
==== Front
J Orthop Surg Res
J Orthop Surg Res
Journal of Orthopaedic Surgery and Research
1749-799X
BioMed Central London

39218922
5030
10.1186/s13018-024-05030-1
Research Article
Salidroside alleviates simulated microgravity-induced bone loss by activating the Nrf2/HO-1 pathway
Wang Nan 1
Zuo Zhuan 1
Meng Tong 2
Liu Yuliang 1
Zheng Xiwei 1
Ma Yongsheng 600546@hrbmu.edu.cn

1
1 https://ror.org/05vy2sc54 grid.412596.d 0000 0004 1797 9737 Department of Orthopedic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China
2 grid.460182.9 Department of Orthopedic Surgery, Xi’an City First Hospital, Xi’an, China
2 9 2024
2 9 2024
2024
19 53111 7 2024
24 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Background

Bone loss caused by microgravity exposure presents a serious threat to the health of astronauts, but existing treatment strategies have specific restrictions. This research aimed to investigate whether salidroside (SAL) can mitigate microgravity-induced bone loss and its underlying mechanism.

Methods

In this research, we used hindlimb unloading (HLU) and the Rotary Cell Culture System (RCCS) to imitate microgravity in vivo and in vitro.

Results

The results showed that salidroside primarily enhances bone density, microstructure, and biomechanical properties by stimulating bone formation and suppressing bone resorption, thereby preserving bone mass in HLU rats. In MC3T3-E1 cells cultured under simulated microgravity in rotary wall vessel bioreactors, the expression of osteogenic genes significantly increased after salidroside administration, indicating that salidroside can promote osteoblast differentiation under microgravity conditions. Furthermore, the Nrf2 inhibitor ML385 diminished the therapeutic impact of salidroside on microgravity-induced bone loss. Overall, this research provides the first evidence that salidroside can mitigate bone loss induced by microgravity exposure through stimulating the Nrf2/HO-1 pathway.

Conclusion

These findings indicate that salidroside has great potential for treating space-related bone loss in astronauts and suggest that Nrf2/HO-1 is a viable target for counteracting microgravity-induced bone damage.

Keywords

Microgravity-induced bone loss
Salidroside
Oxidative stress
Nrf2/HO-1 pathway
Heilongjiang Province Post-doctoral Foundation GrantLBH-Z10075 issue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
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pmcBackground

Currently, with the continuous advancement of human space exploration, bone loss caused by microgravity conditions during spaceflight has emerged as a major health danger for astronauts and limits long-term space travel [1, 2]. In the microgravity environment of space, astronauts undergo severe physiological changes in their skeletal structure due to the absence of regular physical load stimuli [3, 4]. This environment perturbs the dynamic equilibrium between bone formation and bone resorption in astronauts, resulting in marked reductions in bone density. Research has demonstrated that in the microgravity environment, astronauts experience a bone loss rate of approximately 1.4–1.5% per month in the proximal femur and 0.9% in the spine [5]. Several strategies, including physical exercise, mechanical stimulation, drug interventions, and nutrition, have shown efficacy in mitigating astronaut bone loss, but these strategies have limitations [6–8]. High calcium intake and supplementation with vitamins K and D have been reported to prevent increases in serum calcium levels but have a limited impact on restoring cortical bone mass [9]. Additionally, excessive exercise may have detrimental effects on the skeletal system [10]. Hence, it is imperative to investigate innovative pharmaceuticals for the prevention and therapy of bone loss due to spaceflight.

In recent years, numerous traditional Chinese herbs and natural compounds have demonstrated efficacy in preventing and treating bone loss [11]. Notably, icariin, Salvia miltiorrhiza, and Astragalus roots have garnered increased amounts of attention in this field. The significant therapeutic effects of these agents, coupled with their low cost and minimal side effects, render them promising for use in the treatment of osteoporosis. SAL, a chemical compound classified as a phenylpropanoid glycoside, is extracted from the roots of the plant species rhodiola rosea [12]. SAL exhibits many pharmacological benefits, such as antioxidant properties, anti-inflammatory effects, and cardio-cerebrovascular protection [13–15]. Recent research indicates that SAL has a positive impact on preventing bone loss in ovariectomized rats [16]. In vitro, SAL alleviated the suppressive effects of dexamethasone on bone formation through the transforming growth factor-beta/Smad2/3 signalling pathway [17]. However, the impact of SAL on bone loss induced by microgravity has not been investigated.

In this research, we utilized respectively hindlimb unloading (HLU) and the Rotary Cell Culture System (RCCS) to mimic microgravity both in vivo and in vitro [18]. Our objective was to demonstrate the efficacy of SAL to mitigate bone loss in HLU rats by observing changes in bone structure and metabolism. Furthermore, we examined the impact of SAL on osteoblast differentiation in MC3T3E1 cells under simulated microgravity in vitro, thereby evaluating its effectiveness in mitigating bone loss under simulated microgravity; additionally, we explored its mechanism of action by inhibiting relevant receptors, proposing a viable candidate drug to treat bone loss associated with spaceflight.

Materials and methods

Animals and treatment

Eighteen healthy male SD rats (200 ± 20 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Liaoning, China). The animals were grown under optimal temperature conditions, accompanied by a 12-hour alternating pattern of light and darkness. During the experiment, the rats were given normal rodent feed and water. After seven days of adaptation, the hind limbs of the rats were suspended. The rats were allocated randomly into three groups: the control group, the HLU group, and the HLU + SAL group (30 mg/kg/day); each group consisted of six rats. SAL (C14H20O7, 10338-51-9, > 98% purity) was purchased from Chengdu Pfield Biotechnology Co., Ltd. The HLU + SAL group was gavaged once daily for four weeks, while the control group and HLU group were orally administered the same amount of normal saline at the same frequency. The rats were weighted weekly to assess changes and euthanized after four weeks of hindlimb suspension.

HLU rat model

The hind limbs of the rats were unloaded following the Morey-Holton and Globus method [19]. It involved wrapping adhesive surgical tape around the rat’s tail, approximately 5 cm from its base. Subsequently, a slender metal chain was attached to the tape, with the other end fixed to a freely movable support on the cage’s top. This construction maintained the rat at a 30° head-down angle to simulate microgravity. It was imperative to regularly check the tape around the tail area to prevent any tightness, thereby preventing tail damage. All rats subjected to tail suspension were adjusted weekly to ensure that their hind limbs did not contact the ground.

Preparation with Cytodex 3

The dried Cytodex-3 microcarriers (MCs) were soaked in phosphate-buffered saline (PBS) without calcium or magnesium ions. Then, the blend was left to incubate at ambient temperature for more than 3 h to facilitate expansion. Following incubation, the supernatant was discarded, and the MCs were thoroughly washed three times with an equal volume of PBS that didn’t involve calcium or magnesium. The wash solution was replaced with fresh PBS lacking calcium and magnesium ions. Subsequently, the microcarriers were subjected to high-pressure sterilization for 20 min. After removing the supernatant, the MCs were incubated with osteogenic induction differentiation culture medium and kept at 4 °C.

Cell culture and study design

MC3T3-E1 cells were acquired from Suzhou Haixing Biotechnology Co., Ltd. (Jiangsu, China). The cells were cultured in alpha minimum essential media (α-MEM) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin‒streptomycin (Gibco, China) at 37 °C with 5% CO2 in a humidified incubator. The cell culture media was replaced every 2 days. During the induction of osteoblast differentiation, an osteogenic induction differentiation kit (MUXMT-90021, Opicell, China) was used to prepare the osteogenic induction medium pursuant to the manufacturer’s guidelines.

Under simulated microgravity conditions, MC3T3E1 cells were treated with MCs or MCs + SAL (20 µM) or MCs + SAL + ML385 (10 µM) for 72 h. Subsequently, the cells were harvested from the cell-microcarrier complexes by protease digestion. All cell experiments were repeated three times.

In vitro-simulated microgravity

Under the simulated microgravity conditions created by the RCCS, microcarriers and cells were cocultivated in a 10 ml rotary wall vessel bioreactor. This bioreactor was filled entirely with osteogenic differentiation induction medium to prevent air bubbles from stimulating mechanical unloading effects. Initially, the apparatus was incubated at 37 °C in an incubator with 5% CO2 for 1 h without rotation. Following this, the cells acquired the opportunity to adhere to the microcarrier beads. Subsequently, the apparatus was rotated around a horizontal axis perpendicular to the gravitational vector and gradually adjusted to 12–15 rpm, generating microgravity conditions ranging from 10 − 2 G to 10 − 3 G. For comparative purposes, a control group was maintained under identical environmental conditions but without rotation.

Microcomputed tomography (micro-CT) scanning

The left femur, which was immobilized with 4% paraformaldehyde, was positioned and secured on carbon fibre boards. The carbon fibre board was subsequently inserted into the machine. The left femurs were scanned with an Inveon micro-CT (Siemens, Munich, Germany). The scanning parameters were set at 80 kVp for voltage, 500 µA for current, and 9.41 μm for image pixel size. After data acquisition, reconstruction was carried out using Inveon Acquisition Workplace. The reconstructed data were imported into Inveon Research Workplace for analysis. Subsequently, starting from point 100 continuous slices below the distal growth plate of the femur, the region with a height of 2 mm was designated the volume of interest (VOI) for subsequent data processing. The 3D metrics obtained by analysing the VOIs for the trabecular bone were as follows: the bone volume per total volume (BV/TV, %), trabecular number (Tb.N, 1/mm), trabecular thickness (Tb.Th, mm), trabecular separation (Tb.Sp, mm) and cortical thickness (Cr.Th, mm).

Three-point bending mechanical test

Initially, the right femur stored at -20 °C was thawed in physiological saline solution for 3 h at room temperature. Subsequently, it was positioned on a universal material testing machine (AGS-X1KN, Japan) with a 16 mm span, and a three-point bending apparatus was used. A load was centrally applied to the femoral shaft at a 2 mm/min compression rate. The aim of this procedure was to evaluate the femur’s mechanical properties up to the point of fracture. The resulting load‒deformation curve was then analysed to calculate the mechanical properties, including the maximum load (N), stiffness (N/mm), maximum stress (MPa), and Young’s modulus (GPa).

Biochemical serum analysis

Blood was collected through transabdominal aortic puncture and subsequently centrifuged at 3500 rpm for 15 min to isolate the serum. The levels of bone turnover biochemical markers, including PINP, BGP, and TRACP5b, were assessed using rat ELISA kits (Jianglai, China). All testing procedures strictly followed the manufacturer’s recommendations.

Intracellular ROS measurement

The reactive oxygen species (ROS) levels in MC3T3-E1 cells were assessed using a reactive oxygen species assay kit (Beyotime, China). After removing all cells from the rotating-wall vessel bioreactors and separating them from the microcarriers according to the instructions, the cells from each group were collected and suspended in prediluted DCFH-DA (10 µM). This suspension was incubated for 20 min at 37 °C in a cell culture incubator with thorough mixing. Afterwards, the cells received three cycles of washing with serum-free culture media in order to eliminate extracellular DCFH-DA. After washing, the cells were observed using a fluorescence microscope, and the intensity of fluorescence was quantitatively analysed using ImageJ software.

Western blot

Total protein was extracted from lysate buffer with protease inhibitor (Beyotime, China), and the protein concentration was detected via the BCA technique. The total protein was fractionated using 8–12% SDS‒PAGE and subsequently transferred to a PVDF membrane. The membrane was cut horizontally into strips to identify proteins with varying molecular weights. The membranes were incubated with primary antibodies against the following proteins overnight at 4 °C: RUNX2 (1:500; Proteintech, China), Col1a1 (1:2000; Proteintech, China), HO-1 (1:2000; Proteintech, China), and Nrf2 (1:1000; ABclonal, China). Polyclonal goat anti-rabbit IgG (1:2000; ORIGen, China) or goat anti-mouse IgG (1:2000; ORIGen, China) was mixed and incubated with horseradish peroxidase (HRP) at room temperature for 1 h. An ECL chemiluminescent substrate was used in the assay system to detect the immunoreactive bands. The proteins were quantified using ImageJ software.

Statistical analysis

Statistical analysis and graphing were conducted using GraphPad Prism 9.5.0. All the data are presented as the means ± standard deviations from a minimum of three separate experiments. Statistical significance was assessed by a two-tailed t test or one-way ANOVA. P < 0.05 was considered to indicate statistical significance.

Results

Body weights

As revealed in Fig. 1, there were no noticeable variations in the initial body weights across all groups. The body weights of rats in all groups had comparable increase rate throughout the initial one week. From the second week when the animals were tail-suspended, the body weights in the HLU and HLU+SAL groups exhibited significant decreases compared with the control group, whereas the body weights showed no significant differences between the HLU and HLU+SAL groups. These results demonstrate SAL has no effect on the development of rats.

Fig. 1 Body weight changes in the three groups of rats. The results are presented as the means ± SDs (**p < 0.01, ***p < 0.001 vs. the control group; n = 6)

SAL mitigates alterations in the microarchitecture of the distal femur and BMD in hindlimb-unloaded rats

Micro-CT was utilized to evaluate microarchitectural changes in the distal of the femur as an indicator of bone structure. Three-dimensional CT scans of the left distal femur from the three rat cohorts are depicted in (Fig. 2A-C). In comparison to the control group, the HLU group exhibited significant reductions in BV/TV, Tb.N, Tb.Th, and BMD (Fig. 2D-F, H); however, there was a considerable rise in Tb.Sp (Fig. 2G). These findings indicate that hindlimb unloading causes substantial impairment of the microarchitecture of cancellous bone and leads to a decline in bone mass. Conversely, treatment with SAL markedly elevated the trabecular bone metrics, including BV/TV, Tb.N, Tb.Th, and BMD, while the Tb.Sp was reduced contrast to that in the HLU group (Fig. 2D-H). These data indicate that SAL effectively mitigates the adverse effects on the trabecular microstructure and bone mass caused by hindlimb unloading. Additional analysis of the cortical bone revealed a notable decrease in Cr. Th of the HLU group contrast to that of the control group (Fig. 2I). However, Cr. Th in the SAL + HLU group did not significantly increase (Fig. 2I), suggesting that SAL had a modest ability to restore cortical damage caused by hindlimb unloading.

Fig. 2 Effects of salidroside on bone microstructure and BMD. (A) 3D-micro-CT images of the trabecular bone of distal femurs. (B) Section images of the trabecular bones of distal femurs. (C) 3D-micro-CT images of cortical bone in femurs. Quantitative analysis of (D) bone volume per total volume (BV/TV), (E) trabecular number (Tb.N), (F) trabecular thickness (Tb.Th), (G) trabecular spacing (Tb.Sp), (H) BMD and (I) cortical thickness (Cr.Th) of bone. The results are presented as the means ± SDs (**p < 0.01, ***p < 0.001 vs. the control group; #p < 0.05, ###p < 0.001 vs. the HLU group; n = 4)

SAL rescues the decreased mechanical characteristics of the femur in hindlimb-unloaded rats

To elucidate the impact of SAL on the mechanical strength and structural attributes of rat femurs, we employed a three-point bending experiment. The HLU group exhibited a pronounced decrease in biomechanical metrics, including maximum stress, maximum load, Young’s modulus, and stiffness, in contrast to the control group (Fig. 3A-D). In contrast, intervention with SAL effectively reversed the mechanical property impairments induced by HLU. This effect manifested as significant reductions in the maximum stress, stiffness, and Young’s modulus (Fig. 3A-C), although the increase in the maximum load was not statistically significant (Fig. 3D). These results demonstrate the ability of SAL to significantly ameliorate the reductions in bone strength and structure caused by hindlimb unloading.

Fig. 3 Effects of salidroside on bone biomechanics. The following parameters were evaluated in the three groups of rats: (A) maximum stress, (B) Young’s modulus, (C) stiffness, and (D) maximum load. The results are presented as the means ± SDs (***p < 0.001 vs. the control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. the HLU group; n = 6)

SAL alleviates bone loss in hindlimb-unloaded rats by enhancing bone formation and reducing bone resorption

We evaluated bone metabolism among various rat groups by quantifying serum bone turnover biomarkers. Relative to the control group, the HLU group exhibited a substantial reduction in the levels of serum bone formation indicators, such as PINP and BGP, as well as a significantly rise in the level of the bone resorption marker TRACP-5b (Fig. 4A-C). However, after the administration of SAL, the levels of PINP and BGP were effectively restored in the SAL + HLU group, and the elevated TRACP-5b levels induced by hindlimb unloading were significantly decreased (Fig. 4A-C). These findings suggest that SAL effectively alleviates bone loss resulting from hindlimb unloading by simultaneously enhancing bone formation and reducing bone resorption.

Fig. 4 Effect of salidroside on bone turnover markers. The bone turnover biomarker levels in the three groups of rats were as follows: (A) PINP, (B) BGP, and (C) TRACP 5b. The results are presented as the means ± SDs (***p < 0.001 vs. the control group; #p < 0.05, ##p < 0.01 vs. the HLU group; n = 6)

SAL alleviates the increase in intracellular ROS levels induced by microgravity

The increase in intracellular ROS levels induced by microgravity is a significant cause of bone loss. Fluorescence staining with DCFH-DA demonstrated that SAL markedly elevated the levels of ROS in MC3T3-E1 cells (Fig. 5A). The quantitative analysis, revealed a substantial increase in ROS levels in MC3T3-E1 cells under simulated microgravity conditions in contrast with those in the control group (Fig. 5B). Conversely, SAL therapy markedly reduced the increase in ROS levels. However, subsequent treatment with ML385 re-elevated ROS levels. These results demonstrate that the rise of intracellular ROS levels under microgravity can be reduced via SAL.

Fig. 5 Effect of salidroside on MC3T3-E1 cellular ROS levels under microgravity conditions. (A) Intracellular ROS levels in MC3TE-E1 cells (B) and quantitative results. The results are presented as the means ± SDs (***p < 0.001 vs. the control group, ###p < 0.001 vs. the MG group, ^^p < 0.01 vs. the MG + SAL group, n = 3)

SAL protects against the downregulation of osteogenic marker expression induced by microgravity

Increased oxidative stress hinders the osteoblastic differentiation process and the bone formation. As shown in Fig. 6, under simulated microgravity conditions, the expression levels of the osteogenic-related proteins Col1a1 and RUNX2 in MC3T3-E1 cells were markedly lower than those in the control group. This observation demonstrated that osteogenic differentiation in MC3T3-E1 cells was suppressed under microgravity conditions. Conversely, the administration of SAL to MC3T3-E1 cells significantly elevated the levels of the Col1a1 and RUNX2 proteins, indicating that SAL can counteract the suppression of osteogenic differentiation caused by microgravity.

Fig. 6 Effect of salidroside on osteogenic markers in MC3T3-E1 cells under microgravity conditions. Western blot and quantitative analysis of Runx2 and Col1a1 expression. The values are shown as the means ± SDs (***p < 0.001 vs. the control group, ##p < 0.01, ###p < 0.001 vs. the MG group, n = 3)

SAL alleviates microgravity-induced oxidative stress by activating the Nrf2/HO-1 signalling pathway

As a direct downstream pathway of ROS, nuclear factor erythroid-related Factor 2 (Nrf2) is a regulatory factor that controls cellular redox homeostasis [20]. As depicted in Fig. 7, Western blot experiments revealed a significant decrease in the protein expression of Nrf2 and HO-1 under simulated microgravity conditions in contrast with that in the control group. SAL notably enhanced the intracellular protein levels of Nrf2 and HO-1. To verify the modulatory impact of SAL on the Nrf2/HO-1 signalling pathway, we used ML385, a selective inhibitor of the Nrf2 signalling pathway. ML385 markedly attenuated the protein expression of Nrf2 and HO-1 compared with that in the MG+SAL group. These findings suggest that SAL mitigates microgravity-induced oxidative stress by stimulating the Nrf2/HO-1 signalling pathway, consequently exerting bone-protective effects.

Fig. 7 Effect of salidroside on the Nrf-2/HO-1 axis in MC3T3-E1 cells under microgravity conditions. Western blot and quantitative analysis of Nrf2 and HO-1 expression. The values are shown as the means ± SDs (***p < 0.001 vs. the control group, ##p < 0.01 vs. the MG group, ^^p < 0.01 vs. the MG + SAL group, n = 3)

Discussion

In the realm of manned space exploration and advancing space life sciences, microgravity-induced bone loss has garnered increasing scientific attention [21, 22]. Despite this interest, the development of effective pharmacological interventions for bone loss during space missions remains an unmet challenge. In this study, we used HLU and RCCS to establish in vivo and in vitro simulated microgravity environments. Our findings present the first definite evidence supporting the efficacy of SAL in mitigating microgravity-induced bone loss. Furthermore, this study revealed that SAL can modulate the Nrf2/HO-1 pathway to ameliorate the cellular oxidative stress induced by microgravity. This oxidative stress reduction facilitates osteoblast differentiation and mitigates microgravity-induced bone loss. These findings not only indicate that SAL shows promise as a therapeutic agent for microgravity-induced bone loss but also contribute novel insights into the pathogenesis of osteoporosis in microgravity environments.

SAL, as a naturally occurring small molecule compound, has been demonstrated to be an efficacious drug for regulating bone metabolism. Fu et al. demonstrated the regulatory effects of SAL on bone metabolism under physiological condition. They discovered that SAL exerts its function by modulating AMPK to enhance bone formation in MC3T3-E1 cells and suppress bone resorption in mouse osteoclasts [23]. Homeostasis of bone mass is maintained by the dynamic balance between bone resorption by osteoclasts and bone formation by osteoblasts. Hence, we infer that salidroside is beneficial to bone mass accumulation under physiological condition. In addition, previous research has established the efficacy of SAL in treating both glucocorticoid-induced and postmenopausal osteoporosis [24]. However, there is no direct evidence of its protective role in mitigating microgravity-induced bone loss. In our study, animal experiments suggested that SAL was crucial for preserving bone mass in rats with hindlimb unloading by improving bone density, the microarchitecture of trabeculae, and biomechanical properties. Furthermore, the evaluation of serum bone turnover biomarkers has revealed the underlying mechanisms through which SAL mitigates bone damage under microgravity conditions. Serum biochemical analyses of HLU rats revealed a notable elevation in indicators of bone resorption and a reduction in markers of bone formation in comparison to the control group. This phenomenon was likely a consequence of insufficient loading, which promoted an increase in osteoclasts and a reduction in osteoblasts, thereby disrupting the dynamic equilibrium between bone resorption and formation. Consequently, this imbalance leads to the destruction of bone structure and bone depletion [25]. However, after treatment with SAL, the rats exhibited enhanced bone formation and suppressed bone resorption. These findings indicate that SAL potentially counters bone loss induced by hindlimb unloading via the dual mechanisms of promoting osteogenesis and inhibiting osteoclastic activity. Furthermore, SAL upregulated the level of RUNX2 and Col1a1 in MC3T3-E1 cells under simulated microgravity. Collectively, our in vitro and in vivo findings demonstrate the potential of SAL in reducing bone loss associated with microgravity conditions.

We subsequently investigated the potential mechanisms through which SAL alleviates bone loss under simulated microgravity conditions. Evidence from space missions and terrestrial simulation models suggests that bone loss in both humans and animals, induced by spaceflight or simulated microgravity, is related to oxidative stress [26–28]. Oxidative stress, typically characterized by excess free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), is recognized as an independent risk factor for bone-related illnesses, particularly the onset of osteoporosis [29]. Excessive ROS not only inhibit osteogenic differentiation and accelerate osteoblast apoptosis but also escalate bone resorption and the creation of osteoclasts. This disruption in skeletal homeostasis culminates in profound bone loss. Intriguingly, our research revealed that the levels of ROS in MC3T3-E1 cells under simulated microgravity were notably increased in contrast with those in the control group, while the levels of Col1a1 and RUNX2 was markedly decreased. The results are consistent with the findings of Sun et al., who detected that oxidative stress was present in the femur and lumbar spine of rats subjected to simulated microgravity [30]. Collectively, these results reveal that microgravity-induced bone loss is closely linked to oxidative stress. SAL, recognized as an endogenous antioxidant, has been previously reported to ameliorate radiation harm by decreasing oxidative stress in the submandibular gland [31]. Furthermore, recent investigations have verified that SAL relieves oxidative stress in dry eye syndrome through activating autophagy via the AMPK-Sirt1 pathway [32]. However, the potential antioxidative effects of SAL in the context of bone loss under microgravity conditions have not yet been explored. Our research revealed that treatment with SAL markedly mitigated the increase in ROS levels induced by microgravity and enhanced the expression of relevant bone metabolism indices. These observations demonstrate that SAL counteracts microgravity-induced bone loss by inhibiting oxidative stress. Nrf2 is a pivotal transcriptional regulator of oxidative stress-responsive genes that rapidly initiates transcription within seconds following gravitational load alterations [33]. Similar experiments in spaceflight have demonstrated that other tissues, such as the myocardium, exhibit reduced Nrf2 expression levels in microgravity, which subsequently recovers postlanding [34]. In our study, we observed marked reductions in the levels of Nrf2 and HO-1 protein expression in MC3T3-E1 cells under simulated microgravity compared to those in the control group. The result suggested that the expression of Nrf2 was inhibited under simulated microgravity conditions. Previous research has shown that Nrf2 can regulate bone remodeling by influencing antioxidant reactions in osteoclasts and osteoblasts [35, 36]. Therefore, the inhibition of Nrf2 under microgravity conditions may disrupt the bone balance and lead to bone loss. In light of this, we conclude that Nrf2 may be a potential target for oxidative stress in osteoblasts under microgravity conditions.

The Nrf2 signalling pathway is crucial for the antioxidative effects of SAL in treating diseases. Prior research has demonstrated that SAL mitigates the ailments caused by oxidative stress, like pulmonary fibrosis, myocardial injury, and cerebral ischaemia/reperfusion damage by enhancing the transcription of Nrf2-regulated genes [37–39]; it reduces excessive ROS and ameliorates mitochondrial dysfunction. Additionally, SAL can also relieve acetaminophen-induced hepatotoxicity through the Nrf2 pathway [40]. Hence, to clarify whether SAL inhibits oxidative stress in microgravity-induced bone loss via Nrf2 pathway activation, we employed the Nrf2 inhibitor ML385 and observed that the therapeutic effect of SAL was significantly reduced by ML385 treatment. This observation confirmed that SAL exerts its antioxidative effects via the Nrf2/HO-1 signalling pathway. SAL triggers the translocation of Nrf2 to the cell nucleus, and accumulated Nrf2 in the nucleus binds to antioxidant response elements (AREs), thereby enhancing the expression of the downstream antioxidative factor HO-1 [41, 42]. This process rejuvenates antioxidative enzyme activity, reduces the buildup of ROS, and mitigates microgravity-induced oxidative stress in osteoblasts [43]. Hence, SAL exerts its antioxidative effects via the Nrf2/HO-1 signalling pathway. Additionally, mitochondrial oxidative stress is implicated in mediating iron-induced ferroptosis, and the Nrf2 pathway has been recognized as a crucial mediator linking oxidative stress to ferroptosis. Recent research has indicated that microgravity conditions can induce ferroptosis in osteoblasts [44]. Based on these findings, we propose that SAL can regulate oxidative stress in osteoblasts and inhibit ferroptosis via regulating the Nrf2/HO-1 signalling pathway, thereby alleviating bone loss induced by microgravity. However, this line of thought requires further verification in our subsequent work.

Conclusion

In conclusion, our research has demonstrated for the first time through both in vivo and in vitro osteoporosis models that SAL noticeably mitigates bone loss under microgravity conditions. Additionally, we investigated how SAL can attenuate bone loss by stimulating the Nrf2/HO-1 pathway under simulated microgravity conditions, and we speculate that this process may be related to ferroptosis. Our findings indicate that SAL represents an effective prophylactic and therapeutic agent against microgravity-induced bone loss and has potential as a herbal remedy for long-duration spaceflight.

Acknowledgements

We are particularly grateful to all the people who have given us help on our article.

Author contributions

WN and ZZ designed the study and performed the experiment, MT collected the data, ZXW analyzed the data, WN and LYL prepared the manuscript, MYS supervised this study, provided valuable suggestions and revisions. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Heilongjiang Province Post-doctoral Foundation Grant (LBH-Z10075).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

All the animals were handled in compliance with protocols established by the Institutional Animal Care and Use Committee (IACUC) of the First Affiliated Hospital of Harbin Medical University (IACUC permission number: 2,023,061). All animal experiments adhered to instructions in compliance with the statutes for the management of experimental animals.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Abbreviations

BGP Bone Gla-Protein

BMD Bone Mineral Density

BV/TV Bone Volume Per Total Volume

Cr.Th Cortical Thickness

HLU Hindlimb Unloading

HO-1 Heme Oxygenase 1

Nrf2 Nuclear factor erythroid 2-related factor 2

PINP Procollagen I N-Terminal Propeptide

RCCS Rotary Cell Culture System

SAL Salidroside

Tb.N Trabecular Number

Tb.Sp Trabecular Separation

Tb.Th Trabecular Thickness

TRACP5b Tartrate Resistant Acid Phosphatase 5b

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