Long-term inorganic nitrate administration protects against ovariectomy-induced osteoporosis in rats

Abstract

The risk of osteoporotic fractures increases in women after menopause. This study aims at determining the effects of long-term inorganic nitrate administration against ovariectomy-induced osteoporosis in rats. Rats were divided into 4 groups (n=6/group): Control, control+nitrate, ovariectomized (OVX), and OVX+nitrate. Sodium nitrate (100 mg/L in drinking water) was administered for 9 months. Trabecular bone quality in the proximal tibia was measured using a Micro-Computed Tomography (micro-CT) scanner at months 0, 1, 3, and 9. Levels of nitric oxide (NO) metabolites (NOx) and oxidative stress indices, and mRNA expression of endothelial NO synthase (eNOS) were measured at month 9 in the proximal tibia. Compared to controls, OVX rats had lower NOx levels by 47 %, eNOS mRNA expression by 55 %, catalase activity (CAT) by 45 %, total antioxidant capacity (TAC) by 70 %, and higher malondialdehyde (MDA) levels by 327 % in the bone tissue at month 9. OVX rats, compared to controls, had lower bone volume/tissue volume (BV/TV), trabecular number (Tb.N.), and trabecular thickness (Tb.Th.) by 32 %, 58 %, and 17 %, respectively, and higher trabecular separation (Tb.Sp.) by 123 %, at month 9. Nitrate administration to control rats increased TAC by 46 % in the bone tissue at month 9 but did not significantly affect other parameters in serum and bone tissue. Nitrate in OVX rats significantly increased NOx levels by 86 %, eNOS expression by 2.14-fold, CAT activity by 75 %, TAC by 170 %, and decreased MDA levels by 36 % at month 9 in the bone tissue. Nitrate-treated OVX rats at month 9 had higher BV/TV (42 %) and Tb.N. (61 %) and lower Tb.Sp. (15 %). Long-term inorganic nitrate administration at a low dose has protective effects against OVX-induced osteoporosis in rats; this effect is associated with increasing eNOS-derived NO and decreasing oxidative stress in the bone tissue.

Keywords: nitrate; nitric oxide deficiency; osteoporosis; ovariectomy; oxidative stress; rat.

Table 1. Primer sequences used for real-time PCR

Table 2. Verification of ovariectomy in rats

Table 3. Verification of osteoporosis in rats

Figure 1. Experimental design of the study

BV/TV, bone volume/tissue volume; CAT, catalase activity; eNOS, endothelial nitric oxide (NO) synthase; GSH, reduced glutathione; iNOS, inducible NOS; MDA, malondialdehyde; NOx, NO metabolites; TAC, total antioxidant capacity; Tb.N., trabecular number; Tb.Th., trabecular thickness; Tb.Sp., trabecular separation.

Figure 2. Effect of inorganic nitrate on (A) serum nitric oxide (NO) metabolites (NOx) during the study, (B) bone NOx levels, (C) bone mRNA expression of the endothelial NO synthesis (eNOS), and (D) inducible NOS (iNOS) at month 9 in ovariectomized (OVX) and control rats. ^*,† P < 0.05 compared to control and OVX rats, respectively. Results are mean ± SEM (n=6/group).

Figure 3. Effects of inorganic nitrate on trabecular bone volume (BV/TV, A), number (Tb.N., B), thickness (Tb.Th., C), and separation (Tb.Sp., D) in ovariectomized (OVX) and control rats.

^*, † P < 0.05 compared to control and OVX rats, respectively. Results are mean ± SEM (n=6/group).

Figure 4. Effects of inorganic nitrate on catalase activity (CAT, A), total antioxidant capacity (TAC, B), malondialdehyde (MDA, C), and reduced glutathione (GSH, D) levels in serum of ovariectomized (OVX) and control rats. ^{*, †} P < 0.05 compared to control and OVX rats, respectively. Results are mean ± SEM (n=6/group).

Figure 5. Effects of inorganic nitrate on catalase activity (CAT, A), total antioxidant capacity (TAC, B), malondialdehyde (MDA, C), and reduced glutathione (GSH, D) levels in the bone tissue of ovariectomized (OVX) and control rats. ^*,† P < 0.05 compared to control and OVX groups, respectively. Results are mean ± SEM (n=6/group).