Study On The Mechanism Of Low Molecular Weight Hyaluronic Acid Synergistically Promoting Bone Resorption And Osteoclast Formation

BackgroundDue to its characteristics of high water absorption,viscoelasticity,biocompatibility,and non-immunogenicity,hyaluronic acid is regarded as an optimal soft tissue filler and has been extensively utilized in the field of plastic surgery.With the extensive application of HA,various side effects or complications has appeared,including subcutaneous nodules,subcutaneous hematoma,acute infection,chronic granuloma,vascular embolism,ect.In our previous clinical study,we discovered that HA injection could induce bone resorption in the mentum,and histological results revealed that there was a large amount of fragmented hyaluronic acid accumulation around the bone resorbed tissue.The biological properties of HA mainly depend on its molecular weight.High molecular weight of hyaluronic acid has been applied in treatment of osteoarthritis due to its significant inhibition of inflammation,inhibition of angiogenesis,and promotion of osteogenesis,whereas low molecular weight hyaluronic acid(LMW-HA)has proinflammatory and pro-angiogenesis effects.We hypothesize that LMW-HA with inflammatory effect may contribute to bone resorption.However,limited research on the effect of LMW-HA on osteoclast formation and bone resorption was conducted.Therefore,we conducted a series of vivo and vitro experiments to corroborate whether LMW-HA could promote osteoclast formation and bone resorption,as well as to investigate the potential molecular biological mechanism.Objectives1.To determine whether LMW-HA can promote cranial osteolysis and osteoclast formation in vivo,and initially to explore the potential mechanism.2.To explore the direct effect of LMW-HA on osteoclast differentiation in vitro.3.To investigate the changes of TLR4/NF-κB signaling pathway when LMW-HA promotes osteoclast differentiation.Methods1.In the first part,we explored the phenomenon of bone resorption induced by LMWHA and preliminarily understood the mechanism.①To explore the phenomenon of bone resorption,we conducted animal study.We established a mouse cranial osteolysis model,and explored the effect of LMW-HA injection on bone resorption.The C57/BL6J mice were randomly divided into five groups.Subcutaneous injection with the following drugs for 7 consecutive days:NC group(PBS),high-dose LPS group(100 μg LPS/day),low-dose LPS group(10 μg LPS/day),low-dose LPS+LMW-HA group(10 μg LPS+500 μg LMW-HA/day),and LMW-HA group(500 μg LMW-HA).Micro-CT was used to evaluate the bone resorption.TRAP staining was used to evaluate osteoclast formation,and HE staining was used to evaluate bone destruction.The mRNA expressions of osteoclast-related and the inflammatory genes were determined by qRT-PCR.ELISA was used to determine the release of inflammatory cytokines.The infiltration of F4/80 macrophages in the skin around the skull was evaluated by immunohistochemistry.Finally,we investigated whether TAK-242(TLR-4 inhibitor)could inhibit osteoclast formation in vivo.The C57/BL6J mice were randomly divided into four groups:NC group(PBS),low-dose LPS+LMW-HA group(10 μg LPS+500μg LMW-HA/day),TAK-242+low-dose LPS+LMW-HA group(3 mg/kg/day TAK-242+10 μg LPS+ 500 μg LMW-HA/day),TAK242 group(3 mg/kg/day TAK-242).TRAP staining was used to evaluate osteoclast formation.②Cell experiments were conducted to explore the mechanism by which LMW-HA could indirectly promote the formation of osteoclasts by promoting the expression of some cellular inflammatory factors or osteoclast factors.Peritoneal macrophages were extracted,and qRT-PCR was conducted to determine the mRNA expression of inflammation-related genes after treatment with LMW-HA.Bone marrow mesenchymal stem cells were extracted,and the mRNA expression of the osteoclast-related gene after treatment with LMW-HA was determined by RT-qPCR.Primary bone marrow macrophages were extracted to explore whether LMW-HA can directly promote osteoclast formation induced by RANKL or TNF-α in vitro.2.In the second part,RAW264.7-cell induced osteoclast model was used to explore the direct effect of LMW-HA on osteoclasts and to detect the expression of corresponding landmark gene or protein.Firstly,osteoclast precursors were treated with different concentrations of LMW-HA(NC group,20 μg/ml,100μg/ml,500μg/ml,2500(μg/ml).CCK-8 was performed to evaluate the effect of drugs on cell proliferative activity.The expression of proliferation-related genes Ki-67 and C-myc at 6h and 24h were detected by RT-qPCR.The osteoclast precursor RAW264.7 cells were treated with or without RANKL at different concentrations of LMW-HA.The formation of osteoclasts was detected by tartrate-resistant acid phosphatase(TRAP)staining,and the TRAP activity was detected by TRAP enzyme activity kit.The mRNA expression of the osteoclast specific gene at 48h and 96h were detected by RT-qPCR.The osteoclast specific proteins were detected by Western blotting,and the optimal working concentration of the drug was selected for further mechanism study.3.In the third part,the role of TLR4/NF-κB signaling pathway in promoting osteoclast differentiation of LMW-HA was explored.The F-actin ring formation of mature osteoclasts in the NC group,RANKL group,and LMW-HA+RANKL group was detected by phalloidin staining.After treatment with RANKL with or without LMWHA at 0 min,5 min,15 min,30 min,45 min,and 60 min,total proteins were extracted.The expression levels of TLR-4,MYD88,TRAF-6,p-NF-κBp65,NFκBp65,p-IκBα,and IκBα proteins were determined by WB.After adding TAK-242,a TLR-4 inhibitor,immunofluorescence was used to detect whether nuclear translocation of NF-κB p65 could be inhibited,and the expression of NFATC-1,cFos MMP-9 CTSK,a key protein of osteoclast differentiation downstream of TLR4/NF-κB signaling pathway,was detected by WB.Results1.LMW-HA significantly promoted bone resorption in vivo.Micro-CT showed that LMW-HA could increase the LPS-induced cranial osteolysis.Compared with NC group,low dose LPS group and LMW-HA group,the proportion of bone tissue to total tissue(BV/TV)in LMW-HA+low dose LPS group was significantly decreased,while the percentage of porosity and number of porosity in LMW-HA+low dose LPS group were significantly increased.The difference was statistically significant.The number of TRAP-positive osteoclasts in LMW-HA+low-dose LPS group was significantly higher than that in NC group,low-dose LPS group and LMW-HA group.RT-qPCR showed that,compared with low-dose LPS or LMW-HA group alone,LMW-HA combined with low dose LPS significantly promoted the expression of osteoclast-related genes CTSK,MMP-9,TRAP and RANKL and the expression of inflammation-related genes TLR-4,IL-6,TNF-α and IL-1β.Serological tests confirmed the up-regulated expression of inflammatory genes.LMW-HA combined with low-dose LPS significantly increased serum levels of IL-6,TNF-α and IL-1 β.The immunohistochemical results showed that LMW-HA significantly increased the infiltration of F4/80 positive macrophages into skin.Osteoclastic differentiation tests showed that LMW-HA can directly promote osteoclast differentiation induced by RANKL and TNF-α.LMW-HA could significantly increase the expression of inflammatory genes IL-6,TNF-α and IL-1β in PMMs.and promote the expression of osteoclast-related genes RANKL in BMSCs.The high level of expression of inflammatory and osteoclast-related cytokines in vivo may be related to the effect of LMW-HA on PMMs or BMSCs.Finally,TLR-4 receptor inhibitors significantly inhibited the formation of LMW-HA-induced osteoclasts in vivo.2.LMW-HA could synergistically promote RANKL-induced osteoclast differentiation in a dose-dependent manner in vitro.The CCK-8 showed that LMW-HA could promote the proliferation of RAW264.7 cells within 24h compared with NC group,and the difference was statistically significant.The results of RT-qPCR showed that LMW-HA could temporarily increase the proliferation-related genes Ki-67 and c-myc expression at 6h compared with NC group,but there was no statistically significant difference in these genes at 24h.TRAP staining results confirmed that treatment with LMW-HA alone could not directly induce osteoclast formation,but synergistically promote RANKL-induced osteoclast formation in a dose-dependent manner.Compared with the NC,RANKL and LMW-HA group,the number of TRAP-positive osteoclasts in the LMW-HA+RANKL group was significantly increased.The promoting effects was enhanced with the increase of drug dosage,in which the 500μg/ml of LMW-HA could induce the highest number of mature osteoclasts.RT-qPCR results showed that LMW-HA promoted the expression of RANKL-induced early(48h)osteoclast transcription-related genes compared with NC group and RANKL group.In the late stage(96h),the expression of transcription factors NFATC-1,c-Fos were up-regulated,and the expression of bone resorption and fusion related gene MMP-9 CTSK Dc-stamp Atp6v0d2 was up-regulated,and the difference was significant.The promotion of these genes expression was also concentration dependent,.The trend of WB results was consistent with that of RT-qPCR.Compared with the RANKL group alone,LMW-HA showed dose-dependent promotion of key osteoclast protein NFATC-1,c-Fos,MMP-9,CTSK expression,with the strongest effect at 500 μg/ml.3.LMW-HA synergistically promotes osteoclast formation through TLR4/NF-κB signaling pathway.First,the results of F-actin ring staining showed that LMW-HA could significantly increase the number and average area of F-actin rings in mature osteoclasts,and the differences were statistically significant.Subsequent WB results showed that LMW-HA treatment could significantly activate the TLR4/NF-κB signaling pathway,which was manifested as up-regulated expression of upstream proteins TLR4,MYD88 and TRAF6,and increased phosphorylation levels of NFκBp65 and IκB-α,increased the degradation of total IκB-α proteins.It represents the activation of the TLR4/NF-κB signaling pathway.Subsequently,TAK-242,an inhibitor of TLR4,was added to detect whether it could inhibit downstream NFκBp65 nuclear translocation and expression levels of key osteoclast proteins.Immunofluorescence showed that the number of NF-κBp65 nuclear translocated cells was significantly increased in the LMW-HA+RANKL group compared with the RANKL group while the number of NF-κBp65 nuclear translocated cells decreased to the level of the RANKL group after adding with TAK-242.Finally,the expression level of NFATC-1,c-Fos MMP-9 CTSK protein was significantly increased after LMW-HA treatment compared with RANKL group,and TAK-242 could reverse this increase to the level of RANKL group.Conclusion1.Our results suggest that LMW-HA can exacerbate bone destruction in LPS-induced calvaria bone resorption model.The potential mechanisms may be LMW-HA could directly promote the RANKL-and TNF-alpha-induced osteoclast differentiation and increase secretion of inflammatory and osteoclast-associated cytokines to indirectly promote osteoclast differentiation.2.LMW-HA synergistically promote RANKL-induced osteoclastic differentiation of RAW264.7 cells and the expression of specific genes and proteins of osteoclasts in a dose-dependent manner at a concentration of 500 μg/ml.3.LMW-HA activates TLR4/NF-κB signaling pathway,to further promote nuclear translocation of NF-κBp65 and expression of key osteoclastic proteins,thus increasing the number of mature osteoclast formation.