Regulation Of ATF-6 On Cr(Ⅵ)-induced Apoptosis In DF-1 Cells

Hexavalent chromium(Cr(VI))is one of the valence states of metal chromium,which is the most toxic and has carcinogenicity and corrosiveness to the skin and mucous membranes.Studies have found that Cr(VI)can cause liver damage and cell damage.ATF-6(activating transcription factor 6)is one of the three receptor protein of endoplasmic reticulum(ER).As a proximal sensor of ER,ATF-6 can cause the unfolded protein response(UPR)in the endoplasmic reticulum.ATF-6 is an important regulator of apoptosis in ER stress pathway,which can activate the transcription and expression of a variety of endoplasmic reticulum stress and apoptosis-related proteins.Apoptosis is an active mode of cell death under physiological or pathological conditions.The ER is an important factor in cell homeostasis,and mitochondria are the regulatory centers of apoptosis.Both have a very important role in the process of apoptosis.This study aimed to investigate the regulatory effect of ATF6 on apoptosis of DF-1cells induced by Cr(VI),and to inhibit apoptosis by siRNA interference of ATF-6expression,providing a theoretical basis for screening Cr(VI)antidote.Exposure of DF-1cells to Cr(VI)induces cytotoxicity and apoptosis.DF-1 cells were exposed to different concentrations of Cr(VI)to induce cytotoxicity and apoptosis.The cell viability of DF-1cells were measured by CCK-8 assay kit,the results showed that DF-1 cells treated with Cr(VI)could induced cell apoptosis in a time and dose-dependent manner,the inhibition rate of cells treated with 150μM Cr(VI)for 8 hours was 49.7%.The nucleus were stained by Hoechst 33342,the changes of cell morphology and nuclear morphology were observed by laser confocal microscope.The results show that Cr(VI)can cause cell shrinkage and suspension,nuclear enrichment,nuclear internal lines,and apoptotic bodies.Next,ATF-6 in DF-1 cells was silenced by siRNA.The apoptosis rate of DF-1 cells was evaluated by flow cytometry using 7-AAD/Annexin V double staining.The apoptosis rate of cells treated with 150μM Cr(VI)alone for 8h was 40.3%.When treated with ATF-6siRNA and Cr(VI),the apoptosis rate decreased to 25.74%.In addition,the expression ofapoptosis-related protein(Caspase-3,Cleaved Caspase-3)and ER stress protein(GRP78)were detected by Western blotting.The results showed that the amount of GRP78 and Cleaved Caspase-3 expression in Cr(VI)-treated cells was significantly increased,while this increase was inhibited after silencing ATF-6.Next,the expression of different apoptotic pathway proteins was measured.The results showed that cell were treated with Cr(VI)after silencing ATF-6,the expression of PERK and ATF-6 were significantly decreased,the protein levels of Caspase-9 and Bcl-2 were significantly increased,and the expression of Caspase-8 was relatively stable and showed no significant change.These results indicate that Cr(VI)induces apoptosis in DF-1 cells by activating the mitochondrial damage apoptotic pathway and ER stress apoptotic pathway,which may be independent of the death receptor pathway.Moreover,as an ER stress protein,ATF-6could regulate the mitochondrial damage and ER stress apoptotic pathway in DF-1 cells.The JC-1 probe was used to determine changes in MMP and the ROS were stained by DCFH-DA,the results showed that the ROS levels increased significantly and the number of MMP-decreased cells increased significantly when treated with 150μM Cr(VI),and silencing ATF-6 significantly reduced MMP loss and inhibited ROS increase.In summary,this study shows that 150μM Cr(VI)induce ER stress and apoptosis in DF-1 cells,and ATF-6 can regulate the Cr(VI)-induce apoptosis by activating ER stress and mitochondrial apoptosis pathway,reducing MMP and increasing ROS.