阅读说明：本技术 Jmjd3抑制剂gsk-j1 hcl在制备治疗心肌纤维化药物中的用途 (Application of JMJD3 inhibitor GSK-J1 HCL in preparation of drugs for treating myocardial fibrosis ) 是由 刘新华 朱依谆 龙芬 于 2019-10-09 设计创作，主要内容包括：本发明属制药领域,涉及化合物GSK-J1 HCL在制药中的新用途,具体涉及组蛋白去甲基酶JMJD3抑制剂GSK-J1 HCL在制备治疗心肌纤维化药物中的用途。本发明通过建立的小鼠左冠状动脉结扎手术引起的心肌纤维化模型和Ang II诱导NRCFs的体外心肌纤维化模型实验,结果表明GSK-J1 HCL通过抑制JMJD3的表达降低胶原含量,减少纤维化相关蛋白的表达对心肌纤维化起到调控作用。所述化合物GSK-J1 HCL(C22H23N5O2.HCL)及其相关抑制剂可用于制备防治心肌纤维化疾病的药物。(The invention belongs to the field of pharmacy, relates to a new application of a compound GSK-J1 HCL in pharmacy, and particularly relates to an application of a histone demethylase JMJD3 inhibitor GSK-J1 HCL in preparation of a medicine for treating myocardial fibrosis. According to the invention, the established myocardial fibrosis model caused by mouse left coronary artery ligation operation and the in vitro myocardial fibrosis model experiment of Ang II induced NRCFs show that the GSK-J1 HCL can reduce the collagen content by inhibiting the expression of JMJD3 and reduce the expression of fibrosis-related protein to play a role in regulating and controlling myocardial fibrosis. The compound GSK-J1 HCL (C22H23N5O2.HCL) and related inhibitors thereof can be used for preparing medicines for preventing and treating myocardial fibrosis diseases.)

Use of JMJD3 inhibitor GSK-J1 HCL in the manufacture of a medicament for the treatment of myocardial fibrosis; the molecular formula of the GSK-J1 HCL is as follows: c22h23n5o2. hcl.

2. The use of claim 1, wherein said GSK-J1 HCL acts therapeutically on myocardial fibrosis by decreasing collagen deposition, inhibiting expression of myocardial fibrosis-associated proteins.

3. The use of claim 1, wherein GSK-J1 HCL achieves therapeutic effects on myocardial fibrosis by inhibiting Ang II-induced collagen expression levels in rat suckling mouse primary myocardial fibroblasts (NRCFs) and reducing fibrosis-associated protein expression.

Technical Field

The invention belongs to the field of pharmacy, relates to a new application of a compound GSK-J1 HCL in pharmacy, and particularly relates to an application of a histone demethylase JMJD3 inhibitor GSK-J1 HCL in preparation of a medicine for treating myocardial fibrosis.

Background

The prior art discloses that Myocardial Infarction (MI) is myocardial necrosis caused by severe and persistent ischemia of the corresponding myocardium due to a sharp reduction or interruption of coronary blood supply, which may lead to ventricular remodeling and may progress to heart failure, seriously threatening human health and life. Research shows that Myocardial Fibrosis (MF) is a common pathological change of various heart diseases at a certain stage, is one of the most characteristic structural changes, is one of the main manifestations of ventricular remodeling after MI, and can promote myocardial contraction and relaxation dysfunction; it has also been shown that long-term activation of Cardiac Fibroblasts (CFs), i.e. enhanced collagen content, myofibroblast differentiation and concomitant extracellular matrix secretion, can lead to myocardial fibrosis.

The compound GSK-J1 HCL is a potent histone methyltransferase JMJD3 inhibitor, and is much studied in tumor and inflammation; in the related pathological process, JMJD 3-mediated removal of methylation modification of H3K27 site can regulate the active state of gene expression in the process of disease occurrence and development, and related experimental results also reveal that GSK-J1 HCL is involved in improvement of collagen deposition through inhibiting JMJD3 and regulates the effect of myocardial fibrosis, so far, no related report of the effect of GSK-J1 HCL on myocardial fibrosis treatment is available.

Based on the current situation of the prior art, the inventor of the application intends to provide a new application of a compound GSK-J1 HCL in pharmacy, and particularly relates to an application of a histone demethylase JMJD3 inhibitor GSK-J1 HCL in preparation of a medicament for treating myocardial fibrosis.

Disclosure of Invention

The invention aims to provide a new application of a compound GSK-J1 HCL in pharmacy based on the current situation of the prior art, and particularly relates to an application of a histone demethylase JMJD3 inhibitor GSK-J1 HCL in preparation of a medicine for treating myocardial fibrosis.

The invention provides a new pharmacological action and application of a compound GSK-J1 HCL in the aspect of cardiovascular, and particularly relates to an application of GSK-J1 HCL in preparing a medicament for treating myocardial fibrosis.

The molecular formula of the GSK J1 is C22H23N5O2. HCL.

According to the invention, the established myocardial fibrosis model caused by mouse left coronary artery ligation operation and the in vitro myocardial fibrosis model experiment of Ang II induced NRCFs show that the GSK-J1 HCL can reduce the collagen content by inhibiting the expression of JMJD3 and reduce the expression of fibrosis-related protein to play a role in regulating and controlling myocardial fibrosis. The compound GSK-J1 HCL (C22H23N5O2.HCL) and related inhibitors thereof can be used for preparing medicines for preventing and treating myocardial fibrosis diseases.

More specifically, the present invention is to provide a novel,

the invention adopts C57BL/6J to carry out experimental study on the established mouse left coronary artery anterior descending myocardial infarction model, and cardiac ultrasonic detection is carried out on cardiac function parameters; detecting the influence of GSK J1 on the expression level of myocardial collagen (collagen), effector protein Connective Tissue Growth Factor (CTGF) protein and Fibronectin (FN) by a western-blotting method; detecting the influence of GSK J1 on morphology of an infarct area and an infarct marginal zone of a heart by an HE staining method; detecting the influence on the collagen content in the infarct area and the infarct marginal area by a Masson dyeing method; the results show that GSK-J1 HCL can significantly increase the values of ejection fraction (EF%) and left ventricular short axis shortening rate (FS%), decrease myocardial fibrosis area, and effectively inhibit protein expression levels of collagen, CTGF and FN.

In the invention, through establishing an angiotensin (Ang II) induced rat suckling mouse primary fibroblast (NRCFs) myocardial fibrosis model experiment in vitro, the influence of GSK-J1 HCL on the fibrosis effect is detected, and the result shows that the GSK-J1 HCL can obviously inhibit the expression of Ang II induced fibrosis effect protein; the experimental result proves that the compound can inhibit the fibrosis effect of primary myocardial fibroblasts and has a therapeutic effect.

The invention provides a new pharmacological action and application of a compound GSK-J1 HCL in the aspect of cardiovascular, and results of experiments of a mouse myocardial fibrosis model and an in vitro myocardial fibrosis model prove that the GSK-J1 HCL reduces the collagen content by inhibiting the expression of JMJD3 and reduces the expression of fibrosis-related protein to play a role in regulating and controlling the myocardial fibrosis; the GSK-J1 HCL can be used for preparing medicines for preventing and treating myocardial fibrosis diseases.

Drawings

FIG. 1: the effect of GSK-J1 HCL on cardiac function, wherein,

sham, MI, anterior descending coronary artery ligation group, MI + GSK-J1 HCL: the left anterior descending coronary artery ligation operation + GSK-J1 HCL administration group, 20mg/kg/day, #: MI group has p less than 0.05 compared with sham group; p <0.05 in the MI + GSK-J1 HCL group compared to the MI group.

FIG. 2 Effect of GSK-J1 HCL on the expression level of fibrotic effector proteins in cardiac tissue, wherein,

sham, MI, anterior descending coronary artery ligation group, MI + GSK-J1 HCL: left anterior descending coronary artery ligation + GSK-J1 HCL administration group, 20 mg/kg/day.

FIG. 3: the morphological effects of GSK-J1 HCL on the infarct zone and infarct border zone of the heart, wherein,

sham, MI, anterior descending coronary artery ligation group, MI + GSK-J1 HCL: left anterior descending coronary artery ligation + GSK-J1 HCL administration group, 20 mg/kg/day.

FIG. 4: effect of GSK-J1 HCL on collagen content in infarct zone and infarct rim zone of heart, wherein sham: sham group, MI: anterior descending ligation group of left coronary artery, MI + GSK-J1 HCL: left anterior descending coronary artery ligation + GSK-J1 HCL administration group, 20 mg/kg/day.

FIG. 5: the effect of GSK-J1 HCL on Ang II-induced collagen content in NRCFs cells, wherein,

control: normal control group, Ang II: model group, Ang II + GSK-J1 HCL, Ang II + GSK-J1 HCL.

FIG. 6: effect of GSK-J1 HCL on Ang II-induced expression levels of CTGF, an effector protein of cellular fibrosis of NRCFs, wherein control: normal control group, Ang II: model group, Ang II + GSK-J1 HCL, Ang II + GSK-J1 HCL.

Detailed Description

Example 1 GSK-J1 HCL improves EF% and FS% values after myocardial infarction, reduces collagen deposition in tissues, inhibits the expression of fibrotic Effector proteins in cardiac tissues

Male C57BL/6J mice weighing 25-30g were fixed with isoflurane anesthesia in supine position and breast preserved. The chest was opened between the third and fourth intercostals of the left thoracic cavity, and the anterior descending branch of the left coronary artery was permanently ligated between the left atrial appendage and the pulmonary artery cone with a 7-0 gauge suture needle at a distance of about 1mm from the aortic root, and the whitening of the myocardium in the blood supply region was observed as a successful model criterion. Rapidly closing the thoracic cavity, cleaning with sterile normal saline, suturing skin, keeping warm, and feeding water and standard feed in cages after the mice are awake. The sham group performed the same procedure except that the coronary artery was not ligated. GSK-J1 HCL was dissolved in physiological saline. The group was randomly divided into sham (sham, saline, ip.n.: 6), model (MI, saline, ip.n.: 6), and model + GSK-J1 HCL (MI + GSK-J1 HCL, 20mg/kg/day, ip.n.: 6). After continuous administration for 14 days, the cardiac function of the mice is detected by a cardiac ultrasound method, the hearts are taken out quickly after sacrifice, fixed by 4 percent paraformaldehyde, embedded to be paraffin sections, observed by HE staining for histomorphology, and detected by a Masson staining method for the content of tissue collagen.

Compared with the model group, the GSK-J1 HCL group can significantly improve the mouse heart EF% and FS% values (as shown in FIG. 1). The Western-blotting result indicates that GSK-J1 HCL significantly reduces the expression of the fibrosis effector protein CTGF (as shown in figure 2). HE staining results show that GSK-J1 HCL significantly improves residual cardiomyocyte hypertrophy in the myocardial infarction margin region and infiltration of inflammatory cells in the infarct region, so that myocardial tissues are arranged orderly, the boundary is clear, and the cell nucleus is uniform in size (as shown in fig. 3). Masson staining showed that GSK-J1 HCL significantly reduced collagen deposition in the infarct zone and infarct border zone of the heart (as shown in FIG. 4).

Example 2 GSK-J1 HCL reduction of Ang II induced collagen content and Effector levels in NRCFs cells

Rat suckling mouse primary myocardial fibroblasts (NRCFs) are cultured in a DMEM medium containing 10% fetal calf serum, placed in a 5% CO2 incubator at 37 ℃ and cultured, and when the cell confluency reaches 80-90%, the cells are subjected to passage; cells were seeded in a 24-well plate, and a normal Control group (Control, no drug intervention, no Ang II addition), a Model group (Model, no drug intervention, Ang II stimulation for 48h), and a GSK-J1 HCL group (GSK-J1 HCL, 20 μ M, pretreated 4h in advance and stimulated with Ang II) were set; the immunofluorescence method result shows that GSK-J1 HCL can effectively reduce the collagen content in primary myocardial fibroblasts; the cells are planted in a 6-well plate, and each test control group is induced by Ang II for 48 hours and then the cells are lysed to extract protein; the Western-Blotting method detects CTGF and FN, and the result shows that GSK-J1 HCL can obviously reduce the expression level of the fibrosis effector protein.