Selective STAT3 Inhibitor Alantolactone Ameliorates Osteoarthritis via Regulating Chondrocyte Autophagy and Cartilage Homeostasis

Abstract

Osteoarthritis (OA), which is identified by chronic pain, impacts the quality of life. Cartilage degradation and inflammation are the most relevant aspects involved in its development. Signal transducer and activator of transcription 3(STAT3), a member of the STATs protein family, is associated with inflammation. Alantolactone (ALT), a sesquiterpene lactone compound, can selectively suppress the phosphorylation of STAT3. However, the pharmacological effect of ALT on OA is still imprecise. In this study, IL-1β (10 ng/ml) was applied to cartilage chondrocytes, which were treated with different concentrations of Alantolactone for 24 h. The expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2(COX2), matrix metalloproteinases (MMPs) and thrombospondin motifs-5 (ADAMTS5) were detected by western blot. Protein expression of Collagen Ⅱ was observed by western blot, safranin O staining and immunofluorescence. Manifestation of autophagy related proteins such as autophagy-related gene-5 (ATG5), P62, LC3Ⅱ/Ⅰ and PI3K/AKT/mTOR-related signaling molecules were measured by western blot and autophagic flux monitored by confocal microscopy. Expression of STAT3 and NF-κB-related signaling molecules were evaluated by western blot and immunofluorescence. In vivo, 2 mg/kg ALT or equal bulk of vehicle was engaged in the destabilization of medial meniscus (DMM) mouse models by intra-articular injection, the degree of cartilage destruction was classified by Safranin O/Fast green staining. Our findings reported that the enhance of inflammatory factors containing iNOS, COX2, MMPs and ADAMTS5 induced by IL-1β could be ameliorated by ALT. Additionally, the diminish of Collagen Ⅱ and autophagy which was stimulated by IL-1β could be alleviated by ALT. Mechanistically, STAT3, NF-κB and PI3K/AKT/mTOR signal pathways might be involved in the effect of ALT on IL-1β-induced mouse chondrocytes. In vivo, ALT protected cartilage in the DMM mouse model. Overall, this study illustrated that ALT attenuated IL-1β-induced inflammatory responses, relieved cartilage degeneration and promoted impaired autophagy via restraining of STAT3 and NF-κB signal pathways, implying its auspicious therapeutical effect for OA.

Keywords: MMPs; NF-κB; alantolactone; autophagy; osteoarthritis; stat3.

FIGURE 1

The identification of mouse chondrocytes and ALT did not impact the viability of mouse chondrocytes. (A) Phase-contrast micrographs of primary mouse cells after isolation day 1, 3, and 5. (B) Safranin O staining of primary mouse cells. (C) Collagen II immunofluorescence staining of primary mouse cells. Cells were treated by ALT (2.5, 5, and 10 μM) in the presence or absence of IL-1β (10 ng/ml) for 24 h. (D) Chemical structure of ALT. (E) Cell viability was determined by CCK-8 assay.

FIGURE 2

ALT moderated IL-1β-induced iNOS and COX2 expression in mouse chondrocytes. Cells were stimulated by IL-1β (10 ng/ml) in the presence or absence of ALT (2.5, 5, and 10 μM) for 24 h. (A) Expression of iNOS and COX2 were detected by Western blot. (B) Relative protein expression was qualified by ImageJ software, GAPDH was served as the loading control (n = 3). #p < 0.05 vs. control group; *p < 0.05 vs. IL-1β group; **p < 0.01 vs. IL-1β group.

FIGURE 3

ALT alleviated excess MMPs and ADAMTS5 expression in IL-1β-stimulated mouse chondrocytes. Chondrocytes were exposed to ALT (2.5, 5, and 10 μM) with or without IL-1β (10 ng/ml) for 24 h. (A) Western blot was employed to determine the expression of MMP1, MMP3, MMP13, and ADAMTS5. (B) Relative protein expression was qualified by ImageJ software, GAPDH was used as the internal control (n = 3). #p < 0.05 vs. control group; *p < 0.05 vs. IL-1β group; **p < 0.01 vs. IL-1β group.

FIGURE 4

ALT attenuated the degeneration of Collagen II induced by IL-1β in mouse chondrocytes. (A) Western blotting result of Collagen II. (B) Quantification analysis of western blotting results, GAPDH was regarded as an internal control (n = 3). (C) Safranin O staining for proteoglycans deposition in each group after a 7-days incubation (scale bar 100 μm). (D) Collagen II expression was determined by immunofluorescence staining (scale bar 100 μm). #p < 0.05 vs. control group; *p < 0.05 vs. IL-1β group; **p < 0.01 vs. IL-1β group.

FIGURE 5

ALT mitigated impaired autophagy induced by IL-1β in mouse chondrocytes. Cells were stimulated by IL-1β (10 ng/ml) with or without ALT (2.5, 5, and 10 μM) for 24 h. (A) Western blot was employed to detect the expression of ATG5, P62 and LC3Ⅱ/Ⅰ. (B) Relative protein expression was qualified by ImageJ software, GAPDH was used as the internal control, respectively (n = 3). (C) Mouse chondrocytes were exposed to IL-1β (10 ng/ml) at different time points (0, 0.25, 0.5, 1, and 2 h). PI3K/AKT/mTOR-related signaling molecules was determined by Western blot. (D) Cells were exposed to ALT (10 μM) with or without IL-1β (10 ng/ml) for 1 h. Protein levels of P-PI3K, PI3K, P-AKT, AKT, P-mTOR and mTOR were detected by Western blot. (E) Relative protein expression was qualified by ImageJ software, PI3K, AKT and mTOR were used as the loading control, respectively (n = 3) (F) Chondrocytes were transfected with the mRFP-GFP-LC3 adenovirus for 24 h and treated by IL-1β (10 ng/ml) with or without ALT (10 μM) for 24 h. Autophagosomes were represented by yellow puncta and autolysosomes by red puncta in merged images. #p < 0.05 vs. control group; *p < 0.05 vs. IL-1β group; **p < 0.01 vs. IL-1β group.

FIGURE 6

ALT restrained the phosphorylation of STAT3 induced by IL-1β in mouse chondrocytes. (A) Mouse chondrocytes were exposed to IL-1β (10 ng/ml) at different time points (0, 0.5, 1, 2, 4, and 6 h). Phosphorylation of STAT3 was determined by Western blot. (B) Relative protein expression was qualified by ImageJ software, STAT3 was used as the internal control (n = 3). (C) Cells were treated by ALT (2.5, 5, and 10 μM) with or without IL-1β (10 ng/ml) for 2 h. Phosphorylation of STAT3 was determined by Western blot. (D) Relative protein expression was qualified by ImageJ software, STAT3 was used as the loading control (n = 3). #p < 0.05 vs. control group; *p < 0.05 vs. IL-1β group; **p < 0.01 vs. IL-1β group.

FIGURE 7

ALT suppressed IL-1β-induced STAT3 translocation in mouse chondrocytes. Mouse chondrocytes were treated with or without ALT (10 μM) and stimulated with IL-1β (10 ng/ml) for 2 h. (A) Protein level of STAT3 in nucleus was detected by Western blot. (B) Relative protein expression was qualified by ImageJ software, Histone H3 was used as the loading control (n = 3). (C) STAT3 translocation was observed by Immunofluorescence (scale bar 100 μm). #p < 0.05 vs. control group; *p < 0.05 vs. IL-1β group; **p < 0.01 vs. IL-1β group.

FIGURE 8

ALT relieved IL-1β-induced NF-κB signaling pathway activation in mouse chondrocytes. Cells were exposed to ALT (10 μM) with or without IL-1β (10 ng/ml) for 30 min. (A) Protein levels of p- IκB, IκB, p-p65, p65 were detected by Western blot. (B) Relative protein expression was qualified by ImageJ software, IκB and p65 were used as the loading control, respectively (n = 3). (C) p65 translocation was observed by Immunofluorescence (scale bar 100 μm). #p < 0.05 vs. control group; *p < 0.05 vs. IL-1β group; **p < 0.01 vs. IL-1β group.

FIGURE 9

ALT attenuated the vitiation of cartilage on the mouse OA model. (A) Microscopic photos of Safranin-O-Fast green stained mouse knee joint sections of three groups (scale bar 200 and 100 μm). (B) The OARSI scores of each group. **p < 0.01; ***p < 0.001 (C) Immunohistochemistry staining of MMP13. (D) Number of MMP13 positive cells per field under 100-time magnification. (E) Immunohistochemistry staining of Collagen II. (F) Number of Collagen II positive cells per field under 100-time magnification. #p < 0.05 vs. sham group; **p < 0.01 vs. DMM group.

FIGURE 10

Schematic diagram of the effect of ALT on cartilage degeneration. IL-1β induces the expression of pro-inflammatory factors, including iNOS, COX2, MMPs and ADAMTS5. IL-1β stimulates the degradation of Collagen II and the impairment of autophagy. Furthermore, IL-1β functions by activating the PI3K/AKT/mTOR, NF-κB signaling pathways, phosphorylating and translocating STAT3. As shown in the figure, the levels of P-PI3K, P-AKT, P-mTOR, P-IKB, P-P65, P-STAT3 and the translocation of P65 and STAT3 are increasing. However, ALT can attenuate these effects.