Study On The Molecular Mechanism Of Schisantherin B Regulating Osteoclasts In The Treatment Of Osteoporosis Through NF-κB Signal Pathway

Objective:To explore the Molecular Mechanism of Schisantherin B regulating osteoclasts in the treatment of Osteoporosis through NF-κB signal PathwayMethods:(1)In vitro experiment:(1)CCK8 test was used to determine the effects of different concentrations of Schisantherin B(0,0.03,0.1,0.3,1,3,10,30μM)on the growth activity of mouse bone marrow macrophages.(2)The effects of different concentrations of Schisantherin B(0,0.3,1,3,10μM)on the differentiation of mouse bone marrow macrophages and raw cells were detected,and TRAP staining was performed.(3)The actin ring test of mouse bone marrow macrophages treated with different concentrations of Schisantherin B(0,0.3,1,3,10μM)was carried out to observe the effect of different concentrations of Schisantherin B on the fibrous ring of osteoclasts.(4)Bone slices were treated with different concentrations of Schisantherin B(0,3,10μM)and the effects on bone resorption were observed.(5)CCK8 was used to determine the effects of different concentrations of Schisantherin B(0,0.03,0.1,0.3,1,3,10,30μM)on the growth and activity of mouse mesenchymal stem cells.(6)Different concentrations of Schisantherin B(0,0.3,1,3,10 μM)were used to detect the effect on the differentiation of mesenchymal stem cells,and ALP staining was performed.(7)Different concentrations of Schisantherin B(0,0.3,1,3,10μM)were used to detect the effect on the mineralization of mesenchymal stem cells,and silver nitrate mineralization staining was performed.(8)Western Blotting(Westernblot)was used to detect the effect of Schisantherin B on the related proteins of MAPK and NF-κB pathway.(9)Compare the effects of high concentration of Schisantherin B(10μM)on p65 nuclear transfer induced by RANKL.(10)Real-time fluorescence quantitative Q-PCR was used to detect the effect of Schisantherin B on the downstream genes and transcription factors of NF-κB pathway.(2)In vivo experiment:(1)Ovariectomized female mice were used to establish the model of osteoporosis in ovariectomized mice.(2)Intraperitoneal injection of Schisantherin B(20mg/kg),once every other day,for a total of 8 weeks.(3)After the mice were killed,the femurs of the mice were scanned and analyzed by Micro-CT for three-dimensional imaging and the related indexes were counted.(4)After decalcification,the femur specimens were made into wax blocks and sliced.(5)The sections were stained with hematoxylin-eosin and TRAP respectively,and the pictures were observed and collected under positive microscope.Results:1.The results of CCK8 detection showed that Schisantherin B had no toxic effect on the viability of BMMs and BMSCs cells within 30μM.2.With the increase of Schisantherin B concentration(0,0.3,1,3,10μM),the formation and differentiation of osteoclasts were significantly inhibited in a dose-dependent manner,but had no significant effect on the differentiation of osteoblasts.3.Westernblot results showed that Schisantherin B inhibited osteoclast formation by regulating the phosphorylation level of proteins related to NF-κB signal pathway induced by RANK.4.Real-time PCR results showed that the expression level of NFATc1 mRNA gene in osteoclasts treated with Schisantherin B decreased.5.The osteoporosis model of ovariectomized mice was established successfully.Compared with the control group,Schisantherin B can significantly improve osteoporosis.Conclusion: Schisantherin B can inhibit osteoclast formation and bone resorption induced by RANKL in a dose-dependent manner and inhibit the occurrence of osteoporosis in ovariectomized mice by regulating the expression of proteins and genes related to NF-κB signal pathway.