Zhao L, Yu Y, Zhu Z, et al, - "Expressions of protein and mRNA relevant to hedgehog signaling pathway in liver of fluorosis rats" Chinese Journal of Public Health 31(9): 1162-1166 (2015) doi: 10.11847/zgggws2015-31-09-16
Zhao L, Yu Y, Deng C - "Expression of sonic hedgehog signaling pathway and its inhibition by cyclopamine in rat liver with chronic fluorosis" Chinese Journal of Pathology 43(12):814-9 (2014)
https://pubmed.ncbi.nlm.nih.gov/25623978/
Objective: To investigate the expression of sonic hedgehog (Shh) signaling pathway in liver fluorosis and to explore related mechanism.
Methods: To establish animal model, 48 normal SD rats (aged 4-5 weeks) were randomly divided into 4 groups (12 each): control group, fluoriosis group, blocking group and blocking control group. After 6 months, the blocking group and blocking control group were injected intraperitoneally once every 2 days for 3 times with 10 mg/kg cyclopamine or dimethysulfoxide, respectively. Rats were sacrificed at the end of the experiment and the fluoride content in urine and liver function was determined. The expression of Shh and Gli1 protein and mRNA in hepatocytes was detected by immunohistochemistry and real-time fluorescence quantitative PCR, respectively.
Results: The fluoride contents in the urine and the incidence of dental fluorosis increased in the fluoride and blocking control groups as compared with those in the control group, but decreased in the blocking group compared with those of the fluoride and blocking control group. Compared with the control group, the titers of aspartate transaminase (AST) and alanine transaminase (ALT) significantly increased, while the activity of total protein and albumin decreased in the fluoride and blocking control groups. Compared with the fluoride and blocking control groups, the activity of the ALT slightly declined and the AST, total protein and albumin slightly increased in the blocking group. Histologically, the cells were disorganized and swollen with cytoplasmic clearing (balloon cells), compared with the control group. The expression of Shh and Gli1 significantly increased in all but the control group. Compared with the fluoride and blocking control groups, the expression of Shh and Gli1 declined in the blocking group.
Conclusions: The overexpression and cyclopamine inhibition of the Shh signaling pathway are closely related to the content of fluoride in the liver. The Shh signaling pathway plays an important role in the pathogenesis of liver injury caused by fluorosis, suggesting a preventive and therapeutic target of the disease.
Zhao L, Yu Y, Deng C - "Protein and mRNA expression of Shh, Smo and Gli1 and inhibition by cyclopamine in hepatocytes of rats with chronic fluorosis" Toxicol Lett 225(2):318-24 (2014). doi: 10.1016/j.toxlet.2013.12.022
Abstract
In order to investigate the Sonic hedgehog (Shh) signaling pathway and the effect of cyclopamine in rat hepatocytes with chronic fluorosis, 48 Wistar rats were randomly divided into 4 groups. The control group was provided with tap water in which the fluorine concentration was <1mg/L, while the remaining three groups were provided with water containing sodium fluoride (NaF) at a concentration of 50mg/L. After 6 months, the blocking and blocking control groups were injected intraperitoneally once every 2 days for 6 days with 10mg/kg cyclopamine or dimethyl sulfoxide, respectively. The urinary and skeletal fluoride contents were determined by the ion selective electrode method. Levels of aspartate transaminase (AST), alanine transaminase (ALT), total protein (TP) and albumin (Alb) in the serum were determined by using autobiochemical machine. Histological changes in liver tissue were evaluated with Hematoxylin & Eeosin (H&E) staining using light microscopy. The protein and mRNA expression of Shh, Smo and Gli1 in hepatocytes of experimental animals was determined by immunohistochemistry (IHC), Western blotting (Wb) and Real-time quantitative PCR (RT-qPCR). Fluoride content of the urine and bone was increased in the fluorosis and blocking groups compared to those in the control group (P<0.05), while fluoride content in the blocking group was decreased compared to the fluorosis and blocking control groups (P<0.05). The expression of Shh, Smo and Gli1 at the mRNA and protein levels was significantly increased in hepatocytes from the fluorosis and blocking control groups compared with the control group, and expression in the blocking group was lower than that of the fluorosis and blocking control groups. The difference between any two groups was considered to be statistically significant (P<0.05). Taken together, our study indicates that the expression of Shh, Smo and Gli1 at the protein and mRNA level in hepatocytes of rats with chronic fluorosis can be increased by fluoride and may be inhibited by cyclopamine and that the Shh signaling pathway plays an important role in the liver pathogenesis caused by fluorosis.
Zhu Zhijian, Yu Yanni, Tao Xin, Deng Chaonan - "Role of Hh signaling pathway in fluoride-induced primary chondrocyte damage in rats" Chinese Journal of Public Health 5:574-578 (2015)
PFPC Library
Objective: To investigate the roles of sonic hedgehog (Shh), smoothened (Smo), and bone morphogenetic protein-2 (BMP-2) in fluoride-induced primary chondrocyte damage in rats in vitro.
Methods: The primary chondrocytes were obtained from one-week-old healthy Sprague-Dawley rats with a mechanical-enzyme digestion method and identified with immunohistochemical cells. They were then divided into a control group and 5, 10, 20, and 40 mg/L sodium fluoride (NaF) exposure groups. After 48 hours of treatment, the viability of the cells was determined with the methyl thiazolyl tetrazolium test (MTT); the expressions of protein and mRNA of Smo, Shh, and BMP-2 were detected with Western blot and reverse transcription-polymerase chain reaction (RT-PCR), and apoptosis was detected with flow cytometry.
Results: Compared with the control group, the vitality of the cells exposed to NaF of 5 and 10 mg/L significantly increased (113.33 ± 11.74% and 127.25 ± 10.24%, P < 0.05) but decreased significantly for the cells exposed to NaF of 40 mg/L (73.91 ± 9.94%, P < 0.05). The protein and mRNA expressions of Shh, Smo, and BMP-2 increased along with the increment of fluorine concentration. The apoptosis rate of the cells with 40 mg/L NaF exposure was 11.13 ± 1.20%, significantly higher than that of the control group (P < 0.05).
Conclusion: Fluoride can activate the hedgehog (Hh) signaling pathway, and both the Hh signaling pathway and apoptosis may play roles in the pathogenesis of cartilage damage in fluorosis.
Zhu ZJ, Yu YN, Chen R - "Role of Hedgehog signaling pathway on cartilage tissue damage in chronic fluorosis rats" Chin J Public Health. 34(2):241-245 (2018) doi:10.11847/zgggws1115085shu.
PFPC Library
Objective:
To investigate the role of the Hedgehog (Hh) signaling pathway in cartilage tissue damage in rats with chronic fluorosis.
Methods:
Thirty-six healthy Sprague-Dawley (SD) rats were randomly assigned to three groups (6 males and 6 females in each group): a control group (supplied with drinking water containing sodium fluoride [NaF] < 1 mg/L) and two fluorosis groups (supplied with drinking water containing NaF at concentrations of 5 mg/L and 50 mg/L, respectively). After six months of treatment, fluoride content in urine and bone was measured using a fluoride-ion selective electrode method. Histological changes in cartilage tissues were observed under a light microscope following hematoxylin and eosin (HE) staining. Protein components of the Hh signaling pathway, including Sonic Hedgehog (Shh), Smoothened (Smo), and Bone Morphogenetic Protein-2 (BMP-2), as well as apoptosis-regulating proteins B-cell lymphoma/leukemia-2 (Bcl-2) and Bcl-2-associated X protein (Bax), were detected using immunohistochemistry staining and Western blot analysis.
Results:
The fluoride content in urine (3.66 ± 0.43 mg/L and 8.05 ± 0.60 mg/L) and bone (402.38 ± 33.77 mg/kg and 935.12 ± 49.60 mg/kg) of rats in the low- and high-dose fluoride groups were significantly higher than those in the control group (P < 0.05 for all). Histological examination revealed thinning of the metaphyseal cartilage in the stifles, a decreased number of chondrocytes, and features consistent with sclerotic skeletal fluorosis in the fluoride-treated groups. Compared to the control group, the low- and high-dose fluoride groups showed significantly increased protein expression of Shh (0.86 ± 0.09 and 1.11 ± 0.15), Smo (0.92 ± 0.11 and 1.17 ± 0.14), BMP-2 (1.02 ± 0.14 and 1.13 ± 0.12), and Bax (0.91 ± 0.14 and 0.92 ± 0.11), with a concomitant decrease in Bcl-2 expression (0.78 ± 0.03 and 0.57 ± 0.09) (P < 0.05 for all).
Conclusion:
The activation of the Hedgehog signaling pathway and excessive expression of its downstream target genes may play a key role in the pathogenesis of chondrocyte damage in chronic fluorosis in rats.
Zhang Jiejing - "Effects of Hh signaling pathway on cartilage damage in rats with fluorosis" Chinese Journal of Control of Endemic Diseases1:25-27 (2016)
PFPC Library
Objective: To investigate the effect of Hedgehog (Hh) signaling pathway on cartilage damage in rats with fluorosis.
Methods: Sixty healthy Wistar rats were selected and divided into the control group (tap water), low fluorine group (10 mg/L sodium fluoride solution), and high fluorine group (50 mg/L sodium fluoride solution) based on the random number table method. Rats' cartilage cell activity and apoptosis rate were recorded under different strengths of fluoride poison. Chondrocytes Alto speed hedgehog protein (Shh) and Indian hedgehog (IHH), a transmembrane protein (SMO), transmembrane protein receptor (PTCH1), nuclear transcription factor protein Gli1, Gli2, and Gli3 mRNA and protein expression levels of the three groups were compared.
Results: The activity of chondrocytes in the control group, low fluoride group, and high fluoride group were (100 ± 0)%, (116.48 ± 10.27)%, and (76.25 ± 11.38)%, respectively. The apoptosis rate was (4.15 ± 0.78)%, (3.02 ± 0.71)%, and (9.58 ± 1.14)%, respectively. The cartilage expression of Shh, Ihh, Smo, Gli1, Gli2 mRNA, and protein expression of Shh, Ihh, Smo, Ptch1, Gli1, Gli2 in the control, low fluorosis, and high fluorosis group increased in turn, but Gli3 mRNA was gradually decreased (P < 0.05). Smo, Ptch1, Gli1, Gli2, Gli3, and Hh were involved in the activity of cartilage injury in rats.
Deng Chaonan, Zhang Ying, Xu Lin, Zhao Lina, Linghuyan, Yu Yanni - "Expressions of Ihh, Shh and Smo mRNA and protein in rats' bone exposed to different doses of fluoride and the significance" Chinese Journal of Endemic Diseases" Issue 09 (2020)
https://www.airitilibrary.com/Publicati ... z202009003
Objective:
To investigate the changes in the expression of Hedgehog-related factors (Ihh, Shh, and Smo) in the bone of rats with chronic fluorosis and to explore their significance in bone formation.
Methods:
Thirty-six healthy Sprague-Dawley (SD) rats (body weight 100-120 g) were randomly assigned to three groups (n=12 per group, 6 males and 6 females) using a random digits table. The control group was given tap water (NaF < 1 mg/L), while the experimental groups received sodium fluoride (NaF) in their drinking water at concentrations of 5 mg/L (low-dose fluoride group) and 50 mg/L (high-dose fluoride group) to establish a chronic fluorosis model. After six months, 24-hour urine samples were collected, and the femoral metaphysis was harvested. Urine fluoride and bone fluoride concentrations were measured using a fluoride ion-selective electrode method. Bone tissues were stained with hematoxylin-eosin (HE) and observed under a light microscope. Serum bone alkaline phosphatase (BALP) was measured using an enzyme-linked immunosorbent assay (ELISA). The mRNA and protein expression levels of Ihh, Shh, and Smo in bone tissue were analyzed by real-time PCR and immunohistochemistry (IHC).
Results:
Urine fluoride, bone fluoride, and serum BALP levels increased progressively in the control, low-dose, and high-dose fluoride groups. The following significant differences were observed (P < 0.05):
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.
Serum BALP: (27.78 ± 4.09), (46.59 ± 5.75), (57.45 ± 3.99) U/L.
Bone sclerosis was observed in the low- and high-dose fluoride groups under light microscopy. The mRNA expression of Ihh, Shh, and Smo in the high-dose fluoride group (1.39 ± 0.36, 0.56 ± 0.23, 0.40 ± 0.15, respectively) was significantly higher than in the control and low-dose fluoride groups (Ihh: 0.73 ± 0.19, 0.92 ± 0.34; Shh: 0.19 ± 0.04, 0.36 ± 0.16; Smo: 0.14 ± 0.04, 0.24 ± 0.13; P < 0.05). The Shh mRNA expression in the low-dose fluoride group was also significantly higher than that of the control group (P < 0.05).
At the protein level, the expression of Ihh and Smo in the high-dose fluoride group (138.89 ± 3.72, 149.29 ± 7.63, respectively) was significantly higher than in the control (127.39 ± 2.69, 134.81 ± 3.53) and low-dose fluoride groups (129.64 ± 12.62, 139.07 ± 9.30; P < 0.05). Protein levels of Ihh and Smo were also higher in the low-dose fluoride group compared to the control group (P < 0.05). Similarly, Shh protein expression in the high-dose fluoride group (141.26 ± 7.49) was significantly higher than in the control group (130.96 ± 11.10, P < 0.05).
Conclusion:
Chronic fluorosis induces changes in the expression of Hedgehog signaling pathway-related factors (Ihh, Shh, and Smo) in bone tissue. These changes suggest that fluoride activates the Hedgehog signaling pathway, which in turn affects bone formation. This may provide new insights into the molecular mechanisms underlying fluoride-induced bone alterations.
Bcl-2 is a downstream target gene for Shh. The following study by Yang et al. found that high amounts of fluoride downregulated Bcl-2 in ameloblast-like LS8 cells. It is further noteworthy that both retinoic acid and dexamethasone were used to induce maturation of mouse ameloblast-like LS8 cells, as this strongly implicates T3 (triiodothyronine) in the process.
Yang T, Zhang Y, Li Y, Hao Y, Zhou M, Dong N, Duan X - "High amounts of fluoride induce apoptosis/cell death in matured ameloblast-like LS8 cells by downregulating Bcl-2" Arch Oral Biol 58(9):1165-73 (2013). doi: 10.1016/j.archoralbio.2013.03.016
https://www.sciencedirect.com/science/a ... 6913001106
Abstract
Objective: Excessive fluoride intake during enamel formation may result in enamel fluorosis and apoptosis is regarded to be involved in the process by an unclear mechanism. We hypothesize that excessive fluoride might cause apoptosis in the ameloblasts and fluoride-induced apoptosis varies with the maturation stages of ameloblasts.
Methods: We set up an in vitro differentiation model of ameloblasts by using retinoic acid (RA) and dexamethasone (DEX) to induce the maturation of mouse ameloblast-like LS8 cells.
Results: The mRNA and protein levels of two enamel matrix proteins and two enamel proteinases were downregulated and upregulated, respectively, in the RA/DEX induced cells, indicating RA/DEX induced cells possessed the characteristics of matured ameloblasts. The strengthened endocytosis function and decreased intracellular pH value inside RA/DEX treated ameloblasts confirmed the maturation inducing effect of RA/DEX on ameloblasts. Excessive fluoride inhibited cell proliferation of ameloblasts within 72h. High amounts of fluoride also induced more apoptosis/dead cells and reduced the expression of Bcl-2, but to a different degree in the non-induced cells and RA/DEX induced cells.
Conclusions: We inferred that high doses of fluoride may easily target the transitional/early maturation ameloblasts and cause apoptosis or cell death. Bcl-2 might be involved in this process.
Wang Y, Zhang X, Zhao Z, Xu H - "Preliminary Analysis of MicroRNAs Expression Profiling in MC3T3-E1 Cells Exposed to Fluoride" Biol Trace Elem Res 176(2):367-373 (2017). doi: 10.1007/s12011-016-0833-x.
https://link.springer.com/article/10.10 ... 016-0833-x
"The Wnt, insulin, TGF-beta, hedgehog, VEGF, and notch pathways in osteoblasts were those mainly affected by fluoride treatment."
Abstract
Overexposure to fluoride from environmental sources can cause serious public health problems. Disrupted osteoblast function and impaired bone formation were found to be associated with excessive fluoride exposure. A massive analysis of microRNAs (miRNAs) was used to figure out the possible pathways in which fluoride affects osteoblast function. MC3T3-E1 cells were treated with 8 mg/L of fluorine for 7 days. Total RNA of cells was extracted, and their integrity and purity were tested. RNA samples were analyzed by using miRNA array, including miRNA labeling, hybridization, scanning, and expression data analysis to compare the profiling of miRNA expression between control and fluoride-treated group. Transcriptome analysis console and enrichment analysis calculated by miRSystem were used to predict target genes and collect miRNAs pathway maps. Forty-five upregulated and 31 downregulated miRNAs expression were found in the fluoride-treated group, and most of the verified miRNAs were mature. The KEGG pathway enrichment analysis searched out 36 pathways that scored more than 0.1. These pathways mainly included intracellular signaling, cytokines, metabolism, and cytoskeleton-related pathways. Among them, the Wnt, insulin, TGF-beta, hedgehog, VEGF, and notch pathways in osteoblasts were those mainly affected by fluoride treatment. These results have shown a number of higher level systemic pathways activated by overexposure of fluoride in osteoblastic cells and verified that fluoride affected the molecular crosstalk in the osteoblasts.