Molecular Mechanism Research Of Icariin On Supression Of Matrix Metalloproteinases In Osteoarthritis In Vivo And In Vitro

Objective: To study the effects of Icariin at different concentrations oncell viabilities of human SW1353chondrosarcoma cells and theexpressions of MMP-1,3,13in IL-1β-induced SW1353cells in order toscreen the optimal concentration. To observe the effects of Icariin at theoptimal concentration on the mRNA and protein expressions of MMP-1,3,13in IL-1β-induced SW1353cells. To investigate the effects of Icariin onMAPKs signaling pathways, containing p38pathway, ERK pathway andJNK pathway, and Wnt/β-catenin pathway in IL-1β-induced SW1353cells.The in vivo effects of Icariin on an experimental rat model of OA inducedby anterior cruciate ligament transection (ACLT) was examined. To studythe effects of Icariin on articular cartilage histomorphology, MMPs,MAPKs signaling pathwys and Wnt/β-catenin pathway in experimentalosteoarthritic cartilages.Methods:1. Experiments in vitro1.1SW1353cells were treated with seven concentrations of Icariin (i.e. 0,5,10,20,40,80,100μM) for24h, and then the cell viabilities weredetermined by an MTT assay. Futhermore, the expressions of MMP-1,3,13in IL-1β-induced SW1353with pretreatments of Icariin at differentconcentrations (i.e.0,5,10,20μM) were verified by Western Blot.1.2SW1353cells were pretreated with20μM Icariin for1h, thenstimulated with10ng/mL IL-1β for24h. The cells and the supernates wereharvested. The protein expressions of MMP-1,3,13were determined byELISA. The mRNA expressions of MMP-1,3,13were verified byquantitative real-time polymerase chain reaction (q-PCR).1.3SW1353cells were pretreated with20μM Icariin for1h, thenstimulated with10ng/mL IL-1β for24h. Phosphorylated p38,phosphorylated ERK1/2, phosphorylated JNK, and β-catenin were verifiedby Western Blot and Immunofluorescence.1.4cells were pretreated with or without Icariin (20μM), a p38specific inhibitor, SB203580(10μM), a selective ERK1/2inhibitor,PD98059(10μM), a specific JNK inhibitor, SP600125(10μM), aβ-catenin inhibitor, XAV-939(10nM), and then stimulated with IL-1β.MMP-1, MMP-3, MMP-13, P-p38, P-ERK1/2, P-JNK and β-catenin weredetermined by Western Blot.2. Experiments in vivo2.1Seventy8-week-old SD rats were obtained from the AnimalCenter of Chongqing Medical University.60rats underwent ACLT in the right posterior knee joint to generate an experimental model of OA. Theserats were ramdonly divided into six groups (i.e. the Vehicle group,SB203580group, PD98059group, SP600125group, XAV-939group andIcariin group). After four weeks, the rats received weekly intra-articularinjections with DMSO (0.3mL,0.05%), Icariin (0.3mL,20μM), the p38inhibitor, SB203580(0.3mL,10μM), the ERK1/2inhibitor, PD98059(0.3mL,10μM), the JNK inhibitor, SP600125(0.3mL,10μM), or theβ-catenin inhibitor, XAV-939(0.3mL,10nM). These injections weremaintained for six weeks. The remaining ten rats were used as normalcontrols. The articular cartilage histomorphology were analyzed viaGrading of H/E staining according to Mankin Scoring system.2.2The protein expressions of MMP-1,3,13of cartilage in all thegroups were dertermined by Immunohistochemistry and western blot.2.3The protein expressions of P-p38、P-JNK、P-ERK1/2and β-cateninwere verified by Western Blot.Results:1. Experiments in vitro1.1The effects of Icariin at different concentrations on the cellviabilities and IL-1β stimulated MMP-1，3，13in SW1353cellsThe MTT reulsts showed that the cell viabilities were more than90%while SW1353cells were treated with0,5,10,20μM concentrations ofIcariin. But cell viabilities were less than90%while SW1353cells were treated with40,80,100μM concentrations of Icariin. That is to say, theconcentration more than20μM have adverse effects on cell viability.Icariin has been shown to cause dose-dependent decreases of theexpressions of MMP-1,3,13in IL-1β-induced SW1353cells. Therefore,the concentration at20μM was used in subsequent experiments.1.2The effects of Icariin on the mRNA and protein expressions ofMMP-1,3,13in IL-1β-induced SW1353cellsHuman SW1353chondrosarcoma cells were pretreated with orwithout20μM Icariin for1h, then were stimulated with10ng/mL IL-1β.After24h, cells and the supernates were harvested. Compared with that inIL-1β group, the mRNA and the protein expressions MMP-1,3,13significantly decreased in Icariin group.1.3The effects of Icariin on the expressions of P-p38、P-JNK、P-ERK1/2and β-catenin in IL-1β-induced SW1353cellsThe expressions of P-p38、P-JNK、P-ERK1/2and β-catenin increasedafter IL-1β stimulated, compared with that in the control group. Whilecompared with that in IL-1β group, The expressions of P-p38、P-JNK、P-ERK1/2and β-catenin in Icariin were significantly decreased.1.4The effects of Icariin and pathway inhibitors on the expressions ofMMP-1,3,13in IL-1β-induced SW1353cellsCompared with IL-1β group, the expressions of P-p38, MMP-1,MMP-3and MMP-13in SB203580group, the expressions of P-ERK1/2, MMP-1, MMP-3and MMP-13in PD98059group, the expressions ofP-p46, P-p54and MMP-13in SP600125group, the expressions ofβ-catenin and MMP-13in XAV-939group, and the expressions of MMP-1,MMP-3, MMP-13, P-p38, P-p46, P-p54and β-catenin in Icariin group weredown-regulated significantly. Compared with the signaling-specificinhibitors groups respectively, expressions of P-p38、P-p46、P-ERK1/2andβ-catenin were increased, but the expressions of MMP-1,3,13reducedsignificantly.2. Experiments in vivo2.1The effects of Icariin and signaling-specific inhibitors on articularcartilage histomorphology in cartilageH/E staining of knee joint sections showed a smooth surface and aclear laminar structure in the articular cartilage from the normal group，andthe Mankin·s score was low. In contrast, the cartilage surface was roughwith some superficial leakage and ulcers for the vehicle-treated group,moreover, mankin·s score was much higher. Furthermore, Icariin-treatedcartilage and cartilage treated with signaling pathwayspecific Inhibitorsshowed fewer lesions and lower mankin·s scores compared withvehicle-treated cartilage sections.2.2The effects of Icariin on MMP-1, MMP-3, MMP-13, P-p38、P-JNK、P-ERK1/2and β-catenin in cartilageThe expression levels of MMP-1,3,13in normal cartilages were low. In contrast, the expression levels of MMP-1,3,13in Vehicle group weremuch higher, where the expression sites located in the cytoplasm andintercellular matrix. Compared with the vehicle-treated group, theexpressions of MMP-1,3,13in SB203580group were down-regulated, sowere the expressions of MMP-1,3,13in PD98059group, MMP-13inSP600125and XAV-939groups, and MMP-1,3,13in Icariin group.Compared with signaling inhibitors, the expression of MMP-1,3,13were declined2.3The effects of Icariin and pathway inhibitors on MAPKs signalingpathways and Wnt/β-catenin pathwayCompared with the normal group, the expressions of P-p38, P-p46,P-p54, P-ERK1/2and β-catenin were enhanced in Icariin, while comparedwith the vehicle-treated group, the five targets were down-regulated.Compare with the pathway inhibitors respectively, the expressions of P-p38,P-p46, P-ERK1/2and β-catenin were enhanced in Icariin group.Conclusions1. The concentration of Icariin at20μM did not affect the cell viability,and it could supress the increasement of IL-1β-induced expressions ofMMP-1,3,13. Therefore, it was used as the optimal concentration in thesubsequent experiments.2. The protein and mRNA expressions of MMP-1,3,13were enhancedafter being stimulated by IL-1β in SW1353cells, which were down-regulated after the treatment with Icariin. Icariin could inhibit theincreases in MMP-1,3,13in SW1353cells treated with IL-1β.3. Icariin could supress the increasements of P-p38、P-ERK、P-JNKand β-catenin, for which the increasements of MMP-1,3,13wereinhibited.4. Icariin could protect cartilage from degeneration via decreasing theMankin·s scores and damages of the articular cartilage in a rat osteoarthritismodel.5. Icariin could down-regulate the protein expressions of MMP-1,MMP-3, MMP-13,P-p38, P-ERK1/2, P-p46, P-p54and β-catenin in a ratosteoarthritic cartilage.6. Icariin exhibited certain supressions of the p38, ERK, JNK and Wnt/β-catenin pathways, but the supressive effects were inferior to the signalingpathway specific inhibitors.7. The suppressive effects of Icariin on MMP-1,3,13were greater thanthat exhibited by other signaling pathway inhibitors, which might associatewith its supressions of multi-targets.