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Method for preparing medicine for reducing coupling degree of TRPV4 and NOX2

A drug and coupling technology, applied in the field of chemical medicine, can solve the problems of strong side effects and off-target, and achieve the effect of small side effects, strong targeting, and prevention of thrombosis

Pending Publication Date: 2021-02-09
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

All of the above drugs have experimental evidence. Although they have a certain improvement effect on oxidative stress, developing drugs for a single target is prone to off-target and strong side effects. Drugs for stress are important

Method used

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  • Method for preparing medicine for reducing coupling degree of TRPV4 and NOX2
  • Method for preparing medicine for reducing coupling degree of TRPV4 and NOX2
  • Method for preparing medicine for reducing coupling degree of TRPV4 and NOX2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: In vitro coupling activity test of compounds

[0068] Experimental method: HEK-293 cells co-transfected with TRPV4 and NOX2 or co-transfected with AQP1 and NOX2 were treated and planted in the center of the confocal small dish. When the confluence of the cells reached about 80%, DMSO (1‰) was used respectively. Tretinib (10nM), L755507 (10μM), Gambogic acid (1μM), CB-839 (1μM), Bohemine (1μM), M12 (10nM) were incubated for 24 hours, fixed with 4% paraformaldehyde for 20 minutes, Permeabilize with PBS containing 0.1% Triton X-100 for 10 minutes, and block with PBS containing 5% BSA for half an hour.

[0069] Cells were then stained with TRPV4-550 and NOX2 as primary and conjugated secondary antibodies (invitrogen) and with DAPI (Vector Laboratories, Burlingame, CA, USA). Shoot with a laser confocal microscope, and calculate the Fret value. For specific results, see Figure 5 and Table 1.

[0070] Table 1 Effects of different compounds on the coupling of TR...

Embodiment 2

[0076] Example 2: Exploring the Effect of Compounds on Intracellular ROS Levels

[0077] Experimental method: The isolated obese mouse primary endothelial cells were treated and planted in the center of the confocal small dish. When the confluence of the cells reached about 80%, they were incubated with 10Nm M12 for 24 hours, and the medium was discarded and washed with NPSS. After three trips, cells were incubated with NPSS containing CM-H2DCFDA in a dark environment in an incubator at 37 °C. Then use a laser confocal microscope to shoot, and use image J to calculate the fluorescence intensity statistically, which can reflect the level of intracellular ROS. For specific results, see Image 6 and Table 2.

[0078] The influence of table 2 compound on intracellular ROS level

[0079] CM-H2DCFDA fluorescence intensity (multiple compared with DIO) Unincubated DIO group 1.000000 Incubate with M12 0.496864

[0080] Where: DIO means diet-induced obesi...

Embodiment 3

[0082] Example 3: Exploring the effects of compounds on the cellular structure of obese mice:

[0083] Experimental method: Isolate the primary endothelial cells of obese mice, plant them in the center of the confocal small dish after treatment, and when the confluence of the cells reaches about 80%, incubate with 10nM M12 for 24 hours, discard the medium, and wash three times with NPSS Finally, fix with 4% paraformaldehyde for 20 minutes, permeabilize with PBS containing 0.1% Triton X-100 for 10 minutes, and block with PBS containing 5% BSA for half an hour. Cells were incubated with NPSS mixed with rhodamine-Phallotoxins (invitrogen) for half an hour and stained with DAPI (Vector Laboratories, Burlingame, CA, USA). Images were taken with a confocal laser microscope. For specific results, see Figure 7

[0084] Where: BSA refers to bovine serum albumin. PBS is a buffer solution. Rhodamine-Phalloidin is a high-affinity probe of F-actin, which can be used to observe the ch...

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Abstract

The invention discloses a method for preparing a medicine for reducing a coupling degree of TRPV4 and NOX2, and belongs to the technical field of chemical medicines. According to the invention, entrectinib, L755507, gambogic acid and M12 compounds are used as active ingredients and applied to preparing the medicine for reducing the coupling degree of TRPV4 and NOX2, anti-oxidative stress and vascular permeability, in order to realize a reduced coupling degree in obese patients with excessively coupled TRPV4 and NOX2, so that the two excessively coupled proteins are decoupled, and the normal state is recovered, thus oxidative stress and vascular permeability of the obese patients are recovered to normal levels.

Description

technical field [0001] The invention specifically relates to a method for preparing a medicine for reducing the coupling degree of TRPV4 and NOX2, and belongs to the technical field of chemistry and medicine. Background technique [0002] Reactive oxygen species (ROS) are a series of intermediate products derived from the reduction process of molecular oxygen, mainly including superoxide anion (·O2-), hydrogen peroxide (H2O2), hydroxyl radical (·OH), hypochlorous acid (HOCl ) etc. (Antioxid Redox Signal, 2015, 23, 514-32). Among them, free radicals refer to any substances with unpaired electrons, which are easy to react with healthy molecules. They can be produced in different mechanisms in cells and can proliferate indefinitely in the presence of oxygen. Once these free radicals reach a high Concentrations can cause oxidative damage to tissues or organs (Biomed Res Int, 2014, 831-841). In the mitochondrial respiratory chain, free radicals are produced as by-products of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/496A61P39/06A61P9/10A61P7/02A61P39/00A61P25/28A61P43/00A61K38/08A61K31/255
CPCA61K31/496A61P39/06A61P9/10A61P7/02A61P39/00A61P25/28A61P43/00A61K38/08A61K31/255
Inventor 马鑫唐春雷高梦茹韩婧
Owner JIANGNAN UNIV
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