The Mechanisms Of Pulmonary Inflammatory Response And Organelle Dysfunction Induced By Particulate Matter Exposure

Air pollution is a worldwide environmental problem,which is rather serious in China,characterized by large pollution area,long duration and a large number of exposed people.As a major component of air pollutants,particulate matter(PM)can cause a variety of adverse outcomes,including respiratory diseases,cardiovascular diseases and even cancers.The American Cancer Society conducted a 16-year study on 500,000 people and found that the risk of death was increased when the concentration of PM2.5 in the air was higher than 10?g/m3,and the total risk of death was increased by 4%,the risk of death from cardiopulmonary diseases was increased by 6%and the risk of death from lung cancers was increased by 8%when the concentration of PM2.5 was increased by 10?g/m3.MicroRNAs(miRNAs)play an important role in the process of PM exposure inducing adverse health effects.MiRNAs can bind to the 3'non-coding region of the target mRNA,facilitating degradation of the mRNA or inhibiting its translation.MiRNAs are closely related to inflammation and can exert pro-inflammatory or anti-inflammatory effects by up-regulating or down-regulating the levels of inflammatory cytokines.However,the role of miRNAs in the process of PM exposure inducing inflammatory response is not clear.Oxidative stress and inflammation are closely related and interdependent pathophysiological processes.Numerous studies have shown that oxidative stress participates in or even plays a key role in the inflammatory response induced by PM exposure.In addition to its association with innflammation,oxidative stress may further induce other biological events,such as organelle dysfunction.Mitochondria,lysosomes and endoplasmic reticulum are linked with cell apoptosis,endocytosis and homeostasis,respectively.In the adverse effects induced by PM exposure,mitochondria,lysosomes and endoplasmic reticulum often become targets of PM,and their dysfunction appears earlier than obvious adverse outcomes,so they can be used as early markers.At present,there are limited studies on the dysfunction of mitochondria,lysosomes and endoplasmic reticulum caused by PM exposure.To solve the scientific problems above,we selected three representative PMs for research with four parts:First,the inflammatory response of mouse lung was induced by single and repeated intra-tracheal instillation(IT)of diesel exhaust particle(DEP),and the change profiles of IL-6,let-7 microRNA and LIN28B in the lungs were demonstrated.The second was to verify the role of LIN28B/let-7 axis in the DEP-induced inflammatory response in vitro by silencing the LIN28B gene and over-expressimg let-7.The third was to use high-content screening(HCS)technology to investigate the effects of carbon black(CB),DEP and PM2.5 on oxidative stress and organelle function in three lung-related cells,and to compare the toxicity difference of three kinds of PMs and the sensitivity difference of the three cells.The fourth was to verify the role of oxidative stress in the induction of mitochondria and lysosomal damage and apoptosis in vitro upon CB exposure.1.The change profiles of let-7 and related indexes in DEP-induced pulmonary inflammatory response in vivoFor single IT section,90 mice were randomly distributed into 10 groups(n=9 in each group),including 5 experimental groups(administered with 50?l of suspension containing 100?g of DEP per body)and 5 control groups(administered with 50?l of vehicle per body).There were 5 time points including 0.5h,6h,12h,24h and 48h and each time point corresponded to an experimental and a control groups.Mice were sacrificed at their own time point after single IT.For repeated IT section,30 mice were randomly divided into 3 groups(n=10 in each group),which were control group(administered with 50?l of vehicle per body),low-dose group(administered with 50?l of suspension containing 12.5?g of DEP per body)and high-dose group(administered with 50?1 of suspension containing 50?g of DEP per body).Mice were intra-tracheally administered 3 times a week,consecutive 4 weeks in total,and were sacrificed 24h after the last IT.Three mice in each group were chosen for histological assessment.Hematoxylin and eosin(H&E)staining was used to evaluate the pathological changes of lung tissue.Enzyme-linked immunosorbent assay(ELISA)was used to determine IL-6 protein level.Real time polymerase chain reaction(Real-time PCR)was used to quantify the levels of let-7 family members.Western blot was used to determine LIN28B protein level.For single IT section,at 6h,12h and 24h time points in DEP exposure groups,a large amount of neutrophils were infiltrated,epithelial cells of bronchioli mucous desquamated partly and alveoli wall thickened significantly,and the pathological scores were significantly higher than those in corresponding control groups;The protein levels of IL-6 and LIN28B were significantly elevated and the levels of let-7d and let-7g were significantly decreased.For repeated IT section,remarkable abnormalities in the lungs occurred in low-dose and high-dose groups,including substantial neutrophils infiltration,hemorrhage and alveoli wall thickening.Compared with the control,in two DEP exposure groups,the scores and levels of IL-6 and LIN28B protein were significantly higher,but the levels of let-7d and let-7g were significantly lower.2.The role of LIN28B/let-7 axis in DEP-induced inflammatory response16HBE and Beas-2B cell lines were applied and seven groups were set,including control,DEP group,si-LIN28B group,siRNA NC group,let-7d group,let-7g group and mimic NC group.The DEP group was exposed to 100?g/ml of DEP for 24h.For LIN28B silencing,the cells were transfected with 50nM si-LIN28B or siRNA NC in each well.For the over-expression of let-7d or let-7g,the cells were transfected with 50nM let-7d mimic,let-7g mirmic or mimic NC in each well,respectively.After transfection,the cells were exposed to 100?g/ml of DEP for 24h and then all group cells were harvested for the subsequent assays.Total RNA was extracted from the lung tissue using Trizol reagent and reverse transcribed to complementary DNA.Real-time PCR was used to quantify the expression of let-7d,let-7g and IL6 mRNA.The expression profiles of let-7d,let-7g and IL6 were all identical between the two cell lines.Let-7d and let-7g levels were significantly decreased but IL6 mRNA level was notably increased in response to DEP treatment,compared with the control.Let-7d level in si-LIN28B group was much higher than that in siRNA NC group following DEP exposure,and let-7g showed the same trend.In addition,IL6 mRNA level in si-LIN28B group was much lower than that in siRNA NC group following DEP exposure.Similarly,IL6 mRNA level in let-7d or let-7g group was lower than that in mimic NC group following DEP exposure,respectively.3.Three representative PMs induce oxidative stress and organelle dysfunction16HBE,human embryonic lung fibroblasts(HELF)and human umbilical vein endothelial cells(HUVEC)were exposed to different concentrations of CB,DEP and PM2.5 for 24h.The cell viability was measured using CCK-8 Kits.The molecular probes were used to determine the levels of ROS,mitochondrial superoxide,mitochondrial membrane potential(MMP),intracellular ATP,lysosomal pH and intracellular Ca2+.Real-time PCR was used to quantify the mRNA levels of HO-1,NQO1,CHOP and XBP-1s.The cell viability of HUVEC was decreased significantly following three PMs exposure,while the cell viability of three cell lines was all decreased significantly after CB exposure.A significant increase in ROS levels in three cell lines was observed upon exposure to three PMs to some extent.The level of mitochondrial superoxide was significantly increased in three cell lines upon CB exposure while only in HELF after 100?g/ml of PM2.5 exposure.The levels of MMP and intracellular ATP were decreased to some extent in three cell lines after three PMs exposure compared with the control.The lysosomal pH was increased in three cell lines upon CB exposure while only in HUVEC upon DEP or PM2.5 exposure,compared with the control.The intracellular Ca2+ level was remarkably elevated in 16HBE upon CB exposure,and the mRNA levels of CHOP and XBP-1s were elevated in 16HBE and HUVEC compared with the control.4.The role of oxidative stress in organelle dysfunction and apoptosis induced by CB exposureBeas-2B cells were incubated with CB at the concentrations of 0,25,50,or 100?g/ml for 24h,or at the CB concentration of 50?g/ml with 2mM of N-acetyl-L-cysteine(NAC)for 24h.Then cell viability was determined by CCK-8 kits,and the intracellular ROS,MMP and lysosomal pH were detected by different molecular probes and analyzed by HCS system.The apoptosis rate was examined by flow cytometry.The cell viability was decreased significantly at the CB concentration of 50 and 100?g/ml compared with the control.The intracellular ROS,lysosomal pH and apoptosis rate were increased while MMP was decreased in these two groups mentioned above compared with the control.NAC alleviated or neutralized the changes of these endpoints to some extent.Conclusions1.Pulmonary DEP exposure elicited notable pathological injuries with a large amount of neutrophils infiltration,increased levels of IL-6 and LIN28B protein,but decreased levels of let-7d and let-7g.LIN28B/let-7 axis might be involved in the inflammatory response induced by DEP exposure.2.The down-regulation of let-7d and let-7g expression levels and the up-regulation of IL6 mRNA levels after DEP exposure were attenuated by the silence of LIN28B in vitro,indicating that LISN28B is the upstream gene of let-7d,let-7g and IL6.The up-regulation of IL6 mRNA level after DEP exposure was alleviated by the over-expression of let-7d and let-7g,indicating that let-7d and let-7g are the upstream genes of IL6.3.Three types of PMs induced obvious oxidative stress and mitochondrial dysfunction in three cell lines and lysosomal alkalinization in HUVEC while only CB trnggered endoplasmic reticulum(ER)stress response in 16HBE and HUVEC.CB basically exhibited more potent toxicity compared with DEP and PM2.5,which might help to know the complexity of toxicity features of nanometer-sized particles.The finding that PMs-induced toxicity impacts exhibited a cell-type dependent manner might help to understand the sensitivity difference of different tissue in the lung.4.Exposure to CB induced oxidative stress response in Beas-2B cells,which subsequently mediated the dysfunction of mitochondria and lysosomes,and the organelles dysfiunction might further trigger cell apoptosis.The finding may help to understand the pathophysiological mechanism of CB exposure causing respiratory damage.