Effect Of Adenosine Released By Hypoxic Lung Cancer Cells On Brain Metastasis Of Lung Cancer And Its Possible Mechanism

Objectives To clarify the effect of adenosine onbrain metastasis of lung cancer and the possible mechanism of adenosine promoting brain metastasis of lung cancer.Methods At different time points,A549 lung cancer cells were cultured in hypoxia(0h,4h,8h,12 h,24h,48h).Brain microvascular endothelial cells were cultured in normal state,and the blood-brain barrier model was constructed in vitro.After establishing a blood-brain barrier model in vitro,Western blot was used to detect hypoxia inducible factor-1(HIF-1)and tight junction protein ZO-1 on brain microvascular endothelial cells in lung cancer cells;The expression level of adenosine in lung cancer cell culture medium was determined by ELISA;the permeability of the blood-brain barrier model was detected by fluorescence analysis;staining by trypan blue staining and observation by inverted microscope The number of A549 lung cancer cells in the lower chamber of Transwell;LSD-T test analysis to detect the change trend of the experimental results parameters and the correlation of each test parameter.Results 1.Changes in adenosine content in lung cancer cell culture medium: After hypoxic culture of lung cancer cells,the content of adenosine in culture medium began to increase,and the content of adenosine was the highest at 12 h after hypoxia,and then gradually decreased,compared with the control group.Statistical significance(P<0.01),indicating that lung cancer cells can cause the release of adenosine from lung cancer cells after hypoxia culture.2.Expression level of tight junction protein ZO-1 on brain microvascular endothelial cells of blood-brain barrier: After the culture medium of lung cancer cell A549 after hypoxia culture was applied to the constructed model of blood-brain barrier in vitro,the tightness of brain microvascular endothelial cells The expression of connexin ZO-1 began to decrease.The culture medium with hypoxia for 12 h resulted in the lowest expression of tight junction protein ZO-1 and the highest permeability of bloodbrain barrier.The difference was statistically significant(P<0.05),indicating that the blood-brain barrier is Through the tight barrier between brain microvascular endothelial cells in the blood-brain barrier,this major barrier mechanism acts as a barrier.Lung cancer cells release adenosine after hypoxia,and adenosine may reduce the expression of tight junction protein ZO-1 in the blood-brain barrier.Levels increase the permeability of the blood-brain barrier.3.Permeability changes of the blood-brain barrier: the hypoxic lung cancer cell culture medium and sodium fluorescein were applied to the upper blood-brain barrier model,and the blood-brain barrier permeability was indirectly reflected by observing the fluorescence intensity of the lower chamber.The results showed that the hypoxic cultured cell fluid could increase the level of lower fluorescein sodium,and the fluorescence intensity reached the highest in hypoxia culture for 12 hours,indicating that the blood-brain barrier was the most permeable at this time,compared with the control group.The difference was statistically significant(P < 0.01).4.Changes in the number of lung cancer cells in the lower chamber of Transwell: After the lung cancer cell suspension was applied to the upper chamber of the blood-brain barrier model in vitro,the number of cells was observed by trypan blue staining and inverted microscope under different time points of hypoxia.The results showed that the number of cells in the lower chamber culture medium increased after hypoxia,and the number of cells in the lower chamber culture medium was the highest at 12 h after hypoxia.Compared with the control group,the difference was statistically significant(P<0.01).This indicates that the number of lung cancer cells passing through the blood-brain barrier model is the highest,and the permeability of the blood-brain barrier is the largest.5.Expression of HIF-1 in lung cancer cells: The expression of HIF-1 in lung cancer cells was detected by Western blot.The results showed that the expression level of HIF-1 in hypoxic lung cancer cells increased with the prolongation of hypoxia.The expression level reached the peak at 12 h after hypoxia,and the difference was statistically significant compared with the control group(P<0.01).6.Trend analysis of each test parameter change: In order to further clarify the relationship between each test result,we also analyzed the general trend of each parameter made in this experiment,and the results showed that adenosine and HIF-1,adenosine and BBB Permeability,adenosine and number of migrated cells,number of migrated cells and BBB permeability are consistent with the trend of adenosine and ZO-1 expression,ZO-1 expression and BBB permeability.The dynamic trend between the two is exactly the opposite,and the changes in each parameter are most obvious at 12 h.7.Correlation analysis of each test parameter: In order to further analyze the correlation between the experimental results,we also compared the results of each index in the experiment,and the results showed: HIF-1 and adenosine Content,HIF-1 and blood-brain barrier permeability,HIF-1 and number of cells crossing the blood-brain barrier,adenosine content and bloodbrain barrier permeability,adenosine content and number of cells crossing the blood-brain barrier,blood-brain barrier There was a positive correlation between permeability and the number of cells passing through the blood-brain barrier.The expression level of HIF-1 and tight junction protein ZO-1,the expression level of adenosine and tight junction protein ZO-1,the expression level of tight junction protein ZO-1 and blood-brain barrier permeability and tight junction There was a negative correlation between the expression level of protein ZO-1 and the number of cells crossing the blood-brain barrier.Conclusions Hypoxia can cause the release of adenosine from lung cancer cells.Increased adenosine leads to decreased expression of the tight junction protein ZO-1 in the blood-brain barrier,leading to increased permeability of the blood-brain barrier,which may eventually lead to the development of lung cancer brain metastasis.Figure 9;Table 7;Reference 142.