南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (9): 1381-1388.doi: 10.12122/j.issn.1673-4254.2022.09.15

• • 上一篇    下一篇

靛玉红可减轻骨关节炎大鼠的软骨细胞炎症和损伤

陈 昕，齐秀春，曹玉净，李 扬，李浩亮，王前进，艾进伟   

  1. 河南省中医院创伤骨科，河南 郑州 450002
  • 发布日期:2022-09-28

Indirubin relieves inflammatory injury of chondrocytes in a mouse model of osteoarthritis

CHEN Xin, QI Xiuchun, CAO Yujing, LI Yang, LI Haoliang, WANG Qianjin, AI Jinwei   

  1. Department of Orthopedic Trauma, Henan Provincial Hospital of TCM, Zhengzhou 450002, China
  • Published:2022-09-28

RichHTML

38

PDF

518

摘要： 目的 探讨靛玉红对大鼠骨关节炎症和损伤的作用及其机制。方法 IL-1β构建骨关节炎细胞模型；0.1、0.5、1、2 μmol/L靛玉红分别与IL-1β共同处理细胞；NPAS2 siRNA或non-specific siRNA转染细胞；四甲基噻唑蓝染色法检测细胞增殖；流式细胞仪检测细胞凋亡；Western blot检测BAX、Bcl-2、ACAN、COL2A1、MMP-13和NPAS2蛋白含量；ELISA检测细胞培养上清液NO、PGE2和TNF-α含量，实时荧光定量 PCR检测NPAS2、ACAN、COL2A1和MMP-13 mRNA含量；构建C57BL/6小鼠骨关节炎模型，Western blot检测靛玉红对骨关节组织BAX、Bcl-2、ACAN和MMP-13蛋白含量的影响。结果 0.1 μmol/L靛玉红对软骨细胞增殖、凋亡、caspase-3活性、BAX和Bcl-2蛋白表达水平无显著影响；0.5、1和2 μmol/L靛玉红上调细胞增殖量和Bcl-2蛋白含量，降低凋亡、caspase-3活性和BAX蛋白含量（P<0.05）；0.5 μmol/L靛玉红上调NPAS2、ACAN和COL2A1蛋白和mRNA含量，下调MMP-13蛋白和mRNA、NO、PGE2 和TNF-α含量（P<0.05）；干扰NPAS2表达可显著抑制0.5 μmol/L靛玉红对IL-1β诱导的软骨细胞炎症和损伤的保护作用；小鼠骨关节炎模型中BAX和MMP-13蛋白含量上升（P<0.01），而Bcl-2（P<0.05）和ACAN（P<0.01）下降；靛玉红抑制小鼠关节组织中BAX和MMP-13蛋白含量（P<0.01），上调Bcl-2和ACAN蛋白含量（P<0.05）。结论 靛玉红对骨关节炎组织具有保护作用并可能通过NPAS2调节骨关节炎软骨细胞炎症和损伤。

关键词: 靛玉红；软骨细胞；NPAS2；炎症因子；细胞外基质；骨关节炎

Abstract: Objective To investigate the effect of indirubin for relieving joint inflammation and injury in a rat model of osteoarthritis. Methods Articular cartilage chondrocytes were isolated from adult rat knee joint and cultured in the presence of interleukin-1β (IL-1β) and 0.1, 0.5, 1.0, or 2.0 μmol/L indirubin. The cells were transfected with NPAS2 siRNA or a non-specific siRNA, and the cell proliferation and apoptosis were evaluated using tetramethylthiazole blue staining and flow cytometry. The protein expression levels of Bax, Bcl-2, ACAN, COL2A1, MMP-13 and NPAS2 were detected with Western blotting, and the levels of NO, PGE2 and TNF-α in the culture supernatant were determined with ELISA. The mRNA expression levels of NPAS2, ACAN, COL2A1 and MMP-13 were detected using fluorescence quantitative PCR. In a C57BL/6 mouse model of osteoarthritis, the effect of indirubin on BAX, Bcl-2, ACAN and MMP-13 protein expressions in the bone and joint tissues were evaluated with Western blotting. Results Treatment with 0.1 μmol/L indirubin produced no significant changes in chondrocyte proliferation, apoptosis, caspase-3 activity, or BAX and Bcl-2 protein expressions. At higher doses (0.5, 1.0 and 2.0 μmol/L), indirubin significantly promoted cell proliferation, increased Bcl- 2 protein expression, and lowered cell apoptosis rate, caspase-3 activity and Bax protein expression (P<0.05). Indirubin treatment at 0.5 μmol/L up-regulated the protein and mRNA expressions of NPAS2, ACAN and COL2A1, and down-regulated the expressions of MMP-13, NO, PGE2 and TNF-α (P<0.05). Interference of NPAS2 expression significantly attenuated the protective effect of 0.5 μmol/L indirubin against IL-1β-induced chondrocyte injury. The mouse model of osteoarthritis showed obviously increased protein levels of BAX and MMP-13 (P< 0.01) and decreased levels of Bcl-2 (P<0.05) and ACAN (P<0.01) in the knee joint, and indirubin treatment of the mouse models significantly inhibited the increase of BAX and MMP-13 protein expressions (P<0.01) and up-regulated the protein expressions of Bcl-2 and ACAN (P<0.05). Conclusion Indirubin has a protective effect on osteoarthritis tissue and alleviates inflammation and damage of osteoarthritis chondrocytes possibly through NPAS2.

Key words: indirubin; chondrocyte; NPAS2; inflammatory factors; extracellular matrix; osteoarthritis