Objective: To investigate the effect of resveratrol (RES) on inflammation-induced cartilage endplate (CEP) degeneration, and its regulatory mechanism on high mobility group box-1 protein (HMGB1) signaling pathway.
Methods: The intervertebral CEP cells of Sprague Dawley (SD) rats aged 3 weeks were extracted and identified by toluidine blue staining and immunofluorescence staining of rabbit anti-rat collagen type Ⅱ. The cell counting kit 8 (CCK-8) method was used to screen the optimal concentration of RES on intervertebral CEP cells. Gene chip analysis was used to determine the target of RES on intervertebral CEP cells. Interleukin 1β (IL-1β) was used to construct the intervertebral CEP cell degeneration model caused by inflammation and the 7-8-week-old SD rat intervertebral disc degeneration model, and pcDNA3.1-HMGB1 (pcDNA3.1) was used as the control of RES effect. Flow cytometry and TUNEL staining were used to detect the apoptotic rate of intervertebral CEP cells and rat intervertebral disc tissue cells, respectively. ELISA kit was used to detect the content of interleukin 10 (IL-10) and tumor necrosis factor α (TNF-α) in the cell supernatant and rat serum. Western blot was used to detect the expressions of HMGB1, extracellular signal-regulated protein kinase (ERK), phosphorylated ERK (p-ERK), B cell lymphoma/leukemia 2 gene (Bcl-2), and Bcl-2-associated X protein (Bax).
Results: The extracted cells were identified as rat intervertebral CEP cells. CCK-8 method screened out the highest activity of intervertebral CEP cells treated with 30 μmol/L RES. The gene chip analysis confirmed that the HMGB1-ERK signal was the target of RES. Both cell experiments and animal experiments showed that RES treatment can significantly down-regulate the apoptosis rate of intervertebral CEP cells, inhibit the release of TNF-α, and increase the content of IL-10; and down-regulate the expressions of HMGB1, p-ERK, and Bax, and increase Bcl-2; and pcDNA3.1 could partially reverse these effects of RES, and the differences were all significant (P<0.05).
Conclusion: RES can significantly inhibit the apoptosis of intervertebral CEP cells induced by inflammation, which is related to inhibiting the expression of HMGB1.
目的: 探讨白藜芦醇（resveratrol，RES）对炎症所致椎间软骨终板（cartilage endplate，CEP）退变的作用，以及对高迁移率蛋白1（high mobility group box-1 protein，HMGB1）信号的调控机制。.
方法: 提取3周龄SD大鼠椎间CEP细胞，并采用甲苯胺蓝染色、兔抗大鼠Ⅱ型胶原蛋白细胞免疫荧光染色进行鉴定；采用细胞计数试剂盒8（cell counting kit 8，CCK-8）筛选RES对椎间CEP细胞的最佳浓度；采用基因芯片分析确定RES对椎间CEP细胞的作用靶点。分别以IL-1β构建炎症所致椎间CEP细胞退变模型和7～8周龄SD大鼠椎间盘退行性病变模型，并以pcDNA3.1-HMGB1（pcDNA3.1）作为RES作用的对照，分别以流式细胞术和TUNEL染色检测椎间CEP细胞和大鼠椎间盘组织中椎间CEP细胞的凋亡率；ELISA试剂盒检测细胞上清液和大鼠血清中IL-10和TNF-α的含量；Western blot检测HMGB1、细胞外信号调节激酶（extracellular signal-regulated protein kinase，ERK）、磷酸化ERK（phosphorylated ERK，p-ERK）、 B细胞淋巴瘤/白血病-2基因（B cell lymphoma/leukemia 2 gene，Bcl-2）和Bcl-2相关X蛋白（Bcl-2-associated X，Bax）蛋白表达。.
结果: 经鉴定所提取细胞为大鼠椎间CEP细胞；CCK-8法筛选出以30 μmol/L RES处理椎间CEP细胞时活性最高；经基因芯片分析确定HMGB1-ERK信号为RES的作用靶点。细胞实验和动物实验均显示，RES处理均能明显下调椎间CEP细胞的凋亡率，抑制TNF-α的释放，升高IL-10的含量；并明显下调HMGB1、p-ERK和Bax蛋白表达，升高Bcl-2蛋白表达；且pcDNA3.1能部分逆转RES的上述作用，差异均有统计学意义（P<0.05）。.
结论: RES能明显抑制炎症诱导的椎间CEP细胞凋亡，这与抑制HMGB1的表达有关。.
Keywords: Resveratrol; cartilage endplate; degeneration; high mobility group box-1 protein; rat; signaling pathway.