Sema4d regulates the bone metabolism in combination with leptin or melatonin

doi:10.21203/rs.3.rs-2270806/v1

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Sema4d regulates the bone metabolism in combination with leptin or melatonin

https://doi.org/10.21203/rs.3.rs-2270806/v1

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Journal Publication

published 08 Apr, 2023

Read the published version in Journal of Orthopaedic Surgery and Research →

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Purpose

The present study aims to explore the regulatory function of Sema4D on bone metabolism in combination with leptin or melatonin, as well as the underlying mechanism.

Methods

The osteoporosis model was established in rats using the OVX method. The bilateral tibial specimens of rats were taken for Micro-CT scanning analysis and three-dimensional reconstruction. The pathological state of bone tissues was evaluated by the HE staining assay. The concentration of estradiol in the serum was detected by the ELISA assay. Six groups were divided in the present study: Control, OVX, OVX + NL, OVX + Sema4D, OVX + Sema4D + leptin, and OVX + Sema4D + MT groups. According to the above grouping, the Sema4D or leptin overexpressing vectors were injected into rats through the tail vein. 3D bone structure was detected by high-resolution micro-CT system. Serum bone-derived alkaline phosphatase (BALP) and tartrate-resistant acid phosphatase-5b (TRAP-5b) activities were measured by ELISA. TRAP staining was used to calculate the number of osteoclasts in the metaphysis of the upper tibia. The expressions of bone morphogenetic protein-2 (BMP-2) and leptin in bone tissue were detected by immunohistochemistry.

Results

Compared to OVX + NL, the level of V/TV, Tb.N, BMD, and BMC in the OVX + Sema4D + leptin and OVX + Sema4D + MT groups was extremely elevated, accompanied by a declined Tb.Sp level. Compared to the OVX group, in the OVX + Sema4D + leptin and OVX + Sema4D + MT groups, the structure of bone trabeculae was relatively complete and tended to be closely arranged. The number of bone trabeculae was greatly increased and the number of TRAP-positive osteoclasts decreased significantly, accompanied by an upregulation of BMP-2 and leptin, and a declined activity of BALP and TRAP-5b.

Conclusion

The function of Sema4d on the microstructure of trabecular bone, bone formation, and repairment on the trabecular bone damage in osteoporosis rats was improved by leptin or melatonin.

Sema4d

leptin

melatonin

osteoporosis

bone metabolism

Osteoporosis is a common age-related disease. In recent years, with the dramatic increase in the number of patients, osteoporosis has become a global public health issue ^[1]. According to statistics, more than 200 million patients are suffering from osteoporosis worldwide ^[2]. The prevalence of osteoporosis in people over 50 years old in China is about 19.2%, and the prevalence of osteoporosis in people over 65 years old is as high as 32.0% ^[3]. More than 50 million patients over the age of 50 in the United States are suffering from osteoporosis, and nearly 1.5 million patients experience osteoporotic fragility fractures each year, which is expected to increase by one-third by 2030 ^[4]. Osteoporosis is characterized by significant defects in the quality and quantity of bone tissues, resulting in decreased bone strength and fracture resistance, especially at trabecular sites including the spine, ribs, and hip cortex. Decreased bone strength and fracture resistance increase the risk of fragility fracture ^[5], which is highly associated with disability and mortality ^{[6, 7]}. The pathogenesis of osteoporosis includes excessive bone resorption by osteoclasts and reduced new bone formation by osteoblasts. Loss of bone strength due to an imbalance between bone resorption and bone formation mechanisms leads to increased fragility and fracture. It is of great significance for osteoporosis patients to increase the hormones that have a positive regulatory effect on bone metabolism and reduce the hormones that have a negative effect on bone metabolism to improve bone density, so as to provide a new basis for the treatment of osteoporosis ^[8].

Brain signaling protein 4D (Sema4D) is a member of the semaphorin family. It has been proved that members of the semaphorin family have a close relationship with immune regulation, which is also named as immune semaphorin. Sema4D is reported to participate in a variety of signal conduction pathways, so as to play a variety of biological functions, which is not only involved in the regulation on B cells, T cells and other immune cells, but also plays a critical role in regulating the development of the nervous system, platelet function, epithelial cell function, and endothelial cell function ^[9]. In recent years, Sema4D is found expressed on osteoclasts to inhibit bone formation, which may be involved in the development of osteoporosis and osteosclerosis ^[10]. Leptin, a 16kD protein located on chromosome 7q31.3, is composed of 167 amino acids. After a series of modifications, leptin is finally observed in the blood with only 146 amino acid residues ^[11]. Leptin is mainly derived from adipose tissue and regulates energy by acting on the central nervous system, mainly through the negative feedback system to regulate appetite and activate receptors on target cells. Furthermore, leptin is reported to be an intermediate substance regulating bone metabolism ^[12] by participating in the regulation of bone formation. Melatonin is a neuroendocrine hormone synthesized and secreted by the pineal gland at night under the regulation of the suprachiasmatic nucleus of the hypothalamus, which is distributed in various tissues and organs such as gastrointestinal tract, ovary, lymphocytes, macrophages, retina, and skin ^[13]. Melatonin derived from the outside of pineal gland exerts paracrine or autocrine effects, which is superimposed on neuroendocrine hormone responses and acts as a local antioxidant ^{[14, 15]}. Melatonin is found to improve mood disorders, prolong physical performance, and limit skeletal muscle weakness ^[15]. Reiter R et al. reported that the content of melatonin in bone marrow was higher than that in blood, implying that melatonin might play an extremely important role in bone growth and development ^{[16, 17]}.

The present study aims to explore the effects and mechanisms of Sema4D, Sema4D combined with leptin, and Sema4D combined with melatonin on bone metabolism in OVX rat model, which will provide potential molecular targets for clinical treatment of osteoporosis.

OVX modeling and grouping: Under the aseptic condition, a 1–2 cm incision was made on the lateral abdomen, and the skin-fascia-muscle was separated with scissors successively with the white moist cellulite exposed. The bright pink cauliflower-shaped ovary was removed after separating the uterus, followed by cleaning up the wound and suturing the skin. After rats woke up, they were put back into the clean cage and raised in the feeding room. The state and death of the rats were observed regularly and recorded. Three days after surgery, each rat was injected with penicillin at 20 000 U/kg to prevent infection. Six groups were divided in the present study: Control, OVX, OVX + NL, OVX + Sema4D, OVX + Sema4D + leptin, and OVX + Sema4D + MT groups. In the Control group, the incision was made and the skin was sutured without removing the ovary. In the OVX group, no vector was injected and animals were orally dosed with normal saline for 28 days since the 62nd day of modeling. In the OVX + NL group, animals were injected with the adenovirus containing empty vector and orally dosed with normal saline for 28 days since the 62nd day of modeling. In the OVX + Sema4D group, animals were injected with the adenovirus containing Sema4D overexpressed vector and orally dosed with normal saline for 28 days since the 62nd day of modeling. In the OVX + Sema4D + leptin group, animals were injected with the adenovirus containing Sema4D overexpressed vector and leptin overexpressed vector and orally dosed with normal saline for 28 days since the 62nd day of modeling. In the OVX + Sema4D + MT group, animals were injected with the adenovirus containing Sema4D overexpressed vector and orally dosed with melatonin for 28 days since the 62nd day of modeling.

The 3D bone structure was detected using the high-resolution micro-CT system

After sacrificing rats, the soft tissue around the tibia was removed under sterile conditions, and the tibia was scanned and reconstructed by Micro-CT. Then the six groups of indicators were compared, including bone mineral density (BMD, g/cm3), bone mineral content (BMC, g), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular spacing (Th.Sp), and bone volume/total volume (BV/TV, %).

ELISA assay

The level of BALP (MM-0619R1, MEIMIAN, China) and TRAP-5b (MM-70644R1, MEIMIAN, China) was checked using the ELISA assay. The supernatant was collected as testing samples, which were added into the 96-well plate along with the standards, followed by adding the biotin-labeled antibody to be incubated at 37℃ for 1 h. After discarding the solution, the HRP reagent was added to be incubated at 37℃ for 15 min, followed by introducing the stop solution. After 15 min incubation, the OD value was achieved at 450 with a microplate reader (SuPerMax 3100, Shanghai Flash Spectrum Biotechnology Co., LTD, China).

HE staining

After collecting tibia tissues from each animal, tissues were rinsed with water for 2 h. After dehydration using different concentration of ethanol solution, tissues were dehydrated with xylene until transparent, followed by embedding for 1 hour and sliced. Subsequently, slides were roasted, dewaxed, hydrated, immersed in distilled water, and dyed in hematoxylin aqueous solution for 3 min, followed by being differentiated with hydrochloric acid ethanol differentiation solution for 15s. After being slightly washed with water and blue-returning solution for 15 s, slides were rinsed with water and dyed with eosin (G1100, Solarbio, China) for 3 min. Lastly, pictures were obtained using the inverted microscope (OLYMPUS, Japan).

TRAP staining

Paraffin sections were deparaffinized for 5 min, followed by incubation in absolute ethanol for 5 min, in 90% ethanol for 2 min, and in 70% ethanol for 2 min, successively. Subsequently, the TRAP staining solution was added for fixation at 4°C for 60 s. After washing with water, sections were put into TRAP incubation solution at 37°C for 45-60min, followed by stained with methyl green for 2–3 min. Images were taken under the microscope (CX43, OLYMPUS, Japan).

Immunohistochemical assay: Slides were placed in PBS to be rinsed for 1 hour, followed by being incubated with 10% goat serum overnight. Then, slides were introduced with the primary antibody against BMP-2 (1:100, AF5163, Affinity, USA) or leptin (1:100, DF8583, Affinity, USA) at 4℃ for 24 hours, followed by being washed with PBS. The secondary antibody (1:100, ZB-2301, SolelyBio, China) was then added to be incubated at 4℃ for 24 hours, followed by rinsed and stained with DAB dye. Lastly, images were taken using the inverted microscope (CX43, OLYMPUS, Japan).

Statistical analysis

Mean ± SD was utilized to present data, which was analyzed using the one-way ANOVA method with the software of GraphPad prism software 6.0. P < 0.05 was considered to be a statistically significant difference.

The establishment of OVX model in rats and the identification of the transfection of adenovirus

As shown in Fig. 1A, in the control group, the tibia trabeculae was dense and interwoven into a network. The number of bone trabeculae in the OVX group was significantly reduced, and the bone trabeculae was thinned and fractured. The bilateral tibial specimens of rats were scanned by Micro-CT and analyzed. As shown in Fig. 1B, the number of bone trabecula was dramatically reduced in the OVX group, accompanied by a declined bone density. Furthermore, compared to control, dramatically elevated E2 level was observed in OVX rats. These data suggested that the OVX model was successfully established in rats. As shown in Fig. 1D, compared to control, Sema4D and leptin were extremely upregulated in OVX rats.

The impact of Sema4d combined with leptin or melatonin on the microstructure of trabecular bone in tibia of OVX rats

As shown in Fig. 2, compared to the OVX + NL group, the level of V/TV, Tb.N, BMD, and BMC was greatly increased, while the Tb.Sp level was dramatically declined in the OVX + Sema4D + leptin and OVX + Sema4D + MT groups.

The impact of Sema4d combined with leptin or melatonin on the pathological changes in tibia tissues of OVX rats

As shown in Fig. 3, the structure of bone trabecula was stable and arranged orderly in the control group. Disrupted structure of randomly arranged trabecula and decreased number of bone trabecula were observed in the OVX, OVX + NL, and OVX + Sema4D groups. Compared to the OVX group, relatively tightly arranged integrated structure of bone trabecula and increased number of bone trabecula were observed in the OVX + Sema4D + leptin and OVX + Sema4D + MT groups.

The impact of Sema4d combined with leptin or melatonin on the osteoclast differentiation in tibia tissues of OVX rats

TRAP staining was used to evaluate the number of osteoclasts in metaphysis at the upper end of the tibia. Compared to control, the number of TRAP positive osteoclasts was dramatically increased in the OVX, OVX + NL, and OVX + Sema4D groups, among which the highest number of osteoclasts was observed in the OVX + Sema4D group. Furthermore, compared to the OVX group, dramatically declined TRAP positive osteoclasts were observed in the OVX + Sema4D + leptin and OVX + Sema4D + MT groups (Fig. 4).

The impact of Sema4d combined with leptin or melatonin on the level of BMP-2 and leptin in tibia tissues of OVX rats

As shown in Fig. 5, compared to control, extremely declined level of BMP-2 and leptin was observed in the OVX, OVX + NL, and OVX + Sema4D groups. Compared to the OVX group, dramatically elevated level of BMP-2 and leptin was observed in the OVX + Sema4D + leptin and OVX + Sema4D + MT groups.

The impact of Sema4d combined with leptin or melatonin on the activity of BALP and TRAP-5b in OVX rats

As shown in Fig. 6, compared to control, markedly elevated activity of BALP and TRAP-5b was observed in the OVX, OVX + NL, and OVX + Sema4D groups. Compared to the OVX group, dramatically reduced activity of BALP and TRAP-5b was observed in the OVX + Sema4D + leptin and OVX + Sema4D + MT groups.

In bone tissues, Sema4D is only expressed in osteoclasts, and its expression is upregulated during the production of osteoclasts ^[10]. PlexinB1 is expressed in osteoblasts, which is a high-affinity receptor for Sema4D ^[18]. Studies have confirmed that Sema4D combined with PlexinB1 to regulate cell morphology and migration through the regulation of actin reorganization related to RhoA family small GTPases ^[10]. Sema4D gene knockout animal experiments ^[10] suggest that the activity of osteoclasts, the number of osteoclasts, and the degree of bone lacunae are not significantly impacted by Sema4D. However, the number of osteoblasts on the bone surface and the bone mineralization ability of osteoblasts are impacted by Sema4D. In Sema4D-knockdown mice, the phenotype and bone formation rate of osteoblasts are increased, including an increased osteoblastic phenotype and a higher rate of bone formation in the femur. Although Sema4D is exclusively expressed by osteoclasts. Studies have shown that Sema4D not only inhibits bone mineralization through osteoblasts, but also induces osteoclast formation ^[10]. Since the expression of Sema4D is upregulated in the process of osteoclast formation, it is inferred that the osteoblast differentiation will be inhibited by Sema4D secreted by osteoclasts, thus affecting bone mineralization. In the present study, OVX was used to construct an osteoporosis rat model. Pathological results showed that the number of tibial trabeculae in OVX rats was significantly reduced, and the bone trabeculae were thin and fractured with low density, accompanied by an increased serum E2 level. These results were consistent with the research reports of Zhao ^[19] and Wang ^[20]. After the overexpression of Sema4D, the microstructure of tibial trabeculae was significantly damaged, the arrangement was irregular, the number of bone trabeculae was reduced, the number of osteoclasts was significantly increased, and the osteogenic related indicators were significantly decreased. These results suggested that Sema4D might aggravate the degree of osteoporosis by increasing the number of osteoclasts, which was close to results reported by Takako ^[10].

Leptin is reported to promote the proliferation of osteoblasts and the transformation of bone marrow stromal cells into osteoblasts. In previous experiments, an extremely repressed long-bone growth, reduced bone surface area, and decreased mineral and bone density level were observed in leptin deficient mice which were dramatically rescued by the the injection of leptin, indicating that leptin played an important role in the differentiation and maturation of osteoblasts ^[21]. It is reported that the increase of leptin level is positively correlated with the number of bone mineralized nodules in osteoblasts. Furthermore, the targets of leptin are found on osteoblasts, which further indicates that leptin exerts a positive effect on the metabolism of osteoblasts. Bone marrow mesenchymal stem cells (BMSCs) proliferate in large quantities in vivo and differentiate into a variety of cell types, such as osteoblasts and adipocytes. The differentiation of BMSCs to adipocytes and osteoblasts is reported to be facilitated by leptin ^[22], which further increases bone matrix, and affects the metabolism of bone cells. In the present study, after increasing the level of leptin in Sema4D-overexpressed OVX rats, significant improvement of tibia bone trabecular microstructure and arrangement of bone trabecular, increased number of bone trabecular, and elevated level of osteogenesis related factors were observed, suggesting that leptin significantly reversed the pathological changes in tibia of Sema4D-overexpressed OVX rats. The functional mechanism of leptin might be associated with the receptor activator of nuclear factor-κB (RANK)/osteoprotegerin (OPG)/ receptor activator of NF-κB ligand (RANKL) axis. Studies have reported that OPG competitively binds RANK with RANKL to affect osteoclast activity, which is upregulated by leptin, thereby inhibiting the activation and maturation of osteoclasts, reducing bone resorption and bone destruction, and regulating bone metabolism ^{[23, 24]}. In the present study, the number of osteoclasts was significantly reduced after leptin overexpression in Sema4D-overexpressed OVX rats, which further verified that the improvement effect of leptin on osteoporosis might be related to the inhibition of osteoclast activation.

The effect of melatonin on primary osteoporosis has been widely reported. St Hilaire et found that melatonin levels were lower in premenopausal women and were accompanied by increased bone resorption ^[25]. Maria reported that melatonin activated melatonin receptor (MR) by inhibiting RANKL signaling to promote the generation of osteoblasts ^[26]. St Hilaire investigated the effect of melatonin on bone mineral density of postmenopausal women with osteoporosis in the past 10 years. It was found that melatonin increased BMD of femoral neck and lumbar spine in both perimenopausal and postmenopausal women. Combined treatment with melatonin improves scores at the femoral neck and lumbar spine, and melatonin may increase osteocalcin in both perimenopausal and postmenopausal women ^[27]. Due to its unique antioxidant, anti-inflammatory, and immunomodulatory functions, melatonin possesses great medical potential. It is an ideal method to treat osteoporosis by enhancing the activity of osteoblasts and inhibiting the formation of osteoclasts. A series of studies have reported the function of melatonin on bone metabolism. Zhu et al. found that melatonin effectively inhibited the osteolysis around the prosthesis caused by titanium particles ^[28]. Zhang et al. claimed that melatonin restored the osteogenic differentiation ability of stem cells damaged by titanium particles ^[29]. Chen et al. found that melatonin improved the osteogenic differentiation potential of BMSCs derived from osteoporotic rats ^[30]. In the present study, melatonin was used for the treatment on Sema4D overexpressed OVX rats. Results showed that in Sema4D overexpressed OVX rats, the structure of bone trabeculae was relatively complete, the arrangement of the bone trabeculae tended to be neat, the number of bone trabeculae was increased, the number of osteoclasts was decreased significantly, and the secretion level of BMP-2 and other osteogenic indicators was elevated significantly by the administration of melatonin. These results indicated that melatonin significantly reversed the aggravation of lesion caused by Sema4D overexpression in OVX rats.

In conclusion, Sema4D aggravated the pathological process of osteoporosis by activating osteoclasts, which was reversed by leptin and melatonin. The present study provided potential and reliable target molecules for the clinical drug development of osteoporosis.

Ethical Approval

The present study was approved by the Animal Care and Use Committee of the Zvast-Bio (No.2021091001).

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

ZL designed the experiment and conducted the experiment. ZHL 、XCL、XYL and XC helped ZL conduct some experiments and The experimental data is recorded. SX、DX and YL instruct ZL to perform statistical analysis. ZL wrote the paper, and ZL posted a comment, proposing appropriate revisions and corrections. All authors read and approved the final manuscript.

Funding

This work was supported by the Fujian Provincial Natural Science Foundation of China(Grant no. 2020J011188).

Availability of data and materials

The experimental data will be available on the request.

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No competing interests reported.

Download PDF

Journal Publication

published 08 Apr, 2023

Read the published version in Journal of Orthopaedic Surgery and Research →

Editorial decision: Major revision
23 Nov, 2022
Editor assigned by journal
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Submission checks completed at journal
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14 Nov, 2022

You are reading this latest preprint version

Sema4d regulates the bone metabolism in combination with leptin or melatonin

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