Effect Of Puerarin On Type Ⅱ Collagen Expression Cultured With IL-1β In Chondrocytes

Objective:Using cell and molecular biology techniques, this study explored the effect of Puerarin (Pue) on type II collagen in cartilage cells in rats induced by IL-1β and discussed the balance of cartilage cells between proliferation and apoptosis, the mechanism of protecting the cartilage cells and preventing the osteoarticular diseases,to provid the experimental foundation and theoretical basis for using Pueraria in clinical to treat osteoarticular diseases.Methods:1.To establish and identify the vitro cultured system of chondrocytes. The Primary chondrocytes were obtained from four-week old SD articular cartilage with collagenase and trypsin digestion, then identified by toluidine blue staining and observed the biological characteristics of chondrocytes.2.MTT assay was performed to detect the effect of different concentration of IL-1β on cartilage cells in rats, and filter the optimal induced concentration.3.MTT assay was performed to detect the effect of different concentration of Pue on proliferation of cartilage cells in rats, and filter the optimal induced concentration.4.RT-PCR was performed to observe the effect of Pue on genetic expression of COL-II in cartilage cells induced by IL-1β.5. Western Blot was performed to detect Pue’s effect on protein expression of COL-II in cartilage cells induced by IL-1β.Results:1. After establishing the vitro culture system by chondrocytes collagenase and trypsin digestion, the biological characteristics with invert micros cope and identified through toluidine blue staining,which confirmed cartilage cells by isolated culture,and they were steady in3th generation.2.With respect to inhibition effect with the change of different concentration of IL-1β on cartilage cells proliferation, the result induced by10ng/mlIL-1β at the time of48h was strongest.The effect on cartilage cell proliferation showed that all of50μmol/L,100μmol/L, and200μmol/L Pue all can promote proliferation in cartilage cells by normally cultured, and the proliferation effects increased with dose concentration of progressive increase. At the time of48h, for the cartilage cells by normally cultured,200μmol/L Pue is optimal tor promoted cartilage cells proliferation with a remarkable statistical difference compared with control group(P<0.05).3.Results of RT-PCR is as below:the expected length of specific strip in II collagen mRNA RT-PCR products were visible in all four groups’ specimens. The expression of treatment group intervened by IL-1β (3was lower significantly in comparison with normal group(P<0.05). The expression of Pue group sigbificantly increased compared with normal group and10ng/ml IL-1β group with a remarkable statistical difference(P<O.05). Compared with lOng/ml IL-1(3, lOng/mL IL-1β+200μmol/L Pue group’s expression Significantly increased(P<0.05), but there was no remarkable statistical difference compared with the expression of normal group’s mRNA.4.Results of Western Blot showed as follows:cartilage cells can express COL-2protein generally, but after intervened by10ng/mL IL-1β, expression of COL-II protein was significantly weakened(P<0.05). On the contrary, the protein expression in200μmol/L Pue group was significantly increased. After intervened by200μmol/L Pue, COL-II protein expression of cartilage cells in rats, which were suppressed by10ng/mL IL-1β,was significantly increased compared with group without intervention of Pue (P<0.05), resulting in no remarkable difference with protein expression in normal group (P>0.05).Conclusion:1.IL-1β can suppress proliferation of cartilage cells in rats, and its dose is positively correlated to the effect of suppression.2.Pue can promote proliferation of cartilage cells in rats with normally cultured, and dose is positively correlated to the promotion effect.3. Pue can effectively prevent inhibiting effect of IL-1(3to cartilage cells in rats on gene and protein expression in type II collagen.It may attribute for Pue’s effective effects of protection on cartilage cells, defense from inflammatory factors, prevention and treatment of osteoarticular diseases.