| Objectives To investigate the effect of aspirin on brain metastasis of lung cancer and its possible mechanism.Methods A549 lung cancer cells and human brain microvascular endothelial cells were cultured to establish the blood-brain barrier model in vitro.After stimulation with aspirin(8m M/L)for different time points(0 min,30 min,60 min,120 min,180 min and 240 min),the number of A549 lung cancer cells in the lower chamber of the transwell was observed by automatic cell counter,trypan blue staining and inverted microscope,and the survival rate of the cells was analyzed by live-dead staining;the permeability change of the bloodbrain barrier in vitro was determined by horseradish peroxidase flux method,and the morphological change of the cytoskeleton was observed by phalloidin fluorescence staining;the content of tumor necrosis factor-α(TNF-α)in the lung cancer cell culture medium was determined by ELISA;the expression levels of heat shock protein 70(HSP70),tight junction proteins ZO-1 and occludin on the blood-brain barrier brain microvascular endothelial cells were detected by Western blot.Results 1 Determination of cell invasiveness by in vitro cell migration assay: Lung cancer cells were placed in the upper chamber of the in vitro blood-brain barrier model.Compared with the control group,the number of cells in the lower chamber culture medium was reduced after aspirin treatment,and the number of cells in the lower chamber culture medium was the least at 120 min of treatment,with a statistically significant difference compared with the control group(P<0.05);live-dead staining of A549 lung cancer cells after aspirin treatment revealed that the number of cell death was the highest at120 min of aspirin treatment.These results indicate that cells are minimally invasive at this time.2 Changes of permeability of blood-brain barrier: The permeability of blood-brain barrier in vitro was analyzed by HRP flux.After treatment with aspirin(8 m M/L)in the upper chamber of Transwell,the effect of aspirin on blood-brain barrier in vitro was observed at different time points(0min,30 min,60min,120 min,180min and 240 min).The results showed that compared with the control group,the HRP leakage rate began to decrease at 60 min of aspirin treatment,and HRP flux was the lowest at 120 min after administration;the cytoskeleton changed after phalloidin staining.Compared with the control group,the cells showed fascicular cytoskeletal structure and the intercellular space became smaller at 120 min after aspirin administration,indicating that the permeability of blood-brain barrier was the smallest at this time,with statistically significant difference compared with the control group(P<0.05).3 Changes of TNF-α content in lung cancer cell culture medium: The concentration of TNF-α in A549 lung cancer cells culture medium at different time points(0min,30 min,60min,120 min,180min and 240 min)after aspirin administration was determined by ELISA.Compared with the control group,the concentration of TNF-α secreted by A549 lung cancer cells after aspirin administration was significantly reduced,and TNF-α secretion was significantly inhibited 60 min after aspirin administration,1.21～5.03 times lower than that in the control group(P<0.05).These results suggest that lung cancer cells stimulated by aspirin can cause a decrease in the release of TNF-α from lung cancer cells.4 Expression levels of HSP70 in brain microvascular endothelial cells and ZO-1 and occludin proteins in blood-brain barrier endothelial cells: After A549 lung cancer cells culture medium treated with aspirin was applied to the constructed in vitro blood-brain barrier model,the results of Western blot showed that the HSP70 protein concentration was the highest at 60 min;the proteinexpression levels of ZO-1 and occludin were the highest at 120 min,and then decreased with time.Meanwhile,we added TNF-α at 120 min of aspirin treatment,and the results revealed that the protein expression levels of HSP70,ZO-1,and occludin in the ASP+TNF-α group were significantly lower than those in the aspirin 120 min group.Conclusions Aspirin may upregulate the expression of tight junction proteins through TNF-α and HSP70,thereby reducing the permeability of the blood-brain barrier and probably inhibiting the development of brain metastasis in lung cancer.Figure10;Table3;Reference 136... |
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