Effect And Mechanism Of AKBA On Wear Particle-induced Periprosthetic Osteolysis

Part I: Study on Inhibition of Periprosthetic Osteolysis by AKBA Inhibiting Ti ParticlesObjective: To investigate the therapeutic effect of AKBA in inhibiting periprosthetic osteolysis induced by titanium particles.Methods: A total of 56 male C57BL/J6 mice were selected and randomly divided into 4 groups: control group,Ti particle group(Ti group),AKBA low-dose treatment group(L-AKBA)and AKBA High-dose treatment group(H-AKBA),14 mice per group.The Control group received only surgery.A sagittal incision was made in the middle of the skull of the mouse,and then sutured.The mice were injected intraperitoneally with the same volume of sterile PBS every day;Ti particles group(Ti)was implanted with Ti particles on the surface of the mouse skull,and the mice were injected intraperitoneally with equal volume of sterile PBS every day;In L-AKBA and H-AKBA groups,7.5mg/kg and 15mg/kg AKBA were injected into mice intraperitoneally respectively every day after Ti particles were implanted.Two weeks after administration,the mice were sacrificed and the skull and peripheral blood were reserved.Micro-CT,H&E staining,TRAP staining,ELISA,RT-PCR,and routine blood and biochemical tests were performed.Results: Micro-CT results showed that the skeletal osteolysis was significantly reduced in the L-AKBA and H-AKBA groups.The number of lacunae and lacunae area were significantly reduced compared with the Ti group.The bone density and bone volume fraction were significantly increased compared with Ti group(p<0.05).The results of H&E staining showed that there were a lot of macrophage-like cells in the periosteum of Ti group.The inflammatory response of L-AKBA and H-AKBA groups were significantly reduced.The absorption area of sagittal suture was significantly decreased compared with that of Ti group(p<0.05).The results of TRAP staining showed that a large number of TRAP positive cells were seen on the dissolvable side of the skull in the Ti group,and the number of TRAP positive cells in the L-AKBA and H-AKBA groups were significantly reduced,the Oc S/BS decreased by 35.3% and 70.6%,respectively,compared with the Ti particles group(p<0.05).The results of RT-PCR showed that the m RNA expression levels of the osteoclast-associated genes such as TRAP,Cath-K,CTR,and OSCAR were significantly up-regulated in the Ti group,while the m RNA expression levels of these genes were gradually decreased respectively in the L-AKBA and H-AKBA groups compared with the Ti group(p<0.05).The results of ELISA showed that the expression of TNF-α,IL-1β and IL-6 in the culture supernatant of the Ti group were significantly increased.The expression levels of inflammatory factors were significantly decreased respectively in the L-AKBA and H-AKBA groups,and the inhibitory effect was dosedependent(p<0.05).Blood routine and biochemical results showed that hemoglobin,leukocyte,platelet,alanine aminotransferase,aspartate aminotransferase,urea nitrogen,and creatinine levels in the L-AKBA and H-AKBA groups were not significantly different from those in the Ti group(p>0.05).Conclusion: AKBA can inhibit the secretion of inflammatory cytokines induced by Ti particles to reduce the chronic inflammatory response in a dose-dependent manner,inhibit the expression of osteoclast-related genes to reduce the differentiation and formation of osteoclasts,reduce bone loss and increase bone mass,in order to prevent and treat the wear particles induced osteolysis reaction.Part II: Effect of AKBA on RANKL-Induced Osteoclast DifferentiationObjective: To further clarify the inhibitory effect of AKBA on RANKL-induced osteoclast differentiation.Methods: Bone marrow-derived macrophages(BMMs)were used for the research to observe the effect of AKBA on the activation of osteoclasts induced by RANKL(50 ng/ml).CCK8 assay was used for cell proliferation and toxicity analysis;tartaric acid phosphatase(TRAP)staining was used to detect the number of mature osteoclasts;bone plate absorption experiments was used to detect bone resorption capacity of osteoclasts;phalloidin staining was used to detect formation of F-actinin ring;RT-PCR was used to detect the m RNA expression levels of osteoclast-related genes such as TRAP,Cath-K,CTR,OSCAR,NFATc1,MMP-9,and the m RNA expression levels of inflammatory cytokine such as TNF-α,IL-1β and IL-6.Results: The CCK8 test results showed that there was no significant effect on the proliferation of BMMs cells,and there was no toxic effect on the cells when the concentration of AKBA was less than 30 μM.The results of TRAP staining showed that a large number of purple-red multinuclear macrophages were seen in the Control group,while the number of macrophages gradually decreased with the increase of the concentration in the AKBA groups.The results of statistical analysis showed that the number and area of TRAP-positive cells in the AKBA groups were significantly decreased with a concentration dependence compared with that of the Control group,and the difference was statistically significant(p<0.05).Bone plate absorption experiment results showed that the Control group revealed obvious bone resorption lacuna formation,and the bone resorption area ratio was as high as 73.4%,while the AKBA group gradually decreased with the increase of the concentration;the statistical analysis results showed that bone Absorption area ratio(%)was significantly reduced in the AKBA group compared with the Control group,showing a concentration-dependent,statistically significant difference(p<0.05).Phalloidin staining showed that the Control group could be clearly observed the well-differentiated F-actin ring of osteoclasts;and the size and quantity of Factin rings decreased significantly with the increase of concentration in AKBA group.The results of RT-PCR showed that with the increase of AKBA concentration,the m RNA expression levels of osteoclast-associated genes such as TRAP,Cath-K,CTR,OSCAR,NFATc1 and MMP-9 were gradually decreased compared with the Control group;and the m RNA expression levels of inflammatory cytokines such as TNF-α,IL-1β,and IL-6 gradually decreased(p<0.05).Conclusion: In vitro cell culture,AKBA can inhibit the expression of inflammatory factors in a concentration-dependent manner to reduce the chronic inflammatory response,and inhibit the expression of osteoclast-related genes to reduce the differentiation and formation of osteoclasts within a certain concentration range,destroy F-actin rings to reduce osteoclast resorption.Part Ⅲ: Study on the mechanism of AKBA inhibition of osteoclast differentiationObjective: To further define the specific signaling pathways and target locations for AKBA inhibition of osteoclast differentiation.Methods: Mouse mononuclear/macrophage cell line RAW264.7 was used for the research to observe the effect of AKBA on the activation of osteoclasts induced by RANKL(50 ng/ml).The expression of IκBα,p-IκBα,p65,p-p65,ERK,P-ERK,JNK,pJNK,p38,p-p38,AKT1,p-AKT1,MEK,p-MEK,NFATc1 and c-Fos were determined by protein electrophoresis(Western blot),and the relative gray values were determined as well.RT-PCR was used to detect the m RNA expression levels of NFATc1 and c-Fos.Results: With time,the phosphorylation levels of AKT,IκBα,p65,JNK and p38 were not inhibited by AKBA,and there was no significant difference in the relative gray value between AKBA group and Control group(p>0.05).Over time,the phosphorylation level of ERK was gradually inhibited by AKBA;The relative gray values of 15 minutes,30 minutes,and 60 minutes were significantly different between the AKBA group and the Control group(p<0.05).With increasing AKBA concentration,the phosphorylation level of ERK also gradually decreased,and the phosphorylation level of ERK was almost completely suppressed while AKBA was 15 μM;The relative gray values of AKBA 0.1 μM,1 μM and 15 μM decreased significantly compared with 0 μM group respectively(p<0.05).With the passage of time,the phosphorylation level of the upstream factor MEK was not inhibited by AKBA,and the relative gray value of AKBA group was not statistically different from that of the Control group(p>0.05).The phosphorylation levels of downstream pathway factors c-Fos and NFATc1 were gradually inhibited by AKBA,respectively,and the relative gray values on the first and third days were statistically significant compared with the Control group(p<0.05).The degree of inhibition of c-Fos was larger than NFATc1.RT-PCR results showed that with the increase of AKBA concentration,the m RNA expressions levels of NFATc1 and c-Fos were gradually reduced(p<0.05).Conclusion: AKBA mainly regulates RANKL-induced osteoclast differentiation and osteoclast-related gene expression by inhibiting the ERK pathway in MAPK signaling pathway and downstream pathway factors including c-Fos and NFATc1.Inhibition of phosphorylation of ERK is the target site for AKBA action,and these signaling pathways have a synergistic or inhibitory effect.