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Application of natural small molecular compound in inflammation and tumor resistance

A small molecule compound, small molecule technology, applied in the field of biomedicine

Inactive Publication Date: 2015-05-20
YANBIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report of inhibiting NF-κB activity

Method used

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  • Application of natural small molecular compound in inflammation and tumor resistance
  • Application of natural small molecular compound in inflammation and tumor resistance
  • Application of natural small molecular compound in inflammation and tumor resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Implementation 1: Cell Culture Technology

[0029] After the cells entered the logarithmic growth phase, they were digested with 0.25% trypsin, and the number of cells was 5 × 10. 4 The density was evenly divided into 5 cell culture dishes with a diameter of 6 cm, and 3 ml of DMEM medium (containing inactivated 10% calf serum, 100 U / L penicillin) was added to each culture dish and placed in a constant temperature incubator (37 ° C, 5% CO 2, normal oxygen concentration and saturated humidity). After the cells were completely attached to the wall, the cells were divided into negative control group, positive control group, and concentration gradient group (1 μM, 5 μM, 20 μM). After culturing for 4 hours, the positive control group and concentration gradient group were treated with TNFα 3 μl (final concentration 10 ng / ml, Hela cells were treated with TNFα, RAW264.7 cells were treated with LPS), and cultured for 15 minutes.

Embodiment 2

[0030] Implementation 2: Westernblot method

[0031] (1) Preparation of protein samples, including extraction of total protein and nuclear protein.

[0032] (2) Protein denaturation: 15 μl of sample protein was taken and added to corresponding tubes, 5 μl of loading buffer was added, and boiled for 5 minutes.

[0033] (3) Place the boiled protein sample on an SDS-PAGE gel for electrophoresis.

[0034] (4) After the electrophoresis stops, the protein sample on the gel is transcribed onto a methanol-soaked PVDF membrane.

[0035] (5) Put the PVDF membrane into 5% skimmed milk and seal it at room temperature for 1 hour.

[0036] (6) Primary antibody hybridization, secondary antibody hybridization.

[0037] (7) ECL luminescent kit detection.

Embodiment 3

[0038] Implementation 3: PCR method

[0039] (1) Total RNA extraction:

[0040] The drug-treated cells were repeatedly washed with PBS three times, 1ml of Ttiol was added, and the cells were blown repeatedly with a syringe to lyse them all. Add 200 μl CCl 4 The solution was separated into layers, and centrifuged at 12000 r for 15 min at 4°C. Take the upper layer into a new tube, add 500 μl of isopropanol, mix well, and centrifuge at 12000r at 4°C for 10 minutes. Carefully discard the supernatant, add 1ml of 75% ethanol to the tube containing RNA, shake, and centrifuge at 12000r at 4°C for 5min. Fully discard the supernatant, and place the ultra-clean bench to ventilate and dry. After being thoroughly dried, add 30 μl DEPC-DDW to dissolve it completely.

[0041] (2) Using RNA as a template to synthesize cDNA by reverse transcription.

[0042] Table 1

[0043]

[0044] RT-PCR reaction conditions:

[0045]

[0046] (3) PCR reaction: double-stranded DNA is synthesize...

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Abstract

The invention relates to application of a natural small molecular compound in inflammation and tumor resistance. The small molecular compound is separated from a mesquite amorpha fruticosa fruit and is a novel active monomer named Amorfrutin A. Through a series of researches, Amorfrutin A blocks transfer of p50-p65 from cytoplasm to nucleus and inhibits expression of genes such as proinflammatory genes, cell survival genes, proliferation genes, invasive genes, angiogenesis genes and the like as well as expression of up-regulated apoptosis genes caused by activation of NF-kB. The invention provides a method for treating abnormal activation related disease by changing the activity of the transcription factor NF-kB. Moreover, Amorfrutin A has an obvious inhibiting effect on expression of HIF-1alpha induced by CoCl2.

Description

Technical field: [0001] The invention belongs to the field of biomedicine, in particular to the application of Amorfrutin A, a natural small molecule compound extracted from Amorpha fruit in anti-inflammatory and anti-tumor. The compounds are particularly useful in the treatment of inflammatory diseases and cancers involving the mediation of NF-κB activation. Background technique: [0002] It is well known that NF-κB coordinates the expression of multiple functional genes and plays critical roles in acute and chronic inflammatory responses, immune regulation, cell survival and proliferation. Aberrant activation of NF-κB is associated with a variety of human diseases, including cancer. Therefore, research strategies targeting NF-κB have attracted more and more attention. [0003] NF-κB is a family of transcription factor proteins, including five subunits: Rel (cRel), p65 (RelA), RelB and p50 (NF-κB1), p52 (NF-κB2). They all have an N-terminal Rel homology domain (RHD) and ...

Claims

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Application Information

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IPC IPC(8): A61K31/192A61P29/00A61P35/00
Inventor 金学军李晶埈石慧马娟李静米春柳
Owner YANBIAN UNIV
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