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Objective:To explore whether chronic fluorosis can cause liver fibrosis in rats by observing expression changes in type Ⅰcollagen (Col-Ⅰ), type Ⅲ collagen (Col-Ⅲ) and alpha smooth actin (α-SMA) in the liver tissue of chronic fluorosis rats.Methods:According to body weight (90 - 100 g), forty-eight SD rats were randomly divided into control group (drinking water fluoride ion concentration < 0.5 mg/L), low, medium and high concentration fluoride groups (drinking water fluoride ion concentration of 5.0, 50.0 and 100.0 mg/L), with 12 rats in each group (half male and half female), and fed for 6 months. Fluoride ion selective electrode method was used to detect bone fluoride and urinary fluoride levels; hematoxylin-eosin staining (HE staining) and Masson staining were used to observe the pathological and morphological changes and the collagen deposition of liver tissue; quantitative real-time polymerase chain reaction and immunohistochemical staining were used to observe Col-Ⅰ, Col-Ⅲ and α-SMA mRNA and protein expressions.Results:There was significant difference in bone fluoride and urine fluoride between the 4 groups [bone fluoride: (92.52 ± 5.64), (112.21 ± 11.86), (142.99 ± 7.87), (235.63 ± 11.55) mg/kg; urinary fluoride: (5.47 ± 0.88), (17.78 ± 1.48), (54.16 ± 5.96), (121.11 ± 6.32) mg/L, P < 0.001]. Under light microscope, with the increase of fluoride concentration, the degree of hepatic cell edema was aggravated, and the deposition of collagen fiber around the central vein and the portal area increased significantly. The mRNA expression level of Col-Ⅰ in low, medium and high concentration fluoride groups (1.20 ± 0.09, 1.80 ± 0.08, 1.58 ± 0.06) was significantly higher than that in control group (1.00 ± 0.00, P < 0.05); Col-Ⅲ and α-SMA mRNA expression levels in medium and high concentration fluoride groups (Col-Ⅲ: 1.15 ± 0.14, 1.64 ± 0.24; α-SMA: 1.69 ± 0.02, 2.34 ± 0.06) were significantly higher than those of low concentration fluoride group (Col-Ⅲ: 0.59 ± 0.17; α-SMA: 0.80 ± 0.13, P < 0.05). With the increase of fluoride concentration, the liver tissue Col-Ⅰ(0.00 ± 0.00, 0.03 ± 0.01, 0.08 ± 0.01, 0.13 ± 0.02), Col-Ⅲ (17 803.05 ± 3 221.16, 47 523.15 ± 3 490.10, 127 786.35 ± 13 008.86, 237 233.03 ± 47 614.63) and α-SMA (516.83 ± 181.18, 2 885.03 ± 864.92, 11 186.94 ± 2 394.08, 37 182.43 ± 12 390.59) protein levels were also increased significantly ( P < 0.05). Conclusion:Long-term excessive intake of fluorine may cause the production of collagen fibers around the central vein and the portal area of the liver in rats to increase, and then lead to the formation of liver fibrosis.
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Objective:To observe the protein and mRNA expressions of microtubule-associated protein 1 light chain 3 (LC3)B, P62 and Beclin1 in the liver of rats with chronic fluorosis, and to explore the role of autophagy in pathogenesis of liver injury induced by fluorosis.Methods:Using a group design, 54 SD rats were divided into 9 groups according to their weight (100 - 120 g) using a random number table method, each group with 6 rats, half male and half female. They were control group (NC group), low fluoride group (LF group), high fluoride group (HF group), NC + rapamycin (RAP) group, LF + RAP group, HF + RAP group, NC + chloroquine (CQ) group, LF + CQ group, and HF + CQ group. The NC group drank tap water (fluoride concentration was 0.5 mg/L), LF group drank fluoride water (fluoride concentration was 5.0 mg/L), HF group drank fluoride water (fluoride concentration was 50.0 mg/L); NC + RAP group, LF + RAP group and HF + RAP group were fed with corresponding drinking water, respectively, for 3 months, and then RAP (1.5 mg/kg) was intraperitoneally administered for 10 d; NC + CQ group, LF + CQ group and HF + CQ group were fed with corresponding drinking water, respectively, for 3 months, and then CQ (60 mg/kg) was intraperitoneally administered for 10 d. Bone and 24-hour urine samples of rats in each group were collected to detect the contents of bone fluoride and urine fluoride; liver histomorphological changes were observed through hematoxylineosin staining; protein and mRNA expressions of LC3B, P62 and Beclin1 in liver were detected by immunohistochemistry and real-time fluorescence quantitative PCR, respectively.Results:Compared with the NC group [(0.03 ± 0.00) mg/kg, (0.34 ± 0.08) mg/L], the contents of bone fluoride [(3.86 ± 0.08) mg/kg] and urine fluoride [(1.11 ± 0.16) mg/L] in HF group were higher ( P < 0.05). In the NC group, the lobule structure of liver tissue was clear, the hepatic cords were arranged in order, and the cell structure was normal. There were different degrees of hepatocyte edema in LF and HF groups. After intraperitoneal injection of RAP, compared with the corresponding fluoride group, the morphology of hepatocytes did not change significantly. After intraperitoneal injection of CQ, compared with the corresponding fluoride group, the liver cells showed obvious edema, and the degree of edema aggravated with the increase of fluoride concentration. Compared with the NC group, the protein expressions of LC3B and Beclin1 in HF group were higher ( P < 0.05), and the protein expression of P62 was lower ( P < 0.05). After intraperitoneal injection of RAP, the protein expressions of LC3B and P62 in LF + RAP group was lower than that in LF group ( P < 0.05); Compared with HF group, the protein expressions of LC3B and Beclin1 in HF + RAP group were lower ( P < 0.05). After intraperitoneal injection of CQ, protein expression of P62 in LF + CQ group was higher than that in LF group ( P < 0.05); Compared with HF group, protein expression of P62 in HF + CQ group was higher ( P < 0.05). Conclusions:Early (3 month) fluoride intake could promote autophagy and induce edema of hepatocytes in rats, and RAP had similar effects. CQ may induce liver injury by inhibiting autophagy of hepatocytes.
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Objective@#To investigate the change and association of glioma-associated oncogene homolog 1 (Gli1) and β-catenin on bone formation in rats with chronic fluorosis which were inhibited by cyclopamine (Cycl).@*Methods@#Forty-eight Sprague-Dawley rats were evenly divided to four groups, including control, F, F+Cycl and F+DMSO groups. The control group were fed with tap water (NaF<1 ppm). The F, F+Cycl and F+DMSO groups were exposed to NaF (50 ppm) in drinking water as the chronic fluorosis model. Then the rats in F+Cycl or F+DMSO groups were injected by Cycl or DMSO after 6 months, respectively. Urine fluoride concentration was detected using fluorine ion selective electrode. The enzyme-linked immunosorbent assay (ELISA) was used to detect bone alkaline phosphatase (BALP). Bone tissues were stained with hematoxylin-eosin. The mRNA and protein expression of Gli1 and β-catenin in bone tissue were detected using real-time PCR, immunohistochemistry and Western blot.@*Results@#Compared with the controls, the urine fluoride concentration and the width and volume of bone trabeculae were increased in the F, F+Cycl and F+DMSO groups, but no statistical difference among the 3 fluorosis groups. The concentration of BALP was increased in the F group and decreased in F+Cycl group (P<0.05). The expression of Gli1 and β-catenin mRNA and protein was higher in the F and F+Cycl groups than controls, but lower in the F+Cycl group than in the F group. There was positive correlation between the expression of Gli1 and β-catenin (r=0.476, P<0.05). The expression of Gli1 and β-catenin was also associated with BALP concentration and volume of bone trabeculae, respectively (r1=0.457, r2=0.466, r3=0.581, r4=0.554, respectively, P<0.05 for all).@*Conclusions@#The expression of Gli1 can be inhibited by Cycl. It may be involved in the bone formation of rats with chronic fluorosis. It may also affect the expression of β-catenin, which is an osteogenesis factor.
ABSTRACT
To investigate the change and association of glioma-associated oncogene homolog 1 (Gli1) and β-catenin on bone formation in rats with chronic fluorosis which were inhibited by cyclopamine (Cycl). Forty-eight Sprague-Dawley rats were evenly divided to four groups, including control, F, F+Cycl and F+DMSO groups. The control group were fed with tap water (NaF1 ppm). The F, F+Cycl and F+DMSO groups were exposed to NaF (50 ppm) in drinking water as the chronic fluorosis model. Then the rats in F+Cycl or F+DMSO groups were injected by Cycl or DMSO after 6 months, respectively. Urine fluoride concentration was detected using fluorine ion selective electrode. The enzyme-linked immunosorbent assay (ELISA) was used to detect bone alkaline phosphatase (BALP). Bone tissues were stained with hematoxylin-eosin. The mRNA and protein expression of Gli1 and β-catenin in bone tissue were detected using real-time PCR, immunohistochemistry and Western blot. Compared with the controls, the urine fluoride concentration and the width and volume of bone trabeculae were increased in the F, F+Cycl and F+DMSO groups, but no statistical difference among the 3 fluorosis groups. The concentration of BALP was increased in the F group and decreased in F+Cycl group (0.05). The expression of Gli1 and β-catenin mRNA and protein was higher in the F and F+Cycl groups than controls, but lower in the F+Cycl group than in the F group. There was positive correlation between the expression of Gli1 and β-catenin (0.476, 0.05). The expression of Gli1 and β-catenin was also associated with BALP concentration and volume of bone trabeculae, respectively (0.457, (2)0.466, 0.581, 0.554, respectively, 0.05 for all). The expression of Gli1 can be inhibited by Cycl. It may be involved in the bone formation of rats with chronic fluorosis. It may also affect the expression of β-catenin, which is an osteogenesis factor.
ABSTRACT
Objective:To investigate the expression changes of Hedgehog related factors (Ihh, Shh and Smo) in bone of rats with chronic fluorosis, and the significance.Methods:Thirty-six healthy SD rats were divided to three groups with the method of random digits table by body weight (100 - 120 g), 12 rats in each group, half male and half female. The rats of control were fed with tap water (NaF < 1 mg/L), and the experimental rats were exposed to NaF (low dose fluoride group: 5 mg/L, high dose fluoride group: 50 mg/L) added to the drinking water to establish the chronic fluorosis model. After the rats were raised for six months, 24-hour urine samples were collected and the femoral metaphysis of the rats was taken. Urine fluoride and bone fluoride were detected by fluorin ion selective electrode method. Bone tissues were stained with hematoxylin-eosin and observed under light microscope. The content of bone alkalinephosphatase (BALP) in rats' serum was detected by enzyme-linked immunosorbent assay (ELISA). The expressions of Ihh, Shh and Smo mRNA and protein in bone were detected by Real-time PCR and immunohistochemistry (IHC).Results:The contents of urine fluoride, bone fluoride and serum BALP were increased gradually in the control, low and high doses fluoride groups [urine fluoride: (1.37 ± 0.44), (5.96 ± 0.56), (7.60 ± 0.61) mg/L; bone fluoride: (306.04 ± 12.58), (652.91 ± 51.83), (1 094.11 ± 126.34) mg/kg; BALP: (27.78 ± 4.09), (46.59 ± 5.75), (57.45 ± 3.99) U/L, P < 0.05]. It could observed that bone sclerosis by light microscope in low and high doses fluoride groups. The expressions of Ihh, Shh and Smo mRNA in high dose fluoride group (1.39 ± 0.36, 0.56 ± 0.23, 0.40 ± 0.15) were higher than those of the control and low dose fluoride groups (0.73 ± 0.19, 0.92 ± 0.34; 0.19 ± 0.04, 0.36 ± 0.16; 0.14 ± 0.04, 0.24 ± 0.13; P < 0.05). The expression of Shh mRNA in low dose fluoride group was higher than that of the control group ( P < 0.05). The expressions of Ihh and Smo protein in high dose fluoride group (138.89 ± 3.72, 149.29 ± 7.63) were higher than those of the control and the low dose fluoride groups (127.39 ± 2.69, 134.81 ± 3.53; 129.64 ± 12.62, 139.07 ± 9.30), and the low dose fluoride group were higher than those of the control group ( P < 0.05). The expression of Shh protein in high dose fluoride group (141.26 ± 7.49) was higher than that of the control group (130.96 ± 11.10, P < 0.05). Conclusion:The expression of Hedgehog signaling pathway related factors in bone of rats with chronic fluorosis is changed, which indicates that bone formation can be affected by activation of Hedgehog signaling pathway induced by fluoride.
