Quercetin alleviates osteoporosis in rat mandibles induced by ovariectomy through modulation of autophagy and inhibition of NLRP3 pathway

doi:10.21203/rs.3.rs-2772620/v1

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Quercetin alleviates osteoporosis in rat mandibles induced by ovariectomy through modulation of autophagy and inhibition of NLRP3 pathway

https://doi.org/10.21203/rs.3.rs-2772620/v1

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Background With the aging population and the popularity of implant prostheses, more and more postmenopausal patients with osteoporosis (PMOP) have a need for implant restorations, but poor bone condition affects the long-term stability of their implant prostheses. The aim of this study was to investigate the therapeutic effect of quercetin (QR) compared to alendronate (ALN), the primary treatment for PMOP, on mandibular OP induced by ovariectomy in female rats (OVA).

Methods Adult female rats were treated with QR (50 mg/kg/day), ALN (6.25 mg/kg/week) by gavage for 8 weeks, chloroquine CQ (10 mg/kg/twice a week), and MCC950 (10 mg/kg/three times a week) by intraperitoneal injection for 8 weeks after bilateral ovariectomy. Blood samples were collected prior to euthanasia; the mandibles were harvested and subjected to MicroCT and pathological analysis.

Results The results showed that QR administration controlled weight gain and significantly improved bone microstructure in OVA rats, increasing bone mass, and Bone mineral density (BMD), reducing bone trabecular spacing, and decreasing osteoclast numbers. WB, rt-qPCR, and serum markers confirmed that QR inhibited the differentiation of osteoclasts on the NLRP3 pathway that promotes osteoclast differentiation of IL-1β, IL-18, Immunofluorescence, WB also confirmed that QR inhibited autophagy in OVA rats and suppressed the number of Trap-stained positive osteoclasts.

Conclusion QR may protect the skeleton and prevent bone loss in osteoporotic rats by inhibiting the NLRP3 pathway and autophagy in osteoclasts with comparable effects to ALN, thus QR has the potential to be a promising alternative supplement for the preventive and therapeutic treatment of postmenopausal osteoporosis.

quercetin

alendronate

postmenopausal osteoporosis

autophagy

NLRP3

osteoclasts

ovariectomy

Implant prosthesis has gradually become the primary choice for patients with tooth loss due to their advantages of no damage to natural teeth, high chewing efficiency, comfort, and aesthetics [1]. The demand for implant prostheses is increasing, especially in China, where the population is aging, and the proportion of patients with osteoporosis who have tooth loss has increased over the years [2]. Osteoporosis (OP) is a widespread, systemic, metabolic bone disease marked by a decrease in bone mineral density (BMD) and destruction of bone structure, in addition to a substantial increase in bone fragility and fracture susceptibility [3], caused by an imbalance between bone resorption by osteoclasts and bone deposition by osteoblasts [3, 4]. According to WHO, about 200 million people worldwide are suffering from OP disease, and its incidence has jumped to the seventh place of common diseases among the elderly. With social development and increasing aging, the incidence of OP is rising annually. In 2018, the National Health Council of China released the results of an epidemiological survey of OP, which showed that the prevalence of OP in people over 50 years of age was 19.2%, and the percentage of the low bone mass group was as high as 46.4% [5]. Postmenopausal women are at high risk for the disorder known as postmenopausal osteoporosis (PMOP), which is the most common type of OP and is mainly associated with decreased estrogen levels [6]. Female patients over age 50 are a group with strong demand for oral implants. The precondition for a successful implant denture is the formation of good osseointegration between the implant and the surrounding bone, while osteoporosis can easily lead to insufficient bone quantity and unhealthy bone quality decreased initial stability of implant placement, prolonged bone healing time, and weakened regeneration ability of the peri-implant bone, and changes in the local peri-implant bone microenvironment due to trauma and infection after implantation, which affect the success rate of implant surgery [5, 7]. The incidence of PMOP is increasing by the year, which seriously affects the life quality of patients and brings a heavy economic burden to society, and has become a public health problem that needs to be solved worldwide. Moreover, methods to improve osteoporosis in postmenopausal women, increase the rate of implant osseointegration, and shorten the healing time have been the hot spots of research in oral implantology.

Autophagy is involved in the vital activities of osteoblasts, osteoclasts, and osteocytes [8], maintaining the dynamic balance between osteoblasts and osteoclasts in the physiological state and the stress response in the pathological condition [9, 10], closely related to bone loss diseases such as OP, playing an essential role in regulating bone homeostasis in the bone immune system [11, 12]. Nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) protein 3 (NLRP3) inflammatory vesicles, as the body's first line of defense against pathogens, also play an active role in the pathogenesis of osteoporosis, inhibiting bone formation while promoting bone resorption, ultimately leading to bone loss [11, 13–14]. Studies have reported that NLRP3 inflammatory vesicles interact with autophagy to maintain the dynamic homeostasis of the body under stressful conditions. Autophagy for removing damaged proteins and enriched organelles along with destroying intracellular pathogen-induced inflammatory response process can lead to excessive activation of NLRP3 inflammatory vesicles if autophagy is dysfunctional [15].

Conventional treatment options for PMOP include anti-resorptive agents (e.g., bisphosphonates, hormone replacement therapy, selective estrogen receptor modulators, and anti- RankL antibodies) and synthetic drugs (e.g., intermittent low-dose teriparatide and anti-sclerostin antibodies), however, these treatments are also associated with some adverse effects [16–18]. Alendronate is a third-generation amino diphosphonate bone resorption inhibitor with a strong affinity for intraosseous hydroxyapatite, which exerts an anti-resorptive effect by inhibiting osteoclast activity and has been commonly used in the treatment of PMOP. It is well tolerated, with gastrointestinal reactions such as nausea, abdominal distension, abdominal pain, occasional headache, skeletal muscle pain, and rare rash or erythema. However, with the widespread use of this drug, the number of esophageal adverse events has increased, and side effects such as esophageal erosion and esophageal ulceration have occurred [19]. Are there natural compounds with potent osteoprotective properties and fewer side effects that can be alternatives to overcome the shortcomings of existing therapies?

Quercetin (C₁₅H₁₀O₇) is the major monomer component of eucommia flavonoid subclasses, widely exists in plants in nature, and is a natural polar auxin transport inhibitor [20]. The keto carbonyl group can generate basic oxygen atoms of strongly acidic salts, and the presence of phenolic hydroxyl groups and their double bonds also enables the antioxidant effect of quercetin to be enhanced. These molecular structures make it significant in the effective scavenging of free radicals in the body, maintaining the normal structure and function of body cells, preventing tumor mutations, and delaying the onset of aging [21]. Quercetin has been shown to have estrogen-like effects and promote osteogenesis by inhibiting bone resorption and having an anti-osteoporotic effect[22]. The addition of 0.25% quercetin to the diet of rats with ovaries removed was able to slow down bone resorption, and particularly to inhibit bone loss due to estradiol deficiency [23].

Does quercetin exert a therapeutic effect on osteoporosis by regulating autophagy and inhibiting the NLRP3 inflammasome? It has not been reported yet. Since quercetin has been used as a health supplement for many years and has potent antioxidant, anti-inflammatory, and autophagy-promoting effects, we hypothesized that quercetin might be a promising, multi-targeted inhibitor of osteoclast activation, making alveolar bone free from osteoclast/inflammation-mediated bone resorption. This study investigated the anti-osteoporotic effect of quercetin in a bilateral ovariectomy-induced PMOP model in rats, aiming to provide some theoretical basis for the application of quercetin in oral implants in PMOP patients.

Chemicals

Quercetin (10.0g, purity ≥ 95%) was purchased from sigma Chemical Company, CAS No.: 117-39-5. Alendronate Tablets (Fosamax)(Specification: 70mg/tablet), Production Lot No.: J20170085, Merck Sharp & Dohme, Inc. Chloroquine, an autophagy inhibitor (specification: 1.0g; purity: 97%), was purchased from Shanghai Macklin Biochemical Co.CAS lot number: 54-05-7; NLRP3 pathway inhibitor MCC950 (specification: 2.0g; purity ≥ 98%), purchased from Sichuan Weikeqi Biological Technology Co. CAS lot number:256373-96-3. PINP,CTX-1,IL-18,IL-1β ELISA kit was bought from Quanzhou Ruixin Biotech Company.Anti-bodies of Caspase-1 (#24232), IL-1β (#12507), NLRP3 (#15101) were purchased from CTS company. Anti-bodies of LC3 (14600-1-Ap), Beclin1 (11306-1-AP) and p62(18420-1-AP)were purchased from Proteintech company. Anti-bodies of β-Actin (ab8226), IL-18 (ab191860) were purchased from Abcam company. Goat anti-rabbit IgG (E-AB-1003) were purchased from Elabscience company.

Animals

Ninety-six adult female Sprague-Dawley rats were used for this study. These animals were housed in a 12-hour alternating light/dark cycle. Rodent standard chow and ad libitum water were fed. The rats were randomized into nine different groups, including group 1: sham operation (Sham); group 2: ovariectomy (OVX); group 3: ovariectomy + 50 mg/kg/day quercetin (OVX-QR); group 4: ovariectomy + 6.25 mg/kg/week alendronate (OVA + ALN);group 5: ovariectomy + 10 mg/kg/twice a week chloroquine (OVX-CQ); group 6: ovariectomy + 10 mg/kg/twice a week Chloroquine + 50 mg/kg/day Quercetin (OVX-CQ-QR); Group 7: Ovariectomy + 10 mg/kg/three times a week MCC950 (OVX-M); Group 8: Ovariectomy + 10 mg/kg/three times a week MCC950 + 50 mg/kg/day Quercetin (OVX-M-QR). All quercetin and alendronate treatments were administered by gavage, with equal volumes of saline in the sham-operated group, while CQ and MCC950 were administered by intraperitoneal injection for 8 weeks. All treatments were started at twelve weeks after the ovariectomy. The doses and durations of QR, AL,CQ, and M were selected based on previous studies [24–26]. The ethics committee of Guizhou Medical University approved the experimental protocol (approval number: 2201633).

Ovariectomy procedure

After fasting and water fasting for 12 h, the 12-week-old rats were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (10 mg/kg) and fixed on a surgical plate. Bilateral ovariectomy was performed by the dorsal method, in which the dorsal side of the animal was shaved and cleaned with 70% ethanol, a 1-cm-long incision was made under aseptic conditions, both ovaries were ligated and removed, the incision was closed in layers, and lidocaine and penicillin powder were applied locally to the incision area. The sham-operated group excised an equal volume of adipose tissue adjacent to the ovaries.

Sample collection

The body weights of rats were measured one day after ovariectomy and before the start of weekly treatment. After 8 consecutive weeks of treatment, blood samples were collected from the abdominal aorta after anesthetizing the rats with an intraperitoneal injection of 1% sodium pentobarbital (10 mg/kg), and the serum obtained was subjected to biochemical analysis under appropriate storage conditions before increasing the anesthetic dose and killing the rats; the mandibles of each rat were removed and cleaned of cartilage, adherent tissue, and bone marrow, and stored at -80°C on one side for RNA extraction and real-time polymerase Chain reaction (RT-PCR), western blot assay, and the other side of the mandible was partially stored in electron microscopy fixative, decalcified, embedded, sectioned and used for transmission electron microscopy, and partially stored in 4% paraformaldehyde, undecalcified for micro CT scanning and analysis. The remaining mandibles were decalcified using 10% EDTA decalcification solution for approximately 4 weeks until a 10 ml syringe needle could be easily inserted, dehydrated in ethanol step by step, waxed, paraffin-embedded, sectioned (5 µm), and used for staining, immunofluorescence, and immunohistochemistry.

Micro-CT scan and analysis of rat mandible

After completion of calibration, the undecalcified unilateral mandible was taken and placed in 10% neutral buffered formalin seeds for 24 hours of fixation, and excess soft tissue was removed. The femur was scanned using a Skyscan 1276 micro CT instrument (BrukermicroCT Kontich, Belgium) with the following scan settings: source voltage, 55 kV, source current, 200 H a, AI 0.25 mm filter, pixel size 6 m, and rotation step of 0.3 degrees. Images were constructed with NR econ software (Bruker microCT, Kontich, Belgium) with the following settings: ring artifact correction: 5; smoothing: 3; beam sclerosis correction: 30%. The following parameters such as bone volume percentage (BV/TV%), bone trabecular number (Tb. N), bone trabecular thickness (Tb. Th), bone trabecular spacing (Tb. Sp), and bone mineral density (BMD) were analyzed using the CTAn program (Bruker microCT, Kontich, Belgium).

Detection of serum bone metabolism indexes. 3 months after modeling and 8 weeks after drug intervention, rats in each group were anesthetized with 1% sodium pentobarbital 10 mg/kg intraperitoneally, and 3–5 mL of blood was collected from the abdominal aorta, centrifuged at 3,000 r/min for 15 min at 4 ℃, and the serum was extracted and stored in a refrigerator at 4 ℃. The levels of IL-18, IL-1β, PINP, and CTX-1 in the serum were detected according to the standard of the ELISA kit.

Real-time fluorescence quantitative PCR to measure the expression of Caspase-1 and NLRP3 mRNA

After 8 weeks of drug intervention, the rats in each group were anesthetized and executed. The cancellous bone of the jaw was then removed, and ground into powder, and the total RNA was extracted using the triazole technique. The RNA concentration and purity were then determined using the Nanodrop 2000. Reaction system: 2×SYBR Premix Ex Taq Ⅱ 7.5 µL, upstream and downstream primers 1.5 µL, cDNA: 2 µL, plus dH₂O 4.0 µL; 95 ℃ -30 s, 95 ℃ -15 s, 60 ℃ -30 s, 40 cycles; melting curve: 65 ℃ -5 s, 95 ℃ -10 s. With the primer sequences listed in Table 1, the expressions of the Caspase-1 and NLRP3 genes were assessed. GAPDH mRNA levels served as the baseline for other mRNA levels. The relative gene expression levels were calculated via the 2-∆∆Ct technique. The primer sequences were listed in Table I.

Table 1

Primer sequences
Primer	Primer sequence	Length of product(bp)
NLRP3	(S)5’-CTGGAGAAACCTGCCAAGTATG-3’ (A)5’-GGTGGAAGAATGGGAGTTGCT-3	138
Caspase-1	(S)5’-TGCCTGGTCTTGTGACTTGGAG-3’ (A)5’-TGTCCTGGGAAGAGGTAGAAACG-3	132
GAPDH	(S)5’-GATTTCTCCACAACTCACCCAA-3’ (A)5’-AGTCTGGAAGAACAGGCAACAT-3	112

Western blot detection of NLRP3 pathway and autophagy-related protein expression in rat mandibular bone tissue

Weight 50–100 mg of frozen mandibular bone tissue specimens, grind them in liquid nitrogen, and put them into 1.5 ml EP tubes, add RIPA lysis solution containing protease inhibitor to each tube for 200 min, lyse on ice for 40 min, centrifuge at 4°C for 25 min at 12000 T/min, extract the supernatant, and extract the total protein from bone tissue. Following SDS-polyacrylamide gel electrophoresis (PAGE), the proteins were transferred, incubated with IL-1β, IL-18, Caspase-1, NLRP3, LC3, Beclin1, P62, and β-Actin at a 1:2000 dilution overnight at 4°C, and horseradish peroxidase-labeled rat antibodies were added. After incubation for 2 h at room temperature and color development with an ECL color development kit, the protein expression levels were determined by analyzing the grayscale values with β-Actin as the reference, and three replicates were set for each group.

Immunofluorescence assay

The immunofluorescence technique was utilized to assess the expression of LC3 and BECN1 proteins. After rehydration in a warmed alcohol series and phosphate-buffered saline (PBS), the alcohol series and phosphate-buffered saline (PBS) were permeabilized with 0.3% T-2. Permeabilization was performed with 0.3% Triton X-100 for 30 minutes at room temperature. Sections were then blocked with 10% goat serum in PBS for 30 minutes. Sections were incubated with the primary monoclonal antibody to LC3 (14600-1-AP, at dilution of 1:500 in PBS), Beclin1 (11306-1-AP, at dilution of 1:50 in PBS), and p62(18420-1-AP,at dilution of 1:1500 in PBS) were incubated overnight, followed by conjugation with a secondary polyclonal antibody Cy3conjugatedGoatAnti-RabbitIgG(H + L) at 37°C ( GB21303, 1:200 in PBS; Service-bio) for 1.5 hr. Final. After 4 times rinses with PBS, incubated with 4′,6-diamidino-2-phenylindole (DAPI). 2-Phenylindole (DAPI) was incubated for 5 min and observed with a fluorescence microscope. Images were captured at ×400 magnification Fluorescence microscopy (Olympus BX51) equipped with an Olympus DP70 digital camera. Immunofluorescence staining was quantified with ImageJ software (version 1.37; National Institutes of Health, USA). For this purpose, five different microscopic areas were randomly investigated by a blind investigator through a fluorescence microscope.

Histopathological changes of rat mandibles observed by HE, and Trap staining

Each group of decalcified, embedded, and sectioned rats had their alveolar bone dyed under the directions of the HE, and Trap staining kits before being observed under a light microscope and photographed.

Statistical analysis

Statistical analysis was performed using spss22.0 software (IBM Corp., Armonk, NY, USA), and statistical graphs were drawn using GraphPad Prism 7.0. Measures were tested for conformity to a normal distribution and expressed as mean-sergeant deviation (x \(\pm\) S). Comparisons between two groups were made by t-test or rank sum test, and multiple group comparisons were made by ANOVA (One Way ANOVA and Two Way ANOVA) or Kruskal-Wallis test, and data with differences were compared between groups by LSD-t test or rank sum test, and the test level was a = 0.05, and P ≤ 0.05 was considered statistically significant.

Effect of ovariectomy and treatment on changes in body weight of rats

The mean body weight of rats increased significantly with longer rearing time after the removal of bilateral ovaries, with statistically significant differences (P < 0.05). At the 5th month after surgery, the body weight of rats in the OVA group was significantly greater than that of the SHAM group, while treatment with QR, ALN, CQ, and MCC950 controlled the body weight of rats, with no statistical difference compared to the SHAM group. (Fig. 1)

Effect of ovariectomy and treatment on mandibular bone and bone trabecular parameters

In the left mandibular first molar apical compartment alveolar bone area, after 8 weeks of treatment, bone trabeculae in the mandibular alveolar bone were dense and well aligned in the SHAM group; in the OVA group, bone volume was lost after ovariectomy, bone trabeculae became thin and narrow, and the degree of dispersion between trabecular bone increased; after treatment with QR and ALN, bone volume was restored and an increase in the number of bone trabeculae was seen, bone trabeculae were densely and well aligned, and the degree of separation was significantly reduced (Fig. 2AB).

Bone microstructural parameters of the alveolar bone were derived from analysis of the ROI region by Micro-CT scanning. After 8 weeks of treatment, the bone volume fraction (BV/TV), bone trabecular thickness (Tb. Th), and bone mineral density (BMD) decreased and bone trabecular separation (Tb. Sp) increased in the OVA group of rats compared to the SHAM group, with all differences being statistically significant(p < 0.05) except for Tb. Th, indicating successful modeling of the osteoporosis model; compared with the OVX group, the QR and ALN groups showed increased BV/TV, BMD, and trabecular number (Tb. N), decreased Tb. Sp (p < 0.05), but there was no significant difference in Tb. Th compared to the OVA group (P > 0.05); the QR and ALN groups displayed increased BV/TV, decreased BMD and increased Tb. Sp (p < 0.05); there was no significant difference between these two groups (P > 0.05) (Fig. 2C-G).

Effect of oophorectomy and treatment on serum biochemical markers of bone metabolism

After 8 weeks of treatment, the bone resorption index CTX-I and bone formation index PINP were both increased to different degrees in the OVA group compared with the SHAM group, indicating that the rate of bone conversion was higher in the ovariectomized rats and the bone tissue was in a highly reconstructed state. However, both bone conversion indexes decreased to varying degrees when subjected to the effects of QR, ALN, M, or CQ, indicating that the bone tissue reconstruction became stable. There was no significant difference between the treated group compared to the SHAM group and a statistically significant difference compared to the OVA group (Fig. 3AB).

Following 8 weeks of treatment, the serum levels of IL-1β and IL-18 increased to different degrees in the OVA group compared to the SHAM group (p < 0.05), indicating that inflammatory factors increased in the serum of rats after ovarian removal. In contrast, the levels of inflammatory factors IL-1β and IL-18 decreased to different degrees after treatment with QR, ALN, M, or CQ, except for the degree of reduction of IL-18 after ALN treatment was statistically different (p < 0.05) (Fig. 3CD).

Effect of oophorectomy and treatment on mRNA levels of NLRP3 signalling pathway-related genes in rat mandibular alveolar bone tissue

Results from RT-qPCR revealed that Caspase-1 and NLRP3, two genes connected to the NLRP3 signaling pathway, had significantly higher levels of expression in the osteoporotic state of alveolar bone tissue compared to the SHAM group (p < 0.05). In contrast, Caspase-1 expression levels decreased after treatment with QR and ALN, with Caspase-1 decreasing more significantly (p < 0.05). Caspase-1 and NLRP3 responses to the NLRP3 pathway inhibitor MCC950 alone or in combination with QR did not differ noticeably from those to QR (Fig. 4).

Effect of ovariectomy and treatment on the expression of NLRP3 pathway-related proteins as well as autophagy-related proteins

Compared with the SHAM group, NLRP3, Caspase-1, IL-1β, and IL-18, Beclin1 protein expression, LC3II/I were upregulated (p < 0.05), and p62 protein expression was upregulated (p > 0.05) in the OVA group at the same time point of 8 weeks of treatment; compared with the OVX group, the QR group, and the ALN group The expression levels of NLRP3, Caspase-1, IL-1β, IL-18, and Beclin1 decreased in the QR and ALN groups compared to the OVX group (p < 0.05), while the expression levels of IL-1β, Beclin1, and LC3II/I were not significantly different in the SHAM group compared to the QR group. ALN reduced Caspase-1 more effectively than QR, with no significant differences in the remaining indicators (Fig. 5).

In comparison to the SHAM group, Beclin1 (p < 0.05) and p62 (p > 0.05) protein expression was upregulated and LC3II/I was upregulated (p < 0.05) in the OVA group at the same time point at 8 weeks; compared to the OVX group, Beclin1 protein expression levels were decreased (p < 0.05) in the QR, CQ and CQ + QR groups, LC3II/I (p < 0.05) and p62 protein expression only increased with CQ alone or in combination with QR was statistically different; Beclin1 was reduced more significantly with the combination of CQ and QR (Fig. 6A).

Protein expression of NLRP3, caspase-1, IL -1β, and IL -18 was upregulated in the OVA group compared with the SHAM group at the same time point at 8 weeks (p < 0.05); The expression levels of NLRP3, Caspase-1, IL-1β and IL-18 protein were all reduced in the QR, M and M + QR groups when compared to the OVX group (p < 0.05). The combined use of M and QR were more effective in lowering NLRP3 and Caspase-1 than alone, but less effective in reducing IL-1β and IL-18 (Fig. 6B).

Immunofluorescence

Immunofluorescence staining (Fig. 7) revealed red-fluorescent Beclin1, LC3, and p62-positive cells within the mandibular region of OVA rats, with blue-fluorescent nuclei in all cells. When the images are merged, the red and blue fluorescence overlap, and the red fluorescence is mostly located in the osteochondral cancellous of the rat mandible. In the SHAM group, red fluorescence was occasionally seen in Beclin1, LC3, and p62 positive cells, while in the OVA group, the intensity of Beclin1 and LC3 red fluorescence was significantly higher (p < 0.05) and the average fluorescence of p62 was comparable to that of SHAM. After treatment with QR, CQ or a combination of both, Beclin1 and LC3 fluorescence intensity significantly decreased (p < 0.05) and p62 fluorescence intensity increased (p < 0.05), indicating that QR inhibited Beclin1 and LC3 expression and promoted p62 accumulation in the mandibular alveolar bone of OVA rats, with no significant difference from the effect of CQ alone or in combination (p > 0.05).

Effects of QR on histopathological changes in the mandibular alveolar bone of OVA rats

The results of hematoxylin-eosin (HE) staining showed that the alveolar bone trabeculae in the SHAM group were more continuous, neatly arranged, morphologically intact, and without significant pathological changes; in the OVX group, the alveolar bone trabeculae were thinner and narrower, discontinuous, with more free ends, and the bone marrow cavity was widened, with a large number of osteoclasts, vacuolated fat cells, and bone resorption sockets, and a decrease in osteocytes and bone volume; compared with the OVX group, the rest of the treatment Compared with the OVX group, the rest of the treatment groups had relatively more intact bone structure, with slightly more bone trabeculae, lamellar and dense, reduced bone marrow cavity, significantly fewer fatty vacuoles and osteoclasts, more osteoblasts, more osteoclasts, more bone volume, and a small number of new blood vessels. The Micro-CT results were further validated, indicating that QR treatment partially restored the lost bone mass in the alveolar bone of the OVA rats (Fig. 8).

Impact of QR on osteoclasts in the mandibular alveolar bone of OVA rats

The results of anti-tartrate acid phosphatase (TRAP) staining of the mandibular alveolar bone tissues in each group showed that more obvious multinucleated, cytoplasmic red-stained osteoclasts were visible in the alveolar bone tissues of the OVA group, spreading over the surface of the bone trabeculae, compared to the SHAM group, and the number of TRAP-positive osteoclasts was significantly reduced in the remaining treatment groups after treatment compared to the OVX group. The results further suggest that QR may inhibit the number of osteoclasts in the alveolar bone in the osteoporotic state to alleviate bone resorption (Fig. 9).

Natural products are an excellent and reliable means of preventing osteoporosis because they have fewer side effects, are less costly, and are more suitable for long-term application. QR, which has phytoestrogen effects, can induce increased expression of bone-bridging proteins, bone morphogenetic proteins, alkaline phosphatase, and Runx2. In vivo experiments in mice have confirmed the ability of QR to enhance the osteogenic differentiation of cells by activating an estrogen receptor-dependent mechanism, as well as the effect of its induced autophagy on the differentiation and mineralization of osteoblast MC3T3-E1 cells, confirming that QR can increase bone density and improve bone microarchitecture [27, 28]. In vitro experiments confirmed that QR inhibited the formation (detected by TRAP-positive polymorphonuclear cells), proliferation, survival, and maturation of osteoblasts treated with LPS and macrophage colony-stimulating factor (M-CSF) and/or RANKL [24]. Despite its significant antioxidant, anti-inflammatory, and anti-cancer properties and possible activity in inhibiting bone resorption, the therapeutic use of QR is limited by its low bioavailability. By co-culturing osteoblast-osteoclast endothelial cells in different concentrations of hydroxyapatite functionalized with QR, the new material was found to have a positive effect on the bone repair microenvironment by stimulating osteoblast proliferation and activity, reducing osteoclast production, and supporting the micro angiogenic process required for new bone formation [29]. The team also loaded QR onto hydroxyapatite functionalized with ALN to develop a new multifunctional system for the topical administration of QR and ALN. In vitro co-culture of osteoblasts and osteoclasts in a microenvironment altered by oxidative stress showed that the system was capable of sustained release of QR in phosphate buffer and that both ALN and QR significantly reduced osteoclast viability and were able to counteract the negative effects of oxidative stress on osteoblast viability and differentiation [30], providing a new idea for the development of a slow release system in vivo. Our experiment aimed to evaluate the therapeutic effect of QR compared to ALN as the primary treatment for PMOP in ovariectomy-induced female rats with mandibles. It is hoped that through mechanistic studies, it will be possible to investigate in greater depth whether quercetin acts as a preventive and palliative agent against PMOP through its action on autophagy and NLRP3 inflammasome, and provide some theoretical basis for the application of this drug in the clinical setting.

Ovarian hormones have been shown to play a critical role in metabolism, appetite, and body weight regulation in female animals, and reduced hormone levels after ovariectomy increase food intake and body weight, leading to obesity in experimental animals [31]. Treatment with QR, ALN, CQ, and MCC950 reversed the changes in body weight in the OVA rats, and although body weight was still higher in the treated group than in the SHAM group, this was not equivalent to changes in bone mass but was still a positive development in terms of weight control. Both QR and ALN increased BV /TV, Tb. N, Tb. Th and BMD and decrease Tb.Sp in OVA rats by micro-scanning CT. Tb.Th、Tb. N、Tb. Sp reflects changes in bone microstructure and is an important indicator for evaluating osteoporosis, while BMD is the main indicator for evaluating the effect of OP treatment [32]. Our results show that quercetin can improve bone microstructure, enhance bone quality, effectively increase BMD in PMOP rats, and treat PMOP from the structural aspect, which can achieve similar therapeutic effects as ALN.

The International Osteoporosis Foundation (IOF) recommends the use of propeptide of type I procollagen (PINP) and cross-linked C-telopeptide of type 1 collagen (CTX-1) in osteoporosis clinical trials as preferred markers for clinical assessment of bone turnover[33]. Although not used for the diagnosis of osteoporosis, they are valuable for patient assessment and improve the ability to identify certain secondary causes of osteoporosis, and are also effective in monitoring treatment adherence [34]. Therefore, in this study, PINP and CTX-I were selected as indicators to evaluate the effectiveness of using QR and ALN in the management of PMOP. PINP is derived from posttranslational shearing of type 1 procollagen and is expressed mainly during the proliferative phase of osteoblasts, whereas CTX-I is a degradation product of type 1 collagen and reflects the active state of osteoclasts. In this experiment, the serological bone turnover indices (CTX-I and PINP) were high in rats in the untreated OVA group, indicating a markedly progressive and abnormal bone remodeling process and bone resorption activity. High bone turnover is typical of most postmenopausal women, with bone mass decreasing dramatically at this stage [35]. In contrast, QR and ALN decreased PINP, which reflects osteoclast activity, and CTX-I, which indicates the degree of bone turnover. The combination of QR and MCC950 or CQ showed an even more significant reduction in PINP and CTX-I, similar to the results of previous studies [36], suggesting that QR may play a dual role in inhibiting bone resorption and promoting bone formation. However, the exact mechanism needs to be further explored.

Activation of NLRP3 inflammatory vesicles during osteoclast activation can lead to the development of inflammatory diseases.NLRP3 functions as an intracellular multiprotein complex that triggers cell death in response to the combined action of pathogens (PAMPs) and damage-associated molecular patterns (DAMPs). Recognition of endogenous and exogenous factors leads to the formation of the NLRP3/apoptosis-associated speck-like protein containing a CARD (ASC), ASC/pro-Caspase-1 inflammatory complex, which activates Caspase-1 and acts on the processing precursors IL-1β, IL-18, resulting in the release of mature cytokines and triggering an inflammatory response [37]. IL-1β is one of the leading members of the IL -1 family, which plays an essential role in bone loss following estrogen deficiency [38] and is not only a potent stimulator of bone resorption but also a potent inhibitor of osteogenesis. IL-18 and IL-1β are closely related, share a similar 3D structure, and promote osteoclast differentiation through multiple pathways. IL-1β and IL-18 are involved in and prolong the OP inflammatory response [39]. MCC950 is a potent and specific NLRP3 inflammasome inhibitor. In vitro experiments have shown that NLRP3 knockdown and supplementation with MCC950 significantly reduce age-related alveolar bone loss in aged mice, resulting in higher bone mineral density and bone mass per unit tissue. In vitro experiments have also shown that MCC950 indirectly regulates osteoclastogenesis by inhibiting IL -1β secretion from specific sites of macrophages and/or PMNs, and also directly controls osteoclast differentiation independent of IL -1β maturation by inhibiting RANKL-induced caspase-1 activation [11]. In line with the findings, QR was comparable to ALN and MCC950 in reducing inflammatory mediators, and the combination of MCC950 and QR resulted in a more effective reduction of caspase-1.

Autophagy plays a protective role for the organism and is primarily responsible for eliminating damaged or excess organelles to maintain normal metabolism. Abnormal autophagy disrupts bone homeostasis, and proper regulation of autophagy is essential for maintaining normal cellular metabolism and function [40]. p62, also known as Sequestosome1, is a marker protein that reflects autophagic activity [41]. P62/SQSTM1 is an autophagic selective substrate that is severely consumed when autophagic vesicles bind to lysosomes to degrade their contents [42] and act as an autophagic adaptor. Microtubule-associated protein LC3 regulates autophagosome formation. Under normal conditions, LC3 exists as LC3Ⅰ, and when autophagy is activated, a conversion of LC3Ⅰ to LC3Ⅱ can be observed [43], with a gradual increase of LC3Ⅱ/Ⅰ [44]. Beclin1 is another essential protein in autophagosome formation [45], and it is not only a component of the PI3K complex but also a principal autophagy regulator [46]. Thus, during autophagy activation, Beclin1 and LC3II are upregulated, while p62 decreases; conversely, inhibition of autophagy leads to a decrease in Beclin1 and LC3II and an accumulation of p62. Beclin-1 induces RANKL-mediated Atg activation and osteoclast differentiation through ubiquitination during the differentiation of osteoclast progenitor cells into osteoclasts [47]. Deletion of LC3- II leads to the formation of disordered actin structures that severely impair the migratory ability of osteoclasts [48]. Enhanced osteoclast-mediated bone resorption is critical in the early stages of postmenopausal osteoporosis and rapid glucocorticoid-induced bone loss, suggesting that inhibition of osteoclastic autophagy activity may be potent in reversing bone loss. Results from WB and IF revealed a decrease in Beclin1, LC3II/I, and an accumulation of p62. HE and Trap staining also observed that treatment with QR resulted in a tidier arrangement of bone trabeculae, a marked decrease in fat vacuoles and osteoclasts, and an increase in osteoblasts. Rat skeletal pathology was repaired, and bone metabolism remained balanced. However, there was no discernible difference in the protein expression of p62 between the QR and OVA groups, probably because genes related to autophagy can be induced by various triggers of cellular stress, such as oxidative stress, estrogen, and endoplasmic reticulum contingency.

There are some limitations in the study. Due to the limited sample size, the comparison of the effect of QR at different doses and the detection of PMOP markers such as alkaline phosphatase were not performed. The therapeutic effect of quercetin on PMOP was confirmed only from a structural aspect, while no analysis of bone mechanical properties was performed, and further exploration remains to determine whether quercetin can improve bone function.

In conclusion, QR reduces estrogen-deficiency-induced bone loss and protects bone mass in OVA rats with effects comparable to those of ALN, possibly by inhibiting NLRP3-mediated production of inflammatory factors and suppressing osteoclast autophagy. Our findings confirm the potential of QR as a natural product to prevent and treat bone loss in the hope of improving the long-term stability of implant prostheses in PMOP patients.

PMOP Postmenopausal patients with osteoporosis

OP Osteoporosis

QR Quercetin

ALN Alendronate

OVA Ovariectomy

SHAM Sham-operated group

CQ Chloroquine

M MCC950

NLRP3 Nucleotide-binding oligomerization domain-like receptor protein 3

IOF The International Osteoporosis Foundation

CTX-1 Cross-linked C-telopeptide of type 1 collage

PINP Propeptide of type I procollagen

BV/TV Bone volume fraction

Tb. Th Bone trabecular thickness

Tb. Sp Bone trabecular separation

Tb. N Trabecular number

BMD Bone mineral density

HE Hematoxylin-eosin

TRAP Anti-tartrate acid phosphatase

Acknowledgements

We are grateful to the Key Laboratory of Molecular Biology and Animal Laboratory of Guizhou Medical University for providing us with experimental space.

Author contributions

LJ, XY, and HCW designed and conceived the experiments, and confirmed the authenticity of all original data. XY, HCW, SC, and PL performed the experiments, and XY and HCW drafted the manuscript. CYT, SC, PL, and LR analyzed the experimental data. LJ, XY, HCW, CYT, and LR reviewed and revised the manuscript. All authors read and approved the final manuscript.

XY and HCW Contributed equally.

Funding

The work was supported by the Prosthodontics and Implantology, School/Hospital of Stomatology, Guizhou Medical University, in Guizhou, China, and this research was funded by the Natural Science Foundation of China (grant no. 82060207).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

The animal experiments were carried following approval by The ethics committee of Guizhou Medical University (approval no. 2201633).

Consent for publication

Not applicable.

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Author details

¹Department of Prosthodontics and Implantology, School/Hospital of Stomatology, Guizhou Medical University, Guiyang 550004 Guizhou, China ²Aosi car dental, Shantou 515041, Guangdong, China ³Guizhou Medical University, Guiyang 550004, Guizhou, China

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No competing interests reported.

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Quercetin alleviates osteoporosis in rat mandibles induced by ovariectomy through modulation of autophagy and inhibition of NLRP3 pathway

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Abstract

Figures

Introduction

Materials And Methods

Chemicals

Animals

Ovariectomy procedure

Sample collection

Micro-CT scan and analysis of rat mandible

Real-time fluorescence quantitative PCR to measure the expression of Caspase-1 and NLRP3 mRNA

Western blot detection of NLRP3 pathway and autophagy-related protein expression in rat mandibular bone tissue

Immunofluorescence assay

Histopathological changes of rat mandibles observed by HE, and Trap staining

Statistical analysis

Results

Effect of ovariectomy and treatment on changes in body weight of rats

Effect of ovariectomy and treatment on mandibular bone and bone trabecular parameters

Effect of oophorectomy and treatment on serum biochemical markers of bone metabolism

Immunofluorescence

Effects of QR on histopathological changes in the mandibular alveolar bone of OVA rats

Impact of QR on osteoclasts in the mandibular alveolar bone of OVA rats

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

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