软骨细胞膜蛋白低密度脂蛋白受体相关蛋白1低表达的意义

Knockdown of low density lipoprotein receptor-related protein 1 in chondrocytes

背景:肿瘤坏死因子α是骨关节炎主要致病因子,主要通过激活核因子KB信号通路活性实现软骨细胞炎症反应。细胞膜蛋白低密度脂蛋白受体相关蛋白1参与调节多种细胞因子诱导的细胞炎症反应。目的:观察大鼠软骨细胞膜蛋白低密度脂蛋白受体相关蛋白1对炎症因子肿瘤坏死因子α反应中的作用。方法:将慢病毒包装的低密度脂蛋白受体相关蛋白1-sh RNA转染原代培养的软骨细胞。转染3 d后用核因子κB磷酸化抑制剂Bay 11-7082(10μmol/L)预处理的阴性对照组和sh LRP1组软骨细胞30 min,肿瘤坏死因子α(30μg/L)分别作用两组软骨细胞30 min,收集总蛋白并进入后续Western blot的实验,测定相关胞内信号蛋白表达情况,收集总RNA并行Real-time PCR实验检测相关RNA表达情况。收集肿瘤坏死因子α(30μg/L)作用软骨细胞12 h的培养基行ELISA检测基质金属蛋白酶13表达水平。结果与结论:①慢病毒介导敲低低密度脂蛋白受体相关蛋白1后的软骨细胞肿瘤坏死因子α受体1表达增加;②肿瘤坏死因子α作用后的sh LRP1组软骨细胞核因子k B通路较对照组均有明显激活,诱导型一氧化氮合酶表达增加;3ELISA检测提示sh LRP1组胞外基质金属蛋白酶13浓度增加;4结果提示,软骨细胞膜蛋白低密度脂蛋白受体相关蛋白1低表达对炎症因子肿瘤坏死因子α的作用表现为激活核因子k B通路和提高骨关节炎相关的蛋白基质金属蛋白酶13表达以及易凋亡。

BACKGROUND： Tumor necrosis factor α, as a pathogenic factor, induces the inflammatory reaction mainly via the activation of the nuclear factor kappa B signaling pathway. Low density lipoprotein receptor-related protein 1（LRP1） is involved in the regulation of the inflammatory reaction induced by cytokines. OBJECTIVE： To study the effect of knockdown of LRP1 on tumor necrosis factor α-induced inflammatory reaction. METHODS： Primary cultured rat chondrocytes were transfected with lentivirus-mediated RNA interference to knockdown LRP1 gene. Three days after lentivirus transfection, chondrocytes were pretreated with Bay 11-7082（10 μmol/L） for 30 minutes prior to the addition of tumor necrosis factor α（30 μg/L） for 30 minutes. Signaling protein and m RNA expressions in chondrocytes were detected by western blot assay and real-time PCR analysis, respectively. Chondrocytes were pretreated with or not Bay 11-7082（10 μmol/L） 30 minutes prior to the addition of tumor necrosis factor α（30 μg/L） for 12 hours after starvation in DMEM for overnight, and the culture medium was collected for ELISA determination of matrix metalloproteinase 13 level. RESULTS AND CONCLUSION： Tumor necrosis factor α receptor 1 expression was upregulated in chondrocytes after lentivirus-induced knockdown of LRP1. Increased expression of inducible nitric oxide synthase and activation of the nuclear factor kappa B signaling pathway were found after the addition of tumor necrosis factor α in sh LRP1 group. Moreover, increased level of matrix metalloproteinase 13 was determined by ELISA. Taken together, knockdown of LRP1 up-regulates the expression of tumor necrosis factor α-induced inducible nitric oxide synthase and matrix metalloproteinase 13 through the activation of the nuclear factor kappa B signaling pathway.