Effects Of Icariin On Adhesion And Cytoskeleton Of Osteoblasts In Response To The Extreme Mechanical Environment Of Hypergravity

Objectives: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^-7mol/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,q PCR and Western Blot were used to detect the effects of icariin on osteoblasts adhesion protein integrinα5 and integrinβ1and 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 of10G 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 q PCR and Western Blot experiments all confirmed that the expressions of integrinα5,integrinβ1 and F-actin were up-regulated after icariin treatment.10 G loading could promote the expression of integrinα5,integrinβ1 and F-actin,and 40 G loading significantly inhibited the expression of the mRNA and proteins.Conclusion:1.10G mechanical loading can significantly promote the development,adhesion and cytoskeleton stabilization of osteoblasts;while under 40 G mechanical loading,osteoblast activity and related adhesion,cytoskeleton protein and gene expression are significantly inhibited;2.Icariin has obvious significance for promoting the growth,development,cell adhesion and cytoskeleton stabilization of osteoblasts in response to different hypergravity environments.