Perillyl alcohol promotes autophagy by suppressing the PI3K-AKT-mTOR signalling pathway in osteoarthritic chondrocytes in vitro

Abstract

Osteoarthritis (OA) is a common degenerative disease characterized by chondrocyte death and extracellular matrix (ECM) degradation, but the present OA treatments are only symptomatic and not aetiologic. Autophagy is a key protective mechanism for OA through the inhibition of chondrocyte apoptosis and degeneration. Perillyl alcohol (POH) is a monomer derived from Chinese herbal medicines that can regulate autophagy and has anti-inflammatory effects. In this study, cell viability, chondrocyte phenotype and the expression of molecules involved in autophagy and the PI3K-AKT-mTOR signalling pathway were detected to explore the hypothesis that POH may decrease the progression of OA by regulating autophagy. We found that POH significantly inhibited the expression of inflammatory cytokines, maintained the chondrocyte phenotype, and protected against ECM degeneration. We also provide evidence that the anti-inflammatory effect of POH works by regulating autophagy via the PI3K-AKT-mTOR signalling pathway. This study demonstrates for the first time that POH has a therapeutic effect on arthritis, which suggests that POH may be a promising candidate for OA therapy.

Keywords: Autophagy; Osteoarthritis; PI3K-AKT-mTOR signalling pathway; Perillyl alcohol.

Fig. 1

(a) Cytotoxicity was determined by a CCK-8 assay of chondrocytes at various concentrations (0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 µg/mL). (b) Cell viability of Il-1β-induced chondrocytes treated with the abovementioned concentrations of POH was detected by a CCK-8 assay. (c) Live/dead staining of chondrocytes in the control, OA and POH groups. Original magnification × 100, scale bar = 400 μm. (d) Statistical analysis of cell counts from different fields. (e) Flow cytometry was used to determine the viability of the control, OA and POH groups. (f) The percentage of apoptotic cells (Q-UR). (g) The expression of cleaved caspase-3 was determined by Western blot analysis and (h) quantitative analysis. One-way ANOVA was used for statistical analyses, and multiple comparisons were performed for each group. The data represent the mean ± SD of three independent culture experiments, * indicates p < 0.01, ***/### indicates p < 0.001.

Fig. 2

(a) The DNA content of chondrocytes and (b) GAG production normalized to DNA content in the control, OA and POH groups. (c) HE staining and (d) cell counts from different fields of HE staining. (e) Safranin O staining of chondrocytes and (f) statistical analysis of safranin O staining using ImageJ. Original magnification × 100, scale bar = 400 μm. Data from three independent experiments were evaluated, and the means ± SDs are shown. One-way ANOVA was used for statistical analyses, and multiple comparisons were performed for each group. ns indicates p > 0.05, # indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

Fig. 3

(a) mRNA expression of Col2a1, Il-1β, and Mmp13 detected by qRT‒PCR. (b) Immunofluorescence staining and (c) quantitative analysis of COL2A1 and MMP13. Original magnification × 200, scale bar = 200 μm. (d) The expression of the proteins Aggrecan, COL2A1 and MMP13 was determined by Western blot and (e) quantitative analyses. Data from three independent experiments were evaluated, and the means ± SDs are shown. One-way ANOVA was used for statistical analyses, and multiple comparisons were performed for each group. NS indicates no significant difference, **/## indicates p<0.01, ***/### indicates p < 0.001.

Fig. 4

(a) Gene expression of Becn1, P62, Atg5 and Atg7 was detected by qRT‒PCR. (b) Immunofluorescence staining and (c) quantitative analysis of LC3-B. Original magnification × 200, scale bar = 200 μm. (d) Western blotting was used to analyse the protein expression of LC3-B, ATG5 and ATG7 and (e) perform quantitative analysis. (f) Autophagic flux was observed by stubRFP-sensGFP-LC3 lentivirus transfection. Original magnification × 400, scale bar = 100 μm. The data represent the mean ± SD of three independent culture experiments. One-way ANOVA was used for statistical analyses, and multiple comparisons were performed for each group. * indicates p<0.05, **indicates p<0.01, and *** indicates/ ### p<0.001.

Fig. 5

(a) Immunofluorescence staining and (b) quantitative analysis of p-PI3K, p-AKT and p-mTOR. Original magnification × 200, scale bar = 200 μm. (c) Western blot and (d) quantitative analysis of proteins involved in the PI3K-AKT signalling pathway. n = 3. The data represent the mean ± SD. One-way ANOVA was used for statistical analyses, and multiple comparisons were performed for each group. NS indicates no significant difference, */ # indicates p<0.05, **/## indicates p<0.01, ***/### indicates p<0.001.

Fig. 6

Schematic illustration of the mechanism by which POH regulates autophagy via the PI3K-AKT-mTOR signalling pathway to protect chondrocytes from osteoarthritis.