Xanthohumol Inhibits The Degradation Of Osteoarthritis Chondrocytes Extracellular Matrix By Regulates C/EBPβ Signal Pathway

Background:Osteoarthritis(OA)is a chronic degenerative disease,which seriously endangers the health of middle-aged and elderly people.Xanthohumol(XH)is a kind of isopentene flavonoid compound with anti-tumor and anti-inflammatory effects.Previous studies have shown that XH in vivo can alleviate the condition of osteoarthritis and is related to inhibiting the degradation of extracellular matrix.However,the regulatory mechanism of XH inhibiting the degradation of extracellular matrix of chondrocytes in OA is still unclear.Objective:To explore whether XH inhibits the degradation of extracellular matrix of osteoarthritic chondrocytes by regulating C/EBPβ signal pathway.Methods:1.To construct a rat knee osteoarthritis model.Six-week-old SD rats were selected and randomly grouped into normal group(NC group),model group(OA group),xanthohumol low-dose group(OA+50mg/kg XH)and xanthohumol high-dose group(OA+100mg/kg XH).The osteoarthritis was modeled by intra-articular injection of sodium iodoacetate into the knee joint of the model group and the administered group at a dose of 2 mg each,while the rats in the administered group were given xanthohumol by gavage at a dose of 50 mg/kg and 100 mg/kg daily since the beginning of modeling.After 1 month of feeding,rats were sacrificed and knee joint tissues were taken for HE staining and immunohistochemical staining.2.C28/I2 cells were cultured in vitro,and the cells were treated with different gradient concentrations of XH such as 0 μM,2.5 μM,5 μM,10 μM,20 μM,40 μM,80 μM and 160 μM according to the results of the pharmacokinetic study of XH,and finally the proliferative activity of C28/I2 cells was detected using the CCK-8method.3.Human C28/I2 chondrocytes were cultured in vitro,and the protein expression of MMP-13 and Collagen II in chondrocytes was detected by Western-blot.4.Human C28/I2 chondrocytes were cultured in vitro,and the protein expression of PERK,P-PERK,ATF4,C/EBPβ,and P-C/EBPβ in chondrocytes was detected by Western-blot.5.C28/I2 cells were cultured in vitro,ATF4 was knocked down and the expression of MMP-13,Collagen II,ATF4,C/EBPβ,and P-C/EBPβ proteins in chondrocytes was examined.6.C28/I2 cells were cultured in vitro,knockdown of C/EBPβ and detection of MMP-13,Collagen II,C/EBPβ,P-C/EBPβ protein expression in chondrocytes.Results:1.XH alleviates the progression of OA induced by sodium iodoacetate injection in rats.The pictures of the knee joint showed that the cartilage surface of the knee joint of the rats in the OA group was rough and inflammatory compared with the NC group,and obvious cartilage defects were seen.The rats in both XH gavage groups also had minor cartilage defects on the surface of the knee joint,but the degree of arthropathy was reduced in both groups.The HE staining results showed that the cartilage layer of the knee joint of the OA rats was thinned,the arrangement of chondrocytes was disturbed,and the cartilage surface was severely damaged compared with the NC group,while inflammatory cells and connective tissue infiltration were seen.However,XH administration significantly alleviated the tissue structure damage of knee cartilage.2.Determination of the optimal concentration of effective drug for XH in C28/I2 cells.The results showed that XH had no significant drug toxicity to C28/I2 cells in the concentration range of 0-20 μM,and once the concentration was greater than 20μM,XH exhibited inhibition of proliferation activity of C28/I2 cells.Therefore,we chose 5 μM and 20 μM as the dosing concentrations of XH.3.XH ameliorates IL-1β-induced ECM degradation in C28/I2 cellsThe results of IHC experiments showed that MMP-13 protein expression was increased and Collagen II protein expression was decreased in knee cartilage of OA rats compared with NC group,and XH administration reversed this situation,thereby upregulating Collagen II protein expression and decreasing MMP-13 protein expression and improving ECM degradation in chondrocyte.4.XH ameliorates IL-1β-induced ECM degradation in C28/I2 cells by inhibiting the PERK/ATF4/C/EBPβ signaling axisIt was found that the expression of PERK,ATF4 and C/EBPβ proteins was increased in chondrocytes of OA rats compared to normal rats.In contrast,XH administration inhibited the expression of proteins such as PERK,ATF4 and C/EBPβ.Consistent with the results of the IHC assay,in vitro WB experiments also confirmed that XH induced inhibition of expression of active forms of proteins such as PERK,ATF4 and C/EBPβ in IL-1β-stimulated C28/I2 cells.5.XH ameliorates IL-1β-induced ECM degradation in C28/I2 cells through downregulation of ATF4 protein levelsIt was found that the decrease in ATF4 protein expression was accompanied by a decrease in C/EBPβ and MMP-13 protein expression,and an increase in Collagen II protein expression.XH further reduced the protein expression of ATF4,C/EBPβ and MMP-13 and further increased the protein expression of Collagen II.This indicates that ATF4 plays an important role in ECM degradation in C28/I2 cells,and XH can alleviate ECM degradation in C28/I2 cells by inhibiting ATF4 expression.6.XH ameliorates IL-1β-induced ECM degradation in C28/I2 cells through downregulation of C/EBPβ protein levelsThe experimental results showed that the knockdown of C/EBPβ gene was accompanied by a decrease in MMP-13 protein expression and an increase in Collagen II protein expression.The co-treatment of XH further reduced the protein content of C/EBPβ and MMP-13,and further increased the protein expression of Collagen II.This suggests that XH can improve ECM degradation in C28/I2 cells by inhibiting C/EBPβ expression.Conclusion:In summary,XH inhibits C/EBPβ expression by suppressing the PERK/ATF4 signaling axis in chondrocytes,which in turn inhibits extracellular matrix degradation in OA chondrocytes and improves the progression of OA.