| Fluorine (F), as an essential microelement for human body, plays an important role on metabolism; meanwhile, F is a kind of protoplasm poison, which could be easily effect destruction by passing through cytomembrane then combine protoplasm. Human body overdose intake F in a long term, it will result in whole body chronic F accumulation of earth chemical disease, this is called endemicity F intoxication or fluorosis for short. Drinking water fluoride exposure type is the most extensively representation of fluorosis. The injury mechanism which resulted by F intoxication, relates to the body oxidation injury which resulted by F in some time, apoptosis would be an important step. Fluoride accumulation in tissues and organs is the material basic of damage effect, and kidney is main organ of Fluoride accumulation and elimination, therefore it’s one of sensitivity target organ for F toxicity, and mitochondria as the center of cell metabolism, if overdose F intake, that will injury kidney in some time, which injury mitochondria obviously. Dietary calcium could affect the injury resulted by F toxicity, increase or decrease kidney apoptosis rate, thus aggravating or alleviating the organ toxicity effect of F. This research firstly studied the effect of calcium intake on drinking flurosis-induced mitochondrial injury of kidney systematically from sub-cellular and molecular levels, further clarified the mechanism of flurosis-induced mitochondrial injury of kidney (the expression of fission and fusion proteins) and the effect of dietary calcium,and filtered the key link of F-induced kidney injury, to provide new solution for preventing and curing F-induced injury.Chronic exposure were carried out using healthy early weaning (aged three weeks) 50 SD males rats (for reproduction) and 100 female rats of clean level, animals were divided into five groups. The control group (NW):all drinking tap water (water fluoride<0.2mg/L), eating normal diet (the calcium content is 0.79%); fluoride group (NF):all drinking NaF solution (the fluorine content is 100mg/L), eating normal diets (the calcium content is 0.79%); low calcuim group (LW):all drinking tap water (water fluoride<0.2mg/L), eating low calcuim diet (the calcium content is 0.063%); low calcuim fluoride group (LF):all drinking NaF solution of 100mg/L, eating low calcium diet (the calcium content is 0.063%); high calcuim fluoride group (HF):all drinking NaF solution of 100mg/L, eating high calcuim diet (the calcium content is 7%). All male rats drank tap water (fluoride content<0.2mg/kg), and fed by standard diet, of which the content of calcium is about 7-10g/kg, all the above solution used tap water as solvent. Feeding for three months, after that, mated the male and female rat of ratio 1:1, using offsprings of 14 days and 28 days to carry out relevant experiments for two batches. Each experimental group took rat kidney viscera coefficient, kidney damage related blood indexes, renal tubular epithelial cells mitochondrial ultrastructure, apoptosis of kidney, the activity of signal enzyme of kidney mitochondria and lipid peroxidation index, and the expression levels of mitochondria fission and fusion proteins as indicators of observation.The experimental results were as follows:(1) The results of weight and organ coefficients of offsprings:compared with the control group, weight of 14 days rats in NF group and LF group were significantly decreased (p<0.05) and the weight of HF group showed a certain decreasing trend (p>0.05), compared with the control group, the weight and organ coefficients of 14 days rats increased significantly (p<0.05). The weight of 28 days rats in NF group were significantly decreased (p<0.05), and the weight of other treatment groups also had a certain decline (p>0.05), and the organ coefficient of each group declined (p> 0.05).(2)The determination results of kidney damage-related blood indicators of offsprings:compared with the control group, the creatinine, uric acid and urea nitrogen content of offsprings increasd basically, and compared with the NF group, three indicators of 14 days female HF group all increased significantly (p<0.05), apart from that, the levels of these indicators in LF group and HF group had a certain trend of rise and fall (p>0.05).(3)The observation of mitochondrial ultrastructure in rat renal tubular epithelial cells of offsprings:the mitochondria of renal tubular epithelial cells in control group arranged neatly, presented rod-shaped, showed obvious mitochondrial cristae. Compared with the control group, the NF, LW, LF and HF group presented an obvious small ball alveolar membrane structure, showed an increased number of mitochondrial cross section of different sizes, with density increased significantly, and the mitochondria swelled and disappeared at the same time, part of mitochondrial crest turned fuzzy. Compared with the NF group, the mitochondrial damage condition of LF group aggravated, with mitochondrial cristae disappeared completely and mitochondria swelled, while the HF group alleviated, there retained some mitochondria in tubular network structure.(4) The determination of SDH enzyme activity and lipid peroxidation index of offsprings:compared with the control group, the SDH enzyme activity of the male rats of 14 days showed a certain decline (p>0.05), the SDH enzyme activity of 28 days male rats were significantly decreased (p<0.05), while the SDH enzyme activity of females of 14 days in NF and LF group were significantly reduced (p<0.05), females of 28 days in LF decreased significantly (p<0.05); compared with the NF group, the SDH enzyme activity of HF group increased significantly (p<0.05), and the SDH enzyme activity of LF group decreased significantly (p<0.05). Compared with the control group, the content of MDA of all females in NF, LF and HF groups were significantly increased (p<0.05), the MDA content of 14 days males in NF and LF group significantly increased (p<0.05), and 28 days males showed no significant difference (p>0.05), but the mitochondrial MDA content of the fluorosis groups in offsprings showed a trend of rise; compared with the NF group, the MDA content of all groups had no significance (p>0.05), however, the MDA content of LF group showed an increasing trend, while the HF group had a certain trend of decline.(5) The results of renal apoptosis in offsprings:under optical microscope, there were brown yellow or brown particles in the nucleus of apoptosis cells, and chromatic agglutination, karyopyknosis, and nuclear fragmentation could be observed in apoptpsis cells, compared with the control group, the kidney tissue arranged disorderly in fluorosis groups, and the amount of apoptotic cells increased obviously, moreover, compared with the NF group, apoptosis cells of the LF group increased, while the HF group decreased.(6) The expression levels of fission proteins and fusion protein in mitochondria of offsprings:compared with the control group, the expression level of protein Fisl in NF group increased significantly (p<0.05), while other groups had a certain rise trend (p>0.05), division protein Drpl of 14 days females in NF and HF group and 28 days males in LF and HF group increased significantly(p<0.05), and all other groups showed a certain rising trend (p>0.05). There had a reverse trend in the expression level of fusion protein Mfn2, that was, compared with the control group, the expression level of mitochondria fusion protein showed a certain decline (p>0.05), and compared with the NF group, the expression level of fusion protein in LF group had a decreasing trend (p>0.05),while that of HF group was on the rise (p>0.05).In conclusion, drinking water F exposure could enhance the lipid peroxidation in mitochondria, reduce the activity of SDH enzyme, imbalance the fusion and fission of mitochondrial membrane, fracture the mitochondria in renal tubular epithelial cells, induce cell apoptosis and lead to kidney structural damage and dysfunction. The molecular mechanism of kidney mitochondria damage induced by fluorosis might be that it led to the imbalance of mitochondrial dynamics, changed the mitochondrial membrane permeability, thus leading to cell apoptosis of renal cells by regulating the expression of Fisl and Drpl upward and Mfh2 downward. Too low calcium intakes could aggravate fluorosis, act synergistically with F, while appropriate dose of dietary calcium might diminish the mitochondrial lipid peroxidation, regulate the expression of fission protein and fusion protein in mitochondria, thus reducing the occurrence of renal cell apoptosis in kidney mitochondria and alleviate the injury of fluorosis. The mechanisms of dietary calcium’s effect on drinking flurosis-induced mitochondrial injury of kidney are very complicate, the design of calcium dose especially the calcium dose range need to be further discussed in subsequent research. |
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