Effects Of Different Doses Of Calcium On Mitochondrial Apoptotic Pathway Induced In The Bone Of Fluorosis Rats

[Objective] To investigate the effects of different doses of calcium on the bone metabolism related biochemical indicators in rats’serum and the bone histopathological changes. Meanwhile we try to explore the effects on the protein expression of Bax, Bcl-2, Cyto-C, ROCK I, BMP-2and BGP by immunohistochemical staining and the mRNA expression of Bax, Bcl-2, Cyto-C, Apaf-1, Caspase6, Caspase7and ROCKI in the bone of fluorosis rats.[Methods] In the test,160healthy (120±5) g male rats were randomly divided into five groups, and named as normal control group (normal diet, distilled water), fluorosis group (normal diet,150mg/L NaF-water), Test Ⅰ group (0.5%Ca,150mg/L NaF-water), Test Ⅱ group (1%Ca,150mg/L NaF-water) and Test Ⅲ group (2%Ca,150mg/L NaF-water), respectively. After4weeks,8weeks,12weeks and16weeks, we randomly selected8rats from each group. The blood of the rats was collected and the weights were weighed. After16weeks, the bones of the rats were collected. And the serum calcium levels, alkaline phosphatase activity and the resistant tartrate acid phosphatase activity were also detected. The bone histopathological changes were observed by HE stain. In addition, the protein expression of Bax, Bcl-2, Cyto-C, ROCK I, BMP-2and BGP were studied by immunohistochemical staining. The expression levels of Bax, Bcl-2, Cyto-C, Apaf-1, Caspase6, Caspase7and ROCK I were researched by real-time RT-PCR.[Results](1) Compared with the normal control group, the content of serum calcium in rats of fluorosis group significantly decreased (P<0.01). Compared with the fluorosis group, the serum calcium in rats of test groups decreased, and the serum calcium in high dose group decreased significantly after12weeks and16weeks (P<0.05or P<0.01). Compared with the normal control group, alkaline phosphatase activity in rats of fluorosis group increased. Compared with the rats of fluorosis group, alkaline phosphatase activity in test groups decreased, but there was no significant difference. And compared with the normal control group, the resistant tartrate acid phosphatase activity in rats of fluorosis group increased, and there was significantly difference after4weeks (P<0.05). Compared with the fluorosis group, the resistant tartrate acid phosphatase activity in test groups decreased, and that in the high dose group decreased significantly after4weeks (P<0.05).(2) Compared with the normal control group, we observed that the thickness distribution of bone trabecula was uneven. It appeared to be disordered and the shape was irregular. The size of the visible range of the crushing cavity was not all the same. We also observed that the funicular structures of bone trabecula were disappeared. The cell morphological was abnormal and the distribution was uneven in the rats of fluorosis group. Compared with the model group, it showed that the cells presented a significant recovery and the distribution was uniform. The bone trabecula had the funicular structures and got a basic recovery in test groups.(3) Compared with the fluorosis group, the protein expression of ROCK I in rats of the test groups decreased, and that in the middle dose group decreased significantly(P<0.05). Compared with the normal control group, the protein expression of Cyto-C in rats of fluorosis group significantly decreased (P<0.01). Compared with the fluorosis group, the protein expression of Cyto-C in rats of high dose group significantly decreased (P<0.01). Compared with the normal control group, the protein expression of Bcl-2in rats of fluorosis group significantly decreased (P<0.01). Compared with the fluorosis group, the protein expression of Bcl-2in rats of test groups increased, those in the middle and high dose groups increased significantly (P<0.01). Compared with the normal control group, the protein expression of Bax in rats of fluorosis group significantly increased (P<0.05). Compared with the fluorosis group, the protein expression of Bax in rats of the test groups decreased significantly (P<0.05). Compared with the normal control group, the protein expression of BMP-2in rats of fluorosis group significantly decreased (P<0.01). Compared with the fluorosis group, the protein expression of BMP-2in rats of the test groups decreased, and that in the middle dose group decreased significantly (P<0.05). Compared with the normal control group, the protein expression of BGP in rats of fluorosis group significantly decreased (P<0.01). Compared with the fluorosis group, the protein expression of BGP in rats of test groups decreased, those in the middle and high dose groups decreased significantly (P<0.05).(4) Compared with the normal control group, the mRNA expression of Bax, Cyto-C, Apaf-1, Caspase6, Caspase7and ROCK I in rats of fluorosis group significantly increased (P<0.01). The mRNA expression of Bcl-2significantly decreased (P<0.01). Compared with the fluorosis group, the mRNA expression of Bax, Cyto-C, Apaf-1, Caspase6, Caspase7and ROCK I in rats of the test groups significantly decreased (P<0.05or P<0.01), and the mRNA expression of Bcl-2in rats of middle and high dose groups significantly increased (P<0.01).[Conclusion] In conclusion, the results of the present study demonstrated that the calcium could control bone metabolism related biochemical indicators in rats and alleviate the fluorosis. Meanwhile, it could regulate the expression of Bax, Cyto-C, Apaf-1, Caspase6, Caspase7and ROCK I genes. These findings could signify that calcium could relieve the bone injury caused by fluorine.