Protection of Icariin Against Hydrogen Peroxide‐Induced MC3T3‐E1 Cell Oxidative Damage

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 (H(2)O(2); 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, H(2)O(2) (0.2 mM), icariin (0.1 μM) and H(2)O(2) (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 H(2)O(2), 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 H(2)O(2). In addition, icariin could increase the Wnt‐signaling related proteins. The results showed that MC3T3‐E1 cells in the H(2)O(2) (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 H(2)O(2) (0.2 mM) + icariin (0.1 μM) groups. The ALP activity and calcium content were decreased in the 0.2 mM H(2)O(2) + 0.1 μM icariin + DKK‐1 group compared to that observed in the 0.2 mM H(2)O(2) + 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 H(2)O(2) and protect MC3T3‐E1 cells against H(2)O(2)‐induced inhibition of osteogenic differentiation, which may occur through the Wnt/β‐catenin signaling pathway.