Study On The Protective Effect Of Brazilin On Chondrocyte In KOA Cell Model

Objective: To explore the protective effect of Brazilin on chondrocyte proliferation in IL-1β-induced rat knee osteoarthritis cell model and the changes of endoplasmic reticulum stress pathway,so as to provide theoretical basis for the application of Brazilin in clinical treatment of knee osteoarthritis.Methods: 1.The primary chondrocytes of rats were isolated and stained with toluidine blue,safranine and alcian blue,and the chondrocytes were identified.The effect of Brazilin on the proliferation activity of normal chondrocytes was detected by CCK8;2.The experiment was divided into normal chondrocyte group(Control group),The other groups were treated with IL-1β(10ng/ml)for 48 h to establish in vitro KOA cell model,which were divided into model group(IL-1β group),Brazilin low-dose group(2.5μg /ml),Brazilin medium-dose group(5μg /ml)and Brazilin high-dose group(10μg)/ml)to conduct the following experiments: The morphological changes of cells in each group were observed by microscopy,and the expressions of TNF-α,Collagen Type II and MMP-13 were detected by ELISA.The activity of caspase-3 was detected by the caspase-3 activity kit,and the gene and protein expressions of GRP78,IRE1,XBP1,CHOP,Caspase-3,Collagen Type II and MMP-13 were detected by QRT-PCR and WB respectively.Results: 1.Chondrocytes were identified by toluidine blue,safranine and alcian blue staining.The purpose of this experiment was to isolate and obtain primary chondrocytes with high purity from Suckling rats.2.CCK8 was used to detect the proliferation and toxicity of Brazilin on chondrocytes.The results showed that the inhibition of chondrocyte proliferation began at the concentration of 10ug/ m L,and the inhibition rate was statistically different when the concentration was higher than 14ug/ m L(P<0.05).3.In the IL-1β-induced KOA cell model,a large number of cells in the IL-1β group showed shrinkage,chromatin shrinkage,cytoplasm and nuclear fragmentation,indicating the presence of apoptosis,while Brazilin could reduce the occurrence of IL-1β-induced apoptosis and was positively correlated with dose.4.Caspase-3 activity was increased in the IL-1β group using the Caspase-3 activity kit.Enzyme-linked immunosorbent assay(ELISA)was used to detect increased expression of TNF-α and MMP-13 in IL-1β group,but the expression of Collagen Type II was decreased,indicating that chondrocytes were damaged.However,the expression and activation of related factors were effectively reversed after treatment with Brazilin..5.In the IL-1β group,the expression levels of ERS pathway markers molecules GRP78 and CHOP increased,and the IRE1-XBP1 pathway was activated,suggesting that the mechanism of IL-1β-induced cartilage injury may be related to the ERS pathway.However,after treatment with Brazilin,the activation of downstream factors of ERS pathway was inhibited.Thus promoting upstream molecules to play a role in chondrocyte repair.Conclusion: The protective effect of Brazilin on cartilage in IL-1β-induced knee osteoarthritis cell model,and this protective effect may be achieved by activating IRE1-XBP1 pathway in ERS.