Hedgehog通路异常激活对小鼠关节软骨稳态的影响

口腔生物医学 ›› 2024, Vol. 15 ›› Issue (2): 97-103.

Hedgehog通路异常激活对小鼠关节软骨稳态的影响

金泽瑜¹,孙瑶²

1. 同济大学口腔医学院，同济大学附属口腔医院种植科，上海牙组织修复与再生工程技术研究中心
2. 同济大学口腔医学院·同济大学附属口腔医院种植科，上海牙组织修复与再生工程技术研究中心

收稿日期:2023-10-18 修回日期:2023-12-13 出版日期:2024-04-25 发布日期:2024-04-29
通讯作者: 孙瑶 E-mail:yaosun@tongji.edu.cn
基金资助:
国家自然科学基金

Role of Hedgehog pathway in preserving integrity of the mouse articular cartilage

Received:2023-10-18 Revised:2023-12-13 Online:2024-04-25 Published:2024-04-29

摘要/Abstract

摘要： 目的：探索Hedgehog通路在小鼠骨关节炎中的作用。方法：选取10周龄C57BL/6J雄性小鼠，在左侧后肢膝关节建立内侧半月板不稳定模型，右侧后肢膝关节为自身对照。术后8周处死建模组及对照组小鼠，收集其膝关节样本，体视镜观察膝关节破坏程度，显微CT（micro-CT）扫描分析半月板钙化程度，番红O-固绿软骨染色、甲苯胺蓝染色、苏木素-伊红染色和II型胶原蛋白（Col2）免疫荧光染色观察软骨细胞外基质及软骨细胞变化，实时荧光定量PCR检测膝关节软骨Hedgehog信号通路重要分子平滑蛋白（Smo）、补缀同源物1（Ptch1）、神经胶质瘤相关癌基因同源物1（Gli1）基因表达水平及Col2、性别决定区Y框蛋白9（Sox9）、基质金属蛋白酶13（Mmp13）等软骨及其细胞外基质稳态相关分子表达变化。白细胞介素-1β（IL-1β）诱导ATDC5软骨细胞建立骨关节炎细胞模型，使用Smo抑制剂NVP-LDE225作用于细胞，实时荧光定量PCR检测Smo、Ptch1、Gli1、Col2、Sox9、Mmp13基因表达水平。结果：成功构建小鼠骨关节炎模型，建模组膝关节肿大，关节软骨表面出现损伤，钙化半月板体积高于对照组（P＜0.01）；膝关节软骨破坏，细胞外基质蛋白聚糖含量减少，软骨细胞排列紊乱；免疫荧光染色Col2阳性信号不连续；Col2、Sox9基因表达降低，Mmp13及Hedgehog通路相关分子Smo、Ptch1、Gli1基因表达高于对照组（P＜0.05）。对软骨细胞使用Smo抑制剂NVP-LDE225可拮抗IL-1β诱导的Col2、Sox9基因表达降低及Mmp13、Smo、Ptch1、Gli1表达升高（P＜0.05），挽救骨关节炎相关表型。结论：在骨关节炎状态下Hedgehog通路被异常激活，Hedgehog通路参与骨关节炎的发生发展。

关键词: 骨关节炎, 软骨, Hedgehog通路

Abstract: Objective:To investigate the role of Hedgehog pathway in the mice osteoarthritis (OA). Methods:10-week-old male C57BL/6J mice were subjected to destabilization of the medial meniscus (DMM) surgery. The left hindlimb knee was the experimental group, with right hindlimb knee as control. Mice were sacrificed 8 weeks after the surgery and knee joints were harvested for subsequent experiments. Stereomicroscope was used to observe the damage degree of knee joint. Micro-CT was used to detect the meniscus calcification. Safranin O-Fast Green staining, toluidine blue staining, hematoxylin-eosin (HE) staining and immunofluorescence staining of Col2 were used to observe the changes of cartilage extracellular matrix and chondrocytes. The expression of genes related to articular cartilage homeostasis (Col2, Sox9, Mmp13) and Hedgehog signaling pathway (Smo, Gli1, Ptch1) were detected by RT-qPCR. ATDC5 cell model of OA was established using IL-1β, treated with NVP-LDE225 as Smo inhibition. RT-qPCR was used to detect the expression of Smo, Gli1, Ptch1, Col2, Sox9 and Mmp13. Results:Mouse OA model was established successfully after the DMM surgery. The experimental mice present knee joint enlargement, destroyed cartilage layer, higher meniscus calcification volume, knee cartilage deformation, decreased content of extracellular matrix proteoglycan and disordered arrangements of the chondrocytes. The immunofluorescence staining of Col2 indicated an aberrant extracellular matrix. In contrast, Col2 and Sox9 were significantly downregulated. Mmp13 and Hedgehog related genes (Smo, Ptch1, Gli1) was significantly increased in the OA model mice（P＜0.05）. Besides, inhibition of Smo in ATDC5 cell OA model antagonized the downgraded gene expression of Col2 and Sox9 and upgraded gene expression of Mmp13, Smo, Ptch1 and Gli1 induced by IL-1β, thus attenuating the severity of OA（P＜0.05）. Conclusions:Hedgehog signaling was activated in OA and Hedgehog signaling might be involved in the occurrence and development of OA.

Key words: osteoarthritis, cartilage, Hedgehog signaling

金泽瑜孙瑶. Hedgehog通路异常激活对小鼠关节软骨稳态的影响[J]. 口腔生物医学, 2024, 15(2): 97-103.

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