Based On SDF-1/CXCR4 Axis,the Effect Of Astragaloside Ⅳ On The Expression Of ADAMTS4 And ADAMTS5 In Chondrocytes Was Investigated

ObjectiveTo analyse whether Astragaloside Ⅳ(AS-Ⅳ)was a CXCR4 antagonist based on molecular docking.The effects of AS-Ⅳ and AMD3100,a known CXCR4 antagonist,on the expression of ADAMTS4 and ADAMTS5 in vitro with chondrocytes that is stimulated by CXCL12.Methods1.Molecular Docking MethodThe molecular structure of ligand AS-Ⅳ was downloaded from Pub Chem database and saved as.SDF format.The Chem3 D software optimizes the binding energy of the ASⅣ molecular structure and saved as.mol2 format.The crystal structure of CXCR4 was downloaded from PDB protein databank,the PDB protein named 3ODU was selected as the receptor structure.The removal of solvent and organic was performed with Pymol2.5software and saved as.PDB format.Auto Dock Vina v.1.0.2 software was used to molecular simulation docking and the results were visualized analysis by Pymol2.5software.2.In vitro study2.1 RT-qPCR testHuman immortalized chondrocytes C28/I2(SCC043)were purchased and for ADATMS4 and ADATMS5 mRNA expression analysis,C28/I2 cells were seeded in 12-well plates and treated with CXCL12,AS-Ⅳ and CXCR4 antagonist AMD3100 at desired concentrations at 37℃ for 24 h.C28/I2 cells were grown in DMEM and treated with CXCL12,AS-Ⅳ and AMD3100 at desired concentrations at 37℃ for 24 h.(among which CXCL12 50ng/m L,AS-Ⅳ 10μM,AMD3100 10μM).The experiment was divided into four groups:(1)control group: no CXCL12 and experimental drugs;(2)model group: only CXCL12;(3)AMD3100 experimental Group: AMD3100 + CXCL12;(4)AS-Ⅳ experimental Group:AS-Ⅳ + CXCL12.2.2 Western blot testFor ADATMS4、ADATMS5 protein expression analysis,C28/I2 cells were seeded in12-well plates and treated with CXCL12 and AS-Ⅳ at desired concentrations at 37 ℃ for24 h.The cell lysate was collected,and the protein was purified for western blot test.The experiment was divided into two groups which included the group A of AS-Ⅳ and the group B of AMD3100.The group A of AS-Ⅳ were divided into seven subgroups which included control group a(no CXCL12 and AS-Ⅳ),model group A0(50ng/m L CXCL12),experimental group A1(50ng/m L CXCL12+0.3μM AS-Ⅳ),experimental group A2(50ng/m L CXCL12+1.0μM AS-Ⅳ),experimental group A3(50ng/m L CXCL12+3.0μM AS-Ⅳ),experimental group A4(50ng/m L CXCL12+10.0μM AS-Ⅳ)and experimental group A5(50ng/m L CXCL12+30.0μM AS-Ⅳ).The group B of AMD3100 were divided into seven subgroups which included control b(no CXCL12 and AMD3100),model group B0(50ng/m L CXCL12),experimental group B1(50ng/m L CXCL12+0.3μM AMD3100),experimental group B2(50ng/m L CXCL12+1.0μM AMD3100),experimental group B3(50ng/m L CXCL12+3.0μM AMD3100),experimental group B4(50ng/m L CXCL12+10.0μM AMD3100)and experimental group B5(50ng/m L CXCL12+30.0μM AMD3100).Results1.The results of molecular docking show that AS-Ⅳ and CXCR4 exhibited the top docking score 10.6 kcal/mol.In our docking simulation,AS-Ⅳ interacts D97,D187 and Q200 through hydrogen bonds to achieve the inhibition effect.2.The results of RT-qPCR show that ADAMTS4 and ADAMTS5 m RNA were expressed in normal chondrocytes of control group.Compared with the control group,the m RNA levels of ADAMTS4 and ADAMTS5 in the model group after CXCL12 stimulation were significantly increased,with statistically significant differences(P<0.05).After treatment with AMD3100 and AS-Ⅳ,the expression of ADAMTS4 and ADAMTS5 in chondrocytes was significantly lower than that in the model group(P<0.05).3.The results of Western blot,a method of protein content determination show that:(1)Experiment group A: In the control group,only a small amount of ADAMTS4 and ADAMTS5 were expressed in the supernatant of normal chondrocytes.Compared with the control group,the expression levels of ADAMTS4 and ADAMTS5 after CXCL12 stimulation were significantly higher than those in the control group(P<0.05).Compared to the model group,the ADAMTS4 protein of the experimental group A1 which including 0.3μM AS-Ⅳ was decreased and the difference was not statistically significant(P>0.05),while the ADAMTS5 protein was significantly decreased,and the difference was statistically significant(P<0.05).the ADAMTS4 and ADAMTS5 protein of the experimental group A2 which including 1.0μM AS-Ⅳ were significantly decreased(P<0.05).the ADAMTS4 and ADAMTS5 protein of the experimental group A3 which including 3.0μM AS-Ⅳ were significantly decreased,and the difference was statistically significant(P<0.05).the ADAMTS4 and ADAMTS5 protein of the experimental group A4 which including 10.0μM AS-Ⅳ were significantly decreased,and the difference was statistically significant(P<0.05).the ADAMTS4 and ADAMTS5 protein of the experimental group A5 which including 30.0μM AS-Ⅳ were significantly decreased(P<0.05).Compared with experimental group A3,the ADAMTS4 protein of the experimental group A4 which including 10.0μM AS-Ⅳ was significantly decreased,and the difference was not statistically significant(P>0.05),while the ADAMTS5 protein was decreased and the difference was statistically significant(P<0.01).The ADAMTS4 and ADAMTS5 proteins in the other experimental groups with different concentrations of AMD3100 gradually decreased,and there was no statistically significant difference between the groups(P>0.05).(2)Experimental Group B: In the control group,only a small amount of ADAMTS4 and ADAMTS5 were expressed in the supernatant of normal chondrocytes.Compared to the control group,the ADAMTS4 protein of the model group was increased and the difference was statistically significant(P<0.05),while the ADAMTS5 protein was increased,and the difference was not statistically significant(P>0.05).Compared to the model group,the ADAMTS4 protein of the experimental group B1 which including 0.3μM AMD3100 was significantly decreased,and the difference was statistically significant(P<0.01),while the ADAMTS5 protein was decreased and the difference was not statistically significant(P>0.05).the ADAMTS4 and ADAMTS5 protein of the experimental group B2 which including 1.0μM AMD3100 were significantly decreased(P<0.05).the ADAMTS4 and ADAMTS5 protein of the experimental group B3 which including 3.0μM AMD3100 were significantly decreased(P<0.05).the ADAMTS4 and ADAMTS5 protein of the experimental group B4 which including 10.0μM AMD3100 were significantly decreased(P<0.05).the ADAMTS4 and ADAMTS5 protein of the experimental group B5 which including 30.0μM AMD3100 were significantly decreased(P<0.05).Compared with experimental group B1,the ADAMTS4 protein of the experimental group B2 which including 1.0μM AMD3100 was significantly decreased,and the difference was statistically significant(P<0.01),while the ADAMTS5 protein was decreased and the difference was not statistically significant(P>0.05).The ADAMTS4 and ADAMTS5 proteins in the other experimental groups with different concentrations of AMD3100 gradually decreased,and there was no statistically significant difference between the groups(P>0.05)Conclusion1.AS-Ⅳ may affect CXCL12/CXCR4 signaling pathway as a CXCR4 antagonist by molecular docking.2.In vitro cultured chondrocytes,CXCL12 up-regulated the expression of ADAMTS4 and ADAMTS5 enzymes,which may be inhibited by AS-Ⅳ in a dose-dependent manner.