姜黄素调节Notch1/Hes1/Prdx1通路对IL-1β诱导的软骨细胞炎性损伤的影响

The effect of curcumin regulating the Notch1/Hes1/Prdx1 pathway on IL-1β-induced inflammatory injury of chondrocytes

关键词:  姜黄素  缺刻基因1  发状分裂相关增强子1  过氧化物还原蛋白1  白细胞介素-1β  炎性损伤

Key words:  curcumin  Notch 1  hairy and enhancer of split homolog 1  peroxiredoxin 1  interleukin-1β  inflammatory injury

摘要:

[目的] 探讨姜黄素（Cur）调节缺刻基因1（Notch1）/发状分裂相关增强子1（Hes1）/过氧化物还原蛋白1（Prdx1）通路对白细胞介素-1β（IL-1β）诱导的软骨细胞炎性损伤的影响。[方法] C28/I2细胞随机分为对照（Ctrl）组（正常培养）、IL-1β组（10 ng/mL IL-1β）、Cur低剂量（L-Cur）、Cur中剂量（M-Cur）、Cur高剂量（H-Cur）组（IL-1β诱导+10、20、40 μmol/L Cur）、Cur+锯齿蛋白1（Jagged 1）组（IL-1β诱导+40 μmol/L Cur+0.5 μg/mL Notch激活剂Jagged 1）。MTS法和5-乙炔基-2'-脱氧尿苷（EdU）染色检测C28/I2细胞增殖；流式细胞仪检测C28/I2细胞凋亡；2'，7'-二氯荧光黄双乙酸盐（DCFH-DA）荧光探针检测C28/I2细胞中活性氧（ROS）水平；免疫荧光检测C28/I2细胞Ⅱ型胶原（ColⅡ）、聚集蛋白聚糖（Aggrecan）表达；酶联免疫吸附测定（ELISA）试剂盒检测C28/I2细胞中白细胞介素（IL）-6、IL-8、肿瘤坏死因子-α（TNF-α）、基质金属蛋白酶（MMP）-3、MMP-13表达；蛋白免疫印迹（Western Blot）检测C28/I2细胞中增殖细胞核抗原（Ki-67）、微染色体维持复合体组分7（MCM）7、Notch1、Hes1、Prdx1、BCL2拮抗剂（Bak）、存活蛋白（survivin）蛋白表达。[结果] IL-1β组OD₄₉₀值、EdU阳性细胞率、ColⅡ荧光强度、Aggrecan荧光强度、Ki-67、MCM7、survivin低于Ctrl组，ROS荧光强度、凋亡率、Bak、MMP-3、MMP-13、IL-6、IL-8、TNF-α、Notch1、Hes1、Prdx1高于Ctrl组（P<0.05）；L-Cur组、M-Cur组、H-Cur组OD₄₉₀值、EdU阳性细胞率、ColⅡ荧光强度、Aggrecan荧光强度、Ki-67、MCM7、survivin高于IL-1β组，ROS荧光强度、凋亡率、Bak、MMP-3、MMP-13、IL-6、IL-8、TNF-α、Notch1、Hes1、Prdx1低于IL-1β组（P<0.05）；Cur+Jagged 1组OD₄₉₀值、EdU阳性细胞率、ColⅡ荧光强度、Aggrecan荧光强度、Ki-67、MCM7、survivin低于H-Cur组，ROS荧光强度、凋亡率、Bak、MMP-3、MMP-13、IL-6、IL-8、TNF-α、Notch1、Hes1、Prdx1高于H-Cur组（P<0.05）。[结论] Cur通过抑制Notch1/Hes1/Prdx1通路，减轻IL-1β诱导的软骨细胞炎性损伤。

Abstract:

[Objective] To investigate the effect of curcumin（Cur）regulating the Notch1/hairy and enhancer of split homolog 1（Hes1）/ peroxiredoxin 1（Prdx1）pathway on inflammatory injury of chondrocytes induced by interleukin-1 β（IL-1β）. [Methods] C28/I2 cells were randomly separated into Ctrl group（normal culture），IL-1β group（10 ng/mL IL-1β），low-dose Cur （L-CUR），medium-dose Cur （M- Cur），and high-dose Cur （H-Cur）groups（IL-1β induced+10，20，40 μmol/L Cur），and Cur+Jagged 1 group（IL-1β induced+40 μmol/L Cur+0.5 μg/mL Notch activator Jagged 1）. The proliferation of C28/I2 cells was detected by MTS assay and 5-Ethynyl-2'-deoxyuridine （EdU）staining. The apoptosis of C28/I2 cells was detected by flow cytometry. The 2'，7'-dichlorofluorescein diacetate（DCFH-DA） fluorescent probe was used to detect the level of reactive oxygen species（ROS）in C28/I2 cells. Immunofluorescence was used to detect the expressions of type Ⅱ collagen（ColⅡ）and Aggrecan in C28/I2 cells. Enzyme-linked immunosorbent assay（ELISA）kits were used to detect the expression of interleukin（IL）-6，IL-8，tumor necrosis factor-α（TNF-α），matrix metalloproteinase（MMP）-3，and MMP-13 in C28 / I2 cells. Western blot was used to detect the protein expression of proliferating cell nuclear antigen（Ki-67）， minichromosome maintenance complex component 7（MCM）7，Notch1，Hes1，Prdx1，BCL2 antagonist（Bak），and survival protein （survivin）in C28 /I2 cells. [Results] The OD₄₉₀ value，EdU positive cell rate，ColⅡ fluorescence intensity，Aggrecan fluorescence intensity，Ki-67，MCM7 and survivin in the IL-1β group were lower than those in the Ctrl group，while the apoptosis rate，ROS fluorescence，Bak，MMP-3，MMP-13，IL-6，IL-8，TNF-α，Notch1，Hes1 and Prdx1 were higher than those in the Ctrl group（P<0.05）. The OD 490 value，EdU positive cell rate，ColⅡ fluorescence intensity，Aggrecan fluorescence intensity，Ki-67，MCM7 and survivin in the L-Cur group，M-Cur group，H-Cur group were higher than those in the IL-1β group，while the apoptosis rate，ROS fluorescence， Bak，MMP-3，MMP-13，IL-6，IL-8，TNF-α，Notch1，Hes1 and Prdx1 were lower than those in the IL-1β group（P<0.05）. The OD₄₉₀ value，EdU positive cell rate，ColⅡ fluorescence intensity，Aggrecan fluorescence intensity，Ki-67，MCM7 and survivin in the Cur+ Jagged 1 group were lower than those in the H-Cur group，while the apoptosis rate，ROS fluorescence，Bak，MMP-3，MMP-13，IL-6，IL-8， TNF-α，Notch1，Hes1 and Prdx1 were higher than those in the H-Cur group（P<0.05）. [Conclusion] Cur alleviates IL-1β-induced inflammatory injury of chondrocytes by inhibiting the Notch1/Hes1/Prdx1 pathway.