Patchouli Alcohol Inhibits D-Gal Induced Oxidative Stress and Ameliorates the Quality of Aging Cartilage via Activating the Nrf2/HO-1 Pathway in Mice

Figure 3

Influences of patchouli alcohol (PA) on senescence process and extracellular matrix (ECM) homeostasis of chondrocytes induced by D-gal. (a) Effect of PA on cell viability and cytotoxicity was determined by CCK-8 assay. (b) Effect of PA on ECM degradation marker gene expression of collagen 2a1 (Col2a1) and matrix metalloproteinase 13 (Mmp13) in D-gal-caused senescent chondrocytes was analyzed by RT-qPCR. (c) Representative images of alcian blue, safranin O, and SA-β-gal staining in chondrocytes treated with PA (0, 2.5, 5, and 10 μM) and D-gal (5 mg/ml) for 24 h. The scale bar of alcian blue and safranin O staining, 250 μm. The scale bar of SA-β-gal staining, 10 0 μm. (d) Influence of PA on gene expression of Tp53 and Cyclin-Dependent Kinase Inhibitor 1A (Cdkn1a/p21^Cip1/Waf1) in D-gal-caused senescent chondrocytes. (e and f) The immunofluorescence detection of MMP13 and TP53 in D-gal-caused senescent chondrocytes was treated with 10 μM PA for 24 h. Scale bar, 50 μm. (g) The expression and quantitative analysis of aggrecan (ACAN), COL2A1, a disintegrin and metalloproteinase with thrombospondin 5 (ADAMTS 5), and MMP13 proteins in chondrocytes disposed with PA (0, 2.5, 5, and 10 μM) and D-gal (5 mg/ml) for 24 h were determined by western blotting. The protein expression level is the gray value ratio of the target protein to β-actin. (h) The expression and quantitative analysis of TP53 and CDKN1A/p21^Cip1/Waf1 proteins in D-gal-mediated aging chondrocytes administrated with 10 μM PA for 24 h. The protein expression level is the gray value ratio of the target protein to β-actin. . and vs. control group; ^# vs. D-gal group.

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