The present study was approved to use commercially available primary cells by the Clinical Trials and Biomedical Ethics Committee of Sichuan University West China Hospital (approval no. 2021-102).

All chondrocytes used in the present study were primary rat (cat. no. RAT-iCell-s003; each vial containing >5×10⁵ cells in 1 ml volume) or human chondrocytes (cat. no. HUM-iCell-s018; each vial containing >5×10⁵ cells in 1 ml volume), and were provided by iCell Bioscience Inc. The cells were cultured in a specialized primary chondrocyte culture system (cat. no. PriMed-iCell-020; iCell Bioscience Inc.).

The CYTO-ID Autophagy Detection kit (Enzo Life Sciences, Inc.) detects autophagy vesicles using novel dyes that selectively label the accumulation of autophagy vesicles so that they exhibit bright fluorescence. Primary rat and human chondrocytes at the logarithmic growth phase were prepared into a single-cell suspension. The cell density was adjusted to 1×10⁵ cells/ml and 1 ml cell suspension was transferred in each well of 12-well plate. The cells were either left untreated (blank control), or treated with dexamethasone (200 µM; Beijing Solarbio Science & Technology Co., Ltd.), or dexamethasone (200 µM) combined with lithium chloride (MilliporeSigma) at various concentrations (0.01, 0.1, 1 or 10 mM), with four replicates in each group. The concentration of dexamethasone was determined according to a previous study (7) and our preliminary experiments. A previous study showed that 200 µM dexamethasone could increase the autophagy level of chondrocytes and significantly reduce cell viability (7). Similar results have been obtained from our preliminary experiments (unpublished data). After the cells adhered to the walls, each well was added with the drugs corresponding to the aforementioned grouping, followed by incubation for 24 or 48 h at 37°C in an incubator with 5% CO₂. Subsequently, 300 µl pre-configured CYTO-ID staining solution was added to each well, and the cells were stained for 20 min at 37°C in an incubator with 5% CO₂. The cells were then observed and photographed under an inverted fluorescence microscope (Zeiss AG). Under an ×20 magnification field of view, five fields of view were randomly selected and the average fluorescence intensity was semi-quantitatively calculated using ImageJ software (version 1.8.0; US National Institutes of Health).

After screening for the suitable lithium chloride concentration (10 mM) and the duration of action (48 h) (under these conditions, the autophagy level of chondrocytes was changed most significantly), the following experiments were performed under these conditions.

Primary chondrocytes at the logarithmic growth phase were prepared into a single-cell suspension. The cell density was adjusted to 1×10⁵ cells/ml and 2 ml of cell suspension was transferred into each well of three 6-well plates. The cells were either left untreated, or treated with dexamethasone (200 µM), or dexamethasone (200 µM) combined with lithium chloride (10 mM) for 48 h at 37°C in an incubator with 5% CO₂, with four replicates in each group. The cells were washed with PBS twice, digested and collected using trypsin. Subsequently, the cell suspensions were centrifuged at 250 × g for 5 min at room temperature, the supernatants were discarded and 0.5% glutaraldehyde fixation solution was slowly added along the tube wall using a dropper. Following resuspension, the cell suspension was placed in an environment at 4°C for 10 min for fixation, and the cell suspension was then transferred into a 1.5-ml tip bottom EP tube and centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was gently discarded, whereas the precipitate was retained and 3% glutaraldehyde fixing solution was slowly added using a dropper. The samples were then observed under a transmission electron microscope (JEM-1400FLASH, JEOL, Ltd.). A total of five normal cells were randomly selected from each sample and autophagosomes in the cytoplasm were counted. The average number of autophagosomes was compared between groups.

The cell density was adjusted to 1×10⁵ cells/ml and 2 ml of cell suspension was transferred into each well of three 6-well plates. The cells were either left untreated, or treated with dexamethasone (200 µM), or dexamethasone (200 µM) combined with lithium chloride (10 mM) for 48 h at 37°C in an incubator with 5% CO₂, with four replicates in each group to extract RNA. To evaluate the expression levels of autophagy-related genes, including LC3B, AKT and mTOR, total RNA was extracted from the chondrocytes using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Total RNA was then reverse-transcribed into cDNA using the PrimeScript™ RT reagent kit (Promega Corporation). Reactions were performed using a 20 µl final volume with 2 µl cDNA and 10 µl SYBR™-Green PCR Master Mix (Promega Corporation). The nucleotide sequences of the PCR primers are presented in Table I.

Cycle threshold (Ct) values were obtained using Thermo Scientific PikoReal software (version 2.2; Thermo Fisher Scientific, Inc.). Relative expression was calculated using the 2^−ΔΔCq method and normalized to the internal reference gene β-actin (19).

The cell density was adjusted to 1×10⁵ cells/ml and 2 ml of cell suspension was transferred into each well of three 6-well plates. The cells were either left untreated, or treated with dexamethasone (200 µM), or dexamethasone (200 µM) combined with lithium chloride (10 mM) for 48 h at 37°C in an incubator with 5% CO₂, with four replicates in each group to extract the protein. RIPA buffer (Wuhan Servicebio Technology Co., Ltd.) was used to lyse the rat and human chondrocytes from on ice for 10 min. The lysate mixture was then centrifuged at 12,000 rpm for 10 min at 4°C. The BCA Protein Assay kit (Beyotime Institute of Biotechnology) was used to measure the total protein concentration and 20 µg protein/lane was separated by SDS-PAGE on a 10% or 12% gel. The separated proteins were subsequently transferred onto a PVDF membrane and blocked for 2 h at 25°C in a PBS solution containing 5% skim milk powder. Subsequently, the membranes were incubated overnight at 4°C with primary antibodies against LC3B (rabbit; 1:2,000; cat. no. ab192890; Abcam), phosphorylated (p-)AKT (rabbit; 1:1,000; cat. no. 310021; Chengdu Zen Bioscience Co., Ltd.), AKT (rabbit; 1:1,000; cat. no. 382804; Chengdu Zen Bioscience Co., Ltd.), phosphorylated mTOR (mouse; 1:1,000; cat. no. sc-293133; Santa Cruz Biotechnology, Inc.), mTOR (mouse; 1:1,000; cat. no. sc-517464; Santa Cruz Biotechnology, Inc.), and β-tubulin (rabbit; 1:2,000; cat. no. AF7011; Affinity Biosciences). After washing with PBST, the membranes were incubated with the secondary polyclonal goat anti-rabbit/mouse HRP-conjugated antibodies (cat. no. ZDR-5306 and ZDR-5307; both 1:5,000; OriGene Technologies, Inc.) at room temperature for 2 h. Finally, after washing the membranes with PBST, the protein bands were visualized using the ECL method (Torchlight Hypersensitive ECL Western HRP Substrate; cat. no. 17046; Chengdu Zen Bioscience Co., Ltd.) and the Quantity One software (version 4.6.6; Bio-Rad Laboratories, Inc.) was used to analyze the results with β-tubulin as the loading control.

To measure the cell viability, the cell density was adjusted to 3×10⁴ cells/ml and 100 µl of cell suspension was transferred into each well of a 96-well plate. The cells were either left untreated, or treated with dexamethasone (200 µM), or dexamethasone (200 µM) combined with lithium chloride (10 mM) for 48 h at 37°C in an incubator with 5% CO₂, with four replicates in each group. The CCK-8 reagent (Apexbio Technology LLC) was diluted 1:10 and 110 µl diluted CCK-8 solution was added into each well. After 2 h, the optical density (OD) value of each well was examined at 450 nm according to the manufacturer's instructions. Cell viability was calculated as follows: Cell viability (%)=(OD value of observation group-OD value of zero adjustment group)/(OD value of control group-OD value of zero adjustment group) ×100%.

Statistical analysis was performed using SPSS 26.0 software (IBM Corp.). For continuous data, one-way analysis of variance (ANOVA) was used and Tukey's test was used as the post hoc test. Continuous data are presented as the mean ± standard deviation unless otherwise indicated. P<0.05 was considered to indicate a statistically significant difference.

The results of CYTO-ID autophagy fluorescence staining revealed that the autophagy level of rat chondrocytes increased significantly following treatment with dexamethasone for 24 and 48 h (Fig. 1). Following treatment with dexamethasone combined with various concentrations of lithium chloride for 24 and 48 h, it was found that 10 mM lithium chloride for 48 h significantly reduced the autophagy level overactivated by dexamethasone in rat chondrocytes.

The results of CYTO-ID autophagy fluorescence staining revealed that the autophagy level of human chondrocytes did not change significantly following treatment with dexamethasone for 24 h and the autophagy level of human chondrocytes increased significantly following treatment with dexamethasone for 48 h (Fig. 2). Following treatment with dexamethasone combined with various concentrations of lithium chloride for 24 and 48 h, it was found that 10 mM lithium chloride for 48 h significantly reduced the autophagy level overactivated by dexamethasone in human chondrocytes. Therefore, the rat and human chondrocytes were divided into three groups in subsequent experiments: The control group was composed of cells untreated; the dexamethasone group was composed of cells treated with 200 µM dexamethasone for 48 h; the lithium group was composed of cells treated with 200 µM dexamethasone and 10 mM lithium chloride for 48 h.

Compared with the control and lithium groups, a significantly greater number of autophagosomes was observed by TEM in the dexamethasone group of rat chondrocytes (Fig. 3A and C). Compared with the control and lithium groups, a significantly greater number of autophagosomes was observed by TEM in the dexamethasone group of human chondrocytes (Fig. 3B and D).

The results of the CCK-8 assay of rat chondrocytes revealed that the average cell viability of the dexamethasone group was significantly lower than that of the control and lithium groups, and the average cell viability of the lithium group was significantly lower than that of the control group (Fig. 4A). The results of the CCK-8 assay of human chondrocytes revealed that the average cell viability of the dexamethasone group was significantly lower than that of the control and lithium groups, and the average cell viability of the lithium group was significantly lower than that of the control group (Fig. 4B).

The results of RT-qPCR of rat chondrocytes revealed that the relative expression of LC3B in the dexamethasone group was significantly higher than that of the control and lithium groups, while the relative expression of AKT and mTOR in the dexamethasone group was significantly lower than that of the control and lithium groups (Fig. 4C-E). The results of RT-qPCR of human chondrocytes revealed that the relative expression of LC3B in the dexamethasone group was significantly higher than that of the control and lithium groups, while the relative expression of AKT and mTOR in the dexamethasone group was significantly lower than that of the control and lithium groups (Fig. 4F-H).

The results of western blot analysis of rat chondrocytes revealed that the relative expression of LC3II/I in the dexamethasone group was significantly higher than that of the control and lithium groups, while the relative expression of phosphorylated AKT/AKT and phosphorylated mTOR/mTOR in the dexamethasone group was significantly lower than that of the control and lithium groups (Fig. 5A-D). The results of western blot analysis of human chondrocytes revealed that the relative expression of LC3II/I in the dexamethasone group was significantly higher than that of the control and lithium groups, while the relative expression of phosphorylated AKT/AKT and phosphorylated mTOR/mTOR in the dexamethasone group was significantly lower than that of the control and lithium groups (Fig. 5E-H).