Application of Vasorin in preparation of medicine for resisting ischemic cardiomyopathy and fibrosis

Abstract

The invention discloses application of Vasorin in preparation of an anti-ischemic cardiomyopathy and anti-fibrosis medicine, and relates to the field of medicinal chemistry, experiments prove that Vasorin can effectively relieve cardiac fibrosis symptoms after myocardial ischemia and effectively improve cardiac function conditions after myocardial infarction, and therefore the effect of treating ischemic heart diseases is achieved. In addition, occurrence and development of fibrosis can be effectively inhibited, and a basis can be provided for development of anti-ischemic heart disease drugs in the future.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the application of Vasorin in the preparation of drugs for resisting ischemic cardiomyopathy and fibrosis. Background Art

[0002] Ischemic heart disease, also known as coronary heart disease, refers to heart disease caused by myocardial ischemia, hypoxia or necrosis due to coronary atherosclerosis causing stenosis or obstruction of the lumen, including silent myocardial ischemia, angina pectoris, myocardial infarction, ischemic heart failure and cardiac arrest. Usually, when the main coronary artery occlusion occurs due to atherosclerosis or arteriosclerosis, blood flow in the coronary artery is initially maintained because the peripheral resistance far from the occlusion is reduced. Ischemia occurs when more than 75% of the lumen is occluded, especially if the coronary collateral circulation is poor. Fibrosis is scarring and tissue hardening caused by excessive deposition of extracellular matrix proteins by myofibroblasts in a chronic inflammatory response.

[0003] During cardiac ischemia and reperfusion, many endogenous mediators, such as small molecule second messengers, are produced that affect myocardial function. Myocardial contractility decreases within minutes of ischemia, and the complete recovery of contractility depends mainly on the duration of ischemia (Daemen et al., 1999). In addition, cytokines, particularly TNFα and IL-1β (Bolli, 1990), are produced locally. In the intact heart, these cytokines act on ischemia-induced myocardial dysfunction by inducing inducible nitric oxide synthase (iNOS) (Daemen et al., 1999), cyclooxygenase-2 (COX-2) and phospholipase A2, as well as gene expression of vascular adhesion molecules and several chemokines. Therefore, small molecule messengers mediate an immediate decrease in myocardial contractility, followed by cytokine-mediated neutrophil infiltration, which further damages the myocardium. Animal heart studies conducted in the absence of blood or blood products have detailed the role of TNFα (Herskowitz et al., 1995) and IL-1β during ischemic attack. Due to the effects of these endogenous cytokines, cardiomyocytes also lose their contractile force (Meldrum et al., 1998).

[0004] Fibrosis plays a role in the progression of chronic diseases in many organs, including the heart, liver, and lungs. Myocardial infarction and subsequent heart failure are one of the main causes of death in patients with cardiovascular disease. Myocardial cell death after myocardial infarction causes the activation of cardiac fibroblasts, which have a repairing effect. However, when they are overactivated, excessive deposition of secreted extracellular matrix will form fibrotic tissue and eventually lead to heart failure after myocardial infarction.

[0005] Vasorin (VASN) was first discovered and reported as a TGF-β binding protein in 2004. It is mainly expressed in the aorta, the vascular-rich placenta and the kidney. It can regulate the aortic injury response during the repair of vascular damage by participating in the TGF-β pathway. VASN is highly expressed in breast cancer cells, glioma cells, and liver cancer cells. VASN is also contained in the exosomes of urine of patients with early IgA nephropathy. VASN is highly expressed in human brain tumor tissues. When siRNA knocks down the expression level of VASN in glioma cells, the apoptosis rate induced by hypoxia or TNF-α is significantly increased. VASN protein is a typical type I transmembrane glycoprotein with a total length of 673 amino acids. Bioinformatics predicts a relative molecular weight of approximately 72kD. Its extracellular domain can be cleaved and detached by disintegrin and metalloprotease 17 (ADAM17) to become soluble VASN (sVASN). sVASN contains important functional domains such as tandem leucine-rich (LRR) domains, epidermal growth factor (EGF)-like domains and fibronectin III (FN3) domains, which are the structural basis of its biological function. In addition, Vasorin protein is also involved in the negative regulation of epithelial-to-mesenchymal transition, maintaining tissue homeostasis, and may participate in the response of cells to hypoxia and redox state by regulating Notch signaling and other mechanisms. Vasn- / - mice die around 21 days after birth, indicating that Vasorin plays an important physiological role in the development of mice; to explore the in vivo function of Vasorin, the researchers established a rat carotid artery balloon injury model and confirmed that the expression level of Vasorin in the carotid artery was downregulated during the repair process after arterial injury. This finding indicates that Vasorin is involved in the formation of injury-induced vascular lesions. The downregulation of Vasorin was reversed by in vivo administration, which significantly reduced the intima / media area ratio of the injured artery, indicating that Vasorin inhibits pathological conditions such as atherosclerosis or restenosis after carotid artery injury in rats. Pintus et al. found that after Vasorin binds to TGF-β1, it inhibits the fibrotic signal transduction of TGF-β1 in vascular smooth muscle cells. With the progression of aging, the expression of Vasorin in the rat arterial wall decreases, activating matrix metalloproteinase-2 in the vascular smooth muscle cells of the arterial wall of aged rats and enhancing the angiotensin II-mediated pro-fibrotic signal transduction.

[0006] However, to date, the role of vasorin in heart disease has not been described. Summary of the invention

[0007] The present invention aims to provide the use of Vasorin in preparing drugs for resisting ischemic cardiomyopathy and anti-fibrosis.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] The invention provides application of Vasorin in preparing medicine for treating ischemic heart disease.

[0010] Application of Vasorin in preparing medicine for treating cardiac fibrosis.

[0011] Preferably, the dosage form of the drug includes tablets, powders, granules, capsules, oral liquids, injections or sustained-release preparations. Use of Vasorin in the preparation of drugs for treating ischemic heart disease.

[0012] Preferably, an unnatural amino acid FSY is introduced into Vasorin to generate a Vasorin-FSY mutant, a p-VASN (FSY) plasmid is constructed, and the p-PylRS / tRNA Pyl The plasmids were co-transfected into HEK 293T cells, and the cell membranes and platelet cell membranes were extracted 48 hours later to prepare hybrid membrane liposomes.

[0013] The present invention provides the use of Vasorin in the preparation of anti-ischemic cardiomyopathy and anti-fibrosis drugs, which has the following beneficial effects: the Vasorin in the present invention can effectively alleviate the symptoms of cardiac fibrosis after myocardial ischemia, and effectively improve the cardiac function after myocardial infarction, thereby playing a role in treating ischemic heart disease, and can also effectively inhibit the occurrence and development of fibrosis, which can provide a basis for the future development of anti-ischemic heart disease drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the present invention or the technical solutions in the prior art, the drawings required for describing the prior art are briefly introduced below.

[0015] Figure 1 Quantification of myocardial fibrosis in four groups of experimental mice in the present invention;

[0016] Figure 2 Typical short-axis M-ultrasound images of mouse echocardiograms of the four groups of experimental mice in the present invention;

[0017] Figure 3 Ejection fraction (EF) graphs obtained after echocardiographic analysis of four groups of experimental mice in the present invention;

[0018] Figure 4 Fractional shortening (FS) graphs obtained after echocardiographic analysis of four groups of experimental mice in the present invention;

[0019] Figure 5In Example 2 of the present invention, qPCR was used to detect the expression levels of COLI, COLIII, α-SMA and fibronectin;

[0020] Figure 6 In Example 2 of the present invention, Western blot was used to detect the expression levels of COL1, COLIII and α-SMA proteins. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solutions and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention.

[0022] Embodiment 1

[0023] Vasorin can significantly alleviate the symptoms of cardiac fibrosis after myocardial ischemia, thereby playing a role in treating ischemic heart disease, and can be used to prepare drugs for treating ischemic heart disease.

[0024] Pharmacological test methods:

[0025] 1. Establishment of myocardial ischemia model in mice;

[0026] SPF male C57BL / 6J wild-type mice (6-8 weeks old, weighing between 22-27 grams) were used. The mice were anesthetized with isoflurane gas. A skin incision was made between the 3rd and 4th intercostal spaces on the left side of the mouse, and the subcutaneous tissue, pectoralis major muscle and serratus anterior muscle were bluntly separated. The intercostal space was opened with hemostats to expose the heart. The direction of the left anterior descending coronary artery (LAD) was found under a microscope. The suture was held with a needle holder, and the needle was inserted at a certain distance (such as 2 mm) from the lower edge of the left atrial appendage. The suture passed through the LAD and tied to completely block its blood flow. After successful ligation, the color of the ischemic area after ligation turned white, which was observed with the naked eye as a preliminary criterion for successful ligation. Sutures were used to close the chest and skin incisions layer by layer to ensure that there were no gaps or dislocations in the sutures. The electrophysiological changes of the mouse heart were observed by electrocardiogram, and the increase in QRS amplitude, ST segment elevation, and towering or inverted T waves were used as the criteria for successful ischemia.

[0027] 2. Experimental groups and drug intervention

[0028] (1) Blank group (Sham group): 6 rats, normal rats.

[0029] (2) Model group (MI group): 6 rats. After the myocardial ischemia model was successfully established, normal saline was injected into the myocardium and intraperitoneally once every two days for 4 consecutive weeks.

[0030] (3) Vasorin-IP group (MI + intraperitoneal injection of Vasorin group): 6 rats. After the myocardial ischemia model was successfully established, a solution containing Vasorin was intraperitoneally injected once every two days for 4 consecutive weeks.

[0031] (4) Vasorin-IM group (MI + myocardial point injection of Vasorin group): 6 rats, after the myocardial ischemia model was successfully established, Vasorin was injected into the myocardial point, and the Vasorin solution was injected intraperitoneally once every two days for 4 consecutive weeks.

[0032] 3. Masson staining to assess the degree of cardiac fibrosis

[0033] Masson staining steps: After 4 weeks, the myocardial tissue sections of the mice in the above four experimental groups were dewaxed, dehydrated and then rinsed, and the nuclei were stained with Weigert's hematoxylin for 5-8 minutes; washed thoroughly with water, and if overstained, they could be differentiated with hydrochloric acid and alcohol; washed with distilled water; stained with Masson's acid red solution for 5-10 minutes; washed with 0.2% glacial acetic acid for 5 seconds; differentiated with 1% phosphomolybdic acid aqueous solution for 3-5 minutes; without washing, directly stained with light green solution for 5 minutes; immersed in 0.2% glacial acetic acid aqueous solution for 5 seconds; 95% alcohol, anhydrous alcohol, xylene transparent, and neutral gum sealing.

[0034] After the above-mentioned Masson staining, myocardial cells were stained red, myocardial fibrosis was stained green, and the cytoplasm was blue-black. Then 8 fields of view were randomly selected on the Masson slices, and the area and degree of myocardial fibrosis were measured by ImageJ image analysis software. Figure 1 As shown in the figure, it can be clearly seen from the comparison that compared with the model group (MI group), the degree of myocardial fibrosis in the Vasorin-IP group (MI + intraperitoneal injection of Vasorin group) and the Vasorin-IM group (MI + myocardial point injection of Vasorin group) was significantly reduced. In particular, the reduction of cardiac fibrosis in the Vasorin-IM group (MI + myocardial point injection of Vasorin group) was the most obvious.

[0035] 4. Echocardiographic analysis

[0036] After 4 weeks, the mice in the above four experimental groups were subjected to cardiac ultrasound examination. Specifically, the mice in each group were depilated and anesthetized with isoflurane mask at a dose of 1%-2%, and then fixed on a heating pad. The isoflurane flow was carefully controlled to maintain the heart rate at around 500 beats / min. A two-dimensional (2-D) guided M-mode echocardiogram equipped with a 30mHz linear transducer was then used to evaluate the geometric structure and function of the heart. Typical short-axis M-mode echocardiograms of mice were obtained (such as Figure 2As shown in the figure, the heart rate is recorded at the same time. The heart geometry and functional indicators are obtained: ejection fraction (EF), shortening fraction (FS). Each measurement indicator is averaged over at least 5 consecutive cardiac cycles. The results are shown in the figure. Figure 3-4 shown.

[0037] The results of echocardiographic analysis of mice in the four experimental groups showed that compared with the model group (MI group), the ejection fraction (EF) and shortening fraction (FS) values ​​in the Vasorin-IP group and Vasorin-IM group injected with Vasorin were significantly increased, indicating that Vasorin can effectively improve the cardiac function of mice after myocardial infarction.

[0038] Embodiment 2

[0039] Anti-fibrosis testing

[0040] (1) Extracting primary cardiac fibroblasts;

[0041] Quickly remove the SPF male rat heart, place it in a dish containing PBS solution, rinse, cut the heart tissue into pieces, wash the pieces with PBS several times, transfer the washed pieces to another culture dish, soak the tissue with 0.25% trypsin digestion solution, and digest at room temperature for 3-5 minutes. After digestion is completed, use complete culture medium to terminate digestion, add 0.8mg / ml type II collagenase at 37℃ for 10 minutes and collect the supernatant. Repeat the above process 5-6 times until the tissue disappears. After centrifugation of the supernatant, resuspend the cells. After 1.5 hours, the attached cells are primary cardiac fibroblasts.

[0042] (2) Experimental groups and drug intervention

[0043] Blank group: The extracted primary cardiac fibroblasts were cultured in a medium containing 0% or 2% FBS and then 0.1 umol of PBS was added and stimulated for 48 h.

[0044] AngII group: The primary cardiac fibroblasts were cultured in a medium containing 0% or 2% FBS and 0.1 μmol of PBS was added. -6 mol / L AngII stimulation for 48h.

[0045] Vasorin group: 0.1 μmol of Vasorin solution was added to the extracted primary cardiac fibroblasts in the culture medium containing 0% or 2% FBS and stimulated for 48 h.

[0046] AngII+Vasorin group: The primary cardiac fibroblasts were cultured in a medium containing 0% or 2% FBS and 0.1 μmol of Vasorin solution was added. -6 mol / L AngII stimulation for 48h.

[0047] (3) Detection by qPCR and WB

[0048] 1) qPCR detection

[0049] Specific primers were designed for Collagen1, Collagen3, α-SMA and Tubulin. The qPCR primer sequences are shown in Table 1 below.

[0050] Table 1 qPCR primer sequences

[0051]

[0052] Total RNA was then extracted from the four groups of samples to be tested and reverse transcribed to obtain cDNA. cDNA, primers, qPCRmix and other reactants were mixed and PCR amplified. During each round of DNA supplementation, the fluorescence signal was measured and recorded using a fluorometer for quantitative analysis. The results are shown in Figure 5 .pass Figure 5 The qPCR test results shown in the figure show that after AngII-induced fibrosis, administration of vasorin can down-regulate the expression of fibrosis-related genes such as Collagen1, Collagen3, α-SMA, and fibronectin (FN).

[0053] 2) Western blot detection

[0054] The above four groups of test samples were crushed and added to the lysis buffer, and protease inhibitors were added to prevent protein degradation. The cells were fully dissolved by vortexing, and the cell fragments were further precipitated by centrifugation. The supernatant contains the target protein, which can be transferred to a new centrifuge tube, SDS loading buffer was added, and the protein sample was stored at -80°C after protein denaturation in a metal water bath to avoid protein degradation. The tissue protein was separated by SDS-PAGE and transferred to a PVDF membrane by wet transfer. After blocking with 5% skim milk, the primary antibody was incubated at 4°C overnight. After washing, incubate with the corresponding secondary antibody at room temperature for 1 hour. Then color development was performed on a colorimeter; the picture was saved and analyzed. Results are shown in Figure 6 .pass Figure 6 The results of Western blot assay shown in the figure show that Vasorin can inhibit the expression of Collagen1, Collagen3, and α-SMA fibrosis proteins.

[0055] Therefore, through Figure 6 It can be clearly concluded that Vasorin can effectively inhibit the occurrence and development of fibrosis.

[0056] Embodiment 3

[0057] On the basis of Example 1, the present invention also discloses a hybrid membrane liposome for treating ischemic heart disease: first, a three-dimensional structural model of Vasorin was constructed using the AlphaFold 3 prediction tool, and molecular docking analysis was performed using the AutoDock-Vina software. Based on the predicted structure of the Vasorin / TGF-β1 complex, the non-natural amino acid FSY was introduced into the Gln61 and Gln325 sites of Vasorin to generate Vasorin-61FSY and Vasorin-325FSY mutants, respectively, and p-VASN(Gln61FSY)-Flag and p-VASN(Gln325FSY)-Flag plasmids were constructed, and then the above plasmids were respectively combined with p-PylRS / tRNA Pyl The plasmid was co-transfected into HEK 293T cells. After 48 hours, the cell membrane and platelet cell membrane were extracted to prepare hybrid membrane liposomes. The prepared hybrid membrane liposomes were injected into mice after myocardial infarction through the tail vein. The therapeutic effect was stronger than that of unmodified vasorin.

[0058] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of Vasorin in the preparation of medicines for treating ischemic heart disease.

2. Application of Vasorin in the preparation of anti-fibrosis drugs.

3. The use according to claim 1, characterized in that: The dosage form of the drug includes tablets, powders, granules, capsules, oral liquids, injections or sustained-release preparations.

4. The use according to claim 1, characterized in that: The unnatural amino acid FSY was introduced into Vasorin to generate the Vasorin-FSY mutant, and the p-VASN(FSY) plasmid was constructed and combined with p-PylRS / tRNA Pyl The plasmids were co-transfected into HEK293T cells, and the cell membranes and platelet cell membranes were extracted 48 hours later to prepare hybrid membrane liposomes.

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