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Use of podophyllotoxin in activating bmp signaling pathway

A technology of podophyllotoxin and signaling pathway, which is applied in the field of application of podophyllotoxin in activating BMP signaling pathway, achieving good development and application prospects

Active Publication Date: 2019-05-07
SHANGHAI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are currently no reports of podophyllotoxin used in the treatment of osteoporosis, fracture healing, bone repair and other related diseases

Method used

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  • Use of podophyllotoxin in activating bmp signaling pathway
  • Use of podophyllotoxin in activating bmp signaling pathway
  • Use of podophyllotoxin in activating bmp signaling pathway

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: High-throughput screening and evaluation of compounds

[0026] 1. Compound preparation:

[0027] The Small Molecule Compound Library is owned by the applicant and stored in 100% dimethyl sulfoxide at a concentration of 1 mM.

[0028] 2. Experimental materials:

[0029] Free-Style 293-F cell suspension culture expression system was purchased from Invitrogen, including Free-Style293-F cells (Cat. No. R790-07), transfection reagent 293fectin (Cat. No. 12347500), culture medium Free-Style293Expression Medium (Cat. No.: 12338001) .

[0030] The luciferase reporter gene detection kit neolite Reporter Gene Assay System was purchased from PerkinElmer, Cat. No. 6016711.

[0031] Plasmid pGL4.33Luc2P / hID-1 was purchased from Promega.

[0032] Opti-MEM was purchased from Thermo Fisher, Cat. No. 11058021.

[0033] 96-well cell culture plates were purchased from PerkinElmer, Cat. No. 005182.

[0034] Luciferase detection kit was purchased from Promega, catalog numbe...

Embodiment 2

[0044] Example 2: Concentration Gradient Detection of BMP Signaling Pathway Activation

[0045] 1. Compound preparation:

[0046] Dissolve podophyllotoxin (12D8) in dimethyl sulfoxide, the initial concentration point is 100uM, make a 3-fold concentration gradient dilution, 10 concentration points, the concentrations are 100uM, 33.3uM, 11.1uM, 3.7uM, 1.23uM , 0.41uM, 0.137uM, 45.6nM, 15.23nM, 5.07nM, the control group was an equal volume of dimethyl sulfoxide.

[0047] 2. Experimental materials:

[0048] Free-Style 293-F cell suspension culture expression system was purchased from Invitrogen, including Free-Style293-F cells (product number R790-07), Free-Style 293-F cell liposome transfection reagent 293fectin (product number 12347500), culture Liquid Free-Style 293Expression Medium (Cat. No. 12338001).

[0049] The luciferase reporter gene detection kit neolite Reporter Gene Assay System was purchased from PerkinElmer, Cat. No. 6016711.

[0050]Plasmid pGL4.33Luc2P / hID-1 w...

Embodiment 3

[0063] Example 3: Detection of osteogenic differentiation marker alkaline phosphatase activity

[0064] 1. Compound preparation:

[0065] Podophyllotoxin (12D8) was dissolved in dimethyl sulfoxide with an initial concentration point of 10 uM, and diluted in a 2-fold concentration gradient with 8 concentration points. The control group was an equal volume of dimethyl sulfoxide.

[0066] 2. Experimental materials:

[0067] α-MEM culture medium was purchased from Thermo Fisher, Cat. No. 11095-072.

[0068] Common reagents such as ascorbic acid, β-glycerol phosphate and dexamethasone were purchased from Sigma.

[0069] DEA solution (50 mmol / L glycine and 1 mmol / L magnesium chloride, pH 10.5), glycine and magnesium chloride were purchased from Sinopharm.

[0070] PNPP solution was purchased from Sigma-Aldrich, catalog number: N7653.

[0071] BCA reagent solution A was purchased from Pierce Company, product number #23228.

[0072] BCA solution B was purchased from Pierce, Cat. ...

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Abstract

The invention provides application of podophyllotoxin in activation of a BMP signal pathway. It is found through luciferase activity screening that podophyllotoxin has significant activation on hID1 (human ID1) and SBE (Smad-binding element), and EC50 is 88 nM and 14 nM separately. It is found in primary human mesenchymal stem cell osteogenesis experiment detection that podophyllotoxin with the minimum concentration of 4 nM can promote activity of alkaline phosphatase. Therefore, podophyllotoxin can be used for preparing drugs for treating diseases related to osteogenic differentiation and has a good development and application prospect.

Description

technical field [0001] The present invention relates to a new application of podophyllotoxin, in particular to a new application of podophyllotoxin in preparing medicines for promoting osteogenesis and differentiation. Background technique [0002] Bone morphogenetic protein (BMP) is a member of the transforming growth factor (TGF-β) superfamily, and is a very important extracellular signaling molecule that promotes bone formation and induces osteoblast differentiation. BMPs can bind and activate specific receptors on the target cell membrane, and transmit specific signals through Smads. When BMP activates Smads, the activated Smads can transfer the TGF-β signal from the cell membrane to the nucleus, thereby inducing the expression of osteogenic genes in the nucleus. ID1 is a key regulatory protein of cell proliferation and differentiation, and a direct target gene of BMP / Smads signaling pathway. Therefore, the change of ID1 and Smads protein is an important means to detec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K31/365A61P19/10A61P19/00
CPCA61K31/365
Inventor 姜标杨光陈红莉曲志虎
Owner SHANGHAI TECH UNIV
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