SOX5对大鼠成骨细胞增殖及核因子-κB信号通路的影响

冷冬月; 李旭峰; 方兴刚

doi:10.13898/j.cnki.issn.1000-2200.2024.02.002

SOX5对大鼠成骨细胞增殖及核因子-κB信号通路的影响

Effects of SOX5 on rat osteoblast proliferation and NF-κB signaling pathway

摘要

摘要:
目的探究Y-box蛋白5(SOX5)对大鼠成骨细胞(OB)增殖及核因子-κB(NF-κB)信号通路的影响。
方法取新生24 h SPF级SD乳鼠，应用酶消化法分离大鼠颅骨OB。形态学观察和ALP染色鉴定成骨细胞；取生长良好的第4代OB，分为空白对照组、pcDNA3.1组、pcDNA-SOX5组、si-NC组、siRNA-SOX5组。转染48 h后qRT-PCR检测细胞中SOX5 mRNA的表达；MTT法检测细胞增殖活力；ALP活性检测试剂盒测量ALP活性；茜素红染色观察钙结节的形成情况；Western blotting检测OB分化及NF-κB信号通路相关蛋白Runt相关转录因子2(Runx2)、Ⅰ型胶原蛋白(Collagen Ⅰ)、NF-κB p65及其磷酸化蛋白、KB抑制蛋白激酶α(IκBα)、肿瘤坏死因子α(TNF-α)的表达。
结果与空白对照组相比，pcDNA-SOX5组大鼠OB中SOX5 mRNA水平、p-NF-κB p65/NF-κB p65、TNF-α蛋白表达显著升高(P < 0.05)，OB增殖活力、ALP活性、钙化结节数量和面积、Runx2、Collagen Ⅰ、IκBα表达显著降低(P < 0.05)；siRNA-SOX5组大鼠OB中SOX5 mRNA水平、p-NF-κB p65/NF-κB p65、TNF-α蛋白表达明显降低(P < 0.05)，OB增殖活力、ALP活性、钙化结节数量和面积、Runx2、Collagen Ⅰ、IκBα表达明显升高(P < 0.05)。
结论SOX5具有调控OB增殖、分化的作用，其作用机制可能与调控NF-κB信号通路有关。

Abstract:
ObjectiveTo explore the effects of Y-box protein 5 (SOX5) on rat osteoblasts (OB) proliferation and nuclear factor κB (NF-κB) signaling pathway.
MethodsThe newborn 24 h SPF grade SD suckling rats were taken, and the OB of the rat skull was separated by enzyme digestion method.Morphological observation and ALP staining were used to identify osteoblasts.The fourth generation OBs with good growth were divided into blank control group, pcDNA3.1 group, pcDNA-SOX5 group, si-NC group, and siRNA-SOX5 group.After 48 h of transfection, qRT-PCR was used to detect the expression of SOX5 mRNA in the cells; MTT method was used to detect cell proliferation viability; ALP activity detection kit was used to measure ALP activity; alizarin red staining was used to observe the formation of calcium nodules; Western blotting was used to detect OB differentiation and the expression of NF-κB signaling pathway-related proteins Runt-related transcription factor 2 (Runx2), type Ⅰ collagen (collagen Ⅰ), nuclear factor κB p65 (NF-κB p65) and its phosphorylated protein, KB inhibits protein kinase α (IκBα), tumor necrosis factor α (TNF-α).
ResultsCompared with the blank control group, the SOX5 mRNA level, p-NF-κB p65/NF-κB p65, and TNF-α proteins expression in the OB of rats in the pcDNA-SOX5 group were significantly increased (P < 0.05), the OB proliferation activity, ALP activity, the number and area of calcified nodules, Runx2, collagen Ⅰ, and IκBα expression were significantly reduced (P < 0.05);the SOX5 mRNA level, p-NF-κB p65/NF-κB p65, and TNF-α proteins expression in the OB of rats in the siRNA-SOX5 group were significantly reduced (P < 0.05), the OB proliferation activity, ALP activity, the number and area of calcified nodules, Runx2, collagen Ⅰ, and IκBα expression were significantly increased (P < 0.05).
ConclusionsSOX5 can regulate the proliferation and differentiation of OB, and its mechanism may be related to the regulation of NF-κB signaling pathway.

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