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Applications of ROCK2 inhibitor in preparing medicines for treating inflammatory bowel diseases

An inflammatory bowel disease and inhibitor technology, which is applied to ROCK2 inhibitors and the application field of preparing inflammatory bowel disease medicines, can solve the problems of unclear immune response effect and other problems

Inactive Publication Date: 2017-08-08
SHANGHAI TENTH PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its expression in IBD tissues and its role in the immune response in the process of intestinal mucosal inflammation are still unclear.

Method used

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  • Applications of ROCK2 inhibitor in preparing medicines for treating inflammatory bowel diseases
  • Applications of ROCK2 inhibitor in preparing medicines for treating inflammatory bowel diseases
  • Applications of ROCK2 inhibitor in preparing medicines for treating inflammatory bowel diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1. Determination of ROCK2 activity

[0017] 1.1 Extraction of total tissue protein

[0018] 1) Put the intestinal mucosal tissue into an EP tube, add an appropriate amount of IP lysate into the tube, and grind it on ice with a grinding rod until no formed tissue can be seen, then place it on ice for 2 hours.

[0019] 2) After fully lysing, centrifuge, centrifugation conditions: 12000rpm, 4°C, 5min. After the centrifugation is over, carefully take out the EP tube, and carefully transfer the supernatant liquid to another EP tube with a pipette gun.

[0020] 1.2 Determination of protein concentration

[0021] 1) Make a standard curve according to the instructions of the BCA kit. The protein standard curve formulation table is as follows:

[0022] Hole number 0 1 2 3 4 5 6 7 Protein standard solution (μl) 0 1 2 4 8 12 16 20 Deionized water (μl) 20 19 18 16 12 8 4 0 Corresponding protein content (μg) 0 0.5 1.0 ...

Embodiment 2

[0036] Example 2. Enzyme-linked immunosorbent assay (enzyme-linkedimmuno sorbentassay, ELISA)

[0037] (1) Dilute 200× CaptureAntibody (coating antibody) to 1× in the sample tank with coating buffer. Use a pipette to transfer 50 μl of 1× CaptureAntibody to a 96-well plate, and shake the 96-well plate slowly to ensure that the liquid surface covers the bottom of the well. Place in 4°C refrigerator overnight.

[0038] (2) On the second day, the liquid in the wells was removed, and the wells were washed 3 times with buffer solution, each time the buffer solution stayed in the wells for 3 minutes.

[0039] (3) Transfer 100 μl of blocking solution (blocking buffer) to a 96-well plate with a pipette, and shake in a shaker at room temperature for 1 hour.

[0040] (4) After washing the wells according to step (2), dilute the protein standard with blocking solution according to the following concentration gradient: 1000ng, 500ng, 250ng, 125ng, 64ng, 32ng and 16ng. Transfer 50 μl of ...

Embodiment 3

[0046] Example 3. TNBS-induced colitis model in mice and in vivo treatment with SLx-2119

[0047] (1) The Balb / c mice to be tested were fasted for 24 hours and divided into four groups: A, B, C, and D groups; wherein, the Balb / c mice were a commercially available commodity.

[0048] (2) Mix 5% TNBS and 100% ethanol at a ratio of 1:1 to form an appropriate amount of TNBS mixture, and store it in a refrigerator at 4°C in the dark.

[0049] (3) Prepare 1.25% sodium pentobarbital and anesthetize the mice to be tested.

[0050] (2) Grab the tail of the anesthetized mouse, slowly insert the enema tube connected with the syringe from the anus of the mouse to 4 cm, and slowly inject 150 μl of TNBS mixture into the mice of the two groups A and B, while the other group Two groups were infused with 150μl 50% ethanol.

[0051] (4) Lift the mouse upside down for 30 seconds, put the mouse back into the cage, and restore the normal food and water supply.

[0052] (5) After waking up, the ...

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Abstract

The invention provides applications of a ROCK2 inhibitor in preparing medicines for treating inflammatory bowel diseases. By discovering the effects of the ROCK2 activity level in the intestinal inflammation, the invention provides applications of a ROCK2 activity selective inhibitor SLx-2119 in preparing medicines for treating inflammatory bowel diseases.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to the application of ROCK2 inhibitors in the preparation of inflammatory bowel disease medicines. Background technique [0002] Inflammatory bowel disease (BD), including Crohn's disease (CD) and ulcerative colitis (UC), is a chronic nonspecific inflammatory gastrointestinal disease. The pathogenesis and mechanism of IBD are still unclear, and it may be related to persistent intestinal infection, impaired intestinal mucosal barrier, immune regulation disorder in mucosal tissue, genetic susceptibility and environmental factors. The disease has a long course of disease and is prone to relapse, seriously affecting the quality of life of patients, and even causing disability, causing a heavy social medical and economic burden. In recent years, the number of IBD patients in my country has shown an obvious upward trend. Due to the differences in living environment, dietary structure...

Claims

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Application Information

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IPC IPC(8): A61K31/517A61P1/04A61P1/00
CPCA61K31/517
Inventor 刘占举杨文静周广玺余琳余天名方蕾蕾陈良
Owner SHANGHAI TENTH PEOPLES HOSPITAL
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