Inductive Effect of Palmatine on Apoptosis in RAW 264.7 Cells

Abstract

Osteoporosis is a serious public health problem characterized by low bone density and deterioration of the bone microarchitecture. Current treatment options target either osteoclast resorption or osteoblast formation. It has been reported that berberine, a close structural analog of palmatine, inhibited bone loss in an osteoporosis model. In this study, osseous metabolism was observed in vitro with osteoclast bone resorbing cells. We proved that mouse preosteoclastic cell line (RAW 264.7) has a higher sensitivity to palmatine than mouse osteoblastic cell line (MC3T3-E1); the cell survival rates significantly decreased at 40 μM palmatine. The NO2 (-) level, a metabolic product of nitric monoxide (NO), and iNOS mRNA expression, an osteoclast with NO induced enzyme, also increased with higher dosage of palmatine. Furthermore, it was recognized that the cell viability decrease from palmatine was caused by apoptosis rather than necrosis. Additionally, osteoclast apoptosis from palmatine did not occur when iNOS was inhibited with N(G)-nitro-L-arginine methyl ester hydrochloride (pan NOS inhibitor). These results indicate that palmatine plays an important role in osteoclast apoptosis via the NOS system. Hence, palmatine could be considered as a viable pharmaceutical candidate for osteoporosis bone resorption inhibitor.

Figure 1

TRAP-positive cell detection with the coculture method. The differentiation to osteoclast of a RAW 264.7 cell was examined using TRAP staining in culture of a RAW 264.7 cell and an MC3T3-E1 cell for 5 days. Digital images were obtained using an optical microscope. RAW 264.7 cells, which were dyed using TRAP in the cell block section, show differentiation of a RAW 264.7 cell, cytogamy image. (a)–(f) ×100 magnification, (g)–(l) ×600 magnification. (m) The number of mature osteoclasts, multinucleated RAW 264.7 cell, per a field (quadrangle of 2 mm × 2.5 mm) was counted to quantify influence of palmatine. Asterisks indicate statistically significant differences (^∗∗ p < 0.01). Error bars denote ±SD.

Figure 2

Cell viability for palmatine with cytotoxicity CCK-8 assays. The proliferation of cells cultured with different palmatine concentrations for 24 h or 5 days was evaluated using a CCK-8 assay. Seeded cells were incubated with or without palmatine for 24 h or 5 days. The cell viability was calculated using the following formula: cell viability (%) = [OD_experiment − OD_blank]/[OD_control − OD_blank] × 100%. The absorbance was measured at 450 nm. (a) The survival rate of MC3T3-E1 cells that were incubated in palmatine component medium for 5 days. (b) The survival rate of RAW 264.7 cells that were incubated in palmatine component medium for 5 days. (c) The survival rate of RAW 264.7 cells that were incubated in palmatine component medium for 24 h. (d) The survival rate of mature RAW 264.7 cells that were incubated in palmatine component medium for 5 days. Asterisks indicate statistically significant differences (^∗∗ p < 0.01, n.s., no significant difference). Error bars denote ± SD.

Figure 3

Apoptosis detection with an APOPercentage assay. The cell apoptotic rate was measured using an APOPercentage assay after the seeded cells were incubated with each palmatine concentration for 24 h. The absorbance at 550 nm was measured. Asterisks indicate statistically significant differences (^∗ p < 0.05, ^∗∗ p < 0.01). Error bars denote ± SD.

Figure 4

Supernatant NO₂ ⁻ level as a surrogate for NO. Nitrous acid ion (NO₂ ⁻) production in RAW 264.7 cells was measured using the NO₂/NO₃ assay kit FX. The black bars show the supernatant NO₂ ⁻ level that cultured RAW 264.7 cells for 2 h. The white bars show the supernatant NO₂ ⁻ level which cultured RAW 264.7 cells for 4 h. Asterisks indicate statistically significant differences (^∗ p < 0.05, ^∗∗ p < 0.01 versus control). Error bars denote ± SD.

Figure 5

Relative quantity (RQ) of iNOS mRNA expression in RAW 264.7 cells. The experiment was conducted to examine whether addition of palmatine would change the iNOS mRNA expression in RAW 264.7 cells. After culture for 2 h, RAW 264.7 cells were collected and used for the measurement of iNOS mRNA expression by RT-PCR. Asterisks indicate statistically significant differences (^∗ p < 0.05, ^∗∗ p < 0.01). Error bars denote ± SD.

Figure 6

Cell viability and supernatant NO₂ ⁻ level detection for L-NAME treatment. NO release was inhibited by the addition of L-NAME to clarify whether NO affects the survival of RAW 264.7 cells. ((a) and (b)) The cell survival rate after culture and NO₂ ⁻ level were examined to clarify the direct influence of L-NAME on RAW 264.7 cells after 5 days in culture medium containing L-NAME. (c) The proliferation of cells cultured with different palmatine concentrations for 24 h was evaluated using a CCK-8 assay. The cell viability was calculated using the following formula: cell viability (%) = [OD_experiment − OD_blank]/[OD_control − OD_blank] × 100%. Absorbance was measured at 450 nm. Nitrous acid ion (NO₂ ⁻) production in RAW 264.7 cells was measured using the NO₂/NO₃ assay kit FX. Asterisks indicate statistically significant differences (^∗∗ p < 0.01, n.s., no significant difference). Error bars denote ± SD.

Figure 7

Bone resorption evaluation with a CaP-coated plate. Bone resorption ability of a RAW 264.7 cell examined using a bone resorption assay kit in culture of a RAW 264.7 cell and an MC3T3-E1 cell for 5 days. The bone resorption activity was evaluated by measuring the pit formation on a CaP-coated plate and the fluorescence intensity of the conditioned medium. (a)–(l) Digital images of pit formation on a CaP-coated plate were obtained using an optical microscope. The brown pit shows the locus where a mature RAW 264.7 cell (differentiation like osteoclast) dissolved a calcium plate. (a)–(f): ×40 magnification, (g)–(l): ×400 magnification. (m) The total pit area on a CaP-coated plate. (n) The fluorescence intensity of bone resorption dependence in the conditioned medium. Asterisks indicate statistically significant differences (^∗ p < 0.05, ^∗∗ p < 0.01). Error bars denote ± SD.