Application of a histamine H1 receptor antagonist in the preparation of drugs for treating myocardial fibrosis

Abstract

This invention relates to the field of pharmaceutical technology for myocardial fibrosis. Specifically, it provides the application of histamine H1 receptor antagonists in the preparation of drugs for treating myocardial fibrosis. This invention utilizes a stress overload mouse model constructed via TAC surgery to simulate the in vivo process of myocardial fibrosis, and a TGF-β1-induced fibrotic cell model. Combined with network pharmacology analysis and histamine H1 receptor gene knockout mice, the anti-myocardial fibrosis effects and mechanisms of action of the histamine H1 receptor antagonists brompheniramine maleate (BHM) and desloratadine (DLT) were investigated at both the systemic and cellular levels, providing a theoretical and experimental basis for the development of new anti-myocardial fibrosis drugs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology for myocardial fibrosis, and more particularly to the application of a histamine H1 receptor antagonist in the preparation of a drug for treating myocardial fibrosis. Background Technology

[0002] Myocardial fibrosis is closely related to many clinical diseases, including hypertensive heart disease, myocardial infarction, diabetic cardiomyopathy, and atherosclerosis, and is a common pathological feature of various cardiovascular diseases. Myocardial fibrosis is a pathological change characterized by excessive proliferation of cardiac fibroblasts, excessive deposition and abnormal distribution of collagen in the extracellular matrix (ECM), leading to cardiac structural damage and myocardial dysfunction. Myocardial fibrosis can even cause serious complications such as arrhythmias, heart failure, and sudden cardiac death. Activated fibroblasts and myofibroblasts are the central effector cells in myocardial fibrosis and are also the main source of matrix proteins. However, there are currently no drugs that target the etiology of myocardial fibrosis and have a definite therapeutic effect; therefore, the search for new and effective anti-myocardial fibrosis drugs is urgently needed. Summary of the Invention

[0003] The purpose of this invention is to provide an application of histamine H1 receptor antagonists in the preparation of drugs for treating myocardial fibrosis, providing a theoretical and experimental basis for the development of new drugs against myocardial fibrosis.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides the application of a histamine H1 receptor antagonist in the preparation of a drug for treating myocardial fibrosis.

[0006] Preferably, the histamine H1 receptor antagonist is brompheniramine maleate or desloratadine.

[0007] Preferably, the myocardial fibrosis is TGF-β1-induced cell fibrosis.

[0008] The present invention also provides the application of histamine H1 receptor antagonists in the preparation of drugs that inhibit fibroblast activation and reduce collagen secretion, characterized in that the histamine H1 receptor antagonist is brompheniramine maleate or desloratadine.

[0009] This invention also provides the application of desloratadine in the preparation of drugs that reduce the expression of myofibroblast differentiation marker α-SMA and collagen deposition or downregulate the expression levels of ANP and BNP.

[0010] This invention provides the application of histamine H1 receptor antagonists in the preparation of drugs for treating myocardial fibrosis. This invention simulates the in vivo process of myocardial fibrosis by constructing a stress overload mouse model using TAC surgery, and by using a TGF-β1-induced mouse fibrotic cell model. Combined with network pharmacology analysis and histamine H1 receptor gene knockout mice, the anti-myocardial fibrosis effects and mechanisms of action of the histamine H1 receptor antagonists brompheniramine maleate (BHM) and desloratadine (DLT) were investigated at both the systemic and cellular levels, providing a theoretical and experimental basis for the development of new anti-myocardial fibrosis drugs. Attached Figure Description

[0011] Figure 1 Histamine H1 receptor antagonists inhibit TGF-β1-induced collagen synthesis in fibroblasts. (Control: blank control group; TGF-β1: model group; Val: valsartan positive control group; BHM: brompheniramine maleate treatment group; DLT: desloratadine treatment group;) p < 0.05; p < 0.01; p < 0.001).

[0012] Figure 2 Effects of histamine receptor antagonists on TAC-induced cardiac morphology and function in mice. (A. Parasternal long-axis M-section; B. Statistical graph of systolic left ventricular diameter; C. Statistical graph of left ventricular ejection fraction; D. Statistical graph of left ventricular fractional shortening. Sham: sham-operated group; Model: TAC model group; Val: valsartan positive control group; BHM: brompheniramine maleate treatment group; DLT: desloratadine treatment group;) p < 0.05; p < 0.01).

[0013] Figure 3 To alleviate TAC-induced myocardial fibrosis with histamine receptor H1 antagonists. (A. Mouse myocardial HE staining results; B. Mouse myocardial Masson staining results; C. Mouse myocardial Sirius red staining results; D. Masson staining statistics; E. Sirius red staining statistics. Sham: sham-operated group; Model: TAC model group; Val: valsartan positive control group; BHM: brompheniramine maleate treatment group; DLT: desloratadine treatment group;) p < 0.01; p < 0.001; p < 0.0001).

[0014] Figure 4DLT inhibits TGF-β1-induced cell fibrosis. (A. DLT inhibits cellular collagen synthesis; B. DLT inhibits α-SMA mRNA expression in fibroblasts; C. DLT inhibits Collagen I mRNA expression in fibroblasts; D. DLT inhibits Collagen III mRNA expression in fibroblasts;) p < 0.05; p < 0.001; p < 0.0001).

[0015] Figure 5 To demonstrate how DLT improves TAC-induced cardiac dysfunction in mice by acting on histamine H1 receptors. (A. M-mode ultrasound images of the long axis of the mouse sternal border; B. Statistical graph of left ventricular systolic diameter; C. Statistical graph of left ventricular ejection fraction; D. Statistical graph of left ventricular short axis shortening; E. mRNA expression of ANP in mouse heart tissue; F. HRH1) - / - Statistical diagram of systolic diameter of left ventricle in mice; G. HRH1 - / - Statistical graph of left ventricular ejection fraction in mice; H. HRH1 - / - Statistical graph of left ventricular fractional shortening in mice; I. mRNA expression of BNP in mouse cardiac tissue. Sham: sham-operated group; Model: TAC model group; DLT: desloratadine treatment group; HRH1 - / - Histamine H1 receptor gene knockout type. p<0.05; p < 0.01; p < 0.001; p < 0.0001; no significant difference in ns).

[0016] Figure 6 Desloratadine (DLT) reduces myocardial fibrosis by acting on histamine H1 receptors. (A. Mouse myocardial HE staining results; B. Mouse myocardial Masson staining results; C. Mouse myocardial Sirius red staining results; D. Masson staining statistical results; E. Sirius red staining statistical results; Sham: sham-operated group; Model: TAC model group; DLT: desloratadine treatment group; HRH1) - / - Histamine H1 receptor gene knockout type. p < 0.01; p < 0.0001; no significant difference in ns).

[0017] Figure 7DLT was used to inhibit the expression of α-SMA, Collagen I, and Collagen III in mouse myocardium. (A. DLT inhibition of fibrosis marker protein expression; B. α-SMA protein expression results; C. Collagen I protein expression results; D. Collagen III protein expression results; E. DLT inhibition of α-SMA mRNA expression; F. DLT inhibition of Collagen I mRNA expression; G. DLT inhibition of Collagen III mRNA expression.) p < 0.05; p < 0.01; p < 0.001; p < 0.0001).

[0018] Figure 8 DLT inhibits the transcription of myocardial inflammation and oxidative stress-related factors in mice. (A. KEGG enrichment analysis of major signaling pathways of potential target genes; B. PPI network analysis of statistical maps of potential target genes; C. DLT inhibits IL-1β mRNA expression; D. DLT inhibits IL-6 mRNA expression; E. DLT inhibits eNOS mRNA expression; F. DLT inhibits Nrf2 mRNA expression.) p < 0.05; p < 0.01; p < 0.001; p < 0.0001).

[0019] Figure 9 DLT inhibits the phosphorylation of IKK and ERK1 / 2 proteins in mouse myocardial tissue. (A. DLT inhibits the expression of IKK, p-IKK, ERK1 / 2, and p-ERK1 / 2 proteins in mouse myocardium; B. Statistical results of IKK protein expression; C. Statistical results of p-IKK protein expression; D. Statistical results of ERK1 / 2 protein expression; E. Statistical results of p-ERK1 / 2 protein expression.) p < 0.05; p < 0.01; p < 0.001; p < 0.0001). Detailed Implementation

[0020] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0021] drug

[0022] Table 1. Reagents and chemicals used in the experiment

[0023]

[0024]

[0025] Example 1

[0026] 1. Establishment and grouping of fibrotic cell models

[0027] Mouse embryonic fibroblast cell line (NIH3T3) was cultured in high-glucose DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. When cells reached 70% confluence, the culture medium was aspirated, washed with PBS, and then replaced with FBS-free medium for 12 hours of starvation. The culture medium was aspirated again and washed with PBS. The control group was cultured in FBS-free medium for an additional 48 hours, while the model group was cultured in medium containing 15 ng / ml TGF-β1. The treatment groups were stimulated with 15 ng / ml TGF-β1 followed by the addition of Val (1 μM), BHM (5 μM), and DLT (10 μM) for 48 hours, after which cells were collected for analysis.

[0028] 2. Determination of total collagen in cells using Sirius red staining method

[0029] Cells were seeded into 12-well plates. When cells reached 70% confluence, they were treated with TGF-β1 (15 ng / ml) and the corresponding drug concentration for 48 h. After treatment, the cell culture medium was aspirated, and the cells were washed with PBS. 1 ml of 0.05 M acetic acid was added to each well, and cells were scraped off with a cell scraper and transferred to 1.5 ml centrifuge tubes. Standard solutions of 250, 125, 63, 31.5, 16, and 8 μg / ml were prepared. 100 μl of blank, diluted standard solutions, and samples were added to 1.5 ml centrifuge tubes. 500 μl of Sirius red solution was added to each tube, vortexed, and incubated at room temperature for 20 min. Centrifugation at 10000 rpm for 3 min yielded a red collagen precipitate, and the supernatant was carefully removed. 500 μl of wash buffer was added to each tube, and the precipitate was resuspended in the wash buffer by vortexing. Centrifugation at 10000 rpm for 3 min was performed, and the supernatant was aspirated. 250 μl of extraction buffer was added to each tube to dissolve the precipitate. The OD value at 530 nm was read using an ELISA reader.

[0030] 3. Establishment of TAC mouse model and experimental grouping

[0031] A mouse model of myocardial fibrosis induced by pressure overload was established through transverse aortic arch constriction. Two weeks after surgery, hemodynamic parameters at the aortic arch ligation site were assessed using ultrasound imaging to determine the success of the model. In the sham surgery group, only the thoracic cavity was opened and sutured; the aortic arch was not constricted.

[0032] The mice were randomly assigned to groups based on body weight and administered medication by gavage for two weeks (the sham-operated group and the model group received 0.5% sodium carboxymethyl cellulose by gavage). The experimental groups were as follows: sham-operated group (n=6), TAC model group (n=7), Val group (valsartan 10 mg / kg, n=6), BHM group (brompheniramine maleate 30 mg / kg, n=6), and DLT group (desloratadine 10 mg / kg, n=6). The drug dosage for each group was calculated using the conversion factor between mouse and human surface area (0.0026).

[0033] 4. Ultrasound imaging system for detecting cardiac morphology and function

[0034] Following TAC surgery, echocardiography was performed on mice in each group using a Visual Sonics Vevo1100 small animal ultrasound imaging system (probe frequency 30MHz). The specific steps were as follows: Mice were placed in an induction box and anesthetized with isoflurane at a concentration of 2%. They were then placed in a supine position on the physiological information monitoring platform, at which point the isoflurane concentration was adjusted to 0.5%. Eye protection lubricant was applied to the mice's eyes to prevent dryness. The mice's paws were fixed to copper plates on the physiological information monitoring platform coated with a small amount of conductive adhesive to obtain ECG and respiratory information. Hair was removed from the mice's chest using depilatory cream and cleaned with a moistened cotton swab. Pulsed Doppler images of the aortic arch section were acquired to obtain the blood flow velocity at the aortic arch ligation site, determining the success of the TAC modeling surgery. B-mode and M-mode ultrasound images of the left ventricle along the parasternal long axis were acquired. The systolic left ventricular diameter (LVID;s), left ventricular ejection fraction (EF%), and left ventricular fractional shortening (FS%) were measured using the accompanying analysis software to assess the cardiac function of the mice.

[0035] 5. Draw materials

[0036] Mice were weighed and anesthetized with sodium pentobarbital via intraperitoneal injection. Starting from the xiphoid process, the skin was incised along the lower edge of the sternum to open the thoracic cavity and expose the heart. Approximately 400 μl of blood was drawn from the left apex of the heart via a skeletal needle inserted at an angle. This blood was transferred to a blood collection tube containing sodium heparin, and the tube was agitated to ensure thorough contact between the blood and the sodium heparin. The tube was allowed to stand for 10 minutes. The blood was centrifuged at 3000g for 10 minutes, and the supernatant (plasma) was carefully transferred to a 1.5 ml centrifuge tube and stored at -80°C. The right atrial appendage was cut open, and physiological saline was slowly perfused through the blood collection site until the liver turned white. Then, 1 ml of 4% paraformaldehyde was continued perfusion. The heart was removed, and the ventricular portion was fixed in 4% paraformaldehyde.

[0037] 6. Pathological staining

[0038] Mouse hearts were perfused with physiological saline and 4% paraformaldehyde, and the ventricular portion was removed and fixed in 4% paraformaldehyde for 48 hours. The tissue was then immersed in a gradient of 70%, 80%, and 90% ethanol solutions for 30 minutes each, followed by two 20-minute immersions in 95% and 100% ethanol solutions. Next, the tissue was sequentially immersed in a mixture of equal volumes of ethanol and xylene for 15 minutes each, then in different xylene containers for 15 minutes each, until the tissue became transparent. Finally, the tissue was immersed in a mixture of equal volumes of xylene and paraffin for 15 minutes, and then sequentially in two different paraffin containers for 60 minutes each. The forceps and paraffin mold were slightly heated over an alcohol lamp, a small amount of paraffin was poured in, the tissue was picked up and placed cut-side down into the wax mold, arranged neatly. An embedding cassette was placed on top, and molten paraffin was poured in. Before sectioning, the paraffin blocks embedded with tissue were frozen on a freezing stage for 30 minutes, and then sectioned (4μm) using a paraffin microtome. The sections were spread in a 40℃ water bath, removed with an adhesive slide, and dried in a 58℃ oven for 2 hours before use. Before use, the prepared sections were dried in an oven at 58℃ for 1 hour, followed by dewaxing and staining.

[0039] (1) HE staining

[0040] Sections were routinely dewaxed to water (sequentially immersed in two fresh xylene solutions for 10 min each, two fresh anhydrous ethanol solutions for 5 min each, 85% ethanol solution for 5 min each, 75% ethanol solution for 5 min each, and finally washed with distilled water for 2 min). Hematoxylin staining was added for 5 min, followed by rinsing with tap water to remove the stain. Differentiation solution was added for 30 s, followed by rinsing with tap water for 30 s. Blueing solution was applied for 1 min, followed by rinsing with tap water. Eosin staining was applied for 1 min, followed by rinsing with tap water. Finally, the sections were immersed in a gradient of 75%, 85%, 95%, and 100% ethanol solutions for 5 s each to dehydrate, and then cleared in xylene for 1 min. After mounting with neutral resin, the sections were examined under a microscope.

[0041] (2) Masson staining

[0042] After dewaxing to water, the nuclei were stained with freshly prepared Weigert iron hematoxylin staining solution for 5 min. Differentiation was performed with acidic ethanol differentiation solution for 15 s, followed by rinsing with water. Masson's blue solution was added for 3 min to restore blue color, followed by rinsing with water and then staining with Ponceau S and fuchsin for 4 min, then rinsing with weak acid working solution for 1 min. Phosphomolybdic acid solution was added for 2 min of staining, followed by rinsing with weak acid working solution for 1 min. Aniline blue solution was added for 1 min of staining, followed by rinsing with weak acid working solution for 1 min. After routine dehydration and clearing, the sections were mounted with neutral resin and examined under a microscope.

[0043] (3) Improved Sirius red staining

[0044] Dewax the sections to water, add freshly prepared iron-hematoxylin staining solution and stain for 5 minutes, then wash with water for 5 minutes. Add Sirius red staining solution and stain for 12 minutes, then wash the surface stain with water. After dehydration and clearing, mount with neutral resin and observe under a microscope.

[0045] 7. RT-qPCR

[0046] Cells were collected or 20 mg of mouse heart tissue was weighed. RNA was extracted using the Trizol method, and the RNA concentration was determined. Reverse transcription was performed according to the reverse transcription kit instructions, and cDNA was obtained after the reaction. β-actin was selected as an internal control, and the expression levels of Collagen I, Collagen III, and α-SMA were detected using SYBRGREEN I real-time PCR. Primers were designed and synthesized in Primer 5 or BD based on sequences retrieved from NCBI; the sequence numbers are shown in Table 2. The PCR reaction was performed according to the following program: 95℃ for 2 min; 95℃ for 15 s, 58℃ for 30 s, for 40 cycles. The melting curve was established at 60℃~95℃. Each sample was tested in triplicate. Data were analyzed using 2... ^-△△Ct The method is used for analysis.

[0047] 8. Western Blot

[0048] Cells and mouse myocardial tissue were collected, washed twice with PBS, and RIPA lysis buffer was added. PMSF was then added at a specific ratio (PMSF:RIPA = 1:100) and collected for later use. BCA method was used for protein quantification and to plot a standard curve. Total protein from each group of samples was loaded onto SDS-PAGE membranes, transferred, and blocked with 5% BSA. After primary antibody incubation, the membranes were washed with TBST and then incubated with secondary antibody. After washing, ECL chromogenic buffer was added for development, and the grayscale value and protein expression of each band were analyzed using a gel imaging system and software.

[0049] 9. Statistical Analysis

[0050] Data analysis employed Mean ± SD for comparison. GraphPad Prism8 was used for data processing and plotting. Homogeneity of variance was tested using one-way ANOVA, and pairwise comparisons were performed using t-tests. The significance level was set at α = 0.05, and P < 0.05 was considered statistically significant.

[0051] Table 2 Primer sequences

[0052]

[0053] Experimental results

[0054] 1. BHM and DLT inhibit collagen synthesis in fibroblasts.

[0055] To preliminarily explore the effects of the histamine H1 receptor antagonists BHM and DLT on myocardial fibrosis, a fibrotic cell model was constructed by stimulating mouse embryonic fibroblasts (NIH3T3) with 15 ng / ml TGF-β1. After treatment with BHM (5 μM) and DLT (10 μM) for 48 h, total collagen content in the cells was measured using the Sirius red assay. (See attached image). Figure 1 The results showed that after TGF-β1 stimulation, the intracellular collagen content was 308.2±32.6 μg / ml, significantly higher than that of the blank control group (196.8±11.25 μg / ml, p<0.001). Intervention with the histamine H1 receptor antagonist BHM and DLT significantly reduced the total collagen content in the TGF-β1-induced fibrotic cell model. DLT intervention reduced the total collagen content to 233.1±16.84 μg / ml (p<0.01 vs TGF-β1 model group), and its effect in inhibiting total collagen synthesis was comparable to that of the positive control drug Val (212.6±18.51 μg / ml).

[0056] 2. Effects of H1 receptor antagonists on cardiac function in mice with myocardial fibrosis

[0057] Four weeks post-surgery, echocardiography was performed on mice to investigate the effects of the histamine H1 receptor antagonists BHM and DLT on cardiac function in TAC model mice. Figure 2 The results showed that the systolic left ventricular intraventricular diameter (LVIDs) in the sham-operated group was 2.185±0.21 mm, while that in the TAC model group was 2.417±0.14 mm, significantly higher than that in the sham-operated group (p<0.05). The left ventricular ejection fraction (EF%) in the sham-operated group was 62.14±2.43%, and the left ventricular fractional shortening (FS%) was 32.48±1.61%. Four weeks after TAC surgery, the EF% and FS% in mice were 55.81±2.95% and 28.30±1.98%, respectively, both significantly lower than those in the sham-operated group (p<0.05), suggesting that TAC can induce left ventricular systolic dysfunction in mice. After DLT treatment, the LVIDs in mice recovered to 1.769±0.21 mm, significantly lower than those in the model group (p<0.05). Compared with the model group, after DLT treatment, the EF% of mice recovered to 78.32±3.73% (p<0.01), and the FS% also recovered to 45.96±3.53% (p<0.01), both returning to normal levels, indicating that DLT treatment significantly improved cardiac systolic function in mice. After BHM intervention, the LVIDs of mice were 2.37±0.38 mm, EF% was 60.0±7.82%, and FS% also recovered to 31.38±5.28%, indicating recovery of cardiac function indicators.

[0058] 3. BHM and DLT alleviate TAC-induced myocardial fibrosis

[0059] HE staining results showed that ( Figure 3 A) In the sham-operated group, the cardiomyocytes were structurally intact and well-defined, with neat and tightly arranged myocardial fibers. In the TAC model mice, the cardiomyocytes were irregularly shaped, with disordered myocardial fiber arrangement, exhibiting rupture, connective tissue hyperplasia, and inflammatory cell infiltration. Compared to the model group, the Val treatment group showed more orderly arrangement of myocardial fibers and significantly improved fibrosis characteristics. After BHM and DLT treatment, under light microscopy, the cells showed more regular morphology, reduced intercellular spaces, and a small amount of collagen fiber hyperplasia and inflammatory cell infiltration in the interstitium, all showing improvement compared to the model group. Masson staining and Sirius red staining results showed ( Figure 3 In the BE and TAC model groups, cardiomyocytes were disordered, and the myocardial interstitium showed abundant positive collagen fiber staining (Masson staining: blue collagen fibers; Sirius red staining: red collagen fibers), with large areas of fibrosis replacing normal myocardial tissue. In both the BHM and DLT treatment groups, cell morphology was more regular, with a small amount of positive collagen fiber staining. Statistical analysis of the fibrosis area using Masson and Sirius red staining revealed that, compared to the model group, the histamine H1 receptor antagonists BHM and DLT significantly reduced the area of ​​myocardial fibrosis (p < 0.01 vs. model group).

[0060] 4. Research on the mechanism of DLT in the treatment of myocardial fibrosis

[0061] Previous experimental results suggest that both the histamine H1 receptor antagonist BHM and DLT have therapeutic effects on myocardial fibrosis. We further explored the mechanism of action of DLT in improving myocardial fibrosis.

[0062] (1) DLT inhibits TGF-β1-induced fibroblast activation

[0063] A fibrotic cell model was established by inducing mouse embryonic fibroblast differentiation with 15 ng / ml TGF-β1. After treatment with a series of DLT concentration gradients for 48 h, the total collagen content of the cells was measured. The results showed that ( Figure 4 A) DLT can inhibit collagen content in activated fibroblasts, with an IC50 of 2.61 μM.

[0064] α-SMA is a marker of fibroblast differentiation into myofibroblasts. RT-qPCR results show that ( Figure 4(B, C, D) Using β-actin as an internal control, compared with the blank control group, TGF-β1 significantly upregulated the mRNA expression of α-SMA in mouse fibroblasts (p < 0.001). With cell differentiation, the mRNA expression of Collagen I and Collagen III increased, leading to collagen deposition in fibroblasts. DLT concentrations of IC25 (0.56 μM), IC50 (2.61 μM), and IC75 (9.12 μM) were used to intervene in the TGF-β1-induced fibrotic cell model. Within a certain range, DLT significantly inhibited the mRNA expression of α-SMA, Collagen I, and Collagen III in a dose-dependent manner, inhibiting TGF-β1-induced cell fibrosis. The 9.12 μM concentration showed the best effect in inhibiting fibroblast activation and collagen synthesis (p < 0.0001 vs TGF-β1 model group).

[0065] (2) Detection of cardiac morphology and function in mice with myocardial fibrosis

[0066] DLT improves cardiac dysfunction by acting on histamine H1 receptors.

[0067] Four weeks after surgery, cardiac morphology and function in mice were assessed using echocardiography. Figure 5 In wild-type mice () Figure 5 In the sham-operated group, the LVIDs of mice were 1.95±0.12 mm, EF% was 62.88±2.84%, and FS% was 32.91±2.04%. Compared with the sham-operated group, the LVIDs of mice in the TAC model group were significantly increased (2.61±0.17 mm, p<0.0001), while EF% (48.98±2.38%, p<0.0001) and FS% (24.12±1.45%, p<0.0001) were significantly decreased, suggesting that TAC induces cardiac contractile dysfunction in mice. (Regarding HRH1...) - / - Ultrasound results in mice showed ( Figure 5 FH), HRH1 - / - In the sham-operated group, the LVIDs were 1.99±0.20 mm, EF% was 67.69±5.79%, and FS% was 32.25±3.56%. HRH1 - / - The model group mice had LVIDs of 2.06±0.13 mm, EF% of 69.79±5.54%, and FS% of 34.94±1.72%. HRH1 - / - Sham-operated mice and HRH1 - / - Compared with the model group mice, there were no significant changes in cardiac function indicators such as LVIDs, EF%, and FS% (p>0.05), suggesting that knocking out histamine H1 receptors can inhibit TAC-induced cardiac dysfunction in mice. Figure 5 FH). Compared with the TAC model group, mice treated with DLT showed that LVIDs, EF% and FS% all returned to normal levels. Figure 5 AD). ANP and BNP are recognized markers of heart failure, reflecting cardiac function. The expression of ANP and BNP in mouse heart tissue was detected using RT-qPCR technology, and the results showed ( Figure 5 In E and I tests, the mRNA expression levels of ANP (p < 0.001) and BNP (p < 0.01) in the model group mice were significantly higher than those in the sham-operated group, indicating that the model group mice had heart failure. DLT treatment at 10 mg / kg and 20 mg / kg significantly reduced the mRNA expression of BNP in the myocardium of mice (p < 0.01 vs. model group) and improved TAC-induced cardiac dysfunction.

[0068] (3) DLT improves myocardial fibrosis by acting on histamine H1 receptors.

[0069] ①DLT alleviates TAC-induced myocardial fibrosis in mice

[0070] HE staining results showed that ( Figure 6 A) TAC induced structural disorder in mouse cardiomyocytes, resulting in localized connective tissue hyperplasia and inflammatory cell infiltration. After DLT treatment, the cardiomyocytes in mice were more tightly and neatly arranged than those in the model group. HRH1 - / - The structure of myocardial cells after TAC in mice was compared with that in HRH1 mice. - / - No significant changes were observed in the sham-operated group. Masson staining of mouse myocardium (…) Figure 6 B, D) and Sirius red staining results ( Figure 6 (C, E) showed that the TAC model group mice had extensive fibrosis staining in the myocardial interstitium and perivascular area, with a significantly larger fibrosis area than the sham-operated group (p < 0.0001). After DLT treatment, the degree of myocardial fibrosis in mice was significantly reduced compared to the model group (p < 0.001). HRH1 - / - The degree of myocardial fibrosis was mild in both the sham-operated group and the model group, with no significant difference between the two groups.

[0071] ②DLT inhibits the expression of markers of myocardial fibrosis in mice

[0072] Further examination of the expression of myofibroblast markers α-SMA, Collagen I, and Collagen III in mouse myocardium revealed ( Figure 7Compared with the sham-operated group, the mRNA and protein expression levels of α-SMA in the myocardial tissue of mice in the TAC model group were significantly upregulated (p < 0.001), suggesting that cardiac fibroblasts differentiated into myofibroblasts four weeks after TAC surgery. Simultaneously, the mRNA and protein expression of Collagen I and Collagen III in the myocardium of mice in the model group were also significantly upregulated compared with the sham-operated group (p < 0.01), suggesting that TAC surgery induced increased collagen synthesis in the mouse myocardium, leading to myocardial fibrosis. Compared with the model group, the mRNA and protein expression levels of fibrosis markers such as α-SMA, Collagen I, and Collagen III in the mouse myocardium were significantly downregulated after DLT treatment (p < 0.05), suggesting that DLT can inhibit cardiac fibroblast activation and reduce cardiac collagen synthesis.

[0073] (4) DLT inhibits the development of myocardial fibrosis by regulating the expression of potential targets.

[0074] ①DLT inhibits the transcription of myocardial inflammation and oxidative stress-related factors in mice.

[0075] By constructing a network pharmacological map of histamine-regulated targets and genes related to myocardial fibrosis, it was found that 76 out of 92 genes involved in histamine regulation also have regulatory effects on myocardial fibrosis. Enrichment analysis using PPI, GO, and KEGG revealed (…). Figure 8 (A, B) The key regulatory genes of histamine in myocardial fibrosis are mainly concentrated in two pathways: lipid and atherosclerosis, and fluid shear stress and atherosclerosis. Analysis of genes in these two pathways revealed that histamine may participate in the regulation of myocardial fibrosis mainly by affecting the expression of various pro-inflammatory and inflammatory factors.

[0076] Based on network pharmacology analysis results, the expression of oxidative stress and inflammation-related factors in mouse heart tissue was analyzed. Figure 8RT-qPCR results showed that four weeks after TAC surgery, the mRNA expression levels of inflammatory factors interleukin (IL)-1β and IL-6, as well as oxidative stress-related factors endothelial nitric oxide synthase (eNOS) and nuclear factor erythroid 2-related factor 2 (Nrf2) in the heart tissue of model group mice were significantly increased (p < 0.05 vs sham-operated group). Compared with the model group, DLT significantly inhibited the mRNA expression of IL-1β, IL-6, eNOS, and Nrf2 in a dose-dependent manner (p < 0.05). These results suggest that DLT can effectively reduce the transcription of inflammation and oxidative stress-related factors during fibrosis, and has antioxidant and anti-inflammatory effects.

[0077] ②DLT inhibits phosphorylation of IKK and ERK1 / 2 proteins in mouse myocardial tissue

[0078] Western blot was further used to explore the effect of DLT on the MAPK signaling pathway. The results showed that ( Figure 9 The TAC model enhanced the phosphorylation of MAPK-related proteins IKK and ERK1 / 2 in mouse myocardium. After DLT treatment, the phosphorylation of IKK and ERK1 / 2 proteins was inhibited, with 20 mg / kg DLT showing the best effect in inhibiting the phosphorylation of IKK and ERK1 / 2 proteins (p < 0.01 vs model group).

[0079] As can be seen from the above embodiments, this invention provides an application of histamine H1 receptor antagonists in the preparation of drugs for treating myocardial fibrosis. This invention simulates the in vivo process of myocardial fibrosis by constructing a stress overload mouse model using TAC surgery, and by using a TGF-β1-induced mouse fibrotic cell model, combined with network pharmacology analysis and histamine H1 receptor gene knockout mice, to explore the anti-myocardial fibrosis effects and mechanisms of action of the histamine H1 receptor antagonists brompheniramine maleate (BHM) and desloratadine (DLT) at both the whole-body and cellular levels, providing a theoretical and experimental basis for the development of new anti-myocardial fibrosis drugs.

[0080] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of a histamine H1 receptor antagonist in the preparation of a drug for treating myocardial fibrosis, wherein the histamine H1 receptor antagonist is brompheniramine maleate.

2. The application according to claim 1, characterized in that, The myocardial fibrosis described is TGF-β1-induced cellular fibrosis.

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