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Application of Pin1 siRNA in preparation of targeted medicine for treating alcoholic cardiomyopathy

A technology of alcoholic cardiomyopathy and pin1, which is applied in gene therapy, drug combination, cardiovascular system diseases, etc., and can solve problems such as no targeted drugs for alcoholic cardiomyopathy

Inactive Publication Date: 2016-06-08
HARBIN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There are currently no targeted drugs for alcoholic cardiomyopathy

Method used

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  • Application of Pin1 siRNA in preparation of targeted medicine for treating alcoholic cardiomyopathy
  • Application of Pin1 siRNA in preparation of targeted medicine for treating alcoholic cardiomyopathy
  • Application of Pin1 siRNA in preparation of targeted medicine for treating alcoholic cardiomyopathy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Expression and activation of Pin1 in alcohol-treated cardiomyocytes

[0025] Firstly, the expression of Pin1 was analyzed after cardiomyocytes were exposed to different concentrations of alcohol (alcohol: 0, 50, 100 or 200 mM). Such as figure 1 a, b, from the results, it can be seen that ethanol can increase the mRNA and protein levels of Pin1 in a dose-dependent manner. In addition, ethanol dose-dependently ( figure 1 c) Increase the activity of Pin1.

Embodiment 2

[0026] Example 2: Pin1siRNA inhibits alcohol-induced cardiomyocyte apoptosis

[0027] method:

[0028] 1. Cell culture and cell transfection

[0029] Cardiomyocytes from neonatal mice were isolated. Heart tissue was minced and digested, sedimented, fibroblasts and endothelial cells were removed, and cardiomyocytes were cultured in collagen-coated culture dishes (approximately 1.5×10 5 cells). Pin1 siRNA (purchased from Invitrogen (Carlsbad)), Pin1 plasmid (purchased from Addgene (Cambridge)) and ScramblesiRNA were transfected using Lipofectamine RNAiMAX (Invitrogen) transfection reagent. Lipofectamine LTX (Invitrogen) was used according to the instructions. Cardiomyocytes (5×10 4 cells / well) were seeded into 24-well plates and grown overnight to approximately 80% confluency. Then the cells were co-cultured with 30 pmol siRNA or 500 ng plasmid for 48 hours, and the transfection efficiency was identified by western blot.

[0030] 2. Quantitative reverse transcription poly...

Embodiment 3

[0049] Example 3: Knockout of Pin1 (Pin1siRNA) reduces alcohol-mediated mitochondrial oxidative stress in cardiomyocytes

[0050] Alcohol-induced apoptosis is dependent on mitochondrial oxidative signaling, and downregulation of Pin1 protects against mitochondrial oxidative stress in diabetic patients. Therefore, we investigated whether Pin1 downregulation could suppress alcohol-induced mitochondrial oxidative stress in cardiomyocytes. In cardiomyocytes with downregulation of Pin1, mCyt.C, mitochondrial membrane potential, and mitochondrial reactive oxygen species (mROS) assays were performed with and without 200 mM alcohol.

[0051] method:

[0052] 1. Measurement of mitochondrial membrane potential

[0053] Mitochondrial membrane potential (Δψm) was assessed using the TMRE Mitochondrial Membrane Potential Assay Kit.

[0054] 2. Determination of mitochondrial cytochrome c release into the cytoplasm

[0055] Cytochrome C release from mitochondria to cytosol was determined ...

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Abstract

The invention discloses application of Pin1 siRNA in preparation of a targeted medicine for treating the alcoholic cardiomyopathy, and belongs to the field of prevention and treatment of the alcoholic cardiomyopathy. It is proved through experiments that Pin1 takes participate in the cardiac muscle cell apoptosis process caused by alcohol and high-dosage alcohol stimulates the expression and activity of Pin1. By means of Pin1 gene knock-out mediated by Pin1 siRNA, cardiac muscle cell apoptosis mediated by alcohol can be remarkably restrained; due to the over-expression of Pin1, the number of apoptosis muscle cells is further increased. It is further proved that Pin1 promotes mitochondria oxidative stress mediated by alcohol and restrains the expression of endothelial nitric oxide synthase. Through Pin1 gene knock-out mediated by Pin1 siRNA, mitochondria oxidative stress mediated by alcohol, the production decrease of NO and the level decrease of eNOS can be remarkably restrained, and therefore the occurrence and development process of the alcoholic cardiomyopathy is further delayed. An effective technological means is put forward to prevent and treat the alcoholic cardiomyopathy.

Description

technical field [0001] The invention relates to the new medical application of Pin1siRNA, in particular to its application in the preparation of targeted drugs for preventing and treating alcoholic cardiomyopathy, and belongs to the field of prevention and treatment of alcoholic cardiomyopathy. Background technique [0002] To this day, heart failure remains an important public health problem. Chronic heavy alcohol consumption is considered the leading cause of nonischemic dilated cardiomyopathy, known as "alcoholic cardiomyopathy" (ACM), in both men and women in the United States. Typically, asymptomatic ACM may develop symptomatic ACM with signs of heart failure if the alcohol consumption exceeds 90 grams per day for more than 5 years. [0003] In the asymptomatic ACM stage, the LV is usually dilated with increased LV mass and reduced or normal LV wall thickness. Pathologically, previous studies have shown a strong correlation between ACM and cardiomyocyte apoptosis. In...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K31/7088A61K38/52A61P9/00
CPCA61K48/005A61K31/7088A61K38/52C12Y502/01008
Inventor 王越红杨巍李子卓孙晶田娟娟
Owner HARBIN MEDICAL UNIVERSITY
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