本研究旨在探讨黄芩清热脱脂胶囊(HQC)通过调节p53/p21信号通路延缓骨关节炎(OA)大鼠软骨细胞衰老的机制。以单钠碘乙酸盐(MIA)联合外风湿热环境刺激诱导OA大鼠风湿热麻痹模型,分为正常(Con)组,OA模型(MIA)组,OA模型+风湿热刺激模型(MIA-M)组,MIA-M+HQC低剂量(MIA-M+HQC-L)组,中剂量(MIA-M+HQC-M)组,高剂量(MIA-M+HQC-H)组,和MIA-M+葡糖胺(MIA-M+GS)组。成功制备模型,灌胃30d。苏木精-伊红(HE)和番泻叶O固绿(SO)染色观察软骨病理变化。酶联免疫吸附试验(ELISA)检测白细胞介素(IL)-1β和IL-6的表达。流式细胞术(FCM)检测细胞凋亡和细胞周期。MMP13、ADAMTS-5、COlⅡ、RT-qPCR检测TGF-β。p53/p21、p16、Bax的蛋白表达,Westernblot检测Bcl-2。Con组大鼠关节软骨表面光滑,潮汐线很平稳。MIA和MIA-M组软骨层明显受损,软骨基质减少。以上情况在MIA-M组中更严重。HQC高剂量组和MIA-M+GS组软骨表面基本完整,分层明显。与MIA-M+HQC-H组相比,HQC低剂量和中剂量组的Mankin评分较高,MIA-M+GS组变化不明显。与Con组相比,MIA和MIA-M组软骨细胞G_1比例升高,S相和G_2相的比例明显下降。此外,细胞凋亡率增加。与MIA-M相比,HQC组以浓度依赖性方式抑制细胞凋亡并促进细胞增殖。与MIA-M+HQC-H组相比,HQC高剂量组比HQC中低剂量组的效果更显著,而在MIA-M+GS组中不显著。与Con组相比,IL-1β和IL-6在MIA和MIA-M组中升高,MMP13和ADAMTS-5的mRNA水平升高。p53,p21,p16和Bax蛋白升高,COLⅡ和TGF-β的mRNA水平降低。与MIA-M组相比,HQC和GS药物干预后IL-1β和IL-6下降,和MMP13和ADAMTS-5的mRNA水平,以及p53,p21,Bax,p16下降。此外,Bcl-2增加。MIA-M+HQC-H组这些指标的改善明显优于HQC低剂量和中剂量组,与MIA-M+GS组差异无统计学意义。HQC延缓MIA诱导的OA大鼠软骨细胞衰老,抑制炎症反应和细胞外基质(ECM)降解,其机制可能与抑制p53/p21通路有关。
This study aims to investigate the mechanism of Huangqin Qingre Chubi Capsules(HQC) in delaying chondrocyte senescence of osteoarthritic(OA) rats by regulating the p53/p21 signaling pathway. Rheumatic fever paralysis models of OA rats were induced based on monosodiun iodoacetate(MIA) combined with external rheumatic fever environmental stimuli and divided into normal(Con) group, OA model(MIA) group, OA model+rheumatic fever stimulation model(MIA-M) group, MIA-M+HQC low-dose(MIA-M+HQC-L) group, medium-dose(MIA-M+HQC-M) group, and high-dose(MIA-M+HQC-H) group, and MIA-M+glucosamine(MIA-M+GS) group. The models were successfully prepared and administered by gavage for 30 d. The pathological changes of cartilage were observed by hematoxylin-eosin(HE) and Senna O solid green(SO) staining. The expression of interleukin(IL)-1β and IL-6 was detected by enzyme-linked immunosorbent assay(ELISA). Flow cytometry(FCM) was used to detect apoptosis and cell cycle. The mRNA expression of MMP13, ADAMTS-5, COLⅡ, and TGF-β was detected by RT-qPCR. The protein expression of p53/p21, p16, Bax, and Bcl-2 was detected by Western blot. The articular cartilage surface of rats in the Con group was smooth, and the tide line was smooth. The cartilage layer of MIA and MIA-M groups was obviously damaged, and the cartilage matrix was reduced. The above conditions were more severe in the MIA-M group. The cartilage surface of the HQC high-dose group and MIA-M+GS group was basically intact with clear delamination. Compared with the MIA-M+HQC-H group, Mankin\'s score was higher in the HQC low-dose and medium-dose groups, and the change was not obvious in the MIA-M+GS group. Compared with the Con group, the proportion of chondrocytes G_1 was elevated in the MIA and MIA-M groups, and the proportion of the S phase and G_2 phase was significantly decreased. In addition, the apoptosis rate was increased. Compared with MIA-M, HQC groups inhibited apoptosis and promoted cell proliferation in a concentration-dependent manner. Compared with the MIA-M+HQC-H group, the effect was more significant in the HQC high-dose group than in the HQC medium-low dose, while it was not significant in the MIA-M+GS group. Compared with the Con group, IL-1β and IL-6 were elevated in the MIA and MIA-M groups, and mRNA levels of MMP13 and ADAMTS-5 were elevated. p53, p21, p16, and Bax protein were elevated, and mRNA levels of COLⅡ and TGF-β were decreased. Compared with the MIA-M group, IL-1β and IL-6 decreased after drug interventions of HQC and GS, and mRNA levels of MMP13 and ADAMTS-5, as well as protein levels of p53, p21, Bax, and p16 decreased. In addition, Bcl-2 increased. The improvement of these indexes was significantly better in the MIA-M+HQC-H group than in the HQC low-dose and medium-dose groups, and the difference with the MIA-M+GS group was not significant. HQC delayed MIA-induced chondrocyte senescence in OA rats, inhibited inflammatory response and extracellular matrix(ECM) degradation, and its mechanism may be related to the inhibition of the p53/p21 pathway.