Research Of Prevention And Treatment Mechanisms Of Fuyuan Capsule On The Osteoarthritis With Modulating OPG-RANKL-RANK System Via MAPKs Signal Pathways

Part1: Effects of IL-1β and1α.25(OH)2D3on the expressions of OPG–RANKL-RANK systemObjective: To investigate the regulation mechanism of OPG–RANKL-RANKsystem associated with MAPKs pathways.Methods: SW1353Cells were treated with IL-1β,1.25（OH）2D3, p38inhibitorSB203580, ERK1/2inhibitor PD980959and JNK inhibitor SP600125respectively anddivided into9groups. The models was induced with IL-1β or1.25（OH）2D3alone. Theexpressions of p-p38, p-ERK1/2, p-JNK were determined by western blot. Theexpressions of OPG, RANKL and RANK mRNA and protein were determined byqRT-PCR and ELISA.Results: Data from western blot shown an increase of OPG,RANKL,RANK andOPG/RANKL levels in SW1353cell IL-1β induced. The expression of p-ERK1/2wasincreased but p-p38and p-JNK decreased in SW1353cell1.25（OH）2D3, induced. Allpathways inhibitors could inhibit the activation of corresponding pathway. IL-1βsignificantly increase OPG,RANKL, RANKmRNA and protein and OPG/RANKL ratio（p<0.05）The OPG and OPG/RANKLratio were lower in SB203580group（p<0.05）and the RANKL and RANK levels were higher（p<0.05） but ERK1/2shown anopposite effects that the OPG expression was decreased and RANKL and RANK were increased, but the changes were not reached the statistical significance（p>0.05）,except for the decreased OPG/RANKL ratio（p<0.05）.The JNK inhibitor decreasedOPG,RANKL,RANK mRNA and protein but increased OPG/RANKL ratio significantly（p<0.05）.Conclusion: The MAPKs can mediate the expressions of OPG–RANKL-RANKsystem in OA chondrocyte.Part2: The effects of OPG on survival of human chondrocyteObjective: To investigate the effects and mechanism of OPG on chondrocyte.Methods: The cells were stimulated with various concentrations of humanosteoprotegerin(OPG-Fc, cut out the Heparin domain). Subsequently the cellsproliferation and the optimum concentrations and time of OPG-Fc were measuredaccording to A value by MTT assay and applied in the following experiments. After thatthe SW1353were incubated with50ng/ml,100ng/ml OPG-FC,, SB203580orPD980959for72h. The cells were divided into7groups.The levels of p-p38andp-ERK1/2were detected by western blot. Bax, caspase3mRNA and Bcl-2mRNA levelswere tested by qRT-PCR and cell cycles were analysed by flow cytometer.Results: The MTT results indicated that25ng/ml and50ng/ml OPG-Fc promotedthe proliferation of SW1353notably(p<0.05) while100ng/ml OPG-FC accelerated thecell apoptosis prominently by the normal group(p<0.05). Furthermore the50ng/mlOPG-Fc on cells proliferation was superior than25ng/ml(p<0.05) and the effects ofOPG-Fc were strongest at72h. Refer to MAPK pathways,50ng/ml OPG-Fc suppressedthe p-p38protein and promoted the p-ERK1/2in SW1353significantly(p<0.05) while100ng/ml OPG-Fc accelerated the p-p38level and inhabited the p-ERK1/2dramaticllycompared with the normal group(p<0.05).50ng/ml OPG-Fc decreased apoptosis genesBax,caspase3mRNA but increased anti-apoptosis gene Bcl-2mRNA and the proliferation index(PI)significantly(p<0.05) while100ng/ml OPG-Fc reinforceBax,caspase3mRNA expressions but drop off gene Bcl-2mRNA and PI levels evidentlycompared with than normal group(p<0.05) which were blocked by SB203580andexpedite by PD98059(p<0.05).Conclusion: OPG can modulate cell proliferation or apoptosis via MAPKsdepended on its concentration.Part3: Effects of Icariin on the expressions of OPG–RANKL-RANK systemassociated with MAPKs pathways.Objective: To investigate the effects of Icariin on the expressions of OPG–RANKL-RANK system.Methods: The SW1353cell were stimulated with various concen-trations ofIcariin. Subsequently the cells proliferation and the optimum concentrations and time ofIcariin were measured according to A value by MTT assay and applied in the followingexperiments. After that the SW1353were incubated with IL-1β, SB203580orPD980959for48h. The cells were divided into7groups such as model group inducedwith IL-1β alone. The expressions of p-p38and p-ERK1/2were determined bywestern blot. The expressions of OPG, RANKL and RANK mRNA and protein weredetermined by qRT-PCR and ELISA.Results: The MTT results indicated that1.5μg/ml-24μg/ml Icariin can significantlypromote the cells proliferation than normal group（p<0.05）.1.5μg/ml icariin promotedthe cells proliferation mildly (p>0.05). Furthermore, effects of12μg/ml icariin werestrongest at48h. Icariin can inhibited the p38pathway and actived the ERK1/2pathwaysignificantly IL-1β induced.(p<0.05) compared with IL-1β group. When the cellsincubated with IL-1β,the icariin obviously decreased the OPG,RANKL,RANKexpressions and OPG/RANKL ratio (p<0.05).PD98059could inhibit the decreased levels of OPG and OPG/RANKL Icariin induced but expedited by SB203580.Conclusion: Icariin could promote the chondrocytes proliferation in vitro. Icariincan attenuate the expressions of OPG, RANKL, RANK and OPG/RANKL ratio viaMAPKs.Part4: Effects of FYC-CS on the expressions of OPG–RANKL-RANK systemassociated with MAPKs pathways.Objective: To investigate the effects of Icariin on the expressions of OPG–RANKL-RANK system and explore the mechanism of the OAtreatment of FYC.Methods: The cells were stimulated with various concentrations of FYC-CS.Subsequently the cells proliferation and the optimum concentrations and time ofFYC-CS were measured according to A value by MTT assay and applied in thefollowing experiments. After that the SW1353were incubated with IL-1β,1.25（OH）2D3, FYC-CS, CS, or PD980959for48h. The cells were divided into9groups such asmodel group induced with IL-1β or1.25（OH）2D3alone. The expressions of p-p38and p-ERK1/2were determined by western blot. The expressions of OPG, RANKL andRANK mRNA and protein were determined by qRT-PCR and ELISA.Results: The MTT results indicated that5%,10％,15%FYC-CS could promoteproliferation of SW1353. Furthermore, effects of15%FYC-CS were strongest at48h.The expression of p-ERK1/2was elevated in the SW135315%FYC-CS and IL-1βstimulated(p<0．05) but decreased in the SW13531.25(OH)2D3stimulated(p<0．05).The expressions of OPG,RANKL,RANK and OPG/RANKL ratio in IL-1β group wereobviously increased which can be blocked by the15%FYC-CS significantly(p<0．05).On the contrary1.25(OH)2D3can down-regulated the OPG expression andOPG/RANKL ratio and up-regulated the RANK and RANKL which also can be blocked by15%FYC-CS significantly(p<0.05)..The effects of FYC-CS onOPG-RANKL-RANK system were abated by ERK1/2inhibitor PD98059.The MTT assay indicated that the A value of optimal concentration of Icariin groupwas1.92. The Icariin dropped the OPG/RANKL ratio down to1.05IL-1β induced. TheA value of FYC-CS group was2.91. The FYC-CS dropped the OPG/RANKL ratiodown to1.11IL-1β induced. It suggested that FYC-CS occupied the strongerproliferation of human chondrocytes than Icariin and exhibited the superior effectes onOPG-RANKL-RANK system than Icariin.Conclusion: FYC-CS could promote the chondrocytes proliferation in vitro.FYC-CS can modulate the OPG-RANKL-RANK system via ERK pathway. FYC-CSoccupied the stronger proliferation of human chondrocytes and the superior effectes onOPG-RANKL-RANK system than Icariin.