Isorhamnetin ameliorates ovariectomy-induced osteoporosis in female rats by regulating the receptor activator of nuclear factor kappa B ligand, osteoprotegerin, bone morphogenetic protein 2 and runt-related transcription factor 2 signaling pathway

Abstract

Osteoporosis is characterized by reduced bone density and increased fracture risk. The present study assessed anti-osteoporotic effects of isorhamnetin on ovariectomy (OVX)-induced osteoporosis in female rats. Osteoporosis was induced in OVXX-female Sprague-Dawley rats by using Freund's Complete Adjuvant and randomly divided into the following groups (n=15): OVX control, alendronate (3 mg/kg, subcutaneous), and isorhamnetin (10, 20, and 40 mg/kg, p.o.), and received treatment for five weeks after OVX. In results following OVX, significant alterations in behavioral, biochemical, and histological parameters were observed. Conversely, isorhamnetin (20 and 40 mg/kg) treatment significantly improved (P<0.05) OVX-induced alterations in body, femur, and uterine weight, bone mineral content and density, but also effectively mitigated (P<0.05) elevated allodynia and hyperalgesia. It notably improved (P<0.05) changes in serum alkaline phosphatase, osteocalcin, C-terminal telopeptide of type I collagen (CTX-I), serum and urinary calcium, and phosphorus levels. Isorhamnetin markedly attenuated elevated (P<0.05) serum TNF-α, IL-1β, IL-6 levels but increased (P<0.05) serum IL-4 and IL-10 levels. Furthermore, mRNA expression of osteoprotegerin (OPG), runt-related transcription factor 2 (Runx2), and bone morphogenetic protein 2 (BMP-2) was upregulated (P<0.05), whereas receptor activator of nuclear factor kappa B ligand (RANKL) was downregulated (P<0.05). Histological analysis demonstrated that isorhamnetin effectively improved (P<0.05) OVX-induced inflammation, thereby preventing cellular infiltration, synovial hyperplasia, cartilage erosion, and pannus formation in bone specimens. In conclusion, isorhamnetin exerts its anti-osteoporotic potential by modulating pain (allodynia and hyperalgesia), serum biomarkers (osteocalcin, CTX-I, TNF-α, ILs), and bone signaling pathways (RANKL, OPG, BMP-2, Runx2).

Keywords: isorhamnetin; osteocalcin; osteoporosis; osteoprotegerin; pro-inflammatory cytokines; receptor activator of nuclear factor kappa B ligand.

Fig. 1.

Effect of isorhamnetin on body weight (A) and pain-related behavioral parameters, including paw withdrawal threshold (B), paw withdrawal latency (C), and changes in joint diameter (D) in rats with ovariectomy-induced osteoporosis. The results are presented as the mean ± SEM (n=6) and were subjected to one-way ANOVA, followed by Tukey’s multiple range test for analysis. Statistical significance (P<0.05) is indicated when compared to the *OVX control group, ^#Sham group, and ^$between groups. The figures in parentheses indicate the doses of the respective treatments (mg/kg). A, Alendronate; IHN, isorhamnetin; OVX, ovariectomy.

Fig. 2.

Effect of isorhamnetin on femoral OPG (A), Runx2 (B), RANKL (C), and BMP-2 (D) mRNA expression in rats with ovariectomy-induced osteoporosis. One thousand base pair ladder of mRNA expression (lane 1); mRNA expression in the sham group (lane 2); OVX Control group (lane 3); alendronate (3 mg/kg)-treated group (lane 4); and isorhamnetin (10, 20, and 40 mg/kg)-treated groups (Lane 5–7). The results are presented as the mean ± SEM (n=6) and were subjected to one-way ANOVA, followed by Tukey’s multiple range test for analysis. Statistical significance (P<0.05) is indicated when compared to the *OVX control group, ^#Sham group, and ^$between groups. The figures in parentheses indicate the doses of the respective treatments (mg/kg). A, Alendronate; BMP-2, bone morphogenetic protein 2; IHN, isorhamnetin; OPG, osteoprotegerin; OVX, ovariectomy; RANKL, receptor activator of nuclear factor-kappa-B ligand; Runx2, Runt-related transcription factor 2.

Fig. 3.

Simple regression of femoral OPG (A), Runx2 (B), RANKL (C), and BMP-2 (D) mRNA expression levels in OVX rats. Correlation coefficients were determined using a two-sided Fisher’s test. BMP-2, bone morphogenetic protein 2; OPG, osteoprotegerin; OVX, ovariectomy; RANKL, receptor activator of nuclear factor-kappa-B ligand; Runx2, Runt-related transcription factor 2.

Fig. 4.

Effect of isorhamnetin on the histopathology of femur bone in rats with ovariectomy-induced osteoporosis stained with hematoxylin and eosin (H&E) (40 × and inset at 100 × with a scale bar=100 μm) (A–F). Quantitative representation of the histological scores (G). Results are presented as mean ± SEM (n=3) and were subjected to one-way ANOVA, followed by the Kruskal-Wallis test for post-hoc analysis. Statistical significance (P<0.05) is indicated when compared to the *OVX control group, ^#Sham group, and ^$between groups. Histological changes, such as cellular infiltration (red arrow) and cartilage degradation (blue arrow), are indicated with the respective colored arrows. The figures in parentheses indicate the doses of the respective treatments (mg/kg). A, Alendronate; IHN, isorhamnetin; OVX, ovariectomy.