高迁移率蛋白-1在大鼠腰椎软骨终板退变中的作用及相关机制

The role and mechanism of high mobility group box-1 protein in the degeneration of lumbar cartilage endplate in rats

中文关键词:  高迁移率蛋白-1  ERK信号转导途径  软骨终板  大鼠

英文关键词:High mobility group box-1 protein  Extracellular regulated protein kinases signaling pathway  Cartilage endplate  Rat

中文摘要:

  【摘要】 目的：观察高迁移率蛋白-1（high mobility group box-1 protein，HMGB1）在大鼠腰椎软骨终板退变中的作用，探讨其相关机制。方法：选取4周龄SD大鼠处死，在显微镜下取出腰椎软骨终板，消化后提取细胞，再进行培养、分离、纯化，免疫荧光染色检测Ⅱ型胶原鉴定软骨终板细胞（cartilage endplate cell，CEC）。用不同浓度的ＦＢＳ分别培养ＣＥＣ　２４ｈ、４８ｈ，MTT法检测细胞活性。在第三代CEC中加入HMGB1后，分别在３ｈ、6h、12h、24h时检测金属基质蛋白酶-3（proteins of the matrix metalloproteinase，MMP-3）、血管内皮生长因子（vascular endothelial growth factor，VEGF）及白细胞介素-10（interleukin-10，IL-10）的mRNA表达，6h、12h、24h时检测蛋白表达量；CEC中加入100ng/ml HMGB1后分别在0、5、10、30、60和120min，使用Western blot检测细胞外信号调节激酶（extracellular regulated protein kinases，ERK）磷酸化的表达；继而将CEC分为6组：对照组、HMGB1（100ng/ml）组、FPSMZ1（HMGB1抑制剂）组、HMGB1+FPSMZ1组、U0126（ERK抑制剂）组、HMGB1+U0126组），使用Western blot分别检测MMP-3、VEGF和ERK信号转导途径磷酸化的表达及Elisa检测IL-10的表达。全部结果均为重复独立３次实验，计算其均数±标准差。结果：分离纯化的细胞Ⅱ型胶原抗体表达呈阳性，为ＣＥＣ细胞；10%FBS培养48h为ＣＥＣ增殖最佳的时间点和浓度；HMGB1能够诱导ＥＲＫ和ＪＮＫ信号转导途径磷酸化，从开始到10min逐渐升高，10min时达峰值。HMGB1上调了MMP-3（P=0.039）和VEGF的mRNA（P=0.042）表达，下调IL-10（P=0.025）的mRNA表达，三种因子蛋白表达与mRNA有相同的规律；HMGB1+FPSZM1组与HMGB1组相比，ERK信号转导途径的磷酸化明显减少；在CEC中加入U0126后，MMP-3（P=0.041）和VEGF（P=0.042）蛋白表达明显降低，而IL-10（P=0.004）蛋白表达明显增高；在CEC中加入FPSMZ1后，HMGB1对ERK信号转导途径磷酸化表达的作用明显降低（P=0.031）。结论：HMGB1可增加大鼠腰椎CEC中的MMP-3和VEGF的表达，降低IL-10的表达，其可能是通过ERK信号转导途径实现的。

英文摘要:

  【Abstract】 Objectives: To explore the effect and mechanism of high mobility group box-1 protein(HMGB1) on the degeneration of lumbar cartilage endplate in rats. Methods: Four-week-old SD rats were sacrificed and the lumbar cartilage endplates were taken out under the microscope. The supernatant was extracted after digestion. After culture, isolation and purification, immunofluorescence staining was used to detect type Ⅱ collagen and　cartilage endplate cell　(CEC). Different concentrations of FBS were used to culture CEC for 24h and 48h respectively. MTT assay was used to detect cell viability. The expressions of MMP-3, vascular endothelial growth factor and interleukin were detected at 3, 6, 12 and 24 hours after adding human HMGB1 to the third generation CEC. The expression of extracellular signal-regulated kinase phosphorylation was detected by Western blot at 0, 5, 10, 30, 60 and 12０ｍｉｎ after adding 100ng/ml HMGB１ to CEC. CEC was divided into six groups: control group, HMGB1(100ng/ml) group, FPSMZ１(HMGB１ inhibitor) group, HMGB1+ FPSMZ１ group, U0126(ERK inhibitor) group and HMGB1+U0126 group. The expressions of MMP-3, VEGF and ERK signal transduction pathway phosphorylation and Elisa IL-10 were detected by Western blot. All results were repeated independently for three experiments, and their mean + standard deviation was calculated. Results: The expression of type Ⅱ collagen antibody was positive in CEC cells, and the best time and concentration for proliferation of CEC was 48 hours on 10%FBS. HMGB1 could induce phosphorylation of ERK signal transduction pathway, which gradually increased from the beginning to 10min and reached its peak at 10min. HMGB1 up-regulated the expressions of MMP-3(P=0.039) and VEGF(P=0.042), but down-regulated the expression of IL-10(P=0.025). The expression of three factors was the same as that of gene. Compared with HMGB group, phosphorylation of ERK signal transduction pathway was significantly reduced in HMGB１+FPSMZ１ group. After ERK signal transduction pathway inhibitor(U0126) added to CEC, the expressions of MMP-3(P=0.041) and VEGF(P=0.042) decreased significantly, while the expression of IL-10(P=0.004) increased significantly. After adding RAGE inhibitor(FPSMZ１) to CEC, the phosphorylation of ERK signal transduction pathway by HMGB1 decreased significantly(P=0.031). Conclusions: HMGB1 can increase the expression of MMP-3 and VEGF in rat lumbar CEC and decrease the expression of IL-10 possibly by ERK signal transduction pathway.