Metabolic effects of HCHF diet feeding.

<p>(A) Body weight of Wistar rats on CD or HCHF diet (n = 8). (B) Dorsal view of the rats showing the changes in the total abdominal length caused by the two diets after 16 weeks. ELISA analysis of pro-inflammatory (C) and (D) or anti-inflammatory (E) cytokines in serum (n = 6). Data were analyzed by two-tailed Student’s t test. All data are presented as mean ± SD. P < 0.05 (CD vs HCHF at two time point- week 8 and week 16) was considered to be significant. * = <i>p</i> <0.05.</p

Dietary intake and body composition in CD and HCHF rats.

<p>Dietary intake and body composition in CD and HCHF rats.</p

Synovial fluid of 16-week HCHF rats alters macrophage polarization and chondrocyte differentiation in vitro.

<p>Relative qPCR analysis of pro-inflammatory M1-like (A) or anti-inflammatory M2-like (B) genes in BMDMs after synovial fluid stimulation. ELISA analysis of pro-inflammatory (C) or anti-inflammatory (D) cytokines in conditioned medium. (E) Relative qPCR analysis of MMP13, ADAMTS5, COL10, ACAN and SOX9 in micromass cultured ACCs after 7 days of synovial fluid stimulation. (F) GAG release in supernatant of ACCs after synovial fluid stimulation at day 3 and day 7. Data were analyzed by two-tailed Student’s <i>t</i> test. All data are presented as mean ± SD, <i>P</i> < 0.05 was considered to be significant. * = <i>p</i> <0.05. n = 5 independent samples.</p

M1 macrophage negatively affects chondrogenic differentiation of chondrocytes.

<p>Relative qPCR analysis of pro-inflammatory M1-like (A) or anti-inflammatory M2-like (B) genes in CD14+ macrophages after cytokine treatment. ELISA analysis of pro-inflammatory (C) or anti-inflammatory (D) cytokines in conditioned medium. Relative qPCR analysis of MMP2 (E), MMP13 (F), RUNX2 (G), ADAMTS5 (H), SOX9 (I), and ACAN (J) mRNA levels in chondrocytes treated with M1 CM or M2 CM. (K) GAG release in supernatant of chondrocytes treated with 50% M1/M2 CM treatment for 3 and 7 days. Data were analyzed by two-tailed Student’s <i>t</i> test. Values represent the mean ± SD of experimental triplicates, <i>P</i> < 0.05 was considered to be significant. * = <i>p</i> <0.05. n = 5 independent samples.</p

Macrophage-like cells increase in inflamed synovium of 16-week HCHF rats and are predominately iNOS+.

<p>(A) Representative immunohistochemical and immunofluorescence analyses of synovial tissues from CD or HCHF diet rats with anti-CD68, anti-iNOS or anti-Arg1. (B) Quantitative assessment of CD68+, iNOS+ or Arg1+ cells of inflamed synovium. Total positive cells per 100 cells were used as a standard measure to quantify. (C, D) qPCR analysis of pro-inflammatory M1-like (C) or anti-inflammatory M2-like (D) genes in synovium after diet stimulation. Data were analyzed by two-tailed Student’s <i>t</i> test. All data are presented as mean ± SD, <i>P</i> < 0.05 was considered to be significant. * = <i>p</i> <0.05. n = 5. Scale bar = 20 μM.</p

Time-dependent histopathologic changes in the joint of HCHF diet-fed rats (8 and 16 weeks).

<p>Histological evaluation of the knee joints. Tissues were stained with safranin-O and fast green (A-B) to estimate the proteoglycan loss among the two time points in HCHF and CD groups. Scale bar = 20 μM. Extent of articular cartilage degradation was graded using Mankin scoring system (E). Safranin-O and fast green staining shows the difference in thickness of the synovial membrane following, (C)8, and (D)16-week HCHF diet Histological scoring showed increased synovial thickening in 8 and 16-week-diet rats (F). Data were analyzed by Mann-Whitney test. All data are presented as mean ± SD. <i>P</i> < 0.05 (HCHF at week 8 vs HCHF at week 16) was considered to be significant. * = <i>p</i> <0.05. n = 8 per each group at each collection time point. Scale bar = 20 μM.</p