BACKGROUND:Previous studies have shown that minichromosome maintenance 3 is related with fluorosis, but the expression of minichromosome maintenance 3 in fluorosis patients is not clear yet.
OBJECTIVE:To analyze the mRNA expression level of minichromosome maintenance 3 in peripheral blood from patients exposed to fluoride and normal controls.
METHODS:Eleven patients with mild fluorosis by drinking water (exposure group) and 11 cases of control (non-exposure group) were selected for research. SYBRGreen1 real-time quantitative PCR was used to determine the mRNA expression of minichromosome maintenance 3 in peripheral blood mononuclear cel s, and the liver and renal function indicators were detected.
RESULTS AND CONCLUSION:mRNA expressions of minichromosome maintenance 3 in the exposure group and non-exposure group were (0.573 60±0.102 59) and (0.550 0±0.171 81), respectively, and there was no significant difference between two groups (P>0.05). There were no significant differences in the liver and renal function indicators between two groups. The results indicate that mild fluorosis has no significant effect on mRNA expression of minichromosome maintenance 3 in the peripheral blood mononuclear cel s. More indicators are needed to compressively analyze the effect of fluoride on the liver and renal functions.

Objective To observe glucose metabolism in C57 mice treated with different doses of fluoride.Methods Forty male C57 mice (body weight 20-24 g) were divided into four groups which were exposed to 0,50,100 and 150 mg/L sodium fluoride (NaF) by random number table according to body weight,each group had 10 mice.At 2,4,6,8,10 and 12 weeks after fluoride exposure,body weight was measured,blood glucose and glycosylated hemoglobin were detected by blood glucose meter and glycosylated hemoglobin meter,serum insulin and glucagon were detected by enzyme-linked immunosorbent assay (ELISA).Results At 10 and 12 weeks after fluoride exposure,the differences of fasting glucose between groups of C57 mice were statistically significant (F =35.12,21.92,all P ＜ 0.05),the fasting glucose of 100,150 mg/L fluoride groups [(7.7 ± 0.2),(7.3 ± 0.3),(8.6 ± 0.5),(9.1 ± 0.7)mmol/L] were higher than those of the control group [(5.4 ± 0.3),(5.0 ± 0.3)mmol/L,all P ＜ 0.01].The differences of glycosylated hemoglobin,glucagons between groups were statistically significant (F =3.85,8.74,all P ＜ 0.05).The glycosylated hemoglobin of 100,150 mg/L fluoride groups [(7.73 ± 0.76),(7.80 ± 1.15) mmo]/L] were higher than those of the control group [(5.43 ± 1.27) mmol/L,all P ＜ 0.05]; serum glucagon levels of 50,100,150 mg/L fluoride groups [(19.15 ± 11.84),(26.55 ± 15.97),(20.05 ± 7.29)ng/L] were lower than that of the control group [(48.35 ± 2.79)ng/L,all P ＜ 0.01].Conclusion Long term excess fluoride intake can reduce the function of sugar metabolism in C57 mice.

Objective To observe gene different expression of unfolded protein response signaling pathway in human osteoblasts under the excessive fluoride ,and explore the role of endoplasmic reticulum stress in fluorosis .Methods Human osteoblasts were cultured with fluoride ,intervening for 24 h .Cell viability and apoptosis were inspected by MTS assay and flow cytometer respective‐ly .The UPR signaling pathway was examined by real time PCR array ,and protein expressions were detected by Western blot .Re‐sults T he cell survival rates w ere (100 .678 5 ± 2 .830 3 )% ,(105 .393 4 ± 2 .538 4 )% ,(106 .125 7 ± 2 .048 3 )% ,(77 .977 3 ± 2 .544 3)% (P<0 .05) ,(30 .237 7 ± 0 .632 73)% (P<0 .05) treated with sodium fluoride at the concentration 0 ,5 ,10 ,20 ,40 ,80 mg/L respectively .Apoptosis rate inspected by flow cytometer was 4 .8% in 5 mg/L group ,13 .8% in 10 mg/L group ,37 .0% in 20 mg/L group ,58 .9% in 40 mg/L group ,63 .2% in 80 mg/L group (P<0 .05) .Only 1 gene was down regulated and 14 genes were up regulated .Western blot analysis showed BIP ,ATF4 ,CHOP and IRE1 both showed their protein expression gradually up regula‐ted with fluorine dose .XBP1 expression gradually increased in NaF 5-20 mg/L ,and its expression decreased at 40 and 80 mg/L . Conclusion Sodium fluoride can cause osteoblasts endoplasmic reticulum stress pathway through PTEN and IRE1 pathway ,and at high concentrations can cause apoptosis of osteoblast .

Objective To explore excessive fluoride on expression of osteocalcin (OC) in osteoblast and glucolipid metabolism in mice.Methods 3T3-L1 preadipocyte and MC3T3-E1 osteoblast were conventionally cultured,MC3T3-E1 cells were stimulated with O,2,10 and 50 mg/L sodium fluoride (NaF) to establish fluorine cell model,and levels of OC were tested.The corresponding NaF concentration resulted in the highest OC level was used as the optimal fluorine concentration.Cell experiments were divided into four groups:3T3-L1,3T3-L1 + NaF,3T3-L1 + MC3T3-E1 and 3T3-L1 + MC3T3-E1 + NaF.The treatment groups were respectively or jointly treated with the optimal concentration of NaF (50 mg/L) or MC3T3-E1 osteoblast,enzyme linked immunosorbent assay (ELISA) was used to detect OC and adiponectin (APN) levels.At the same time,40 C57BIL/6 mice were numbered by weight,randomly divided into control and fluoride groups,20 per group,control group drunk pure water,fluoride group drunk 100 mg/L NaF solution,changes of teeth and body weight [M (P25,P75)] of the mice were observed.Serum OC and APN levels were tested by ELISA at 12 weeks after modeling.The fasting plasma glucose (FPG) and the fasting plasma insulin (FINS) were detected by glucose oxidase and chemiluminescence methods,and insulin resistance (HOMA-IR) was evaluated.Results The APN levels of 3T3-L1,3T3-L1 + NaF,3T3-L1 + MC3T3-E1 and 3T3-L1 + MC3T3-E1 + NaF groups were (0.94 ± 0.18),(1.07 ± 0.21),(1.76 ± 0.20),and (2.49 ± 0.43) μg/L,MC3T3-E1 osteoblast with NaF had promote collaborative interaction effect on APN levels (F =14.519,P ＜ 0.01).The body weight of mice in fluoride group [27.5 (25.8,28.3) g] was significantly lower than that of the control group [31.4 (30.3,32.6) g,Z =-4.695,P ＜ 0.01].The levels of FPG [(7.78 ± 1.86) mmol/L],FINS [(3.22 ± 0.75) mU/L],OC [(6.11 ± 1.49) μμg/L],APN [(8.65 ± 1.78) μg/L] and HOMA-IR (1.15 ± 0.49) were higher than those of control groups [(5.40 ± 1.51) mmol/L,(2.45 ± 0.64) mU/L,(3.14 ± 0.92) μg/L,(4.03 ± 1.45) μg/L,0.62 ± 0.31],the differences were statistically significant (t =-4.466,-3.518,-7.560,-9.002,-4.182,P ＜ 0.01).OC levels in mice were positively correlated with FPG,FINS,APN and H-OMA-IR (r =0.868,0.707,0.911,0.818,P ＜ 0.01).Conclusion The OC of osteoblast in mice exposed to fluoride is increased significantly,OC levels in mice are closely related to blood glucose and APN,it is one of the key molecules in lipid metabolism.

Objective To observe the effects of excessive fluoride on osteocalcin and glucose metabolism in mice.Methods Thirty-two male C57 mice (body mass:18-24 g) were selected as study subjects which were randomized into four groups (8 mice in each group) according to their body mass by random number table method:0 mg/L group (control group),50 mg/L fluorine group,100 mg/L fluorine group and 150 mg/L fluorine group.Sodium fluoride in distilled water was freely taken by these animals to replicate fluorosis animal model.After 12 weeks,total osteocalcin,uncarboxylated osteocalcin,insulin and glucagon were measured by enzyme-linked immunosorbent assay,fasting blood glucose and glycated hemoglobin were measured by chemiluminescence.Results After 12 weeks of intervention with sodium fluoride,serum total osteocalcin,uncarboxylated osteocalcin,fasting blood glucose,glycated hemoglobin,insulin,and glucagon were significantly different between different groups (F =17.23,22.29,4.43,45.57,4.45,55.21,P ＜ 0.05).Total osteocalcin,uncarboxylated osteocalcin,fasting blood glucose,glycated hemoglobin,and insulin in the 100,150 mg/L fluorine groups were higher than those of control group [(30.02 ± 5.35),(35.22 ± 4.98) vs (20.23 ± 3.22) μg/L;(8.72 ± 1.34),(11.01 ± 1.02) vs (5.80 ± 1.60) μg/L;(7.53 ± 2.29),(8.53 ± 2.81) vs (4.99 ± 1.60) mmol/L;(6.74 ± 0.68),(7.12 ± 0.25) vs (4.95 ± 0.28) mmol/L;(2.65 ± 0.25),(2.74 ± 0.47) vs (2.13 ± 0.28) mU/L,P＜ 0.05].The serum glucagon levels in the 50,100,150 mg/L fluorine groups were lower than that in the control group [(20.90 ± 3.00),(23.68 ± 2.58),(21.63 ± 2.42) vs (38.61 ± 3.73) ng/L,P ＜ 0.05].Conclusion Excessive fluoride can lead to elevated osteocalcin level and abnormal glucose metabolism in mice.

Objective:To investigate the effects of osteocalcin (BGP) on bone growth and development and the expression levels of related hormones in offspring rats under the intervention of sodium fluoride.Methods:Twenty-four clean female SD rats and 24 clean male SD rats were selected, weighing 180-220 g, and the rats were mated in a 1∶1 cage for 10 d. The fluorosis rat model was established by drinking the fluorosis water method, female rats were divided into 3 groups according to body weight by random number table method, each group of 8 rats, including the high-dose, low-dose and control groups, with sodium fluoride of 200, 100, 0 mg/L in drinking water. The female rats were exposed to fluoride from the 0th day of pregnancy to the 3rd week after the offspring rats were born (before weaning). After weaning, 10 male offspring rats were selected from each group and continued to be exposed to fluoride in the same amount and manner until the 12th week after birth. The body weight and length of the offspring rats were measured every week before weaning and every two weeks after weaning. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum BGP, parathyroid hormone (PTH), calcitonin (CT), and alkaline phosphatase (ALP) contents in the offspring rats of each group exposed to fluoride for 12 weeks.Results:In the 2nd week of fluoride exposure, the body weights [(27.25 ± 3.57), (26.27 ± 4.48) g] and body lengths [(6.92 ± 0.46), (6.50 ± 0.54) cm] of the low-dose and high-dose groups were lower than those of the control group [(31.32 ± 3.62) g, (7.19 ± 0.26) cm, P < 0.05], but there were no significant differences in body weights and lengths between the high-dose group and the low-dose group ( P > 0.05). From the 3rd week of fluoride exposure, the body weight and length of the high-dose group were lower than those of the low-dose group and the control group ( P < 0.05). Serum BGP, PTH and ALP contents [(5.42 ± 0.26) mg/L, (157.53 ± 32.21) ng/L, (36.62 ± 6.01) U/L] in the control group were lower than those of the low-dose group [(6.15 ± 0.29) mg/L, (212.26 ± 51.97) ng/L, (50.68 ± 6.11) U/L] and high-dose group [(7.31 ± 0.77) mg/L, (274.21 ± 60.32) ng/L, (74.99 ± 9.08) U/L], and CT content [(182.40 ± 17.39) ng/L] was higher than those of the low-dose and high-dose groups [(135.77 ± 14.06), (70.09 ± 13.49) ng/L], and the differences were statistically significant ( P < 0.05); serum BGP, PTH and ALP contents in the high-dose group were higher than those in the low-dose group, and the CT content was lower than that in the low-dose group ( P < 0.05). Conclusion:Sodium fluoride may be involved in regulating the expression of related hormones by promoting the secretion of BGP, thereby affecting the bone growth and development of offspring rats.

Objective:To investigate the effects of different doses of fluoride on vascular endothelial injury in rats.Methods:Thirty clean male SD rats of 2 - 3 months old were selected, with body mass of 180 - 220 g. According to body mass, they were divided into three groups by random number table. The rat model of fluorosis was established by drinking water intoxication. According to the concentration of sodium fluoride in drinking water, the three dosage groups were blank control group (0 mg/L of sodium fluoride), low dose group (100 mg/L of sodium fluoride), and high dose group (200 mg/L of sodium fluoride), with 10 rats in each group. The rats were continuously exposed to fluoride for 12 weeks, and their body mass was measured every 2 weeks. After 12 weeks of fluoride exposure, the levels of serum osteocalcin (BGP), endothelin-1 (ET-1), intercellular adhesion factor-1 (ICAM-1), interleukin-6 (IL-6) and adiponectin (APN) were measured by enzyme-linked immunosorbent assay (ELISA). The ultrastructure of vascular endothelium in rats was observed by transmission electron microscopy.Results:At the 4th week of fluoride exposure, the body mass of rats in the blank control group was higher than that of the fluoride-exposed groups [(306.90 ± 19.13), (280.31 ± 18.44), (269.03 ± 17.47) g, P < 0.05], but there was no significant difference between the low dose group and the high dose group ( P > 0.05). From the 6th week of fluoride exposure, the body mass of rats in the blank control group was higher than that in the fluoride-exposed groups [(377.40 ± 23.72), (329.50 ± 21.78), (306.75 ± 27.09); (422.89 ± 32.23), (368.90 ± 23.79), (343.00 ± 18.41); (450.00 ± 29.26), (395.17 ± 28.22), (362.99 ± 21.77); (473.20 ± 28.43), (409.27 ± 29.95), (371.76 ± 21.65) g, P < 0.05], while that in the high dose group was lower than that in the low dose group ( P < 0.05). After 12 weeks of fluoride exposure through drinking water, the levels serum BGP, ET-1, ICAM-1 and IL-6 in blank control group were lower than those in fluoride-exposed groups ( P < 0.05), and the serum APN was higher than that in fluoride-exposed groups ( P < 0.05); while the levels of serum BGP, ET-1, ICAM-1 and IL-6 in high dose group were higher than those in low dose group, and the level of serum APN was lower than that in low dose group ( P < 0.05). Compared with the blank control group, the external processes of vascular endothelial cells in the low dose group were not standardized, the endothelial cells were not closely connected with the basement membrane, and the vacuoles were obvious. In the high dose group, the endothelial cells became short or detached; the content of heterochromatin increased significantly, the endothelial cells dropped into the vascular cavity, and with endothelial cells apoptosis. Conclusion:High fluoride can cause vascular endothelial injury.