Study On FSHR In The Progression Of Periapical Periodontitis And OSCC

PART I FSH aggravates Bone Loss in Ovariectomised Rats with Experimental Periapical PeriodontitisPeriapical bone loss is one of the prominent pathologic and clinical features of peri-apical periodontitis, which begins as a bacterial infection in the dental pulp; inflam-matory cells are then recruited and cytokines are subsequently generated.lt was found that the regulatoy mechnisms of apical periodontitis were complex and poorly known.Post-menopausal osteoporosis has been considered as one of the risk factor of bone loss from periapical periodontitis. Oestrogen deficiency has been considered as the main cause of post-menopausal osteoporosis. Accompanied by sex steroid with-drawal in mammals, serum levels of the pituitary hormone follicle-stimulating hormone (FSH) are sharply increased. Recently it was reported that FSH exhibits osteoclastogenic and proresorptive actions in a Gi2a-coupled FSH receptor (FSHR) identified in human and mouse osteoclasts and their oestrogen-independent precursors.This research was aimed to assess the effect and the related mechanisms of FSH on apical periodontitis related bone loss. In this study, enzyme histochemical examination, radioimmunoassay and immunohistochemical staining were conducted to detect the following:FSHR, a novel positive biomarker of osteoclasts; RANKL, a positive regulator of osteoclastogenesis and osteoclastic activity; OPQ a negative regulator of osteoclastogenesis and osteoclastic activity; and TNF-a and IL-1?, inflammatory cytokines.EXPERIMENT I FSH may enhance the progression of OVX rat periapical periodontitisObjective:The purpose of the present study was to build up the induced rat periapical lesions combined with OVX and injection of gonadotropins or gonadotropins inhibitor model, harvesting samples, identifying the model by using Highresolution X-ray Imaging and histological analysis.Methods:Thirty Twelve-week-old female Sprague-Dawley rats were randomly assigned into the following six groups with 5 rats each:(1) sham surgery+vehicle, (2) bilateral OVX+vehicle, (3) bilateral OVX+1.6 mg/kg LE, (4) bilateral OVX+1.6 mg/kg LE+1.85 ?g/kg LH, (5) bilateral OVX+1.6 mg/kg LE+3?g/kg FSH and (6) bilateral OVX+3 jig/kg FSH. The experimental protocols in this study were approved by the Institutional Animal Care and Use Committee of Wuhan University.The rats in Groups 2 through 6 were subjected to bilateral ovariectomies (OVX). Meanwhile, the rats in Group 1 were subjected to sham surgeries. After surgery, LEwere subcutaneously injected into the rats in Groups 3 to 5.At 7th day after surgery, all rats were subjected to bilateral mandibular first molars pulp exposure.After pulpal exposure, FSH, LH, LE and vehicle were subcutaneously injected in the back of rats daily as designed.On the 0th,7th,14th and 21th day after pulpal exposure, the venous blood samples were collected and the serum was separated and acquired to test E2, FSH and LH levels.On the 21th day after pulpal exposure, bilateral mandibles were harvested for X-ray imaginghistological observation. The areas of periapical lesions were double-blinded measured by IMAGE PRO PLUS software, v6.0.Results:From 7 days after ovariectomy, compared to SHAM group, levels of E2 in serum of all OVX groups were decreased significantly and reached stable about one fourth of that in SHAM group (p<0.01), which had no statistical differences among all OVX groups (p>0.05). Levels of FSH and LH in serum were increased significantly than SHAM group after ovariectomy, which were prevented after application of LE (p<0.05). Levels of FSH were increased sharply after application in FSH-treated group, which were about ten times higher than that in OVX group (p<0.05). Level of LH in the OVX+LE+LH group were increased sharply and reached stable from third week after administration of LH (p<0.05). Compared to those in SHAM group, specimens in OVX groups showed a significant increase in bone loss of periapical lesions (p<0.05), which were reversed by administration of LE or LE+LH (p>0.05). Compared to those in SHAM and OVX groups, bone loss of periapical lesions were significantly increased after administration of FSH (p<0.05). ConclusionsrFSH accelerated the progression of induced experimental periapical lesions.EXPERIMENT ? The mechanism of FSH in pathologic process of OVX rat periapical periodontitisObjective:The purpose of the present study was to investigate the expression of FSHR in rat periapical lesions and its relationship with osteoclasts, TNFa, IL-1?and RANKL/OPG.Methods:Ninety Twelve-week-old female Sprague-Dawley rats were randomly assigned into the following six groups with 15 rats each:(1) sham surgery+vehicle, (2) bilateral OVX+vehicle, (3) bilateral OVX+1.6 mg/kg LE, (4) bilateral OVX+ 1.6 mg/kg LE+1.85 ?g/kg LH, (5) bilateral OVX+1.6 mg/kg LE+3?pg/kg FSH and (6) bilateral OVX+3 ?g/kg FSH. Periapical lesions were induced and rats were sacrificed on day 7,14,21 and their mandibles were harvested for enzyme histo-chemistry and immunohistochemistry analysis (TNF?,IL-1? and RANKL/OPG)Results:On the 7th,14th and 21th day after pulpal exposure, the numbers of osteoclasts and FSHR, RANKL, TNF-a and IL-1?-positive cells in OVX-group were higher than that of SHAM group (p<0.05). In the FSH-treated groups, the number of the numbers of osteoclasts and FSHR, RANKL, TNF-a and IL-1?-positive cells were increased significantly than those in SHAM, OVX or OVX+LE groups (p<0.05). There was no difference between OVX+LE and OVX+LE+LH groups (p>0.05).There were no significant differences found between FSH-treated groups and corresponding groups in number of OPG-positive cells.Conclusions:FSH accelerated the progression of induced experimental periapical lesions, which might be mediated by increasing secretion of osteoresorptive cytokines (TNF-a and IL-1?) and altered RANKL/OPG ratios.[Keywords] follicle-stimulating hormone, leuprorelin, periapical lesion, ovariectomyPART II Study on the Expression and Function of FSHR in OralSquamous Cell CarcinomaOral squamous cell carcinoma (OSCC) is one of the ten most frequently diagnosed cancers in the world, and is the most common malignant tumor in head and neck. The prognosis of OSCC is not well due to the relatively limited scope of surgery and the invasive, metastatic and recurrent biological characters of tumor. So it is particularly important to explore the mechanisms of malignant phenotype, and to find the effective blocking target for clinical therapy.FSHR is a glycosylated transmembrane protein that belongs to the G protein-coupled receptor family. Upon binding follicle stimulating hormone (FSH), FSHR transduces signal primarily via the adenylyl cyclase-cAMP-protein kinase A pathway, patients with EOCs expressing the FSHR, but not the LHR, had a poorer prognosis, whereas patients whose tumours expressed the LHR, but not the FSHR, had overall better survival.Targeting FSHR may be of particular interest in CaP, where FSHR may also be expressed by CaP cells. The follicle-stimulating hormone receptor (FSHR) was recently found to be expressed in the vascular endothelium of a wide range of solid tumors. A variety of characteristics suggest that FSHR in the neovasculature is a promising novel target for cancer therapy and imaging. There is reason for optimism, but much work remains to fully exploit this exciting new target for cancer.However, to our knowledge a comparative analysis of oral normal mucosa and OSCC FSHRexpression in patients has not been performed. In this study, we investigated FSHR expression in OSCC tissues using IHC, analyzed the relationship of them with clinicalpathological features and cancer-related survival of patients, and explored the association between them.Section I Expression of FSHR in OralSquamous Cell Carcinoma and its clinical significanceObjectivesTo explore FSHR expression on OSCC tissues and its clinical significance.Materials and methodsRetrospective cohort study comprised 100 OSCC cases diagnosed at the Department of Pathology, Wuhan University Stomatological Hospital Between December 2001 and February 2009.Following institutional review board approval, the histological slides were retrieved and whole sections reviewed. Representative areas were selected and tissue microarrays were constructed.Each case includes adjacent normal mucosa and tumor region. Immunohistochemistry was used to detect FSHR xpression on tumor cells. Histoscores were achieved using Imagescope. The average expression intensity in each group was then subjected to statistical analysis with one-way analysis of variance(P<0.05).ResultsIHC results showed that FSHR protein expressed mainly oncell membrane and cytoplasm of tumor cells and adjacent normal oral mucosa. The expression of FSHR was obviously stronger in OSCC tissuesthanin normal oral mucosa tissues (P<0.05). While there were no correlation between FSHR expression with patients' gender, age, grade, and tumor size. The expression level of FSHR in OSCC samples with metastasis was stronger than those without metastasis. Overallsurvival analysis revealed that overexpression of FSHR in OSCC tissueswas associated with shorter cancer-related survival. (P<0.05)ConclutionThere were expressions of FSHR protein inOSCC tissues. The expression level of FSHR in OSCC samples with metastasis was stronger than those without metastasis. Overexpression of FSHRin tumor region was associated with poor clinical outcomes and shorter cancer-related survival.Key words:Oral squamous cell carcinoma; FSHR;Metastasis; PrognosisSection II The effect of FSHR overexpression on the biological behavior of the oral squamous cell carcinomacellsObjective1.To detect the effect of FSHR overexpression on proliferation of scc25 and scc9 cells;2.To detect the effect of FSHR overexpression on invasion and migration of scc25 and scc9 cells.Materials and methodsThe cDNA sequence of FSHR was obtained by PCR. The vector construction and virus packaging was finished.Lentiviral vector was then co-transfected into 293T cells with packaging plasmids. The lentiviruses carrying FSHR were then harvested and used to transfect oral squamous cell lines:scc25 and scc9. After 48hs, assay the cells by flowcytometry. Following transduction, expression of FSHR in these cells was detected using real time PCR analysis. CCK8 was used to detect cell proliferation in LV-FSHR, LV-vector and Blank groups.A cell invasion assay was carried out using Transwell cell culture chambersin LV-FSHR, LV-vector and Blank groups.Nude Mouse Model was established to identify the invasive capacity of FSHR overexpression OSCC cells. And real time PCR on FSHR relative oncogens (FOSB, IL-6, IL-8, NFKB2, MMP2, MMP14, TGFB2)was performed to explore potential mechanism.Results1.Transfection of FSHR overexpression Lenti-virus vector could enhance the expression level of FSHR both in mRNA level, using RT-PCR analysis.2.CCK8 result showed that proliferative activity among the LV-FSHR,LV-vector and Blank groups had no obvious difference.3.Transwell invasion assay results showed that the count of the invaded cells was obviously higher in LV-FSHR group than in LV-vector and Blank groups (P<0.05). There was no significant difference between LV-vector and Blank groups.Nude mousemodel showed that scc25+FSHR cells could form more and largertumornodules in lung.4. FSHR ovexpression may result in IL6 and IL8 upregulated.Conclusion1.Transfection of FSHR overexpression Lenti-virus vector could obviously enhance the expression level of FSHR in mRNA level.2.Overexpression of FSHR in scc25 and scc9 cells had no effects on cell proliferation.3.Overexpression of FSHR in scc25 and scc9 cells could enhance the ability of cell invasion.4.FSHR may promote invasion of OSCC cells by up-regulation of IL6 and IL8.