Abstract: BACKGROUND: Previous in vitro experiments have shown that the compound Miao medicine Jiuxian Luohan Jiegu Decoction may activate autophagy by regulating the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway, thereby promoting the proliferation and differentiation of osteoblasts.
OBJECTIVE: To investigate the effect of Jiuxian Luohan Jiegu Decoction on the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway in rats during fracture healing.
METHODS: Thirty Sprague-Dawley rats were used to establish a femoral fracture model in the right hind limb. After modeling, the rats were randomly divided into five intervention groups, with six rats in each group: the model group received intragastric administration of normal saline; the low-, medium-, and high-dose Jiuxian Luohan Jiegu Decoction groups received intragastric administration of 2.95, 5.9, and 11.8 g/(kg·d) of the decoction, respectively; and the Shangke Jiegu Tablet group received intragastric administration of 0.24 g/(kg·d) of Shangke Jiegu Tablets. All administrations were given twice daily for 28 consecutive days. After 2 and 4 weeks of administration, fracture healing was assessed by X-ray. After 4 weeks of administration, fracture healing was evaluated by Micro-CT; mRNA expression of phosphatidylinositol 3-kinase, protein kinase B, mammalian target of rapamycin, microtubule-associated protein 1 light chain 3II, and P62 in the fracture callus was detected by q-PCR; protein expression of phosphorylated phosphatidylinositol 3-kinase, phosphatidylinositol 3-kinase, phosphorylated protein kinase B, protein kinase B, phosphorylated mammalian target of rapamycin, mammalian target of rapamycin, microtubule-associated protein 1 light chain 3II, and P62 in the fracture callus was detected by western blot; and fracture healing and callus formation were observed by hematoxylin-eosin staining.
RESULTS AND CONCLUSION: After 2 and 4 weeks of administration, X-ray examination showed that fracture healing and callus formation in the high-dose Jiuxian Luohan Jiegu Decoction group were superior to those in the other four groups. Micro-CT analysis showed that fracture healing in the high-dose Jiuxian Luohan Jiegu Decoction group was better than that in the other four groups, characterized by callus circumferentially surrounding the fracture ends, denser and more regularly arranged trabecular bone structure, and blurred fracture lines. Compared with the model group, the mRNA expression of phosphatidylinositol 3-kinase, protein kinase B, mammalian target of rapamycin, and P62 was decreased in the other four groups, while microtubule-associated protein 1 light chain 3II mRNA expression was increased; protein expression of phosphorylated phosphatidylinositol 3-kinase, phosphorylated protein kinase B, phosphorylated mammalian target of rapamycin, and P62 was decreased, while microtubule-associated protein 1 light chain 3II protein expression was increased. The most significant changes were observed in the high-dose Jiuxian Luohan Jiegu Decoction group. Hematoxylin-eosin staining showed that the number of osteoblasts, the amount of new bone tissue, and the quality of bone formation at the fracture site in the high-dose Jiuxian Luohan Jiegu Decoction group were superior to those in the other four groups. To conclude, these findings indicate that Jiuxian Luohan Jiegu Decoction may enhance the level of autophagy in osteoblasts by regulating the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway, thereby accelerating the process of fracture healing in rats.

Key words: compound Miao medicine Jiuxian Luohan Jiegu Decoction, PI3K/AKT/mTOR signaling pathway, fracture healing, autophagy, callus formation