| Cadmium (Cd) is an extremely toxic metal, which is commonly found、long biological half-lifed and could cause severe damage. To human, it damage the nervous system, cardiovascular system, immune system, affect the endocrine system, reproductive system, as well as the skeletal system. The skeletal toxicity mechanism needs exactly a further study. Experiments in vitro and vivo have already proved that Cd induced cellular oxidatived damage, DNA damage and apoptosis, which was correlated with oxidative stress, intracellular calcium homeostasis, mitochondrial damage, the activation of MAPK, Caspase and Caspase independent pathway et al. In our experiment, the model of osteoblasts cultured in vitro was used, which were obtained from foetal Sprague-Dawley rats at18days of gestation. The potential mechanism of cytotoxicity and apoptosis induced by cadmium, and the protection of NAC was investigated by cytobiological and molecular biological methods, which will offer theoretic evidences for future exploring on the mechanism of skeletal toxicity induced by cadmium.1. The cytotoxicity induced by Cd and the protection of NAC in OBsTrypsin and collagenase type II were used for digestion to obtain OBs from parietal bones of the fetus of Sprague-Dawley rats at18days of gestation. To explore the cytotoxicity induced by Cd and the protection of NAC, OBs were treated with0、0.125、0.25、0、1、2、5、10、20μmol/L CdAc2and incubated with0、100、500、μmol/L、1、2mmol/L NAC. The morphology damage was observed by a reverse microscope. OBs were identified using alkaline phosphatase (ALP). Cell viability was evaluated by cck-8assay and the leakage of lactate dehydrogenase (LDH) was measured with colorimetric method.With the dosage increasing of Cd, cell viability was decreased significantly (P<0.05、 P<0.01), the leakage of LDH increased significantly (P<0.05、P<0.01). NAC alone did not affect the cell morphology, cell viability and the leakage of LDH. Cotreatment of NAC and Cd increased cell viability and decreased the leakage of LDH significantly (P<0.05、P<0.01). NAC can effectively attenuate the cytotoxicity induced by Cd in OBs. 2. Morphology damage and apoptotic induced by Cd and the protection of NAC in OBsThe changes of cell morphology and structure can reflect the degree of injury directly, and influences its function. OBs were treated with0、1、2、5μmol/LCdAc2and500μmol/L NAC. Optical microscope, fluorescence staining, transmission electron microscope were used to observe the changes of cell morphology、nuclear morphology and ultrastructure of OBs induced by Cd and NAC. Meanwhile, cell apoptosis was detected by flow cytometry as a basis for further exploring on the mechanism of cadmium damage in OB structure and function.The morphology damage of cells、nuclear and mitochondrial, as well as typical features of apoptosis can be observed in the experiment. The statistical results of flow cytometry showed that cell apoptosis rate increased with the exposure dose, which shows a dose effect relationship. NAC can effectively alleviate the damage of Cells, nuclear and mitochondrial induced by Cd, but the apoptosis rate had no significant difference with the exposed groups.3. The oxidative damage induced by Cd and the protection of NAC in OBsOxidative stress plays an important role in the oxidative damage induced by Cd. In this part, OBs were treated with0、1、2、5μmol/LCdAc2and500μmol/L NAC. Activity of Superoxide dismutase(SOD)、Glutathione reductase (GR)、content of glutathione (GSH) were measured with Xanthine oxidase and DTNB colorimetric method. Level of reactive oxygen species(ROS) and mitochondrial membrane potential (ΔΨm) was detected by fluorescent dyes DCFH-DA and rhodamine123.The data showed that the activity of SOD increased significantly (p<0.01or p<0.05), the activity of GR increased significantly (p<0.05) at24h incubation with1μmol/LCd, then decreased sharply(P<0.01).The content of GSH increased significantly (p<0.01) while those of ROS and ΔΨm decreased significantly (p<0.01). The results suggested that0-5μmol/L Cd triggered the antioxidant system in vivo, the enzymes and inenzymatic antioxidants formed an effective protection mechanism which cleaned up ROS and alleviate oxidative damage caused by low concentrations of Cd. Cotreatment of NAC and Cd decreased the activity of SOD(P<0.01)、alleviated the decreasion of GR and ROS(P<0.01),and increased the content of GSH significantly (p<0.01), which confirmed a protection of NAC in cadmium induced oxidation damage in OBs.4. Role of Caspase pathway in Cd-induced OB apoptosisIn the signal transduction of apoptosis, cysteine protease (Caspase) is an important starter and performer. In this part, Activity of Caspase-3was measured by spectrophotometry. Z-VAD-fmk, a Caspases inhibitor, was also used to find whether the process of OB apoptosis depend on Caspase pathway, to provide materials for its molecular mechanism in cadmium induced OB apoptosis.We could see that with1,2,5μmol/L Cd, the activity of Caspase-3increased significantly (p<0.05、p<0.01); cotreatment with Z-VAD-fmk had no obvious influence on the survival rate in OBs(P>0.05), as well as on the morphology and number of apoptotic cells, which suggests that both the Caspase pathway and the Caspase independent pathway play roles in Cd-induced OB apoptosis.5. Effect of the disequilibrium of calcium homeostasis in Cd-induced OB apoptosisThe disequilibrium of calcium homeostasis is a common phenomenon in apoptosis. In this study, Bapta-AM, the calcium chelator, was used to investigate the effect of disequilibrium of calcium homeostasis in Cd-induced OB apoptosis through the changes of OB morphological and nucleus, the survival rate,[Ca2+]i, ROS level and ΔΨm collapse. OB apoptosis was also detected by flow cytometry.Results showed that the level of intracellular calcium increased significantly with Cd at1.5h (P<0.01), while Bapta-AM could alleviate the morphological changes in cells and the nucleus, inhibite Cd-induced increase of intracellular calcium, increase ROS level, ΔΨm collapse and cell survival rate significantly (P<0.01); decreased the percentage of apoptosis cells, which confirmed that the disequilibrium of calcium homeostasis is an important cause in OB apoptosis.6. Role of MAPK pathway in Cd-induced OB apoptosisMAPK is an important signal transduction system to transfer extracellular stimulation to intracellular reaction. MAPK take part in cell proliferation, apoptosis, and other process by control gene expression through phosphorylation of transcription factors. In this study, western blot was used to measure the phosphorylation of SB202190, SP600125and U0126, which are inhibitors of key proteins in MAPK, to clarify the role of MAPK pathway in Cd-induced OB apoptosis.We could see raises of the phosphorylation levels in ERK、p38MAPK, and a drop in that of JNK. Inhibitors of p38and ERK can effectively prevent the raises of p-p38and p-ERK induced by Cd, but the inhibitor of JNK did not suppress the drop of p-JNK, which Indicated that cadmium can activate the MAPK pathway in OBs, in which ERK, p38MAPK may promote apoptosis, while JNK may play the opposite role.7. Effect of Cd on RANKL/OPG in OBsRANKL/RANK/OPG system is an important signal transduction pathway in bone metabolism. Many factors and hormones control bone formation and resorption by adjusting the ratio of RANKL/OPG. We measured the protein expression level of RANKL and OPG in OBs with0-5μmol/L cadmium by western blot, to explore its role in bone metabolism.Results indicated that cadmium decreased the ratio of RANKL/OPG in OBs, suggesting that cadmium could inhibit the differentiation of osteoclast by blocking the combine of RANK and RANKL, and then inhibit bone resorption in fetal rat. |
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