| Objective:To study the effect of EGCG on the senescence of chondrocytes,and to explore the role of autophagy in the inhibition of chondrocyte senescence by EGCG Methods:(1)Take 4-week-old SD rat hind limb articular cartilage,and use type ?collagenase stepwise digestion method to isolate and culture SD rat chondrocytes.(2)Toluidine blue and type II collagen immunocytochemical staining were performed for cell staining identification of P2 generation cells.(3)After 24 hours of treatment of chondrocytes in the IL-1? group with different concentrations of EGCG,the CCK-8 method was performed to detect the survival of the chondrocytes after the pretreatment,and the cell survival rate was calculated to determine the final concentration of EGCG in the experiment.(4)The experiment is divided into 4 groups.The first group is the blank control group(Control group);the second group: 10ng/m L IL-1? induced aging group(IL-1?group);the third group: EGCG+10ng/m L IL-1? co-culture group(EGCG group);The fourth group: EGCG+10ng/m L IL-1?+5m Mol/L 3-MA group(EGCG+3-MA group).(5)The chondrocytes in the Control group,IL-1? group and EGCG group were cultured for 72 hours.The ?-galactosidase staining method was used to detect the senescence of chondrocytes.(6)The chondrocytes in the Control group,IL-1? group and EGCG group were cultured for 72 hours.Real-time PCR was performed to detect the m RNA expression levels of type II collagen(Col2),MMP3 and MMP13 in each group of cells.(7)After the above 4 groups of chondrocytes were cultured for 72 hours.Western blot was used to detect the protein expression of Beclin-1,Col2,MMP3 and MMP13 in each group of cells.Results:(1)The primary chondrocytes taken from 4-week-old SD rats have a large number of cells adhered to the wall within 48 hours,and they are triangular or polygonal under microscopy,with clear nuclei and rich cytoplasm.They can adhere to the wall in about 3 days.Up to more than 90%.(2)The P2 generation chondrocytes were stained separately.Toluidine blue stained cytoplasm was blue,and nucleus was dark blue;type II collagen immunocytochemical staining cytoplasm was brown and the nucleus was dark brown.All three identification methods are positive.(3)After 24 hours of treatment of chondrocytes with different solubility of EGCG(6.25?12.5?25?50?100?200?mol/L),CCK-8 results showed that compared with the Control group,the cell viability of IL-1? group was significantly reduced.Compared with IL-1? group,EGCG group has significantly increased cell viability,and it is determined that the final solubility of EGCG in this experiment is 100?mol/L.(4)The results of ?-galactosidase staining showed that a large number of chondrocytes were seen in Control group,but the cells were not stained.(5)The IL-1? group had a large number of cells,basically all of them had a staining reaction,which was dark blue,the cells shrank and rounded,and lost the basic morphology of chondrocytes.Only a few cells were unstained.The number of cells in the EGCG group was basically normal,most of the cells maintained the normal morphology of chondrocytes,and only a few cells had staining reactions.(6)Real Time PCR detection showed that the m RNA expression of Col2 in the Control group and EGCG group was significantly higher than that in the IL-1? group.The expression of MMP3 and MMP13 m RNA in IL-1? group was significantly higher than that in Control group and EGCG group.(7)Western blot results showed that the protein expression of Col2 and Beclin-1 in the Control group and EGCG group was significantly higher than that in the IL-1? group and EGCG+3-MA group.The protein expression of MMP3 and MMP13 in IL-1? group and EGCG+3-MA group increased significantly.Conclusion:EGCG can effectively inhibit the senescence of chondrocytes,and autophagy plays an important role in this process. |
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