Abstract
Purpose: To investigate the effect of 7-H-pyrrolo[2,3-d]pyrimidine derivative (7-HPPD) on the viability of chondrocytes in vitro, and to elucidate the associated mechanisms.Methods: Chondrocyte proliferation was determined by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay while cell cycle was assessed by flow cytometry. Western blot and immunohistochemical staining assays were used to determine protein levels.Results: The results show that 7-HPPD significantly increased the proliferation rate of chondrocytes in a concentration-dependent manner (p < 0.05). The proportion of chondrocytes in S phase increased significantly with subsequent reduction in G0/G1 phase of cell cycle on treatment with 7-HPPD (p <0.05). Thus, 7-HPPD increased the rate of chondrocyte proliferation by promoting transition through G1/S phase of cell cycle. Reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis revealed that treatment of chondrocytes with 7-HPPD caused a marked increase in the level of cyclin D1, cyclin dependent kinase (CDK)-4 and CDK6 proteins (p < 0.05). Western blot and immunohistochemical staining assays showed that 7-HPPD treatment caused a significant increase in the levels of type II collagen matrix in the chondrocytes.Conclusion: Proliferation of chondrocytes is increased by 7-HPPD and this occurs by facilitating G1/S phase transition and increasing expression of cyclin D1, CDK4 and CDK6 proteins. Therefore, 7-HPPD may be developed as a chemotherapeutic agent for the treatment of osteoarthritis.
 Keywords: Immunohistochemistry, Type II collagen, G1/S phase, Cyclin D1, Osteoarthritis
Highlights
Osteoarthritis is the disease of joints associated with the degradation of articular cartilage and bones [1,2]
The level of type II collagen matrix in the chondrocytes was determined using western blot and immunohistochemical staining assays (Figure 5). 7-HPPD treatment markedly increased the level of type II collagen matrix in chondrocytes in concentration based manner
Osteoarthritis is the disorder of joints caused by the ECM degradation and apoptosis of bone cells leading to the cartilage damage [13]
Summary
Osteoarthritis is the disease of joints associated with the degradation of articular cartilage and bones [1,2]. In the present study the effect of 7-H-pyrrolo[2,3-d]pyrimidine derivative (7-HPPD) on chondrocyte proliferation and cell cycle progression was investigated. The study demonstrated that 7-HPPD increased chondrocyte proliferation and promoted cell cycle progression by up-regulation of cyclin protein and type II collagen matrix level in chondrocytes. The chondrocytes at 1.5 x 104 cells per well density were put into the 96-well plates containing DMEM mixed with 10 % FBS. The cells were incubated with 7HPPD for 72 h following which medium was removed and 30 μL MTT solution (0.5 %) was added to the wells. The chondrocytes were incubated for 72 h with 7-HPPD followed by digestion with type II collagenase. The chondrocytes at 2.5 x 105 cell density in 3 mL of medium were put into the 6-well plates and incubated for 72 h with 80, 100, 150 and 200 μM concentrations of 7-HPPD. Differences were considered statistically significant at p < 0.05
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