Application of andrographolide in inhibition of osteoclast formation and activation
A technology of andrographolide and osteoclasts, which is applied in the field of biomedicine, can solve problems such as bone loss diseases where andrographolide has not been found, and achieve the effects of improving bone mass loss, preventing osteoporosis, and inhibiting differentiation and maturation
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Embodiment 1
[0041] Example 1: Effect of andrographolide on osteoclast differentiation of mouse bone marrow mononuclear cells
[0042] The experimental method is as follows:
[0043] 1) Take a C57BL\6 mouse, kill it by dislocation of the neck, soak in 75% alcohol for 5 minutes;
[0044] 2) Separate the long bones of the two hindlimbs and the humerus of the forelimbs under sterile conditions;
[0045] 3) Remove the metaphysis in the ultra-clean bench, extract the a-MEM medium with a sterile syringe and gently rinse the bone marrow cavity repeatedly until the bone marrow cavity turns white;
[0046] 4) Centrifuge at 1200r / min for 5min after filtration with a 100um cell filter;
[0047] 5) Discard the supernatant, add 10 times the volume of sterile erythrocyte lysate, pipette to mix, lyse on ice for 5 minutes, centrifuge at 1000r / min for 5min, discard the red supernatant to remove erythrocytes;
[0048] 6) Resuspend the pellet with serum-free a-MEM medium and wash twice;
[0049] 7) Resus...
Embodiment 2
[0053] Example 2: Effect of andrographolide on the expression of genes related to osteoclast differentiation
[0054] The experimental method is as follows:
[0055] According to the operation in Example 1, primary mouse mononuclear cells were extracted for osteoclast differentiation, and the cells were divided into 4×10 5 / mL were inoculated in 24-well plates, and the experiments were grouped as follows: the negative control group (Control group) only added 40ng / mL M-CSF, the positive control group (RANKL group) and the drug group both added 40ng / mL M-CSF and 100ng / mL RANKL, the drug group was added with 2 μM andrographolide at the same time, and each group had 3 replicate wells. After induction for 3 days, the medium was discarded, washed with PBS, and Trizol was added to lyse the cells, the total RNA in the cells was extracted, and cDNA was prepared by reverse transcription. The system is shown in Table 1 below:
[0056] Table 1
[0057]
[0058] Reverse transcription...
Embodiment 3
[0070] Example 3. Detection of the effect of andrographolide on the activity of the GLS promoter induced by ERRa / PGC1-β
[0071] The experimental method is as follows:
[0072] 293T cells were divided into 2×10 5 cells / mL were inoculated into 96-well plates, and after the cells adhered to the wall, the pGL3 reporter plasmid containing the GLS gene promoter was co-transfected into the cells with the ERRa and PGC-1β overexpression plasmids, and the phRL-TK plasmid was transfected at the same time as an internal reference Plasmid, add andrographolide of different drug concentration after transfection 6h, detect after 18h, detection method is as follows:
[0073] 1) Discard the culture medium, add 100 μl PBS to the wells inoculated with cells in the 96-well plate to wash once, operate gently, and aspirate the PBS completely as much as possible;
[0074] 2) After washing, add 50 μl 1×PLB, shake and lyse on a shaker at room temperature for 20 minutes, and observe under a microscop...
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