Norisoboldine, a Natural Isoquinoline Alkaloid, Inhibits Diaphyseal Fracture Healing in Mice by Alleviating Cartilage Formation

Abstract

Norisoboldine (NOR), the major isoquinoline alkaloid constituent of a Chinese traditional medicine Radix Linderae, has been demonstrated to inhibit osteoclast differentiation and improve arthritis. The aim of this study is to examine the effect of NOR on bone fracture healing and the underlying mechanisms correlated with bone marrow stromal cells (BMSCs) differentiation to chondrocytes. Our results showed that NOR inhibits the tibia fracture healing process by suppressing cartilage formation, which leads to less endochondral ossification, indicated by less osterix and collage I signaling at the fracture site. Moreover, NOR significantly reduced the differentiation of primary BMSCs to chondrocytes in vitro by reducing the bone morphogenetic protein 2 (BMP2) signaling. These findings imply that NOR negatively regulates the healing of the tibial midshaft fracture, which might delay the union of the fractures and should be noticed when used in other treatments.

Keywords: BMP2; endochondral ossification; fracture healing; norisoboldine.

Figure 1

NOR treatment inhibited callus formation in mice tibia fracture healing; (A) chemical structure of NOR; (B) body weight change at 7 and 14 days post-fracture; (C) schematic of a mouse tibial fracture following intramedullary pinning; (D) representative computer renderings of bone structure of the fracture sites at days 7 and 14 post-fracture; (E–H) quantitative analysis of callus after micro-CT scanning (male, n = 5–6): (E) BV/TV (%); (F) Tb. N (1/mm); (G) trabecular thickness (Tb. Th) (mm); (H) Tb. Sp (mm). * p < 0.05.

Figure 2

NOR suppresses cartilage formation and chondrogenic differentiation of BMSCs. (A) Safranin O staining of the fracture site at 7 and 14 days post-fracture. (Scale bar, 500 μm). (B) Relative cartilage area in the fracture callus at 7, 14, and 21 days after fracture. (C) Relative bone mineral area in the fracture callus at 7 and 14 days after fracture. * p < 0.05.

Figure 3

NOR suppresses the ability of BMSCs differentiation into chondrocytes. (A) Wound healing assay representing cell migration ability in control and NOR group. (Scale bar, 100 μm). (B) Alcian blue staining of the BMSCs incubated in chondrogenic medium for 14 days. (Scale bar, 200 μm). (C–H) Bmp2, Aggrecan, Col2a1, Alk2, IL-1β and IL-6 mRNA levels in 7 and 14 days differentiated BMSCs. * p < 0.05.

Figure 4

NOR suppressed OSX and COL1 signaling at the fracture site. Tibias from fracture model were co-stained for OSX (green) and COL1 (red) at 7 or 14 days after surgery. (Scale bar: 100 μm).