| Background and ObjectivesSilicosis,one of the most serious pneumoconiosis,is a systemic disease caused by fibrosis of lung tissue due to inhalation of crystalline silica particles.It accounts for 50% cases of pneumoconiosis in our country.The cellular pathogenetic mechanism in silicosis has not yet been totally understood.It was reported in several studies that the pathogenesis of silicosis was related to lysosome.The inhaled silica leads to the damage of lysosome in alveolar macrophages(AMs)and the release of lysosomal enzyme into the cytoplasm,which play important roles in the pathogenesis of silicosis. Lysozyme is one of the major hydrolyases in lysosome of AMs.Lysozyme in AMs can stimulate lung fibroblast proliferation and lead to excessive synthesis and deposition of collagen.Thus,lysozyme probably acts as a fibrogenic agent,which accelerates the process of lung fibrosis.It has been indicated by studies that LSZ is easier to be released than other lysosomal hydrolases,which makes lysozyme a proper marker enzyme of alveolar macrophages in this study.The silica can also induce the expression of various kinds of hydrolyases in AMs, which may accelerate the damage to AMs and lungs,and promote lung fibrosis during the development of silicosis.We presume that some medicine,such as indomethacin in differnent concentrations,be used to lower the expression of lysozyme in alveolar macrophages and thus relieve the damage to the macrophages and the lung tissue. This study was therefore designed to detect the levels of LSZ expression in AMs at different time points after injection of quartz dust and indomethacin suspension by western blotting method. We intend to find out the effects of indomethacin on the expression of lysozyme induced by silica in rat AMs in vitro,which may provide the help to the further understanding of pathogenic mechanisme of silicosis at cellular and molecular levels.Materials and MethodsThe experiment was divided into two parts.Part 1:The Cellular ExperimentAMs were obtained from healthy SD rats by bronchoalveolar lavage(BAL) according to the improved Myrivk method in the sterile circumstances.The BAL fluid was collected together and centrifuged at 1500 rpm for 15 minutes.The supernatant was discarded,and then we rinsed the precipitation with PBS once.Finally the cells precipitation were added into the medium of RPMI 1640 containing 10%calf serum. The AMs were then switched to RPMI1640 without calf serum after cultivation 2 hours.According to whether adding SiO2 or indomethacin in the cultural medium, three groups,the control group,the model group and the intreventive group,were divided.And the interventive group has three levels,10-5μg/ml,10-4μg/ml,10-3μg/ml, from low to high concentration.AMs in this new medium were cultured at 37℃for 8 hours respectively.Finally we took the cell picture through the inverted microscope.Part 2.Protein Immunoblotting Assay:Western BlottingThe methods of culture and purification of the AMs of SD rats were as above. The purified AMs were switched to PRMI1640 without calf serum.According to the three groups,three levels of AMs in this new medium were cultured at 37℃for 8 hours,16 hours respectively,and collected at each time point.Total cellular protein of AMs were extracted from the groups.The extracted protein underwent the protein immunoblotting assay to detect the expressions of LSZ in AM.Image analysis was performed through Quantity One analysis system aided by the computer.ResultsPart 1:The Cellular ExperimentAfter purified and treated by silica and indomethacin for 8 hours,the cell adherence of Ams in the control group was in good growth.Cellular swelling and distortion appeared in the model group,even with some cell debris being found in Ams.While the shape of the AM cells in the interventive group appeared relatively complete,compared with the model group,due to the use of indomethacin concentration.Part 2:Protein Immunoblotting Assay:Western BlottingThe expression of lysozyme protein was up-regulated in AMs exposed to silica for 8 hours,16 hours.With the use of indomethacin,the expression of lysozyme protein in the intreventive group showed the tendency of decline.The expressions of lysozyme in AMs of the model group at 8 hours and at 16 hours,were higher than that of the control group,which was 1.945 and 2.285 higher than the control group respectively.The difference of the two group was significant(PC＜0.0125).LSZ protein expressions of the interventive group were down-regulated compared with the model group,depending on the concentration of indomethacin.The more indomethacin,the less expression of lysozyme protein.At 8 hours time point [Compared with cotrol group,1.586,1.055,0.864 respectively],compared with model group,significant difference appeared in both the middle and the high concentration group(P＜0.0125).At 16 hours time point[Compared with cotrol group,1.544,1.481,0.908 respectively],compared with model group,only the high concentration group showed significant difference(P＜0.0125).ConclusionsBased on the analysis of pathogenesis of silicosis,we first present in this study the presumption that silica could induce high protein expression of lysozyme in AMs,and that adding indomethacin into the cultural medium could prevent or lower the development of silicosis disease.Through western blotting assay,we detected the expression of lysozyme protein were up-regulated in AMs exposed to silica at 8 hours, 16 hours.With the use of indomethacin,the protein expression of lysozyme in Ams tended to decline,which indicates that indomethacin has a depressant effect on protein expression,and that the depressant effect demonstrates the dose-effect relation.This study presents a new idea for the further investigation on the pathogenesis and prevention of silicosis.
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