PMC

Cytotoxic activity of Rosa extract on βTC6 cells. βTC6 cells were cultured on a 96-well plate, at a concentration of 5 × 10⁴ cells/mL in the absence or presence of different concentrations of Rosa extract (0.00001 to 1 mg/mL). Cell viability was measured using a cell proliferation kit (MTT), 24 hours after seeding. *Values are significantly different compared with control cells (P < .05).

Effect of botanical extracts on signaling proteins and cartilage-specific transcription factor SOX-9 in chondrocytes. Serum-starved chondrocytes (0.1 × 10⁶ cells/mL) were cultured for 24 h and then treated with 10 ng/mL IL-1β for 48 h, botanical extracts (each 10 μg/mL) for 72 h, or pretreated with botanical extracts (10 μg/mL each) for 24 h and then cotreated with 10 ng/mL IL-1β for 48 h or left untreated and evaluated after 72 h. Results of western blot analysis revealed down-regulation of Shc (I), ERK1/2 (II), and SOX-9 (III) in chondrocytes with IL-1β. Cotreatment of chondrocytes preincubated with botanical extracts and IL-1β relieved the IL-1β-induced inhibition of Shc (I), ERK1/2 (II), and SOX-9 (III). In untreated and in positive control cultures, expression of Shc, ERK1/2, and SOX-9 were equally strong in chondrocytes (I–III). Expression of β-actin was not affected by IL-1β and/or botanical extracts (IV). Data shown are representative of three independent experiments. Treatments: C (untreated control); IL-1β; RC (Rosa canina); SA (Salix alba); UD (Urtica dioica).

Effects of botanical extracts on IL-1β-induced upregulation of proinflammatory enzymes in chondrocytes. Serum-starved chondrocytes (0.1 × 10⁶ cells/mL) were cultured for 24 h and then treated with 10 ng/mL IL-1β for 48 h, botanical extracts (each 10 μg/mL) for 72 h, or pretreated with botanical extracts (10 μg/mL each) for 24 h and then cotreated with 10 ng/mL IL-1β for 48 h or left untreated and evaluated after 72 h. IL-1β-stimulation leads to an increase in synthesis of COX-2, MMP-9, and MMP-13 (I, II, III). However, COX-2, MMP-9, and MMP-13 upregulation was blocked in chondrocytes pre-incubated with botanical extracts (10 μg/mL each) for 24 h and then co-treated with IL-1β (10 ng/mL) for 48 h (I, II, III). In untreated and in botanical extracts alone treated control cultures, expression of COX-2-, MMP-9 and MMP-13 were not seen in chondrocytes (I–III). Expression of the housekeeping gene β-actin was not affected by treatment with IL-1β and/or botanical extracts (IV). Data shown are representative of three independent experiments. Treatments: C (untreated control); IL-1β; RC (Rosa canina); SA (Salix alba); UD (Urtica dioica).

Botanical extracts block the IL-1β-induced phosphorylation and nuclear translocation of p65 in chondrocytes. Western blot analysis of IL-1β-treated nuclear extracts. Serum-starved chondrocytes (0.1 × 10⁶ cells/mL) were pretreated with botanical extracts (10 μg/ML each) for 4 hours followed by cotreatment with 10 ng/mL IL-1β and botanical extracts for 1 h. Some chondrocyte cultures remained either untreated or were treated with 10 μg/mL botanical extracts (each alone) or with 10 ng/mL IL-1β alone for 1 h. Nuclear extracts were probed for phospho p65, (I) by western blot analysis using antibodies to p65, phospho-specific p65, and PARP (II, control). Treatment of chondrocytes with IL-1β (10 ng/mL) revealed a clear increase in expression of phospho-p65 in the nuclear extracts (I). Co-treatment of chondrocytes with botanical extracts (all three) completely abolished the IL-1β-dependent activation of phospho p65 in the nucleus (I). Synthesis of PARP remained unaffected in nuclear extracts (II). Data shown are representative of three independent experiments. Treatments: C (untreated control); IL-1β; RC (Rosa canina); SA (Salix alba); UD (Urtica dioica).