The Role Of Autophagy In Cadmium-induced Apoptosis Of Primary Rat Osteoblasts

Cadmium (Cd) is a heavy metal pollutant widely present in the environment. Due to the cumulative toxicity, it can cause great damage to the body. It can cause damage to multiple organs, including the kidney and bone which are the major target organs of cadmium. There are many reports about the bone toxicity exposed to cadmium, but the reports of apoptosis and autophagy in osteoblasts with cadmium exposure are fewer. Therefore, this study sets up an in vitro model of primary rat osteoblasts (OBs) to investigate the effect of cadmium exposure on the autophagy and apoptosis in osteoblasts and the role of autophagy in cadmium-induced apoptosis. To survey whether cadmium can induce apoptosis, OBs were treated with CdAc2 of different concentrations (0,1,2,5 μmol/L) for 12 h and 2 μmol/L for various times (0,1,2,3,6,12 h). Bax, Bcl-2 and Cleaved-PARP were measured by western blotting. After exposed to cadmium of different concentrations (0,1,2.5 μmol/L) for 12 h, nuclear morphology was analyzed by Hochest 33258 staining. To study the effects of Cd treatment on autophagy in OBs, LC3-Ⅱ and Beclin-1 were measured by western blotting after CdAc2 of different concentrations (0,1,2,5 μmol/L) treated osteoblasts for 1 h and 2 μmol/L for different times (0,1,2,3,6,12h). We detected the autophagy by cytometry using AO staining after CdAc2 (0,1,2,5 μmol/L) treated osteoblasts for 1 h. MDC staining was also used to observe autophagic vacuoles. Furthermore, the punctate LC3 was observed by immunofluorescence to monitor autophagy. To explore the role of autophagy in cadmium-induced apoptosis in OBs, we detect the apoptosis rate by cytometry after cells were treated with CdAc2 (2 μmol/L), or CQ (5 μmol/L) for 3 h alone or taken together. OBs were also treated with CdAC2 (2 μmol/L), or RAP (100 nmol/L) for 12 h alone or taken together. RNA interference was also used to study the effect of autophagy on the apoptosis.The results showed that:① When OBs were treated with CdAc2 of different concentrations (0,1,2,5 μmol/L) for 12 h and 2 μmol/L for various times (0,1,2,3,6,12 h), compared with the control group, Cd exposure significantly increased Bax/Bcl-2 ratio and the expression of Cleaved-PARP (P<0.05 or P<0.01). Cadmium can cause changes in nuclear morphology of osteoblasts showing nuclear shrinkage, chromatin condensation and so on.When the cadmium concentration increased, the number of cells with the changes of nuclear morphology increased. ② Compared with the control group, after cadmium (2 μmol/L) treated osteoblasts for 1 h, the fluorescence intensity of MDC staining enhanced, appearing bright punctate structures. Cells also showed obvious phenomenon of LC3 puncta in cadmium-treated OBs. Compared with the control group, in Cd-treated group (1,2 μmol/L), the red fluorescence intensity of AO staining significantly enhanced (P<0.05 or P<0.01), while in 5 μmol/L Cd-treated group significantly decreased (P<0.01). Different concentrations of cadmium treated osteoblasts for 1 h, with increasing concentrations of cadmium, the expression of LC3-II first increased, then decreased, and compared with the control group, in 1 μmol/L and 2 μmol/L Cd-treated group the expression of LC3-II significantly increased. So is Beclin-1. When 2 μmol/L cadmium treated OBs at different times, the expression of LC3-II first increased, then decreased along the extension of exposure time, and compared with the control group, exposed after 3 h (including 3 h) the expression levels of LC3-II were significantly reduced (p<0.01). So is Beclin-1. ③ Compared with the respective individual cadmium group, the protein expression levels of LC3-II of the combined treatment group significantly increased. The apoptosis rate of RAP and cadmium combined treatment group was significantly decreased (p<0.01), but CQ and cadmium combined treatment group was significantly increased (p<0.01). Compared with Cd alone group, Beclinl RNA interference can reduced the expression levels of LC3-II and Beclinl (p<0.01), while apoptosis rate was also significantly increased (p<0.01).Conclusions:Cadmium can induce apoptosis in OBs, causing changes in the level of autophagy. Cadmium could inhibit apoptosis by inducing autophagy, thus autophagy has a protective role in Cd-treatment OBs.