Effects Of Silica On The Expression Of Lysozyme In Rat Alveolar Macrophages

Silicosis is one of the most serious occupational diseases caused by inhalation of crystalline silica particles. Pulmonary fibrosis is the prominent pathological feature. Cellular and molecular mechanisms in silicosis are not yet totally understood. It is reported by several studies that the pathogenesis of silicosis are related to lysosome. Ingestion of silica by alveolar macrophages(AMs) leads to lysosome damage and release of lysosomal enzyme into the cytoplasm, which plays an important role in the pathogenesis of silicosis. Lysozyme is an important hydrolyases in lysosome in AMs. Lysozyme in AMs can stimulate lung fibroblast proliferation and lead to excessive synthesis and deposition of collagen. So lysozyme probably acts as a kind of flbrogenic agent, finally promotes lung fibrosis.We presume that silica can also induce the expression of various kinds of hydrolyases in AMs, which may accelerate damage to AMs and lung, and promote lung fibrosis during the development of silicosis. The current study was therefore designed to detect the levels of LSZ expression in AMs at different time points after injection of quartz dust suspension by immunohistochemical/immunocytochemical method and image analysis system. RT-PCR assay was also used to evaluate the levels of lysozyme mRNA expression at different time points after interact with silica. We intend to find the effects of silica on the expression of lysozyme in rat AMs at organic, cellular and molecular levels for ourfurther understanding the pathogenesis of silicosis.Materials and MethodsThe experiment was divided into three parts. Parti. The experiment of animalHealthy and male SD rats (weight 200~220g) were supplied by the center of laboratory animal in the medical school of Zhejiang University. Forty-eight experimental rats were randomly divided into two groups, control group and silica group. Rats of silica group and control group were injected lml quartz dust suspension(40g/L) and lml normal saline intratracheally respectively, and sacrificed at the 15th,3Oth and 60th day after injection. Samples of lung tissue were collected. The level of LSZ expression in AMs was detected by immunohistochemical method and image analysis system.Part 2. The experiment of cellAMs were obtained from healthy SD rats by bronchoalveolar lavage(BAL) according to improved Myrivk methods 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 thrice. Finally the cells precipitation were added into the medium of RPMI 1640 containing 10% calf serum. The AMs were then switched to RPMI 1640 without calf serum. According to the concentration of SiC>2 in the cultural medium, the two groups, Oug /ml and 50|ig/ml, were divided. AMs in this new medium were cultured at 37°C for 3h, 7h> 10h> 151k 20h respectively and collected at each time point for immunocytochemistry determination. The level of LSZ expression in AMs was detected by immunocytochemical method and image analysis system.Part 3. RT-PCR assayThe method of culture and purify of the AMs of SD rats was as above. The purified AMs were switched to PRMI1640 without calf serum. According to theconcentration of SiO2 in the cultural medium, the two groups, Oug /ml and 50(ig /ml, were divided. AMs in this new medium were cultured at 37°C for 3 Ik 71k 10 Ik 15 Ik 20h respectively and collected at each time point. Total cellular RNA of AMs were isolated by Trizol method. Reverse transcription-polymerase chain reaction(RT-PCR) was performed to detect the gene expression of lysozyme.ResultsPart 1. The experiment of animalThe products of area and gray value of LSZ in AMs of silica-treated rats at the 15th ,30th and 60th day after intratracheal injection of quartz dust suspension were(2063.22± 1302.31) , (3117.03±2445.63) and (3417.22±2744.19) respectively, which were significantly higher than those in control group [ (859.63±365.37),(763.82±326.83) and (771.41±284.50) respectively] (P<0.01)oPart 2. The experiment of cellThe products of area and gray value of LSZ in AMs exposed to silica at 3h, 7h, lOh, 15h and 20h were increased markedly [ (1146.07±41.91) , (1441.63±98.81) ,(1772.35±112.27) , (1634.34±149.78) and (1487.21±123.13) respectively], and reached the peak at lOh ( 1772.35±112.27 ) . Compared with control group[ (417.32±16.27) , (405.23±22.54) , (438.01±21.43) , (400.99±21.24) ,(429.65±26.70) respectively], the difference at every time point was significant (PPart 3. RT-PCR assayThe expression of lysozyme gene were up-regulated in AMs exposed to silica at3h, 7h, lOh, 15h and 20h[(0.54±0.06), (0.87±0.10)> (1.03±0.12X (1.41±0.12)>(1.11 ±0.08) respectively], and reached the peak at 15h (1.41 ±0.12 ). Compared withcontrol groupf (0.19±0.06), (0.25±0.08) , (0.22±0.04), (0.2O±O.05X (0.17±0.06) respectively], the difference at every time point was significant (P <0.01) .ConclusionsOn the basis of analysis of pathogenesis of silicosis, we first raised the presumption in present study that silica could induce high expression of lysozyme in AMs, and then proved it through immunohistochemical method, immunocytochemical method and RT-PCR assay. Our study offered a new idea for the further investigation of pathogenesis and prevention of silicosis.