Effects of icariin on adhesion and cytoskeleton of osteoblasts in response to the extreme mechanical environment of hypergravity

Objective To establish a hypergravity loading model with a high-acceleration centrifugal loading device and to investigate the effects of different hypergravity loading and icariin on osteoblast adhesion and cytoskeleton. Methods MC3T3-E1 cells were seeded in the dishes of cell culture at a density of 2×105/cm2. And the experiment was divided into 6 groups: control group (without icariin and loading); simple administration group (only icariin); 10 G loading group (only loading); 10 G administration group (with icariin and loading); 40 G loading group (only loading); 40 G administration group (with icariin and loading). The experimental loading group was loaded with MC3T3-E1 cells using a high-acceleration centrifugal loader. And continuous loading for 3 d, 30 min per d. The control group and the simple administration group were exposed to normal gravity, and the remaining conditions were not different from the experimental group. Icariin was used at a concentration of 10-7 mol/L in all administration groups, and the experiments were carried out according to the method of preventive administration. At the same time, the related molecular biological techniques such as alizarin red staining, alkaline phosphatase (ALP) activity measurement, CCK-8 cell proliferation experiment, cytoskeleton phalloidin staining, qPCR and Western Blot were used to detect the effects of icariin on osteoblasts adhesion protein integrin α5 and integrin β1 and cytoskeleton protein F-actin under hypergravity extreme mechanical environment. Results All models were successfully prepared. The alizarin red staining: The icariin could significantly promote the formation of osteoblastic calcified nodules. And the 10 G loading could also promote the mineralization of osteoblasts and increase the number of mineralized nodules, while the mineralization and the number of mineralized nodules of osteoblasts are significantly reduced in 40 G loading. ALP activity test: The OD values of simple administration group, 10 G loading group and 40 G loading group were 0.246, 0.331 and 0.163, respectively. Compared with 0.207 in the control group, the differences were statistically significant (P<0.05). The 10 G administration group and the 40 G administration group were 0.373 and 0.180, and the differences were statistically significant (P<0.05). The results of CCK-8 proliferation experiments: The OD value of simple administration group were 0.650, which was statistically significant compared with 0.551 of control group (P=0.031). The 10 G loading group and 40 G loading group were 1.193 and 0.245, and their differences with the control group were both statistically significant (P<0.05). The OD value of 10G administration group and the 40 G administration group were 1.300 and 0.310, which were significantly different from the respective loading groups (P<0.05). Phalloidin staining: 10 G loading significantly increased the number of cells, but the changes in cells morphology and skeleton were not obvious. 40 G loading significantly inhibited the increase of the number of cells, meanwhile, made the pseudopods of cells more shorter and even disappeared. 40 G loading made the seriously damage of the cytoskeleton and even cause the cells to death. Icariin had no effect on the cells morphology, but it did has a certain repair effect after the cells loading. The results of qPCR and Western Blot experiments all confirmed that the expressions of integrin α5, integrin β1 and F-actin were up-regulated after icariin treatment. 10 G loading could promote the expression of integrin α5, integrin β1 and F-actin, and 40 G loading significantly inhibited the expression of the mRNA and proteins. Conclusion Both 10 G condition and icariin can promote the development, cell adhesion and the cytoskeleton's stability of osteoblasts, while 40 G has a significant inhibitory effect. Key words: Osteoblast; Hypergravity; Cell adhesion; Cytoskeleton