Activation Of Mitochondrial P53 Apoptotic Pathway Is Involved In Cadmium Induced Osteoblast Injury

Cadmium is a widespread heavy metal pollutant that can cause damage to multiple organs and systems by accumulating in the body through the respiratory and digestive tract.Bone is one of the most important target organs of cadmium.Cadmium exposure leads to low bone mass,osteoporosis and even to an increased incidence of bone fractures,while the specific molecular mechanism remains unclear.p53 plays an important role in regulating cell cycle arrest,apoptosis and DNA repair.However,the role of p53 in osteoblast apoptosis has not been reported.Therefore,the primary rat osteoblasts were used to investigate the effects of cadmium on p53 activation,mitochondrial apoptosis pathway and oxidative DNA damage,and further explore the relationship between DNA oxidative damage and mitochondrial p53 apoptosis pathway.This study aims to provide scientific basis for the molecular mechanism of cadmium-induced osteoblast toxicity.1.Effect of cadmium on rat osteoblasts proliferation and apoptosisTo explore the effect of cadmium on apoptosis and proliferation of osteoblasts,osteoblasts were treated with different concentrations of cadmium(0,1,2 and 5?mol/L)for 6 h or 12 h.Cell morphology was observed under phase-contrast microscope;apoptosis rate and cell cycle were detected by flow cytometry;cell proliferation was monitored by Real-time Cell Analyzer;expression of apoptosis-related proteins were detected by western blotting.The results showed that,compared with the control group,the round,floating cells,and apoptosis rates increased in the 2 ?mol/L and 5 ?mol/L cadmium-treated groups.The expressions of Cleaved caspase-9,Cleaved caspase-3,p53,PUMA and Noxa were significantly up-regulated in all treatment groups(p<0.05 or p<0.01).In addition,both Bax/Bcl-2 ratio and cytoplasmic Cyto c expression were up-regulated significantly in 2 ?mol/L and 5 ?mol/L cadmium treatment groups(p<0.01).Osteoblasts proliferation reduced significantly,the proportion of cells in G0/G1 phase cells increased,and the proportion of cells in S phase decreased(p<0.05 or p<0.01)after exposed to 2 ?mol/L and 5 ?mol/L cadmium.Furthermore,the number of cells in G2/M phase increased after treated with 5 ?mol/L cadmium for 12 h(p<0.05).These data indicated that cadmium inhibits the proliferation,activates p53,promotes expression of proteins in mitochondrial apoptosis pathway,and induces apoptosis in osteoblasts.The p53 and mitochondrial apoptosis pathway may be involved in regulating cadmium-induced rat osteoblast apoptosis.2.Cadmium induces osteoblast apoptosis by activating p53-mediated mitochondrial apoptosis pathwayTo clarify the role of p53-mediated mitochondrial apoptosis pathway in cadmium-induced osteoblast apoptosis,immunofluorescence was used to detect the nuclear translocation of p53 after osteoblasts treated with 2 ?mol/L cadmium for different times(0,1,3 and 6 h).Then,cells were treated with p53 transcription activity inhibitor(PFT-?)alone or in combination with cadmium for 6 h or 12 h.Apoptosis rate and mitochondrial membrane potential were detected by flow cytometry;cell proliferation was analyzed by Real-time Cell Analyzer;expression of p53 and proteins in mitochondrial apoptosis pathway were measured by western blotting.The results showed that,with the prolonged time of cadmium exposure,the fluorescence aggregation of p53 in the nucleus gradually increased.Compared with cadmium-treated group,the apoptosis rate reduced significantly(p<0.01),the cell proliferation was increased significantly,and the expression of p53,PUMA,Noxa,Bax,and Cleaved caspase-3 decreased significantly(p<0.05 or p<0.01)in PFT-? and cadmium co-treatment group.In addition,the mitochondrial membrane potential was clearly restored compared to cadmium-treated group(p<0.01).These data indicated that p53-mediated mitochondrial apoptosis pathway plays a critical role in regulating cadmium-induced osteoblast apoptosis.3.Effect of oxidative DNA damage and apoptosis on cadmium-induced osteoblasts injuryTo study the role of oxidative DNA damage in p53 activation and apoptosis,cells were treated with NAC alone or in combination with cadmium.The contents of H2O2 and O2-were detected by flow cytometry;the level of malondialdehyde(MDA)was detected by the kit;the nuclear translocation of p-H2AX and p53 were detected by immunofluorescence;the expression of p-H2AX,p53 and apoptosis-related proteins were determined by western blotting.The results showed that,compared with the control group,the H2O2 content increased significantly(p<0.05 or p<0.01)in all groups after cadmium treatment for 15 min.The H2O2 content decreased significantly(p<0.01)in all groups treated with cadmium for 6 h,while the intracellular O2-level clearly elevated(p<0.05 or p<0.01).There is no significant difference in MDA level(p>0.05).In addition,compared with cadmium-treated group,the expressions of p-H2AX,p53,and apoptosis-related proteins reduced significantly(p<0.01),and the p-H2AX and p53 fluorescent aggregation spots also reduced in NAC and cadmium co-treatment group.These data indicated that cadmium induces over production of ROS and DNA damage,promotes p53 expression,and eventually results in apoptosis.NAC has a significant protective effect during this process.