Application of artesunate to preparation of anti-osteoclast differentiation medicine
An osteoclast and artesunate technology, which can be used in drug combinations, bone diseases, pharmaceutical formulations, etc., can solve problems such as osteonecrosis of the jaw and high price
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experiment example 1
[0027] Experimental example 1. The cell survival effect of artesunate on RAW264.7 cells:
[0028] RAW264.7 cells 1×10 4 or 1×10 3 Each well was planted in a 96-well plate, and the drug artesunate was added after overnight at a concentration of 3.125 μM, 6.25 μM, and 12.5 μM. 3 replicate wells in each group. After incubating for 8h, 24h, 48h, and 7d respectively, remove the supernatant, add 0.5mg / ml MTT 100μl to each well, incubate in a 37°C incubator for 4h, discard the supernatant, add DMSO 150μl, shake for 10 minutes , Measure the absorbance at 450 nm with a Genios Pro Tecan microplate reader.
[0029] Depend on figure 1 It can be seen that the in vitro concentration ≤ 12.5 μM has no significant effect on the survival of RAW264.7 cells.
experiment example 2
[0030] Experimental example 2. The effect on the differentiation of osteoclast precursor Raw264.7 cells into osteoclasts:
[0031] Select RAW264.7 cells in good growth state and press 1×10 3 Inoculate in a 96-well plate at a density of / ml, and add 100 μl / well of DMEM medium containing 10% FBS, 100 U / ml penicillin G and 100 μg / ml streptomycin, at 37°C, 5% CO 2 Incubator incubation. After the cells were stabilized overnight, in addition to the negative control group (Control group), the positive control group (RANKL group) and the drug group were all added RANKL with a final concentration of 100 ng / ml. 6.25 μM and 12.5 μM (respectively Art1.56 μM group, Art 3.125 μM group, Art 6.25 μM group and Art 12.5 μM group), each group had 3 replicate wells. Change the medium every 2 days. On the 4th day of induction, TRAP staining was observed, photographed under a microscope, and counted as osteoclasts with more than 3 nuclei positive for TRAP. Such as figure 2 .
experiment example 3
[0032] Experimental example 3. Effect on the differentiation of mouse bone marrow macrophages (BMMs) into osteoclasts:
[0033] Take the femur and tibia of C57BL / 6 mice, peel off the muscle and cut off both ends, use a syringe to absorb ice-cold a-MEM and repeatedly wash the bone marrow cavity of the femur and tibia until the bone marrow cavity turns white. %CO 2 After 30 minutes in the incubator, the supernatant suspension was taken. cells by 2 x 10 5 / ml density inoculated in 96-well plate, in addition to the negative control group (Control group), the positive control group (RANKL group) and the drug group were all added M-CSF and RANKL, the final concentration was 50ng / ml, and the drug group was added green Artesunate 1.56 μM, 3.125 μM, 6.25 μM and 12.5 μM (Art1.56 μM group, Art 3.125 μM group, Art 6.25 μM group and Art 12.5 μM group respectively), each group had 3 replicate wells. Change the medium every 2 days. On the 4th day of induction, TRAP staining was observed,...
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