Hematoxylin eosin h e

Maltol and cisplatin were manufactured by Sigma-Aldrich (St. Louis, MO, USA). DMSO, MTT were purchased from Sigma Chemicals Co. (St. Loius, MO, USA). The commercial assay kits of BUN, CRE, GSH, SOD, CAT, MDA, hematoxylin-eosin (H&E) and Periodic Acid-Schiff (PAS) kit were bought from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). ELISA kits of mouse TNF-α and IL-1β, iNOS, NF-κB were measured using R&D systems (Minneapolis, MN, USA). Immuno-Histological Staining Kit was obtained from the Boster Biological Technology Co. Ltd (Wuhan, China). Antibodies for Bax, Bcl-2, iNOS, COX-2, caspase 3, 8, 9 and GAPDH were provided by BOSTER Biological Technology (Wuhan, China) or Cell Signaling Technology (Danvers, MA, USA). The antibodies against AMPK, phospho-AMPK (p-AMPK), Akt, phospho-Akt (p-Akt), mTOR, phospho-mTOR (p-mTOR), PI3K, phospho-PI3K (p-PI3K) and p53 were purchased from Wanlei Bio (Shenyang, China). All other chemicals and reagents, unless indicated, were provided by Beijing Chemical Factory (Beijing, China).

The rats were sacrificed and the knee joints were collected and fixed in 4% paraformaldehyde for 48 h, then decalcified in 10% ethylenediaminetetraacetate (EDTA) for 4 weeks. After serial dehydration, the joints were embedded in paraffin and sagittally sectioned at 3 μm thickness. The sections were de-waxed and stained with hematoxylin–eosin (HE, Jian Cheng Biotech, China) and safranin O/fast green (Solarbio, Beijing, China). Then, three independent observers graded the sections based on the scoring criteria reported by Osteoarthritis Research Society International (OARSI) [45 (link)].
The immunohistochemical staining was used to analyze the secretion of MMP-13. The dewaxed sections in the different groups were washed with PBS and exposed to 3% (v/v) hydrogen peroxide H₂O₂ to block endogenous peroxidase activity for 15 min at room temperature. After blocking with normal goat serum for 20 min at room temperature, primary MMP-13 antibodies were added (1:200 dilution, Abcam, USA) and incubated at 4 °C overnight. The sections were incubated with secondary antibody for 15 min and then added with biotin-labeled horse radish peroxidase for 15 min. A 3, 3′-diaminobenzidine tetrahydrochloride (DAB) kit and hematoxylin were used for color development and nuclei dye. Tissues sections were observed and photographed with a microscope.

The cellular morphology of articular chondrocytes was observed with optical microscope and fluorescent microscope. For optical microscope, cells were cultured for 2, 4, and 6 days, followed by fixing in 95% alcohol. Cells were then subjected to Hematoxylin-eosin (HE) (Jiancheng Biotech, China) staining, followed by washing with PBS, naturally dried, and sealed with neutral gum. After that, cells were observed under an inverted phase contrast microscope and photographed with an adapted camera.
Fluorescent microscope was employed to investigate the structure of cellular matrix. Firstly, cells were fixed with 4% paraformaldehyde (PFA, Sigma) for 10 min at room temperature, rinsed with PBS, and then treated with 0.5% Triton X-100 (Sigma Aldrich) for 5 min. Cells were then treated with rhodamine phalloidin (Invitrogen, Carlsbad, CA, USA) for 30 min at room temperature in dark followed by double-staining with Hoechst 33258 (Beyotime, Santa Cruz, CA, USA) for 5 min. Finally, the labeled-articular chondrocytes were observed with a laser scanning confocal microscope (Nikon A1, Tokyo, Japan).