Oroxin B alleviates osteoarthritis through anti-inflammation and inhibition of PI3K/AKT/mTOR signaling pathway and enhancement of autophagy

Abstract

Background: Osteoarthritis (OA) is a common aging-related degenerative joint disease with chronic inflammation as its possible pathogenesis. Oroxin B (OB), a flavonoid isolated from traditional Chinese herbal medicine, possesses anti-inflammation properties which may be involved in regulating the pathogenesis of OA, but its mechanism has not been elucidated. Our study was the first to explore the potential chondroprotective effect and elucidate the underlying mechanism of OB in OA.

Methods: In vitro, primary mice chondrocytes were stimulated with IL-1β along with or without the administration of OB or autophagy inhibitor 3-methyladenine (3-MA). Cell viability assay was measured with a cell counting kit-8 (CCK-8). The phenotypes of anabolic-related (Aggrecan and Collagen II), catabolic-related (MMP3, MMP13, and ADAMTS5), inflammation-related (iNOS, COX-2, TNF-α, IL-6, and IL-1β), and markers of related signaling pathways in chondrocytes with different treatment were detected through western blot, RT-qPCR, and immunofluorescent staining. In vivo, the destabilized medial meniscus (DMM) operation was performed to establish the OA mice model. After knee intra-articular injection with OB for 8 weeks, the mice's knee joints were obtained for subsequent histological staining and analysis.

Results: OB reversed the expression level of anabolic-related proteins (Aggrecan and Collagen II) and catabolic-related (MMP3, MMP13, and ADAMTS5) in IL-1β-induced chondrocytes. Mechanistically, OB suppressed the inflammatory response stimulated by IL-1β, as the inflammation-related (iNOS, COX-2, TNF-α, IL-6, and IL-1β) markers were downregulated after the administration of OB in IL-1β-induced chondrocytes. Besides, the activation of PI3K/AKT/mTOR signaling pathway induced by IL-1β could be inhibited by OB. Additionally, the autophagy process impaired by IL-1β could be rescued by OB. What's more, the introduction of 3-MA to specifically inhibit the autophagic process impairs the protective effect of OB on cartilage. In vivo, histological staining revealed that intra-articular injection of OB attenuated the cartilage degradation, as well as reversed the expression level of anabolic and catabolic-related proteins such as Aggrecan, Collagen II, and MMP13 induced in DMM-induced OA models.

Conclusions: The study verified that OB exhibited the chondroprotective effect by anti-inflammatory, inhibiting the PI3K/AKT/mTOR signaling pathway, and enhancing the autophagy process, indicating that OB might be a promising agent for the treatment of OA.

Keywords: PI3K/AKT/mTOR; autophagy; chondrocytes; inflammation; oroxin B; osteoarthritis.

Figure 1

Viability of chondrocytes were not affected by IL-1β/OB/3-MA. (A) Molecular structure of OB. (B) The viability of chondrocytes treated with IL-1β (5 ng/ml), OB (160 μM), 3-MA (5 mM) for 24 h was measured by a CCK-8 kit. Data were presented as means ± SD (n = 3). ns, no significance.

Figure 2

OB upregulated the anabolism in IL-1β–induced chondrocytes. Chondrocytes were treated with IL-1β (5 ng/ml) or/and OB (160 μM) for 24 h. (A) Western blot analysis and (B) quantitative data for the key anabolic-related proteins (Aggrecan, Collagen II). (C, E) Immunofluorescence staining and (D, F) quantitative data of the expression levels of Aggrecan and Collagen II. Data were presented as means ± SD (n = 3). ns, no significance; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

OB downregulated the catabolism in IL-1β–induced chondrocytes. Chondrocytes were administered with IL-1β (5 ng/ml) or/and OB (160 μM) for 24 h. (A) Western blot analysis and (B) quantitative data for the key catabolic-related proteins (MMP13, MMP3, and ADAMTS5). (C) Quantitative analysis of the catabolic-related (MMP13, MMP3, and ADAMTS5) mRNA expression by using RT-qPCR analysis. (D) Immunofluorescence staining and (E) quantitative data of the protein expression of MMP13. Data were presented as means ± SD (n = 3). ns, no significance; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4

OB attenuated the inflammatory responses in IL-1β–induced chondrocytes. Chondrocytes were administered with IL-1β (5 ng/ml) or/and OB (160 μM) for 24 h. (A) Western blot analysis and (B) quantitative data for inflammatory proteins (iNOS, COX-2, TNF-α, IL-6, and IL-1β) in chondrocytes. (C) Immunofluorescence staining and (D) quantitative analysis of the expression levels of COX-2. Data were presented as means ± SD (n = 3). ns, no significance; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

IL-1β triggered the early activation of PI3K/AKT/mTOR signaling pathway and OB inhibited the PI3K/AKT/mTOR signals in IL-1β–induced chondrocytes. (A) Western blot and (B) quantitative analysis for proteins associated with the PI3K/AKT/mTOR pathway in chondrocytes stimulated with 5 ng/ml IL-1β at different time durations (0, 0.25, 0.5, 1, and 2h). (C) Western blot analysis and (D) quantitative data the above proteins in chondrocytes which were administered with IL-1β (5 ng/ml) for 1 h or/and OB (160 μM) for 24 h. Data were presented as means ± SD (n = 3). ns, no significance; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6

OB rescued the impaired autophagy process in IL-1β–induced chondrocytes. Chondrocytes were administered with IL-1β (5 ng/ml) or/and OB (160 μM) for 24 h. (A) Western blot and (B) quantitative analysis showed the impaired autophagy induced by IL-1β could be rescued by the treatment of OB (160 μM). (C) Chondrocytes were transfected with tandem GFP-RFP-LC3 adenovirus, and the strength of autophagic flux was captured with a confocal microscope. (D, E) Quantitative analysis of autolysomes (red puncta) and autophagosomes (yellow puncta) among groups. Data were presented as means ± SD (n = 3). ns, no significance; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 7

3-MA reversed the anti-cartilage degradation and the anti-inflammatory effects of OB in IL-1β–induced chondrocytes. Chondrocytes were administered with IL-1β (5 ng/ml) or/and OB (160 μM) or/and 3-MA (5 mM)for 24 h. (A) Western blot analysis and (B) quantitative data for autophagy-related proteins (Atg3, Atg7, Beclin-1, P62, and LC3 I/II) in chondrocytes. (C) Western blot analysis and (D) quantitative data for anabolic and catabolic-related proteins (Aggrecan, Collagen II, MMP3, and MMP13). (E) Western blot analysis and (F) quantitative data for inflammatory proteins (iNOS, COX-2, TNF-α, and IL-6). Data were presented as means ± SD (n = 3). ns, no significance; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 8

OB alleviated cartilage destruction in DMM–induced mice OA model. (A) H&E staining and (B) SOFG staining of the mice knee articular cartilage. (C) Quantitative analysis of the degree of cartilage destruction among groups by the OARSI scoring system (n=8). (D) Immunohistological staining and (E) quantitative analysis of anabolic and catabolic-related proteins (Aggrecan, Collagen II, and MMP13) (n=3). Data were presented as means ± SD (n = 3). ns, no significance; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 9

Schematic diagram of the chondroprotective effect of OB on mice OA. IL-1β–induced OA chondrocytes were employed in in vitro experiments. The anabolism–inhibiting, catabolism–enhancing, and inflammation-promoting effects induced by IL-1β could be reversed after the treatment of OB. Mechanistically, OB inhibted the activation of PI3K/AKT/mTOR signaling and restored the reduced autophagy process in IL-1β induced chondrocytes. However, 3-MA, an autophagy inhibitor, abolished the protective effect of OB on cartilage, indicating that OB plays a protecting role in articular cartilage through the autophagy process.