Platelets Rich Plasma Increases Antioxidant Defenses of Tenocytes via Nrf2 Signal Pathway

Abstract

Tendinopathies are common disabling conditions in equine and human athletes. The etiology is still unclear, although reactive oxygen species (ROS) and oxidative stress (OS) seem to play a crucial role. In addition, OS has been implicated in the failure of tendon lesion repair. Platelet-rich plasma (PRP) is rich in growth factors that promote tissue regeneration. This is a promising therapeutic approach in tendon injury. Moreover, growing evidence has been attributed to PRP antioxidant effects that can sustain tissue healing. In this study, the potential antioxidant effects of PRP in tenocytes exposed to oxidative stress were investigated. The results demonstrated that PRP reduces protein and lipid oxidative damage and protects tenocytes from OS-induced cell death. The results also showed that PRP was able to increase nuclear levels of redox-dependent transcription factor Nrf2 and to induce some antioxidant/phase II detoxifying enzymes (superoxide dismutase 2, catalase, heme oxygenase 1, NAD(P)H oxidoreductase quinone-1, glutamate cysteine ligase catalytic subunit and glutathione, S-transferase). Moreover, PRP also increased the enzymatic activity of catalase and glutathione S-transferase. In conclusion, this study suggests that PRP could activate various cellular signaling pathways, including the Nrf2 pathway, for the restoration of tenocyte homeostasis and to promote tendon regeneration and repair following tendon injuries.

Keywords: Nrf2; oxidative stress; platelet-rich plasma; protein oxidation; tendinopathy; tenocytes.

Figure 1

Fluorescent images of tenocytes immunolabeled with tenomodulin (red) (200× magnification), collagen 1 (green) (400× magnification), vimentin (green) (200× magnification), and SOX9 (red) (200× magnification). Nuclear counterstaining was performed with DAPI (blue).

Figure 2

Effect of H₂O₂ on tenocyte viability in medium supplemented with (a) 10% FBS or (b) 10% PRP. Cells were treated with different concentrations of H₂O₂ (0, 0.1, 0.5, 1, 2 mM) for 24 h. Cell viability was measured via MTT assay. Data of four independent experiments performed in triplicate are expressed as the mean ± SD. Cell viability was calculated as percentage of ratio between the OD (optical density) of the samples treated with H₂O₂ and OD of the respective control. ** p < 0.01. **** p < 0.0001.

Figure 3

Protective effect of PRP on H₂O₂-induced protein oxidation. Cells were treated with/without H₂O₂ (1 mM) for 24 h in medium with FBS (10%) (CTR) or PRP (10%). (a) Carbonylated protein detected as DNP-protein adducts: Bar graph and representative image of immunoblotting and relative blue Coomassie-stained PVDF membrane used for data normalization. Data obtained from three independent experiments were reported as means ± SD. * p < 0.05, ** p < 0.01. (b) 4-HNE- proteins: bar graph and representative image of immunoblotting and relative blu Comassie-stained PVDF membrane used for data normalization. Data obtained from three independent experiments were reported as means ± SD. * p < 0.05, ** p < 0.01.

Figure 4

PRP effect on cytosolic (a) and nuclear (b) levels of Nrf2. Cells treated with or without H₂O₂ (1 mM) for 24 h in medium with FBS (10%) (CTR) or PRP (10%) were lysed to extract cytoplasmic and nuclear proteins. The protein levels Nrf2 in both fractions were evaluated via Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Lamin B were used as cytosolic and nuclear housekeeping proteins, respectively. Results are presented as means ± SD of three separated experiments. * p < 0.05; ** p < 0.01, *** p < 0.001.

Figure 5

Effect of PRP of levels and activity of antioxidant enzymes. Cells treated with or without H₂O₂ (1 mM) for 24 h in medium with FBS (10%) (CTR) or PRP (10%) were lysed to analyze the levels of antioxidant enzymes, including GSTP, CAT, SOD2, GCLC, NQO1, and HO-1. β-actin, α-tubulin, and GAPDH are used as housekeeping protein. Representative Western blotting images (a) and correspondent relative bar graphs (b) of data obtained from three independent experiments are shown. The data are expressed as mean ± SD. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05. (c) Enzyme activities of GST and CAT measured on cellular lysates obtained from tenocytes exposed for 24 h to (1 mM) H₂O₂ in medium supplemented with 10% FBS or 10% PRP. The data expressed as mean ± SD are derived from three different experiments. *** p < 0.001; ** p < 0.01; * p < 0.05.