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Journal of Chinese Integrative Medicine ›› 2012, Vol. 10 ›› Issue (12): 1413-1418.doi: 10.3736/jcim20121213

• Original Experimental Research • Previous Articles     Next Articles

Inhibition effect of osthole on proliferation of rat chondrocytes

Dao-fang Ding1,2, Song-pu Wei1,2, Xiao-feng Li3,4, Xiao-gang Zhang1,2, Hong-sheng Zhan1,2, Tie-li Duan1,2, Yue-long Cao1,2()   

  1. 1. Institute of Traumatology and Orthopedics, Shanghai Academy of Traditional Chinese Medicine, Shanghai 201203, China
    2. Shi’s Center of Orthopedics and Traumatology, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
    3. Institute of Spine, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China
    4. Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China
  • Received:2012-06-09 Accepted:2012-07-05 Online:2012-12-20 Published:2018-12-15

Objective: To investigate the effects of osthole on chondrocyte proliferation in vitro.
Methods: Primary rat chondrocytes were isolated from the femoral head of newborn rats using collagenase digestion and cultured in Dulbecco’s modified Eagle’s medium. The proliferation of primary chondrocytes was assessed in second-passage cultures using cell counting kit-8 and the growth curve was drawn. Type Ⅱ procollagen gene (Col2a1) expression in chondrocytes was also identified using cell immunofluorescence assay. The second-passage chondrocytes were divided into five groups, including control group and osthole groups at 6.25, 12.5, 25 and 50 μmol/L. The growth property of rat chondrocytes was observed after 24, 48 and 72 h of culture with osthole at corresponding dose. Both protein and mRNA expression of proliferating cell nuclear antigen (PCNA) and cyclin D1 was measured by Western blot and polymerase chain reaction methods.
Results: The second-passage chondrocytes were viable and showed Col2a1 expression in the cytoplasm. The proliferation of rat chondrocytes was inhibited by osthole in a dose-dependent manner. Meanwhile, there were significant decreases in both protein and mRNA expression of PCNA and cyclin D1 in the osthole groups compared with the control group.
Conclusion: Osthole exhibits inhibitory effect on proliferation of rat chondrocytes by down-regulating PCNA and cyclin D1 expression.

Key words: plant extracts, osthole, chondrocytes, cell proliferation, proliferating cell nuclear antigen, cyclin D1, rats

Figure 1

Identified Col2a1 expression in chondrocytes observed by immunofluorescence staining (Olympus IX71, ×200) Col2a1: type Ⅱ procollagen gene; DAPI: 4′,6-diamidino-2-phenylindole."

Figure 2

Growth curve of rat chondrocytes detected by cell counting kit-8"

Figure 3

Effects of osthole on proliferation of chondrocytes after 24 or 48 h of culture (light microscopy, ×100)"

Table 1

Proliferation of chondrocytes in five groups (x±s)"

Group n 24 h 48 h 72 h
OD450 Inhibition rate OD450 Inhibition rate OD450 Inhibition rate
Control 6 0.344±0.005 0.539±0.009 1.260±0.025
6.25 μmol/L osthole 6 0.323±0.002* 6.10% 0.472±0.007* 12.43% 1.145±0.025* 9.13%
12.5 μmol/L osthole 6 0.316±0.002*△ 8.14% 0.455±0.004*△ 15.58% 1.062±0.031*△ 15.71%
25 μmol/L osthole 6 0.302±0.004*△ 12.21% 0.426±0.004*△ 20.96% 0.915±0.013*△ 27.38%
50 μmol/L osthole 6 0.295±0.004*△ 14.26% 0.397±0.006*△ 26.35% 0.768±0.032*△ 39.05%

Figure 4

Expression of PCNA and cyclin D1 proteins in primary chondrocytes treated with osthole at different concentrations The specific bands and the relative protein levels of PCNA (A) and cyclin D1 (B) in rat chondrocytes after 24 h of treatment with 6.25, 12.5, 25 and 50 μmol/L osthole respectively as determined by Western blotting. PCNA: proliferation cell nuclear antigen; GAPDH: glyceraldehyde-3-phosphate dehydrogenase."

Table 2

Inhibition rate of PCNA and cyclin D1 protein expression in chondrocytes treated with osthole"

Group n PCNA Cyclin D1
Relative to GAPDH Inhibition rate Relative to GAPDH Inhibition rate
Control 3 1.406±0.009 0.615±0.010
6.25 μmol/L osthole 3 1.330±0.019* 5.67% 0.514±0.008* 16.39%
12.5 μmol/L osthole 3 1.047±0.043*△ 25.53% 0.393±0.011*△ 36.07%
25 μmol/L osthole 3 0.985±0.010*△ 30.50% 0.374±0.009*△ 39.34%
50 μmol/L osthole 3 0.546±0.009*△ 61.00% 0.076±0.008*△ 88.52%

Figure 5

mRNA transcription levels of PCNA and cyclin D1 in primary chondrocytes treated with osthole at different concentrations mRNA levels of PCNA and cyclin D1 were decreased in a concentration-dependent manner when chondrocytes were cultured with osthole. PCNA: proliferation cell nuclear antigen; GAPDH: glyceraldehyde-3-phosphate dehydrogenase."

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