Cibotium barometz polysaccharides stimulate chondrocyte proliferation in vitro by promoting G1/S cell cycle transition

Abstract

Cibotium barometz polysaccharides (CBPS) are one of the most important bioactive components extracted from the Cibotium barometz plant, which belongs to the Dicksoniaceae family. It has been widely used for the treatment of orthopedic diseases in traditional Chinese medicine. However, the molecular mechanisms behind the therapeutic effects of CBPS remain to be clarified. In the present study, the concentration of CBPS was detected by phenol-vitriol colorimetry. Furthermore, the effects stimulated by CBPS on the viability and G1/S cell cycle transition in primary chondrocytes from Sprague-Dawley rats were investigated. A cell viability assay demonstrated that chondrocyte proliferation may be enhanced by CBPS in a dose‑ and time‑dependent manner. The mechanism underlying the promotion of chondrocyte cell cycle was suggested to involve the stimulation of G1 to S phase transition. To further confirm the results, reverse transcription‑quantitative polymerase chain reaction and western blot analyses were used to detect the expression of mRNA and protein levels of cyclin D1, cyclin‑dependent kinase 4 and retinoblastoma protein. The results suggested that CBPS may stimulate chondrocyte proliferation via promoting G1/S cell cycle transition. Since osteoarthritis is characterized by deficient proliferation in chondrocytes, the present study indicates that CBPS may potentially serve as a novel method for the treatment of osteoarthritis.

Figure 1.

Standard curve for detecting the content of the CBPS by phenol-sulfuric acid colorimetry. CBPS, Cibotium barometz polysaccharides.

Figure 2.

Morphological changes of chondrocytes were observed and images were collected by inverted phase contrast microscope (magnification, ×200). Images of primary cells cultured for (A) 24 h, (B) 4 days, (C) and 8 days, and (D) chondrocytes at passage 2.

Figure 3.

Effect of CBPS on the proliferation of chondrocytes. MTT assay was used to detect the chondrocyte proliferation following treatment with different concentrations of the CBPS (0, 100, 200, 400 and 800 µg/ml) for (A) 48 h or at (B) 200 µg/ml (0, 24, 48 and 72 h). The data are presented as mean ± standard deviation. *P<0.05, **P<0.01, vs. the control group (0 µg/ml). CBPS, Cibotium barometz polysaccharides.

Figure 4.

Morphological changes of the chondrocytes following CBPS treatment at 0, 100, 200 and 400 µg/ml for 48 h (magnification, ×200). CBPS, Cibotium barometz polysaccharides.

Figure 5.

Flow cytometry results following CBPS treatment. (A) Cell cycle changes of chondrocytes. (B) Proportion of chondrocytes in the G0/G1 phase following CBPS treatment. (C) Proportion of chondrocytes in the S phase following CBPS treatment. The data are presented as mean ± standard deviation, *P<0.05, **P<0.01 vs. the control group (0 µg/ml). CBPS, Cibotium barometz polysaccharides.

Figure 6.

mRNA expression of CDK4, cyclin D1 and RB determined using reverse transcription-polymerase chain reaction. (A) Expression of CDK4, cyclin D1, RB and β-actin mRNA in the chondrocytes following CBPS treatment. (B) Relative mRNA expression of CDK4. (C) Relative mRNA expression of cyclin D1. (D) Relative mRNA expression of RB. The data are presented as mean ± standard deviation. *P<0.05, **P<0.01, vs. the control group (0 µg/ml). CDK4, cyclin-dependent kinase 4; RB, retinoblastoma; CBPS, Cibotium barometz polysaccharides.

Figure 7.

Protein expression of CDK4, cyclin D1 and pRB using western blot analysis. (A) The expression of CDK4, cyclinD1, pRB and β-actin protein in the chondrocytes via CBPS. (B) Relative protein expression levels of CDK4. (C) Relative protein expression levels of cyclin D1. (D) Relative protein expression levels of pRB. The data are presented as mean ± standard deviation. *P<0.05, **P<0.01, vs. the control group (0 µg/ml). CDK4, cyclin-dependent kinase 4; pRB, retinoblastoma protein; CBPS, Cibotium barometz polysaccharides.