淫羊藿苷对高重力环境下成骨细胞黏附和细胞骨架的影响

目的通过高加速度离心加载装置建立高重力加载模型,探讨不同高重力加载环境对成骨细胞黏附及细胞骨架的影响,并且分析淫羊藿苷对高重力加载后细胞黏附和细胞骨架的影响。方法将MC3T3-E1细胞按2×105个/cm2的密度接种于细胞培养皿中。实验共分为6组:对照组、单纯给药组、10 G加载组、10 G给药组、40 G加载组、40 G给药组。加载组应用高加速度离心加载机对细胞施行加载,连续加载3 d,每天30 min。对照组和单纯给药组暴露于正常重力情况下,其余条件与实验组无差别。给药组淫羊藿苷均采用10-7 mol/L浓度,且按预防给药的方法实验。通过茜素红染色、碱性磷酸酶(alkaline phosphatase,ALP)活性测定、CCK-8细胞增殖实验、细胞骨架鬼笔环肽染色、qPCR和Western Blot等相关分子生物学技术检测淫羊藿苷对高重力环境下成骨细胞integrinα5、integrinβ1和F-actin的影响。结果所有模型均成功制备。茜素红染色:淫羊藿苷可促进成骨细胞钙化结节形成,10 G加载可促进成骨细胞矿化,而40 G加载则抑制成骨细胞的矿化。ALP活性检测:单纯给药组、10 G加载组、40 G加载组的检测OD值分别为0.246、0.331、0.163,与对照组的0.207相比,差异均有统计学意义(P<0.05);10 G给药组和40 G给药组分别为0.373和0.180,与各自加载组的差异有统计学意义(P<0.05)。CCK-8增殖实验:单纯给药组OD值为0.650,与对照组0.551的差异有统计学意义(P=0.031);10 G加载组和40 G加载组OD值分别为1.193和0.245,与对照组的差异均有统计学意义(P<0.05);且10 G给药组和40 G给药组1.300和0.310,与各自加载组的差异有统计学意义(P<0.05)。鬼笔环肽染色:10 G加载细胞促进的数量增加,但细胞形态及骨架未见明显变化,40 G加载则抑制,淫羊藿苷对细胞形态无影响,但对加载损伤后的细胞有一定修复作用。QPCR和Western Blot实验结果一致证实,淫羊藿苷作用后,integrinα5、integrinβ1和F-actin的mRNA和蛋白表达上调,10 G加载可促进integrinα5、integrinβ1和F-actin mRNA和蛋白的表达,40 G加载则明显抑制其mRNA和蛋白的表达。结论10 G条件和淫羊藿苷干预均可促进成骨细胞的生长发育、细胞黏附及细胞骨架的稳定,而40 G则具有明显抑制作用。

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 integrinα5 and integrinβ1 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 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 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.