关键词: 筋骨痛消丸, 含药血清, 髌下脂肪垫间充质干细胞, 外泌体, 膝骨关节炎, 软骨细胞, PI3K/AKT/mTOR信号通路, 自噬

Abstract: BACKGROUND: Both exosomes derived from infrapatellar fat pad and Jingu Tongxiao Pill have good therapeutic effects on knee osteoarthritis, but the mechanism and synergistic effect remain to be studied.
OBJECTIVE: To explore the effect and mechanism of the drug-containing serum of Jingu Tongxiao Pill on autophagy of chondrocytes in rabbits with knee osteoarthritis through exosomes derived from adipose-derived mesenchymal stem cells.
METHODS: (1) Rabbit subpatellar fat pad mesenchymal stem cells were cultured in vitro. In vitro intervention was carried out using phosphate buffer saline, 10 mmol/L glucosamine, and 10% Jingu Tongxiao Pill drug-containing serum respectively. Exosomes were isolated and extracted from the supernatants and detected by transmission electron microscopy and nanoparticle tracking analysis technology. (2) Thirty healthy female New Zealand rabbits aged 8-9 months were randomly assigned to five groups, namely the blank group, the model group, the exosomes-phosphate buffer saline group, the exosomes-glucosamine group, and the exosomes-Jingu Tongxiao Pill group, with six rabbits in each group. The blank group was not treated. The other four groups adopted the left intra-knee injection of type II collagenase method to establish knee osteoarthritis models. After the modeling was verified to be successful, the exosomes-phosphate buffer saline group, the exosomes-glucosamine group, and the exosomes-Jingu Tongxiao Pill group were respectively injected into the knee with exosomes-phosphate buffer saline, exosomes-glucosamine, and exosomes-Jingu Tongxiao Pill for intervention once a week, 1 mL each time, for 5 consecutive injections. One week after the intervention ended, the knee joint grade of each animal was evaluated using the modified Lequesne MG. The femoral condylar cartilage and tibial plateau cartilage were taken for pathological observation. The femoral condylar cartilage was stained with hematoxylin-eosin staining. Mankin's score was performed after light microscopy observation. The expression of phosphatidylinositol 3-kinase, protein kinase B, mammalian target of rapamycin, matrix metalloproteinase 13 gene and related proteins in the cartilage tissue of the tibial plateau was detected by western bolt assay and real-time PCR.
RESULTS AND CONCLUSION: (1) Flow cytometry showed that the surface markers CD73, CD90, and CD44 of the extracted rabbit infrapatellar fat pad mesenchymal stem cells were specifically highly expressed. (2) Transmission electron microscopy showed that exosomes derived from infrapatellar fat pad mesenchymal stem cells had a typical cup or spherical structure. Nanoparticle tracking analysis showed that the particle size distribution of these exosomes was 79.7-175.8 nm, the average diameter was 126.5 nm, and the particle concentration was 6.8×10⁶ particles/mL. (3) Rabbit knee osteoarthritis models were successfully established. The knee grade scores of the three groups intervened by intra-knee injection of exosomes decreased to varying degrees. Compared with the model group, the modified Lequesne MG score and Mankin’s score, and the mRNA and protein expression levels of mammalian target of rapamycin, phosphatidylinositol 3-kinase, protein kinase B, and matrix metalloproteinase 13 in cartilage of exosomes-Jingu Tongxiao Pill group were significantly lower (P < 0.05). The results showed that the intervention of Jinju Tongxiao Pill containing serum on exosomes derived from mesenchymal stem cells in the infrapatellar fat pad could promote the autophagy of chondrocytes in osteoarthritis by regulating the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway.

Key words: Jingu Tongxiao Pill, drug-containing serum, infrapatellar fat pad mesenchymal stem cell, exosome, knee osteoarthritis, chondrocyte, PI3K/AKT/mTOR signaling pathway, autophagy