SDH5 down-regulation mitigates the damage of osteoporosis via inhibiting the MyD88/NF-κB signaling pathway

Abstract Background Osteoporosis has become a serious public health problem especially in postmenopausal women. This work aims to assess both the function and mechanism of SDH5 in osteoporosis. Methods The animal model of osteoporosis in Sprague-Dawley rats was established by utilizing ovariectomy (OVX). The trabecular bone morphometry had been determined by micro-CT, and tibia injury of rats was detected through HE and alcian blue staining. Meanwhile, the levels of oxidative stress factors, including malondialdehyde, catalase, glutathione peroxidase (GSH-Px), and superoxide (SOD), were detected by ELISA. The proliferation and apoptosis of osteoblasts isolated from OVX-induced rats were found out by CCK-8 and flow cytometry, respectively. The expression of SDH5, Osterix, Type I collagen (CoL1A1), osteocalcin (OC), SOD1, SOD2, p-MyD88/MyD88, and p-NF-κB p65/NF-κB p65 was assessed by Western blot. The effect and mechanism of SDH5 knockdown on osteoporosis were verified by lipopolysaccharide treatment. Results In the osteoporosis rat model, the expression of SDH5 had an up-regulated tend. A higher bone mineral density value was found in the SDH5 knockdown group. SDH5 inhibition ameliorated bone loss, mitigated bone histopathological injury, alleviated oxidative stress, and elevated osteogenic marker protein expression in vivo and in vitro. SDH5 down-regulation also promoted the proliferation and restrained apoptosis of osteoblasts extracted from OVX-induced rats. Furthermore, we found that the underlying mechanism was associated with the inhibition of the MyD88/NF-κB pathway. Conclusion Down-regulation of SDH5 mitigates the damage of osteoporosis both in vivo and in vitro via inhibiting the MyD88/NF-κB signaling activation.


Introduction
Osteoporosis is a kind of widespread bone disease in humans. It is a disease with low bone mass and impairment of bone tissue microstructure so as to cause bone fragility, which poses a great threat to the occurrence of fractures [1]. Epidemiological studies suggest that the incidence of osteoporotic fractures may be increasing exponentially in Asia [2]. Osteoporosis is common in women, mainly in postmenopausal women. Postmenopausal osteoporosis is featured by decreased bone mineral density (BMD) and bone microstructure destruction with decreased estrogen levels [3]. The current view is that osteoporosis occurs due to an instable balance between bone formation and bone resorption [4]. The pathogenesis of bone-associated diseases is complex, involving intercellular communication between diverse cells in bone microenvironment, and is regulated by a variety of cytokines [5]. Receptor activator of NF-jB ligand signaling is critical for the proliferation and differentiation of osteoclasts [6,7]. Osteocytes regulate bone mass and homeostasis by noncell autonomous mechanisms affecting bone remodeling, that is, osteoclast-mediated bone resorption followed by osteoblast-mediated bone formation [8]. The bone remodeling without a corresponding increase in bone formation alters the bone trabecular structure and increases fragility of bones [9]. Current drugs used to treat postmenopausal osteoporosis such as bisphosphonates, estrogen, calcitonin, selective estrogen receptor modulators, and recombinant human parathyroid hormone fragments. Adverse effects, such as typical femur fracture, hypercalcemia, thromboembolism, bisphosphonate-associated osteonecrosis of the jaw, vaginal bleeding, and even breast and endometrial cancers, has greatly reduced these drugs' widespread use [10]. Therefore, safer treatment methods are eagerly needed.
Previous results have shown that oxidative stress can inhibit osteoblast differentiation, stimulate osteoclast differentiation and promote the occurrence and development of osteoporosis [11]. The superoxide produced by osteoclasts directly participates in bone degradation. Osteoclasts synthesize superoxide free radicals at the osteoclast-bone interface, and inhibiting the production of superoxide free radicals by osteoclasts can reduce bone absorption [12]. Damage to the antioxidant defense system can result in osteoporosis. The exact mechanism by which oxidative stress inhibits osteoblast differentiation and induces osteoblast apoptosis remains unclear. Research has showed that NF-jB plays a significant role in inhibiting osteoblast differentiation and apoptosis [13,14]. Although it has been reported that oxidative stress-induced osteoporosis may be related to the NF-jB pathway, the specific mechanism remains unclear [15].
Succinate dehydrogenase 5 (SDH5), also called SDHAF2 or PGL2, has been reported to affect the flavination of SDHA [16]. SDH5 gene is involved in the mitochondrial electron transport chain through its function of flavination of SDHA [17]. It has been identified to be involved in the development of several cancers and is mutated in paragangliomas and gastrointestinal stromal tumors [18,19]. However, the functional significance of SDH5 and possible molecular mechanisms in osteoporosis are still unclear.
In our work, we evaluated the function and mechanism of SDH5 in estrogen deficiency-induced osteoporosis model. Sprague-Dawley (SD) rats were applied to set up the ovariectomy (OVX) in vivo model. We assessed the BMD, bone morphometry, and several bone strength parameters in the animal model. Meanwhile, the changes in oxidative stress and osteoblast proliferation and apoptosis were evaluated. In rats with osteoporosis affected by estrogen deficiency, the trabecular bone morphometry was abnormal, bone-specific transcription factor expression was reduced, oxidative stress occurred, and the MyD88/NF-jB signaling pathway was also actuated. We also established an in vitro model using lipopolysaccharide (LPS)-induced osteoblasts isolated from rats. Notably, SDH5 knockdown mitigated these abnormalities, and LPS treatment reversed those inhibiting effects. Thus, this study indicated that SDH5 could be a new aim for osteoporosis treatment.

Animal model establishment
The study was carried out by using nonparous, female, SD rats (200-280 g, 6-8 weeks, SLAC Laboratory Animal Co., Ltd., Shanghai, China) that were housed in a tidy room (22 C-24 C, light cycle of 12/12 h) with free food and water intake. For sham-operated rats (Sham group, N ¼ 6), the ovaries were only exposed but not removed. The ovariectomized female osteoporosis rat model (OVX) was established in the remaining 18 rats. Anesthesia with 2% pentobarbital sodium was administered to each rat; an incision of 1 cm was made on either side of the spinal cord at the junction of 1 cm below the costal margin and 1 cm on either side of the ventral costal margin. Subsequently, bilateral ovaries were ligated and removed. To prevent infection, 400,000 units of penicillin were administered intraperitoneally to the rats. Lentiviral constructs carrying shRNA negative control vector or lentiviral-shRNA-SDH5 vector (GeneChem Gene Technology, Shanghai, China) were injected into ovariectomized rats using Entranster TM in vivo Infection Reagent (Engreen Biosystem, Beijing, China) following 8 weeks of surgery. To be specific, we anesthetized rats with intraperitoneal injections of 2% pentobarbital sodium (0.5 mg/kg). Then, the surgical site was disinfected with iodophor, and 20 lL of lentiviral was injected on both sides of the spine. The rats were then divided into OVX group (N ¼ 6, ovariectomized rats injected with phosphate buffer saline, PBS), OVX þ sh-NC group (N ¼ 6, ovariectomized rats injected with vector carrying lentivirus-shRNA negative control), and OVX þ sh-SDH5 group (N ¼ 6, ovariectomized rats injected with vector carrying lentivirus-shRNA-SDH5). After normal feeding for 4 weeks, tissues were brought together under anesthesia coming exsanguination. Intend to access the effect of SDH5 in osteoblasts, we conducted a LPS induction experiment to establish an LPS model of osteoporosis [20]. Rats (N ¼ 6) that were treated the same as OVX þ sh-SDH5 group were intraperitoneally injected with LPS (20 lg) after 1-week feeding. In the following 3 weeks, injections were made again three times a week. When the last injection for 3 days happened, tissues were brought together under anesthesia following exsanguination. The tibias were instantly fixed with 4% (W/V) paraformaldehyde. The whole animal experiment procedures were supported by the Ethics Committee of Karamay Central Hospital and were conducted in line with the guidelines about the care and use of laboratory animals of the International Association for the Study of Pain.

Bone mineral density
The left tibia of rats was dissected and examined to observe biomechanical properties of bone. Tibial BMD was measured using a BMD detector (Aptima Assays, Hologic Diagnostics, MA, USA) according to manual instructions [21].

Micro-computed tomography (CT) analysis
After dissection of rat tibias, the soft tissue was removed. The tibias were fixed with 4% paraformaldehyde for 24 h and fully scanned by micro-CT. Parameter Settings: voltage 60 kV, current 220 lA, exposure time 1500 ms, valid pixel size 8.89 lm [22]. Morphometric measurement of trabecular bones was assessed using high-resolution Inveon microtomography (Siemens, Munich, Germany). Further quantitative analysis was made to analyze bone volume/total volume (BV/TV), bone surface area/BV bovine serum albumin (BSA/BV), trabecular thickness (Tb. Th), trabecular bone number (Tb. N), trabecular separation (Tb. Sp), and bone surface area/BV (BS/BV) by Inveon Research Workplace. Morphometric measurements of cartilage were evaluated by applying high-resolution Inveon microtomography (Siemens, Munich, Germany).

Histological analysis
The left tibia bone of rats was placed in 10% formaldehyde fixative at room temperature for 24 h. These samples were decalcified in 10% formic acid for 28 days and regularly examined with an optical microscope. Then, the decalcified and fixed right tibias were cut into 3 lm thick pieces with a microtome. Under a light microscope, Hematoxylin and Eosin (H&E) staining and Alcian blue staining were undertaken to examine the tibial injury.

ELISA
Blood samples from experimental animals were collected in sterile tubes. The samples were then centrifuged at 4 C for 10 min at 1200 g to obtain serum. Levels of malondialdehyde (MDA), catalase (CAT), superoxide (SOD), and glutathione peroxidase (GSH-Px) in serum of ovariectomized rats were detected by different ELISA kits (R&D Systems, Minneapolis, MN, USA) following the manufacturer's instructions.

Extraction of osteoblasts
Cancellous bone was cleaned with sterile PBS under aseptic conditions for three times, chopped into bone particles of about 1 mm 3 , and rinsed with PBS until they turned white. Bone particles were separated with 0.1% collagenase in 1:10 at 37 C water bath for 20 min and then separated with collagenase again for 1 h. The obtained suspension was centrifuged at 1200 rpm/min and washed with PBS for three times. Next, the culture medium (Dulbecco's Modified Eagle's medium, Gibco, Grand Island, NY, USA) containing 15% fetal bovine serum (Gibco, Grand Island, NY, USA), penicillin (100 U/mL), and streptomycin (100 U/mL) was added to finish a suspension. Osteoblasts were incubated at a temperature of 37 with 5% CO 2 to keep a saturated condition; the medium was updated every 3 days.

Cell transfection
The osteoblasts were transfected with 20 lM of small interfering RNA (siRNA) control or SDH5 siRNA (Invitrogen, Carlsbad, CA, USA) using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) according to the protocol.

Flow cytometry
The cells were cultured for 24 h and digested with trypsin without ethylenediaminetetraacetic acid. Annexin-V-FITC/propidium iodide (PI) solution was prepared from Annexin-V-FITC, PI, and HEPES buffer in a ratio of 1:2:50 (Beyotime, Shanghai, China). Then, cells were suspended with 100 lL of dyeing (1 Â 10 6 ) and shaken well. After incubating for 15 min, cells were added with 1 mL HEPES buffer and mixed by shaking. Cell apoptosis was measured by a flow cytometer (BD-Aria FACS Calibur, Beckman Coulter Inc., USA).

Cell counting kit-8 (CCK-8) assay
Osteoblasts at the logarithmic growth stage were uniformly inoculated into 96-well plates at the rate of 2 Â 10 4 cells per well and were transfected with siRNAs. The cells were cultured in an incubator for a further 24 h. Next, 10 mL of CCK-8 reaction solution (Beyotime, Shanghai, China) was put into each well. The mixture was incubated at 37 C for 2 h in a dark place. The absorbance measurement at 450 nm wavelength was performed by a microplate reader (Detie, Nanjing Detie, Nanjing, China).

Statistical analysis
The experiments were conducted again at least for three times. The research data were listed as mean ± standard deviation (SD). In order to evaluate data between two groups, a t-test was performed, and to evaluated data among multiple groups of parametric data (GraphPad Prism 8.0), a one-way ANOVA was assessed followed by a Dunnett test. We considered p < 0.05 to be statistically significant.

SDH5 is up-regulated in the bones of ovariectomized rats
First, we measured BMD and the relative expression of bone trabecular morphology of the Sham group and OVX group, respectively. In ovariectomized rats, BMD was lowered than that in normal rats, showing a statistically significant difference (Figure 1(A)). Compared with Sham group, BV/TV, Tb.Th, and Tb.N values were lower in OVX group, while Tb.Sp and BS/BV values were higher (Figure 1(B-F)). Relative mRNA level and protein expression of SDH5 in OVX group were significantly elevated compared with Sham group (Figure 1(G,H)). These results suggested that abnormal elevation of SDH5 might be associated with OVX-induced osteoporosis in rats.

SDH5 knockdown ameliorates OVX-induced osteoporosis in vivo
To explore the role of SDH5 in rats with osteoporosis, the SDH5 shRNA was injected into model rats. As depicted in Figure 2(A,B), the mRNA and protein expression levels of SDH5 were dramatically decreased after injection of sh-SDH5 in osteoporosis rats. The BMD of tibia increased notably by SDH5 knockdown compared with the corresponding control group (Figure 2(C)). OVX rats had decreased BV/TV, Tb.Th, and Tb.N values and promoted Tb.Sp and BS/BV values, however, SDH5 knockdown markedly reversed the decreases and prevented the elevation of Tb.Sp and BS/BV values in osteoporosis rats (Figure 2(D-H)). These findings indicated that SDH5 knockdown ameliorated OVX-induced osteoporosis in vivo.

SDH5 knockdown ameliorates OVX-induced cartilage damage in vivo
Histological analysis was conducted to further evaluate the function of SDH5 knockdown on osteoporosis rats. In the Sham group, the nucleus pulposus was hydrated, the cartilage was smooth and there was an extracellular matrix around it. However, nucleus pulposus atrophy, cluster formation, and cleft formation were put into an observation in the OVX group. This abnormal histological phenomenon was restored by the knockdown of SDH5. The nucleus pulposus shows a normal number of notochord cells and slight mucoid degeneration (Figure 3(A)). As indicated in Figure  3(B), the percentage of cartilage area was dramatically decreased in OVX group compared with the Sham group. Notably, this trend could be reverse by SDH5 knockdown. Moreover, Western blot showed that the expression of bone remodeling-associated cytokines, such as Osx, CoL1A1, and OC, were markedly enhanced by suppressing SDH5 in osteoporosis rats. Moreover, IHC was performed to determine the presentation of Osx, RUNX2, and OC in the tibia tissues of rats. After silencing of SDH5, the expression of RUNX2, Osx, and OC was obviously increased in OVX rats (Supporting Information Figure S1). Taken together, SDH5 knockdown could ameliorate OVX-induced cartilage damage in vivo.

Knockdown of SDH5 alleviates OVX-induced oxidative stress in rats
Next, we probed into the effect of SDH5 knockdown on the OVX-induced oxidative stress in rats. The results identified that OVX treatment raised the accumulation of MDA and lessened the levels of CAT, GSH-Px, and SOD in the serum of rats with osteoporosis. However, SDH5 knockdown significantly abolished these trends (Figure 4(A-D)). Furthermore, the results of Western blot expounded that OVX induction notably weakened the protein expression of SOD1 and SOD2 in the tibia of osteoporosis rats; while SDH5 inhibition dramatically reversed these effects (Figure 4(E)). These findings corroborated that SDH5 knockdown could restrain the oxidative stress induced by OVX in rats.

SDH5 regulates the OVX-induced MyD88/NF-jB pathway in rats with osteoporosis
In order to explore the related pathway of the functions of SDH5 on osteoporosis, the expression of pathway-associated proteins in the tibia of rats was detected. IHC showed that p-NF-jB p65 was markedly enhanced in osteoporosis rats by OVX induction, while SDH5 knockdown notably lessened its expression ( Figure 5(A)). Western blot displayed that the expression of p-MyD88 and p-NF-jB p65 was obviously increased in the OVX group compared with the Sham group. The overexpression and ratios of p-MyD88/MyD88 and p-NF-jB p65/NF-jB p65 were remarkably reversed in the OVX þ sh-SDH5 group ( Figure 5(B)). To further confirm the mechanism of SDH5 in osteoporosis progression, an LPS model was established. The expression and ratios of p-MyD88/MyD88 and p-NF-jB p65/NF-jB p65 were markedly enhanced again after LPS treatment ( Figure 5(C)). As shown in Figure 5(D), the overexpression of bone-specific transcription factors, including Osx, COL1A1, and OC, was notably decreased by the induction of LPS ( Figure 5(D)). Compared with OVX þ sh SDH5 group, LPS led to a significant elevation of MDA level and a down trend of SOD level in the tibia of rats. Collectively, SDH5 could regulate the MyD88/NF-jB pathway to modulate the damage in rats with osteoporosis.

SDH5 regulates progression of OVX-induced rat osteoblasts in vitro
To further clarify the effects of SDH5 in osteoporosis, in vitro experiment was performed in osteoblasts extracted from ovariectomized rats. Outcomes of qRT-PCR and Western blot reveals that the mRNA and protein levels of SDH5 in osteoblasts isolated from rats with osteoporosis were significantly inhibited by the transfection of SDH5 siRNA (Figure 6(A,B)). CCK-8 assay unraveled that SDH5 suppression dramatically promoted the cell viability in osteoblasts in contrast with the corresponding control group (Figure 6(C)). The apoptosis of osteoblasts was observed by flow cytometry, and it also should be noted that the apoptosis rate was notably repressed by suppressing SDH5 in the cells (Figure 6(D)). The role of SDH5 knockdown on oxidative stress and related pathway in osteoporosis in vitro was also examined. As expected, SDH5 knockdown effectively reduced oxidative stress in osteoblasts of rats with osteoporosis, as indicated by the enhanced expression of SOD1 and SOD2 ( Figure  6(E,F)). Suppression of SDH5 also inhibited the activation of MyD88/NF-jB signaling pathway in osteoblasts from rats with osteoporosis, as manifested by the reduced expression of p-MyD88/MyD88 and p-NF-jB p65/NF-jB p65 ( Figure  6(G)). Taken together, SDH5 regulates the MyD88/NF-jB pathway to affect the progression of osteoblasts from rats with osteoporosis in vitro.

Discussion
Osteoporosis is a category of a metabolic bone disease described by chronic excessive bone resorption as opposed to bone formation, as a result of bone loss and bone microstructure' deterioration [23]. Under normal circumstances, trabecular bone is periodically absorbed by osteoclasts, and then osteogenesis occurs in the bone cavity. This process is called bone remodeling. Excessive bone resorption by osteoclasts can lead to pathological bone loss diseases, including postmenopausal osteoporosis [24]. At present, the treatment of postmenopausal osteoporosis is mostly hormone regulation, but the therapeutic effect is strictly limited because of its side-effect.
In the present study, in vivo osteoporosis model was established by using OVX rats. The values of BMD, BV/TV, Tb/ Th, and Tb/N were thought to be reduced, and Tb/Sp and BS/BV values were elevated in the osteoporotic rats, suggesting that the osteoporosis animal model was successfully established. However, down-regulation of SDH5 reversed these abnormal trabecular bone morphometry values.
Several bone-specific transcription factor expressions, RUNX2, Osx, CoL1A1, and OC, were decreased in the animal model. RUNX2 is one of the major osteogenic differentiation markers that regulate bone formation [25]. Osx is necessary for osteoblast differentiation and bone formation, and Osx knockout mice were completely bone deficient [26]. The importance of OC in bone biology should not be underestimated and its shortage may be related to bone fragility [27]. CoL1A1 was indicated to be associated with decreased BMD in patients' osteoporosis [28]. In this work, knockdown of SDH5 rescued the expression of these aberrant bone remodeling-associated cytokines in OVX-induced rats. Atrophy of the nucleus pulposus and formation of cluster and cleft were observed in the OVX-induced rats. Of note, SDH5 down-regulation remarkably mitigated these adverse effects induced by osteoporosis. According to other references [29,30], the successful establishment of OVX model was usually accompanied by the decreases of BMD, BV/TV, Tb.Th, and Tb.N, and the increases of Tb.Sp and BS/BV, which were consistent with our experimental results. In our study, these indices were significantly reversed by SDH5 knockdown compared to OVX-induced model group. In addition, similar to previous work [20], SDH5 knockdown also significantly enhanced the expression of Osx, CoL1A1, and OC and improved cartilage repair of rats. Therefore, we believe that these results are sufficient to prove that OVX model is successfully established and SDH5 knockdown can attenuate osteoporosis to a certain extent.
Studies firmly suggested that estrogen deficiency accelerated bone aging and remarkably damaged the defense mechanism against oxidative stress [31,32]. Estrogen deficiency leads to oxidative stress, reduced levels and activities of antioxidant enzymes in bone tissues of mice, and impaired osteoblast bone formation and osteogenesis [33]. One study proved that the serum level of OC, and antioxidant activity, including GSH-Px, were lower in aged female rats with osteoporosis [34]. Lower GSH-Px and SOD enzyme activities and a higher MDA level were observed in women with osteoporosis [35]. Our current work verified that estrogen deficiency-induced oxidative stress is referred to reduced antioxidase levels and an elevated peroxidation production, whereas down-regulation of SDH5 could alleviate oxidative stress in OVX-induced rats and osteoporosis.
Although cartilage tissue is easy in composition, complex in structure, which is difficult to repair after damage. Due to the lack of revascularization, adult articular cartilage contributes little ability which is to be fully repaired [36]. In our work, Compared with that in ovariectomized rats, the percentage of cartilage area in rats from OVX þ sh-SDH5 group were dramatically increased, which proved that down-regulation of SDH5 contributes to cartilage repair. It has been found that mesenchymal stem cell exosomes mediate cartilage repair through promoting proliferation, suppressing apoptosis, and regulating immune response [37]. Previous work showed that SDHA desuccinylation (K547R) promoted cell proliferation in clear cell renal cell carcinoma by inhibiting the activity of SDH5 [38]. In lung cancer, SDH5 acts as a regulator of p53, leading to p53 activation to increase apoptosis after radiation [39]. Consistent with prior results, we observed that SDH5 knockdown could markedly promote proliferation and suppress apoptosis in osteoblasts extracted from ovariectomized rats, which provides more evidence of the protecting role SDH5 down-regulation in osteoporosis.
LPS treatment can successfully induce inflammation [40,41], apoptosis [42,43], and oxidative stress [44,45] in osteoporosis in vitro. Moreover, LPS has been presented to trigger TLR4-mediated signaling pathways, consisting of inflammation-related NF-jB pathway, apoptosis-related PI3K/ Akt pathway, and oxidative stress-related mitogen-activated protein kinases pathway [46]. Therefore, LPS induction was chosen to examine the effects of SDH5 knockdown on osteoblast proliferation, apoptosis, and oxidative stress. The cytokine production induced by LPS depends chiefly on NF-jB activation [47]. The major subunit of NF-jB, p65, is transferred to the nucleus, more likely explained by osteoclast formation and activation [48]. In our work, we found that the MyD88/NF-jB signaling activation restrained by SDH5 knockdown was triggered again by LPS treatment. This further proves that down-regulation of SDH5 alleviates osteoporosis by inhibiting the MyD88/NF-kB pathway.
Bone remodeling is largely undertaken by osteoblasts and osteoclasts. Osteoclasts absorb old bone and advance its degradation, while osteoblasts synthesize new bone and maintain new bone sources. The balance between bone resorption and bone formation is critical to upholding healthy bone mass and reducing the possibility of osteoporosis risk [49]. MyD88 signaling exerts a crucial role in osteoclast development. Sato et al. demonstrated that both bone resorption and bone formations were reduced in MyD88 -/mice [50]. In addition, oxidative stress has been shown to encourage cell senescence and apoptosis through different signaling pathways activation including NF-jB [51]. Activation of NF-jB promotes osteoclast differentiation and maturation, thereby increasing bone destruction and reducing bone formation, and inhibition of NF-jB activation remarkably inhibits osteoclast differentiation and bone resorptive activity [52]. The TLR-4/MyD88/NF-jB signaling pathway has been shown to contribute to osteoclast development, and methionine therapy can ameliorate postmenopausal osteoporosis via down-regulating the TLR-4/MyD88/ NF-jB pathway [53]. The study should also be noted that berberine-mediated release of surfactant protein D promotes cartilage repair via modulating immune responses by restraining TLR4/NF-OEB signaling pathway [54]. Similarly, further data results reflected that SDH5 down-regulation ameliorated the damage of osteoporosis at least partially through inhibiting the MyD88/NF-jB signaling pathway. However, due to our existing experimental conditions, there are some limitations to our work. The results of 3 D reconstructions of sagittal section of the femur, trabeculae, and cortical bone will be provided in the future study.

Conclusion
In conclusion, the findings of this study demonstrated that SDH5 down-regulation mitigated bone histopathological injury, ameliorated bone loss, alleviated oxidative stress, and enhanced osteogenic marker protein expression in ovariectomized rats. SDH5 knockdown also promoted proliferation and inhibited apoptosis of osteoblast extracted from OVXinduced rats. The underlying mechanism was related to the inhibition of the MyD88/NF-jB pathway. Notably, this outcome could be based on the fact that it was conducted in an animal model with relatively little sample size. To some extent, the preventive effect of SDH5 down-regulation on osteoporosis needs to be further validated.

Author contributions
Yi Liao designed the study. Hongzi Wu, Dehua Zhang, Haijun Xia, Yongqi Li, and Feng Mao collated the data and carried out data analyses. Hongzi Wu produced the initial draft of the manuscript. All authors have read and approved the final submitted manuscript.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author(s) reported there is no funding associated with the work featured in this article.