Objective To study the change of bone ?-carboxyglutamic acid containing protein(BGP) in serum of rats at the initial stage of skeletal fluorosis caused by fluoride in coal.Methods SD rats were randomly divided into 6 groups (the number of female and male in each group was the same respectively):the control group,the low-dose fluoride group,the middle-dose fluoride added nutrition group,the middle-dose fluoride group,the high-dose fluoride added nutrition group,the high-dose fluoride group.All rats in the experimental groups were fed on the corn collected from the prevalent areas and contained different contents of fluoride respectively for 90-100 days.Content of fluoride in the urine,bone,kidney,BGP in serum,bone mineral density (BMD)and calcium in the bone and urine were determined.Results The fluorosis of the rats became more serious as fluoride intake increased.On the condition of same fluoride intake,the fluorosis could be relieved if nutrients added.BGP in serum of rats in each experimental groups had a increase trend,at the earlier stage,BGP of the high-dose group was higher than that of the control group (P

Objective To study the influence of fluorine on signaling pathway of osteoprotegerin(OPG)/ receptor activator of NF-κB ligand(RANKL) in cultured rat osteoblasts.Methods Osteoblasts were isolated from skull of neonatal rats(＜ 24 hours) by enzyme digestion,and fluorine of different concentrations [0 (control),1 × 10-3,1 × 10-4,1 × 10-5,1 × l0-6 and 1 × 10-7 mol/L] were added into the culture medium of second generation of osteoblasts.The expressions of OPG and RANKL mRNA were determined using real-time PCR 24 and 48 hours after culturing.The expressions of OPG and RANKL protein were measured by Western blotting.Results ① After exposed to fluorine for 24 hours,the differences of RANKL and OPG mRNA expression had statistical significance between groups(F =30.95,22.62,all P ＜ 0.01),the expression of RANKL mRNA(5.99 ± 0.39) in the 1 × 10-5 mol/L group and the expressions of OPG mRNA(3.52 ± 0.09,4.81 ± 0.15,3.68 ± 0.04) in the 1 × 10-4,1 × 10-5 and 1 × 10-6 mol/L groups were higher than those of the control group(3.20 ± 0.19,3.09 ± 0.58,all P ＜ 0.05),but in the 1 × 10-3 mol/L group,RANKL mRNA(2.29 ± 0.18) was lower than that of the control group(P ＜ 0.05).After exposed to fluorine for 48 hours,the differences of RANKL and OPG mRNA expression had statistical significance between groups(F =26.62,5.72,all P ＜ 0.01),the expressions of RANKL and OPG mRNA(6.67 ± 0.49 and 5.05 ± 0.51) in the 1 × 10-5 mol/L group were higher than those of the control group(4.29 ± 0.07 and 4.34 ± 0.12,all P ＜ 0.05),and in the 1 × 10-3 mol/L group the expression of OPG mRNA(3.63 ± 0.49) was lower than that of the control group(P ＜ 0.05).② The expression of RANKL protein was not statistically significant between 24 hours and 48 hours groups (F =0.07,0.49,all P ＞ 0.05) ; the differences of OPG protein expression had statistical significance between groups(F =3.26,P ＜ 0.05),the expression of OPG protein in the 1 × 10-5 mol/L group(1.45 ± 0.10) was higher than that of the control group(1.05 ± 0.06,P ＜ 0.05) at the 24 hours.After 48 hours,the expression of OPG protein was not statistically significant(F =0.44,P ＞ 0.05).Conclusions At lower fluorine concentrations,bone formation is the main activity.But when fluorine concentration increased and time prolonged,the osteoclast differentiation and maturation are promoted,and the bone resorption is the main thing.

Objective To investigated the association between bone morphogenetic proteins-2 (BMP-2)gene mutation and coal-burning skeletal fluorosis of children in Zhijin Guizhou.Methods In 2010,121 cases of children with skeletal fluorosis were diagnosed based on the standard X-ray Diagnosis of Skeletal Fluorosis (WS192-1999) in coal-burning skeletal fluorosis areas in Zhijin Guizhou,and 50 cases of them were selected as skeletal fluorosis group.Thirty healthy children free of skeletal fluorosis,rickets and other bone related diseases excluded by X-ray were selected as a control group in the same area.Using polymcrase chain reaction combined with DNA sequencing technology,all three exons of BMP-2 gene were conducted sequence screening in skeletal fluorosis and the control groups to detect gene mutations.Results ①The T insertion mutation on exon 1 between 401-402 bp:the T insertion mutantion genotype frequencies of skeletal fluorosis group and the control group were 27.7％ (13/47)and 7.1％ (2/28),and the difference was statistically significant (x2 =4.600,P ＜ 0.05),adjusted OR value of 4.62(1.94-10.90).②)The 894 bp T→G mutation on exon 2:the TG genotype frequencies of skeletal fluorosis group and the control group were 14.0％ (7/50) and 16.7％(5/30),and the difference were not statistically significant (x2=0.103,P＞ 0.05).③The 1 046 bp A→G mutation on exon 2:the AA,AG,GG genotype frequencies of skeletal fluorosis group and the control group was 30.0％ (15/50),24.0％ (12/50),46.0％ (23/50) and 50.0％ (15/30),20.0％(6/30),30.0％ (9/30),and the differences were not statistically significant (x2 =3.099,P ＞ 0.05).Conclusion Exon 1at 401-402 bp,T insertion mutation and skeletal fluorosis are closely related.The relationship between A→Gmutation in exon 2 at 1 046 bp and skeletal fluorosis is not significant.

Objective To observe the influence of coal-burning type of fluorosis on hypothalamic-pituitaryovary axis function and to explore possible mechanism in female rats.Methods Sixty SD rats were divided into two groups according to body weight with the method of random number table:control group and fluorosis group,30 rats in each group.Fluorosis group was feed with corn powder baked by high fluorine coal from Zhijin area.Changes of female rats' teeth during fluorine exposure were observed.After feeding for 180 days,24 h urine was collected in estrus and fluorine level was tested using fluoride ion-selective electrode; rats were executed and bone fluorine level was tested with high-temperature ashing-fluorine ion-selective electrode.Femoral artery blood was collected and serum was separated to test the contents of follicle stimulating hormone (FSH),luteinizing hormone (LH),testosterone (T),estradiol (E2) and progesterone (P) with electrochemiluminescence radioimmunoassay and gonadotropin-releasing hormone (GnRH),inhibin (INH) with enzyme-linked immunosorbent assay (ELISA),respectively.Organs,including hypothalamus,pituitary gland and ovary were weighted,and organ coefficients were calculated.Pathological morphology of hypothalamus,pituitary gland and ovary was observed after staining and ultrastructure of ovary was examined by electron microscopy.Results Coal-burning induced fluorine poisoning rat model was established successfully.There were no significant differences statistically in organ coefficients between fluorosis groups (0.032 ± 0.004,0.014 ± 0.008,0.037 ± 0.009) and controls (0.035 ± 0.005,0.012 ± 0.006,0.035 ± 0.004,t =0.46,0.87,0.64,all P ＞ 0.05).Rats serum GnRH,FSH,LH and T levels [(21.654 ± 4.765),(29.580 ± 5.221),(53.988 ± 6.506),(23.962 ± 2.255)μg/L] of fluorosis groups were all higher than those of controls [(10.384 ±2.250),(19.217 ± 4.743),(30.314 ± 4.443),(7.883 ± 1.973)μg/L,t =6.762,4.646,9.503,16.971,all P ＜ 0.05].But the level of P,INH [(12.635 ± 3.841),(18.926 ± 3.465)μg/L] were all lower than those of controls [(21.045 ±4.768),(48.076 ± 3.525)μg/L,t =4.344,18.649,all P ＜ 0.05].Serum E2 levels of control group and fluorosis group were (35.375 ± 10.662) and (27.500 ± 12.783)μg/L,respectively.The difference between groups was not statistically significant (t =1.821,P ＞ 0.05).No pathological changes were observed in the two groups of female hypothalamus,pituitary tissue by light microscopy and electron microscopy.Under light microscope,in the control group of normal ovarian tissue,more corpus luteum and different developmental stages of follicles were seen,granulosa cells were neatly arranged in a monolayer or multilayer.In fluorosis group,severe edema of ovarian interstitial cells and follicle degeneration increased.Cell structure and cell contours were blurred and unclear with occasional mature follicles.Under transmission electron microscope,in control group,normal ovarian granulosa cell ultrastructure was observed,nuclei were round,nuclear chromatin was uniform distributed,cytoplasm was rich in mitochondria and endoplasmic reticulum,and normal morphology.In fluorosis group,granulosa cells and interstitial cells showed apoptotic characters,such as nucleoli disappearing,mitochondrial swelling and chromatin aggregating at the nuclear membrane.Conclusions Fluorosis can induce ovarian tissue apoptosis,severely damage the micro environment.Reduction of P and INH affects ovarian,maturation and ovulation and leads to secretion of GnRH,FSH and LH.Fluorosis caused by coal-burning may induce the injury of ovary and cause abnormal secretion of hypothalamic-pituitary-ovary axis.Fluorosis has affected parts of female axis which may not be in the hypothalamus,pituitary,but causes ovarian tissue damage.

Objective To observe the impact of coal-burning-borne fluorosis on the opening day of vagina,and estrogen cycle and endocrinology of female rat.Methods Ninety weaned two weeks and healthy female SD rats,weighing 60-80 g were selected,and divided into three groups by body weight using random number table:control group,moderate fluoride exposed group,and higher fluoride exposed group,thirty rates in each group.Rats in fluoride exposed groups were fed with baked corn collected from coal-burning-borne fluorosis areas containing different levels of fluorine(fluorine contents were 47.8 and 96.0 mg/kg,respectively) to establish coal-burning-borne fluorosis models.The corn fed to control group rats were collected from non-endemic areas (fluoride content was 5.2 mg/kg).After exposed to fluoride for 60,120 and 180 days,the rats were put to death during estrus in three batches (10 rats in every batche).The situation of female vaginal opening was observed and recorded every morning at 8:00 at the beginning of the experiment.Ten female rats in each group were randomly selected to observe vaginal exfoliated cells for 10 days continuously every morning,and HE staining was selected to discriminate estrogen cycle.Femoral artery blood was collected to observe the impact of estradiol(E2) in estrus.Results The vaginal opening day age of the longer fluoride exposed group [(49.0 ± 4.5)d] was longer than that of the control group[(45.4 ± 2.3)d] and the moderate fluoride exposed group[(46.7 ± 1.9)d,all P ＜ 0.05].Estrogen cycle of the longer fluoride exposed group[(5.8 ± 0.8)d] after fluoride exposure for 120 d was higher than that of the control group [(4.4 + 0.9)d] and the moderate fluoride exposed group [(4.9 ± 0.7)d,all P ＜ 0.05].Estrogen cycle increased with increasing dosage of fluoride after fluoride exposure for 180 d in three groups [control group:(4.3 ± 0.7)d,moderate fluoride exposed group:(5.4 ± 1.1)d,higher fluoride exposed group:(6.7 + 0.9)d,all P ＜ 0.05].Estrogen cycle in higher fluoride exposed group at 180 d[(6.7 ± 0.9)d] was higher than that of 60 d and 120 d[(5.2 ± 1.1),(5.8 ± 0.8)d,all P ＜ 0.05].At 60 d after exposed to fluoride,E2 levels of fluoride exposed groups were increased[control group:(30.125 ± 94.17)ng/L,moderate fluoride exposed group:(438.75 ± 71.80) ng/L],higher fluoride exposed group (456.25 ± 57.80)ng/L with increasing dosage of fluoride (all P ＜ 0.05).E2 level in rats exposed to fluoride for 180 d [(275.00 ± 127.83)ng/L] was lower than that of 60 d and 120 d [(456.25 ± 57.80),(392.50 ± 102.50)ng/L,all P ＜ 0.05] in higher fluoride exposed groups.Conclusions Coal-burning-borne fluorosis has a significant effect on vaginal opening day age,estrogen cycle and endocrinology of female rats.The higher the fluoride dose,and the longer the exposure time,the more severe the impact.

Objective To evaluate the treatment effect of compound Chinese medicine on skeletal fluorosis in rats by Micro-CT.Methods Eighty-eight Wistar rats which had been weaned for two weeks were divided into four groups according to body weight [(91.1 ± 10.0)g] by the method of random number table:control group(16 mts),middle fluorine(MF)group(24 rats),high fluorine(HF) group(24 rats),and high fluoride and low calcium low protein (HF-LC-LP) group (24 rats).The amounts of fluorine of MF,HF and HF-LC-LP groups were 50,100 and 100 mg/kg,respectively.The contents of calcium and protein in HF-LC-LP group were half of MF and HF groups.Six months after treatment with fluoride,eight rats of each group were put to death with femoral artery bleeding.The rest 16 rats of each fluorosis group were divided into two groups,one was the control group and the other was fed with both fluorine and the compound Chinese medicine which simulated the actual situation of fluorosis area.Each rat of the treatment group was given the medicine 194 mg/100 g for six days every week.Daily urine samples were collected when the medicine had been used for 0,30 and 60 days.All the rats were put to death with femoral artery bleeding after the medicine had beengiven for 90 days,and limbs bones were dissected.Urine fluoride was tested by the method of fluoride ion selective electrode ; bone fluoride was tested by the method of high temperature ashing-fluoride ion selective electrode; bone mineral density(BMD),tissue mineral density(TMD),structure model index (SMI),trabecular thickness (Tb.Th),trabecular separation (Tb.Sp),anisotropy (a1/a3),trabecular connection density(Conn.D),the volume ratio of trabecular and bone tissue,the ratio of bone surface area and volume(BS/BV),and trabecular number(Tb.N) were detected by Micro-CT technology.Results The level of urinary fluoride of high fluoride and low calcium low protein treatment group [(11.01 ± 3.67)mg/L] was lower than that of its control group [(34.32 ± 9.50)mg/L,t =3.13,P ＜ 0.05] when rats were remedied with the compound Chinese medicine for 60 days.The level of bone fluoride of high fluoride treatment group[(275.38 ± 171.65)mg/kg] was lower than that of its control group[(701.67 ± 178.16)mg/kg,t =5.42,P ＜ 0.05] when rats were remedied withy the compound Chinese medicine for 90 days; bone fluoride of high fluoride and low calcium low protein treatment group[(313.26 ± 124.51)mg/kg] was lower than that of its control group[(794.66 ± 261.35)mg/kg,t =3.25,P ＜ 0.05].The differences of Tb.Th,Tb.Sp,a1/a3,Conn.D,BV/TV,BS/BV and Tb.N among groups were statistically significant(F =2.785,2.681,3.039,27.231,2.595,2.854,5.050,all P ＜ 0.05).Tb.Th[(0.04 ±0.01)mm] and Tb.Sp[(0.03 ± 0.01)mm] of middle fluorine treatment group were higher than those of their control groups[(0.02 ± 0.00),(0.02 ± 0.00)mm,all P＜ 0.05]; al/a3,Corm.D,BV/TV and Tb.N[(0.77 ±0.61),(510.91 ± 304.99)mm-3,(0.42 ± 0.06) and (13.58 ± 2.48)mm-1] were lower than those of their control groups[(1.11 ± 0.01),(2 403.69 ± 124.02)mm-3,(0.46 ± 0.03) and (18.12 ± 0.69)mm-1,all P ＜ 0.05].BV/TV(0.44 ± 0.04) of high fluoride treatment group were lower than those of their control groups(0.49 ± 0.00,P ＜ 0.05) ; Tb.Th[(0.04 ± 0.01) mm] was higher than that of its control group [(0.03 ± 0.00)mm,P ＜ 0.05].Conclusion The compound Chinese medicine may has therapeutic effect on rat skeletal fluorosis.