Anti-Osteoporosis Effect of Perilla frutescens Leaf Hexane Fraction through Regulating Osteoclast and Osteoblast Differentiation

Abstract

Osteoporosis is the result of an imbalance in the bone-remodeling process via an increase in osteoclastic activity and a decrease in osteoblastic activity. Our previous studies have shown that Perilla frutescens seed meal has anti-osteoclastogenic activity. However, the role of perilla leaf hexane fraction (PLH) in osteoporosis has not yet been investigated and reported. In this study, we aimed to investigate the effects of PLH in osteoclast differentiation and osteogenic potential using cell-based experiments in vitro. From HPLC analysis, we found that PLH contained high luteolin and baicalein. PLH was shown to inhibit RANKL-induced ROS production and tartrate-resistant acid phosphatase (TRAP)-positive multi-nucleated osteoclasts. Moreover, PLH significantly downregulated the RANKL-induced MAPK and NF-κB signaling pathways, leading to the attenuation of NFATc1 and MMP-9 expression. In contrast, PLH enhanced osteoblast function by regulating alkaline phosphatase (ALP) and restoring TNF-α-suppressed osteoblast proliferation and osteogenic potential. Thus, luteolin and baicalein-rich PLH inhibits osteoclast differentiation but promotes the function of osteoblasts. Collectively, our data provide new evidence that suggests that PLH may be a valuable anti-osteoporosis agent.

Keywords: Perilla frutescens; RANKL; TNF-α; osteoblast; osteoclast; osteoporosis.

Figure 1

HPLC chromatograms of mixed standard and PLH. The peaks reveal (a) gallic acid, (b) caffeic acid, (c) rutin, (d) rosmarinic acid, (e) luteolin, (f) quercetin, (g) apigenin, (h) kaempferol, and (i) baicalein. The mobile phase consisted of 30% acetonitrile in 0.1% acetic acid and de-ionized water at a flow rate of 1.0 mL/min. The content peaks were detected by a UV detector at 325 nm.

Figure 2

Viability of RAW264.7, MG-63, and SAOS-2 cells incubated with various concentrations of PLH using MTT assay. Each value is the mean ± SD of three independent experiments. # p < 0.05 vs. control (0).

Figure 3

RANKL-induced ROS production by PLH treatment. Untreated cells were used as a negative control (Con). N-acetylcysteine 80 μM (NAC) and vitamin C 250 μM (Vit C) were used as positive controls. Each value is the mean ± SD of three independent experiments, ### p < 0.001 vs. control (Con), *** p < 0.001 vs. RANKL treatment (0).

Figure 4

RANKL-induced TRAP-positive osteoclast-like cell formation by PLH treatment. (A) Osteoclast differentiation: Multinucleated osteoclasts were visualized in 100× magnification under light microphotography. Scale bars, 100 mm. (B) TRAP-positive multinucleated cells were counted as osteoclasts. (C) TRAP activity was measured using the TRAP solution assay. Luteolin (Lut) and baicalein (Bai) were active compounds of PLH. Each value is the mean ± SD of three independent experiments. ### p < 0.001 vs. control (Con), *** p < 0.001 vs. RANKL treatment (0).

Figure 5

RANKL-induced MMP-9 expression by PLH treatment. RAW264.7 cells were co-treated with RANKL and PLH for 72 h. Culture supernatants were collected, and the secretion of MMP-9 was analyzed using gelatin zymography. Each value is the mean ± SD of three independent experiments. ### p < 0.001 vs. control (Con), *** p < 0.001 vs. RANKL treatment (0).

Figure 6

RANKL-induced NF-κB, MAPK, and NFATc1 signaling by PLH treatment. RAW 264.7 cells were pretreated with PLH (0, 12.5, 25, and 50 µg/mL) for 12 h and then exposed to RANKL (100 ng/mL) for 10 min. The whole cell extract was used to determine phosphorylation levels of NF-κB, JNK, and p38. The nuclear extracts were prepared and NFATc1 expression was analyzed. (A) Protein expression of NF-κB, MAPKs, and NFATc1 was measured using the Western blot method. (B) The expression of p-p65, p-JNK, p-p38, and NFATc1 was normalized to total p65, JNK, p38, and PARP, respectively. Each value is the mean ± SD of three independent experiments. ### p < 0.001; ## p < 0.01; # p < 0.05 vs. control (Con), *** p < 0.001; ** p < 0.01; * p < 0.05 vs. RANKL treatment (0).

Figure 7

ALP activity and MMP-2 expression in osteoblast-like cells by PLH treatment. (A) MG-63- and (B) SAOS-2-treated cells were tested for ALP using colorimetric analysis. (C) MG-63 and (D) SAOS-2 culture supernatants were collected and analyzed for MMP-2 secretion by gelatin zymography. Luteolin (Lut) and baicalein (Bai) were active compounds of PLH. Each value is the mean ± SD of three independent experiments. ### p < 0.001; ## p < 0.01; # p < 0.05 vs. control (Con).

Figure 8

PLH restored TNF-α-suppressed osteogenic potential. Cells were co-treated with TNF-α (20 ng/mL) and various concentrations of PLH (0–50 μg/mL) for 48 h (cell proliferation) and 72 h (ALP activity). (A) MG-63- and (B) SAOS-2-treated cells were measured for viability using MTT assay. (C) MG-63- and (D) SAOS-2-treated cells were tested for ALP using colorimetric analysis. Each value is the mean ± SD of three independent experiments. ### p < 0.001; # p < 0.05 vs. control (Con), *** p < 0.001; ** p < 0.01 vs. TNF-α treatment (0).