Intra-Articular Lactate Dehydrogenase A Inhibitor Oxamate Reduces Experimental Osteoarthritis and Nociception in Rats via Possible Alteration of Glycolysis-Related Protein Expression in Cartilage Tissue

Abstract

Osteoarthritis (OA) is the most common form of arthritis and joint disorder worldwide. Metabolic reprogramming of osteoarthritic chondrocytes from oxidative phosphorylation to glycolysis results in the accumulation of lactate from glycolytic metabolite pyruvate by lactate dehydrogenase A (LDHA), leading to cartilage degeneration. In the present study, we investigated the protective effects of the intra-articular administration of oxamate (LDHA inhibitor) against OA development and glycolysis-related protein expression in experimental OA rats. The animals were randomly allocated into four groups: Sham, anterior cruciate ligament transection (ACLT), ACLT + oxamate (0.25 and 2.5 mg/kg). Oxamate-treated groups received an intra-articular injection of oxamate once a week for 5 weeks. Intra-articular oxamate significantly reduced the weight-bearing defects and knee width in ACLT rats. Histopathological analyses showed that oxamate caused significantly less cartilage degeneration in the ACLT rats. Oxamate exerts hypertrophic effects in articular cartilage chondrocytes by inhibiting glucose transporter 1, glucose transporter 3, hexokinase II, pyruvate kinase M2, pyruvate dehydrogenase kinases 1 and 2, pyruvate dehydrogenase kinase 2, and LHDA. Further analysis revealed that oxamate significantly reduced chondrocyte apoptosis in articular cartilage. Oxamate attenuates nociception, inflammation, cartilage degradation, and chondrocyte apoptosis and possibly attenuates glycolysis-related protein expression in ACLT-induced OA rats. The present findings will facilitate future research on LDHA inhibitors in prevention strategies for OA progression.

Keywords: chondrocytes; glycolysis; lactate dehydrogenase A; osteoarthritis; oxamate.

Figure 1

Effects of intra-articular oxamate injection on ACLT-induced OA. The effects of oxamate on (A) ACLT-induced hind limb weight-bearing deficits and (B) knee swelling were studied over time. Rats in the ACLT + oxamate groups were intra-articularly injected with oxamate (0.25 or 2.5 mg/kg per week, black arrow) from the 10th to 14th week after ACLT. Data are expressed as means ± SEM for each group. (* p < 0.05, compared with the sham group; # p < 0.05, compared with the ACLT group). ACLT: anterior cruciate ligament transection.

Figure 2

Histopathological evaluation of knee joints after oxamate treatment in ACLT-rats. (A) Histological sections of knee joints from the sham, ACLT, and ACLT + oxamate (0.25 or 2.5 mg/kg) groups were stained with Safranin O/Fast Green. (B) Histopathological quantitative changes in the knee joints of the four studied groups were evaluated using the OARSI scoring system. The histogram shows the OARSI scores of the sham, ACLT, and ACLT + oxamate (0.25 or 2.5 mg/kg) groups. Data are expressed as means ± SEM for each group. The scale bar represents 100 μm. (* p < 0.05, compared with the sham group; # p < 0.05, compared with the ACLT group). OARSI: Osteoarthritis Research Society International.

Figure 3

The effect of oxamate on glucose transporter 1 and glucose transporter 3 expression in cartilage tissues after ACLT. Immunohistochemical analysis of (A) glucose transporter 1 and (B) glucose transporter 3 in knee joint sections from the sham, ACLT, and ACLT + oxamate (0.25 or 2.5 mg/kg) groups. The quantitative analysis of the ratio of (C) glucose transporter 1-positive and (D) glucose transporter 3-positive cells in joint sections is presented. Data are expressed as means ± SEM for each group. The scale bar represents 100 μm. (* p < 0.05, compared with the sham group; # p < 0.05, compared with the ACLT group). GLUT: glucose transporter.

Figure 4

Effect of oxamate on hexokinase-II and pyruvate kinase M2 expression in cartilage tissues after ACLT. Immunohistochemical analysis of (A) hexokinase-II and (C) pyruvate kinase M2 in joint sections from the sham, ACLT, and ACLT + oxamate (0.25 or 2.5 mg/kg) groups. The quantitative analysis of the ratio of (B) hexokinase II-positive and (D) pyruvate kinase M2-positive cells in joint sections is presented. Data are expressed as means ± SEM for each group. The scale bar represents 100 μm. (* p < 0.05, compared with the sham group; # p < 0.05, compared with the ACLT group). HK: hexokinase; PK: pyruvate kinase.

Figure 5

Effect of oxamate on pyruvate dehydrogenase kinase 1 and pyruvate dehydrogenase kinase 2 expression in cartilage tissues after ACLT administration. Immunohistochemical analysis of (A) pyruvate dehydrogenase kinase 1 and (C) pyruvate dehydrogenase kinase 2 in joint sections from the sham, ACLT, and ACLT + oxamate (0.25 or 2.5 mg/kg) groups. The quantitative analysis of the ratio of (B) pyruvate dehydrogenase kinase 1-positive and (D) pyruvate dehydrogenase kinase 2-positive cells in joint sections is presented. Data are expressed as means ± SEM for each group. The scale bar represents 100 μm. (* p < 0.05, compared with the sham group; # p < 0.05, compared with the ACLT group). PDK: pyruvate dehydrogenase kinase.

Figure 6

Effect of oxamate LDHA expression and TUNEL staining in cartilage tissues after ACLT administration. (A) Immunohistochemical analysis of LDHA and (C) TUNEL staining in joint sections from the sham, ACLT, and ACLT + oxamate (0.25 or 2.5 mg/kg) groups. The quantitative analysis of the ratio of (B) LDHA-positive and (D) TUNEL-positive cells in joint sections is presented. Data are expressed as means ± SEM for each group. The scale bar represents 100 μm. (* p < 0.05, compared with the sham group; # p < 0.05, compared with the ACLT group). LDHA: lactate dehydrogenase A; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling.