions:1Exposure to PM_2.5can exaggerate BLM-induced fibrotic changes in thelung of rats.2Exposure to PM_2.5could impact the concentration of IL-6, TNF-α andTGF-β1by increasing TLR2and TLR4expession, by which exaggerate BLM-induced inflammatory and fibrotic changes in the lung...