2022, Vol. 26 ›› Issue (32): 5155-5161

Emodin promotes fracture healing in osteoporotic fracture rats by inhibiting miR-338-3p expression

Li Haoliang, Li Dongfang   

Abstract: BACKGROUND: Osteoporosis is characterized by low bone mass and osseous degenerative changes. These conditions increase the risk of fractures. Emodin has potent anti-inflammatory effects and can affect bone metabolism, but its specific mechanism of action is unknown.
OBJECTIVE: To investigate the effects and related mechanisms of emodin on the healing of osteoporotic fracture rats.
METHODS: Fifty-three female Sprague-Dawley rats were randomly divided into sham (n=5) and osteoporotic (n=48) groups. An osteoporotic model was created by ovariectomy and then the right femur of each model rat was crosscut. The model rats in the osteoporotic group were further randomized into six groups: simple fracture group, low-, middle-, and high-dose emodin groups, high-dose emodin+NC mimic group, and high-dose emodin+miR-338-3p mimic group, with 8 rats in each group. Emodin was intragastrically administered at a dose of 10, 20, and 40 mg/kg, once daily for 8 weeks; NC mimic and miR-338-3p mimic were injected into the callus at a dose of 2 mg/kg, once a day, for 7 continuous days. After 8 weeks of intragastrical administration, the bone mineral density of the rat femur at the midshaft and lumbar vertebrae was analyzed. The levels of serum inflammatory factors including NO, tumor necrosis factor α, and interleukin 1β were detected using ELISA. Histomorphological examination was performed using hematoxylin-eosin staining. The level of miR-338-3p in the rat callus was determined with RT-qPCR. The effects of different doses of emodin on osteogenesis-related genes and proteins involved in the ERK/BMP/Smad5 signaling pathway in the rat callus were determined by western blot analysis.
RESULTS AND CONCLUSION: Compared with the simple fracture group, emodin increased the bone mineral density of the femoral midshaft and lumbar vertebrae in a dose-dependent manner (P < 0.05). The bone mineral density of the femoral midshaft and lumbar vertebrae in the high-dose emodin+miR-338-3p mimic group was significantly higher than that in the simple fracture group but lower than that in the high-dose emodin group (P < 0.05). Hematoxylin-eosin staining results showed that compared with the simple fracture group, emodin increased the trabecular bone area in the femoral tissue in a dose-dependent manner (P < 0.05). The trabecular bone area in the high-dose emodin+miR-338-3p mimic group was larger than that in the simple fracture group but lower than that in the high-dose emodin group. Compared with the simple fracture group, emodin increased serum nitric oxygen level and decreased serum tumor necrosis factor α and interleukin 1β levels in a dose-dependent manner (P < 0.05). The levels of serum inflammatory factors in the high-dose emodin+miR-338-3p mimic group were higher than those in the simple high-dose emodin group but lower than those in the fracture group. Compared with the simple fracture group, emodin decreased the expression of miR-338-3p mRNA in the callus in a dose-dependent manner (P < 0.05). Compared with the simple fracture group, emodin dose-dependently increased the protein expression of alkaline phosphatase, osteocalcin, Runx2, bone morphogenetic protein 2, and phosphorylated Smad5 (P < 0.05) and reduced the protein expression of phosphorylated ERK1/2 and phosphorylated JNK in the callus. To conclude, emodin could dose-dependently promote fracture healing in osteoporotic fracture rats, probably by suppressing the expression of miR-338-3p and activating the ERK/JNK/BMP/Smad signaling pathway.
Key words: emodin, osteoporosis, miR-338-3p, fracture, ERK/BMP/Smad5 signaling pathway