Extra-virgin olive oil diet and mild physical activity prevent cartilage degeneration in an osteoarthritis model: an in vivo and in vitro study on lubricin expression  Giuseppe Musumeci, Francesca Maria Trovato, Karin Pichler, Annelie Martina Weinberg, Carla Loreto, Paola Castrogiovanni  Journal of Nutritional Biochemistry  Volume 24, Issue 12, Pages 2064-2075 (December 2013) DOI: 10.1016/j.jnutbio.2013.07.007 Copyright © 2013 Elsevier Inc. Terms and Conditions

Fig. 1 (A–D) Histomorphometric and histological analysis of cartilage. (A) Explanted femur bone, from control group (Group 1) without ACLT and common diet. No sign of cartilage degeneration with an intact and normal cartilage structure. The black arrow indicates the normal articular cartilage without signs of cartilage degeneration. (B) H&E staining in control chondrocytes (Group 1) at third passage of culture. The chondrocytes from the control cartilage did not show any sign of cellular suffering, demonstrated by an intense staining. (C) Explanted femur bone from OA group (Group 2), with experimentally induced osteoarthritis and common diet. Serious pathological changes in the cartilage are shown. The black arrow indicates the OA articular cartilage with relevant signs of cartilage degeneration. (D) H&E staining in OA chondrocytes (Group 2) at third passage of culture. The chondrocytes from OA cartilage showed clear signs of cellular suffering, demonstrated by a slight staining. Journal of Nutritional Biochemistry 2013 24, 2064-2075DOI: (10.1016/j.jnutbio.2013.07.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Fig. 2 (A–B) Analysis of histomorphometric parameters. (A) Kraus’ modified Mankin score. (B) histopathology OARSI system. Both A and B confirmed the development of articular degenerative processes in Group 2 compared to Group 1 (P<.01), Groups 3 and 4 still showed articular degenerative processes compared to Group 1 (P<.05), while Group 5 was equated to Group 1 (P>.05). Results are presented as the mean±S.E.M. ANOVA was used to evaluate the significance of the results. *P<.05 and **P<.01, when compared to the control group. Journal of Nutritional Biochemistry 2013 24, 2064-2075DOI: (10.1016/j.jnutbio.2013.07.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Fig. 3 (A–D) Morphological and histological analysis of liver samples. (A) Explanted liver, from Group 1 (rats without ACLT and common diet), demonstrated no sign of hepatic steatosis. (B) H&E staining in liver tissue from Group 1 (rats without ACLT and common diet), demonstrated no sign of tissue degeneration, with a good preservation and absence of lipid droplets. (C) Explanted liver, from Group 5 (rats with ACLT, extra-virgin olive oil supplemented diet and treadmill training), demonstrated no sign of hepatic steatosis. (D) H&E staining in liver tissue from Group 5 (rats with ACLT, extra-virgin olive oil supplemented diet and treadmill training), demonstrated no sign of tissue degeneration, with a good preservation and absence of lipid droplets. Journal of Nutritional Biochemistry 2013 24, 2064-2075DOI: (10.1016/j.jnutbio.2013.07.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Fig. 4 (A–F) Lubricin immunocytochemistry in chondrocytes from all groups. (A) very strong (ES: ++++; IS: 4) lubricin immunoreaction was observed in control chondrocytes (Group 1), without ACLT and common diet. (B) Weak (ES: +; IS: 1) lubricin immunoreaction was observed in chondrocytes from OA chondrocytes (Group 2), with ACLT and common diet. (C) Moderate (ES: ++; IS: 2) lubricin immunoreaction was observed in chondrocytes from Group 3, with ACLT, common diet and treadmill training. (D) Strong (ES: +++; IS: 3) lubricin immunoreaction was observed in chondrocytes from Group 4, with ACLT and extra-virgin olive oil supplemented diet. (E) Very strong lubricin immunoreaction, as in the control group, (ES: ++++; IS: 4) was observed in chondrocytes from Group 5, with ACLT, extra-virgin olive oil supplemented diet and treadmill training. (F) No immunoreaction (ES: 0; IS: 0) was observed in the negative control treated with PBS without the primary antibody. Colour of positive staining was defined as the presence of a fluorescence detection on the edge of the black background. Cytoplasmic or membrane staining by FITC (green fluorescence) (original magnification ×40); scale bars: 50 μm. Journal of Nutritional Biochemistry 2013 24, 2064-2075DOI: (10.1016/j.jnutbio.2013.07.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Fig. 5 (A–F) Lubricin immunohistochemistry in chondrocytes from rat femoral articular cartilage of Groups 1, 2 and 3. (A) Very strong lubricin staining (ES=++++; IS=4) observed in control cartilage (Group 1, without ACLT and common diet) where almost all chondrocytes were immunolabeled (original magnification ×20); scale bars: 100 μm. (B) Magnification of the figure A (original magnification ×40); scale bars: 50 μm. (C) Weak lubricin immunoreaction (ES=+; IS=1) observed in chondrocytes from Group 2 (with ACLT and common diet) where few cells were immunostained (original magnification ×20); scale bars: 100 μm. (D) Magnification of the figure C (original magnification ×40); scale bars: 50 μm. (E) moderate lubricin immunostaining (ES=++; IS=2) observed in chondrocytes from Group 3 (with ACLT, common diet and treadmill training) (original magnification ×20); scale bars: 100 μm. (F) magnification of the figure E (original magnification ×40); scale bars: 50 μm. Journal of Nutritional Biochemistry 2013 24, 2064-2075DOI: (10.1016/j.jnutbio.2013.07.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Fig. 6 (A–F) Lubricin immunohistochemistry in chondrocytes from rat femoral articular cartilage of Groups 4 and 5. (A) strong lubricin immunostaining (ES=+++; IS=3) observed in chondrocytes from Group 4 (with ACLT and extra-virgin olive oil supplemented diet) (original magnification ×20); scale bars: 100 μm. (B) magnification of the figure A (original magnification ×40); scale bars: 50 μm. (C) very strong lubricin immunostaining (ES=++++; IS=4) observed in chondrocytes from Group 5 (with ACLT, extra-virgin olive oil supplemented diet and treadmill training); specimens were comparable to those of the control group (original magnification ×20); scale bars: 100 μm. (D) magnification of the figure C (original magnification ×40); scale bars: 50 μm. (E) no immunoreaction (ES=0; IS=0) was observed in the negative control treated with PBS without the primary antibodies (original magnification ×40); scale bars: 100 μm. (F) magnification of the figure E (original magnification ×40); scale bars: 50 μm. Journal of Nutritional Biochemistry 2013 24, 2064-2075DOI: (10.1016/j.jnutbio.2013.07.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Fig. 7 (A–B) Immunocytochemistry and immunohistochemistry evaluation: percentage of lubricin positive cells out of the total number of cells counted in the control group and in treated groups. (A) in immunocytochemistry, the percentage of lubricin-positive cells was much lower in Groups 2 and 3 (P<.01), lower in Group 4 (P<.05) compared to Group 1 and in Group 5 was equated to Group 1 (P>.05). (B) in immunohistochemistry the percentage of lubricin-positive cells was much lower in Groups 2 and 3 (P<.01), lower in Group 4 (P<.05) compared to Group 1 and in Group 5 was equated to Group 1 (P>.05). Results were presented as the mean±S.E.M. ANOVA was used to evaluate the significance of the results. *P<.05 and **P<.01, when compared to the control group. Journal of Nutritional Biochemistry 2013 24, 2064-2075DOI: (10.1016/j.jnutbio.2013.07.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Fig. 8 (A–D) Lubricin and IL-1 levels in the synovial fluid from all groups determined by ELISA, at 2 and 7 weeks after ACLT. (A) lubricin levels, at 2 weeks after ACLT, significantly decreased in Groups 2, 3, 4 and 5 in comparison to Group 1 (P<.01). In Group 3, levels of lubricin decreased more than in Group 2. In Groups 4 and 5, levels of lubricin were slightly higher compared to Groups 2 and 3. (B) lubricin levels, at 7 weeks after ACLT, significantly decreased in Groups 2, 3 (P<.01) and 4 (P<.05) compared to Group 1 but they increased with respect to the same groups at 2 weeks after ACLT. In Group 5, the levels of lubricin were comparable to Group 1 (P>.05). (C) IL-1 levels, at 2 weeks after ACLT, drastically increased in Groups 2, 3, 4 and 5 in comparison to Group 1 (P<.01). In Group 3, levels of IL-1 increased more than Group 2. In Groups 4 and 5, levels of IL-1 were slightly lower in comparison with Groups 2 and 3. (D) levels of IL-1, at 7 weeks after ACLT, although lower compared with the same groups at 2 weeks after ACLT, remained significantly increased in Groups 2, 3 (P<.01) and 4 (P<.05) in comparison with Group 1. In Group 5 the level of IL-1 was equated to the Group 1 (P>.05). Results are presented as the mean±S.E.M. ANOVA test was used to evaluate the significance of the results. *P<.05 and **P<.01, when compared to the control group. Journal of Nutritional Biochemistry 2013 24, 2064-2075DOI: (10.1016/j.jnutbio.2013.07.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Fig. 9 Lubricin protein expression in all groups was determined by Western blot analysis. Lubricin expression was the strongest in Group 1 and the weakest in Group 2 (P<.01); in Groups 3 and 4 the expression of lubricin increased compared to Group 2, but it was significantly lower than Group 1 (P<.05); in Group 5 lubricin expression significantly increased when compared to Group 2, equated to Group 1 (P>.05). Data showed the relative expression (mean±S.E.M.) of lubricin protein calculated as arbitrary densitometric units (A.D.U.), collected from three independent experiments. Representative immunoblots were obtained using 50 μg of cell homogenates for each group. β-Tubulin was used as loading control in each experiment. *P<.05 and **P<.01, when compared to the control group. Journal of Nutritional Biochemistry 2013 24, 2064-2075DOI: (10.1016/j.jnutbio.2013.07.007) Copyright © 2013 Elsevier Inc. Terms and Conditions