Application of rutaecarpin derivative to preparation of medicine for treating osteoporosis
A technology of evodiamine and osteoporosis, which is applied in drug combinations, bone diseases, and pharmaceutical formulations to achieve the effect of inhibiting osteoclast differentiation and promoting osteoblast differentiation
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experiment example 1
[0044] Experimental example 1 Evodiamine derivatives up-regulate the expression of OPG
[0045] 1.1 Experimental method
[0046] Cell culture: UOP cells were cultured in 500 μg mL -1 G418 and 10% fetal bovine serum in McCoy's 5A medium; mouse osteoblast precursor cells MC3T3-E1 were cultured in α-MEM medium containing 10% fetal bovine serum; mouse mononuclear macrophage Raw264 .7 Cells were cultured in DMEM medium containing 10% fetal bovine serum; human osteosarcoma cells U-2OS were cultured in McCoy's 5A medium containing 10% fetal bovine serum. All cells were stored at 37°C, 5% CO 2 under conditions. All cells are maintained by the laboratory.
[0047] Determination of the up-regulation fold of the compound on the OPG expression up-regulation model: UOP cells in the logarithmic growth phase were divided into 5×10 4 The number of cells was seeded into 96-well clear bottom white plates (Costar). After the cells adhered to the wall, the original medium was removed, and R...
experiment example 2
[0057] Experimental example 2 Western blot analysis and enzyme-linked immunosorbent assay (ELISA)
[0058] 2.1 Experimental method
[0059] Western blot analysis: different concentrations of RUT (0, 1.0 and 10.0 μmol L -1 ) were applied to MC3T3-E1 cells and U-2OS cells for 24 hours, and then the cells were collected. The cells were lysed with RIPA cell lysate (Pulilai Gene Technology Co., Ltd.), and the total protein of the cells was extracted; the total protein concentration was measured with a BCA kit (Pierce), and each sample was adjusted to the same concentration according to the measured total protein concentration, and added A certain amount of 5×protein sample buffer, boiled for 10min. The prepared protein samples were subjected to SDS-PAGE electrophoresis, and semi-dry transfer to PVDF membrane; 5% skimmed milk powder was blocked for 1 h, and the primary antibody was incubated overnight at 4°C; the membrane was washed 3 times with TBST (10 min / time), and then incuba...
experiment example 3
[0066] Experimental Example 3 Osteoblast Differentiation Detection
[0067] 3.1 Experimental method
[0068] Alizarin red staining: 5×10 per well of MC3T3-E1 cells 5 The number of cells was inoculated into a 12-well plate, and the bone differentiation medium was replaced after the cells were fully adhered to the wall, and the medium was changed every other day. At the same time, different concentrations of RUT (1.0 μmol L -1 , 10.0μmol·L -1 ), after 21 days of induction, the medium was discarded, and the cells were washed twice with PBS. Cells were fixed with 95% ethanol for 10 min, and then rinsed 3 times with distilled water. Use 40mmol·L -1 Alizarin red S solution (pH 4.2) was used for staining at room temperature for 10 minutes, rinsed with distilled water for 3 times; then added hematoxylin for staining at room temperature for 1 minute, and rinsed with distilled water for 3 times. Placed under a microscope (Leica, DMIL) to take pictures, and used Image J software to...
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