OVX Group Research Articles

Morin Improves the Bone Histomorphology and Biochemical Markers in an Animal Model of Ovariectomy‐Induced Osteoporosis by Suppressing Autophagy and Apoptosis

ABSTRACTOsteoporosis (OP) is the most prevalent metabolic bone disease and an important postmenopausal consequence. This study aimed to investigate the effects of morin, a flavonoid with beneficial properties, on ovariectomy‐induced OP. Animals were ovariectomized (OVX) and treated with different doses of morin (15, 30, and 45 mg/kg/day) or estradiol (10 μg/kg/day) for 10 weeks by gavage. Then bone histo‐stereology, bone‐related biochemical indicators, and gene and protein levels of autophagy and apoptosis‐related markers were analyzed. In comparison to controls, OVX significantly decreased the number of osteoblasts (5.78 × 106 vs. 1.66 × 106) and osteocytes (32.55 × 106 vs. 11.92 × 106), whereas increasing the number of osteoclasts (83.38 × 103 vs. 392.1 × 103). Moreover, OVX caused a remarkable decrease in bone structures and Ca, P, and estradiol levels while increasing ALP and OC (p < 0.001). The administration of 45 mg/kg/day morin restored the effects of OP on bone histomorphology and biochemical markers (p < 0.05). Further studies revealed that morin caused a 7.1% and 36.6% decrease in the bone level of LC3 and BECN1 proteins, respectively, compared to the OVX group. Also, morin caused a significant decrease of 47.4% in the CASP3 level and a significant increase of 23.6% in the BCL‐2 level compared to OVX animals (p < 0.001). The present findings showed that morin is potentially able to improve the bone‐related histomorphological and biochemical changes caused by osteoporosis, which is probably attributed to the suppression of apoptosis‐ and autophagy‐caused cell death.

Ramadan fasting and exercise combination therapy: A novel approach for osteoporosis prevention in ovariectomized rats

BackgroundOsteoporosis is a chronic bone metabolic disease that affects millions of people worldwide, particularly the elderly and postmenopausal women. It is characterized by weakened bones, increasing the risk of fractures and leading to significant morbidity and mortality. The goal of the current study is to examine the reported osteo-preservative effects of exercise and/or fasting in the Ramadan fasting model (RFM) in ovariectomized (OVX) rats.MethodsThe experimental intervention started 1 month following the ovariectomy procedure and consisted of five 15-min exercise sessions per week at 18–25 m/min and/or an approximately 13-h fast from sunrise to sunset (6:00 AM–19:00 PM). Serum bone metabolism biomarker levels were measured, and mineral concentrations in femoral ashed bones and digested serum were determined. Additionally, serum bone alkaline phosphatase (b-ALP), parathyroid hormone, osteocalcin, calcitonin, and vitamin D3 concentrations were measured using the competitive enzyme immunoassay technique.ResultsCalcium, magnesium, and phosphorus showed a notable decrease in mineral concentration among OVX rat femurs compared with the combination group (OVX + RFM + E) and control groups. In addition, homeostasis of serum concentrations of calcium, magnesium, and phosphorus was observed to increase in the OVX + RFM + E group rather than in the OVX group without intervention when compared with a control group. Furthermore, fasting and exercise, either alone or concurrently with ovariectomy, induced a non-significant elevation in osteocalcin, parathyroid hormone, and vitamin D3, whereas b-ALP and calcitonin increased significantly compared with those in control rats.ConclusionThe combination of the Ramadan fasting model and moderate intensity exercises among OVX rats manifested advantageous effects in bone biomarkers compared with OVX rats without intervention. This could be recommended as a lifestyle modification that is protective against osteoporosis, especially in the context of depleted estrogen hormone after menopause.

Bone-targeted ultrasound-responsive nanobubbles for siRNA delivery to treat osteoporosis in mice

This project aimed to study the efficacy of a bone-targeted ultrasound-responsive nanobubble (NB) platform to deliver gene-silencing cathepsin K (CTSK) siRNA into the bone for osteoporosis treatment using in vitro and in vivo studies. To this end, characterization of CTSK siRNA loaded NB functionalized with alendronate (NB-CTSK siRNA-AL) was performed using transmission electron microscopy (TEM) imaging, and a release profile was obtained through fluorescent spectroscopy. In vitro studies were conducted by culturing NB-CTSK siRNA-AL with osteoclasts to evaluate siRNA uptake, CTSK expression, and the expression of tartrate-resistant acid phosphatase (TRAP). A control group and an NB-CTSK siRNA-AL treated group of ovariectomized (OVX) mice (n = 4) were tested. The OVX group that received treatment underwent weekly sessions for 4 weeks, during which they were exposed to low-intensity pulsed ultrasound (LIPUS) stimulation following administration of NB-CTSK siRNA-AL, prior to being sacrificed. Both groups underwent a series of tests to evaluate the bone targeting, safety, and efficacy of the nanoplatform. These tests included biodistribution studies conducted at 4 h and 24 h post-injection, a 3-point bending test of the femurs, nano-computed tomography analysis, as well as Hematoxylin & Eosin histological staining, Masson's Trichrome staining, and CTSK staining. The biodistribution showed the accumulation of NB-CTSK siRNA-AL in the bone and liver. Results showed that the OVX mice treated with NB-CTSK siRNA-AL had increased distal cortical bone thickness (174.4 ± 5.28 μm vs. 144.3 ± 10.66 μm, p > 0.05)) and bone volume fraction (16.5 ± 3.96 % vs. 6.55 ± 0.13 % (p > 0.05)). A reduced collagen degradation and downregulated CTSK expression were evident in the staining procedures. No adverse effects were recorded within histological assessments on the liver, kidney, and heart post-treatment. Morphology was shown to be normal and healthy within muscle cells post-LIPUS stimulation of NB-CTSK siRNA-AL. From these results, it can be concluded that an ultrasound-mediated NB-CTSK siRNA-AL can serve as a reliable, safe CTSK siRNA carrier to bone-specific targets for in vivo osteoporosis treatment.

Bio-Calcium from Skipjack Tuna Frame Attenuates Bone Loss in Ovariectomy-Induced Osteoporosis Rats.

Bio-calcium derived from fish frames may offer several advantages for osteoporosis prevention. This study aimed to evaluate the effects of bio-calcium derived from skipjack tuna frames on bone loss in ovariectomized rats. Tuna bio-calcium was prepared through enzymatic hydrolysis, defatting, bleaching, and grinding processes. The bioavailability of calcium was tested using the Caco-2 cell monolayer model, showing that 13% of tuna bio-calcium was absorbed, compared to 10% for calcium carbonate. Rats were divided into the five following groups: (1) OVX, (2) sham-operated, (3), OVX + estrogen-treated (4) OVX + calcium carbonate-treated, and (5) OVX + tuna bio-calcium-treated. All groups were raised for eight weeks. Tuna bio-calcium was able to increase BV/TV by 26% in the femur and 29% in the tibia, compared to 13% and 17% in the OVX group, respectively. Trabecular thickness in the femur upsurged to 360 µm in the tuna group, while a thickness of 290 µm was observed in the control. Additionally, osteoclast numbers were reduced to 5 N.Oc/mm in the femur and 6 N.Oc/mm in the tibia in the tuna group, compared to 35 and 45 N.Oc/mm in the control. Overall, tuna bio-calcium effectively prevented bone loss and can serve as a promising natural alternative for managing osteoporosis.

Dihydromyricetin ameliorate postmenopausal osteoporosis in ovariectomized mice: Integrative microbiomic and metabolomic analysis.

The gut microbiota may help mitigate bone loss linked to postmenopausal osteoporosis by affecting the immune and inflammatory responses and the gut-bone axis. Dihydromyricetin (DMY), a natural flavonoid, has some anti-inflammatory and antioxidant properties. This study aimed to investigate the mechanisms underlying the amelioration of bone loss in ovariectomized (OVX) mice treated with various doses of DMY. Eight-week-old C57/BL6 mice underwent ovariectomy and received varying DMY doses over 8 weeks. Thereafter, femoral bone microarchitecture, serum biomarker levels, and colon samples were analyzed to assess bone metabolism and inflammatory and hormonal responses. Fecal samples were subjected to 16S rDNA sequencing, and short-chain fatty acids were quantified. An untargeted metabolomics approach was applied to both serum and fecal samples to investigate alterations in the intestinal microbiota and metabolic profiles following DMY treatment in the OVX mice. The results show high-dose DMY has anti-osteoporotic effects. Compared to the OVX group, the DMY-treated group showed enhanced bone mineral density and reduced inflammation and colonic damage levels. The DMY treatment altered the gut microbiota composition, including the relative abundances at both the phylum and genus levels. In addition, DMY treatment increased the production of acetate and propionate. Metabolomic analysis revealed differential regulation of 37 and 70 metabolites in the serum and feces samples, respectively, in the DMY-treated group compared to those in the OVX group, affecting the serotonergic signaling, arachidonic acid metabolism, and unsaturated fatty acid biosynthesis pathways. In conclusion, these findings indicate that DMY can ameliorate bone loss in OVX mice via the gut-bone axis.

The effects of simvastatin on the bone microstructure and mechanics of ovariectomized mice: a micro-CT and micro-finite element analysis study

BackgroundOsteoporosis is a major health concern for postmenopausal women, and the effect of simvastatin (Sim) on bone metabolism is controversial. This study aimed to investigate the effect of simvastatin on the bone microstructure and bone mechanical properties in ovariectomized (OVX) mice.Methods24 female C57BL/6J mice (8-week-old) were randomly allocated into three groups including the OVX + Sim group, the OVX group and the control group. At 8 weeks after operation, the L4 vertebral bones were dissected completely for micro-Computed Tomography (micro-CT) scanning and micro-finite element analysis (µFEA). The differences between three groups were compared using ANOVA with a LSD correction, and the relationship between bone microstructure and mechanical properties was analyzed using linear regression.ResultsBone volume fraction, trabecular number, connectivity density and trabecular tissue mineral density in the OVX + Sim group were significantly higher than those in the OVX group (P < 0.05). For the mechanical properties detected via µFEA, the OVX + Sim group had lower total deformation, equivalent elastic strain and equivalent stress compared to the OVX group (P < 0.05). In the three groups, the mechanical parameters were significantly correlated with bone volume fraction and trabecular bone mineral density.ConclusionsThe findings suggested that simvastatin had a potential role in the treatment of osteoporosis. The results of this study could guide future research on simvastatin and support the development of simvastatin-based treatments to improve bone health.

The Effects of Rice Bran on Neuroinflammation and Gut Microbiota in Ovariectomized Mice Fed a Drink with Fructose.

Rice bran, which is abundant in dietary fiber and phytochemicals, provides multiple health benefits. Nonetheless, its effects on neuroinflammation and gut microbiota in postmenopausal conditions are still not well understood. This study investigated the effects of rice bran and/or tea seed oil supplementation in d-galactose-injected ovariectomized (OVX) old mice fed a fructose drink. The combination of d-galactose injection, ovariectomy, and fructose drink administration creates a comprehensive model that simulates aging in females under multiple metabolic stressors, including oxidative stress, estrogen deficiency, and high-sugar diets, and allows the study of their combined impact on metabolic disorders and related diseases. Eight-week-old and 6-8-month-old female C57BL/6 mice were used. The mice were divided into six groups: a sham + young mice, a sham + old mice, an OVX + soybean oil, an OVX + soybean oil with rice bran, an OVX + tea seed oil (TO), and an OVX + TO with rice bran diet group. The OVX groups were subcutaneously injected with d-galactose (100 mg/kg/day) and received a 15% (v/v) fructose drink. The rice bran and tea seed oil supplementation formed 10% of the diet (w/w). The results showed that the rice bran with TO diet increased the number of short-chain fatty acid (SCFA)-producing Clostridia and reduced the number of endotoxin-producing Tannerellaceae, which mitigated imbalances in the gut-liver-brain axis. Rice bran supplementation reduced the relative weight of the liver, levels of hepatic triglycerides and total cholesterol; aspartate transaminase and alanine aminotransferase activity; brain levels of proinflammatory cytokines, including interleukin-1β and tumor necrosis factor-α; and plasma 8-hydroxy-2-deoxyguanosine. This study concludes that rice bran inhibits hepatic fat accumulation, which mitigates peripheral metaflammation and oxidative damage and reduces neuroinflammation in the brain.

O08 TESTOSTERONE CAN INDUCE CARDIAC HYPERTROPHY IN OVARIECTOMIZED SPONTANEOUSLY HYPERTENSIVE RATS THROUGH THE ACTIVATION OF THE mTORC2/Atg9a

Objectives: The estrogen level of postmenopausal women decreased sharply, while the androgen level did not decrease significantly, which was characterized by the inversion of estrogen / androgen ratio. Therefore, it could be observed that relative excess androgen in the circulation of postmenopausal women, which may be one of the reasons for the increase of cardiovascular disease in postmenopausal women. In this study, the model of postmenopausal hypertension was established by bilateral ovariectomy in spontaneously hypertensive rats (SHR), and the specific mechanism of testosterone-induced myocardial hypertrophy in ovariectomized SHR was explored. Methods: 24 SHR were randomly divided into four groups: WKY group (n=6), sham operation group (Sham group, n=6), bilateral ovariectomized group (OVX group, n=6) and testosterone propionate intervention group (OVX+T group, 6 mg/kg/Weekly, im for 4 weeks, n=6). Western Blot was used to detect the protein expression of β-MHC, ANP, Androgen receptor, Akt, p-Akt, mTORC2 and its subunits Rictor and LC3 and mitophagy related proteins. Results: 1.The expression of β-MHC and ANP was significantly increased in the OVX and OVX+T groups when compared to the WKY and Sham groups. Additionally, the expression of androgen receptors was markedly upregulated in both the OVX and OVX+T groups. These findings strongly suggest that testosterone primarily induces myocardial hypertrophy in SHR after ovariectomy through androgen receptors. 2. Although no significant difference in mTORC2 expression was noted between the WKY, Sham, and OVX groups, testosterone exhibited a notable capacity to activate mTORC2 expression and the p-Rictor subunit in the OVX+T group. 3. The expression of LC3II/LC3I and DRP1 were increased in the OVX+T group when compared to the WKY, Sham and OVX groups. While the expression of MFN2 in the four groups shows no significant difference. Meanwhile, the expression of Atg9a was increased in OVX+T group. Conclusions: In bilateral ovariectomized SHR, testosterone has the potential to activate mitophagy by activating the mTORC2/Atg9a pathway, which, in turn, leads to cardiac hypertrophy.

Abstract P232: Estradiol Supplementation Prevents Ovariectomy-Induced Arterial Stiffness and Vascular Fibrosis

Background: Arterial stiffness is an independent predictor of cardiovascular events and mortality. Clinical data shows arterial stiffness increases rapidly after menopause, but mechanisms that mediate this change are not well known. The effect of ovariectomy (ovx) in rodent models is inconsistent in the literature. We hypothesize that the loss of estradiol (E2) contributes to ovx-induced arterial stiffening in a mouse model via fibrotic mechanisms, and that restoration of E2 will prevent these changes. Methods: Eight-week-old female C57/Bl6J mice received bilateral ovx or sham operation. Subcutaneous placebo or E2 (0.36 mg/day) pellets were implanted 2 weeks later to achieve three groups (sham+placebo, ovx+placebo, and ovx+E2). Aortic pulse wave velocity (PWV) was measured as an index of arterial stiffness. Blood pressure (BP) was measured by radiotelemetry. Aortic fibrosis was assessed by histological quantification of Masson’s trichrome staining and expressed as fold change in %fibrosis from sham. Pro-fibrotic genes were assessed in aortic tissue via quantitative PCR. Data presented are mean ± SEM. Results: Successful ovx was confirmed by reduction in uterine weight in the ovx group compared with other groups ( p &lt;0.0001). At 6-weeks-post-surgery (N=16-18/group), PWV was increased in ovx (5.2±0.2 mm/ms) versus sham (3.4±0.1 mm/ms, p &lt;0.0001) and ovx+E2 (3.5±0.1 mm/ms, p &lt;0.0001) groups. In biweekly measures (N=4-5/group), PWV was greater in the ovx group at 4- and 6-weeks-post-surgery compared with sham ( p &lt;0.0001) and ovx+E2 ( p &lt;0.0001) groups, while PWV in the ovx+E2 group did not differ from sham at any time ( p ≥0.6). At 6-weeks post-surgery (N=5/group), there were no observed differences between groups for dark or light cycle systolic or diastolic BP, mean arterial pressure, or pulse pressure. Aortic fibrosis was increased in the ovx group (1.6-fold) compared with sham ( p =0.03) and ovx+E2 (0.9-fold, p =0.02) groups (N=10-12/group). Aortic fibrosis correlated with PWV at 6-weeks-post-surgery (R 2 =0.25, p &lt;0.01). Aortic mRNA expression of collagen1a1 was increased in ovx versus ovx+E2 mice ( p =0.04, N=4-6/group). Conclusion: In conclusion, the restoration of E2 prevents ovx-driven arterial stiffening in female mice via a reduction in fibrotic remodeling of the aorta. The results also support that ovx is an appropriate model to study the mechanisms of menopause-induced arterial stiffening and that anti-fibrotic therapeutics may be beneficial in aging women.

Follicle-stimulating hormone promotes atrial fibrosis in menopausal women with atrial fibrillation

BackgroundPostmenopausal women with atrial fibrillation (AF) exhibit a higher level of atrial fibrosis and a higher recurrence rate after ablation compared with men. However, the underlying mechanism remains unclear. ObjectiveThe purpost of this study was to investigate the mechanism through which menopause promotes atrial fibrosis. MethodsIn a prospective cohort of women with AF, regression analyses were conducted to assess the relationship between low-voltage area (LVA) and sex hormone levels. CREM-IbΔC-X mice, a spontaneous AF model, underwent bilateral ovariectomy (OVX). Electrocardiograms, echocardiograms, and Masson staining were performed. Follicle-stimulating hormone (FSH) stimulation was applied in male mice for 3 months. OVX was also applied in an angiotensin II (Ang II)–induced pressure overload mouse model, after programmed electrical stimulation and structural analyses. Bulk RNA sequencing (RNA-seq) was performed to elucidate potential mechanisms. ResultsWomen demonstrated a significantly higher LVA burden than men (P < .001). A positive correlation was observed between LVA burden and FSH level (P = .002). Mice in the OVX group exhibited a significantly higher incidence of AF (P = .040) and atrial fibrosis (P = .021) compared with the Sham group, which could be attenuated by adeno-associated virus encoding small interfering RNA against Fshr. In male CREM-IbΔC-X mice, FSH stimulation promoted the occurrence of AF (P = .035) and atrial fibrosis (P = .002). In Ang II–induced female mice, OVX prompted atrial fibrosis, increased AF inducibility, and shortened atrial effective refractory period, which could be attenuated with knockdown of Fshr. RNA-seq indicated mitochondrial dysfunction. ConclusionPostmenopausal women exhibited a higher LVA burden than men, which was positively correlated with FSH level. FSH promoted atrial fibrosis through oxidative stress.

Relationship between autophagy and NLRP3 inflammasome during articular cartilage degradation in oestrogen-deficient rats with streptozotocin-induced diabetes

BackgroundEstrogen deficiency and Diabetes mellitus (DM) cause joint tissue deterioration, although the mechanisms are uncertain. This study evaluated the immunoexpression of autophagy and NLRP3-inflammasome markers, in rat articular cartilage with estrogen deficiency and DM. MethodsTwenty rats were sham-operated (SHAM) or ovariectomized (OVX) and equally allocated into four groups: SHAM and OVX groups administered with vehicle solution; SHAM and OVX groups treated with 60mg/kg/body weight of streptozotocin, intraperitoneally, to induce DM (SHAM-DM and OVX-DM groups). After seven weeks, the rats were euthanized, and their joint knees were processed for paraffin embedding. Sections were stained with haematoxylin-eosin, toluidine blue, safranin-O/fast-green or subjected to picrosirius-red-polarisation method; immunohistochemistry to detect beclin-1 and microtubule-associated protein 1B-light chain 3 (autophagy markers), NLRP3 and interleukin-1β (IL-1β) (inflammasome activation markers), along with matrix metalloproteinase-9 (MMP-9), Nuclear factor-kappa B (NFκB), and Vascular endothelial growth factor A (VEGF-A) were performed. ResultsDeterioration of articular cartilage and subchondral bone were greater in SHAM-DM and OVX-DM groups. Higher percentages of immunolabeled chondrocytes to NLRP3, IL-1β, MMP-9, NFκB, and VEGF-A, as well as lower percentages of chondrocytes immunolabeled to autophagy markers, were noticed in estrogen-deficient and diabetic groups. These differences were greater in the OVX-DM group. Percentages of immunolabeled chondrocytes showed negative correlation between autophagy markers v.s IL-1β, NLRP-3, MMP-9, NFκB, and VEGF-A, along with positive correlation between VEGF-A vs. MMP-9, NFκB, IL-1β, and NLRP3, and MMP-9 vs. NFκB. ConclusionsIn conclusion, autophagy reduction and NLRP3 inflammasome activation in chondrocytes may be implicated in articular cartilage degradation, under estrogen-deficient and DM conditions. Moreover, the combination of estrogen deficiency and DM may potentiate those effects.

Ovariectomy exacerbates the disturbance of excitation- inhibition balance in the brain of APP/PS-1/tau mice.

The prevalence of Alzheimer's disease (AD) is significantly gender-differentiated, with the number of female AD patients far exceeding that of males, accounting for two-thirds of the total prevalence. Although postmenopausal AD mice have been shown to have more prominent pathologic features and memory impairments than normal AD mice, the relevant molecular mechanisms leading to these outcomes have not been well elucidated. In the present study, we used the disturbance of excitation-inhibition balance in the postmenopausal brain as an entry point to explore the link between estrogen deficiency, disorders of the glutamatergic-GABAergic nervous system, and memory impairment. Wild-type (WT) mice and APP/PS1/tau (3 × Tg-AD) mice (10 months old) were randomly divided into four groups: WT+Sham group, WT+OVX group, 3 × Tg-AD+Sham group and 3 × Tg-AD+OVX group. Ovariectomy (OVX) was performed in the WT+OVX group and the 3 × Tg-AD+OVX group, and sham surgery was performed in the WT+Sham group and the 3 × Tg-AD+Sham group. The learning and memory ability and the anxiety and depression-like behavior changes of mice were evaluated by behavioral experiments, and the association between estrogen-estrogen receptors pathway and glutamatergic/GABAergic nervous system and female AD was evaluated by neurochemical experiments. In WT and 3 × Tg-AD mice, OVX resulted in impaired learning and memory abilities and anxiety and depression-like behaviors; reduced estrogen levels and downregulated the expression of estrogen receptors; upregulated the expression of amyloid-β, amyloid precursor protein, presenilin 1, and p-tau; upregulated the expression of Bcl-2-associated X protein and downregulated the expression of B-cell lymphoma-2, promoting cell apoptosis; reduced the number of neuronal dendrites and downregulated the expression of postsynaptic density protein-95; more importantly, OVX increased brain glutamate levels but downregulated the expression of N-methyl-D-aspartate receptor-2B, excitatory amino acid transporter 1, excitatory amino acid transporter 2, γ-aminobutyric acid receptor-A and γ-aminobutyric acid receptor-B. Our results suggested that OVX-induced estrogen-estrogen receptors pathway disruption caused learning and memory impairment and anxiety and depression-like behaviors, upregulated the expression of AD pathological markers, promoted apoptosis, destroyed neuronal structure, and most importantly, caused glutamatergic/GABAergic nervous system disorders.

Ovariectomy and Estradiol Supplementation Prevents Cyclophosphamide- and Doxorubicin-Induced Spatial Memory Impairment in Tumor-Bearing MMTV-PyVT Mice.

Chemotherapy-related cognitive impairments (CRCIs) encompass cognitive deficits in memory, attention, and executive function that arise during and following chemotherapy. CRCI symptoms are predominantly reported by female cancer patients but also occur in males. These impairments may involve reduced estradiol levels, which then increases vulnerability to the impact of tumors and chemotherapy on cognition. This study utilized the MMTV-PyVT mouse model of breast cancer to test the hypothesis that impaired ovarian function and associated estradiol levels play a critical role in CRCI susceptibility. Mice were either ovariectomized (OVX) or underwent sham surgery. The OVX group then received supplemental estradiol (E2) ad libitum in the drinking water to maintain physiological hormone levels. After tumor development, mice were trained in the Morris water maze to assess spatial memory, and subsequently, they received weekly injections of either saline or a combination of cyclophosphamide (CYP; 66.7 mg/kg, i.v.) and doxorubicin (DOX; 6.7 mg/kg, i.v.) for 4 weeks. Spatial memory was reassessed 10 d and then 35 d, after the final injections. Results demonstrated a significant disruption of normal ovarian cycling in sham-operated mice treated with CYP + DOX, as well as significant spatial memory impairments when compared with OVX mice supplemented with E2 This study suggests that chemotherapy-induced ovarian damage and the consequent drop in circulating estrogens significantly contribute to vulnerability to CRCIs, underscoring the importance of estradiol in mitigating CRCI risks.

Estrogen Deficiency Exacerbates Traumatic Heterotopic Ossification in Mice.

Traumatic heterotopic ossification (HO) is a devastating sequela of orthopedic surgeries and traumatic injuries; however, few studies have explored the effects of the estrogen-deficient state on HO formation. In the present study, we investigated the impact of estrogen deficiency on ectopic cartilage and bone formation in tendon after Achilles tenotomy in an ovariectomized mouse model. A total of 45 female C57BL/6 mice were randomly divided into three groups: sham-operated (control), estrogen depletion by ovariectomy (OVX) and OVX with 17β-estradiol supplementation (OVX + E2), with 15 animals in each group. Three weeks after OVX, all mice were subjected to an Achilles tenotomy using a posterior midpoint approach to induce HO. At 1, 3 and 9 weeks after tenotomy, the left hind limbs were harvested for histology, immunohistochemistry and immunofluorescence evaluations. The volume of ectopic bone was assessed by micro-CT. Mice in the OVX group formed more ectopic cartilage 3 weeks after tenotomy, as well as ectopic bone 9 weeks after tenotomy, compared to the control group. Estrogen deficiency resulted in more severe inflammatory infiltration at the injury sites 1 week after tenotomy, involving the recruitment of more macrophages and mast cells, as well as increasing the expressions of pro-inflammatory mediators, including IL-1β, IL-6, and TNF-α. Moreover, the local TGF-β/SMAD signaling pathway was dysregulated after OVX, which manifested as upregulated expressions of TGF-β and pSMAD2/3. E2 supplementation protected against OVX-induced HO deterioration, inhibited inflammatory infiltration, and downregulated the TGF-β/SMAD signaling pathway. Estrogen deficiency exacerbated HO formation in the Achilles tenotomy model. These findings might be attributable to the disturbance of the inflammatory response and the activation of TGF-β/SMAD signaling at the injury sites during the early stages of HO development.

Regorafenib Attenuates Osteoclasts Differentiation by Inhibiting the NF-κB, NFAT, ERK, and p38 Signaling Pathways.

Osteolytic diseases such as osteoporosis and neoplastic bone metastases are caused by the excessive activation of osteoclasts. Inhibiting the excessive activation of osteoclasts is a crucial strategy for treating osteolytic diseases. This study investigated the roles and mechanisms of regorafenib, a tyrosine kinase inhibitor, on osteoclasts and osteolytic diseases. We first identified the potential targets and mechanisms of regorafenib on osteoclast-related osteolytic diseases using network pharmacological analysis and molecular docking techniques. Then, we verified its role and mechanism on osteoclasts via cellular and animal experiments. Network pharmacology analysis identified 89 common targets shared by regorafenib and osteoclast-related osteolytic diseases. Enrichment analysis suggested that regorafenib may act on osteoclast-related osteolytic diseases by modulating targets such as AKT1, CASP3, MMP9, and MAPK3, regulating biological processes such as cell proliferation, apoptosis, and phosphorylation regulation, and influencing signaling pathways such as MAPK, PI3K/AKT, and osteoclast differentiation. The molecular docking results indicated that regorafenib and AKT1, CASP3, MMP9, MAPK3, and MAPK14 were stably docked. Cell experiments demonstrated that regorafenib significantly inhibited osteoclast differentiation and bone resorption in RAW 264.7 cells and bone marrow macrophages in a dose-dependent manner, with up to 50% reduction at 800 nM concentration without exhibiting cytotoxic effects. Furthermore, Western blot and RT-qPCR results demonstrated that regorafenib inhibited osteoclast differentiation by blocking the transduction of RANKL-induced NF-κB, p38, ERK, and NFAT signaling pathways. In vivo studies using an ovariectomized mouse model showed that regorafenib significantly improved bone volume fraction (BV/TV), bone surface to total volume (BS/TV), and number of trabeculae (TB.N), as well as reduced trabecular separation (Tb.Sp) compared to the OVX groups (P < 0.05). TRAcP staining results revealed a reduction in the number of osteoclasts with regorafenib treatment (P < 0.01). These results indicate that regorafenib exerts its protective effects against osteoclast-related osteolytic disease by inhibiting the RANKL-induced NF-κB, NFAT, ERK, and p38 signaling pathways. This study proves that regorafenib may serve as a potential therapeutic agent for osteoclast-related osteolytic diseases.

Tanshinone ⅡA Ameliorates Cartilage Degeneration in Ovariectomized Rats by Regulating TGF-β1/Smad2/MMPs Signaling Pathway

To investigate the ameliorative effect of tanshinone ⅡA (Tan) on osteoarticular degeneration in ovariectomized rats (a postmenopausal estrogen deficiency model) and the mechanisms involved. Eight-week-old female Sprague Dawley (SD) rats were randomly allocated to 5 groups (n=10 each), including a Sham operation group (Sham), an ovariectomy group (OVX), and low, medium, and high-dose Tan groups. Eight weeks after bilateral ovariectomy, the rats in the low, medium, and high-dose Tan groups were treated with Tan at the doses of 5, 10, and 20 mg/kg for a duration of 28 days. Evaluation of the rat articular cartilage was performed using X-ray imaging, anatomical observation, hematoxylin and eosin (H&E) staining, and toluidine blue staining. Immunohistochemistry was performed to assess the expression levels of transforming growth factor β1 (TGF-β1), phosphorylated-smad2 (p-Smad2), type Ⅱ collagen (CⅡ), matrix metalloproteinase 9 (MMP-9), and MMP-13 in the cartilage tissue. The knee joints of the OVX rats exhibited narrowed joint spaces, osteophyte formation, cartilage erosion or even localized cartilage cracks, faded methylene blue staining on the cartilage surface, disordered arrangement of chondrocytes, unclear or interrupted tidal line, and increased Kellgren-Lawrence grading, Pelletier grading, Mankin grading, and OARSI scores compared to those of the Sham group (P<0.01), as revealed by X-ray imaging, anatomical observation, and histological examination results. Tan ameliorated the degenerative changes in the knee joint caused by OVX in a dose-dependent manner while improving Kellgren-Lawrence grading, Pelletier grading, Mankin grading, and OARSI scores. Immunohistochemistry findings showed that TGF-β1, p-Smad2, and CⅡ expression levels were significantly increased (P<0.01), while MMP-9 and MMP-13 expression levels were significantly decreased (P<0.01) in the articular cartilage of the Tan group compared to those of the OVX group, with all these effects being dose-dependent. Tan mitigates articular cartilage degeneration in ovariectomized rats, which may be related to the regulation of TGF-β1/Smad2/MMPs signaling pathway.

New mechanistic understanding of osteoclast differentiation and bone resorption mediated by P2X7 receptors and PI3K-Akt-GSK3β signaling

ObjectiveOsteoporosis is a global health issue characterized by decreased bone mass and microstructural degradation, leading to an increased risk of fractures. This study aims to explore the molecular mechanism by which P2X7 receptors influence osteoclast formation and bone resorption through the PI3K-Akt-GSK3β signaling pathway.MethodsAn osteoporosis mouse model was generated through ovariectomy (OVX) in normal C57BL/6 and P2X7f/f; LysM-cre mice. Osteoclasts were isolated for transcriptomic analysis, and differentially expressed genes were selected for functional enrichment analysis. Metabolite analysis was performed using liquid chromatography-tandem mass spectrometry (LC–MS/MS), and multivariate statistical analysis and pattern recognition were used to identify differential lipid metabolism markers and their distribution. Bioinformatics analyses were conducted using the Encyclopedia of Genes and Genomes database and the MetaboAnalyst database to assess potential biomarkers and create a metabolic pathway map. Osteoclast precursor cells were used for in vitro cell experiments, evaluating cell viability and proliferation using the Cell Counting Kit 8 (CCK-8) assay. Osteoclast precursor cells were induced to differentiate into osteoclasts using macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-beta ligand (RANKL), and tartrate-resistant acid phosphatase (TRAP) staining was performed to compare differentiation morphology, size, and quantity between different groups. Western blot analysis was used to assess the expression of differentiation markers, fusion gene markers, and bone resorption ability markers in osteoclasts. Immunofluorescence staining was employed to examine the spatial distribution and quantity of osteoclast cell skeletons, P2X7 protein, and cell nuclei, while pit assay was used to evaluate osteoclast bone resorption ability. Finally, in vivo animal experiments, including micro computed tomography (micro-CT), hematoxylin and eosin (HE) staining, TRAP staining, and immunohistochemistry, were conducted to observe bone tissue morphology, osteoclast differentiation, and the phosphorylation level of the PI3K-Akt-GSK3β signaling pathway.ResultsTranscriptomic and metabolomic data collectively reveal that the P2X7 receptor can impact the pathogenesis of osteoporosis through the PI3K-Akt-GSK3β signaling pathway. Subsequent in vitro experiments showed that cells in the Sh-P2X7 + Recilisib group exhibited increased proliferative activity (1.15 versus 0.59), higher absorbance levels (0.68 versus 0.34), and a significant increase in resorption pit area (13.94 versus 3.50). Expression levels of osteoclast differentiation-related proteins MMP-9, CK, and NFATc1 were markedly elevated (MMP-9: 1.72 versus 0.96; CK: 2.54 versus 0.95; NFATc1: 3.05 versus 0.95), along with increased fluorescent intensity of F-actin rings. In contrast, the OE-P2X7 + LY294002 group showed decreased proliferative activity (0.64 versus 1.29), reduced absorbance (0.34 versus 0.82), and a significant decrease in resorption pit area (5.01 versus 14.96), accompanied by weakened expression of MMP-9, CK, and NFATc1 (MMP-9: 1.14 versus 1.79; CK: 1.26 versus 2.75; NFATc1: 1.17 versus 2.90) and decreased F-actin fluorescent intensity. Furthermore, in vivo animal experiments demonstrated that compared with the wild type (WT) + Sham group, mice in the WT + OVX group exhibited significantly increased levels of CTX and NTX in serum (CTX: 587.17 versus 129.33; NTX: 386.00 versus 98.83), a notable decrease in calcium deposition (19.67 versus 53.83), significant reduction in bone density, increased trabecular separation, and lowered bone mineral density (BMD). When compared with the KO + OVX group, mice in the KO + OVX + recilisib group showed a substantial increase in CTX and NTX levels in serum (CTX: 503.50 versus 209.83; NTX: 339.83 versus 127.00), further reduction in calcium deposition (29.67 versus 45.33), as well as decreased bone density, increased trabecular separation, and reduced BMD.ConclusionP2X7 receptors positively regulate osteoclast formation and bone resorption by activating the PI3K-Akt-GSK3β signaling pathway.

Silkworm (Bombyx mori L.) Has Beneficial Effects on Menopausal Symptoms by Enhancing Estrogen Receptor Signaling in Ovariectomized Mice.

Existing hormone replacement therapy for menopause has drawbacks, necessitating new treatment agents. Silkworms have demonstrated estrogenic properties, offering promising alternatives. We assessed the therapeutic effects of freeze-dried silkworm powder (SWP) on menopausal symptoms using an ovariectomized (OVX) mouse model. The experimental design comprised a sham surgery group (Sham), an OVX control group, a low-dose SWP group post-OVX (80 mg/kg, OVX-SWP-L), a high-dose SWP group post-OVX (160 mg/kg, OVX-SWP-H), and an estradiol treatment group post-OVX (OVX-E2). Treatments were administered orally thrice weekly over eight weeks; body weight was monitored weekly. The SWP-treated groups (SWP-L and SWP-H) exhibited less weight gain and increased uterine thickness than the OVX control. Molecular analyses demonstrated that SWP significantly enhanced the phosphorylation of estrogen receptor alpha (ERα), ERK, and AKT. Furthermore, biochemical assays revealed reduced serum neutral lipids across all SWP treatment groups. Notably, HDL-cholesterol levels were significantly increased in the SWP-L group compared to the OVX group. Serum estradiol concentrations were elevated in all the SWP groups, with significant increases in the high-dose group. These findings indicate that SWP may promote the activation of estrogen receptor signaling and improve symptoms associated with estrogen deficiency during menopause.

Exploration of the miR-187-3p/CNR2 pathway in modulating osteoblast differentiation and treating postmenopausal osteoporosis through mechanical stress.

This study aimed to explore how mechanical stress affects osteogenic differentiation via the miR-187-3p/CNR2 pathway. To conduct this study, 24 female C57BL/6 mice, aged 8 weeks, were used and divided into four groups. The Sham and OVX groups did not undergo treadmill exercise, while the Sham + EX and OVX + EX groups received a 8-week treadmill exercise. Post-training, bone marrow and fresh femur samples were collected for further analysis. Molecular biology analysis, histomorphology analysis, and micro-CT analysis were conducted on these samples. Moreover, primary osteoblasts were cultured under osteogenic conditions and divided into GM group and CTS group. The cells in the CTS group underwent a sinusoidal stretching regimen for either 3 or 7 days. The expression of early osteoblast markers (Runx2, OPN, and ALP) was measured to assess differentiation. The study findings revealed that mechanical stress has a regulatory impact on osteoblast differentiation. The expression of miR-187-3p was observed to decrease, facilitating osteogenic differentiation, while the expression of CNR2 increased significantly. These observations suggest that mechanical stress, miR-187-3p, and CNR2 play crucial roles in regulating osteogenic differentiation. Both invivo and invitro experiments have confirmed that mechanical stress downregulates miR-187-3p and upregulates CNR2, which leads to the restoration of distal femoral bone mass and enhancement of osteoblast differentiation. Therefore, mechanical stress promotes osteoblasts, resulting in improved osteoporosis through the miR-187-3p/CNR2 signaling pathway. These findings have broad prospect and provide molecular biology guidance for the basic research and clinical application of exercise in the prevention and treatment of PMOP.

NOX4 and its association with myeloperoxidase and osteopontin in regulating endochondral ossification.

Endochondral ossification plays an important role in skeletal development. Recent studies have suggested a link between increased intracellular reactive oxygen species (ROS) and skeletal disorders. Moreover, previous studies have revealed that increasing the levels of myeloperoxidase (MPO) and osteopontin (OPN) while inhibiting NADPH oxidase 4 (NOX4) can enhance bone growth. This investigation provides further evidence by showing a direct link between NOX4 and MPO, OPN in bone function. This study investigates NOX4, an enzyme producing hydrogen peroxide, in endochondral ossification and bone remodeling. NOX4's role in osteoblast formation and osteogenic signaling pathways is explored. Using NOX4-deficient (NOX4-/-) and ovariectomized (OVX) mice, we identify NOX4's potential mediators in bone maturation. NOX4-/- mice displayed significant differences in bone mass and structure. Compared to the normal Control and OVX groups. Hematoxylin and eosin staining showed NOX4-/- mice had the highest trabecular bone volume, while OVX had the lowest. Proteomic analysis revealed significantly elevated MPO and OPN levels in bone marrow-derived cells in NOX4-/- mice. Immunohistochemistry confirmed increased MPO, OPN, and collagen II (COLII) near the epiphyseal plate. Collagen and chondrogenesis analysis supported enhanced bone development in NOX4-/- mice. Our results emphasize NOX4's significance in bone morphology, mesenchymal stem cell proteomics, immunohistochemistry, collagen levels, and chondrogenesis. NOX4 deficiency enhances bone development and endochondral ossification, potentially through increased MPO, OPN, and COLII expression. These findings suggest therapeutic implications for skeletal disorders.