| Part I The effect of theaflavin-3,3’-digallate in preventing bone loss in ovariectomized miceObjective: To evaluate the effect of theaflavin-3,3’-digallate(TFDG)in preventing bone loss in ovariectomized(OVX)mice.Methods: Thirty-two 8-week old female C57BL/6 mice were randomly classified into 4 groups(each group has 8 mice): 1.Sham Group(no ovariectomized operation,intraperitoneal injection of isodose saline instead of TFDG);2.OVX Group(ovariectomized operation,intraperitoneal injection of isodose saline instead of TFDG);3.OVX+low TFDG Group(ovariectomized operation,intraperitoneal injection of TFDG with the dose of 1mg/kg/day for consecutive 8 weeks and 6 days in one week);4.OVX+ high TFDG Group(ovariectomized operation,intraperitoneal injection of TFDG with the dose of 10mg/kg/dayfor 6 dayseach week,consecutively 8 weeks).All mice were sacrificed by excessive anesthesia and bilateral tibia and femur were prepared for evaluation.Micro-CT was used to analyze parameters about bone morphology,such as bone mineral density(BMD),bone volume fraction(bone volume/total bone volume,BV/TV),trabecular thickness(Tb.Th),trabecular number(Tb.N),trabecular separation(Tb.Sp).H&E staining was performed to analyze the number of trabecular bones.The number of mature osteoclasts was evaluated using Tartrate Resistant Acid Phosphatase(TRAP)staining.Immunohistochemistry was used to evaluate the expression of Receptor Activator of Nuclear Factor Kappa B Ligand(RANKL)and Cathepsin K(CTSK).Results: Micro-CT showed that rare trabecular bones with significant osteoporotic bone tissues were seen in OVX group;the number of trabecular bones increased and the trabecular space decreased in OVX mice after intraperitoneal TFDG injection.The BMD,BV/TV,Tb.Th,Tb.N and Tb.Sp of cancellous bone were 0.082±0.008g/cm3,5.093±0.930%,0.062±0.007 mm,0.820±0.090mm-1 and 0.444±0.050 mm in OVX Group and 0.115±0.015g/cm3,10.161±1.985%,0.080±0.007 mm,1.341±0.198mm-1,0.347±0.027 mm in Sham Group,respectively.The BMD and bone morphological parameters of cancellous bone had statistically significant differences between OVX Group and Sham Group(p<0.05).OVX+low TFDG Group and OVX+ high TFDG Group had improved values of the BMD and bone morphological parameters(BV/TV,Tb.Th,Tb.N and Tb.Sp)of cancellous bone compared to OVX group,and these values were statistically significant between OVX+high TFDG Group and OVX group(p<0.05).No statistically significant differences of BV/TV and Tb.Th of cortical bone were detected among 4 groups(p>0.05).The results of H&E staining presented osteoporosis and discontinuity of trabecular bones in OVX Group and the number of trabecular bones significantly decreased in OVX Group when compared to Sham Group.The number of trabecular bones was more in TFDG injection groups(OVX+low TFDG Group and OVX+ high TFDG Group)compared to OVX group,and less compared to Sham Group.The results of TRAP staining showed large-scale purple areas in OVX Group which indicated increased number of mature osteoclasts.The purple areas were rarely seen in Sham Group and they were significantly decreased after TFDG injection in OVX+low TFDG Group and OVX+ high TFDG Group.Immunohistochemistry evaluation results showed significantly increased expression of RANKL and CTSK large-scale in distal femur in OVX Group which were presented as large-scale claybank areas microscopically.The expression of RANKL and CTSK significantly decreased after TFDG injection in OVX+low TFDG Group and OVX+ high TFDG Group.Conclusion: TFDG can reduce the expression of RANKL and CTSK,reduce the number of mature osteoclasts which indicate TFDG can prevent bone loss after ovariectomized operation.Preventative treatment using TFDG may be a new therapeutic strategy for osteoporosis.Part II Theaflavin-3,3’-digallate inhibits RANKL-induced activation of osteoclastsObjective: To analyze the effect of theaflavin-3,3’-digallate(TFDG)on the activation of RANKL-induced osteoclastsMethods: We selected murine bone marrow-derived macrophages(BMMs)as the object of the study,and RANKL and TFDG used as intervention agent.The BMMs were classified into two groups: control group and TFDG group.Cell Counting Kit-8(CCK-8) was used to evaluate the changes of activation of cell proliferation under different concerntration of TFDG(0,0.01,0.1,1,5,10,20,30,40μM).The number of mature osteoclasts was evaluated by TRAP staining.Osteo Assay Surface(OAS)and phalloidine staining were performed to evaluate bone resorptive function of mature osteoclasts.Reverse transcription polymerase chain reaction(RT-PCR)was performed to evaluate the content of m RNA of TRAP、Nuclear factor of activated T-cells cytoplasmic 1(NFATc1)、C-Fos、Cathepsin K(CTSK)and Osteoclast-associated receptor(Oscar).Results: BMMs were separated and cultured from femur and tibia of C57BL/6 mice.Results of CCK-8 showed that TFDG with a concentration less than 10μM had no effect on the proliferation of BMMs(p>0.05),and while the concentration was more than 20μM,TFDG significantly inhibit the proliferation of BMMs(p<0.05).Five days after induction of RANKL,the results of TRAP staining presented a large number of purple multinucleus cells in control group whereas they were rare in TFDG group.Statistical analysis showed the number of TRAP-positive cells was 280.33±2.52 in control group,and 259±4.58,93.33±3.51 and 29.67±2.52 in TFDG group with concentration of 0.01,0.1 and 1μM,respectively.The area of TRAP-positive cells was 0.9316±0.0029 in control group,and 0.6649±0.0095,0.2005±0.0081,0.0891±0.0021 in TFDG group with concentration of 0.01,0.1 and 1μM,respectively.The number of TRAP-positive cells and area of TRAP-positive cells significantly decreased in TFDG group(0.1,1μM)compared to control group(p<0.05),which indicated that the inhibition effect of TFDG on activation of osteoclasts was concentration-dependent.Further study showed that the number of TRAP-positive cells and area of osteoclasts were 31.67±1.53 and 0.1110±0.0062 when TFDG(1μM)was intervened on day 0(116.33±4.04 and 0.2059±0.0115 on day 2),which showed a statistically significant difference compared to the values in control group(265.67±4.16、0.9373±0.0066)(p<0.05).However,compared to control group,the number of TRAP-positive cells and the area of osteoclasts showed no statistically signigicant difference when TFDG was intervened(1μM)on day 4(p>0.05).The results of OAS evaluation showed the area of bone resorption pits decreased with increase of the concentration of TFDG.There was a significant difference of the area of bone resorption pits between TFDG group(1μM,0.1144±0.0107)and control group(0.8250±0.0071)(p<0.01).The results of phalloidine staining showed that the morphology and size of F-actin rings of osteoclasts significantly decreased with increase of the concentration of TFDG.RT-PCR evaluation presented that TFDG can significantly inhibit the gene expression of TRAP,NFATc1,C-Fos,CTSK and Oscar m RNA which were related to RANKL-induced activation of osteoclasts,and the inhibition effect was time-and dose-dependent.Conclusion: TFDG can inhibit RANKL-induced activation of osteoclasts,reduce the gene expression which is related to activation of osteoclasts,and inhibit bone resorption of osteoclasts.Part III Theaflavin-3,3’-digallate inhibit RANKL-induced activation of osteoclasts by regulating ERK signaling pathwayObjective: To evaluate the effect of TFDG on the signaling pathways of NF-κB,PI3k/Akt and MAPK during the procedure of RANKL-induced activation of osteoclasts.Methods: We used mice RAW264.7 cell line as the object of the study.All cells were classified into two groups: control group and TFDG group.RAW264.7 cells were plated on 6-well plates at the density of 5′105 cells/well.When they were fully confluence,cells were pre-treated with or without 1 μM TFDG for 4 hours and then stimulated with 50 ng/ml RANKL for 0,5,15,30 or 60 minutes.Western blot was performed to evaluate the expression of p65,P-p65,IκBα,P-IκBα,AKT,P-AKT,JNK,P-JNK,p38,P-p38,ERK,P-ERK,MEK and P-MEK.To further confirm the inhibitory effect of TFDG on the ERK signaling pathway,we treated RAW264.7 cells with various concentrations of TFDG(0,0.01,0.1,or 1 μM)for 4 hours,and then treated them with RANKL for 15 minutes.Western blot was then performed to evaluate the expression of ERK and P-ERK.In addition,to explore the effect of TFDG on the downstream regulator MMP9 and NFATc1,RAW264.7 cells were pre-treated with or without 1 μM TFDG for 4 h,and then stimulated with 50ng/ml RANKL for 0,1 or 3 days.Subsequently,the cells were lysed and collected for Western blot analysis of MMP9 and NFATc1.Results: Western blot analysis showed that TFDG significantly inhibited RANKL-induced activation of ERK signaling pathway while it had little effect on activation of NF-κB and PI3k/Akt,JNK and p38 signaling pathways.Further study presented that the inhibition effect of TFDG on ERK signaling pathway was concentration-dependent.TFDG further inhibited the expression of downstream factor MMP9 and NFATc1 whereas it had no significant effect on activation of upstream factor MEK.Conclusion: TFDG inhibited RANKL-induced activation of osteoclasts by inhibiting ERK signaling pathway and the expression of its downstream factor MMP9 and NFATc1. |
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