Objective: The aim of the present study was to evaluate the potential protective mechanism of icariin against oxidative damage caused by hydrogen peroxide in MC3T3-E1 cells.
Methods: MC3T3-E1 cells were treated with different concentrations of icariin to explore the optimal dose of icariin. MC3T3-E1 cells were divided into groups treated with various concentrations of hydrogen peroxide (H2 O2 ; 0, 0.1, 0.2, 0.5, 1, and 2 mM) for 24 h to induce oxidative damage and cell viability was assessed by Cell Counting Kit-8 (CCK-8) assay. Then, cells were divided into five groups: control, H2 O2 (0.2 mM), icariin (0.1 μM) and H2 O2 (0.2 mM), + icariin (0.1 μM). Cell viability was detected by CCK-8 assay. In addition, the content of glutathione and superoxide dismutase and the activity level of malondialdehyde in these treatment groups were determined. Alkaline phosphatase (ALP) and alizarin red S (ARS) staining were also performed to measure the early and late osteogenesis, respectively. Protein expression of β-catenin and cyclin D1 was measured by western blot assay. Then, we used an antagonist of Wnt/β-catenin signaling pathway (DKK-1) and western blot analysis to further explore potential mechanism.
Results: After 24 h of exposure to 0.2 mM H2 O2 , the viability of MC3T3-E1 cells was significantly decreased compared to that of the control cells. We first found that icariin can promote cell proliferation of MC3T3-E1 cells in a dose-dependent manner, with the dosage 0.1 μM showing the best pro-proliferative effect. Furthermore, icariin could promote the protein expression of OSX and RUNX2. The results showed that icariin can reverse the inhibitory osteogenic effects of MC3T3-E1 caused by H2 O2 . In addition, icariin could increase the Wnt-signaling related proteins. The results showed that MC3T3-E1 cells in the H2 O2 (0.2 mM) + icariin (0.1 μM) + Wnt-signaling antagonist (DKK-1) group had weaker ALP and ARS staining compared with that observed in the control and H2 O2 (0.2 mM) + icariin (0.1 μM) groups. The ALP activity and calcium content were decreased in the 0.2 mM H2 O2 + 0.1 μM icariin + DKK-1 group compared to that observed in the 0.2 mM H2 O2 + 0.1 μM icariin group.
Conclusion: The results showed that icariin can increase the viability of MC3T3-E1 cells, reverse the oxidative stress induced by H2 O2 and protect MC3T3-E1 cells against H2 O2 -induced inhibition of osteogenic differentiation, which may occur through the Wnt/β-catenin signaling pathway.
Keywords: H2O2; Icariin; MC3T3-E1; Osteogenesis.
© 2021 The Authors. Orthopaedic Surgery published by Chinese Orthopaedic Association and John Wiley & Sons Australia, Ltd.