The Mechanism Of Endoplasmic Reticulum Stress And Apoptosis In Alveolar Macrophages Induced By SiO₂

Background and Objective:Silicosis is a disease caused by inhaling free silica dust in long time, and lead to lung widely nodular fibrosis. It is the most common and serious, progress fastest type of pneumoconiosis. The main characteristic of Endoplasmic Reticulum Stress (ERS) is unfoled or misfolded protein aggregation in the ER lumen and calcium disturbances. Short-term ERS is a protection to cells, under severe and long-term ERS, activation of unfolded protein response (UPR) leads to apoptosis pathway. Our study is to explore whether SiO2could induce ERS and apoptosis in Aveolar Macrophages (AM).Methods:(1) We made use of RAW264.7induced by200μg/ml SiO2as a model, and the time-point was0h/6h/12h/24h/48h. The morphological changes of cells were observed by transmission electron microscopy (TEM). Real-time PCR and Western blot were used to detect the protein expression of BiP and CHOP.(2) MTT was used to count cell proliferation, while DAPI staining and Flow Cytometry(FCM) and cleaved caspase-3were used to detect apoptosis.(3) Added with different concentrated4-PBA, the ERS inhibitor, we measured the change of ERS and apoptosis.Results:(1) With200μg/ml SiO2induced, TEM showed RAW264.7cells changed into irregular shapes, mitochondria swelling, ER lumen enlarged.(2) Real-time PCR and Western blot demonstrated that BiP expression was increased at first6h and slightly decreased at12h, while the induction of CHOP was increased from24h and peaked at48h.(3) MTT showed cell proliferation curve was lower with SiO2treatment group comparing with control group. DAPI staining appeared pyknosis of early apoptosis and apoptosis body at24h-48h. FCM revealed cell apoptosis rate increased at a time-dependent. Cleaved caspase-3of western blot was increased at24h and peaked at48h.(4) Given4-PBA, we could found the expression of BiP and CHOP were decreased.(5) Given4-PBA, MTT showed cells proliferation curve approached to control group. DAPI staining showed cell nuclear round and well-distributed. FCM revealed cell apoptosis rate decreased. And also cleaved caspase-3was reduced.Conclusions:SiO2induce Endoplasmic Reticulum Stress and Apoptosis in RAW264.7.