Table 1.
qRT-PCR primer sequences.
Table 2.
Primary antibody.
Fig 1.
P-II counteracts the suppressive effect of LPS on chondrocyte proliferation.
(A) Depicted is the chemical structure of Picroside II (P-II). (B) The impact of various concentrations of P-II (5, 10, 25, 50, 100, 200μM) on cell viability after a 24-hour incubation period is illustrated. (C) Shown is the effect of P-II (at concentrations of 5, 10, 25, 50μM) on cell viability over 24 hours under conditions with and without the addition of Lipopolysaccharide (LPS). (D) Representative picture of chondrocytes after toluidine blue staining. Data are presented as mean ± standard deviation (SD). Statistical significance is denoted by asterisks (* P < 0.05, ** P< 0.01, *** P < 0.001) and the symbol ## indicates P < 0.01, ns signifies no statistical significance.
Fig 2.
P-II attenuates ECM degradation in chondrocytes.
(A) Representative Western blot images for Col2 and MMP3. (B, C) Quantitative analysis of the Western blot results for Col2 and MMP3. (D, E) qRT-PCR for Col2 and MMP3 gene expression. (F) Immunofluorescence Col2 (green) and dapi staining nuclear fluorescence (blue) are visible under fluorescence microscope. (G, H) Expression levels of Col2 and MMP3 in chondrocytes by immunofluorescence assay (Scale bar = 50μm). Data are presented as mean ± standard deviation (SD). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns signifies no statistical significance.
Fig 3.
P-II suppresses NLRP3 inflammasome activation and alleviates chondrocyte pyroptosis.
(A) Representative Western blot images for NLRP3, IL-1β, IL-18, caspase-1. (B-E) Quantitative analysis of the Western blot results for NLRP3, IL-1β, IL-18, caspase-1. (F-I) Quantitative analysis of qRT-PCR for NLRP3, IL-1β, IL-18, caspase-1. (J) Immunofluorescence NLRP3 (red) and dapi staining nuclear fluorescence (blue) are visible under fluorescence microscope. (K, L) Representative images of immunohistochemistry for caspase-1 and NLRP3(Scale bar = 25μm). (M, N) Quantitative analysis of caspase-1 and NLRP3 immunohistochemistry. (Data are presented as mean ± standard deviation (SD). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns signifies no statistical significance.
Fig 4.
P-II reduced osteophyte formation, attenuated bone destruction, and attenuated DMM-induced OA in mice.
(A). Representative Micro-CT 3D images of mice subchondral bone. (B) Representative Micro-CT 3D images of mice knee joint. (C-F) Quantitative analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp of subchondral bone.(G, H) Representative images of H&E staining (Scale bar = 50μm), Safranin-O/Fast Green staining (Scale bar = 20μm). (I) OARSI score. Data are presented as mean ± standard deviation (SD). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns signifies no statistical significance.
Fig 5.
P-II inhibition of the MAPK/NF-κB signaling pathway.
(A-G) Representative Western blot images for ERK, p-ERK, JNK, p-JNK, P38, p-P38, p65, p-p65 and quantitative analysis. Data are presented as mean ± standard deviation (SD). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns signifies no statistical significance.
Fig 6.
Potential mechanism of action of Picroside II in osteoarthritis.