Second Department of Orthopedics, Zhejiang Hospital, Hangzhou, China.
Chem Biol Drug Des. 2023 Jun;101(6):1322-1334. doi: 10.1111/cbdd.14215. Epub 2023 Mar 5.
Mitophagy is related to chondrocyte homeostasis and plays a key role in the progress of osteoarthritis (OA). Baicalin has a protective effect on OA chondrocytes, the aim of this study was to explore whether the effect of Baicalin on IL-1β-induced chondrocyte injury is related to the regulation of mitophagy. The expression of collagen II in chondrocytes was detected to identify chondrocytes. The effects of different concentrations of Baicalin (10, 20 and 40 μM), autophagy inhibitor (3-Methyladenine), autophagy activator (rapamycin) and Baicalin combined with PI3K agonist (740Y-P) on the viability (cell counting kit 8), apoptosis (flow cytometry), autophagy activation (Monodansylcadaverine staining) and mitochondrial membrane potential (JC-1 kit) of IL-1β-induced chondrocytes were evaluated. The co-localization of autophagosome and mitochondria was determined by immunofluorescence. Apoptosis-, autophagy-, PI3K/AKT/mTOR pathway- and mitophagy-related proteins were detected by western blot. Our result revealed that Baicalin and rapamycin facilitated cell viability, autophagy and mitophagy, elevated mitochondrial membrane potential and suppressed apoptosis of IL-1β-induced rat chondrocytes. In addition, Baicalin and rapamycin upregulated the levels of Bcl-2, Beclin 1, LC3-II/LC3-I, p-Drp1, PINK1 and Parkin as well as downregulated the levels of Bax, cleaved caspase-3, P62, p-PI3K/PI3K, p-mTOR/mTOR and Drp1 in IL-1β-induced rat chondrocytes. However, 3-Methyladenine did the opposite effects of Baicalin and 740Y-P reversed the effects of Baicalin on IL-1β-induced rat chondrocytes. In conclusion, Baicalin activated mitophagy in IL-1β-induced chondrocytes by inhibiting PI3K/AKT/mTOR pathway and activating PINK1/Parkin and PINK1/Drp-1 pathway, thereby reducing the chondrocyte injury.
自噬与软骨细胞稳态有关，在骨关节炎（OA）的进展中发挥关键作用。黄芩素对 OA 软骨细胞具有保护作用，本研究旨在探讨黄芩素对 IL-1β诱导的软骨细胞损伤的作用是否与调节自噬有关。通过检测软骨细胞中胶原 II 的表达来鉴定软骨细胞。评估不同浓度的黄芩素（10、20 和 40 μM）、自噬抑制剂（3-甲基腺嘌呤）、自噬激活剂（雷帕霉素）以及黄芩素与 PI3K 激动剂（740Y-P）联合对 IL-1β诱导的软骨细胞活力（细胞计数试剂盒 8）、凋亡（流式细胞术）、自噬激活（单丹磺酰戊二胺染色）和线粒体膜电位（JC-1 试剂盒）的影响。通过免疫荧光法测定自噬体和线粒体的共定位。通过 Western blot 检测凋亡、自噬、PI3K/AKT/mTOR 通路和自噬相关蛋白。结果表明，黄芩素和雷帕霉素促进了 IL-1β诱导的大鼠软骨细胞的活力、自噬和自噬，提高了线粒体膜电位，抑制了细胞凋亡。此外，黄芩素和雷帕霉素上调了 Bcl-2、Beclin 1、LC3-II/LC3-I、p-Drp1、PINK1 和 Parkin 的水平，下调了 Bax、cleaved caspase-3、P62、p-PI3K/PI3K、p-mTOR/mTOR 和 Drp1 的水平在 IL-1β诱导的大鼠软骨细胞中。然而，3-甲基腺嘌呤的作用与黄芩素相反，740Y-P 逆转了黄芩素对 IL-1β诱导的大鼠软骨细胞的作用。综上所述，黄芩素通过抑制 PI3K/AKT/mTOR 通路和激活 PINK1/Parkin 和 PINK1/Drp-1 通路激活 IL-1β 诱导的软骨细胞中的自噬，从而减少软骨细胞损伤。
