Metformin attenuates cartilage degeneration in an experimental osteoarthritis model by regulating AMPK/mTOR

Abstract

Background: It is generally thought that the occurrence and progression of osteoarthritis (OA) results from multiple causes, including degradation and destruction of the cartilage matrix and aging of chondrocytes. Metformin is a first-line drug for the treatment of diabetes, and has great potential for the treatment of other disorders. However, the role of metformin in OA is unknown.

Results: Metformin displayed a protective effect against OA. There were lower OARSI scores and fewer MMP-13-positive cells in DMM mice and cartilage explants after treatment with metformin. In addition, metformin treatment decreased p16^INK4a levels in OA chondrocytes, and enhanced polarization of AMPK and inhibition of mTORC1 in OA mice and chondrocytes in a dose-dependent manner.

Conclusions: Metformin effectively alleviated cartilage degradation and aging through regulation of the AMPK/mTOR signaling pathways, suggesting that it could be an effective treatment for OA.

Methods: The effects of metformin on cartilage degradation and chondrocyte aging was determined in a destabilization of the medial meniscus (DMM)-induced OA mouse model and in IL-1β-treated mouse chondrocytes and cartilage explants. Articular cartilage degeneration was graded using the Osteoarthritis Research Society International (OARSI) criteria. Immunostaining, RT-PCR, and western blot analyses were conducted to detect the relative expressions of protein and RNA.

Keywords: AMPK/mTOR; cartilage injury; metformin; osteoarthritis.

Figure 1

Histological scoring of the destabilization of the medial meniscus (DMM), DMM + Metformin joints, and cartilage explants. (A) Representative photographs of the joints of male mice at 2, 5, and 10 weeks after surgery. (B) Osteoarthritis Research Society International (OARSI) scoring of the joints of DMM, DMM + metformin male mice at 2, 5, and 10 weeks after surgery, as well as cartilage explants (D). (C) Representative Safranin O staining of paraffin section of mouse cartilage explants treated with IL-1β + metformin (4 mM and 8 mM) for 5 days. Sections were stained with Safranin O/Fast Green. Two weeks: n = 24; 5 weeks: n = 24; 10 weeks: n = 24. Cartilage explants: (n = 6 per condition). Values are expressed as the mean ± SEM. ^***p < 0.001 compared with the control.

Figure 2

Metformin reduces the degradation of cartilage matrix and stimulates its synthesis, while also affecting the formation of osteophytes. (A) Immunohistochemical detection of MMP-13 in tibial cartilage at 2, 5, and 10 weeks after destabilization of the medial meniscus surgery. (B) Quantification of cells positively stained for matrix metalloproteinase-13 (MMP-13). (C) The mRNA expression levels of MMP-13, SOX9, COLX and COL2al, and (D) Western blot analyses of the protein expression levels of MMP-13 and COL2al. Primary chondrocytes were induced with IL-1β (10 ng/mL), and then co-cultured with metformin (1, 2, 4, and 5 mM) for 72 h. Two important cartilage matrix degrading proteins, MMP-13 and COLX, were decreased during administration, while other important cartilage matrix synthetic proteins, SOX9 and COL2al, were promoted during administration at 72 h. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001 between two groups.

Figure 3

Expression of the p16^INK4a protein in articular cartilage of the destabilization of the medial meniscus (DMM) model in mice. (A) Immunohistochemical detection of p16^INK4a in tibial cartilage at 2, 5, and 10 weeks after DMM surgery. (B) Quantification of cells positively stained for p16^INK4a. ^***P < 0.001 between the two groups. And (C) the western blot analyses of the protein expression levels of p16^INK4a protein in primary chondrocytes which were induced with IL-1β (10 ng/mL) and co-cultured with metformin (1, 2, 4, and 5 mM) for 24 h 48 h and 72 h.

Figure 4

Metformin reduces the expression of p-S6 protein and affects the expression of other related pathway proteins. (A) Immunofluorescence detection of p-S6 in tibial cartilage at 2, 5, and 10 weeks after destabilization of the medial meniscus (DMM) surgery. (B) Quantitation of cells positively stained for p-S6. ^***P < 0.001 between the two groups. (C) Immunofluorescence detection of p-AMPK in tibial cartilage at 2, 5, and 10 weeks after DMM surgery. (D) Quantitation of cells positively stained for p-AMPK. ^***P < 0.001 between the two groups. And (E) the western blot analyses of the protein expression levels of selected proteins in primary chondrocytes which were induced with IL-1β (10 ng/mL) and co-cultured with metformin (1, 2, 4, and 5 mM) for 24 h, 48 h and 72 h. (NC = normal control, C = IL-1β).

Figure 5

The change of protein expression in cartilage explants. (A) Immunohistochemical detection of MMP-13 and p16^INK4a in cartilage explants which were stimulated with IL-1β (50 ng/mL) for 48 h, and then co-cultured with metformin (4 mM and 8 mM) for 5 days. (B) Quantification of cells positively stained for matrix metalloproteinase-13 and p16^INK4a. ^***P < 0.001 between the two groups.