Role Of Hippo-YAP/NF-κB Signaling Pathway Crosslinking In Regulating Biological Behaviors Of Macrophages Under Titanium Ions Exposure

Part Ⅰ Effect of titanium exposure on the proliferations,migrations and immune response of RAW264.7 cellsObjective To study the effect of titanium exposure on the proliferations,migrations and immune response of RAW264.7 cells.Methods RAW264.7 cells were co-cultured with different concentrations of titanium ions for 1,3 and 5 days,and the effects of different concentrations of titanium ions on the proliferations of RAW264.7 cells were compared by CCK-8.1ppm、5ppm and 10 ppm titanium concentrations were selected as the follow-up experimental concentrations.RAW264.7 cells were treated with 0,1,5 and 10 ppm titanium ions respectively for 4 hours,and then the migrations of RAW264.7 cells in different treatment groups was detected by Transwell technique.The expressions level of inflammations-related genes such as TNFa,IL6,IL1 β,CCL2 were then detected by PCR.Results The proliferations of RAW264.7 cells was not significantly changed when the concentrations of titanium ions was below 10 ppm,but promoted when the concentrations was 10 ppm.However,titanium ions above 20 ppm significantly inhibited macrophage proliferation and promoted macrophage death.Transwell experiment indicated that 1 ppm titanium ions can significantly promote the migrations ability of macrophages.When the concentrations of Ti ions increased,the migrations ability of macrophages did not increase significantly.Ti ions treatment induced macrophages releaseing TNFa,IL6,IL1 β,CCL2 in a dose-dependent manner.Conclusions 10 ppm titanium ions can promote the proliferations of macrophages,significantly improve the migrations ability of macrophages and activate macrophage to release inflammatory factors.Part Ⅱ Effect of titanium exposure on Hippo /YAP and NF-кB signaling pathway in RAW264.7 cellsObjective To study the effect of titanium ions exposure on Hippo/YAP and NF-кB signaling pathway in RAW264.7 cellsMethods RAW264.7 cells were co-cultured with 10 ppm titanium for 4 hours.Western blot was used to detect the expressions levels of the key indicators of Hippo/YAP signaling pathway YAP,and the markers of NF-κB signaling pathway P-p65,P-IκBα.The distributions of YAP in the cytoplasm and nucleus of macrophages was detected after 0,1,5,10 and 30 minutes of co-culture with 10 ppm titanium by western blot.After RAW264.7 cells and 10 ppm titanium ions were co-cultured on cell slides for 12 hours,the YAP molecules and nuclei in macrophages were labeled by immunofluorescence staining.The distributions of YAP in macrophages was observed by confocal laser microscopy.Results The expressions level of YAP decreased while the expressions levels of p-p65 and P-IκBα,the markers of NF-κB signaling pathway,increased after 10 ppm titanium ions treated RAW264.7 cells for a period of time.Titanium ion decreased the ratio of p-YAP / YAP in macrophages,increased the dephosphorylation of Yap,activated yap and promoted its transfer from cytoplasm to nucleus.Conclusions Titanium ions can activate NF-κB signal pathway and improve the distributions of YAP molecules in the nucleus which play a biological role in macrophages.Part Ⅲ Role of Hippo-YAP/NF-κB signaling pathway crosslinking in regulating biological behaviors of macrophages under titanium ions exposureObjective To study the role of Hippo-YAP/NF-κB signaling pathway crosslinking in regulating biological behaviors of macrophages under titanium ions exposure.Methods Bay-117082,a specific blocker of NF-κB pathway,was used to inhibit NF-κB signaling pathway in macrophages.The expression of YAP and NF-κB signaling pathway markers P-p65 and P-IκBα were detected by Western blot after 4 hours of co-culture with 0 ppm and 10 ppm titanium ions in blank group and blocking group,respectively.The overexpression plasmid targeting YAP was constructed to enhance the expression of YAP in macrophages.Real-time quantitative PCR was used to detect the transfection efficiency of YAP.Then 0 ppm and 10 ppm titanium ions were co-cultured with transfection group and negative control group for 4 hours respectively.Westren blot was used to detect the protein expression of YAP,P-p65,P-IκBα and inflammation related gene level of TNFa,IL6,IL1β was dectcted by PCR.Results Bay-117082 could effectively inhibit the expression of NF-κB pathway markers such as P-p65 and P-IκBα,and after inhibition,the expression of YAP showed functional recovery.In addition,the NF-κB pathway was further activated by 10 ppm titanium ion after YAP overexpression plasmid was transfected into macrophages,as well as increase inflammatory factors such as TNFa,IL6 and IL1β.Conclusion NF-κB and Hippo/YAP signaling pathway are involved in regulating the biological behavior of macrophages under titanium ions exposure.The activation of NF-κB pathway can promote the degradation of YAP in macrophages,and the activation of YAP can further promote the expression of NF-κB pathway.