Intermittent administration sodium valproate has a protective effect on bone health in ovariectomized rats

doi:10.21203/rs.3.rs-613756/v2

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Intermittent administration sodium valproate has a protective effect on bone health in ovariectomized rats

https://doi.org/10.21203/rs.3.rs-613756/v2

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Objective: the present work was aimed to evaluate the effect of different administration modes of sodium valproate(VPA) on bone strength, bone mass and bone mineral density in ovariectomized(OVX) rats and further investigation of the possible mechanism.

Methods: 60 female SD rats were randomly divided into 4 groups: Sham group (Sham, n=15), OVX group (OVX, n=15), OVX rats received intermittent VPA treatment group(IVPA, n=15) and OVX rats received daily VPA treatment group(EVPA, n=15). After 12 weeks of treatment, the rats were sacrificed, and serum and femur samples were harvested. DEXA, Micro-CT, histology, biomechanical testing, biochemical index and western blot analysis were used to observe the therapeutic effect and explore the possible mechanism.

Results: Micro-CT and DEXA analysis of bones revealed better BMD and higher BV/TV, Tb.Th, Tb.N, Conn. D and lower Tb.Sp at femoral metaphysis with evaluated in IVPA when compared with OVX and EVPA group(P＜0.05). Histological, fluorescent analysis and biological strength revealed more trabecular bone and higher relative mineral apposition rate, maximal load, elastic modulus and energy at break with evaluated in IVPA when compared with OVX and EVPA group(P＜0.05). The levels of P1NP, estrogen, CTX, TRAP5b and RANKL of IVPA group showed a significant increase when compared with the OVX and EVPA group(P＜0.05). We confirm adverse effects on protein expressions including Notch1, Jagged1, HEY1, Wnt 1, β‐catenin and RUNX2 following daily VPA treatment in OVX female rats.

Conclusions: Our current study demonstrated that intermittent administration sodium valproate has a protective effect on bone health in ovariectomized rats and these effects may be achieved by activating Notch/Wnt/β-catenin/ RUNX2 signal axis.

Nutrition & Dietetics

Cellular Metabolism

Osteoporosis

Bone defect

Valproic acid

Bone repair

Bone mineral density

Epilepsy is a serious and life-threatening neurological disease characterized by unpredictable and recurrent seizures and is one of the most common chronic disorders[1]. It was estimated that more than report 50% bone abnormalities are affected by epilepsy worldwide[2]. Due to seizure-related falls, ataxia, immobility leading to falls and long-term use of anti-epileptic drugs causing bone loss and abnormal bone metabolism, the incidence of osteoporosis and osteoporotic fracture in these patients is significantly higher than that of normal people[3, 4]. The relationship between epilepsy, antiepileptic drugs and bone mineral density has been explored for more than 30 years[3-5]. Previous studies have reported that antiepileptic drugs represented by carbamazepine have a notable impact on vitamin D metabolism and bone density reduction[6]. However, some studies based on new antiepileptic drugs did not find a statistically significant changes in calcium, vitamin D, BMD in patients with epilepsy[7].

Valproic acid (VPA) is an inhibitor of the CYP450 enzyme characterized by minimal effects on hepatic metabolic enzymes that has been used as an antiepileptic drug for many years[8]. Previous studies have reported that VPA functions as a histone deacetylase inhibitor (HDACi), with the specific inhibiting activity by binding to the catalytic centre of HDACs , which stimulate apoptosis and inhibit the proliferation of cancer cells[9]. Besides, VPA could cause tubular renal dysfunction and subsequent abnormal metabolism and loss of calcium and phosphorus[10]. Interestingly, clinical and animal experiments have shown the decreased bone mineral density(BMD) with VPA treatment[11, 12], but some studies have failed to show such harmful effects on BMD[13, 14], and even several cellular, and animal experiments have shown VPA’s beneficial effects on bone health[15-17].

Currently, there are several studies examining the potential effects of VPA on bone mass. However, the effect of VPA on bone health is still controversial and conflicting, especially for the bone lose in osteoporosis. At present, the results of animal studies are based on different modes of administration [12, 15, 17]. Therefore, we hypothesized that different administration modes of VPA have different effects on bone remodeling, resulting in different results in bone mass, bone mineral density and bone strength. Therefore, the present work was aimed to evaluate the effect of different administration modes of VPA on bone strength, bone mass and bone mineral density in ovariectomized(OVX) rats and further investigation of the possible mechanism.

Model establishment, treatment, and sample collection

60 female SD rats characterized by 3 months of age and weighing 200-250 g were employed in the study. These rats maintained on pellet feed with free access to food and water, as well as under regulated conditions of temperature (25±1°C) and relative humidity (55-65%) and 12:12h light/dark cycle. After a week of adaptation period, the animals underwent sham-operation (Sham) or bilateral ovariectomy (OVX) following the standard surgical procedures as previously described[18, 19]. Then, the surgical rats were randomly divided into 4 groups: Sham group (Sham, n=15), OVX group (OVX, n=15), OVX rats received intermittent VPA treatment group(IVPA, n=15) and OVX rats received daily VPA treatment group(EVPA, n=15). Animals in the IVPA group receive gavage treatment with three-day consecutive treatment of VPA(300 mg/kg) followed by four-day saline treatment each week, and rats in the EVPA group receive gavage treatment with VPA(300 mg/kg)once a day until 12 weeks. The dosage and mode of VPA used in this experiment refer to the previous studies[12, 15, 17].Two intraperitoneal injections of calcein(20 mg/kg) were injected at the 3rd and 10th day before the rats were sacrificed. Body weight changes of all rats were examined before and after experiment. After 12 weeks of treatment, the rats were sacrificed using an overdose of chloral hydrate. Serum and femur samples were harvested. Femurs were fixed at 4°C with 4% paraformaldehyde. Whole blood were frozen at − 80°C for later use. The experiments and procedures were approved by the Animal Ethics Committee of the First Affiliated Hospital of Wannan Medical College, Yijishan Hospital.

DEXA and Micro-CT scan

The BMD of the femur was measured using by dual-energy X-ray absorptiometry (DEXA, DPX-ALPHA LUNAR^TM1, Lunar Corporation, Madison, USA).The distal femur was analyzed with anisotropic voxel size of 10μm through the Micro‐CT (Bruker Skyscan 1272 system, Kontich, Belgium). The parameter is set to 55 kV and 114 m A with a thickness of 0.048 mm per slice in medium-resolution mode, 1024 reconstruction matrix, and 200 ms integration time. These images and parameters of trabecular bone parameters with a distance of 1 mm proximal from the end of the growth plate in femoral metaphysis were compared between different. After 3D reconstruction, bone volume fraction(BV/TV), trabecular number(Tb.N), trabecular thickness(Tb.Th), trabecular separation(Tb.Sp) and the mean connective density (Conn.D) were automatically determined for identification of osteoporosis model as previously described[20, 21].

Histomorphometric analysis

Part of the femora were decalcified in 10% EDTA (pH 7.4) for 4 weeks and then embedded in paraffin. Four-micrometer-thick longitudinally oriented along the defect sections were used for staining. HE staining was performed to observe the trabecular bone as previously described[22]. The others femurs were dehydrated and embedded in methylmethacrylate solution. Afterwards, thin sections (about 50μm in thickness) were prepared using the diamond saw (Leica Microtome, Wetzlar, Germany). Calcein double labelling in undecalcified bone slices were observed under a fluorescence microscope(FLUOVIEW FV300, Olympus) to quantify the bone mineralization as previously described[23, 24].

Biomechanical testing

The left femurs were thawed at room temperature for the three-point-bending test, which was performed after Micro-CT scan. The center of the shaft of femur was placed in coronal plane on two anvils, with a 20 mm distance. Deflection was performed by lowering a third anvil onto the midshaft of the femur. An Electron E1000 (Instron, High Wycombe, UK) biomechanical machine exert force with load rate of 1 mm pr. minute with 250 N. Load displacement and stress strain curves were generated, and maximal load, elastic modulus and energy at break were recorded or calculated.

Serum index detection

Blood sample taken at the end of the experiment was analyzed for procollagen I N-terminal propeptide (PINP), receptor activator of nuclear factor kappa B ligand (RANKL), tarteresistant acid phosphatase 5b (TRAP5b), estrogen andcollagen type 1 cross-linked C-telopeptide(CTX). Commercial ELISA-based kits(Sincere biotech, Beijing, People’s Republic of China) were used to detect the levels of CTX(intra-assay coefficient of variance (CV): 5.6%; and inter-assay CV: 8.7%), PINP(intra-assay: 5.4%; and inter-assay CV: 8.3%) ,TRACP-5b(intra-assay: 6.2%; and inter-assay CV: 8.7%) ,RANKL(intra-assay: 5.4%; and inter-assay CV: 6.0%) and estrogen(intra-assay: 5.6%; and inter-assay CV: 8.3%) according to the manufacturer’s instructions.

Western blot analysis

The femoral condyle was pulverized, and 50 mg of tissue was obtained from each group. The tissue was thoroughly ground in liquid nitrogen and homogenized buffer and then were dissolved in ice-cold cell lysis buffer (Beyotime) containing protease inhibitors; the protein concentration in cell extracts was quantified using a BCA protein assay kit (Beyotime). Then, 20 μg of total cell lysate protein was prepared in Pro-PREPTM Protein Extraction Solution (Boca Scientific Inc., Boca Raton, FL) and electrophoresed. The primary antibodies against the following proteins: Notch1 (Abcam, ab52627, 1:1000), RUNX family transcription factor 2(RUNX 2, Abcam, ab236639, 1:1000), Jagged1 (Abcam, ab109536,1:1000), HEY1 (Abcam, ab154077, 1:1000), Wnt1(Abcam, ab15251, 1:1000), β‐catenin(Abcam, ab32572, 1:1000). Protein expression levels were normalised to Glyceraldehyde 3 phosphate dehydrogenase (GAPDH; Abcam, ab8245, 1:2000) protein levels. HRP-conjugated goat anti-rabbit (Santa Cruz Biotechnology, Santa Cruz, CA) were used as secondary antibodies. Blots were imaged using an iBrightCL1000 (Invitrogen, Carlsbad, CA) as previously described[25, 22].

Statistical analysis

Data were expressed as mean±SD. Statistical significances among four groups were determined by one-way analysis of variance and the Student s t-test. A value of p<0.05 was considered statistically significant.

Changes in body weight and estrogen levels

A total of 6 rats died during the experiment, including anesthetic accidents, infection and surgical accidents. The death of rats occurred during or after the operation, including OVX group (n = 2), IVPA group (n = 2) and EVPA group (n = 2). Twelve weeks after the ovariectomy, the body weight of OVX group was significantly higher than that of Sham group, while the serum estrogen level was significantly lower than that of Sham group (P＜0.05, Fig 1 A), which indicated that the ovariectomized model was established successfully. At the same time, it was found that the changes of body weight and estrogen in EVPA group were more obvious than those in IVPA group (P＜0.05 , Fig 1 B) , which indirectly indicated that the improvement of osteoporosis in IVPA group was better than that in EVPA group.

Bone mineral density of rats in each group

The results of BMD from each group revealed a significant decrease in OVX at femur when compared with the Sham group(P＜0.05, Fig 2). Interestingly, only the BMD of IVPA group showed a significant increase when compared with the OVX group(P＜0.05). However, daily treatment with VPA further reduced the BMD at femur.

Micro-CT evaluation

The 2D scan images(Fig3. A-D) and 3D reconstruction images(Fig3. a-d) of Micro-CT clearly show us the trabecular bone microstructure at femoral metaphysis after 12 weeks of treatment with different intervention methods. As we expected, the small amount of trabecular bone was observed in the OVX group tissue, while large amounts of trabecular bone was found in the Sham and IVPA group, but it was difficult to find trabecular bone in the EVPA group. The quantitative results were expressed as BV/TV, Tb.Th, Tb.N, Conn. D and Tb.Sp (Fig 4). Intermittent administration with VPA showed a positive effects on all micro-CT parameters, however, the opposite result was observed in the EVPA group. Compared to groups OVX and EVPA, intermittent administration with VPA shows the best bone microscopic parameters including the highest BV/TV, Tb.N, Conn.D, Tb.Th, and a lowest Tb.Sp (P<0.05).

Histological and Fluorescent analysis

Histological and fluorescent images showing the trabecular bone microstructure at femoral metaphysis for different treatment, as shown in Fig 5 and Fig 6. At 12 weeks, a large amount of trabecular bone fills the medullary cavity of femoral metaphysis in the Sham group and IVPA group. In the OVX and EVPA group, a very small amount of trabecular bone and a large amount of fat vacuole can be observed. In fluorescent analysis, intermittent administration with VPA showed the largest relative mineral apposition rate (p<0.05), compared to that of the OVX group and EVPA group.

Biochemical bone turnover markers

The results of biochemical bone turnover markers were detected after 12 weeks of treatment with different intervention methods, as shown in Fig 7. As we expected, a significant increase levels of CTX, TRAP5b and RANKL were observed in the OVX group when compared with the OVX group, except for P1NP (P＜0.05). Interestingly, only the levels of P1NP, CTX, TRAP5b and RANKL of IVPA group showed a significant increase when compared with the OVX group(P＜0.05). However, daily treatment with VPA further reduced the levels of P1NP but CTX, TRAP5b and RANKL.

Biomechanical testing

The results of maximal load, elastic modulus and energy at break from each group revealed a significant decrease in OVX group of femur when compared with the Sham group(P＜0.05, Fig 8). Interestingly, only the biomechanical parameters of IVPA group showed a significant increase when compared with the OVX group(P＜0.05). However, daily treatment with VPA further reduced the maximal load, elastic modulus and energy at break from of femur.

Related protein expression

The protein expressions including Notch1, Jagged1, HEY1, Wnt 1, β‐catenin and RUNX2 of OVX group were significantly lower than that of Sham group(P<0.05, Fig 9). Interestingly, the protein expressions of Notch1, Jagged1, HEY1, Wnt 1, β‐catenin and RUNX2 of IVPA group showed a significant upregulate when compared with the OVX group(P＜0.05). However, daily treatment with VPA further reduced the expressions of Notch1, Jagged1, HEY1 , Wnt 1, β‐catenin and RUNX2.

At present, there is no consensus whether this VPA has any relationship with bone health in osteoporosis. Furthermore, the effect and mechanism of VPA on osteoporosis is complex and the research is limited. Therefore, the purpose of this study was to investigate how frequency of VPA administration affect the microarchitectural properties and BMD of bone in OVX rats. To better determine the drug effects in vivo, biological experiments were carried out and can reflect more apparent and intuitive behavior of the drug. As we hypothesized, the frequency of VPA administration were associated with greater bone microstructural properties. Interestingly, intermittent administration of VPA can increase bone mass and BMD at skeletal sites with rich cancellous bone in OVX rats. By contrast, continuous VPA administration induces bone mass loss and lower bone strength in the experimental osteoporosis model state.

Postmenopausal osteoporosis is a type of bone loss caused by estrogen deficiency with menopause. Estrogen confine bone turnover and affect the osteogenesis of BMSCs, and play a key role in maintaining bone metabolic equilibrium. Lower levels estrogen may affect numerous processes involved an interplay between osteoclastic bone resorption and osteoblastic bone formation. Runx2 is a key transcription factor necessary for osteogenic differentiation and maturation of osteoblasts[26]. Estrogen deficiency was shown to downregulate the expression of Runx2, resulting in bone loss and increased fracture risk[27]. In this study, female SD rats were used to establish an osteoporosis model after bilateral ovariectomy and mimics bone loss in the physiological state of female postmenopausal estrogen deficiency[28, 29]. In this mature animal model, we confirmed that decreased BMD and bone loss by DEXA and Micro-CT occurred in femur at 12 weeks following OVX, consistent with our previous observation[22, 30]. In addition, a significant decrease in bone turnover index and estrogen level were detected in the OVX rats compared with Sham rats. These results suggest that this model used in our study was successful development.

Owing to likely reveal markedly true results by elimination of confounding effects including lifestyle habits, genetic and other individual characteristics, researchers have been put forward the benefits from antiepileptic drugs-related bone fragility studies using animal models[31]. In this study, the ovariectomized rat model was administration with VPA to simulate the postmenopausal osteoporosis women using VPA scenario, which can better reflect the clinical experience. In current study, we also found that uninterrupted administration with VPA greatly affect the microarchitectural properties of the femoral diaphysis in ovariectomized rats, resulting in decreased trabecular volume and bone mineral density supporting our finding of VPA-induced bone loss [32]. Interestingly, intermittent administration of VPA could protect and prevent the loss of bone mass and the decrease of bone mineral density in ovariectomized rats, which was significantly higher than that of OVX, and the trabecular structure detected by Micro-CT and 3D reconstruction was significantly better than that of OVX group, including higher trabecular bone parameters such as BV/TV, Tb. N, Conn. D and Tb.Th. In addition, we found that biomechanical properties for the femur shaft were better in the IVPA group under surveillance compared with OVX rats with saline intervention.

Biochemical markers, such as CTX, P1NP and TRAP5b reflect the bone resorption and bone formation under certain conditions that affect bone metabolism[33]. The specific biochemical markers of bone turnover were chosen and measured in the serum to investigate the pathophysiology of VPA effect on bone. TRAP5b and CTX reflects the number of osteoclasts and function of the osteoclasts, while P1NP is measurement of bone formation by the osteoblasts[34]. RANKL, a member of the TNF family, play a key role in osteoclast formation and induction of resorptive function [35]. In this study, it was found that the levels of serum CTX, P1NP and RANKL and TRAP5b in OVX group were significantly higher than those in Sham group after operation, which confirmed that estrogen deficiency accelerated bone turnover[36, 37]. Moreover, we observed that the bone turnover indexes of IVPA group and EVPA group were significantly higher than that of OVX group. Among them, the bone formation marker P1NP of IVPA group increased the most, while the osteoclast-related indexes CTX, TRAP5b and RANKL of EVPA group changed most significantly. These results seem to indicate that intermittent and continuous VPA treatment has different effects on osteoblasts and osteoclasts; intermittent treatment is beneficial to improve osteoblast function, while continuous VPA intervention will strongly stimulate osteoclast activity. These changes significantly affected the results of the parameters of biomechanics testing and the levels of bone metabolism indexes[38].

In order to further explore possible causes and mechanism of the observed results in this study, protein expression was performed by Western blot. The canonical Wnt/β-catenin signaling pathway plays a crucial role in modulating bone metabolism and bone remodeling, and regulate osteoblasts and osteoclasts biological function such as cell differentiation, cell migration and cell proliferation[39, 40]. In recent years, increasing studies have reported that Notch signaling plays an essential role in BMSCs osteogenic differentiation and osteogenesis differentiation[41, 42]. Previous studies have shown that VPA can activate Notch signaling pathway in different tissues[43, 44] and showed could remarkably increase mineralization and osteogenesis in vitro cell culture experiment[45]. In this study, we recorded that the expression of Notch1, Jagged1, HEY1, RUNX2, Wnt-1 and β-catenin in bone tissue of IVPA group was significantly higher than that of OVX group, but the expression of Notch1, Jagged1, HEY1, RUNX2, Wnt-1 and β-catenin of EVPA group was significantly lower than that of IVPA group. Similar to the results of a previous study[17], intermittent administration of VPA may activate osteoblast activity by Notch/Wnt/β-catenin/ RUNX2 signal axis and mediate the increase of RANKL expression, resulting in an increase in osteoclast activity, but the effect on osteoblasts is significantly greater than that of osteoclasts, resulting in an increase in bone mass. Uninterrupted administration of VPA leads to a high level of VPA in vivo. High level of VPA has a limited effect on promoting the function of osteoblasts and further increases the stimulation of osteoclasts. The increase of RANKL level is the best evidence. Eventually, bone resorption is greater than bone formation, resulting in further loss of bone mass. In previous cell experiments, it was also observed that osteoblasts and osteoclasts showed different biological characteristics of dose-dependent tolerance to VPA[16].

As far as we know, this is the first study of the effect of systemic administration with VPA under different administration modes on the bone mass and BMD in osteoporotic conditions. Nevertheless, this study had several deficiencies. The mechanisms underlying the effects of VPA under different administration modes on osteogenic differentiation of MSCs should be elucidated. The optimal dosage of VPA should be determined for protect and prevent bone loss by using animal studies. Besides, no normal bone mass and healthy animals were used in this study.

In summary, our study suggests that systemic administration with VPA under different administration modes may acquire different effects on bone mass, bone mineral density and bone strength in osteoporotic rats. In addition, under the condition of clinical converted dose, intermittent administration of VPA has a protective effect on bone mass, while uninterrupted administration of the drug reflects an adverse reaction to bone mass, which may be consistent with the different effects of the two modes of administration on Notch/Wnt/β-catenin/ RUNX2 signal axis-mediated osteoblasts and osteoclasts.

This study was supported by a grant from National Natural Science Foundation of China (82002322), Funding of “Peak” Training Program and “Panfeng” Innovation Team Project for Scientific Research of Yijishan Hospital, Wannan Medical College (grant no. GF2019G04, PF2019005, GF2019T02 and PF2019007) and Young and Middle-aged Key Project of Wannan Medical College(WK2020ZF16).

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Intermittent administration sodium valproate has a protective effect on bone health in ovariectomized rats

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