The Study Of The Regulatory Effects Of Macrophage Cells On HAP Induced The Expression Of Relative Inflammatory Factors Of HK-2 Cells In Vitro Co-cultured System

Purpose:This study developed an in vitro system by co-culturing HK-2cells with different concentration of Hydroxyapatite(HAP)and/or macrophage cells to simulate the internal environment of urolithiasis as far as possible,investigating the regulatory effects of macrophage cells on HAP induced the expression of relative inflammatory factors of HK-2 cells.Methods:The control group(H group)was only comprised of HK-2 cells.Experimental groups included co-culturing HK-2 cells and macrophage cells(H+M group),co-culturing HK-2 cells and HAP(H+A group),co-culturing macrophage cells and HAP(M+A group),co-culturing HK-2 cells and macrophage cells with HAP(H+M+A group).In the H+A,M+A and H+M+A group,we set the concentration of HAP as 5 ug/cm~2(A1)and 10 ug/cm~2(A2).After co-culturing for 2,4,and 6 hours,we detected the expression of CCL-2 in the liquid by ELISA.We tested the expression of LDH and ROS to evaluate the damage of HK-2 cells.We assessed the apoptosis of HK-2 cells by using DAPI staining assay,Flow Cytometry and the rate of Bax/Bcl-2.Western Blotting detected OPN,Fetuin-A,Bax,Bcl-2 and MAPK pathways of HK-2 cells.Results:The expression of CCL-2 in the medium of H+A1 and H+A2group increased significantly compared with the control(P<0.05),CCL-2 of M+A1 and M+A2 group was higher than the H+A1 and H+A2 group(P<0.05).The expression of CCL-2 in H+M+A1 and H+M+A2 group was also higher than M+A1 and M+A2 group(P<0.05).Compared with control,the expression of OPN,LDH release,the ratio of BAX/BCL-2 and the generation of ROS in HK-2cells increased in a dose-and time-dependent manner.Compared with the control,the expression of Fetuin-A decreased in various degrees at different incubation periods.Especially when co-culturing for 6 hours,Fetuin-A decreased most seriously in the H+M+A1 group.Meanwhile,the MAPK pathways were activated in different degrees.In addtion,after 6 hours treatment of the MAPK’s inhibitors,the expression of Fetuin-A did not change.Conclusion:1.The HAP can induce the HK-2 cells oxidative stress and inflammatory damage and apoptosis,when adding the macrophages to co-culture,macrophage cells can aggravate the damage and apoptosis of the HK-2 cells.2.After the stimulation of HAP,the expression of OPN in HK-2 cells increased in a time-and dose-dependent manner,macrophage cells can aggravate the increase of OPN in HK-2 cells.3.In the HAP and HK-2 cells co-cultured system,the low level Fetuin-A of HK-2 cells may be related to the excessive consumption of Fetuin-A in the process of HAP induced renal tubular epithelial cell excessive oxidative stress,inflammatory injury and cell apoptosis.When adding macrophage cells to co-cuture,Fetuin-A decreased even more seriously,it remind us macrophage cells can slightly regulate the expression of Fetuin-A in the HK-2 cells.4.The expression of Fetuin-A in the HK-2 cells,macrophage cells and HAP co-cultured system may not be regulated by MAPK pathways.