Abstract:

Objective To discuss the effects of aurone on osteoblast differentiation and its ameliorative effect on glucocorticoid-induced osteoporosis （GIOP） in rats， and to clarify its mechanism. Methods For in vitro experiments， MC3T3-E1 osteoblasts were selected. CCK-8 method was used to detect the cell activities in various groups after treatment with different concentrations of dexamethasone （Dex） and aurone to determine the optimal treatment concentrations of Dex and aurone. MC3T3-E1 osteoblasts were induced with 1 μmol·L^-1 Dex to establish a cell injury model. The cells were divided into control group， Dex group， Dex+aurone group， Dex+GNF351 group， and Dex+aurone+GNF351 group. Kit method was used to detect the level of malondialdehyde （MDA） and the activity of superoxide dismutase （SOD） in the cells in various groups； 2'，7'-dichlorofluorescein diacetate （DCFH-DA） fluorescence method was used to detect the level of reactive oxygen species （ROS） in the cells in various groups； enzyme-linked immunosorbent assay （ELISA） method was used to detect the levels of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， and interleukin-6 （IL-6） in the supernatants of the cells in various groups； alkaline phosphatase （ALP） kit method was used to detect the ALP activity in the cells in various groups； Western blotting method was used to detect the expression levels of Runt-related transcription factor 2 （RUNX-2）， osteocalcin （OCN）， collagen type Ⅰ alpha 1 chain （COL1A1）， osteopontin （OPN）， aromatic hydrocarbon receptor （AHR）， nuclear factor erythroid 2-related factor 2 （NRF2）， and NAD（P）H： quinone oxidoreductase 1 （NQO1） proteins in the cells in various groups. For in vivo experiments， 20 female rats were selected and randomly divided into sham operation group， GIOP group， GIOP+low dose of aurone （7 mg·kg^-1） group， and GIOP+high dose of aurone （14 mg·kg^-1） group， with 5 rats in each group. Except for sham operation group， the rats in various groups were intragastrically administered with 1 mg·kg^-1 Dex to establish GIOP model. The rats in various groups were intragastrically administered with drugs from 2 h after modeling； after continuous intervention for 7 weeks， blood was collected and the rats were sacrificed， and femoral tissue was collected. HE staining method was used to observe the pathological morphology of femoral tissue of the rats in various groups. Histomorphometry method was used to detect the bone mineral density （BMD） of the femoral tissue and trabecular bone volume fraction （BV/TV）， trabecular thickness （Tb.Th）， trabecular number （Tb.N）， and trabecular separation （Tb.Sp） of the rats in various groups. Western blotting method was used to detect the expression levels of AHR， NRF2， and NQO1 proteins in the femoral tissue of the rats in various groups. Results Compared with 0 μmol·L^-1 Dex group， the cell activity of MC3T3-E1 cells in 1 μmol·L^-1 Dex group was significantly decreased （P<0.01）， and 1 μmol·L^-1 Dex was determined for subsequent cell treatment. Compared with Dex group， the cell activity of MC3T3-E1 cells in Dex+5 μmol·L^-1 aurone group was significantly increased （P<0.01）， and 5 μmol·L^-1 aurone was used for subsequent cell treatment. Compared with control group， the cell activity of MC3T3-E1 cells in Dex group was significantly decreased （P<0.01）； the levels of MDA and ROS in the cells and the levels of TNF-α， IL-1β， and IL-6 in the supernatants were significantly increased （P<0.01）； the SOD activity was significantly decreased （P<0.01）； the ALP activity in the cells was significantly decreased （P<0.01）； the expression levels of RUNX-2， OCN， COL1A1， and OPN proteins in the cells were significantly decreased （P<0.01）； and the expression levels of AHR and NRF2 proteins in the nucleus and the expression level of NQO1 protein in the cells were significantly decreased （P<0.01）. Compared with Dex group， the cell activity of MC3T3-E1 cells in Dex+aurone group was significantly increased （P<0.01）； the levels of MDA and ROS in the cells and the levels of TNF-α， IL-1β， and IL-6 in the supernatants were significantly decreased （P<0.01）； the SOD activity was significantly increased （P<0.01）； the ALP activity in the cells was significantly increased （P<0.01）； and the expression levels of RUNX-2， OCN， COL1A1， and OPN proteins in the cells were significantly increased （P<0.01）. Compared with Dex+aurone group， the ALP activity in the cells in Dex+aurone+GNF351 group was significantly decreased （P<0.01）， and the expression levels of AHR and NRF2 proteins in the nucleus as well as the expression levels of NQO1， RUNX-2， and OCN proteins in the cells were significantly decreased （P<0.01）. The in vivo experiment results showed that compared with sham operation group， the trabecular bones in the femoral tissue of the rats in GIOP group were thinned， reduced in number， and diminished in area， the BMD of the femoral tissue， trabecular BV/TV， Tb.Th， and Tb.N of the rats were significantly decreased （P<0.01）， the Tb.Sp was significantly increased （P<0.01）， and the expression levels of AHR， NRF2， and NQO1 proteins in the femoral tissue of the rats were significantly decreased （P<0.01）. Compared with GIOP group， the pathological damage in femoral tissue of the rats in GIOP+low dose of aurone group and GIOP+high dose of aurone group was alleviated， the BMD of the femoral tissue， trabecular BV/TV， Tb.N， and Tb.Th were significantly increased （P<0.05）， the Tb.Sp was significantly decreased （P<0.05）， and the expression levels of AHR， NRF2， and NQO1 proteins in the femoral tissue were significantly increased （P<0.05 or P<0.01）. Conclusion Aurone promotes osteoblast differentiation and alleviates GIOP in rats， and its mechanism is potentially associated with promoting the nuclear translocation of AHR and activating the AHR/NRF2/NQO1 signaling pathway.

Key words: Osteoporosis, Osteoblast differentiation, Aurone, MC3T3-E1 cell, Aromatic hydrocarbon receptor