| Post-menopausal osteoporosis has been attributed solely to declining estrogen levels in the past. It was state that follicle-stimulating hormone receptors (FSHRs) knockout mouse did not lose bone, activation of MEK/Erk, NF-κB, and Akt pathways by precursors possess Gi2a-coupled FSHRs resuLted in enhanced osteoclast formation and function, suggested that high-circuLating FSH causes hypogonadal bone loss, independent of estrogen. Recent research indicated that experimental periodontitis rats with bilateral ovariectomized (OVX) had more severe alveolar bone resorption than periodontitis-only rats. Furthermore, leuprorelin (LE), an FSH inhibitor, had a protective effect on alveolar bone loss in OVX rats, indicated that FSH after menopause may be a systemic condition that contributed to the progression of periodontitis.The cyclooxygenase (COX, also called prostaglandin endoperoxide synthase) enzymes catalyze the stepwise conversion of arachidonic acid into two short-lived intermediates, prostaglandin G2 (PGG2) and prostaglandin H2 (PGH2), which is then metabolized to various PGs by the activity of specific synthases. Usually, COX-1 is constitutively expressed in most cells and reguLates normal physiological responses, whereas COX-2 is undetectable under normal condition, and its expression is increased by pathological stimuLation including interleukin-1, tumor necrosisfactor-a, and lipopolysaccharide (LPS). Therefore, COX-2 seems to be the primary COX that controls prostaglandin E2 (PGE2) synthesis in response to inflammation. COX-2 and COX-2-mediated PGE2 played a key role in periodontitis, including stimulating inflammation factors and increasing osteoclast differentiation. It was indicated that FSH couLd reguLate the expression of COX-2 and PGE2 production in ovarian cancer. However, whether a link existed between FSH and COX-2 expression in periodontal system remained unknown. CCL2 (MCP-1), CCL3 (MlP-la) andCCL5 (RANTES) are three important chemokines produced by many different cell types stimulating the recruitment of white blood cells to the site of inflammation.PDLCs have been reported to express mRNA for CCL2 and CCL5 upon stimulation with viable Porphyromonas gingivalis.It’s a unknown question whether FSH improve the expression of CCL in PDLCs.Therefore, in this thesis, we designed two different experiments according to the problems:(1) we investigate the effect of FSH on alveolar bone resorption and COX-2 and PGE2 expression, the potential mechanism was also investigated;(2) we investigated the effects of FSH on CCL2,CCL3,CCL5 expression in PDLCs.PART ONE FSH enhances alveolar bone resorption via upreguLation of COX-2CHAPTER ONE Effects of FSH on alveolar bone resorption and COX-2 expression in animal model.Exp.1 Establishment of OVX and experimental periodontitis rat model.Objective:The purpose of this study was to establish OVX and experimental periodontitis rat model.Materials and methods:Eight-week-old female Sprague-Dawley rats from 200 and 220 g in weight were selected from the same lot for this experiment. The rats were randomly assigned to 4 groups as follows:Group 1 (n=8), bilateral OVX+Ligatured (L); Group 2 (n= 7), bilateral OVX+L+1.6 mg/kg triptorelin; Group 3 (n=6), sham surgery (SHAM)+L; Group 4 (n= 6); SHAM+L+1.6 mg/kg triptorelin. Rats were anesthetized by subcutaneous injection of 4% chloral hydrate mixture (0.35 mL/ 100 g). Bilateral ovariectomies were performed in groups 1 and 2, and rats in groups 3 and 4 were subjected to SHAM, and the same size of adipose tissue was excised near the ovaries. Immediately after surgery, triptorelin (gonadotropin-releasing hormone agonist) was injected subcutaneously. Once injected, triptorelin maintains a biologically effective blood concentration and inhibits circuLating FSH levels by up to 4 weeks. A sterile thread ligature was placed around the cervix of the first mandibuLar molar to induce experimental periodontitis prior to OVX. Surgeries were performed according to previous studies. All rats were euthanized by administration of an overdose of anesthetic (4 weeks after surgery), and the mandibles were extracted and fixed in 4% paraformaldehyde. The left mandible specimens were decalcified and dehydrated, followed by wax embedding.Results:The experimental data show that this method can successfully constructed OVX and experimental rat model. Tissue sections were prepared for the following experiment.Exp.2 The effect of FSH on rat alveolar bone destructionObjective:The purpose of this experiment was to verify the effect of FSH on alveolar bone resorption.Materials and methods:After all rats were euthanized by administration of an overdose of anesthetic (4 weeks after surgery), and the mandibles were extracted and fixed in 4% paraformaldehyde. A fixed radius and the length of the cylindrical region was chosen to calculate the bone volume fraction (BV/TV) from the furcation region and between the medial and distal roots of the first mandibuLar molar. Bone loss around the right first mandibuLar molars were evaluated by a micro-CT scanning system. After reconstruction, the distance from the CEJ to the ABC was measured along the buccal and lingual long axis of the root surfaces of the first molars. Six sites were established per tooth, and the mean of the six values was taken as the bone loss for each rat. The sample number was 6.Results:The OVX+L group exhibited a significantly lower BV/TV ratio compared with the other groups (P<0.05). There was a significant difference between the OVX+L rats and the L-only rats (P<0.05). There were significant differences between the OVX+L+triptorelin group and the OVX+L group (P<0.05). The Results showed that FSH can increase alveolar bone loss and GnRH-a has a protective effect on alveolar bone loss.Exp.3 The effect of FSH on COX-2 expression in rats modelObjective:The purpose of this study was to confirm that FSH increase the expression of COX-2 in rats.Materials and methods:Serial sections were cut at a 4-μm thickness. Firstly, double immunofluorescence was carried on slices to detect whether FSH is associated with COX-2 expression. Immunofluorescence double staining was used to evaluate the relationship between the FSHR and COX-2. Then immunohistochemistry was used to detect the quantitative expression of COX-2.Results:Double immunofluorescence staining. Results were taken under the same light source and exposure time. The expression of FSHR and COX-2 on the same tissue section was marked with different fluorescence signals (FSHR:green fluorescence signal, COX-2:red fluorescence signal), and finally the images were synthesized to observe the expression of FHSR and COX-2. The OVX group had more red and green fluorescence signal overlap than other groups-yellow fluorescence signal. Immunohistochemical staining was observed in the periodontium, especially in the periodontal ligament under root furcation. The number of COX-2-positive cells in OVX+L rats was significantly higher than in L-only rats (P<0.01). Furthermore, injection with triptorelin significantly decreased the expression of COX-2 in the OVX+L rats (P<0.05). No significant difference was found between SHAM groups.CHAPTER TWO Effect of FSH on the expression of COX-2 in human PDLCsExp.1 CuLture and characterization of human PDLCsObjective:The purpose of this experiment is to develop human PDLCs from premolar teeth.Materials and methods:Primary human PDLCs were obtained from the School of Stomatology of Wuhan University. The human PDLCs was taken from the premolar teeth for orthodontic reasons, with informed consent obtained. Then, under aseptic conditions, the PDL tissue attached to the middle third of the roots was removed and cut into pieces of about 1 mm2, then placed in T25 cuLture bottles. Tissues were cuLtured in DuLbecco’s modified medium (DMEM) with 10%fetal bovine serum (FBS) at 37℃ in a humidified atmosphere of 5%CO2 in compressed air. When the cell coverage area reached 80%, PDLC was trypsinized and used in the subsequent 4th or 5th generation in subsequent experiments.The PDLCs were seeded on the slides at an appropriate density, were fixed with 4%(w/v) paraformaldehyde, blocked with 3% bovine serum albumin, and incubated with primary antibodies against vimentin, keratin, COLI and fibronectin overnight at 4℃. The slices were then incubated with fluorescent secondary antibody for 1 h at room temperature. Finally, 4’,6-diamidino-2-phenylindole (DAPI) was used to stain the nuclei. Phosphate-buffered saline (PBS) was used as a negative control. Normal secondary antibody was used instead of fluorescent secondary antibody to characterize FSHR in PDLCs, and DAB instead of DAPI was used to stain the nuclei.Results:After cuLtured for 7 to 30 days, there were young PDLCs came out from the tissue block under microscope. PDLCs were fibroblast-like, and highly transparent and proliferative, and showed a short spindle type, with large transparency and plump cytoplasm, round or oval cell nucleus which were located in the center of the cell. The cells showed positive expression for COLI, fibronectin and vimentin, but were negative for keratin indicating a mesenchymal origin. No specific staining was visualized in the negative control.Exp.2 Influences of high levels of FSH on the expression of COX-2 in vitroObjective:Effect of FSH on the expression of COX-2 in PDLCsMaterials and methods:The PDLCs were allowed to attach for 12 h after seeded in six-well plates at a density of 1×106 cells. The cells were silenced by treatment with phenol red -free DMEM overnight.PDLCs were subsequently treated with human recombinant FSH for 0,2,4,6,8,12 or 24 h. To eliminate the effect of different types of hormones, phenol red-free DMEM with 5% charcoal-stripped FBS was used. After RNA and protein were collected, qPCR and western-blotting was used to evaluate the mRNA and protein expression of COX-2.Results:FSH couLd increase the mRNA (P<0.05) and protein expression of COX-2 on a time-denpend mammer in human PDLCs. Exp.3 The effect of FSH on PGE2 in PDLCsObjective:FSH increased the expression of PGE2 in PDLCsMaterials and methods:The PDLCswere allowed to attach for 12 h, after seeded in six-well plates at a density of 1×106 cells. The cells were then silenced by treatment with phenol red-free DMEM overnight. PDLCs were subsequently treated with human recombinant FSH for 0 6 or 24 h. To eliminate the effect of different types of hormones, phenol red-free DMEM with 5% charcoal-stripped FBS was used. After protein was collected, the levels of prostaglandin E2 (PGE2) was measured by enzyme-linked immunosorbent assay.Results:PGE2 was significantly increased on a time-denpend mammer in PDLCs when treated with FSH (P<0.01).CHAPTER THERE To explore the potential pathways involved in the promotion of COX-2 expression in PDLCs when treated with FSHExp.1 To explore the pathways activated by FSHObjective:The purpose of this study was to explore the pathways activated by FSHMaterials and methods:The PDLCswere allowed to attach for 12 h, after seeded in six-well plates at a density of 1 × 106 cells. The cells were then silenced by treatment with phenol red -free DMEM overnight. After treatment with FSH for 0,10,15,30 or 60 min, both the activated and total protein of Erk, p38, and Akt were examined by western-blot.Results:Results showed that FSH stimuLated p-Erk, p-p38, and p-Akt activation, while expression of Akt and p38 expression remained unchanged, although Erk expression increased over time and p-Erk was in a increased expression based on Grayscale analysis.Exp.2 The effect of FSH on COX-2 expression after blocking the Erk, p38, and Akt pathwaysObjective:To investigate the effect of FSH on COX-2 expression after blocking the Erk, p38, and Akt pathways.Materials and methods:PDLCs were then preincubated with PD98059 and SB203580, specific inhibitors of the Erk, p38, and Akt pathways, for 2 h before treating them for 6 h with FSH. After RNA and protein were collected, the mRNA and protein expression levels of COX-2 were measured by qPCR and western-blotting.Results:The selective inhibitors of Erk, p38, and Akt markedly inhibited FSH-induced COX-2 expression (P<0.01), indicating that activation of the Erk, p38, and Akt pathways participate in the signaling cascades that mediate the up-regulation of COX-2 expression in PDLCs.Exp.3 The effect of FSH on PGE2 expression after blocking the Erk, p38, and Akt pathwaysObjective:To investigate the effect of FSH on PGE2 expression after blocking the Erk, p38, and Akt pathways.Materials and methods:PDLCs were preincubated with PD98059 and SB203580, specific inhibitors of the Erk, p38, and Akt pathways, for 2 h before treating them for 6 h with FSH. After protein were collected, the levels of PGE2 were detectived by ELISA.Results:Blocking these pathways markedly repressed the expression of PGE2 (P<0.01). The trend for PGE2 production was the same as that for COX-2 expression. These Results suggest that COX-2 is related to PGE2 production in PDLCs treated with FSH.PART TWO Influence of FSH on CCL expression in PDLCsExp.l Effect of FSH on CCL2, CCL3, CCL5 expression in PDLCsObjective:The purpose of this study was to investigate the effect of FSH on CCL2, CCL3, CCLSmRNA and protein expression in PDLCsMaterials and methods:PDLCs were seeded in six-well plates at a density of 1 x 106 cells per well and were allowed to attach for 12 h. The cells were then silenced by treatment with phenol red -free DMEM overnight. PDLCs were subsequently treated with different concentrations of FSH(100,30,10,1 ng/mL), LPS (20 ug/mL) was used as positive control. To eliminate the effect of different types of hormones, phenol red-free DMEM with 5% charcoal-stripped FBS was used. After RNA and cell supernatant were collected, the mRNA and protein expression levels of CCL2, CCL3, CCL5 were measured by qRT-PCR and ELISA.Results:CCL2 mRNA expression did not show any statistical difference among the groups. The mRNA expression of CCL3 in 100 ng/mL FSH group was lower when compared with the negative control group (P<0.05), significant difference was found in the comparison. The mRNA expression of CCL3 in 30 ng/mL FSH group was significantly higher than the negative control group (P<0.05). The mRNA expression of CCL5 in 100 ng/mL FSH group was statistically higher than that in the negative control group (P<0.05), while the mRNA expression of CCL5 in 30 ng/mL FSH group was lower than that in the negative control group, other groups had no significant difference when compared with the negative control group. The level of CCL2 protein was detected by ELISA. The results showed that the level of CCL2 in the FSH1ng/mL was lower than that in the negative control group (P<0.05), and the difference was statistically significant. The protein expression of CCL2 in LPS group was higher than that of negative control group (P<0.05) and the difference was statistically significant.Conclusion1. FSH could increase alveolar bone loss which in accord with the finding of the previous study indicating that FSH could increase alveolar bone loss and that the specific inhibitors of FSH had a protective effect on bone loss.2. FSH increases alveolar bone loss through a COX-2-upregulated mechanism, which play an important part in periodontitis.3. The analysis of signaling pathways revealed the activation of COX-2-mediated pathways including Erk, p38, and Akt.4. The present results can not yet prove that FSH is involved in CCL2, CCL3, CCL5 on PDLCs immune chemotaxis. |
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