Icariin Ameliorates Thioacetamide-Induced Osteoclast Differentiation Through Inhibiting RANKL-p38/ERK-NFAT Pathway

Objective In this study,a thioacetamide(TAA)-induced bone loss rat model was established to analyze the protective effect of Icariin(ICA)on growth,bone metabolism,bone microstructure and osteoclast-related molecules.The osteoclast differentiation of RAW264.7 cells was induced by TAA to explore the protective mechanism of ICA on TAA-induced osteoclast differentiation.Methods 1.TAA and ICA were docked with tartrate-resistant acid phosphatase(TRAP)proteins respectively based on AutoDock software.2.SD rats were divided into groups and treated:Control group,TAA group(intraperitoneal injection of 300 mg/kg TAA),ICA group(oral treatment with 600 mg/kg ICA),TAA+ICA group(intraperitoneal injection of 300 mg/kg TAA and oral treatment with 600 mg/kg ICA)were administered every other day for 6 weeks.The status was observed,and the weight was recorded.3.Mandibular blood was collected from SD rats in each group,and serum was separated to detect the changes of alkaline phosphatase(ALP),type Ⅰ collagen crosslinked Ntelopeptides(NTX-I),calcium(Ca),phosphorus(P)and magnesium(Mg)in serum of SD rats.4.Hematoxylin-eosin(HE)and TRAP staining were used to observe the pathological changes and and injuries of femur in each group.5.The damage of femur structure in each group were detected by Three-point bending test and Micro-CT.6.Q-PCR was used to detect the effect of the expression of genes related to femoral bone differentiation in each group.7.Western blot was used to detect the effect of ICA on the expression of osteoclast differentiation-related protein in each group.8.CCK-8 assay was used to detect the effects of TAA and ICA on RAW264.7 cell proliferation.9.TRAP staining was used to detect the effect of ICA on osteoclast differentiation of RAW264.7 cells induced by TAA.10.Western blot and immunofluorescence assay were used to detect the effect of ICA on the expression of osteoclast differentiation-associated proteins in RAW264.7 cells induced by TAA.Results 1.Molecular docking results showed that TAA and ICA had binding sites to TRAP proteins,respectively.2.Compared with the control group,body weight and femur length were decreased in the TAA group,and significantly increased in the TAA+ICA group.3.Serum biochemical test results showed that the activity of ALP and NTX-I were significantly increased,and the concentrations of Ca,P and Mg were significantly decreased in the TAA group compared with the control group.The activity of ALP in the TAA+ICA group was significantly recovered in the early stage,and the concentration of NTX-I and minerals in the TAA+ICA group was significantly recovered in the late stage.4.Histopathological staining results showed that the trabecular structure was disordered,the trabecular area of the TAA group was significantly reduced,and the TRAP positive area was increased in TAA group compared with the control group,and the TAA+ICA group was recovered.5.The results of Three-point bending test and Micro-CT test showed that the elastic modulus and maximum load were significantly decreased,bone parameters of bone trabeculae,such as bone mineral density(BMD),trabecular pattern factor(Tb.Pf),structure model index(SMI),trabecular thickness(Tb.Th)and trabecular number(Tb.N)were significant differences in TAA group compared with the control group,and the TAA+ICA group was recovered.6.Q-PCR results showed that the gene expression of TRAP and PPAR-y was significantly increased in the TAA group compared with the control group,and the expression of these genes was significantly decreased in the TAA+ICA group.7.Western blot results showed that compared with the control group,the expression of osteoclast-related protein TRAP and cathepsin K were significantly increased in TAA group,and significantly decreased in the TAA+ICA group compared with the TAA group.In addition,the protein expression of RANK,RANKL,p38,ERK,c-Fos and NFATc1 was significantly activated in the TAA group,and the protein expression was inhibited to varying degrees in the TAA+ICA group compared with the TAA group.8.CCK-8 results showed that the proliferation of RAW264.7 cells was not significantly inhibited when TAA concentration was 0.8 mg/mL,and the cell proliferation was promoted most significantly when ICA concentration was 1.0 μM.9.TRAP staining results showed that there were a lot of TRAP positive cells in the RANKL group,and the formation of TRAP positive cells could also be induced by TAA,and the positive cells were reduced in the TAA+ICA group compared with the TAA group.10.Western Blot and immunofluorescence staining showed that expressions of TRAP,Cathepsin K c-Fos and NFATc1 in RAW264.7 cells were significantly up-regulated,and NFATc1 nuclear translocation was significantly increased in TAA group,while the expression of these proteins and NFATc1 nuclear translocation were significantly decreased in the TAA+ICA group.Conclusion 1.ICA alleviated the reduction of weight and femur length,growth inhibition induced by TAA in SD rats.2.ICA ameliorated TAA-induced excessive bone resorption and bone structure damage in rat femur.3.ICA ameliorated the bone resorption of rat femur induced by TAA through inhibiting RANKL-p38/ERK-NFAT signaling pathway.4.ICA inhibited the production of TRAP positive cells in TAA-induced RAW264.7 cells.5.ICA inhibited TAA-induced osteoclast differentiation of RAW264.7 cells by reducing NFATc1 protein expression and nuclear translocation.