时钟基因 Per1 通过 NF-κB 通路调控大鼠颞下颌骨关节炎：一项体外和体内研究。

PURPOSE: Temporomandibular joint osteoarthritis (TMJOA) is a common disease that negatively affects the life quality of human beings. Circadian rhythm acts an important role in life activities. However, whether the clock genes are rhythmic expressed in mandibular condylar chondrocytes, or the clock genes have an effect on the progression of TMJOA remains unknown. In this study, we aim to explore expression of clock genes and regulatory mechanism of TMJOA in rat mandibular condylar chondrocytes. METHODS: After synchronized by dexamethasone, the expression of core clock genes Per1, Per2, Clock, Cry1, Cry2 and Bmal1 and cartilage matrix degrading factor gene Mmp13 were analyzed in mandibular condylar chondrocytes every 4 h with RT-qPCR. The mandibular condylar chondrocytes were stimulated with IL-1β, and expression of Per1, Mmp13, P65 and p-P65 was assessed by RT-qPCR and Western blot. Sh-Per1 lentivirus was used to assess the effect of clock gene Per1 in IL-1β-induced chondrocytes, and expression of Mmp13, P65 and p-P65 was measured. After establishing a rat TMJOA model using unilateral anterior crossbite (UAC), micro-CT, H & E, Alcian Blue & Nuclear Fast Red and Safranin O & Fast Green, cartilage thickness was utilized to assess the damage of cartilage and subchondral bone. Immunohistochemistry of PER1, MMP13 and P65 was performed in condylar sections. RESULTS: All core clock genes and Mmp13 were rhythmically expressed. And Mmp13 expression curve was closed in phase and amplitude with Per1. After stimulation with IL-1β, the expression of MMP13, PER1 and P65 and ratio of p-P65/P65 increased in condylar chondrocytes. After Per1 was down-regulated in condylar chondrocytes, the expression of MMP13 and P65 and ratio of p-P65/P65 decreased. Compared with the condyles of Sham group, the bony parameters of UAC group were significantly worse. The thickness of cartilage in UAC group significantly reduced. The modified Mankin scores and the expression of PER1, MMP13 and P65 in cartilage of UAC group significantly increased compared with Sham group. CONCLUSION: Core clock genes and Mmp13 are rhythmic expressed in rat mandibular condylar chondrocytes. PER1 can regulate the expression of MMP13 through NF-κB pathway in IL-1β-induced mandibular condylar chondrocytes.

目的：颞下颌关节骨关节炎（TMJOA）是一种常见疾病，严重影响人类的生活质量。昼夜节律在生命活动中起着重要作用。然而，时钟基因是否在髁突软骨细胞中呈节律性表达，或者时钟基因是否对 TMJOA 的进展有影响尚不清楚。本研究旨在探讨时钟基因在大鼠髁突软骨细胞中的表达及调控机制。

方法：用地塞米松同步化后，每隔 4 h 用 RT-qPCR 分析核心时钟基因 Per1、Per2、Clock、Cry1、Cry2 和 Bmal1 以及软骨基质降解因子基因 Mmp13 的表达。用 IL-1β刺激髁突软骨细胞，用 RT-qPCR 和 Western blot 检测 Per1、Mmp13、P65 和 p-P65 的表达。用 sh-Per1 慢病毒转染评估时钟基因 Per1 在 IL-1β诱导的软骨细胞中的作用，测量 Mmp13、P65 和 p-P65 的表达。建立单侧前牙反牙合（UAC）大鼠 TMJOA 模型后，用 micro-CT、H&E、Alcian Blue & Nuclear Fast Red 和 Safranin O & Fast Green 评估软骨和软骨下骨的损伤，用软骨厚度评估软骨的损伤。对髁突切片进行 PER1、MMP13 和 P65 的免疫组化染色。

结果：所有核心时钟基因和 Mmp13 均呈节律性表达。Mmp13 表达曲线与 Per1 相位接近且振幅相似。用 IL-1β刺激后，髁突软骨细胞中 MMP13、PER1 和 P65 的表达以及 p-P65/P65 的比值增加。用 sh-Per1 转染下调髁突软骨细胞中的 Per1 后，Mmp13 和 P65 的表达以及 p-P65/P65 的比值降低。与 Sham 组相比，UAC 组的骨性参数明显较差。UAC 组的软骨厚度明显变薄。UAC 组改良 Mankin 评分以及软骨中 PER1、MMP13 和 P65 的表达明显高于 Sham 组。

结论：大鼠髁突软骨细胞中核心时钟基因和 Mmp13 呈节律性表达。PER1 可通过 NF-κB 通路调节 IL-1β诱导的髁突软骨细胞中 MMP13 的表达。