β-蜕皮甾酮干预白细胞介素-1β诱导的骨关节炎软骨细胞的效果及其作用机制研究OA

Objective:To investigate the effect of β-ecdysterone on interleukin(IL)-1β-induced osteoarthritis(OA)chondrocytes and its mechanism.Methods:①Screening of intervention concentration of β-ecdysterone.Chondrocytes of rats in logarithmic growth phase were divided into control group and β-ecdysterone groups with concentrations of 20,40,60,80 and 100 μmol·L-1,respectively.Except for the control group,the chondrocytes in the other groups were treated with 20,40,60,80 and 100 μmol·L-1 β-ecdysterone for 24 hours,and the viability of chondrocytes in each group was detected by CCK-8 method.②Analysis of effect and mechanism of β-ecdysterone on IL-1β-in-duced OA chondrocytes.The rat chondrocytes in logarithmic growth phase were divided into blank control group,model group,20 μmol·L-1β-ecdysterone group and 40 μmol·L-1β-ecdysterone group.Except for the blank control group,IL-1β with a final concentration of 10 ng·mL-1 was added to the chondrocyte culture medium of the other groups.At the same time,β-ecdysterone with a final concentration of 20 and 40 μmol·L-1 was added to the chondrocyte culture medium of the 20 μmol·L-1β-ecdysterone group and the 40 μmol·L-1β-ecdysterone group,respectively,and incubated for 24 hours.The viability of chondrocytes in each group was detected using CCK-8 meth-od.The apoptosis rate of chondrocytes in each group was evaluated using TUNEL assay.The mRNA expression levels of matrix metallopro-teinase(MMP)-13 and collagen Ⅱ α1(COL2A1)in chondrocytes of each group were detected by real-time quantitative PCR.The protein ex-pression levels of hypoxia-inducible factor-1α(HIF-1α)and vascular endothelial growth factor(VEGF)in chondrocytes were detected by Western Blot.③Verification of HIF-1α mRNA overexpression system in chondrocytes.Chondrocytes in logarithmic growth phase were divid-ed into blank control group,HIF-1α overexpression group and HIF-1α overexpression negative control group.HIF-1α overexpression group and HIF-1α overexpression negative control group were transfected into rat chondrocytes with liposome 2000 containing HIF-1α overexpres-sion plasmid and liposome 2000 containing empty plasmid,respectively.The mRNA expression level of HIF-1α in chondrocytes of each group was detected by real-time quantitative PCR.④Verification of the mechanism of β-ecdysterone in interventing IL-1 β-induced OA chondrocytes.Rat chondrocytes in logarithmic growth phase were divided into blank control group,model group,40 μmol·L-1 β-ecdyste-rone group,HIF-1α overexpression group and HIF-1α overexpression negative control group.Chondrocytes of HIF-1α overexpression group and HIF-1α overexpression negative control group were transfected with liposome 2000 containing HIF-1α overexpression plasmid and empty plasmid,respectively.Except for the blank control group,IL-1 β with a final concentration of 10 ng·mL-1 was added to the chondrocyte cul-ture medium of the other groups.At the same time,β-ecdysterone with a final concentration of 40 μmol·L-1 was added to the chondrocyte culture medium of the 40 μmol·L-1β-ecdysterone group,HIF-1α overexpression group and HIF-1α overexpression negative control group,and incubated for 24 hours.Chondrocyte viability,apoptosis rate,mRNA expression levels of MMP-13 and COL2A1,and protein ex-pression levels of VEGF in each group were assessed using the methods described in Part 2.Results:①Screening results of intervention concentration of β-ecdysterone.There was no significant difference in chondrocyte viability between 20 μmol·L-1 β-ecdysterone group and control group(P=0.993),and between 40 μmuol·L-1β-ecdysterone group and control group(P=0.753).The chondrocyte viability of 60,80 and 100 μmol·L-1 β-ecdysterone groups was lower than that of control group(P=0.000,P=0.000,P=0.000).The concentra-tions of 20 and 40 μmol·L-1 were determined as the intervention concentration of β-ecdysterone.②The analysis result of effect and mech-anism of β-ecdysterone on IL-1 β-induced OA chondrocytes.The viability of chondrocytes in the model group was lower than that in the blank control group(P=0.000),and the apoptosis rate was higher than that in the blank control group(P=0.000).The viability of chon-drocytes in the 20 and 40 μmol·L-1 β-ecdysterone groups was higher than that in the model group(P=0.012,P=0.000),and the apop-tosis rate was lower than that in the model group(P=0.000,P=0.000).The relative expression level of MMP-13 mRNA in chondrocytes of the model group was higher than that of the blank control group(P=0.000),and the relative expression level of COL2A1 mRNA was lower than that of the blank control group(P=0.000).The relative expression level of MMP-13 mRNA in chondrocytes of the 20 and 40 μmol·L-1β-ecdysterone groups was lower than that of the model group(P=0.021,P=0.000),and the relative expression level of COL2A1 mRNA was higher than that in the model group(P=0.000,P=0.000).There was no significant difference in the relative expres-sion level of MMP-13 mRNA between the 40 μmol·L-1 β-ecdysterone group and the 20 μmol·L-1 β-ecdysterone group(P=0.257).The relative mRNA expression level of COL2A1 was higher than that of the 20 μmol·L-1 β-ecdysterone group(P=0.016).The relative expression levels of HIF-1α and VEGF in chondrocytes of the model group were higher than that of the blank control group(P=0.000,P=0.000).There was no significant difference in the relative expression level of HIF-1α protein in chondrocytes between the 20 μmol·L-1β-ecdysterone group and the model group(P=0.067),while the relative expression level of VEGF protein was lower than that in the model group(P=0.000).The relative expression levels of HIF-1α protein and VEGF protein in chondrocytes of 40 μmol·L-1β-ecdysterone group were lower than those of model group and 20 μmol·L-β-ecdysterone group(P=0.000,P=0.000,P=0.002,P=0.000).③The verification results of HIF-1α mRNA overexpression system in chondrocytes.The relative expression level of HIF-1α mRNA in chondrocytes of HIF-1α overexpression group was higher than that in blank control group and HIF-1α overexpression negative control group(P=0.000,P=0.000).There was no significant difference in the relative expression level of HIF-1α mRNA between HIF-1α overexpression negative control group and blank control group(P=0.773).④The verification results of the mechanism of β-ecdysterone on IL-1 β-induced OA chondrocytes.The viability of chondrocytes in the model group was lower than that in the blank control group(P=0.000),and the apoptosis rate was higher than that in the blank control group(P=0.000).The viability of chondrocytes in 40 μmol·L-1β-ecdysterone group and HIF-1 α overexpression negative control group was higher than that in model group(P=0.000,P=0.000),and the apoptosis rate was lower than that in model group(P=0.007,P=0.010).There was no significant difference in chondrocyte viability and apoptosis rate between 40 μmol·L-1β-ecdysterone group and HIF-1α overexpression negative control group(P=0.999,P=0.999).There was no significant difference in chondrocyte viability and apoptosis rate between HIF-1α overexpression group and model group(P=0.999,P=0.458).The relative expression level of MMP-13 mRNA in chondrocytes of the model group was higher than that of the blank control group(P=0.000),and the relative expression level of COL2A1 mRNA was lower than that of the blank control group(P=0.000).The relative ex-pression level of MMP-13 mRNA in chondrocytes of 40 μmrol·L-1 β-ecdysterone group and HIF-1α overexpression negative control group was lower than that of model group(P=0.000,P=0.000),and the relative expression level of COL2A1 mRNA was higher than that of model group(P=0.000,P=0.000).There were no significant difference in the relative mRNA expression levels of MMP-13 and COL2A1 in chondrocytes between the 40 μmol·L-1β-ecdysterone group and the HIF-1α overexpression negative control group(P=0.993,P=0.999).There was no significant difference in the relative expression level of MMP-13 mRNA in chondrocytes between the HIF-1α overex-pression group and the model group(P=0.121),and the relative expression level of COL2A1 mRNA was higher than that in the model group(P=0.043).The relative expression level of VEGF protein in chondrocytes in the model group was lower than that in the blank con-trol group(P=0.000).The relative expression levels of VEGF protein in chondrocytes in the 40 μmol·L-1β-ecdysterone group and the HIF-1α overexpression negative control group were lower than that in the model group(P=0.000,P=0.000).There was no significant difference in the relative expression level of VEGF protein in chondrocytes between the 40 μmol·L-1 β-ecdysterone group and the HIF-1αoverexpression negative control group(P=0.996).The relative expression level of VEGF protein in chondrocytes of HIF-1α overexpression group was higher than that in model group(P=0.000).Conclusion:β-ecdysterone can enhance the viability of IL-1β-induced OA chondro-cytes and inhibit their apoptosis,and its mechanism is closely related to the inhibition of the activation of HIF-1α-VEGF signaling pathway.