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Asperuloside alleviates osteoporosis by promoting autophagy and regulating Nrf2 activation

Journal of Orthopaedic Surgery and Research volume 19, Article number: 855 (2024) Cite this article

Abstract

Background

Osteoporosis is a metabolic bone disease that has a common occurrence in postmenopausal women. Asperuloside (ASP) has been reported to exert anti-inflammatory and anti-oxidative effects in numerous diseases, such as rheumatoid arthritis and acute lung injury. However, whether ASP plays a role in osteoporosis has not been addressed.

Methods

In vivo, ovariectomy (OVX) was used to induce mouse osteoporosis. Then, the mice were treated with 20 and 40 mg/kg ASP. In vitro, MC3T3-E1 cells were treated with 0, 1, 10, 20, 40 and 80 µM ASP. We chose 20 and 40 µM for further experiments due to no significant effects on cell viability.

Results

The data indicated that ASP reduced osteoporosis in OVX mice and promoted osteogenic differentiation and mineralization in MC3T3-E1 cells. In addition, we explored that ASP protected against osteoporosis via inducing autophagy and activating Nrf2.

Conclusion

ASP alleviates OVX-induced osteoporosis by promoting autophagy and regulating Nrf2 activation.

Introduction

Osteoporosis is a systemic bone metabolic disease characterized by loss of bone mass and deterioration of microarchitecture, leading to increased risks of fracture or fragile fracture [1,2,3,4]. Generally, postmenopausal women are prone to osteoporosis [4]. Osteoporosis is mainly attributed to inadequate bone formation by osteoblasts to compensate for bone resorption by osteoclasts [5, 6]. Osteoporosis therapies are divided into two classes, retarding bone resorption and stimulating bone formation [7]. Currently, commonly approved pharmacological drugs for postmenopausal osteoporosis are anti-absorption drugs, including bisphosphonates, selective estrogen receptor modulators (SERM) and the RANKL inhibitor denosumab [8,9,10]. Nevertheless, research on the treatment of osteoporosis remains a challenging topic due to variations in drug efficacy among patients and the side effects of drugs [11, 12]. Continuing to investigate the effective drugs and the underlying mechanisms is paramount for the progression of osteoporosis treatment.

Autophagy, a catabolic process of cellular self-protection, exerts a crucial role in maintaining osteoblast-osteoclast homeostasis [13, 14]. Accumulating evidence suggests that autophagy contributes to maintaining bone mass and strength and promoting the differentiation of osteoblasts [15,16,17]. Conversely, knocking out the autophagy genes results in the disruption of osteoblast mineralization and differentiation [18]. Osteoporosis is closely related to the autophagy pathway [19]. For example, Leonurine facilitates osteoblast differentiation through activating autophagy, thereby alleviating ovariectomy (OVX)-induced osteoporosis in rat bone marrow-derived mesenchymal stem cells [20]. Consistently, ginsenoside Rg3 triggers autophagy to protect rats from OVX-induced osteoporosis [21]. Notably, some bisphosphonates and SERM have been reported to regulate osteoclast or osteoblast autophagy to treat osteoporosis [22, 23]. Targeting autophagy has emerged as an essential therapy for the prevention and treatment of osteoporosis [24].

Nuclear factor erythroid2-related factor 2 (Nrf2), a member of a small family of basic leucine zipper proteins, binds to antioxidant response element sequences and activates the expression of antioxidant genes [25, 26]. Nrf2 is a key factor in osteoporosis progression. For instance, a Nrf2 activator dimethyl fumarate is found to restore the bone loss phenotype and accelerate osteocytic gene expression in OVX-treated mice [27]. Nrf2 can be activated by eldecalcitol, thereby alleviating osteoporosis [28]. Besides, the Nrf2 pathway can also be activated by melatonin to ameliorate the osteogenic capacity of MC3T3-E1 osteoblastic cells in rats with diabetic osteoporosis [29]. Thus, it is of great significance to seek novel medicines targeting the Nrf2 pathway to treat osteoporosis patients.

Asperuloside (ASP), an iridoid glycoside, is mainly derived from Eucommiaceae and Rubiaceae [30]. ASP has excellent anti-cancer and anti-inflammatory properties [30, 31]. ASP has been reported to be involved in some diseases, such as peri-implantitis and acute lung injury [32, 33]. However, the effects of ASP on osteoporosis have not been clear. Interestingly, it has been demonstrated that ASP possesses protective effects on the colon injury in dextran sulfate sodium-induced chronic colitis mice through activating Nrf2/heme oxygenase-1 signaling pathway [34]. Therefore, the therapeutic effect of ASP on osteoporosis might be associated with the Nrf2 signal pathway.

At present, we have determined the protective role of ASP in an OVX-induced osteoporosis mouse model and elucidated the possible mechanisms related to autophagy and the Nrf2 pathway.

Materials and methods

Animals

The 8-week-old female C57BL/6 mice were housed in an atmosphere where the temperature was 22 ± 1℃, and the relative humidity was 45–55%. The mice were kept in an alternating 12-hour light/dark cycle and received food and water ad libitum for a week. The mice were randomly divided into four different groups, including the sham group, OVX, OVX + ASP-20 and OVX + ASP-40. Sham group mice underwent a sham operation without abscission of bilateral ovaria. The others were ovariectomized to induce osteoporosis. ASP (Aladdin, Shanghai, China) administration was started the next day after OVX. The mice were injected intraperitoneally once every other day for 8 consecutive weeks with 20 mg/kg and 40 mg/kg ASP and classified as OVX + ASP-20 and OVX + ASP-40 groups, respectively. Meanwhile, the same doses of physiological saline were injected intraperitoneally into the mice of the sham and OVX groups. The dosages were selected based on the previous studies [32, 33]. After the treatment, all mice were euthanized by isoflurane (3% for induction and 2% for maintenance) and exsanguination from the abdominal aorta. The blood and femur tissues were collected for subsequent experiments. All experiments were approved by the Ethics Committee of Shenzhen Third People’s Hospital and performed in accordance with the approval guidelines.

MicroCT scanning

The trabecular bones were wrapped in sealing film and mounted in the microCT system. The bones were imaged at 80 kV using the QuantumGX MicroCT imaging system (PerkinElmer, Shanghai, China). To assess bone mass, we measured the bone mineral density (BMD, g/cm3), bone volume ratio (BV/TV, %), trabecular number (Tb.N, 1/mm), bone volume (BV, mm3), trabecular separation (Tb.Sp, mm) and femur cortical thickness (ct.Th, mm). Caliper software was used to obtain and analyze the data.

Cell culture and treatment

The osteoblastic cell line MC3T3-E1 (iCell, Shanghai, China) was cultured in minimum essential medium α (MEMα; iCell, Shanghai, China) with 10% fetal bovine serum (FBS; Tianhang, Huzhou, China) in 5% CO2 at 37℃. Trypsin (Sigma, St. Louis, MO, USA) was used to digest the cells. The cells that have not yet differentiated are named parental cells. For osteogenic differentiation (od), the collected cells were maintained in a medium containing 50 µg/mL ascorbic acid (Macklin, Shanghai, China) and 10 mM β-glycerophosphate (Macklin, Shanghai, China). Next, the cells were treated with the different doses of ASP (20 µM and 40 µM), which were determined by the Cell Counting Kit-8 (CCK-8; KeyGene, Nanjing, China) below.

For exploring the effects of Nrf2 on osteogenesis by ASP, 7.5 µM Nrf2 inhibitor ML385 (Yuanye, Shanghai, China) was added to the MC3T3-E1 cells at the first half hour after administering ASP. Additionally, autophagy inhibitor 3-Methyladenine (3-MA) was used to study the effects of autophagy on osteogenesis by ASP. The cells with 40 µM ASP were treated using 5 mM 3-MA [35].

CCK-8 assay

Briefly, the cells were seeded in a 96-well culture plate (3 × 103/well) and incubated with 0, 1, 10, 20, 40 and 80 µM ASP for 7 d in 5% CO2 at 37℃, and then cultured with 10 µL CCK-8 for 2 h. The OD values at 450 nm were detected using a microplate reader (BioTek, Winooski, VT, USA).

Hematoxylin-eosin (HE) staining

The femur tissues were decalcified in 10% EDTA Na2 (Biosharp, Hefei, China). To ensure the effectiveness of decalcification, we maintained a volume ratio of 1:50 or above and replaced the decalcification solution every 3 days. When inserting needles into the tissues without obstruction, it indicates successful decalcification. The collected proximal femur tissues were fixed with 4% paraformaldehyde. The fixed tissues were dehydrated using graded alcohol dilutions. Subsequently, the tissues were embedded in paraffin blocks and sliced into 5 μm sections. The sections were stained with hematoxylin (Solarbio, Beijing, China) and eosin (Sangon, Shanghai, China). The femur tissues were observed through a microscope (Olympus, Tokyo, Japan).

Tartrate resistant acid phosphatase (TRAP) staining

TRAP staining was performed according to the manufacturer’s protocol using the TRAP staining kit (Servicebio, Wuhan, China). The kit was used to display osteoclasts in the femur tissues.

Immunofluorescence (IF) staining

IF staining was performed to assess the LC3 and Beclin1 protein expression in the trabecular bones. The 5 µm femur tissue slices were incubated with anti-mouse LC3 antibody (1:50; Santa Cruz, Dallas, TX, USA) or anti-rabbit Beclin1 antibody (1:100; Affinity, Changzhou, China) at 4℃ overnight, followed by FITC-labeled goat anti-mouse IgG (1:200; Abcam, Cambridge, UK) or goat anti-rabbit IgG (1:200; Abcam, Cambridge, UK) at 37℃ for 60 min. In addition, IF staining was used to detect Nrf2 nuclear distribution in the MC3T3-E1 cells. The cells were fixed in 4% paraformaldehyde for 15 min and permeabilized by 0.1% Triton X-100. The cells were blocked with 1% BSA for 15 min, and then incubated with anti-rabbit Nrf2 antibody (1:200; Affinity, Changzhou, China). The next day, the cells were incubated with Cy3-labeled goat anti-rabbit IgG (1:200; Invitrogen, Carlsbad, CA, USA). These femurs and cells were stained with 4’, 6-diamidino-2-phenylindole (DAPI) and observed with fluorescence microscopy (Olympus, Tokyo, Japan).

Alizarin red staining

The cells were obtained after 14 days of culture in osteogenic medium. After fixation in 4% paraformaldehyde for 15 min, they were stained with Alizarin red (Solarbio, Beijing, China) for 20 min to examine the mineralization area of osteoblasts. The cells were washed using double-distilled water for 3 times and imaged under the microscope.

ROS detection

Dihydroethidium (DHE; KeyGene, Nanjing, China) was used to determine the ROS levels. The cells were incubated with 10 µM DHE at 37℃ for 30 min in the dark. The cells were washed and subsequently photographed in microscope.

ALP activity

Biochemical markers of bone turnover, such as bone alkaline phosphatase (ALP), are reliable tools for therapy monitoring in postmenopausal osteoporotic patients [36, 37]. The activities of ALP in mouse serum and cells were tested using the alkaline phosphatase assay kit (Jiancheng Bioengineering Institute, Nanjing, China) on the basis of the manufacturer’s protocols. The protein concentration of the samples was assessed by the BCA protein assay kit (Beyotime, Shanghai, China).

Real-time PCR

Total RNA was extracted from the cells through TRIpure reagent (Bioteke, Beijing, China). Reverse transcription is constructed using BeyoRT II M-MLV reverse transcriptase (Beyotime, Shanghai, China). The expression of osterix (OSX) and osteoprotegerin (OPG) mRNA was determined by SYBR Green-based real-time PCR. The specific primers were as follows: OSX forward: 5’-GGGAAAGGAGGCACAAAGA-3’, OSX reverse: 5’-GAAATGAGTGAGGGAAGGGT-3’; OPG forward: 5’-CTTCTTGCCTTGATGGA-3’, OPG reverse: 5’-TTGGGAAAGTGGGATGT-3’. β-actin forward: 5’-CATCCGTAAAGACCTCTATGCC-3’, β-actin reverse: 5’-ATGGAGCCACCGATCCACA-3’. All mRNA levels were standardized to β-actin mRNA levels. The 2−∆∆Ct method was applied to analyze relative expression levels.

Western blot

Total protein was extracted from the proximal femur tissues and MC3T3-E1 cells by radio-immunoprecipitation assay (RIPA; Solarbio, Beijing, China) buffer containing protease inhibitor phenylmethanesulfonyl fluoride (PMSF; Solarbio, Beijing, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Solarbio, Beijing, China) was used to separate the samples, which were subsequently transferred to polyvinylidene fluoride (PVDF; Millipore, Billerica, MA, USA) membranes. The membranes were incubated with Tris Buffered Saline with Tween 20 (TBST), and then blocked by 5% (M/V) skim milk (Sangon, Shanghai, China) for 1 h. The samples were incubated with anti-rabbit primary antibody OSX (Affinity, Changzhou, China), OPG (Affinity, Changzhou, China), LC3 II/I (ABclonal, Wuhan, China), p62 (ABclonal, Wuhan, China), Nrf2 (Affinity, Changzhou, China), Keap1 (Affinity, Changzhou, China), Beclin1 (Affinity, Changzhou, China), control antibody Histone H3 (Gene Tex, CA, USA), as well as anti-mouse β-actin (Santa Cruz, Dallas, TX, USA) at 4℃ overnight. Histone H3 was diluted to 5000 fold and the others were diluted to 1000 fold with 5% (M/V) skim milk. The membranes were washed using TBST four times for 5 min. After that the samples were incubated with HRP-labeled goat anti-rabbit IgG and goat anti-mouse IgG (1:3000) from Solarbio Science & Technology Co., Ltd. (Beijing, China) at 37℃ for 1 h. The proteins were examined using enhanced chemiluminescence (ECL; Solarbio, Beijing, China).

Transmission electron microscopy (TEM)

The cells were fixed in 2.5% glutaraldehyde (Servicebio, Wuhan, China) in 0.01 M phosphate buffer at 4℃ after centrifugation and medium removal. They were rinsed with 0.1 M phosphate buffer and pre-embedded in agar. Subsequently, the cells were fixed in 1% osmic acid in 0.1 M phosphate buffer at room temperature for 2 h, and then dehydrated in a graded series of ethyl alcohol. The samples were permeated and embedded using acetone (Sinoreagent, Shanghai, China) and epoxy resin (West Chester, PA, USA). The 60–80 nm ultrathin sections were cut with a Leica UC7 Ultramicrotome (Leica, Nussloch, Germany) and collected on formvar coated copper slot grids. These slices were stained using 2% uranyl acetate and 2.6% lead citrate. Autophagosome formation was observed and analyzed by an H-7650 transmission electron microscope (Hitachi, Tokyo, Japan) at 20,000 × and 40,000 × magnification.

Statistical analysis

All values are presented as the means ± SD. The GraphPad Prism software 8.0.2 was used for statistical analysis. The differences between multiple groups were analyzed by a one-way ANOVA. *P < 0.05 represents a statistically significant difference. The data have been checked for normalcy and variance.

Results

ASP protects the mice from OVX-induced osteoporosis

MicroCT images showed that OVX caused bone loss, which was prevented by ASP treatment in the mice (Fig. 1A). High-dose ASP (40 mg/kg) has a better restraining effect on OVX-stimulated osteoporosis than low-dose ASP (20 mg/kg). Quantitative analysis of BMD, BV/TV, Tb.N, BV, Tb.Sp and ct.Th further confirmed the results (Fig. 1B). In addition, ASP constricted the effect that OVX inhibited ALP activity in mouse serum (Fig. 1C). ASP elevated the decreased protein levels of OSX and OPG induced by OVX in the proximal femurs (Fig. 1D). HE staining to some extent reflected that OVX reduced the number of osteoblasts and increased the number of osteoclasts, which were reversed by ASP in the mice (Fig. 1E). TRAP staining revealed that ASP diminished OVX-caused increased osteoclasts in the femurs (Fig. 1F). These results suggested that ASP alleviated OVX-caused bone loss in mice.

Fig. 1
figure 1

ASP displayed a protective effect in OVX-induced osteoporosis mice. (A) Representative microCT images of each experimental group femur. (B) Bone parameters including BMD (g/cm3), BV/TV (%), Tb.N (1/mm), BV (mm3), Tb.Sp (mm) and ct.Th (mm) in these femurs. n = 6. (C) The ALP activity in serum was detected to assess osteoblastic differentiation. n = 6. (D) Western blot assays quantified the protein levels of OSX and OPG in the femurs. n = 3. (E) HE staining of femur trabecular bone. Scale bar = 50 μm. (F) TRAP staining of femur trabecular bone. Scale bar = 100 μm. “ns” represents no significance. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. the OVX group

Full size image

ASP accelerates autophagy and regulates Nrf2 activation in OVX-induced osteoporosis mice

As shown in Fig. 2A and B, IF results revealed that OVX decreased the protein levels of the autophagy-related protein LC3 and Beclin1, whereas ASP increased them in the OVX mouse model. Furthermore, western blot assay displayed that ASP reversed the OVX-caused changes in the LC3 II/I and p62 levels in the proximal femur tissues (Fig. 2C). It was observed that ASP treatment restored down-regulated Nrf2 expression in the nuclear and up-regulated Nrf2 expression in the cytoplasm in the OVX mice (Fig. 2D). These results demonstrated that ASP was capable of promoting autophagy and regulating Nrf2 activation in OVX-treated mice.

Fig. 2
figure 2

ASP facilitated autophagy and regulated Nrf2 levels in OVX-treated osteoporosis mice. (A) The autophagy-related protein LC3 and (B) Beclin1 was examined using IF staining in the mouse trabecular bones. n = 6. Scale bar = 50 μm. (C) LC3 II/I and p62 levels in the trabecular bones were determined by western blot. n = 3. (D) The expression of Nrf2 in nuclear and cytoplasm was tested in the trabecular bones. n = 3. “ns” represents no significance. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. the OVX group

Full size image

ASP facilitates osteogenic differentiation and mineralization in MC3T3-E1 cells

CCK-8 assays were performed to detect the MC3T3-E1 cell viability in the ASP treatment at different concentrations. We selected the doses of 20 µM and 40 µM ASP for subsequent experiments, which exhibited no significant impact on MC3T3-E1 cell viability (Fig. 3A). We determined cell osteogenic differentiation by measuring ALP activity, OSX and OPG levels. It was indicated that ASP markedly enhanced ALP activity in MC3T3-E1 cells in the osteogenic induction medium for 3, 5 and 7 days (Fig. 3B). The results of alizarin red staining proved that ASP administration advanced MC3T3-E1 cell mineralization at 14 days after differentiation (Fig. 3C). The augmented OSX and OPG expressions were presented after 7 days of differentiation in ASP-treated MC3T3-E1 cells (Fig. 3D and E). These results manifested that ASP played a positive role in osteogenic differentiation and mineralization in MC3T3-E1 cells.

Fig. 3
figure 3

ASP accelerated osteogenic differentiation and mineralization in MC3T3-E1 cells. (A) CCK-8 assay presented the cell viability at the different doses of ASP treatment. (B) ALP activities in osteogenic differentiation for 3, 5 and 7 days. (C) Alizarin red staining showed mineralization of the MC3T3-E1 cells. Scale bar = 200 μm. (D, E) The OSX and OPG protein and mRNA levels were tested in the cells by western blot and real-time PCR. n = 3. “Od” represents osteogenic differentiation. “ns” represents no significance. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. the parental + Od group

Full size image

ASP promotes autophagy and regulates Nrf2 activation in the osteoblast-differentiation MC3T3-E1 cells

We observed the presence of autophagic vacuoles in MC3T3-E1 cells with osteogenic differentiation for 7 days through TEM (Fig. 4A). The results reflected that ASP expedited the production of autophagic vacuoles in the cells (Fig. 4A). We found that ASP promoted the Nrf2 nuclear translocation after 7 days of MC3T3-E1 cell differentiation according to IF staining (Fig. 4B). Moreover, the Nrf2 nuclear translocation was observed after 3 and 5 days of MC3T3-E1 cell differentiation (Figure S1A). An interesting result was that as the ASP processing time increases, the Nrf2 nuclear distribution also was increased. Besides, western blot was conducted to further demonstrate that ASP drove an increase in nuclear Nrf2 expression and a decrease in cytoplasmic Nrf2 expression in the cells (Fig. 4C). Keap1 expression was reduced by ASP in the cells, suggesting that Keap1-Nrf2 pathway might be involved in the effects of ASP (Figure S2A). In addition, we revealed that ROS levels were diminished by ASP. These data suggested that ASP might also affect oxidative stress levels (Figure S2B). The ascending protein levels of LC3 II/I and Beclin1 as well as the descending p62 levels in the cells were encouraged by ASP treatment (Fig. 4D). It was indicated that ASP accelerated cell autophagy and Nrf2 nuclear translocation in the osteoblast-differentiated MC3T3-E1 cells.

Fig. 4
figure 4

ASP advanced autophagy and Nrf2 nuclear translocation in the MC3T3-E1 cells. (A) TEM images of the cells at 20,000 × showed the number of autophagosomes. Scale bar = 1 μm. (B) The nuclear distribution of Nrf2 was observed based on IF staining after 7 days of cell differentiation. Scale bar = 50 μm. (C) The nuclear and cytoplasmic Nrf2 expression levels (D) as well as LC3 II/I, Beclin1 and p62 levels of each group were tested by western blot. n = 3. “Od” represents osteogenic differentiation. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. the parental + Od group

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Autophagy is required for the effects of ASP on MC3T3-E1 cell osteogenic differentiation

As shown in Fig. 5A, the autophagy inhibitor 3-MA inhibited MC3T3-E1 cell mineralization treated with ASP at 14 days after differentiation (Fig. 5A). Moreover, 3-MA repressed ALP activity and the expression of OSX and OPG in ASP-treated MC3T3-E1 cells in the osteogenic induction medium for 7 days (Fig. 5B-C). These results indicated that the protective effects of ASP on the cells required the involvement of autophagy.

Fig. 5
figure 5

Autophagy inhibition reversed ASP-induced osteogenic differentiation and mineralization in MC3T3-E1 cells. (A) Alizarin red staining of the MC3T3-E1 cells after 14 days of cell differentiation. Scale bar = 200 μm. (B) ALP activities after 7 days of cell differentiation. (C) The OSX and OPG mRNA levels. n = 3. “Od” represents osteogenic differentiation. *p < 0.05 and **p < 0.01 vs. the Od + ASP 40 group

Full size image

Nrf2 inhibition reverses the effects of osteogenesis induced by ASP

As the TEM images show, ASP promoted the production of autophagic vacuoles, while they were eliminated using the Nrf2 inhibitor ML385 in the cells (Fig. 6A). Nrf2 inhibition blocked cell mineralization induced by ASP in the MC3T3-E1 cells with osteogenic induction for 14 days (Fig. 6B). ML385 repressed the upregulated expressions of LC3 II/I and Beclin1 induced by ASP in the cells (Fig. 5C). As expected, the activities of ALP as well as the levels of OSX and OPG were also increased in ASP-treated MC3T3-E1 cells with osteogenic induction for 7 days. However, ML385 prevented the effects (Fig. 6D and E). These results illuminated that suppressing Nrf2 restored the ASP-induced osteogenesis in the MC3T3-E1 cells.

Fig. 6
figure 6

Nrf2 inhibition reversed the effects of ASP in the osteoblast-differentiation MC3T3-E1 cells. (A) The production of autophagic vacuoles was shown using the TEM images at 20,000 × magnification. Scale bar = 1 μm. (B) The cell mineralization was presented through Alizarin red staining in the cells. Scale bar = 200 μm. (C) LC3 II/I and Beclin1 expression of the cells. (D) The activities of ALP. (E) The mRNA levels of OSX and OPG. n = 3. “ns” represents no significance. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. the parental + Od group or the Od + ASP 40 group

Full size image

Discussion

Osteoporosis poses a serious threat to human health, especially for postmenopausal women [4]. It has been presented that autophagy and Nrf2 activation are significant for the maintenance of bone homeostasis [18, 27]. Currently, the OVX-induced mouse model has been widely utilized in studying osteoporosis [38,39,40]. In this study, we applied OVX to cause mice to have insufficient estrogen levels to simulate postmenopausal women with osteoporosis. We found that ASP was capable of ameliorating OVX-caused osteoporosis in mice. In vitro, ASP expedited osteogenic differentiation and mineralization in MC3T3-E1 cells. Furthermore, we demonstrated that ASP stimulated autophagy and Nrf2 nuclear expression in the mice and osteoblast-differentiation MC3T3-E1 cells. In general, ASP might serve as a new medicine to mitigate osteoporosis by contributing to autophagy and Nrf2 activation in vivo and in vitro.

Autophagy is a lysosomal degradation pathway to degrade and recycle unnecessary organelles and proteins [41, 42]. Dysregulation of autophagy has been proven to be involved in the progression of osteoporosis [43,44,45]. It has been discovered that autophagy enhances osteoblast survival, modulates osteoblast differentiation, and improves bone strength [21, 46]. Leonurine contributes to bone marrow-derived mesenchymal stem cell proliferation and osteogenic differentiation via the activation of autophagy by the PI3K/Akt/mTOR pathway in rats [20]. Resveratrol accelerates osteoblast differentiation in rats with osteoporosis by advancing autophagy in osteoblasts [47]. Moreover, resveratrol augments mitophagy through the upregulation of sirtuin 1, thereby protecting osteoblasts in osteoporosis rats [48]. Autophagy inducer rapamycin diminishes osteoporosis through activating autophagy in osteocytes in senile rat models [49]. Low-dose dexamethasone induces osteoblast viability via autophagy [50]. Thus, targeting the activation of autophagy serves as a promising approach for preventing and treating osteoporosis. Consistent with the previous results, we observed that ASP reversed OVX-induced downregulation of Beclin1 and LC3 II/I as well as the upregulation of p62 expression, which embodied that ASP exhibited a protective function against osteoporosis by stimulating autophagy in vivo. In vitro, ASP promoted osteogenic differentiation and mineralization by encouraging autophagy in osteoblast-differentiated MC3T3-E1 cells.

Nrf2 is a nuclear transcription factor with antioxidant effects [51, 52]. Nrf2 plays a great role in bone homeostasis [53]. It has been investigated that the Nrf2 signaling pathway participates in osteoporosis development in numerous studies. Corynoline is able to attenuate osteoporosis by inhibiting osteoblast damage evoked by H2O2 via activating the Nrf2 pathway [54]. Pyrroloquinoline quinone prevents aging-induced osteoporosis by increasing Nrf2 protein levels to suppress cellular senescence and elevate osteoblastic bone formation [55]. Overall, various drugs with therapeutic effects on osteoporosis involve the activation of Nrf2. Notably, ASP is found to increase the levels of Nrf2 protein, thereby repressing dextran sulfate sodium-induced oxidative stress and inflammation in chronic colitis mice [34]. Herein, we found that ASP reversed the decreased Nrf2 nuclear expression and increased Nrf2 cytoplasmic expression provoked by OVX in mice. Furthermore, ASP facilitated Nrf2 nuclear translocation in osteogenic differentiation MC3T3-E1 cells. ASP inhibited the ROS levels, indicating that ASP might exert the roles through affecting redox status in the cells. Previous research suggests that dipeptidyl peptidase 3 maintains osteoblastic differentiation in high glucose-stimulated diabetic osteoporosis MC3T3-E1 cells, which is reversed by the blockade of Nrf2 using ML385 [56]. Consistently, we used ML385 to induce Nrf2 inhibition, which reverses the protective effects of ASP in the MC3T3-E1 cells. These findings illustrated that ASP might serve as an effective candidate for osteoporosis treatment based on the effect of Nrf2 activation. It’s interesting that oxidative stress has been reported to block osteoblast differentiation, thereby suppressing bone formation, in which the Keap1-Nrf2 signaling plays a significant role [57, 58]. In our study, ASP reduced the ROS levels in the osteogenic differentiation MC3T3-E1 cells. Keap1 expression was decreased by ASP in the cells. It was indicated that ASP might negatively regulate oxidative stress levels via modulating Keap1-Nrf2 signaling.

Our research has some limitations: For the OVX-induced mouse osteoporosis model, because mice and humans have significant physiological differences, which can result in the implementation dose and experimental results on mice not being directly translated into clinical applications. Additionally, OVX primarily affects trabecular bone in mice, which cannot reflect cortical changes observed in post-menopausal women. Further exploration is needed in the future to determine the dosage and efficacy of human medication in clinical practice; Secondly, our experiment lacks a positive control, which affects the scientific validity and credibility of the research; Thirdly, although we explored the protective effect of ASP on osteoporosis by promoting autophagy and affecting the Keap1-Nrf2 pathway, further exploration is needed for more molecular mechanisms. ASP has been reported to play a role in various diseases by affecting pathways such as NF-κB, AMPK/mTOR and NLRP3 pathway [33, 59, 60]. Further research is needed to determine which receptors are affected and which cellular functions are affected. These mechanisms may also play important roles in osteoporosis and require more research; Fourthly, this study suggested that ASP not only contributed to increase bone density but also improved bone quality, similar to the effects of known bone synthesis metabolic agents, like Abaloparatide, which has been reported to treat osteoporosis [61]. Although ASP has therapeutic potential for osteoporosis treatment, the potential side effects have not been elucidated.

Conclusion

Taken together, our present study manifested that ASP effectively reduced osteoporosis in mice, which was associated with the activation of autophagy and the Nrf2 pathway. In addition, ASP was capable of promoting osteogenic differentiation and mineralization in MC3T3-E1 cells by accelerating autophagy and stimulating Nrf2 nuclear translocation. Our study revealed that ASP served as a suitable therapeutic option for osteoporosis and the potential mechanisms.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

ASP:

Asperuloside

OVX:

Ovariectomy

Nrf2:

Nuclear factor erythroid2-related factor 2

BMD:

Bone mineral density

BV/TV:

Bone volume ratio

Tb.N:

Trabecular number

BV:

Bone volume

Tb.Sp:

Trabecular separation

ct.Th:

Femur cortical thickness

MEMα:

Minimum essential medium α

FBS:

Fetal bovine serum

CCK-8:

Cell Counting Kit-8

HE:

Hematoxylin-eosin

IHC:

Immunohistochemistry

H2O2 :

Hydrogen peroxide

BSA:

Bovine serum albumin

IF:

Immunofluorescence

DAPI:

4’, 6-diamidino-2-phenylindole

ALP:

Alkaline phosphatase

OSX:

Osterix

OPG:

Osteoprotegerin

RIPA:

Radio-immunoprecipitation assay

PMSF:

Phenylmethanesulfonyl fluoride

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

PVDF:

Polyvinylidene fluoride

TBST:

Tris Buffered Saline with Tween

ECL:

Enhanced chemiluminescence luminous

TEM:

Transmission electron microscopy

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Acknowledgements

The authors would like to thank all the staff of the participating departments.

Funding

This work was supported by the National Natural Science Foundation of China (82104657) and Shenzhen Postdoctoral Research Funding.

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Authors and Affiliations

  1. Department of Outpatient, Shenzhen University General Hospital, Shenzhen, 518055, China

    Fenglan Huang & Ye Cheng

  2. Department of Sports Medicine, Central Hospital of Dalian University of Technology, Dalian, 116021, China

    Yiteng Wang

  3. Department of Orthopedics, Shenzhen Third People’s Hospital, Shenzhen, 518112, China

    Jinzhu Liu, Xiaonan Zhang & Haoli Jiang

  4. Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging, School of Biomedical Engineering, Shenzhen University Medical School, Shenzhen, 518060, China

    Xiaonan Zhang

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  4. Ye Cheng
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  5. Xiaonan Zhang
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  6. Haoli Jiang
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Contributions

XZ and HJ contributed to the conceptualization and project administration of the study; FH wrote the manuscript; FH, YW and JL conducted the experiments; FH and YC revised the manuscript; FH, XZ and HJ analyzed the data.All authors reviewed the manuscript.

Corresponding authors

Correspondence to Xiaonan Zhang or Haoli Jiang.

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The animal experiments were approved by the Ethics Committee of Shenzhen Third People’s Hospital and performed in accordance with the approval guidelines.

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Huang, F., Wang, Y., Liu, J. et al. Asperuloside alleviates osteoporosis by promoting autophagy and regulating Nrf2 activation. J Orthop Surg Res 19, 855 (2024). https://doi.org/10.1186/s13018-024-05320-8

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Keywords

Journal of Orthopaedic Surgery and Research

ISSN: 1749-799X