The Research Of Curcumine On The Proliferation Of The Articular Chondrocyte Based Upon" Wnt/β-catenin " Signal Pathway

Background:Osteoarthritis(OA) is a kind of chronic joint disease, which is characterized by articular cartilage degeneration and secondary bone hyperplasia. Many middle-aged or senile patients suffer from it and recently it tends to be younger susceptibility. For a long time, the traditional Chinese medicine (TCM) has a unique advantage in treatment of bone diseases. With the advantages of TCM to combat OA widely accepted by society, it has been a hot area of research for several years. Turmeric is one kind of the Chinese medicine for orthopedics diseases in the prescriptions,which has a good effect on OA. During the development of osteoarthritis, many signaling pathways are activated or inhibited in the articular chondrocytes. Wnt/β-catenin signal pathway is the more important one. It plays a significant role in the process of joint growth, protection and repair.Objective:To explore the mechanisms of curcumin on the proliferation of the articular chondrocyte based upon " Wnt/β-catenin " signal pathway. Further to explain the mechanism of the TCM treatment of osteoarthritis at the molecular biological level.To Confirm the high application value of the extraction from Chinese herbs on the tissue engineering and molecular biology in the medical world.Methods:1.One male and one female clean New Zealand white rabbits at 4 weeks old was used for establishing the chondrocytes in vitro culture. Pattern identification was tested by the application methods such as cell morphological observation and toluidine blue staining; 2.To analyze the proliferation of 1-5 generations of chondrocytes within 10 days with a determined by MTT method and drawing growth curves; 3.With CCK-8 to test the concentration of curcumin on the proliferation of chondrocytes for 24 h; 4.With Western blot to test Wnt 2、 GSK-3β、β-catenin related protein expression in the Wnt/β-catenin signaling pathway by 5μmol·L-1、10μmol·L-1 concentrations of curcumin on the chondrocytes for 12h and 24h at 10μl.Results:(1) At the two days from each generation of chondrocytes cultured, the OD value was similar (P>0.05).They were all growing slowly. Until the third day proliferation became faster. The proliferation achieved logarithmic growth at the fourth day.After 5 days,the proliferation of chondrocytes started to slow down.7 days later,the speed of growth became smooth. The stagnate phase was from the 7th day. After 9 days, the proliferation rate decreased by degrees,and chondrocytes growth arrested.(2) The drug had few colors affect at 25、20、10、5μmol·L-1.The index is relatively accurate. Compared to the control group, there was a statistically significant difference (P<0.05 or P<0.01). The OD value was increasing with the decreasing concentration of curcumin.The OD value was at the peak when curcumin was 10μmol·L-1.Then the OD value was decreasing with the decreasing concentration of curcumin. We found the highest OD value was at 10μmol·L-1, without 160,80,40μmol-L-1 high error groups.(3) The expression of Wnt 2 and β-catenin enhanced obviously in the 24h curcumin group than that in the DMSO control group(P<0.05). The expression of 10μmol·L-1 group was higher than 5μmol·L-1. The expression of 10μmol·L-1 group was higher than 5μmol·L-1.At the same concentration, the expression of 24h group was higher than 12h group (P<0.05) However,the expression of Wnt 2 and β-catenin enhanced unobviously in the 12h curcumin group than that in the DMSO control group (P>0.05).The expression of GSK-3P led to the opposite results.Conclusion:(1) The third generation of cultured chondrocytes had the optimal stability.(2)The curcumin can lead to the positive proliferation of chondrocytes. And the chondrocytes with curcumin at 10μmol·L-1 worked best.10μmol·L-1 curcumin on the cultured chondrocytes for 24h proliferated superior to 12h on the same conditions.(3)The mechanism of curcumin leading to the positive proliferation of chondrocytes may be related to the activation of Wnt/β-catenin signaling pathway. Curcumin may result in GSK-3β deactivation by impeling the Wnt signaling molecules combining with specific protein receptors (Frizzled) on the cell membrane to activate intracellular disheveled (Dsh) protein. Then there is an accumulation of β-catenin in the cytoplasm. More and more β-catenin signaling molecules make the way into the nucleus to induce the transcription of gene. More proteins are producted and chondrocytes proliferate quickly. So it plays an important role in protecting and repairing the joint.