Application of hydroxychloroquine in preparation of medicine for preventing and treating immune myocarditis, inflammatory cardiomyopathy and complications of immune myocarditis and inflammatory cardiomyopathy

Abstract

The invention relates to the field of biological medicines, and particularly discloses application of hydroxychloroquine in preparation of medicines for preventing and treating immune myocarditis, inflammatory cardiomyopathy and complications thereof. Immune myocarditis is caused by immune response caused by infectious or non-infectious diseases, and inflammatory cardiomyopathy is cardiomyopathy caused by chronic myocarditis. The hydroxychloroquine can reduce chemotaxis from inflammatory cells to the heart, inhibit macrophages from generating chemotactic factors CXCL16 and infiltration of Th17 cells, NKT cells and T lymphocytes, inhibit cardiac inflammatory response, fibrosis and cardiac function damage, and improve cardiac remodeling and ventricular dilatation. Hydroxychloroquine can significantly reduce cardiovascular composite endpoint events of patients with inflammatory cardiomyopathy, improve cardiac functions of the patients, and reduce hs-cTnI, NT-proBNP, hs-CRP and levels of various plasma inflammatory factors. Clinical and basic research results show that the hydroxychloroquine can be used for treating and / or preventing human immune myocarditis and inflammatory cardiomyopathy, and has the potential of being developed into the medicine for clinically preventing and treating immune myocarditis and inflammatory cardiomyopathy.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to application of hydroxychloroquine in preparing medicines for preventing and treating immune myocarditis, inflammatory cardiomyopathy and complications thereof. Background Art

[0002] Myocarditis is an inflammatory disease of the heart, and its pathological characteristics are inflammatory cell infiltration and cardiomyocyte death in the heart. Myocarditis can be caused by a variety of factors such as infection, autoimmune disease, drugs or toxins. It has become an important cause of sudden cardiac death in young people. Although most patients with myocarditis can recover, about 20% of patients develop cardiac fibrosis due to persistent inflammation, which leads to left ventricular remodeling and inflammatory dilated cardiomyopathy. However, there is currently a lack of effective treatment for myocarditis, resulting in about 50% of dilated cardiomyopathy patients eventually requiring heart transplantation. Given the burden of myocarditis on global health security, how to improve the treatment of myocarditis patients and improve patient survival rates has been a core issue in the diagnosis, treatment and research of myocarditis. There is no special treatment for immune myocarditis in the clinic, and it is usually supportive conventional treatment, including corticosteroid therapy, cardiac drugs such as beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, low-salt diet, diuretic therapy to treat fluid overload, antibiotic therapy to prevent bacterial infection, etc. We urgently need effective clinical interventions to reduce the incidence of myocarditis and reduce the risk of poor prognosis.

[0003] One of the important characteristics of myocarditis is the infiltration of multiple immune cells in the heart and the production of a large number of pro-inflammatory factors. The inflammatory storm caused by the complex immune response in myocarditis leads to a variety of cardiac histological and clinical manifestations in patients. In order to reveal the complex immune mechanism, the researchers used a mouse model called experimental autoimmune myocarditis (EAM) to trigger a cardiac injury response under the combined action of myocardial globulin or troponin I injection and adjuvants. This model effectively simulates the pathophysiological process of human myocarditis and inflammatory cardiomyopathy.

[0004] Inflammatory cardiomyopathy is a long-term sequelae caused by the chronicity of acute and fulminant myocarditis, namely post-myocarditis cardiomyopathy, which is mainly characterized by chronic heart failure caused by myocarditis, including inflammatory dilated cardiomyopathy and non-dilated cardiomyopathy subtypes. The causes of inflammatory cardiomyopathy include infection, immune disorder, physical and chemical damage, etc. Due to the persistent inflammatory state of myocardial tissue, patients with inflammatory cardiomyopathy usually show symptoms and signs of chronic heart failure, usually accompanied by myocardial damage (elevated troponin) and various arrhythmias. The diagnosis of inflammatory cardiomyopathy mainly depends on the history of myocarditis, persistent myocardial damage and cardiac contractile dysfunction, and histological evidence of endomyocardial biopsy. Histologically, inflammatory cardiomyopathy is characterized by focal or diffuse myocardial fibrosis with chronic inflammatory cell infiltration. Epidemiological data show that about 10%-20% of myocarditis patients will eventually develop chronic inflammatory cardiomyopathy, and the five-year survival rate of patients is only 52.7%-70.3%, and standardized and effective treatment is urgently needed. At present, the clinical and basic research results of inflammatory cardiomyopathy are very limited and insufficient to meet the needs of clinical diagnosis and treatment. Although the clinical results of several foreign research teams have confirmed the therapeutic effect of immunosuppressive therapy (mainly glucocorticoid combined with azathioprine) on inflammatory cardiomyopathy, not all patients with inflammatory cardiomyopathy respond to immunosuppressive therapy, and the safety of glucocorticoid combined with azathioprine in the treatment of patients with inflammatory cardiomyopathy has not been fully elucidated. Therefore, more effective clinical drugs and treatments are still needed to guide the personalized treatment of patients with inflammatory cardiomyopathy.

[0005] Hydroxychloroquine (HCQ) is a drug that was once widely used to treat malaria. Because HCQ can modulate innate and adaptive immune responses in multiple ways, it is currently being repurposed to treat a variety of autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and antiphospholipid syndrome. However, no reports have explored the potential of HCQ in the treatment of autoimmune myocarditis and inflammatory cardiomyopathy.

[0006] In the present invention, the applicant has originally discovered the potential role and mechanism of hydroxychloroquine in the treatment of myocarditis and inflammatory cardiomyopathy. This is a new treatment strategy that is beneficial to improving the treatment effect and prognosis of patients with myocarditis and inflammatory cardiomyopathy. Summary of the invention

[0007] The invention aims to provide an application of hydroxychloroquine in preparing a medicine for preventing and treating immune myocarditis, inflammatory cardiomyopathy and complications thereof, so as to provide a new therapeutic medicine for preventing and treating immune myocarditis and inflammatory cardiomyopathy.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a use of hydroxychloroquine in the preparation of a medicament for preventing and treating immune myocarditis and inflammatory cardiomyopathy.

[0010] As a further optimization scheme of the present invention, the immune myocarditis is myocarditis caused by immune response caused by different infections or non-infectious diseases; the inflammatory cardiomyopathy is cardiomyopathy caused by chronic myocarditis.

[0011] As a further optimization scheme of the present invention, the infection is caused by viruses, bacteria, fungi or other pathogens; the non-infectious disease is caused by autoimmune diseases, vaccines, drugs, poisons or other physical or chemical factors.

[0012] As a further optimization scheme of the present invention, the targets of hydroxychloroquine in inhibiting immune response of immune myocarditis include macrophage production of chemokine CXCL16 and infiltration of Th17 cells, NKT cells and T lymphocytes into the heart.

[0013] As a further optimization scheme of the present invention, hydroxychloroquine prevents and treats immune myocarditis and inflammatory cardiomyopathy by reducing the chemotaxis of inflammatory cells to the heart, reducing the production of chemokines by macrophages in the heart, reducing the infiltration of Th17 cells, NKT cells and T lymphocytes, inhibiting cardiac inflammatory response, fibrosis and cardiac function damage, and improving cardiac remodeling and ventricular dilatation of dilated heart failure.

[0014] As a further optimization scheme of the present invention, the hydroxychloroquine can significantly reduce the cardiovascular composite endpoint events of patients with inflammatory cardiomyopathy, improve the patients' cardiac function and reduce the levels of hs-cTnI, NT-proBNP and hs-CRP.

[0015] As a further optimization scheme of the present invention, the hydroxychloroquine can treat inflammatory cardiomyopathy by reducing the levels of multiple plasma inflammatory factors, including IL8, VEGFA, CXCL11, IL-2RB, OSM, CCL4, IL18, TGF-alpha, MCP-4, TNFSF14, IL-18R1, CCL3, CXCL10, CASP-8, ST1A1, and ADA.

[0016] As a further optimization scheme of the present invention, the dosage of hydroxychloroquine injected intraperitoneally or orally gavaged into mice is 1 to 5 mg / kg.

[0017] As a further optimized solution of the present invention, hydroxychloroquine prevents and treats immune myocarditis and inflammatory cardiomyopathy by inhibiting the production and release of inflammatory mediators.

[0018] As a further optimization scheme of the present invention, the drug is administered by subcutaneous injection, intramuscular injection, intravenous injection, oral administration or spray inhalation.

[0019] In a second aspect, the present invention provides a use of hydroxychloroquine in the preparation of a medicament for preventing and treating complications of immune myocarditis and complications of inflammatory cardiomyopathy.

[0020] As a further optimization scheme of the present invention, the complications of immune myocarditis include decreased left ventricular pressure, left ventricular hypertrophy, left ventricular dilatation, decreased left ventricular systolic function, decreased left ventricular diastolic function or heart failure; the complications of inflammatory cardiomyopathy include chronic heart failure caused by persistent inflammatory infiltration and fibrosis of the heart.

[0021] As a further optimization scheme of the present invention, the inflammatory cardiomyopathy is a cardiac dysfunction caused by chronic myocarditis due to various reasons.

[0022] As a further optimization scheme of the present invention, the inflammatory cardiomyopathy is mainly characterized by chronic heart failure after myocarditis, and the clinical manifestations include decreased ejection fraction, enlarged left ventricular internal diameter, persistent increase of hs-cTnI and NT-proBNP, and arrhythmia.

[0023] In a third aspect, the present invention provides a drug for preventing and treating immune myocarditis and inflammatory cardiomyopathy, with hydroxychloroquine as the main ingredient.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] The application of hydroxychloroquine provided by the present invention in the preparation of a drug for preventing and treating immune myocarditis is to induce a mouse cardiac immune inflammation model by subcutaneous injection of α-MHC, and the model animal is given hydroxychloroquine by intraperitoneal injection or oral gavage and the therapeutic effect is observed, and it is found that hydroxychloroquine limits immune myocarditis, inhibits cardiac remodeling, and improves damaged cardiac function. The key mechanism of hydroxychloroquine in treating immune myocarditis is clarified through experiments: by reducing the chemotaxis of inflammatory cells to the heart, reducing the production of chemokine CXCL16 by macrophages in the heart, reducing the infiltration of Th17 cells, NKT cells and T lymphocytes, inhibiting cardiac inflammatory response, fibrosis and cardiac function damage, improving cardiac remodeling and ventricular dilatation of dilated heart failure to prevent and treat immune myocarditis. Hydroxychloroquine can significantly reduce the cardiovascular composite endpoint events of patients with inflammatory cardiomyopathy, improve the cardiac function of patients, and reduce the levels of hs-cTnI, NT-proBNP, hs-CRP and multiple plasma inflammatory factors, and has good safety. The present invention can provide a new treatment strategy for preventing and treating immune myocarditis and inflammatory cardiomyopathy, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings:

[0027] Figure 1 The experimental results show that hydroxychloroquine treatment significantly alleviated the left ventricular dysfunction caused by the acute phase of autoimmune myocarditis in mice; A: left ventricular ejection fraction, B: left ventricular short-axis shortening rate, C: left ventricular anterior wall thickness during systole, D: left ventricular anterior wall thickness during diastole, E: E / A, F: cardiac contractile function index dp / dtmax measured by catheter, G: cardiac diastolic function index dp / dtmin measured by catheter, ultrasound detection: number of mice in each group n=8, catheter detection: Control group and HCQ group, n=6; EAM group and EAM+HCQ group n=8), data are expressed as mean ± standard deviation and analyzed by one-way analysis of variance.

[0028] Figure 2 The experimental results show that hydroxychloroquine treatment significantly alleviated the left ventricular dysfunction caused by the chronic stage of autoimmune myocarditis in mice; A: left ventricular ejection fraction, B: left ventricular short-axis shortening rate, C: left ventricular end-systolic diameter, D: left ventricular end-systolic volume, E: E / A, F: cardiac contractile function index dp / dtmax measured by catheter, G: cardiac diastolic function index dp / dtmin measured by catheter, ultrasound detection: Control group and HCQ group, n=8; EAM group and EAM+HCQ group n=13, catheter detection: n=4, data are expressed as mean ± standard deviation and analyzed by one-way analysis of variance.

[0029] Figure 3 The experimental results show that hydroxychloroquine treatment significantly reduced the cardiac inflammatory infiltration and fibrosis caused by the acute phase of autoimmune myocarditis in mice; wherein, A: a representative HE staining image for analyzing the inflammation score of mouse heart sections (scale bar = 200 μm), B: the mean inflammation score of mouse myocardial tissue (n = 8), C: a representative image of picrosirius red staining of mouse heart sections (scale bar = 200 μm), showing the degree of myocardial fibrosis, D: quantitative analysis of the percentage of myocardial fibrosis area in mouse myocardium (n = 5), data are expressed as mean ± standard deviation, the cardiac inflammation score was analyzed using the Kruskal-Wallis test, and the degree of fibrosis was evaluated using one-way analysis of variance.

[0030] Figure 4The experimental results show that hydroxychloroquine treatment significantly reduced cardiac inflammatory infiltration and fibrosis caused by the chronic stage of autoimmune myocarditis in mice; wherein, A: representative HE staining images for analyzing the inflammation score of mouse heart sections (scale bar = 200 μm), B: mean inflammation score of mouse myocardial tissue (n = 6), C: representative images of picrosirius red staining of mouse heart sections (scale bar = 200 μm), showing the degree of myocardial fibrosis, D: quantitative analysis of the percentage of myocardial fibrosis area in mouse myocardium (n = 6), data are expressed as mean ± standard deviation, the Kruskal-Wallis test was used for cardiac inflammation scoring, and the one-way analysis of variance was used to evaluate the degree of fibrosis.

[0031] Figure 5 The results of the experiment of hydroxychloroquine inhibiting the chemotaxis of the heart of mice with autoimmune myocarditis to inflammatory cells in the blood; A: the number of CD45+ inflammatory cells in the blood, B: neutrophil, monocyte, T lymphocyte (CD3+Tcell), B lymphocyte (Bcell), natural killer cell (NKcell), natural killer T lymphocyte (NKTcell), macrophage (Macrophage) in the blood, Control group and HCQ group, n = 6; EAM group, n = 11, EAM + HCQ group, n = 10. C: Single cell sequencing analysis of GO pathway enrichment of genes downregulated after HCQ treatment, data are expressed as mean ± standard deviation, and comparisons between the same cell types were analyzed using one-way analysis of variance, *P < 0.05, **P < 0.01, ***P < 0.001.

[0032] Figure 6 The results of the experiment of hydroxychloroquine inhibiting the production of chemokine CXCL16 by mouse macrophages; A: down-regulated genes in macrophages after HCQ treatment and the top 10 genes among them. B: Western blot of CXCL16 in heart tissue of mice in each group, C: the level of CXCL16 protein relative to GAPDH (n=3).

[0033] Figure 7The experimental results of hydroxychloroquine inhibiting the chemotaxis of macrophages to NKT cells and T lymphocytes in the heart; A: immunofluorescence image of Th17 cells in the heart, Th17 cell marker gene (IL17a+) is green fluorescence, T cell marker gene (CD3+) is green, DAPI indicates cell nucleus, which is blue, scale bar = 50μm, B: the proportion of Th17 cells in the heart to CD3+T lymphocytes (n = 3), C: natural killer cell marker gene (Integrinalpha2+), T cell marker gene (CD3+) is green, DAPI indicates cell nucleus, which is blue, scale bar = 50μm, D: the proportion of natural killer cell T lymphocytes in the heart (n = 3). Data are expressed as mean ± standard deviation and analyzed by one-way ANOVA.

[0034] Figure 8 The results of hydroxychloroquine's therapeutic effect on patients with inflammatory cardiomyopathy are shown in the figure; AE: the effect of HCQ treatment for 12 months on the cardiovascular composite endpoint of inflammatory patients (including cardiovascular death and heart transplantation, hospitalization due to recurrence of heart failure or myocarditis, and permanent pacemaker or ICD implantation) (A), cardiovascular death and heart transplantation (B), hospitalization due to recurrence of heart failure or myocarditis (C), and permanent pacemaker or ICD implantation (D); EJ: the levels of LVEF (E), LVIDd (F), hs-cTnI (G), NT-proBNP (H), hs-CRP (I) and ESR (J) in patients with inflammatory cardiomyopathy after 12 months of HCQ treatment.

[0035] Fig. 9 To investigate the improvement of 16 plasma inflammatory factors in patients with inflammatory cardiomyopathy treated with hydroxychloroquine. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with the accompanying drawings, embodiments and experimental examples. Of course, the protection scope of the present invention is not limited to the following embodiments. Professional and technical personnel in the field can understand that various changes and modifications can be made to the present invention without departing from the spirit of the present invention. The present invention generally and / or specifically describes the materials and test methods used in the experiment. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described as detailed as possible here. The following examples further illustrate the present invention, rather than limiting the present invention. Any equivalent transformation that is only formal and not substantial based on the concept of the present invention should be regarded as the scope of the technical solution of the present invention.

[0037] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0039] Example 1. Construction of mouse autoimmune myocarditis (EAM) model

[0040] Male BALB / c mice aged 6 to 8 weeks were randomly divided into four groups according to body weight, namely, Control group, HCQ group, EAM group, and EAM+HCQ group. First, the mice were divided into modeling group and non-modeling group according to body weight. For the modeling group, MyHC-α peptide (sequence: Ac-RSLKLMATLFSTYASADR-OH) was dissolved in 0.9% sterile NaCl aqueous solution, and the peptide and Freund's Complete Adjuvant (FCA) were mixed in equal volumes at a ratio of 1:1 with the help of a three-way tube, and the peptide was finally completely emulsified. Then the emulsion was injected subcutaneously at multiple points on the back of the mouse on the 0th and 7th days, and each mouse was injected with 200ul of emulsion containing 300ug of peptide. For the non-modeling group, sterile saline and Freund's Complete Adjuvant were mixed in equal volumes at a ratio of 1:1. The injection method was the same as the modeling group.

[0041] Example 2. Pharmacological efficacy of HCQ on EAM model mice

[0042] This experiment was divided into four groups: Control group, HCQ group, EAM group, and EAM+HCQ group, with 8 mice in each group. Mice in the HCQ group and EAM+HCQ group were given HCQ 52 mg / kg intraperitoneally or orally once a day after modeling, and mice in other groups were given a calculated amount of normal saline, which was continuously administered until the end of the experiment, which was the 21st day (acute stage of myocarditis) and the 60th day (chronic stage of myocarditis), respectively.

[0043] Example 3. Small Animal Cardiac Ultrasound and Catheterization

[0044] At the end of the experiment, mice were anesthetized with 1.5% to 2% isoflurane, and a 30MHz high-frequency ultrasound detection probe specially designed for mice was selected to collect images of two sections, namely the parasternal long-axis section and the cardiac short-axis section. Then, at least three consecutive cardiac cycles were delineated to measure the heart rate (HR), left ventricular ejection fraction (LVEF), left ventricular short-axis shortening (LVFS), left ventricular internal dimension InSystole (LVID, s), left ventricular end-systolic volume (LVESV), left ventricular anterior wall thickness in systole (LVAW; s), left ventricular anterior wall thickness in diastole (LVAW; d), and other indicators. At the same time, the diastolic function of the heart, namely E / A, was detected. The measurement results are shown in Figure 1 A~ Figure 1 E, Compared with the control group mice, the left ventricular ejection fraction and left ventricular axis shortening rate of the mice in the myocarditis group were reduced, E / A, increased, the left ventricular short axis shortening rate was reduced, and the left ventricular anterior wall thickness during systole and diastole was increased. After HCQ treatment, the above indicators were improved. Figure 2 A~ Figure 2 E, Compared with the control group mice, the left ventricular ejection fraction and left ventricular axial shortening rate of the mice in the myocarditis group were decreased, E / A, increased, and the left ventricular end-systolic diameter and volume increased. All of the above indicators were improved after HCQ treatment.

[0045] At the end of the experiment, the mice were subjected to invasive cardiac catheterization. After anesthetizing the mice, the right common carotid artery was isolated, and the distal end of the right common carotid artery was ligated with 4-0 silk thread, and then the silk thread was fixed at the head end. The proximal end of the right common carotid artery was clamped with an artery clamp, and under a continuous magnification stereomicroscope, a guide wire was inserted into the right common carotid artery to measure the left ventricular hemodynamics. Among them, dp / dtmax reflects the contractile function of the left ventricle, and dp / dt mix reflects the diastolic function of the left ventricle. The measurement results are shown in Figure 1 F~ Figure 1 G and Figure 2 F~ Figure 2 G, Both dp / dt max and dp / dt min were decreased in mice with myocarditis, and hydroxychloroquine reversed this change.

[0046] Depend on Figure 1 and Figure 2 It can be seen that hydroxychloroquine can improve the cardiac function of mice with autoimmune myocarditis.

[0047] Example 4. Histopathological detection

[0048] Mouse heart tissue sections were cut transversely, fixed with 4% paraformaldehyde, and then dehydrated, infiltrated and embedded in paraffin. The thickness of each paraffin section was 4 μm.

[0049] HE staining: paraffin sections of mouse hearts were taken and baked in an oven at 65°C for 1 hour before dewaxing and rehydration. The tissue sections were stained with hematoxylin-eosin dye. Hematoxylin can stain the cell nucleus and eosin can be used for counterstaining. After sealing, the sections were observed and photographed under a microscope to evaluate the tissue pathological morphology and perform inflammation scoring. The results are shown in Figure 2 A~ Figure 2 B (acute phase of myocarditis), Figure 3 A~ Figure 3 B (chronic myocarditis), caused by Figure 2 A~ Figure 2 B, Figure 3 A~ Figure 3 B shows that there are a large number of inflammatory cell infiltrations in the hearts of mice in the myocarditis group, and hydroxychloroquine can reduce cardiac inflammatory infiltration in mice with autoimmune myocarditis.

[0050] Sirius red staining, paraffin sections, placed in a 65℃ oven for 0.5h, dewaxed, rehydrated, stained with 0.1% Sirius red staining solution for 1.5h, and finally sealed with glycerol. Observed and photographed under a microscope, and the content of collagen fibers was analyzed using Image J software. Results are shown in Figure 2 C~ Figure 2 D (acute phase of myocarditis), Figure 3 C~ Figure 3 D (chronic stage of myocarditis).

[0051] Depend on Figure 2 C~ Figure 2 D, Figure 3 C~ Figure 3 D shows that a large amount of fibrillar collagen is deposited in the hearts of mice with myocarditis, and hydroxychloroquine can reduce cardiac fibrosis in mice with autoimmune myocarditis.

[0052] Example 5. Effect of HCQ on chemotaxis of inflammatory cells

[0053] Flow cytometry: Flow cytometry was used to analyze cell populations by incubation or staining with different antibodies or fluorescent markers. Prior to staining, single cell suspensions were incubated with anti-CD16 / 32 antibodies (Biolegend, San Diego, CA, USA) to block nonspecific binding to Fc receptors. Cells were incubated at 4 °C for 30 min for surface staining.

[0054] The following antibodies were used: APC-Cy7 anti-CD45, PE-CF594 anti-CD3, BV605 anti-CD4, BV650 anti-CD8a, PE-Cy7 anti-Ly-6C, BV421 anti-CD49b Pan-NKCells, BV510 anti-CD19, BV786 anti-LY-6G, BB515 anti-CD11b (BD Biosciences, San Jose, CA, USA), PE anti-F4 / 80 (BioLegend, San Diego, CA, USA), Fixable Viability Kit (Biolegend, San Diego, CA, USA). For results, see Figure 5 A and 5B, Figure 5 A. In the myocarditis group, the number of inflammatory cells in the blood increased significantly. HCQ treatment can reduce the total number of inflammatory cells in the blood. Figure 5 B, The numbers of various inflammatory cells in the blood of mice with myocarditis increased. HCQ could significantly reduce the numbers of monocytes, T lymphocytes, B lymphocytes, and macrophages, and had a tendency to reduce the numbers of other inflammatory cell types.

[0055] Langendorff perfusion device to isolate non-cardiomyocytes in the heart: The heart was quickly removed and retrogradely perfused with a calcium-free perfusion buffer at 37°C through a Langendorff system. The components of the perfusion buffer included NaCl 125mmol / L, HEPES 15mmol / L, KCl 5mmol / L, taurine 6mmol / L, MgCl2 mmol / L, glucose 10mmol / L and 2,3-butanedione 7.5mmol / L, and the pH was adjusted between 7.35-7.38. The flow rate was maintained at 2ml / min for five minutes. Subsequently, the heart was perfused with an enzyme buffer containing 0.67mg / ml type II collagenase (LS004176, Worthington, NJ, USA) at the same flow rate at 37°C for about 20-25 minutes. The collected cells were centrifuged at 300×g for 3 minutes to collect non-cardiomyocytes for single-cell sequencing. The sequencing results were subjected to GO enrichment analysis ( Figure 5 C), the results showed that multiple inflammatory pathways were activated in the hearts of mice with myocarditis, and HCQ effectively inhibited the activation of inflammatory cells and the chemotaxis of the heart to inflammatory cells.

[0056] Depend on Figure 5 It can be seen that HCQ reduces inflammatory infiltration of the heart by reducing the chemotaxis of the heart to inflammatory cells in the blood.

[0057] Example 6. Effect of HCQ on the production of chemokine CXCL16 by macrophages

[0058] Venn diagram drawing: Macrophage gene expression data were obtained from the EAM group and samples after EAM+HCQ treatment. The data were quality controlled and preprocessed, including steps such as removal of low-quality reads, standardization and normalization. Gene differential analysis was performed using the R package DESeq2 to determine upregulated and downregulated genes and to draw Venn diagrams using R. In the Venn diagram, the number of upregulated and downregulated genes and genes in the overlapping region were marked to highlight the size of the set. In addition, the TOP10 changed genes, i.e., the top 10 genes with the most significant differences in the genes in the overlapping region, were additionally marked. Results can be found in Figure 6 A, Compared with the control group, the number of upregulated genes in the EAM group was 427, the number of downregulated genes after HCQ treatment was 308, and the number of genes in the intersection was 130. Among the top 10 genes, Cxcl16 was the only gene encoding a chemokine.

[0059] Western Blot Analysis: Homogenize the obtained mouse heart tissue samples with ice-cold IP lysis buffer. Determine the protein concentration using the Bio-Rad protein assay. Mix 50ug of protein with sample buffer and denature by heating to 95°C for 5min. Separate the proteins on a 10% SDS polyacrylamide gel and transfer to a nitrocellulose membrane and block with 10% bovine serum albumin for 1 hour. Develop after antibody incubation. The housekeeping protein GAPDH was used as an internal reference. The intensity of the target blot was measured by quantitative scanning densitometer image software (Image J). Results can be found in Figure 6 B and 6C, the expression of cardiac CXCL16 protein was significantly increased in the EAM group, and the expression of CXCL16 was significantly decreased after HCQ treatment.

[0060] Depend on Figure 6 It can be seen that HCQ can reduce the content of key molecules in the heart that have important chemotactic effects on inflammatory cells, namely the CXCL16 protein produced by macrophages.

[0061] Example 7. Effect of HCQ on cardiac NKT cell and T lymphocyte infiltration

[0062] Tissue immunofluorescence staining: After dewaxing and rehydration of tissue sections, antigen retrieval was performed. Then, blocking was performed with 10% goat serum at room temperature for 1 hour, followed by incubation with antibodies at 4°C overnight. Species-specific fluorescent-conjugated secondary antibodies were used, incubated in the dark for 1 hour, and then nuclear staining was performed with DAPI, followed by sealing and observation under a fluorescence microscope. The following antibodies were used: IL17a (ABclonal, Wuhan, China), CD3 (Santa Cruz, CA, USA), Integrin alpha2 (ab181548, Abcam, UK). For results, see Figure 7The numbers of infiltrating Th17 cells and natural killer T lymphocytes in the hearts of mice in the EAM group increased, and HCQ treatment could effectively reduce the infiltration of Th17 cells and natural killer T lymphocytes in the heart.

[0063] Example 8. Effect of HCQ on cardiovascular composite endpoints, cardiac function and myocardial injury markers in patients with inflammatory cardiomyopathy

[0064] Through a prospective randomized controlled study, 50 patients with inflammatory cardiomyopathy were enrolled and randomly assigned to the hydroxychloroquine combined with prednisolone treatment group (HCQ+PDN) and the prednisolone alone treatment group (PDN). The patients were followed up for one year, and the primary endpoint was a cardiovascular composite endpoint including cardiovascular death and heart transplantation, hospitalization for heart failure or recurrent myocarditis, and permanent pacemaker or ICD implantation. The secondary endpoints were improvements in LVEF, LVIDd, hs-cTnI, NT-proBNP, hs-CRP, and ESR.

[0065] Depend on Figure 8 It can be seen that HCQ can significantly reduce the cardiovascular composite endpoint events in patients with inflammatory cardiomyopathy, improve patients' cardiac function and reduce the levels of hs-cTnI, NT-proBNP and hs-CRP.

[0066] Example 9. Effect of HCQ on Plasma Inflammatory Factor Levels in Patients with Inflammatory Cardiomyopathy

[0067] The Olink Target 96 Inflammation panel was used to measure the levels of inflammatory factors in patients with inflammatory cardiomyopathy and healthy controls, and to compare the changes in inflammatory factors in patients with inflammatory cardiomyopathy treated with hydroxychloroquine and those not treated with hydroxychloroquine. Fig. 9 HCQ treatment can significantly reduce 16 inflammatory factors (IL8, VEGFA, CXCL11, IL-2RB, OSM, CCL4, IL18, TGF-alpha, MCP-4, TNFSF14, IL-18R1, CCL3, CXCL10, CASP-8, ST1A1, ADA) to normal levels.

[0068] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. The application of hydroxychloroquine in the preparation of drugs for preventing and treating immune myocarditis and inflammatory cardiomyopathy.

2. The use according to claim 1, characterized in that: The immune myocarditis is myocarditis caused by immune response caused by different infections or non-infectious diseases; the inflammatory cardiomyopathy is cardiomyopathy caused by chronic myocarditis.

3. The use according to claim 2, characterized in that: The infection is caused by viruses, bacteria, fungi or other pathogens; the non-infectious disease is caused by autoimmune diseases, vaccines, drugs, poisons or other physical or chemical factors.

4. The use according to claim 1, characterized in that: The targets of hydroxychloroquine for inhibiting the immune response of immune myocarditis include macrophages producing chemokine CXCL16 and Th17 cells, NKT cells and T lymphocytes infiltrating into the heart.

5. The use according to claim 1, characterized in that: The hydroxychloroquine can significantly reduce the cardiovascular composite endpoint events of patients with inflammatory cardiomyopathy, improve the patients' cardiac function, and reduce the levels of hs-cTnI, NT-proBNP, hs-CRP and multiple plasma inflammatory factors.

6. The use according to claim 5, characterized in that: The plasma inflammatory factors include IL8, VEGFA, CXCL11, IL-2RB, OSM, CCL4, IL18, TGF-alpha, MCP-4, TNFSF14, IL-18R1, CCL3, CXCL10, CASP-8, ST1A1, and ADA.

7. The use according to claim 1, characterized in that: The drug is administered by subcutaneous injection, intramuscular injection, intravenous injection, oral administration or spray inhalation.

8. The use of hydroxychloroquine in the preparation of drugs for preventing and treating complications of immune myocarditis and inflammatory cardiomyopathy.

9. The use according to claim 8, characterized in that: The complications of immune myocarditis include decreased left ventricular pressure, left ventricular hypertrophy, left ventricular dilatation, decreased left ventricular systolic function, decreased left ventricular diastolic function or heart failure; the complications of inflammatory cardiomyopathy include chronic heart failure caused by persistent inflammatory infiltration and fibrosis of the heart.

10. A drug for preventing and treating immune myocarditis and inflammatory cardiomyopathy, characterized in that: With hydroxychloroquine as the main ingredient.

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