Effect Of Different Supplement Of Calcium On Bone Injury And Endoplasmic Reticulum Stress Induced By Fluoride In Rat

[Objective] In this study, we sought to investigate the effects of exogenous calcium supplement on fluorosis-induced bone injury and endoplasmic reticulum stress-related proteins and genes expression involved in the process of osteoblast apoptosis, reveal the mechanism for the alleviation effect of extra calcium supplement on fluorosis, and then provide the theoretical basis for preventing the unreasonable supplement of calcium in fluorosis clinically.[Methods] 160 healthy young adult male SD rats were randomly divided into five groups. Each group were managed as follows: the control group rat were administrated with ultrapure water (Milli-Q Water purification system), F-exposed treated with 150 mg/L NaF in ultrapure water and fed with normal feed, F-exposed+CaⅠtreated with 150 mg/L NaF in ultrapure water and fed with 0.5% diet CaCO3, F-exposed+CaⅡ treated with 150 mg/L NaF in ultrapure water and fed with 1% diet CaCO3, F-exposed+CaⅢ treated with 150 mg/L NaF in ultrapure water and fed with 2% diet CaCO3. The test period is 30 days,60 days, 90days and 120 days. After exposure, collecting serum and femur used for determining the relevant indices including using a fluoride Ion selective electrode to measure the bone fluoride content, using radioimmunoassay to measure bone metabolism hormone levels in serum, using transmission electron microscopy to observe the changes of tissue structure, using real-time PCR and Westernblotting to detect the mRNA and protein expressions of endoplasmic reticulum stress in the process of osteoblast apoptosis, etc.[Result] 1. Compared with the control group, The bone fluoride content significantly increased in fluoride treated groups.2. The result of bone metabolism relevant hormone in serum as follow: the levels of OC and PTH hormone significantly increased in the F-exposed group compared with control group, while the level of CT hormone significantly reduced compared with control group. The appropriate supplement of calcium can make the levels of OC and PT hormone lower in F-exposed group, and then, the level of CT hormone is significantly increased and closed to control group.3. The damage of fluorine on bone is obviously through the index of material property and structural mechanics in bone biomechanics. The supplement of middle calcium have helped offset some of relief on bone damage from fluorosis, therefore, the supplement of low and high calcium is no effect on bone damage, while the supplement of high calcium group even aggravate bone injury.4. The changes of bone cell ultrastructure were observed in 120 days of rats through transmission electron microscopy. The control group is visible organelles complete and structure clear, abundant mitochondria, rough endoplasmic reticulum developed. F-exposed group, a large number of organelles in cells lost and dissolve, mitochondrial matrix thinner, mitochondrial disappear or vacuoles degeneration, rough endoplasmic reticulum shed particles, depolymerize. When the appropriate supplement of calcium it is can be seen that the osteoblast organelles relatively complete, abundant mitochondria, rough endoplasmic reticulum minor expansion and fall off a small amount of particles, and the damage of bone caused by fluorosis significantly reduced.5. The results of real-time PCR and western blotting showed that, compared with the control group, endoplasmic reticulum stress-related caspase-12 and calpain expression at mRNA and protein level were all up-regulated in the process of osteoblast apoptosis, but significant decreases were observed in rats administered with appropriate calcium supplement in fluorosis.[Conclusion] In our present study, bone injury-induced by fluoride was observed by bone biomechanics and TEM analyses at the macro level, endoplasmic reticulum stress-related caspase-12 and calpain expression at mRNA and protein level were markedly increased in high fluoride group by the means of RT-PCR and western-blot at the micro level; appropriate supplement of calcium could alleviate bone damage and endoplasmic reticulum stress induced by fluorosis, mitigating the toxicity effect of fluoride on bone.