| Objective To discuss the effect of the Hh’s specificity repressorCyclopamine (Cycl) by observe the change of Hedgehog (Hh) signalingmolecules and its downstream osteogenesis and apoptosis factors in vivo and invitro. Then the expression of factors related to bone microenvironment in thebone of rats with chronic fluorosis was detected to explore the relationshipbetween change of Hh signaling in injury bone of rats and the excessive boneformation further, and the experimental basis for the research of target treatmentof skeletal fluorosis was provided. Methods1. SD rats were freely drunk bydifferent concentrations of sodium fluoride (NaF) in water for six months. Therat model was established successfully judged by the incidence of dentalflourosis and the fluoride concentration of urine. And the fluoride concentrationof bone in all groups of rats were detected. Bone tissues were stained withhematoxylin-eosin and observed under light microscope. The number ofosteoclast was identified and counted by tartrate-resistant acid phosphatasestaining (TRAP).The concentration of bone alkaline phosphatase (BALP) andtartrate-resistant acid phosphatase (TRACP5b) in rats’ serum was detected byenzyme-linked immunosorbent assay (ELISA). The expressions andimmunoreactivities of Hh signaling pathway and osteogenesis and boneresorption related factors were observed by immunohistochemistry (IHC).2. ThemRNA and protein expression of the key factors in Hh signaling pathways weredetected by real-time fluorescence quantitative PCR (Real-time PCR), IHC andWestern Blot to explore the change of Hh signaling influenced by low or highfluoride concentration.3. Cycl were administered by intraperitoneal injection in the rats fed with high fluoride in water. Furthermore, we investigated the changeof incidence of dental fluorosis, the fluoride concentration of urine and bone,and the concentration of BALP in serum. To screen the best appropriate fluorineand Cycl concentration by the cell counting kit8(CCK8). The activation ofalkaline phhosphatase (ALP) in cultured osteoblasts was detected byp-nitrophenyl phosphate (pNPP). Apoptosis osteoblasts in vivo were located andcounted by transferase-mediated dUTP-bintin (TUNEL). Apoptosis osteoblast invitro was detected by flow cytometry. The mRNA and protein expression ofsome important Hh signaling molecules, ostegenesis factors and apoptosisrelated factors were detected by Real-time PCR, IHC and Western Blot in vivoand in vitro.4. The mRNA and protein expression of factors related to bonemicroenvironment in bone of rats with chronic fluorosis were detected byReal-time PCR, in situ hybridization (ISH) and Western Blot. The order was toexplore the relationship between Hh signaling and the change of bonemicroenvironment in injury bone of rats with chronic fluorosis. Results1. Theincidence of detal fluorosis, the fluoride concentration of urine and bone and theconcentration of BALP in serum were increased with the increasingconcentration of fluoride. It showed that chronic fluorosis rat model wasestablished successfully. It could observed that bone sclerosis by opticalmicroscope.2. The protein expression of Hh signaling pathwaymolecules——Indian hedgehog (Ihh), Sonic hedgehog (Shh) and osteogenesisfactors——Runx2, Osx in fluorosis group was increased with the increasingfluoride concentration. Compared with the high fluoride group, however, thenumber of osteoclast, the concentration of TRACP5b in serum and the proteinexpression of RANKL and NF-κBp50were higher in the low fluoride group.Exposed to fluoride, there was positive correlation between the change of Hhsignaling factors and the bone formation.3. Compared with the control, themRNA and protein expression of ligand (Ihh and Shh), receptor (Smo) andnuclear transcription factors (Glioma-associated oncogene homolog1(Gli1) andGli2) were increased with fluoride in a dose dependent. The mRNA and proteinexpression of receptor (Ptch1) and nuclear transcription factor (Gli3) were nosignificant difference in all groups.4. There was no significant differencebetween the fluorosis group and the fluorosis+Cycl group in incidence of dental fluorosis, the fluoride concentration of urine and bone. The concentration ofBALP in serum of rats in the fluorosis+Cycl group was lower than that in thefluorosis group. The activation of ALP in osteoblast exposed to fluorosis+Cyclwas also lower than that only exposed to fluoride. The mRNA and proteinexpression of Ihh, Shh, Smo, Gli1and Gli2in bone of rats with fluorosis+Cycland the osteoblast exposed to fluorosis+Cycl were lower than those only exposedto fluoride, so did the osteogenesis factors downstream of Hh signaling pathwaysuch as Runx2and β-catenin. But the mRNA and protein expression of Osx inbone of rats and osteoblast exposed to fluorosis+Cycl were same to thoseexposed to fluorosis.5. The apoptotic rate of osteoblasts with fluorosis wasincreased, and it was similar to that exposed to fluorosis+Cycl. The apoptoticrate of cultured osteoblasts exposed to2.5mg/L NaF was higher than that in the5mg/L NaF group. The apoptotic rate of osteoblast exposed to fluorosis+Cyclwas higher than that exposed to fluorosis. Compared with the control, the mRNAand protein of Bcl-2and Bcl-2/Bax on osteoblasts exposed to fluorosis weredecreased and those of Bax was increased in vivo and in vitro. Those in fluorosisgroup were similar to the fluorosis+Cycl group. But the mRNA and proteinexpression of B-cell Lymphoma-2(Bcl-2) and Bcl-2/Bcl-2accociated X protein(Bax) on osteoblasts were higher than those exposed to NaF only, and it wasopposed to those of Bax in vitro.6. There was positive immunoreactivities oftransforming growth factor-β1(TGF-β1), interleukin-1β (IL-1β) and IL-6onosteoblasts in bone of rats with chronic fluorosis. The mRNA and proteinexpressions of TGF-β1, IL-1β and IL-6were increased with the increasingfluoride concentration. There were no significant difference in the mRNA andprotein expression of IL-1β and IL-6between the low fluoride group and thehigh fluoride group. It was showed no positive immunoreactivities of tumornecrosis factor α (TNFα) on osteoblasts, but weakly positive immunoreactivitieson megakaryocyte in bone of rats with chronic fluorosis. There were nosignificantly difference in the mRNA and protein expression of TNFα in allgroups. There were significantly positive relationship between the expression ofIhh, the concentration of bone fluoride and the expression of TGF-β1, IL-1β andIL-6. There was no significantly relationship between the expression of Ihh andTNFα, or the concentration of bone fluoride and the expression of TNFα. Conclusions1. Bone information is superior to bone resorption exposed tofluoride to some extent. The bone sclerosis is observed by optical microscope.BALP in serum of rats can be used as an early biological index to estimate theseverity of bone formation in skeletal fluorosis.2. The expression of Ihh isincreased on osteoblasts of rats stimulated by fluorosis. The signal can activateGli2through Smo, then transcribe the signal into nucleus, and induce theexpression of Runx2. It leads to proliferation of the osteoblast, activition ofBALP and bone sclerosis eventually. At the same time, the activation of Ihh caninduce the apoptosis of osteblast by Bcl-2. The apoptosis product may enhancethe process of osteoblast proliferation.3. Cycl can block the expression of Ihhinduced by fluorosis to some extent, and inhibit the proliferation of osteoblast invitro. It provides a experimental basis for the selection of treatment of skeletalfluorosis.4. Change of microenvironment was influenced by Ihh signalingpathway, it participates in mechanism of bone sclerosis of rat with chronicfluorosis. |
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