高胆固醇对小鼠原代成骨细胞Akt/NF-κB信号通路的影响

Effects of high-level cholesterol on Akt/NF-κB signaling pathway of mouse primary osteoblasts

摘要:

目的  观察高胆固醇对原代成骨细胞Akt/NF-κB信号通路的影响。方法  将小鼠原代成骨细胞按1×105接种于6孔板上，待细胞融合至80%左右，分别加入0μg/ml、12.5μg/ml、25μg/ml和50μg/ml的胆固醇溶液培养基继续培养24 h。采用蛋白质免疫印迹法（Western blot）的方法，检测成骨细胞核转录因子kappa B（NF-κB）和蛋白激酶B（Akt）的蛋白表达；采用实时荧光定量PCR方法，检测成骨细胞中人白细胞介素1α（IL-1α）、人白细胞介素6（IL-6）和肿瘤坏死因子α（TNF-α）的信使RNA（mRNA）表达；采用酶联免疫吸附法，检测细胞培养上清中IL-1α、IL-6和TNF-α的蛋白水平。结果  胆固醇诱导成骨细胞中NF-κB的增加和Akt磷酸化水平降低，增加IL-1α、IL-6与TNF-α的mRNA和蛋白表达，且呈一定的剂量依赖性。结论  高胆固醇可以通过Akt/NF-κB信号通路及其介导的炎症因子表达参与成骨细胞的调节。

Abstract:

Objective To observe the effects of high-level cholesterol on Akt/NF-κB signaling pathway of primary osteoblasts. Methods Mouse primary osteoblasts were seeded at 1×105 to 6-well plates. At 80% fusion, the cells were stimulated with 0 μg/ml, 12.5 μg/ml, 25 μg/ml and 50 μg/ml cholesterol solution culture medium respectively for 24 hours. The protein levels of NF-κB and phosphorylation of Akt were detected by Western blot. The levels of IL-1α, IL-6 and TNF-α were detected by RT-qPCR and ELISA. Results High-level cholesterol increased the NF-κB protein level and decreased phosphorylation of Akt. High-level cholesterol increased not only the mRNA levels but also protein levels of IL-1α, IL-6 and TNF-α. Conclusions Together, these results have uncovered a role of cholesterol in osteoblasts and provided the evidence that treatment with cholesterol at a high dosage may influence the levels of inflammatory factors through Akt/NF-κB signaling pathway in osteoblasts.