27-Hydroxycholesterol contributes to hypercholesterolemia-associated aggravation of apical periodontitis in ovariectomized rats and raloxifene counteracts its action.

AIM: The influence of hypercholesterolemia on the development of apical periodontitis (AP) is inconclusive. Recent studies revealed that cholesterol metabolite 27-hydoxycholesterol (27HC) can affect cellular responses to bacterial infections and oestrogen status and raloxifene may influence its action. Herein, we aimed to examine the impact of 27HC on production of inflammatory mediators by macrophages and the regulatory function of raloxifene. The contribution of 27HC to AP development and the therapeutic effect of raloxifene were evaluated in a rat model. METHODS: Murine macrophages J774 cells were used. The expression of inducible nitric oxide synthase (iNOS) was examined by Western blot. The concentrations of C-C motif chemokine ligand (CCL) 2 and 27HC were assessed by enzyme-linked immunosorbent assay. Colorimetric assay was used to evaluate cholesterol levels. Experimental AP was induced in ovariectomized (OVX) or un-operated rats receiving high-fat/high-cholesterol diet (HFHCD) or normal diet (ND). Micro-computed tomography and immunohistochemistry were employed to evaluate disease severity and the therapeutic effect of raloxifene. RESULTS: Cholesterol enhanced 27HC production in macrophages. 27HC induced iNOS and CCL2 synthesis by macrophages and estradiol suppressed the responses. In our animal model of AP, HFHCD plus OVX significantly augmented serum and lesion tissue levels of 27HC (p < .05 versus the ND group). Lesion size, infiltration of CD68+ cells, and iNOS+ monocytes were increased in parallel with 27HC accumulation. Raloxifene inhibited pro-inflammatory effects of 27HC on macrophages and suppressed AP progression in HFHCD/OVX rats (p < .05 versus the vehicle control group). CONCLUSIONS: Our results suggested that 27HC contributes to AP aggravation associated with hypercholesterolemia. Oestrogen deficiency may both enhance 27HC production and exacerbate its downstream action.

The effects of E'Jiao on body composition, bone marrow adiposity and skeletal redox status in ovariectomised rats.

Background: Menopause is accompanied by increased oxidative stress, partly contributing to weight gain and bone marrow adiposity. Traditional Chinese medication, E'Jiao, has been demonstrated to reduce excessive bone remodelling during oestrogen deprivation, but its effects on body composition and bone marrow adiposity during menopause remain elusive. Objective: To determine the effects of E'Jiao on body composition, bone marrow adiposity and skeletal redox status in ovariectomised (OVX) rats. Methods: Seven groups of three-month-old female Sprague Dawley rats were established (n=6/group): baseline, sham, OVX control, OVX-treated with low, medium or high-dose E'Jiao (0.26, 0.53, 1.06 g/kg, p.o.) or calcium carbonate (1% in tap water, ad libitum). The supplementation was terminated after 8 weeks. Whole-body composition analysis was performed monthly using dual-energy X-ray absorptiometry. Analysis of bone-marrow adipocyte numbers and skeletal antioxidant activities were performed on the femur. Results: Increased total mass, lean mass, and bone marrow adipocyte number were observed in the OVX control versus the sham group. Low-dose E'Jiao supplementation counteracted these changes. Besides, E'Jiao at all doses increased skeletal catalase and superoxide dismutase activities but lowered glutathione levels in the OVX rats. Skeletal malondialdehyde level was not affected by ovariectomy but was lowered with E'Jiao supplementation. However, peroxisome proliferator-activated receptor gamma protein expression was not affected by ovariectomy or any treatment. Conclusion: E'Jiao, especially at the low dose, prevented body composition changes and bone marrow adiposity due to ovariectomy. These changes could be mediated by the antioxidant actions of E'Jiao. It has the potential to be used among postmenopausal women to avoid adiposity.

Self-emulsified annatto tocotrienol improves bone histomorphometric parameters in a rat model of oestrogen deficiency through suppression of skeletal sclerostin level and RANKL/OPG ratio.

Menopause is the leading cause of osteoporosis for elderly women due to imbalanced bone remodelling in the absence of oestrogen. The ability of tocotrienol in reversing established bone loss due to oestrogen deficiency remains unclear despite the plenitude of evidence showcasing its preventive effects. This study aimed to investigate the effects of self-emulsified annatto tocotrienol (SEAT) on bone histomorphometry and remodelling in ovariectomised rats. Female Sprague Dawley rats (n=36) were randomly assigned into baseline, sham, ovariectomised (OVX) control, OVX-treated with annatto tocotrienol (AT) (60 mg/kg), SEAT (60 mg/kg) and raloxifene (1 mg/kg). Daily treatment given through oral gavage was started two months after castration. The rats were euthanised after eight weeks of treatment. Blood was collected for bone biomarkers. Femur and lumbar bones were collected for histomorphometry and remodelling markers. The results showed that AT and SEAT improved osteoblast numbers and trabecular mineralisation rate (p<0.05 vs untreated OVX). AT also decreased skeletal sclerostin expression in OVX rats (p<0.05 vs untreated OVX). Similar effects were observed in the raloxifene-treated group. Only SEAT significantly increased bone formation rate and reduced RANKL/OPG ratio (p<0.05 vs untreated OVX). However, no changes in osteoclast-related parameters were observed among the groups (p>0.05). In conclusion, SEAT exerts potential skeletal anabolic properties by increasing bone formation, suppressing sclerostin expression and reducing RANKL/OPG ratio in rats with oestrogen deficiency.

Oestrogen-deficiency induces bone loss by modulating CD14+ monocyte and CD4+ T cell DR3 expression and serum TL1A levels.

BACKGROUND: Oestrogen-deficiency induced by menopause is associated with reduced bone density and primary osteoporosis, resulting in an increased risk of fracture. While the exact etiology of menopause-induced primary osteoporotic bone loss is not fully known, members of the tumour necrosis factor super family (TNFSF) are known to play a role. Recent studies have revealed that the TNFSF members death receptor 3 (DR3) and one of its ligands, TNF-like protein 1A (TL1A) have a key role in secondary osteoporosis; enhancing CD14+ peripheral blood mononuclear cell (PBMC) osteoclast formation and bone resorption. Whether DR3 and TL1A contribute towards bone loss in menopause-induced primary osteoporosis however, remains unknown. METHODS: To investigate this we performed flow cytometry analysis of DR3 expression on CD14+ PBMCs isolated from pre- and early post-menopausal females and late post-menopausal osteoporotic patients. Serum levels of TL1A, CCL3 and total MMP-9 were measured by ELISA. In vitro osteoclast differentiation assays were performed to determine CD14+ monocyte osteoclastogenic potential. In addition, splenic CD4+ T cell DR3 expression was investigated 1 week and 8 weeks post-surgery, using the murine ovariectomy model. RESULTS: In contrast to pre-menopausal females, CD14+ monocytes isolated from post-menopausal females were unable to induce DR3 expression. Serum TL1A levels were decreased approx. 2-fold in early post-menopausal females compared to pre-menopausal controls and post-menopausal osteoporotic females; no difference was observed between pre-menopausal and late post-menopausal osteoporotic females. Analysis of in vitro CD14+ monocyte osteoclastogenic potential revealed no significant difference between the post-menopausal and post-menopausal osteoporotic cohorts. Interestingly, in the murine ovariectomy model splenic CD4+ T cell DR3 expression was significantly increased at 1 week but not 8 weeks post-surgery when compared to the sham control. CONCLUSION: Our results reveals for the first time that loss of oestrogen has a significant effect on DR3; decreasing expression on CD14+ monocytes and increasing expression on CD4+ T cells. These data suggest that while oestrogen-deficiency induced changes in DR3 expression do not affect late post-menopausal bone loss they could potentially have an indirect role in early menopausal bone loss through the modulation of T cell activity.

Influence of oestrogen deficiency on the development of apical periodontitis.

AIM: To evaluate the effects of a long period of oestrogen deficiency on the development of apical periodontitis in rats. METHODOLOGY: Wistar rats (n = 24), 3 months old, evaluated by vaginal cytology, were included in the study. Twelve animals were ovariectomized (OVX group) and the other 12 were sham operated (control group). One hundred and twenty days after castration, the pulps of the left mandibular first molars were exposed to induce the development of apical periodontitis. Body mass was verified on a weekly basis. Following 21 and 40 days of lesion induction, the animals were sacrificed. Blood was collected for biochemical analysis, and mandibles were removed for radiographic analysis. Comparative analysis of the data was performed by the nonparametric Kruskal-Wallis and Dunn's multiple-comparisons tests. The t-test was applied to compare the oestrogen levels between control and OVX groups. RESULTS: Radiographs revealed that apical periodontitis lesions were significantly larger in the 40-day OVX group when compared with both 40-day (P < 0.05) and 21-day (P < 0.001) control groups. Serum oestrogen levels were significantly lower in the OVX group (P < 0.01), confirming the efficacy of castration. Oestrogen deficiency resulted in significantly greater body mass gain (P < 0.01) in 40-day OVX group when compared with 40-day control group. Serum concentrations of calcium were similar between groups (P > 0.05). Alkaline phosphatase levels, although higher in the OVX groups (21 and 40 days), were not significantly different. CONCLUSIONS: Ovariectomized rats had significantly larger apical periodontitis lesions after 40 days of pulp exposure when compared with controls. These findings suggest that bone alterations as a result of long periods of oestrogen deficiency can influence the progression of apical periodontitis.

An in silico approach to elucidate the pathways leading to primary osteoporosis: age-related vs. postmenopausal.

Numerical models of bone remodelling have traditionally been used to perform in silico tests of bone loss in postmenopausal women and also to simulate the response to different drug treatments. These models simulate the menopausal oestrogen decline by altering certain signalling pathways. However, they do not consider the simultaneous effect that ageing can have on cell function and bone remodelling, and thus on bone loss. Considering ageing and oestrogen decline together is important for designing osteoporosis treatments that can selectively counteract one or the other disease mechanism. A previously developed bone cell population model was adapted to consider the effect of ageing through: (1) the decrease of TGF- ß contained in the bone matrix and (2) an increased production of sclerostin by non-skeletal cells. Oestrogen deficiency is simulated in three different ways: (a) an increase in RANKL expression, (b) a decrease in OPG production, and (c) an increase in the responsiveness of osteoclasts to RANKL. The effect of ageing was validated using the cross-sectional study of (Riggs et al. in J Bone Miner Res 19: 1945-1954, 2004) on BMD of trabecular bone of the vertebral body of men. The joint effect of ageing and oestrogen deficiency was validated using these same clinical results but in women. In ageing, the effect of the increasing production of sclerostin is more important than the decrease of TGF- ß , while the three mechanisms used to simulate the effect of oestrogen deficiency produce almost identical responses. The results show that an early menopause leads to a lower average density in the fifth decade, but after the sixth decade the average density is independent of the age at menopause. Treatment of osteoporosis with denosumab was also simulated to conclude that the drug is not very effective if started before 10 years after menopause or before age 60.

Immune regulation of bone loss by Th17 cells in oestrogen-deficient osteoporosis.

BACKGROUND: The role of T helper (Th) 17 cells in autoimmune diseases has been extensively studied recently, but its function in oestrogen-deficient osteoporosis remains undefined. This review aimed to explore the role of Th17 cells in regulating bone loss induced by oestrogen deficiency. MATERIALS AND METHODS: We searched PubMed, Embase, Google Scholar and Biosis up to 1 February 2013 for immune regulation of oestrogen-deficient osteoporosis by Th17 cells. Terms relevant to immunity, oestrogen deficiency, osteoporosis and Th17 cells were used for database searching. Seventy-five papers met the predetermined searching criteria. RESULTS: Studies indicate Th17 lineage to be a potent osteoclastogenic mediator in oestrogen-deficient osteoporosis. Oestrogen deficiency promotes osteoclastogenesis by up-regulating Th17 cell populations in bone marrow and IL-17 levels in peripheral blood. Meanwhile, transcription factors involved in Th17 cell differentiation, such as RORγt and RORα, are highly expressed in ovariectomized animals. Treatment with IL-17 significantly promotes production of RANKL, TNF and IL-6 as well as TRAP(+) cells in culture; blocking IL-17 pathway significantly reduces abundance of Th17 cells and effectively prevents bone loss in ovariectomized mice. CONCLUSIONS: The main body of literatures suggests Th17 to be a critical modulator in the pathogenesis of oestrogen-deficient osteoporosis, which supports the notion that oestrogen-deficient osteoporosis is a complex interplay between oestrogen, osteoclastogenic cytokines and osteoclasts. In addition, therapeutic strategies targeting IL-17 networks may be clinically useful in treatment for postmenopausal osteoporosis.

Extracellular vesicles and high-density lipoproteins: Exercise and oestrogen-responsive small RNA carriers.

Decreased systemic oestrogen levels (i.e., menopause) affect metabolic health. However, the detailed mechanisms underlying this process remain unclear. Both oestrogens and exercise have been shown to improve metabolic health, which may be partly mediated by circulating microRNA (c-miR) signalling. In recent years, extracellular vesicles (EV) have increased interest in the field of tissue crosstalk. However, in many studies on EV-carried miRs, the co-isolation of high-density lipoprotein (HDL) particles with EVs has not been considered, potentially affecting the results. Here, we demonstrate that EV and HDL particles have distinct small RNA (sRNA) content, including both host and nonhost sRNAs. Exercise caused an acute increase in relative miR abundancy in EVs, whereas in HDL particles, it caused an increase in transfer RNA-derived sRNA. Furthermore, we demonstrate that oestrogen-based hormonal therapy (HT) allows the acute exercise-induced miR-response to occur in both EV and HDL particles in postmenopausal women, while the response was absent in nonusers.

Chronic oestrogen deficiency induced by ovariectomy may cause lung fibrosis through activation of the renin-angiotensin system in rats.

CONTEXT: Oestrogen deficiency is linked with pulmonary fibrosis. Additionally, it may lead to over-activation of the renin-angiotensin system (RAS), which worsens lung fibrosis. OBJECTIVE: The present study aims to investigate the role of RAS on lung fibrosis associated with oestrogen deficiency in ovariectomised rats. MATERIALS AND METHODS: Serum 17ß-oestradiol (E2), arterial blood gases, plasma angiotensin II levels, lung tissue hydroxyproline content, and transforming growth factor beta 1 (TGF-ß1) concentration, the mRNA expression of angiotensin type 1 receptor (AT1R), and angiotensin-converting enzyme (ACE1) were evaluated. Moreover, lung tissues were examined by histopathology and immunohistochemistry. RESULTS: Hydroxyproline content, TGF-ß1 concentration, plasma angiotensin II, the relative mRNA expression of ACE1, and AT1R is found to increase in ovariectomised rats. The mentioned changes can be largely rescued by administration of RAS blockers. CONCLUSION: Oestrogen deficiency activates RAS, which consequently increases the expression of pro-fibrotic factors and stimulates the fibrotic cascade causing lung fibrosis.

Probiotics ameliorate alveolar bone loss by regulating gut microbiota.

OBJECTIVES: Oestrogen deficiency is an aetiological factor of postmenopausal osteoporosis (PMO), which not only decreases bone density in vertebrae and long bone but also aggravates inflammatory alveolar bone loss. Recent evidence has suggested the critical role of gut microbiota in osteoimmunology and its influence on bone metabolisms. The present study aimed to evaluate the therapeutic effects of probiotics on alveolar bone loss under oestrogen-deficient condition. MATERIALS AND METHODS: Inflammatory alveolar bone loss was established in ovariectomized (OVX) rats, and rats were daily intragastrically administered with probiotics until sacrifice. Gut microbiota composition, intestinal permeability, systemic immune status and alveolar bone loss were assessed to reveal the underlying correlation between gut microbiota and bone metabolisms. RESULTS: We found administration of probiotics significantly prevented inflammatory alveolar bone resorption in OVX rats. By enriching butyrate-producing genera and enhancing gut butyrate production, probiotics improved intestinal barrier and decreased gut permeability in the OVX rats. Furthermore, the oestrogen deprivation-induced inflammatory responses were suppressed in probiotics-treated OVX rats, as reflected by reduced serum levels of inflammatory cytokines and a balanced distribution of CD4+ IL-17A+ Th17 cells and CD4+ CD25+ Foxp3+ Treg cells in the bone marrow. CONCLUSIONS: This study demonstrated that probiotics can effectively attenuate alveolar bone loss by modulating gut microbiota and further regulating osteoimmune response and thus represent a promising adjuvant in the treatment of alveolar bone loss under oestrogen deficiency.

The Effect of Menopause Hypoestrogenism on Osteogenic Differentiation of Periodontal Ligament Cells (PDLC) and Stem Cells (PDLCs): A Systematic Review.

(1) Background: Menopause is a physiological condition typified by drastic hormonal changes, and the effects of this transition have long-term significant clinical implications on the general health, including symptoms or physical changes. In menopausal women, the periodontium can be affected directly or through neural mechanism by oestrogen (E2) deficiency. The majority of the biological effects of E2 are modulated via both oestrogen receptor-α (ERα) and oestrogen receptor- ß (ERß). There is evidence that hypoestrogenism has a substantial impact on the aetiology, manifestation and severity of periodontitis, via the regulation of the expression of osteoprogesterin and RANKL in human periodontal ligament cells through ERß. However, the mechanistic understanding of oestrogen in periodontal status has been partially clarified. The aim of this paper was to synopsize the recent scientific evidence concerning the link between the menopause and periodontitis, through the investigation of physio-pathological impact of the oestrogen deficiency on osteogenic differentiation of PDLSCs and PDLSC, as well as the dynamic change of ERα and ERß. (2) Methods: Search was conducted for significant studies by exploring electronic PubMed and EMBASE databases, and it was independently performed by two researchers. All studies on the impact of oestrogen level on alveolar bone resorption were searched from 2005 to July 2020. Data selection was in concordance with PRISMA guidelines. (3) Results: Eight studies met the criteria and were included in this systematic review. All studies reported that oestrogen deficiency impairs the osteogenic and osteoblastic differentiation of PDL cells and oestrogen affects the bone formation capacity of cells. Seven studies were conducted on animal samples, divided into two groups: the OVX animals and animals who received the sham operation. (4) Conclusions: There is a multitude of data available showing the influence of menopause on periodontal status. However, the evidence of this line to investigation needs more research and could help explain the physiological linkage between menopause state and periodontal disease.

Management of urethral caruncle - A systematic review of the current literature.

Urethral caruncle is a benign fleshy outgrowth at the urethral meatus that occurs mainly at posterior lip of urethra and is most commonly seen in postmenopausal women. They are typically asymptomatic and are mostly reported as incidental findings during pelvic examination. When symptomatic, they commonly present with bleeding, haematuria, pain, dysuria or a lump. Despite being a recognised condition in medicine for more than two centuries, urethral caruncle is still a very poorly understood condition and the current literature is largely deficient to guide the general practitioners and gynaecologists in the appropriate management of the condition. The aim of the paper is to systematically review the current literature on the management options available for urethral caruncle and highlight the need for high quality research to develop better management strategies for this condition. We searched the following electronic databases from 1946 until January 2019: MEDLINE, EMBASE, COCHRANE and WEB OF SCIENCE. The search identified a total of 41 articles on the treatment of the condition in pre- and postmenopausal women, out of which only eight studies were relevant for our review. Most of these studies were retrospective case series or small cohort studies, mainly on the surgical treatment of the lesion. We found no systematic studies in the current literature on the conservative management of urethral caruncles. The most common method of surgical treatment encountered in these studies was simple excision. In conclusion, the current literature is largely deficient to guide us in the appropriate management of this common condition and there is need for more robust studies to develop evidence based management of this distressing condition that can guide the general practitioners and gynaecologists.

Sargassum integerrimum inhibits oestrogen deficiency and hyperlipidaemia-induced bone loss by upregulating nuclear factor (erythroid-derived 2)-like 2 in female rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Oestrogen deficiency, high incidences of hyperlipidaemia (HLP) and accelerated bone loss frequently occur in postmenopausal women. There is an urgent need to develop functional foods or specific drugs to protect against bone loss induced by oestrogen deficiency with HLP. AIM OF THE STUDY: In this study, we investigated the potential inhibitory effects of Sargassum integerrimum (SI) on bone loss in an ovariectomized rat model with HLP. MATERIALS AND METHODS: The rats were treated for 12 weeks, and then, bone mineral density, bone biomechanical, bone microstructure, bone morphology, biomarkers of HLP oxidative stress and side effects were determined. Immunohistochemical staining and Western blot were performed to evaluate related protein expression. RESULTS: The femur bone mineral density increased (P < 0.05), and the microscopic structures (ratio of bone volume to total volume [BV/TV], connectivity density [Conn.D], trabecular number [Tb.N] and trabecular thickness [Tb.Th]) of the bone trabecula and mechanical properties (maximum and breaking load [ML and BL, respectively]) improved after SI treatment (P < 0.05). Furthermore, the levels of HLP biomarkers (total cholesterol, triglyceride and low-density lipoprotein) were significantly decreased (P < 0.05), whereas the levels of antioxidant markers (superoxide dismutase and total antioxidant capacity) were increased (P < 0.05). Similar results were obtained with immunohistochemical staining, whereas the Western blot assay showed that SI stimulated the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in bone. CONCLUSION: Our data indicate that rats exposed to SI treatment for 12 weeks did not exhibit noticeable side effects. In conclusion, SI suppressed bone loss induced by ovariectomized and the associated HLP in rats by activating Nrf2, which could be a promising treatment option for osteoporosis induced by oestrogen deficiency and HLP in postmenopausal women. TRANSLATIONAL SCOPE STATEMENT: Our study verified that SI prevented bone loss in rats with oestrogen deficiency with HLP by upregulating nuclear factor (erythroid-derived 2)-like 2. Furthermore, no side effect was observed after the long-term administration of SI. Those results suggested SI could be developed as a functional food or drug for postmenopausal osteoporosis induced by oestrogen deficiency with HLP.