Unveiling the potential of Butylphthalide: inhibiting osteoclastogenesis and preventing bone loss

Abstract

Osteoporosis, resulting from overactive osteoclasts and leading to elevated fracture risk, has emerged as a global public health concern due to the aging population. Therefore, inhibiting osteoclastogenesis and bone resorption function represents a crucial approach for preventing and treating osteoporosis. The purpose of this study was to examine the effects and molecular mechanisms of Butylphthalide (NBP) on the differentiation and function of osteoclasts induced by RANKL. Osteoclastogenesis was assessed through TRAP staining and bone slice assay. An animal model that underwent ovariectomy, simulating postmenopausal women's physiological characteristics, was established to investigate the impact of Butylphthalide on ovariectomy-induced bone loss. To delve deeper into the specific mechanisms, we employed Western blot, PCR, immunofluorescence, and immunohistochemical staining to detect the expression of proteins that are associated with the osteoclast signaling pathway. In this study, we found that Butylphthalide not only suppressed osteoclastogenesis and bone resorption in vitro but also significantly decreased TRAcP-positive osteoclasts and prevented bone loss in vivo. Further mechanistic experiments revealed that Butylphthalide reduces intracellular ROS in osteoclasts, inhibits the MAPK and NFATc1 signaling pathways, and downregulates the key genes and proteins of osteoclasts. This inhibits osteoclast formation and function. The reduction in ROS in osteoclasts is intricately linked to the activity of Butylphthalide-modulated antioxidant enzymes. Overall, NBP may offer a alternative treatment option with fewer side effects for skeletal diseases such as osteoporosis.

Keywords: Butylphthalide; ROS; osteoblast; osteoclast; osteoporosis.

FIGURE 1

NBP repress RANKL-induced osteoclastogenesis in vitro. (A) An illustration of NBP’s molecular structure. (B,C) Cell activity was determined by the CCK-8 assay following exposure to various concentrations of NBP on BMMS for 48 and 96 h. (D) Representative images of TRAcP demonstrated that NBP suppressed the osteoclastogenesis within 7 days of RANKL (50 ng/mL) stimulation (scale bar = 1000 µm). (E) The number of cells with positive TRAcP markers was quantitatively analyzed (nuclei ≥ 3). (F) Representative images of TRAcP revealed that NBP (20 µM) inhibited the process of osteoclastogenesis during specific time periods (scale bar = 1000 µm). (G) TRAcP⁺ cells in each well at different time points (nuclei ≥ 3). *p < 0.05, **p < 0.01, ***p < 0.001 relative to the 0 µM NBP. All data of the bar are presented as the mean ± SD (n = 3 per group).

FIGURE 2

NBP attenuates podosome belt formation and osteoclasts resorption in vitro. (A) Representative images of NBP inhibition of podosome belt formation (scale bar = 1000 µm). (B) Quantification of the average area of osteoclast in different groups of osteoclasts. (C) Quantification of the number of nuclei in each group of osteoclasts. (D) Representative images of microscopic scans of bone slice (scale bar = 200 µm). (E) The resorbed area of bone slices was quantitatively analyzed. *p < 0.05, **p < 0.01, ***p < 0.001 relative to the 0 µM NBP. All data of the bar are presented as the mean ± SD (n = 3 per group).

FIGURE 3

NBP prevents OVX-induced bone loss. (A) The representative images of Micro-CT of proximal femur per group. (B–E) The parameters related to the bone microstructure (Tb.Th, Tb. Sp, Tb.N, BV/TV) were quantified using Micro-CT software. (F) Representative Micro-CT representative images of cortical femur were acquired in various groups. (G–I) Quantification of the parameters related to the microstructure of the bone (BV/TV, B.Ar, B.Pm) in the cortical bone of the proximal femur in different groups. *p < 0.05, **p < 0.01, ***p < 0.001 relative to the 0 mg/kg. All data of the bar are presented as the mean ± SD (n = 6 per group).

FIGURE 4

NBP prevents OVX-induced bone loss and reduce osteoclasts. (A) Representative HE staning images of femurs in different groups. (B) Quantification the percentage of bone trabecular. (C) Representative TRAcP staining images of femur. (D) Quantification of osteoclast number/bone surface. *p < 0.05, **p < 0.01, ***p < 0.001 relative to the 0 mg/kg. All data of the bar are presented as the mean ± SD (n = 3 per group).

FIGURE 5

The NBP attenuates ROS in RANKL-stimulated osteoclasts, and elevates the antioxidant enzymes expression both in vitro and in vivo. (A) Representative images of intracellular ROS after RANKL stimulation of osteoclasts. (scale bar = 400 µm) (B) Quantification of DCF fluorencence intensity. (C) Quantification of the amount of ROS⁺ cells. (D) Representative images of Western blot demonstrate the impact of NBP on antioxidant enzymes. (E–H) Quantification of the ratios of GSR, Nrf2, HO-1, CAT relative to β-actin. (I) Representative images of histologic staining for HO-1 in femoral sections. (J) Quantification of the HO-1⁺ area on femoral sections. (K) Representative histological staining images for Nrf2 of femurs after treatment with NBP. (L) Quantification of the Nrf2⁺ area on femoral sections. *p < 0.05, **p < 0.01, ***p < 0.001 relative to the 0 μM NBP. All data of the bar are presented as the mean ± SD (n = 3 per group).

FIGURE 6

NBP repress the activity and translocation of NFATc1 (A–F) Expression of mRNA for Nfatc1, Fos, Ctsk, Mmp9, Atp6v0d2, Acp5 in osteoblasts after RANKL induction. (G) Representative images of Western blot demonstrate the impact of NBP on osteoclast-specific proteins (CTSK, c-Fos, NFATc1). BMM were interfered with RANKL in the absence or presence of 20 μM NBP for 0, 1, 3, 5 days. (H–J) The expression of the above mentioned proteins was analyzed quantitatively in relation to the β-actin. (K) Representative images of NFATc1 nuclear translocation in osteoclasts induced by RANKL (scale bar = 200 μM). (L) Quantification of the translocation of NFATc1. *p < 0.05,**p < 0.01,***p < 0.001 relative to the 0 μM NBP. All data of the bar are presented as the mean ± SD (n = 3 per group).

FIGURE 7

NBP inhibits MAPK signaling in osteoblasts following RANKL induction. (A) The expression of MAPK signaling pathway (p-ERK, ERK, p-P38, P38, JNK, p-JNK) and β-actin following NBP intervention were evaluated by Western blot. (B–D) The expression of the above mentioned proteins was analyzed quantitatively in relation to the β-actin. (E) The expression of NF-κB signaling pathway (IκB-α, P65, and p-P65) and β-actin following NBP intervention were evaluated by Western blot. (F,G) The expression of the NF-κB signaling pathway was analyzed quantitatively in relation to the β-actin. *p < 0.05, **p < 0.01, ***p < 0.001 relative to the 0 μM NBP. All data of the bar are presented as the mean ± SD (n = 3 per group).

FIGURE 8

NBP is not involved in forming and mineralizing bone. (A) Representative ALP staining images of MC3T3-E1 cells after intervention with different concentrations of NBP (scale bar = 1000 μM). (B) Quantification of ALP in osteoblasts after NBP intervention. (C) Representative ARS staining images of MC3T3-E1 cells after intervention with different concentrations of NBP (scale bar = 1000 μM). (D) Quantification of ARS in osteoblasts after NBP intervention. *p < 0.05, **p < 0.01, ***p < 0.001 relative to the 0 μM NBP. All data of the bar are presented as the mean ± SD (n = 3 per group).

FIGURE 9

The mechanism by which NBP inhibits osteoclastogenesis is demonstrated. This study demonstrates that NBP inhibits MAPK pathway phosphorylation, reduces ROS, increases antioxidant enzyme levels, and ultimately decreases osteoclast formation and bone resorption by down-regulating the NFATc1 signaling pathway.