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Application of LiCl in preparation of drug for resisting myocardial apoptosis induced by excessive accumulation of lipids

A technology of excessive accumulation and myocardial apoptosis, applied in the application field of myocardial apoptosis drugs, to achieve the effect of broad scientific research and clinical development prospects

Inactive Publication Date: 2016-04-20
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the mechanism of cell death caused by excessive lipid accumulation and high glucose and ischemia-reperfusion is not the same, so the search found that the role and influence of lithium chloride on cardiomyocyte apoptosis induced by excessive lipid accumulation, or its potential Whether it is used in the development of anti-myocardial apoptosis drugs related literature or patents, there are no reports at home and abroad

Method used

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  • Application of LiCl in preparation of drug for resisting myocardial apoptosis induced by excessive accumulation of lipids
  • Application of LiCl in preparation of drug for resisting myocardial apoptosis induced by excessive accumulation of lipids
  • Application of LiCl in preparation of drug for resisting myocardial apoptosis induced by excessive accumulation of lipids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Pretreatment with different concentrations of LiCl has an effect on the morphology of cardiomyocytes that are normally cultured and induced by PA to induce apoptosis.

[0029] Preparation of LiCl storage solution: Dissolve LiCl powder in three-distilled water to prepare a 10 mol / l storage solution, adjust the pH to 7.4, filter with a 0.22μm microporous membrane, and then aliquot and store at -20°C.

[0030] Contains 5% CO at 37°C 2 Under the circumstance, use DMEM / 10% fetal bovine serum medium to culture H9c2 cardiomyocytes for use. Before performing morphological observation, the cells in the logarithmic growth phase should be counted with a hemocytometer according to 8×10 4 Cells / ml were seeded in small dishes, pre-divided into BSA control group, PA alone treatment group and BSA+LiCl, PA+LiCl group.

[0031] After the cells grew to a coverage rate of 90%, the drug-added group was pretreated with LiCl at concentrations of 10mM, 20mM, 40mM, and 60mM respectively for...

Embodiment 2

[0033] Example 2: Hoechst33258 staining combined with fluorescence microscopy to detect the effect of LiCl (40mM) on the chromatin condensation of H9c2 cells caused by PA.

[0034] In the case of PA alone treatment for 12h or LiCl pretreatment for 1h and then PA treatment for 12h, Hoechst33258 staining after fixing H9c2 cells is as follows:

[0035] (1) Wash the culture plate once with PBS, aspirate and discard the PBS in a fume hood, and add 4% paraformaldehyde for fixation for 10 minutes.

[0036] (2) Wash twice with PBS, 2min / time

[0037] (3) Add 200ul of Hoechst33258 staining solution (2ug / ml) to each well to fully cover the cells, and incubate at 37°C for 1h. Incubate at a temperature suitable for cell culture for 20-30 minutes.

[0038] (4) Wash twice with PBS, and finally leave 100ul PBS in each well to prevent drying out.

[0039] (5) Ultraviolet light excitation and detection under a fluorescence microscope.

[0040] (6) Judgment of the results: normal cell nuclei were stained ...

Embodiment 3

[0042] Example 3: Flow cytometry to detect the effect of LiCl at a concentration of 40 mM on the apoptosis rate of H9c2 cardiomyocytes induced by PA.

[0043] In the cultured H9c2 cardiomyocytes, 40mM LiCl was added for pretreatment for 1h and then PA was added for another 12h to induce myocardial apoptosis. After AnnexinV-FITC / PI staining, the apoptotic cells were detected by flow cytometry, and the apoptosis rate was counted. The specific method is as follows:

[0044] (1) Cell collection: After the H9c2 cardiomyocytes are treated with drugs, they are digested with 0.25% trypsin without EDTA, suspended, and collected in a 10ml centrifuge tube. The number of cells per sample is (1~5)×10 6 Centrifuge at 900g / min for 5 min, and discard the culture solution.

[0045] (2) Wash once with 1mlstainingbuffer, 900g / min, centrifuge for 5min.

[0046] (3) Resuspend the cells with 50μl bindingbuffer, add 5μl FITC, incubate in the dark for 10-15min at room temperature, flick and mix every 5 minu...

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Abstract

The invention discloses an application of LiCl in preparation of a drug for resisting myocardial apoptosis induced by excessive accumulation of lipids. Experiments prove that LiCl can effectively inhibit myocardial apoptosis induced by long-chain saturated fatty acid palmitate, specifically, LiCl inhibits cutting of palmitate induced apoptosis executor caspase 3 precursors, and the apoptosis rate of cells and pyknosis of nucleus chromatin are remarkably reduced; the experiments further determine that 40 mM concentration can be taken as drug concentration for effectively and specifically inhibiting palmitate induced H9c2 myocardial apoptosis by LiCl, and LiCl is expected to be applied to the preparation of the drug for treating the myocardial apoptosis induced by excessive accumulation of the lipids accompanied by diseases such as obesity, diabetes mellitus and the like in clinical research and has broad scientific research and clinical development prospect.

Description

Technical field [0001] The invention relates to the pharmaceutical application of an antidepressant inorganic lithium compound-lithium chloride (LiCl), in particular to the application of lithium chloride in the preparation of a drug for preventing myocardial apoptosis induced by excessive lipid accumulation. Background technique [0002] In obesity and related diseases, excessive accumulation of lipids in cardiomyocytes can lead to myocardial apoptosis. Cardiomyocytes are the main unit of the structure and function of the heart. Their apoptosis can lead to serious heart-related diseases. Studies have shown that cardiomyocyte apoptosis is closely related to myocarditis, arrhythmia, coronary heart disease, heart failure, and myocardial infarction. contact. Long-chain saturated free fatty acids, especially palmitate (PA), also known as palmitic acid, are the main factors leading to lipid apoptosis. Looking for potential drugs that inhibit palmitic acid-induced myocardial apoptosi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K33/14A61P9/00
CPCA61K33/14
Inventor 尹德领赵静张尚立常芬
Owner SHANDONG UNIV
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