The Effect Of Puerarin On Polymethylmethacrylat-Induced Osteoclastogenesis And Osteolysis In Mouse Model

Artificial joint arthroplasty is the primary mean for the treatment of joint damage,severe joint degenerative diseases,and bone and joint destruction induced by other diseases.The main cause for the failure of arthroplasty was prosthetic aseptic loosening.However,little is known about the underlying mechanisms that contributes to this phenomenon,and it is currently proposed that osteolysis is involved in wear debris that generates from joint surface.Existing evidence revealed that inflammatory reactions-caused by wear debris in the tissues surrounding the prosthesis could induce the excessive formation of osteoclasts and bone resorption,triggering osteolysis and pseudo-membrane formation around the prosthesis,finally resulting in prosthesis loosening.Hence,alleviating wear debris induced inflammatory response and osteoclast-genesis might represent an effective strategy for preventing prosthesis loosening.Puerarin,a natural isoflavone isolated from Chinese traditional plant pueraria lobata,has attracted considerable attention due to its important biological and pharmacological activities.However,its effects on lesion of peri-implant and related mechanism of action are still not clear,which require further investigation.In this study,we evaluated the effects of puerarin on polymethylmethacrylate(PMMA)-induced lesion of peri-implant in vitro and in vivo,and explored its possible mechanism of action.Part one The effect of puerarin on the differentiation of PMMA-induced osteoclast precursor cells RAW264.7Methods1.PMMA particles were added into RAW264.7 cell culture system for indicatedtime.Then cell viability and cell apoptosis were detected using cell counting kit-8 and Flow cytometry.While the differentiation of RAW264.7 cells into osteoclasts were evaluated by TRAP staining.2.The four treatment groups were as follows:(1)Control group;(2)PMMA particles treatment group(PMMA);(3)25 μM puerarin and PMMA particles co-treatment group(PMMA+25 PR);(4)50 μM puerarin and PMMA particles co-treatment group(PMMA+50 PR).3.The expression of MMP-9,TNF-α,IL-6,RANKL,RANK,and OPG were detected by q RT-PCR and Western blotting.4.Western blotting was used to detect the expression of related proteins in the NF-κB pathway(p65)and MAPK signaling pathway(ERK1/2,JNK,and p38)5.The expression of p65 was detected by immunofluorescence staining.Results1.Compared with PMMA treatment group,puerarin treated at the dose of 20 and 50 μM significantly reduced the differentiation of RAW 264.7 cells to osteoclasts(P<0.01,P<0.001),and the effect of 50 μM puerarin was better than 20 μM(P<0.05).2.PMMA treatment notably enhanced the m RNA and protein expression of MMP-9,TNF-α,and IL-6 compared with the control group(all P<0.05),but puerarin administration abolished this effect(all P<0.05).3.PMMA treatment significantly promoted the expression of RANKL and RANK in RAW 264.7 cells compared with the control group(all P<0.05).In addition,puerarin treatment significantly decreased the expression of RANKL and RANK induced by PMMA(all P<0.05).4.Compared with the control group,PMMA treatment significantly activated induced NF-κB and AMPK signaling pathway(P<0.05),while puerarin treatment could effectively alleviate this effect(P<0.05).ConclusionsPuerarin could inhibit PMMA-induced osteoclastogenesis in RAW264.7 cellswith a dose-dependent manner in vitro and effectively down-regulate m RNA and protein expressions of MMP-9,TNF-α,IL-6,RANKL and RANK,which were mediated by the inactivation of NF-κB and MAPK signaling.Part two To verify the effect of puerarin on osteoclast formation and osteolysis induced by PMMA particles in mice Methods1.To establish murine calvarial osteolysis model using PMMA treated with the dose of 300 μl.2.The four treatment groups in mice were as follows:(1)Control group;(2)PMMA particles treatment group(PMMA);(3)0.5 mg/ml puerarin combined with PMMA particles co-treatment group(PMMA+0.5 PR);(4)1.0 mg/ml puerarin combined with PMMA particles co-treatment group(PMMA+1 PR).3.HE staining and TRAP staining were applied to evaluate the osteoclast formation and osteolysis4.qRT-PCR was used to detect the expression of RANKL and RANK.Results1.Compared with the control group,the trabecular structure disorder and the area of destruction and dissolution of skull sagittal line in PMMA treated mice increased significantly(P<0.001).besides,0.5 and 1.0 mg/ml puerarin treatment was attenuated PMMA-induced murine calvarial osteolysis.2.The number of osteoclasts in the murine calvarial treated with PMMA was significantly increased compared with the control group(P<0.01).After 0.5 and 1.0 mg/ml puerarin treatment,the formation of osteoclasts induced by PMMA was significantly alleviated(all P<0.05),and the effect of 1 mg/ml puerarin was better than 0.5 mg/ml(P<0.05).3.PMMA treatment significantly promoted the expression of RANKL and RANK compared with the control group(all P<0.05).In addition,puerarin treatment significantly decreased the expression of RANKL and RANK induced by PMMA(all P<0.05).ConclusionsPuerarin was observed to attenuate PMMA-induced osteoclastogenesis,osteolysis,and m RNA expressions of RANKL and RANK in a murine calvarial osteolysis model.