Fullerenol inhibits tendinopathy by alleviating inflammation

Abstract

Tendinopathy is a common disease in orthopaedics, seriously affecting tendon functions. However, the effects of non-surgical treatment on tendinopathy are not satisfactory and surgical treatments possibly impair the function of tendons. Biomaterial fullerenol has been proved to show good anti-inflammatory effects on various inflammatory diseases. For in vitro experiments, primary rat tendon cells (TCs) were treated by interleukin-1 beta (IL-1β) combined with aqueous fullerenol (5, 1, 0.3 μg/mL). Then inflammatory factors, tendon-related markers, migration and signaling pathways were detected. For in vivo experiments, rat tendinopathy model was constructed by local injection of collagenase into Achilles tendons of rats and fullerenol (0.5, 1 mg/mL) was locally injected 7 days after collagenase injection. Inflammatory factors and tendon-related markers were also investigated. Fullerenol with good water-solubility showed excellent biocompatibility with TCs. Fullerenol could increase expression of tendon-related factors (Collagen I and tenascin C) and decrease expression of inflammatory factors (matrix metalloproteinases-3, MMP-3, and MMP-13) and reactive oxygen species (ROS) level. Simultaneously, fullerenol slowed the migration of TCs and inhibited activation of Mitogen-activated protein kinase (MAPK) signaling pathway. Fullerenol also attenuated tendinopathy in vivo, including reduction of fiber disorders, decrease of inflammatory factors and increase of tendon markers. In summary, fullerenol is a promising biomaterial that can be used to treat tendinopathy.

Keywords: MAPK; ROS; fullerenol; inflammation; tendinopathy.

FIGURE 1

Characterization of fullerenol. (A) Transmission electron microscope (TEM) of fullerenol powder. Scale bar = 0.5 μm (left), 50 nm (right) (B) FTIR of fullerenol powder. (C) Image of aqueous fullerenol (50 mg/mL). (D) Hydrodynamic size of fullerenol in aqueous solution.

FIGURE 2

Biocompatibility of fullerenol with different concentrations. (A) Optical density (OD) value of TCs treated with different-concentration (0, 0.1, 0.3, 0.5, 1, 3, 5, 10 μg/mL) fullerenol at 1 and 3 days tested by CCK-8. (B) Live/dead cell staining of TCs treated with 0, 0.3, 1, 5 μg/mL fullerenol at 3 days. Scale bar = 25 μm. (Data are presented as the mean ± standard deviation. *p < 0.05, **p < 0.01).

FIGURE 3

Effects of fullerenol on inflammation of TCs induced by IL-1β. (A) mRNA level of COL1A1, TNC, MMP-3 and MMP-13 of TCs after fullerenol and IL-1β treatment tested by qRT-PCR. (B) Protein level of TNC, COL1A1 and MMP-13 of TCs after fullerenol and IL-1β treatment tested by Western Blot (Left) and quantitative results (Right). (C) ROS level of TCs after fullerenol and IL-1β treatment tested by ROS assay kit.

FIGURE 4

Migration of TCs after IL-1β (50 ng/mL) and fullerenol treatment. (A) Migration of TCs in control (Ctrl), IL-1β, IL-1β+0.3 μg/mL, IL-1β+1 μg/mL, IL-1β+5 μg/mL groups tested by scratch assay. Scale bar = 250 μm. (B) Migration of TCs in Ctrl, IL-1β, IL-1β+0.3 μg/mL, IL-1β+1 μg/mL, IL-1β+5 μg/mL groups tested by transwell assay. Scale bar = 100 μm. (C) Quantitative results of scratch assay. (D) Quantitative results of transwell assay. (Data are presented as the mean ± standard deviation. *p < 0.05, **p < 0.01).

FIGURE 5

Expression of MAPK signaling pathway after IL-1β (50 ng/mL) and fullerenol (0.3, 1, 5 μg/mL) treatment. (A) The phosphorylation levels of p38 in TCs of Ctrl, IL-1β, IL-1β +0.3 μg/mL, IL-1β+1 μg/mL, IL-1β+5 μg/mL groups were examined by Western Blotting. (B) Quantitative results of phosphorylation levels of p38. (C) The phosphorylation levels of Erk1/2 in TCs of Ctrl, IL-1β, IL-1β +0.3 μg/mL, IL-1β+1 μg/mL, IL-1β+5 μg/mL groups were examined by Western Blotting. (D) Quantitative results of phosphorylation levels of Erk1/2. (E) The phosphorylation levels of JNK in TCs of Ctrl, IL-1β, IL-1β +0.3 μg/mL, IL-1β+1 μg/mL, IL-1β+5 μg/mL groups were examined by Western Blotting. (F) Quantitative results of phosphorylation levels of JNK. (Data are presented as the mean ± standard deviation. *p < 0.05, **p < 0.01)

FIGURE 6

Inhibition of fullerenol (0.5, 1 mg/mL) on tendinopathy in vivo. (A) HE and Masson staining of tendons in Ctrl, Collagenase, Collagenase+0.5 mg/mL, Collagenase+1 mg/mL groups (Left). Statistical results of fiber alignment (Right). Scale bar = 50 μm. (B) Immunohistochemical staining (COL I, COX-2, IL-6) of tendons in Ctrl, Collagenase, Collagenase+0.5 mg/mL, Collagenase+1 mg/mL groups. Scale bar = 20 μm.