The Effect And Mechanism Of Isoliquiritigenin On Osteoarthritis Based On NF-?B And RANKL-RANK-TRAF6 Signaling Pathway

Objective:Osteoarthritis(OA)is a progressive and degenerative joint disease,and represents one of the largest socioeconomic healthcare burdens in the world due to prevention,diagnosis and treatment.Even so,there is still no ideal treatment for osteoarthritis.The total knee arthroplasty has become the only choose for patients with end-stage of osteoarthritis.The trauma and complications of this type of orthopedic surgery discouraged some elderly patients.Currently,no agent has been approved for OA management by the FDA or any other agencies worldwide.Available drugs provide only temporarily symptomatic relief but with numerous side-effects.Therefore,there is a dire need for effective and widely available approaches in OA management.(1)The first aim of this study was to evaluated the effects of Isoliquiritigenin on NF-?B signaling pathway in chondrocyte-like ATDC5 cells of osteoarthritis.(2)The second aim of this study was to evaluated the effects of Isoliquiritigenin on RANKL-RANK-TRAF6 signaling pathway and TGF-?1 in early subchondral bone of osteoarthritis.(3)The third aim of this study was to evaluate the effects of Isoliquiritigenin on angiogenesis in subchondral bone based on RANKL-RANK-TRAF6 signaling pathway.Methods:(1)The effects of Isoliquiritigenin on NF-?B signaling pathway in chondrocyte-like ATDC5 cells of osteoarthritis.1.Cell differentiation and treatment:The differentiation medium was changed every two days for 3 weeks to induce differentiation into chondrocyte-like cells.Differentiation was confirmed by the expression level of m RNAs for COL II and COL X.The production of glycosaminoglycan was visualized by 1% Alcian Blue staining.2.Cell viability assay and Quantitative analysis of apoptosis cells by CCK-8 assay and PE-Annexin V/7-Amino-Actinomycin(7-ADD)double-fluorescence labeling and flow ytometry: the cells pre-treated with ISL(2.5,5,10,20,40 ?mol/L)for 1h,then co-incubated with IL-1?(10 ng/m L)for a further 24,48,72 h,respectively and Cell Counting Kit-8 was used to monitor cell viability.1×106 cells were harvested and washed three times with pre-cold PBS,then re-suspended in binding buffer followed by apoptosis detection kit.The samples were analyzed using FACSAria TM II flow cytometer after PE-Annexin V and 7-ADD marking at indoor temperature for 15 min in the dark.3.Isoliquiritigenin suppresses IL-1? induced degradation reaction: The m RNA and protein expressions level of COL II,cyclooxygenase-2(COX-2)and matrix metalloproteinases-13(MMP-13)were evaluated by Real-time PCR and Western Blot.4.The effect of Isoliquiritigenin on NF-? B singling pathway: Western Blot was used to access chondrocyte apoptosis-related markers such as : Bcl-2,Bax,caspase-3,cleaved-caspase-3,caspase-9,cleaved-caspase-9,and the marker of NF-?B singling pathway,included NF-?B-p65,phospho-p65.(2)The effects of Isoliquiritigenin on RANKL-RANK-TRAF6 signaling pathway and TGF-?1 in early subchondral bone of osteoarthritis.1.Anterior cruciate ligament transaction(ACLT)model in mice: C57BL/6 male mice of 3 months old was purchased from Vital River.Preliminary experiment was performed firstly,the optimal dose(40 mg/kg)was identified by dividing the mice into Sham group,vehicle-treated and multiple concentrations of ISL-treated group(10,20 and 40 mg/kg;n=10 per group).Anterior cruciate ligament of the right knee were transected to generate a destabilized OA animal model.Sham operation was done by opening the joint capsule and then suturing the incision in the right knee of independent mice.Beginning the second day after ACLT surgery,ISL or equivalent volume of vehicle was injected intraperitoneally every other day for 60 days.2.ISL preserved articular cartilage in ACLT mice: By the time the mice were euthanized,the right knee joints of mice were dissected,fixed in 10% buffered formalin for 48 h,and decalcified in 10% EDTA(p H 7.4)for 3 weeks.Specimens were embedded in paraffin.Longitudinal-oriented sections of the medial compartment of the joint were cut with 4?m thick and processed for H&E and Safranin O and fast green staining.The thickness of the hyaline cartilage and calcified cartilage were measured with HE staining.Safranin O and fast green staining was used to evaluate the loss of proteoglycan.The Osteoarthritis Research Society International scoring system was used to quantitative analysis for destruction of articular cartilage.The immunofluorescent staining and immunohistochemical staining were performed to determine the markers in cartilage,included lubricin,Col X,MMP-l3.3.To evaluate the indexes of subchondral bone in the knee of mice after modeling: the specimens were scanned using high-resolution micro-CT(Sky Scan 1176)with the voltage of 50 k Vp,filter of 0.5 mm AI and resolution of 9 ?m per pixel.The data was then reconstructed(NRecon v1.6),analyzed(CTAn,v1.9)and build for 3D model visualization(CTVol,v2.0).The following parameters were measured:(1)Bone volume/tissue volume(BV/TV,%),(2)trabecular pattern factor(Tb.pf,mm-1)as a parameter of bone resorption,(3)subchondral bone plate thickness(SBP Th,mm)as a parameter of bone formation.Immunohistochemical staining were performed to determine TRAP+ osteoclast,Osterix,p Smad2/3.Immunofluorescent staining were performed to determine RANKL,TRAF6,Nestin+ MSCs.In addition,the expression of osteoclast-related factors in mouse systemic circulation was detected by Elisa kit.(3)Effect of isoliquiritigenin on the angiogenesis of subchondral bone based on RANKL-RANK-TRAF6 signaling pathway.1.Micro CT-based microangiography was used to evaluate the effect of isoliquiritigenin on the angiogenesis of subchondral bone,which included the volume and quantity of blood vessels.2.Effects of isoliquiritigenin on angiogenesis-related factors of subchondral bone in mice after modeling: immunofluorescent staining were performed to determine H type vessel which represented CD31+Endomucin+.Additionally,changes in matrix metalloproteinase 2(MMP-2)were detected in each group.Results:(1)The effects of Isoliquiritigenin on NF-?B signaling pathway in chondrocyte-like ATDC5 cells of osteoarthritis: 1.The cells were stimulated with ITS for 21 days and 1% Alcian blue staining were performed at 0,7,14,21 d.The results showed that staining intensities gradually increased in a time-dependent manner from 0 to 21 days in ATDC5 cells cultured with ITS.COL II m RNA increased significantly after 7 day of chondrogenic induction and further increased with a maximum elevation at 14 days,which indicates early-stage differentiation of chondrocytes.m RNA expression of collagen X gradually increased and exceeded COL II at 21 days,indicating late-stage differentiation of chondrocytes.Therefore,ATDC5 cells with differentiation of 14 d were selected as following experimental cells.2.The results showed a significant decrease of IL-1?-induced chondrocytes viability compared with the normal control(p<0.05),which was reversed by ISL at low concentrations(2.5,5,10 ?mol/L)in a concentration-dependent manner(p<0.05).However,higher doses of ISL(20,40 ?mol/L),did not reverse the viability of chondrocyte-like ATDC5 cells.Correspondingly,the cells were treated with only ISL at different concentrations indicated that the viability of the cells was not remarkable change within the scope of 2.5-10 ?mol/L(p>0.05),while significant decrease at high concentrations(20,40 ?mol/L)(p<0.05).3.The results showed that in the IL-1?-induced ATDC5 cells treated by ISL,the m RNA levels of COX-2 and MMP-13 were evidently decreased dose-dependently(p<0.05),whereas the level of COL was increased significantly(p<0.05).The results of Western blot consistently demonstrated the anti-catabolic effect of ISL.4.The results demonstrated that the expression of anti-apoptotic protein Bcl-2 dose-dependent increased with the increasing concentration of ISL while the expression level of pro-apoptotic protein Bax decreased in a concentration manner when ADTC5 cells were stimulated with IL-1?(p<0.05).To provide further evidence for anti-apoptotic effects of ISL on IL-1?-stimulated chondrocyte-like ATDC5 cells,the downstream protein levels of caspase-3 cleavage and caspase-9 cleavage which in mitochondria apoptosis pathway were assessed.Western blot data revealed that ISL had few influence on expression of caspapse-3 and caspapse-9(p>0.05).However,the expression of cleaved-caspapse-3 and cleaved-caspapse-9 was prominent in chondrocyte-like ATDC5 cells stimulated with IL-1?(p<0.05)and was blocked in the cells treated with ISL(p<0.05).(2)The effects of Isoliquiritigenin on RANKL-RANK-TRAF6 signaling pathway and TGF-?1 in early subchondral bone of osteoarthritis: 1.From the Safranin O and fast green staining,significant loss of proteoglycan in vehicle-treated ACLT mice compared with the sham control(p<0.05),which was retention by administrating ISL(40 mg/kg)at 30 day and 60 day after operation(p<0.05).It is also supported by OARSI scores which were improved in ISL-treated group relative to vehicle group(p<0.05),whereas no difference was noted in ISL versus sham controls(p>0.05).Relative to vehicle-treated ACLT mice at 60 day postoperation,decreased thickness of calcified cartilage zone in ISL-treated group was observed from HE staining(p<0.05).Abnormal expression of lubricin,MMP-13 and collagen X(Col X)were found in vehicle group compared with the sham control(p<0.05),which was normalised by administrating ISL as assessed by immunostaining(p<0.05).2.High-resolution Micro-CT was used to access whether the protective role of ISL on the articular cartilage is associated with its potential effect on the microarchitecture of tibial subchondral bone.The results showed that in the vehicle group,the value of bone volume/tissue volume(BV/TV,%)reduced post ACLT(p<0.05),which was abrogated by receiving ISL(p<0.05).Additionally,ISL significantly reduced trabecular pattern factor(Tb.pf)(a parameter of bone resorption)(p<0.05)and increased SBP thickness(a parameter of bone formation)(p<0.05)post ACLT compared with vehicle treatment and there was no statistically significant difference in these parameters compared with sham controls(p>0.05).Correspondingly,ISL significantly reduced the number of TRAP-positive osteoclast cells and osteoprogenitor osterix-positive cells postoperation relative to vehicle treatment(p<0.05).It is also noted that the majority of osterix-positive cells were found in subchondral bone marrow in the vehicle group and relocated to the bone surface in the ISL-treated mice.The results of these data demonstrated that ISL could normalize aberrant subchondral bone remodeling in ACLT mice.Immunofluorescence double staining showed a significant increase of RANKL and TRAF6 in the subchondral bone marrow of vehicle-treated mice as early as 2 weeks postoperation(p<0.05),whereas ISL-treated mice had equivalent expression compared with sham controls(p>0.05).However,no difference in the expression level of CTX I in peripheral blood was observed regardless of vehicle or ISL-treated mice 14 days after ACLT operation compare to sham group by using EIA kit(p>0.05).The results indicated ISL inhibits osteoclastogenesis by suppression RANKL-RANK-TRAF6 singling pathway in the subchondral bone.3.Immunohistochemistry staining indicated that p Smad2/3-positive cells in the subchondral bone of vehicle-treated group were significantly increased(p<0.05)and reversed to similar levels comparable with sham control by administering ISL(p>0.05).With the same developments,TRAP positive osteoclast cells and p Smad2/3-positive cells both increased in subchondral bone as early as 7 d after ACLT.Then the continued osteoclastic bone resorption results in p Smad2/3-positive cells remained at high concentrations until 30 d.These results suggest that ISL could inhibit TGF-? activity in bone marrow MSCs by suppression osteoclastogenesis.(3)Effect of isoliquiritigenin on the angiogenesis of subchondral bone based on RANKL-RANK-TRAF6 signaling pathway: 1.After ACLT operation 30 days,the significantly increased the number of microvascular were observed in the subchondral bone of ACLT mice in vehicle group as well as the volume of microvascular(p<0.05),whereas the aberrant blood vessel formation was prevented by ISL(p<0.05),which retained vessel number and volume similar to sham controls(p>0.05).2.Immunofluorescence double staining for CD31 and endomucin was performed to investigate the potential mechanism of how ISL affect the subchondral bone angiogenesis.Consistently,a significant increase of CD31 and endomucin in the subchondral bone marrow of vehicle-treated mice was found(p<0.05),whereas ISL-treated mice had equivalent expression compared with sham controls(p>0.05).3.The expression level of MMP-2 were also statistically increased in vehicle group(p<0.05).ISL reduced the expression in subchondral bone to similar level compared with sham controls(p>0.05).The data above indicated that ISL could prevent aberrant blood vessel formation in subchondral bone.Conclusion:(1)Isoliquiritigenin suppresses IL-1? induced apoptosis and inflammation in chondrocyte-like ATDC5 cells by inhibiting NF-?B.(2)Increased subchondral bone resorption release more active TGF-? which stimulate increases in the number of MSCs and osteoprogenitors in the bone marrow,which lead to aberrant bone formation and angiogenesis for osteoarthritis progression.Isoliquiritigenin(ISL)inhibits RANKL-RANK-TRAF6 singling pathway in preosteoclast to reduce release of TGF-? in the subchondral bone.(3)ISL prevents aberrant blood vessel formation in subchondral bone by direct suppression of MMP-2,indirect inhibition of TGF-? signaling.