Effects of nicotine on markers of bone turnover in ovariectomized rats

Introduction Osteoporosis is characterized by low bone mass and density, as well as change in microarchitecture of bone tissue leading to decreased bone strength. In vitro research shows nicotine can increase osteoblast activity and proliferation, also suppress osteoclast activity. Therefore we explore nicotine anti-resorptive property by in vivo true experimental and randomized posttest only controlled group research that was conducted in 18-20 weeks old Rattus norvegicus. Methods Twenty-five female rats were divided into five groups, with 5 rats per group. The first group represented normal rats (Sham), while the second to fifth group underwent bilateral ovariectomy. The second group serves as positive control group (ovariectomy-only/OVX). The third to fifth group serve as dose 1 (P1-0.25mg/kg), dose 2 (P2-0.5 mg/kg), and Dose 3 (P3-0.75 mg/kg) treatment group receiving daily per-oral nicotine for 28 days, started 3 weeks post- ovariectomy. After 28 days treatment, the serum was checked. Results Nicotine has dose-dependent manner on serum osteocalcin and serum DPD level. Level of osteocalcin in P2 group was significantly lower (Mann-Whitney, p = 0.008) compared to OVX group (59.4% lower). Level of DPD in all group was not significantly different (ANOVA, p < 0.05) but shows lowest level in P2 group. For serum calcitonin level, there's no significant different between groups. Conclusion Nicotine at right low-dose might be able to inhibit osteoclast activity, thus open a possibility of anti-resorptive property of nicotine.


Introduction
Osteoporosis is a disease of bone characterized by low bone mass and density, as well as change in microarchitecture of bone tissue that leads to decreased of bone strength. This disease increases the bone fragility to fracture [1]. Osteoporosis affects about 75 million people, in the United States, Europe and Japan. In worldwide, osteoporosis causes about 9 million fractures annually [2]. In osteoporosis, there is an imbalance between the activity of osteoclasts (resorption agent) and osteoblasts (bone-forming agent).
This imbalance can increase bone fragility [3]. Based on in vitro research, osteoblast and osteoclast activity can be influenced by nicotine. Nicotine can increase osteoblast activity and proliferation, also suppress osteoclast activity. α4nAChR stimulation in osteoblasts can increase osteoblast proliferation rates [4]. Activation of α3 and α5nACH receptors will increase concentration of bone morphogenic protein-2 [protein with important role in bone induction regulation], and maintenance and repair during fracture healing in mouse models [5]. nAChR ligands reduced the number of osteoclasts.
Stimulation of nAChR can increase osteoclast apoptosis so that it can prevent bone loss. In previous studies it was found that mice with damaged α2nAChR had osteoporosis due to up-regulation of osteoclasts [6]. Therefore, nicotine holds potency as anti-resorptive agent. The potency of nicotine as an anti-resorptive agent for osteoporosis is still poorly studied. In this study, we wanted to see the effectiveness of nicotine as a preventive agent for osteoporosis by examining bone turnover marker: serum osteocalcin, serum deoxypyridinoline (DPD) and serum calcitonin in ovariectomized wistar strain rats as model of postmenopausal osteoporosis. Deoxypiridinoline (DPD) serum levels was measured using rat DPD (deoxypyridinoline) ELISA kit from elabscience (E-EL-R0327).

Study design and animals
Obtaining data and analysis: data was obtained after surgery.
Statistical analysis was carried out using SPSS v. 16 software. All data were expressed as means ± standard error of mean (SEM). The data were analyzed by one-way analysis of variance (ANOVA) followed by Page number not for citation purposes 3 Tukey HSD test to determine difference between groups. The data also analyzed by Kruskall-Wallis and Mann-Whitney method if the data is not suitable for parametric analysis. P values of less than 0.05 were considered statistically significant.

Bone turnover marker levels and statistics
Average serum osteocalcin levels are shown in Figure 1.    (Figure 3).

Discussion
Osteoporosis in rats can be observed since 2 weeks after ovariectomy [7]. It was reported that in the proximal tibia, a significant decrease in trabecular bone volume is observed 2 weeks after ovariectomy [7]. At the femoral neck, a significant decrease in trabecular bone volume occurred at 4 weeks afer ovariectomy. The lumbar vertebrae were much more resistant to ovariectomy-induced changes in trabecular bone volume than either the proximal tibia or femoral neck. It was not until 7 weeks afer ovariectomy that a decrease in trabecular bone volume was significant and reached a plateau between 39 and 77 weeks after ovariectomy [8].  [10]. It was also reported that nicotine can increases cholinergic parasympathetic nerve activity which can prevent a decrease in bone mass so that the cutting of the vagus nerve in mice causes a decrease in bone mass in the lumbar vertebra [11]. Nicotine can also reduce osteoclast activity and bone resorption by inhibiting the expression of cathepsin K, collagenase Another marker of bone turnover is deoxypyridinoline (DPD) [13].
DPD is released from collagen degradation in bone resorption. It reflects the level of osteoclastic activity in the bone-remodeling process. This research shows similar trend between serum osteocalcin Page number not for citation purposes 4 and serum DPD. Although statistical analysis shows the level of serum DPD in All group was not significantly different (p < 0.05), but OVX group has 38.5% higher level of DPD compared to sham group. P1 and P2 group shows lower levels of serum DPD compared to OVX group with both was 22.8% and 31.9% lower, respectively. But higher level of serum DPD was shown in P3 group compared to OVX group. It is suggesting that nicotine has dose-dependent manner as previously reported [4]. Previous study reported that only low dose of nicotine improves posterior spinal fusion in an in vivo rabbit model [14]. In menopause conditions also occur increases in the production of Reactive Oxygen Species (ROS) [15]. When ROS production exceeds the body´s capacity to produce anti-oxidants, the associated oxidative stress can cause bone damage and fragility which eventually leads to osteoporosis [16]. At low doses, nicotine can inhibit the process of lipid peroxidation caused by H2O2 and Fe2 + .
Nicotine inhibits the formation of H2O2 and Fe2 + . Lee et al [17]