L-Glutamine alleviates osteoarthritis by regulating lncRNA-NKILA expression through the TGF-β1/SMAD2/3 signalling pathway

Abstract

Osteoarthritis (OA) is a heterogeneous condition characterized by cartilage degradation, subchondral sclerosis, and osteophyte formation, and accompanied by the generation of pro-inflammatory mediators and degradation of extracellular matrix. The current treatment for early OA is focused on the relief of symptoms, such as pain, but this treatment cannot delay the pathological process. L-Glutamine (L-Gln), which has anti-inflammatory and anti-apoptotic effects, is the most abundant amino acid in human blood. However, its role in OA has not been systematically studied. Therefore, the objective of this work was to explore the therapeutic effect and molecular mechanism of L-Gln on OA. In vitro, we found that L-Gln could up-regulate the expression of the long non-coding RNA NKILA, which is regulated by the transforming growth factor-β1/SMAD2/3 pathway, and inhibit the activity of nuclear factor-κB, thereby decreasing the expression of nitric oxide synthase, cyclooxygenase-2, and matrix metalloproteinase-13 (MMP-13). This led to a reduction in the generation of nitrous oxide, prostaglandin E-2, tumour necrosis factor-α, and degradation of the extracellular matrix (i.e. aggrecan and collagen II) in rat OA chondrocytes. Moreover, intragastric administration of L-Gln reduced the degradation of cartilage tissue and expression of MMP-13 in a rat OA model. L-Gln also relieved the clinical symptoms in some patients with early knee joint OA. These findings highlight that L-Gln is a potential therapeutic drug to delay the occurrence and development of OA.

Keywords: chondrocytes; large intervening non-coding RNA; nuclear factor kappaB; osteoarthritis.

Figure 1. Effect of L-Glutamine (L-Gln) on chondrocytes viability

(A) Molecular structure of L-Gln. (B,C) Chondrocytes were cultured with different concentrations of L-Gln (0, 5, 10, 20 mM) for 24 and 48 h. (D) Chondrocytes were pretreated with IL-1β, and cell viability was determined. #P<0. 05 versus the Control. *P<0.05, **P<0.01 versus the IL-1β group; n=3.

Figure 2. L-Gln inhibits IL-1β-induced cyclooxgenease-2 (COX-2), induced nitric oxide synthase (iNOS), and matrix metalloproteinase-13 (MMP-13) expression, and NO, prostaglandin E-2 (PEG-2), and tumour necrosis factor (TNF)-α release in rat osteoarthritis (OA) chondrocytes

(A) Nitrite evaluated using the Griess test. (B,C) The levels of PGE-2 and TNF-α as determined by enzyme-linked immunosorbent assay. (D–F) The mRNA levels of iNOS, COX-2, and MMP-13. (G–L) The protein levels of iNOS, COX-2, and MMP-13. (M,N) MMP-13 as determined by immunofluorescence and analyzed with Image-Pro6.0; scale bar = 50μm. #P<0.05 versus the control group. *P<0.05, **P<0.01 versus the IL-1β group; n=3.

Figure 3. L-Gln inhibits the degradation of extracellular matrix in rat OA chondrocytes

(A–D) Protein levels of Collagen II and Aggrecan. (E,F) mRNA levels of Aggrecan and Collagen II. (G–J) Fluorescence intensity of Aggrecan and Collagen II as determined by immunofluorescence and analyzed with Image-Pro 6.0; scale bar = 50 μm. #P<0.05 versus the Control. *P<0.05, **P<0.01 versus the IL-1β group; n=3.

Figure 4. L-Gln inhibits over-activation of nuclear factor-κB (NF-κB) in rat OA chondrocytes

(A–E) Protein levels of p65, p-IκB, IκB, and p-P65. (F,G) Nuclear translocation of p65 visualized by immunofluorescence and quantified with Image-Pro6.0; scale bar = 50 μm. #P<0.05 versus the control group. *P<0.05, **P<0.01 versus the IL-1β group; n=3.

Figure 5. L-Gln inhibits NF-κB over-activation in rat OA chondrocytes by regulating TGF-β1/SMAD2/3 pathway-induced NKILA

(A–C) Protein levels of p-SMAD2, SMAD2/3, and p-SMAD3. (D) RNA level of NKILA. (E,F) Nuclear translocation of SMAD2/3 visualized by immunofluorescence and quantified with Image-Pro6.0; scale bar = 50 μm. #P<0.05 versus the control group. *P<0.05, **P<0.01 versus the IL-1β group; n =3.

Figure 6. Intragastric administration of L-Gln can delay the development of OA in rats

(A) The medial and lateral femoral condyle and tibial plateau of rats. (B) H&E staining. (C) Safranin-O/Fast Green staining. (D–F) Immunohistochemical staining of MMP-13, Aggrecan, and Collagen II. (H–J) Quantification with Image-Pro6.0; scale bar = 50 μm. (G) OARSI score. (K, L) The serum levels of IL-1β and L-Gln. #P<0.05 versus the control group. *P<0.05, **P<0.01 versus the OA group; Control: n=3, OA: n=4, OA+L-Gln: n=5.

Figure 7. L-Gln improves the symptoms of some patients with early OA

(A) visual analogue scale (VAS), (B) WOMAC and (C) Lequesne scores at 0 and 4 week after oral administration of L-Gln, respectively. **P<0.01 versus week 0; n=41.