Intra-Articular Injection of (-)-Epigallocatechin 3-Gallate to Attenuate Articular Cartilage Degeneration by Enhancing Autophagy in a Post-Traumatic Osteoarthritis Rat Model

Abstract

(-)-Epigallocatechin 3-gallate (EGCG) is the main active green tea catechin and has a wide variety of benefits for health. Post-traumatic osteoarthritis (PTOA) occurs as a consequence of joint injuries that commonly happen in the young population. In this study, we investigated the effects of EGCG on PTOA prevention by using the anterior cruciate ligament transection (ACLT)-OA model and further investigated the roles of autophagy in OA treatment. Our results showed that intra-articular injection of EGCG significantly improved the functional performances and decreased cartilage degradation. EGCG treatment attenuated the inflammation on synovial tissue and cartilage through less immunostained cyclooxygenase-2 and matrix metalloproteinase-13. We further noted EGCG may modulate the chondrocyte apoptosis by activation of the cytoprotective autophagy through reducing the expression of the mTOR and enhancing the expression of microtubule-associated protein light chain 3, beclin-1, and p62. In conclusion, intra-articular injection of EGCG after ACL injury inhibited the joint inflammation and cartilage degradation, thereby increasing joint function. EGCG treatment also reduced the chondrocyte apoptosis, possibly by activating autophagy. These findings suggested that EGCG may be a potential disease-modifying drug for preventing OA progression.

Keywords: (-)-epigallocatechin 3-gallate (EGCG); apoptosis; autophagy; cartilage; mTOR; post-traumatic osteoarthritis.

Figure 1

The anti-allodynic effect of EGCG. (a) The temporal pattern of weight distribution of the osteoarthritis (OA) joint. Rats injected with 10 μM EGCG (EGCG group) or saline (OA group and control group) in the right knee were examined for the change in hind paw weight distribution for 5 weeks after anterior cruciate ligament transection (ACLT). A significant difference was noted between OA and EGCG-treated groups at 2, 3, 4, and 5 weeks after ACLT. (b) The results of running endurance at 5 weeks after ACLT. The running endurance was tested one week before and every one week after ACLT for 5 weeks. The running endurance was significantly decreased in the OA group compared with the control. There was a significant improvement in running endurance after EGCG treatment. (** p < 0.01 OA vs. control; ## p < 0.01 OA vs. EGCG).

Figure 2

The histological analysis of osteoarthritis (OA) and the quantitative assessment of glycosaminoglycan (GAG) loss in the anterior cruciate ligament transection (ACLT) rats. (a) The representative micrographs of the proximal tibial cartilage with safranin O-fast green staining. (b) The quantitative analysis of GAG loss. No obvious arthritic changes or GAG loss was observed in the control group, but marked GAG loss was observed in the knee joints of ACLT rats. Meanwhile, the GAG loss was reduced after EGCG treatment. (** p < 0.01 OA vs. control; ## p < 0.01 OA vs. EGCG).

Figure 3

Immunohistochemistry (IHC) of collagen type II (Col II) and collagen type X (Col X) in the articular cartilage of control, osteoarthritis (OA), and OA+EGCG joints. (a) The representative micrographs of IHC analysis for Col II and Col X. (b) The quantitative analysis of immunostained Col II. (c) The quantitative analysis of immunostained Col X. The immunostained Col II was reduced and the immunostained Col X was enhanced after anterior cruciate ligament transection (ACLT). There was significantly more staining of Col II and less staining of Col X in the EGCG-treated rats compared with ACLT rats. (** p < 0.01 OA vs. control; ## p < 0.01 OA vs. EGCG).

Figure 4

Representative sections of synovial membrane from both knee joints of rats. (a) Sample from a rat after sham operation (control group). Note the low-grade inflammatory cell infiltrate and the mild increases of synovial hyperplasia and stroma activation. (b) Sample from a rat after anterior cruciate ligament transection (OA group), in which there is more infiltration by inflammatory cells. (c) Sample from a rat after ACLT and treated with EGCG (10 μM EGCG group) shows similar sham-operated joints with a fewer infiltrating cells than the OA joint.

Figure 5

Immunohistochemistry (IHC) of cyclooxygenase-2 (COX-2) and matrix metalloproteinases-13 (MMP-13) in the articular cartilage of control, osteoarthritis (OA), and OA + EGCG joints. (a) The representative micrographs of IHC analysis for COX-2 and MMP-13. Magnification: 10×. (b) The quantitative analysis of immunostained COX-2. Magnification: 40×. (c) The quantitative analysis of immunostained MMP-13 Magnification: 40×. Sections of osteoarthritic cartilage demonstrated more staining of COX-2 and MMP-13 protein compared to samples from control group. In the EGCG-treated joint, the densities of immunostained COX-2 and MMP-13 were significantly reduced. (** p < 0.01 OA vs. control; ## p < 0.01 OA vs. EGCG).

Figure 6

(a) The representative micrographs of TUNEL staining showing an increase of TUNEL-positive cells after osteoarthritis (OA) induction. Notably, fewer TUNEL-positive cells were observed in EGCG-treated OA joints. (b) Quantitative analysis of chondrocyte apoptosis in three groups. (** p < 0.01 OA vs. control; ## p < 0.01 OA vs. EGCG).

Figure 7

Immunohistochemistry (IHC) of autophagy-related proteins in articular cartilage of control, osteoarthritis (OA), and OA+EGCG joint. (a) The representative micrographs of the mechanistic target of rapamycin (mTOR)-, beclin-1-, light chain 3 (LC3)-, and p62-immunostained articular cartilages from the joints of control, osteoarthritis (OA), and OA+EGCG groups. The positive stained cell ratio of autophagy-related proteins were measured and compared among groups. The results of mTOR were shown in (b), beclin-1 were shown in (c), LC3 were shown in (d), and p62 were shown in (e) (** p < 0.01 OA vs. control; ## p < 0.01 OA vs. EGCG).