CN115969839A - Application of acacetin or acacetin water-soluble prodrug in preparation of medicine for preventing and treating immune myocarditis - Google Patents

Abstract

The invention discloses application of acacetin or an acacetin water-soluble prodrug in preparation of a medicine for preventing and treating immune myocarditis, and belongs to the technical field of medicines. For rat and mouse autoimmune myocarditis caused by myosin heavy chain-alpha peptide, the injection of acacetin water-soluble prodrug or oral administration of gold and acacetin can obviously inhibit the immune myocarditis and improve the function of the left ventricle; the effect is that the acacetin inhibits the activation and proliferation of heart-specific autoreactive CD4+ T lymphocytes and the differentiation of Th17 cells, reduces the activity of a CD4+ T cell mitochondrial complex II, thereby inhibiting mitochondrial respiration and mitochondrial reactive oxygen cluster generation in the CD4+ T cells, inhibiting myocardial inflammatory response and myocardial YAP/TAZ expression and inhibiting subsequent delayed myocardial fibrosis. Experimental results show that the acacetin and the acacetin water-soluble prodrug can be used for treating and/or preventing human immune myocarditis and have the potential of being developed into a clinical medicine for preventing and treating the immune myocarditis.

Description

Application of acacetin or acacetin water-soluble prodrug in preparation of medicine for preventing and treating immune myocarditis

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of acacetin or a water-soluble acacetin prodrug in preparation of a medicine for preventing and treating immune myocarditis.

Background

Immune myocarditis is a polymorphic, immune-mediated inflammation of the heart muscle that is mostly due to infection. Myocarditis is an autoimmune inflammatory disease characterized by myocardial cell necrosis and inflammatory cell infiltration, a common cause of sudden juvenile death, which can often progress to dilated heart failure, leading to poor prognosis.

There is no specific clinical treatment for immune myocarditis, and it is usually a supportive routine therapy including corticosteroid therapy, cardiac drugs such as beta blockers, angiotensin Converting Enzyme (ACE) inhibitors, low-salt diets, diuretic therapy to treat fluid overload, antibiotic therapy to prevent bacterial infections, and the like. Recent studies have shown that heart-specific autoreactive CD4+ T cells play an important role in the pathogenesis of myocarditis, where the reactive effector CD4+ T is the major driver for the progression of myocardial injury and dilated heart failure, such as IL-17-producing Th17 cells. Effector CD4+ T cells exacerbate myocarditis by promoting viral replication, cytokine secretion, myeloid infiltration, and myocardial fibrosis. Thus, targeted inhibition of CD4+ T cell activation may be crucial for the development of novel methods of treatment of myocarditis.

In most cases, immune myocarditis regresses spontaneously, but in susceptible people, immune myocarditis progresses to a chronic stage eventually leading to pathological cardiac remodeling. Pathological remodeling includes tissue fibrosis, hypertrophy, and dilated heart failure with apoptosis of myocardial cells and resulting in impaired contractility.

Acacia is widely present in a variety of natural plants and has antioxidant, anti-inflammatory and antiproliferative effects. The research also shows that the water-soluble prodrug formed by the restructuring of the farnesoid can be directly converted into the farnesoid in bodies of beagle dogs, mice and rats to exert pharmacological effects. This is directly effective in improving the availability of acacetin in vivo, and also enables the clinical administration of both gold and acacetin for the treatment of emergency patients (see Water-soluble acacetin precursor formulations diagnostic and infusion in scientific reports. DOI:10.1038/srep36435. And Synthesis of aqueous Water-soluble acacetin precursor for treating experimental impurities CN 115337300A. Scientific reports. DOI. Although the component farnesoid is involved in the composition disclosed in the Chinese patent CN112423726A, the composition treats diseases related to inflammatory skin diseases, but the therapeutic action and mechanism of the farnesoid and the water-soluble prodrug thereof on immune myocarditis are not reported.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide the application of the acacetin or the acacetin water-soluble prodrug in preparing the medicine for preventing and treating the immune myocarditis and provide a new treatment medicine for preventing and treating the immune myocarditis.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the invention discloses application of acacetin or an acacetin water-soluble prodrug in preparation of a medicine for preventing and treating immune myocarditis.

Preferably, the immune myocarditis is myocarditis caused by immune response due to different infectious or non-infectious diseases.

Further preferably, the infection is caused by a virus, a bacterium or a mould.

Preferably, the non-infectious disease is caused by a systemic autoimmune disease, a vaccine or a drug.

Preferably, the symptoms of immune myocarditis include decreased left ventricular pressure, left ventricular hypertrophy, left ventricular dilation, decreased left ventricular systolic function, decreased left ventricular diastolic function, or heart failure.

Preferably, the target of acacetin to suppress the myocarditis immune response includes mitochondrial energy metabolism of CD4+ T cells.

Preferably, farnesoid prevents and treats immune myocarditis by reducing the activity of mitochondrial complex II, inhibiting mitochondrial respiratory function, energy metabolism and immune response in CD4+ T cells, inhibiting CD4+ T lymphocyte activation, proliferation and Th17 cell differentiation, further inhibiting YAP, TAZ or MST1 activity and expression, inhibiting cardiac inflammatory response, fibrosis and cardiac functional damage, improving cardiac remodeling and ventricular dilation of dilated heart failure.

More preferably, the mode of acacetin inhibition of mitochondrial energy metabolism of CD4+ T cells is: by reducing mitochondrial respiratory reserve, T cell activation energy is limited.

Preferably, farnesoid inhibits CD4+ T cell activation in association with inhibition of CD4+ T cell mitochondrial ROS production and mitochondrial membrane potential.

Preferably, farnesoid inhibits CD4+ T cell activation, proliferation and Th17 cell differentiation and reduces cell cycle, electron transport and fatty acid beta oxidation-related gene expression, thereby affecting the cell cycle, mitochondrial respiration and lipid metabolism of CD4+ T cells.

Preferably, the dosage of the mouse oral administration of the Acacia gavage or the water-soluble prodrug of the Acacia gavage is 5-100 mg/kg.

Preferably, farnesin prevents immune myocarditis by inhibiting activation and release of late inflammatory mediators.

Preferably, the medicament is administered by subcutaneous injection, intramuscular injection, intravenous injection, oral administration or inhalation spray.

Preferably, the animal model of immune myocarditis is induced by subcutaneous injection of myocardium-specific alpha-MHC.

Preferably, the acacetin water-soluble prodrug is formed by modifying a fat-soluble molecule of acacetin, and the acacetin water-soluble prodrug exerts a pharmacological effect by being converted into acacetin in vivo.

The invention also discloses application of acacetin or an acacetin water-soluble prodrug in preparation of a medicine for preventing and treating immune myocarditis complications.

The invention also discloses a medicine for preventing and treating the immune myocarditis, which takes the acacetin or the water-soluble prodrug of the acacetin as the main component.

Compared with the prior art, the invention has the following beneficial effects:

the acacetin or acacetin water-soluble prodrug provided by the invention is applied to preparation of a medicine for preventing and treating immune myocarditis, an alpha-MHC (major histocompatibility complex) is injected subcutaneously to induce formation of a rat and mouse heart immune inflammation model, and the acacetin water-soluble prodrug is injected subcutaneously or orally administered with gavage and acacetin to observe curative effects, so that cash and acacetin are used for limiting immune myocarditis, inhibiting myocardial remodeling and improving damaged heart functions. The key mechanism of acacetin in the treatment of immunological myocarditis is clarified by experiments: the mitochondrial respiration and energy metabolism functions of CD4+ T cells are inhibited by affecting the activity of the heart-specific CD4+ T cell mitochondrial complex II, thereby inhibiting CD4+ T cell activation, cell cycle and Th17 cell differentiation. The traditional Chinese medicine composition can inhibit myocardial immune response and inflammatory reaction, limit immune myocarditis, inhibit expressions of YAP, TAZ and MST1 which are related to lower cardiac fibrosis, and inhibit myocardial remodeling, left ventricular hypertrophy and expansion, thereby playing a role in improving the treatment effect of damaged cardiac function, providing a new treatment strategy for preventing and treating the immune myocarditis, and having good application prospect.

Drawings

FIG. 1 is a chemical structure diagram of acacetin and a water-soluble prodrug of acacetin;

FIG. 2 is a graph showing the experimental results of farnesoid prodrug on improvement of cardiac dysfunction in rats with immune myocarditis; wherein, A: the administration scheme is as follows: the α -MHC emulsion was injected subcutaneously on days 0 and 7, and different doses of the acacetin prodrug or vehicle were injected subcutaneously on days 0 to 21 of the experiment, B: the detection result of the rat hemodynamic index shows that b1 is left ventricular pressure, b2 is left ventricular maximum contraction rate, b3 is electrocardiogram, and C: heart rate mean (n = 6-7) for different experimental groups, D: mean blood pressure of different experimental groups, E: left ventricular systolic mean values, F: mean left ventricular systolic maximal rates (n = 6-7) for different experimental groups, data are expressed as mean ± standard deviation and analyzed by one-way analysis of variance;

FIG. 3 is a graph showing the results of experiments in which farnesoid prodrug ameliorated cardiac function impairment in rats with myocarditis in another immune rat model with myocarditis; wherein, A: subcutaneous injections of α -MHC were given on days 0 and 7, and subcutaneous injections of saline (myocarditis vehicle group) or acacetin prodrug group (10 mg/kg, b.i.d.) were given on days 0 to 21 of the experiment, representative echocardiograms of rats, B: control, myocarditis vehicle and myocarditis gold and albizim treated groups rats left ventricular echocardiography ejection fraction mean (n = 15), C: mean left ventricular echocardiography left ventricular axis shortening rates for rats in different experimental groups, D: heart index mean (n = 15) for different experimental groups, data are expressed as mean ± standard deviation and analyzed with one-way analysis of variance;

FIG. 4 is a graph showing the results of experiments in which Au and Albizzim prodrugs inhibited myocardial inflammatory response and myocardial fibrosis in rats with immune myocarditis; wherein, A: representative HE staining images (scale bar =50 μm), B: mean rat myocardial tissue inflammation score (n = 9-10), C: masson stained representative images of rat ventricular sections (scale =50 μm) showing the degree of myocardial fibrosis, D: quantitative analysis of myocardial fibrosis area percent mean (n = 10-11) in rats, data are expressed as mean ± standard deviation and analyzed by one-way analysis of variance, followed by Tukey's test;

FIG. 5 is a graph showing the results of experiments in which gold and albizin prodrugs inhibited the activation of myocarditis-associated proteins in rats; wherein, A: the myocardial TNF- α protein activation results for each group of rats, a1 for western blot, a2 for relative TNF- α levels (n = 5-6), B: results of cardiac TLR4 protein activation in various groups of rats, b1 is western blot, b2 is relative TLR4 level (n = 4), C: results of myocardial NK- κ B protein activation in rats of each group, c1 is western blot, c2 is relative NK- κ B level (n = 4-6), D: the results of IFN- γ protein activation in the myocardium of each group of rats, d1 being the western blot and d2 being the relative IFN- γ level (n = 4), data expressed as mean ± standard deviation and analyzed by one-way analysis of variance followed by Tukey's test;

FIG. 6 is a graph showing the results of experiments on inhibition of myocardial fibrosis protein activation by Au and Albizzia julibrissin in rats with myocarditis; wherein, A: control, myocarditis model (vehicle) and myocarditis gold and albizzia (10 mg/kg) treated rat myocardium YAP protein activation experimental results, a1 is YAP protein and GAPDH for blots, a2 is relative YAP level (n = 4), B: results of the experiment on myocardial TAZ protein activation in rats of different experimental groups, b1 is TAZ for western blot, b2 is relative TAZ level (n = 4), C: results of experiments on myocardial MST1 protein activation in rats of different experimental groups, c1 is MST1 for western blot, c2 is relative MST1 level (n = 6), D: results of experiments on myocardial cytoplasmic and nuclear protein activation in rats of different experimental groups, d1 is TAZ, GAPDH, histone stands for western blot, d2 is relative myocyte TAZ level (n = 4), d3 is relative nuclear TAZ level (n = 4), data are expressed as mean ± standard deviation and analyzed by one-way analysis of variance, followed by Tukey test;

FIG. 7 is a graph of the results of an acacetin assay to reduce myocardial damage in mice with myocarditis; wherein, A: the administration scheme is as follows: alpha-MHC was injected subcutaneously on days 0 and 7, and farnesin (100 mg/kg/day) or vehicle was administered orally on days 0 to 21 of the experiment, B: control mice, myocarditis vehicle mice, and myocarditis farnesoid-treated mice representative echocardiograms, C: echocardiography-measured mouse left ventricle indices, c1 is the percent EF (n = 5-7), c2 is the percent FS (n = 5-7), D: control mice, myocarditis vehicle mice, and myocarditis farnesoid treated mice representative cardiac images (scale bar =5 mm), E: HW/BW ratio (n = 11-12), F: representative HE staining images (scale bar =50 μm), G: mouse myocardial tissue inflammation score mean (n = 11-12), H: mason staining representative images of mouse ventricular sections (scale bar =50 μm), I: mouse myocardium quantification fibrosis area percent mean (n = 11), data expressed as mean ± standard deviation, and analyzed by one-way analysis of variance, then Tukey test or Kruskal-Wallis Dunn multiple comparison test P <0.05, P <0.01, P <0.001, P <0.0001;

FIG. 8 is farnesoid reduces inflammatory cytokines or activated inflammatory immunity in mice with myocarditisA map of relevant cells; wherein, A: from left to right, the mean expression values of genes Il1B, il6, tnf, ccl3, ccl5, ifng, il17a, il17f (relative to GAPDH, n = 5-6) in ventricular tissues of control mice, myocarditis vehicle mice, and farnesoid myocarditis mice, respectively, from top to bottom (B: CD4+ T cell-related index, b1 is a representative CD4+ T cell flow chart (n = 5-12), b2 is a percentage of effector CD4+ T lymphocytes (n = 5-7) in mouse spleen cells, and b3 is a percentage of effector CD4+ T lymphocytes in mouse spleen cells

CD4+ T percentage value, C: th17 cell-associated index, c1 is Th17 cell flow chart (n = 5-7), c2 is Th17 cell percentage value in mouse spleen cells, D: macrophage-related index, d1 is macrophage flow chart, d2 is percent value of macrophages in mouse spleen cells, E: the Ly6C monocyte related index, e1 is a Ly6C monocyte flow chart, e2 is a Ly6C monocyte percentage value, F: the related indexes of the neutrophils, f1 is a neutrophil flow chart, f2 is a neutrophil percentage value, the data are mean values plus or minus standard deviation, unidirectional variance analysis is adopted, and Tukey test analysis is carried out on P<0.05，**P<0.01，***P<0.001，****P<0.0001；

Fig. 9 is a graph showing the results of experiments on inhibition of T cell activation, proliferation and Th17 cell differentiation in vitro by farnesoid; wherein, A: cell CD44 after continuous incubation with different concentrations of acacetin; a1 is a cell flow chart, a2 is a proliferation percentage value, B: after continuous incubation of different concentrations of acacetin, the cells CD25, b1 are cytogram, b2 is percentage proliferation value, C: cells CFSE after continuous incubation with different concentrations of acacetin, c1 is the cytoflow diagram, c2 is the percentage proliferation value, D: th17 (expression of IL-17A) of cells after continuous incubation with different concentrations of farnesoid, d1 is the cytogram, d2 is the percentage proliferation value, measured in vitro against CD3/CD28 activation in the absence or presence of different concentrations of farnesoid for 72 hours

CD4+ T cells (n = 3), data expressed as mean ± standard deviation and analyzed with one-way anova followed by Tukey test analysis, P<0.05，**P<0.01，***P<0.001 or P<0.0001vs. vehicle (0 μ M acacetin);

FIG. 10 is a diagram of RNA-seq revealing transcriptional profiles of farnesoid incubated CD4+ T cells; wherein, A: incubation with 0 (vehicle), 5 or 10 μ M acacetin (acacdtin) for 72 hours, activated with anti-CD 3/CD28 incubation

Principal Component Analysis (PCA) profile of total gene expression of CD4+ T cells, B and C: volcano plots of differences in gene expression (5vs.0 μ M acacetin) (B) and (10 μ M vs.0 μ M acacetin) (C), the color of the dots corresponding to the size of (-log 10q. Value), and the genes at the first 20 positions of (-log 10q. Value), D and E: analysis of 5vs.0 μ M acacetin (D) and 10vs.0 μ M acacetin (E) using c2 (c 2.Cp.v7.5.1. Symbols.gmt) in the molecular signature database of GSEA algorithm (MSigDB), F: mitochondrial respiratory electron transport pathway (5vs.0 μ M acacetin), f1 is GSEA map, f2 is gene expression heatmap, G: DNA replication, g1 is GSEA map, g2 is gene expression heatmap, H: fatty acid bio-oxidation pathway (10 μ M vs. vehicle (0 μ M acacetin), h1 is GSEA profile, h2 is gene expression heat map;

FIG. 11 is a graph of the results of experiments in which farnesoid inhibited the mitochondrial respiration rate, ROS production and membrane potential of CD4+ T cells; wherein, A: cells were assayed for activation by anti-CD 3/CD28 antibodies after 48 hours incubation with 0, 5 or 10 μ M acacetin

CD4+ T cell Oxygen Consumption Rates (OCRs), a1 is cell oxygen consumption rate, a2 is basal respiration, a3 is maximal respiration, a4 is reserve respiratory capacity, a5 is ATP production, B: effect of farnesin on mROS production, b1 is cellular mitochondrial ROS flow cytogram loaded with MitoSOX, b2 is cellular mitochondrial ROS level mean, C: effect of farnesoid on mitochondrial membrane potential, c1 is the cell flow cytogram loading JC-1, c2 is the mean value of the mitochondrial membrane potential of the cell, D: load mitoTracker cell results, d1 is the cytoflow chart, d2 is the mean of the cellular mitochondrial mass levels (n = 3-4), data are expressed as mean ± sd,and analyzed by one-way analysis of variance followed by Tukey's test<0.05，**P<0.01，***P<0.001，****P<0.0001；/>

FIG. 12 is a graph of the results of molecular docking analysis of the interaction of farnesin with mitochondrial complex II; wherein, A: three lowest binding conformations of acacetin to mitochondrial complex II, calculated by Autodock Vina, are represented in yellow, purple and cyan, respectively, B: acacetin (red) binds to the amino acid residue site of mitochondrial complex II (blue), C: in activation with anti-CD 3/CD28 antibodies

CD4+ T cells were assayed for complex II activity and incubated with 5 or 10 μ M acacetin for 48h (n = 5), D: incubation for 72h in the absence or presence of the highly specific mitochondrial Complex II inhibitor Atpen A5 (1 or 2. Mu.M), activation with anti-CD 3/CD28 antibody>

CD4+ T cells, determining the flow cytogram and percentage values (n = 4) of CD44 cells and CFSE cells, d1 being the CD44 cytogram, d2 being the percentage value of CD44 cells, d3 being the flow cytogram of CFSE cells, d4 being the percentage value of CFSE cells, E: on activation with anti-CD 3/CD 28->

CD4+ T cells were assayed for flow cytogram and percent values of CD44 cells and CFSE cells and treated in the presence of 5 or 10 μ M Acacetin (Acacetin) with or without DES for 72h (n = 4), e1 for CD44 cytogram, e2 for CFSE cytogram, e3 for CD44 cell percent values, e4 for CFSE cell percent values, data expressed as mean ± standard deviation, and analyzed with one-way ANOVA followed by Tukey's test (C and D) or two-way ANOVA followed by Bonferroni's test, P<0.05，**P<0.01，***P<0.001，****P<0.0001；

Fig. 13 is a graph of the experimental results of farnesin therapeutic oral gavage administration to improve the progression of dilated heart failure in myocarditis mice; wherein, A: animal experimental protocol: alpha-MHC was injected subcutaneously on days 0 and 7, and farnesin (100 mg/kg/day) or vehicle was administered from day 7 to day 21 of the experiment. B: mean values of the left ventricular indices (n = 8-10) from echocardiography analysis of control, myocarditis vehicle and farnesoid myocarditis treated mice, b1 being EF%, b2 being FS%, b3 being lvdd, b4 being LVEDD, C: representative cardiac images of mice from different experimental groups (scale =5 mm), D: mean HW/BW ratio, E: mason staining (scale =50 μm) representative images of myocardial sections of mice of different experimental groups, F: percent left ventricular fibrosis (n = 8-10) for mice from different experimental groups, data were expressed as mean ± standard deviation and analyzed by one-way variance analysis and Tukey test at P <0.05, P <0.01, P <0.001, P <0.0001.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the accompanying drawings:

the acacetin water-soluble prodrug and the oral administration of gold and acacetin are adopted to treat the immune myocarditis, and the acacetin water-soluble prodrug can be converted into the acacetin in vivo, so that the relative curative effect of a model after the administration of the acacetin water-soluble prodrug is evaluated, and the action mechanism of the acacetin converted in vivo in treating the immune myocarditis is clarified.

1. Study materials and experimental animal groups

1) Preparing a medicine for treating the immune myocarditis: the structure of acacetin and the water-soluble acacetin prodrug is shown in figure 1, and the acacetin and the water-soluble acacetin prodrug can be directly converted into acacetin in beagle dogs, mice and rats. The Water-soluble prodrug of farnesin was dissolved in sterile saline to prepare an injection solution of 40mg/mL concentration according to the literature (Water-soluble acetate in drugs of diagnosis and infusion in therapy. Scientific reports. DOI:10.1038/srep36435, and Synthesis of a high Water-soluble acetate in drugs for treating experimental infection in mice. Scientific reports. DOI:10.1038/srep 25743).

2) Experimental animals: experiments were performed using Lewis male rats (200-220 g) and 7 week old male BALB/c mice for different parameter testing, and animals were purchased from SPF Biotechnology Ltd (Beijing) and maintained in the SPF facility of the college of medicine. The animal protocol was approved by the animal Care Committee of the college of Hospital medical college of science and technology university in Huazhong. The experimental procedure followed the guidelines of the european parliament on the instructions No. 2010/63/EU to protect animals for scientific purposes.

3) Autoimmune myocarditis model: heart-specific alpha-myosin heavy chain peptide (alpha-MHC) is the most commonly used agent for the modeling of autoimmune myocarditis. 2mg of α -MHC (Shanghai GL Biochemical Co.) were dissolved in saline and emulsified with complete Freund's adjuvant in a ratio of 1. On the 0 th day and the 7 th day of the experiment of Lewis rats, 1mL of the alpha-MHC emulsion is injected into Lewis rats subcutaneously to establish an immune rat myocarditis model. On the other hand, on BALB/c mice, on the 0 th day and the 7 th day of the experiment, 0.2mL of the alpha-MHC emulsion is injected into the mice subcutaneously to establish an immune mouse myocarditis model.

4) Grouping experiments: the experimental animals were divided into a control group, a myocarditis model group (vehicle group) and a myocarditis drug-treated group.

In Lewis rat myocarditis model, the therapeutic effect of the water-soluble acacetin prodrug on immune myocarditis is studied, rat hemodynamic changes are detected by using direct intraventricular intubation, cardiac function is detected by using echocardiogram (echocardiography), and myocardial histology inflammatory reaction, fibrosis and changes of myocardial tissue inflammation and fibrosis related protein are detected. Lewis rats in a myocarditis model group and a myocarditis drug treatment group, wherein the rats in the drug treatment group are injected with different dosages (5, 10 or 20mg/kg, b.i.d.) of gold and acacetin prodrugs (acacetin for short) for 3 weeks to observe the effect of the acacetin on treating the myocarditis of immune rats; rats in the control group and the myocarditis model group were injected subcutaneously with an equal volume of physiological saline.

In BALB/c mouse myocarditis model, the treatment effect of oral acacetin on immune myocarditis is researched, the cardiac function is detected by an echocardiogram, the myocardial inflammatory reaction and the fibrosis condition are detected by a histological method, and the myocardial immune inflammatory reaction related gene and related immune cell change are detected by a biochemical method. On the basis of myocarditis modeling of BALB/c mice in a myocarditis model group and a myocarditis drug treatment group, the mice in the myocarditis drug treatment group are intragastrically perfused (100 mg/kg/day) with a suspension of aureoand albizzin and hydroxypropyl-beta-cyclodextrin for 2 weeks or 3 weeks, while the mice in a control group and the myocarditis drug treatment group are intragastrically perfused with an equal-volume hydroxypropyl-alpha-cyclodextrin solution.

5) Detecting the hemodynamic index of the rat: groups of Lewis rats were anesthetized and fixed with pentobarbital (30 mg/kg i.p.), then intubated with an trachea and ventilated manually. The rat body temperature was maintained at 37 ℃ using a temperature control system. A polyethylene catheter (0.8 mm outer diameter) filled with 0.5% heparin physiological saline solution and connected to a pressure transducer was inserted into the left common carotid artery of a rat, and after arterial blood pressure was measured, the left ventricle was introduced to measure hemodynamic indices of contractile function (e.g., left ventricular systolic pressure LVSP, LV + dP/dT), which were recorded with a WYS (R) multi-channel electrophysiology recorder (RM 6240E).

6) Echocardiography: echocardiography examinations were performed using a Vevo 1100 ultrasound system (Visual Sonics, toronto, canada) to assess cardiac function in rats or mice anesthetized with 1.5% isoflurane, and two-dimensional view analysis of the short and parasternal long axes in the ventricles was obtained. Rat or mouse heart function was analyzed for EF% and FS% using Vevo LAB software.

7) And (3) histopathological detection: after echocardiography, the animal was anesthetized and the heart was isolated. Ventricular tissues of each group of rats or mice were fixed in 4% formalin and embedded with paraffin to make left ventricular sections. Traditional hematoxylin/eosin (HE) and Masson trichrome stains were used to determine myocardial inflammation and fibrotic status, respectively. Inflammation was scored using a scale 0-4 method. Myocardial fibrosis was measured by Image Pro Plus software as the proportion of blue stained area on sections. Two independent personnel are used to evaluate the slices, which are coded to limit potential deviations.

8) Western blot analysis: western blot analysis of different proteins in rat myocardium the resulting heart tissue samples from the ischemic areas of rats were homogenized with ice-cold RIPA buffer. Protein concentration was determined using the Bio-Rad protein assay (Bio-Rad Laboratories, hercules, calif.). 50 μ g of protein was mixed with sample buffer and denatured by heating to 95 ℃ for 5 min. Proteins were separated on a 10% SDS polyacrylamide gel and transferred to nitrocellulose membranes.

Nitrocellulose membranes were first blocked with a solution of 5% skim milk in 0.1% tween in Tris-buffered saline (TTBS) for 2 hours, and then probed with the primary antibody overnight at 4 ℃. After 3 washes of TTBS, the nitrocellulose membrane was incubated with the secondary antibody in TTBS for 2 hours at room temperature. The nitrocellulose membrane was washed 3 more times with TTBS and then developed on X-ray film using a chemiluminescent detection system (ECL, GE Healthcare). To exclude differences in protein loading, housekeeping protein GAPDH was probed as an internal control. The intensity of the target blot was measured by quantitative scanning densitometer imaging software (Image J). The target blot intensities in the acacetin group were compared against the same blots in the vehicle group, and the relative intensities of the target western blots were used for quantitative and statistical analysis.

9) T lymphocyte isolation and culture: by using

CD4+ T cell isolation kit (Vancouver STEMCELL, canada), isolation of ^ H from spleen and lymph nodes of normal mice>

CD4+ T cells and were incubated for 2-4 hours on plates pre-coated with anti-mouse CD3 or CD28 (both 5. Mu.g/mL, biolegend, san Diego, calif., USA).

CD4+ T cells were incubated in IMDM medium containing 10% FBS (Wingo Sciencell, canada), 2mM L-glutamine (Gibco, N.Y.), 0.1mM nonessential amino acids (Gibco, U.S.), 1% penicillin/streptomycin (Servicobio, wuhan, china) and 55. Mu.M β -mercaptoethanol (Gibbo, N.Y.). For Th17 cell differentiation, 50ng/mL IL-6, 2.5ng/mL TGF-. Beta.5. Mu.g/mL anti-IFN-. Gamma.and 5. Mu.g/mL anti-IL-4 were added to the medium (Biolegend, san Diego, calif., USA). Gold and albizin were dissolved in dimethyl sulfoxide (DMSO) in the absence or presence of dimethyl succinate (DES, 5 mM) and the mitochondrial complex II inhibitor atpen A5 (1 and 2 μ M) and added to the medium at the start of the culture. Fluorescence was detected by flow cytometry on the second or third day of culture.

Flow cytometry: flow cytometry is used to analyze cell populations by incubation or staining with different antibodies or fluorescence. Prior to staining, the single cell suspension was incubated with anti-CD 16/32 antibody (Biolegend, san Diego, calif., USA) to block non-specific binding to Fc receptors. Cells were incubated at 4 ℃ for 30 minutes for surface staining. For intracellular cytokine staining, cells were incubated with the stimulation/blocking mixture for 4 hours prior to surface staining and cytokine staining was performed after treatment with the fixation/permeation kit.

The following antibodies were used: FITC anti-CD 45, APC-Cy7 anti-CD 11b, brilliant Violet 421 anti-Ly 6C, PE anti-CD 44, PE anti-F4/80, APC anti-Ly 6G, APC anti-CD 62L, APC anti-CD 25, PE anti-IL-17A, and Zombie Aqua Fixable viablility Kit (bioleged, san Diego, calif., USA).

For CFSE staining, cells were incubated with 1.25 μ M CFSE (Biolegend, san Diego, calif., USA) for 10 min prior to incubation. For mitochondrial reactive oxygen species (mROS) assays, cultured cells were incubated with 5 μ M MitoSOX (Invitrogen, carlsbad, USA) for 30 minutes and 5 μ M mitoTracker (Invitrogen, carlsbad, USA) for 30 minutes. Fluorescence was measured by flow cytometry.

Real-time quantitative PCR (RT-qPCR): mouse myocardial tissue total RNA was extracted using TRIzol reagent (Invitrogen, carlsbad, USA) and 1000ng of RNA was reverse transcribed using PrimeScript RT kit (Vazyme, nanjing, china) to generate cDNA. The sequences of primers used in the real-time quantitative PCR process are shown in Table 1 and data analysis was performed using the 2- Δ Δ CT method. The amplified specific cDNA was quantified using AceQ Universal SYBR qPCR Master Mix (Vazyme, nanjing, china) in the Bio-Rad CFX CONNECT detection system.

TABLE 1 primer sequences for RT-qPCR

RNA sequence analysis: total RNA was isolated from cultured CD4+ T cells using TRIzol reagent (Invitrogen, carlsbad, USA) on day 3 according to the manufacturer's protocol. Libraries were prepared using the TruSeqTM RNA sample preparation kit (Illumina, san Diego, CA). mRNA was reverse transcribed into cDNA using SuperScript double stranded cDNA synthesis kit and then sequenced using Illumina HiSeqxten/NovaSeq 6000.

The quality of the raw and trimmed Fastq readings was checked using Hisat 2. The original data was trimmed using the Stringtie tool. The R package "DESeq2" was used to analyze genes differentially expressed between the two groups. Genes with an absolute fold change >1.5 and an adjusted p-value <0.05 were considered differentially expressed. The R package "clusterProfile" was used to implement the GSEA algorithm using c2 (c 2.Cp. V7.5.1.Symbols. Gmt) in the molecular signatures database (MSigDB) to analyze the path change between the two groups. All sequencing data are available through the NCBI GEO database under the accession number (GSE 221244, security token: ghmniomnlnndet)

Molecule docking: gold and albizin compound names, molecular weights and three-dimensional (3D) structures were obtained from the PUBCHEM database. The 3D structures of complexes I-V were obtained from the protein database (Complex I:5XTD, complex II:3AEF, complex III:5XTE, complex IV:5Z62, complex V:6J 5J). Subsequently, the ligands and proteins required for molecular docking were prepared using AutoDock-Vina software (http:// Vina. Script. Edu /). And finally, analyzing the docking result by using an AutoDock tool and PyMOL. The binding capacity of gold and albizim to complex I-V was assessed by affinity (kcal/mol) values.

10 Hippocampal mitochondrial respiration rate assay: mitochondrial respiration rates were evaluated by a mitochondrial pressure test kit and a hippocampal bioscience XF24 extracellular flux analyzer (purchased from santa clara, california, usa). Cells were seeded into hippocampal XF24 well plates (500000 cells per well). The assay medium (supplemented with 10mM glucose, 1mM pyruvate, 2mM glutamine and 5mM HEPES) was then CO-free at 37 deg.C ₂ The incubator was stable for 1 hour. After baseline measurements, oligomycin, FCCP and rotenone/antimycin a were added in sequence to detect oxygen consumption rates.

2. Results of the experiment

1) Gold and albizin prodrugs ameliorate cardiac dysfunction in immune myocarditis rats: the protective effect of gold and albizin prodrugs (5, 10 or 20mg/kg, s.c., b.i.d.) on myocarditis was studied in a rat model of myocarditis (a in fig. 2) established with heart-specific alpha-MHC subcutaneous injection. Hemodynamic index examination of ratThe results are shown in FIG. 2, panel B, and the Left Ventricular Systolic Pressure (LVSP) and left ventricular systolic rate (LV + dP/dT) of representative myocarditis rats _max ) Significantly reduced, and significantly elevated Electrocardiogram (ECG) ST segments, whereas in rats with myocarditis using gold and albizzin at 10mg/kg, reduced left ventricular systolic pressure and left ventricular systolic rate, and elevated ECG ST segments were significantly improved. In the test results of the rats in the myocarditis vehicle group, the heart rate (HR, C in FIG. 2) and the mean blood pressure (MAP, D in FIG. 2) were decreased, LVSP (E in FIG. 2) and LV + dP/dT _max (F in fig. 2) severely impaired (n =6-7,p<0.01 Gold and Albizzim dose-dependently increase decreased heart rate and mean blood pressure, and increase impaired cardiac function (i.e., increase LVSP and LV + dP/dT) _max )(n＝6-7，P<0.05 or P<0.01 It shows that Jinjuhuan and Albizzid can obviously improve heart rate and blood pressure reduction and heart function damage caused by immune myocarditis.

2) In another group of myocarditis rats, different experimental groups of rats were analyzed for cardiac function using echocardiography (a in fig. 3), myocarditis vehicle group of rats significantly decreased in left ventricular ejection fraction percentage (EF%) (B in fig. 3) and left ventricular short axis shortening percentage (FS%) (C in fig. 3) (n =15,p < -0.01), while cardiac indices significantly increased (D in fig. 3) (n =15,p < -0.01); however, in myocarditis rats given subcutaneous injections of both the au and the albizin prodrugs (10 mg/kg, s.c., b.i.d.), the percent of left ventricular ejection fraction and the percent of left ventricular short axis shortening, the increased cardiac index was also improved (P < 0.05). These results further demonstrate that jin and albizzin significantly improve immune myocarditis cardiac function.

3) Gold and albizin prodrugs improved myocardial damage in rats with myocarditis: HE staining showed significant inflammatory cell infiltration (a in fig. 4) in myocarditis rat myocardium, which was significantly improved in myocarditis rat myocardium treated with 10mg/kg (B in fig. 4, n =10, p-restricted 0.01). Masson staining revealed significant fibrosis of myocarditis rats (C in fig. 4), and myocardial fibrosis in myocarditis rats treated with 10mg/kg was significantly improved (D, n =9, p-restricted 0.01 in fig. 4). These results indicate that farnesoid improves myocardial dysfunction may be associated with anti-inflammation and inhibition of myocardial fibrosis.

4) The gold and albizin prodrugs improve the molecular mechanism of myocardial injury of myocarditis rats: FIG. 5 shows that the expression of inflammatory-related proteins TNF-. Alpha.TLR 4, NF-. Kappa.B, IFN-. Gamma.in myocardium, TNF-. Alpha.in myocardium of rat myocardium (A in FIG. 5), TLR4 (B in FIG. 5), NF-. Kappa.B (C in FIG. 5), IFN-. Gamma. (D in FIG. 5) were significantly increased (n =4-6, P < -0.01), and increased concentration-dependent decrease of inflammatory-related proteins by farnesoid (5, 10 and 20 mg/kg) (P <0.05 or P < 0.01). These results indicate that myocarditis inflammatory cell infiltration is associated with increased expression of these inflammatory-related proteins, whose expression is significantly downregulated by farnesin.

YAP/TAZ has been demonstrated to be a sensor of structural and mechanical features of the cellular microenvironment, activated abnormally during tissue fibrosis; in addition, MST1 activation is involved in myocardial fibrosis, excessive inflammatory response, and myocardial cell death. Therefore, we determined the protein expression of YAP, TAZ and miss 1 in myocardial tissue. Fig. 6 shows that YAP, TAZ and MIST1 were significantly increased in myocarditis myocardium (n =4-6, P-lin-0.01vs control), elevated YAP (a in fig. 6), TAZ (B in fig. 6) and MIST1 (C in fig. 6) were significantly reversed in farnesoid (10 mg/kg) treated myocarditis rat myocardium (P <0.01 or P <0.05vs vehicle). In addition, the increase in nuclear TAZ was more pronounced in myocarditis myocardium by measuring cytoplasmic and nuclear TAZ separately (D in fig. 6), indicating that more cytoplasmic TAZ was transferred to the nucleus in myocarditis myocardium. These results indicate that myocarditis myocardial fibrosis is associated with YAP, TAZ and miss 1 activation.

5) Gold and albizzia improved cardiac dysfunction and cardiac injury in myocarditis mice: in order to further research whether the oral farnesoid has the effect of treating the immune myocarditis, a myocarditis mouse model is established by subcutaneously injecting alpha-MHC, and the potential protective effect and the action mechanism of the myocarditis are researched by orally intragastrically administering gold and the farnesoid to the mouse.

During the experiment from day 0 to day 21, mice with myocarditis receiving drug treatment were gavaged with aureoline (100 mg/kg/day), while the control group and myocarditis model group were gavaged with an equal volume of vehicle (a in fig. 7). At the end of the experiment (day 21), the heart function of the anesthetized mice was assessed with echocardiography (B in fig. 7). Significant reductions in left ventricular ejection fraction (EF%) and left ventricular short axis contraction fraction (FS%) were observed in the myocarditis vehicle group (n =5-7, p-slash 0.05vs. control), while reductions in EF% and FS% were reversed in the farnesoid-treated group (n =7, p-slash 0.05vs. vehicle) (C in fig. 7). These results indicate that oral administration of aureoand albizim significantly improved cardiac dysfunction in myocarditis mice.

After echocardiographic analysis, the heart was isolated under animal anesthesia. The heart volume of the vehicle animals in myocarditis was significantly greater than that of the control or acacetin-treated animals (fig. 7D). In myocarditis mice with vehicle, the ratio of heart weight to body weight (HW/BW) increased (n =11-12, p-was <0.01vs. control), while in oral farnesoid-treated myocarditis mice did not (n =12, p-was <0.001vs. vehicle) (fig. 7E).

In the heart ventricle sections of mice stained with HE (F in fig. 7) or Masson (H in fig. 7), inflammatory lesions and fibrotic regions of myocardium were increased in mice with myocarditis vehicle, while in myocarditis mice treated with farnesoid, inflammatory lesions and fibrosis of myocardium were significantly improved. Myocarditis vehicle mice had significantly increased myocardial inflammatory scores (n =11-12, P < -0.001vs control) (G in fig. 7), as well as myocardial fibrosis percentage areas (n =11-12, P < -0.0001vs control) (I in fig. 7), but significantly decreased in oral farnesoid-treated myocarditis animals (P =0.05 or P <0.01vs vehicle). These results indicate that oral administration of farnesoid also significantly improved myocardial injury and fibrosis in mice with myocarditis.

7) Farnesin suppressed the immune response in mice autoimmune myocarditis: in fig. 8 a, it is shown that the pro-inflammatory cytokines Il1b, il6 and Tnf and the chemokines Ccl3 and Ccl5 mRNA levels were significantly elevated in the ventricular tissue of the myocarditis vehicle group mice, however these increases were reversed in the myocarditis mice treated with acacetin. Cardiac-specific CD4+ T cell and Th17 cell effector cytokines Ifng, il17a and Il17f mRNA levels were upregulated in myocardial tissue in mice with vehicle myocarditis, and decreased in myocardial tissue in mice with oral gavage of farnesoid. These results indicate that farnesoid inhibits the production of myocardial inflammatory factors in mice with myocarditis.

To confirm whether farnesoid protection in myocarditis mice was associated with involvement of effector CD4+ T and Th17 cell-mediated immune responses, spleen cells were isolated from different experimental animals and analyzed by flow cytometry for effector CD4+ T cells (CD 62L-CD44+ NCD4+ cells), and effector T cells,

(naive) CD4+ T cells (CD 62L + CD44-CD4+ cells, th17 cells, myeloid cells (e.g., monocytes, macrophages, and neutrophils), etc. in mice with myocarditis in vehicle, the proportion of effector CD4+ T cells was increased, as opposed to the proportion in mice with farnesoid-treated myocarditis>

CD4+ T cells were unchanged and increased in mice with farnesin myocarditis (B in fig. 8).

In myocarditis mice with vehicle, the percentage of splenic Th17 cells increased significantly, whereas in myocarditis mice with oral farnesin the percentage of increase was significantly reversed (C in fig. 8). Furthermore, farnesin treatment significantly reduced the increase in macrophage proportion (D in fig. 8) in myocarditis mice, but did not reduce Ly6C hyperinflammatory monocytes (E, P =0.07 in fig. 8) and neutrophils (F, P = ns in fig. 8) percentage values. These results indicate that the protective effect of farnesoid on myocarditis mice is associated with inhibition of CD4+ T cell activation and Th17 cell differentiation in myocarditis mice.

8) Acacetin inhibits CD4+ T cell activation, proliferation and Th17 cell differentiation in vitro: after isolation from the spleen of normal mice

CD4+ T cells, the effect of farnesoid on CD4+ T lymphocyte activation, proliferation and Th17 differentiation was determined. Both 5 and 10 μ M acacetin significantly reduced the percentage values of cells expressing CD44 and CD25 (T cell activation marker) (a in fig. 9 and B in fig. 9).

Furthermore, farnesin concentration-dependently inhibited T cell proliferation (C in fig. 9). Importantly, farnesoid inhibited Th17 cell differentiation (D, IL-7A + cells in fig. 9). These results indicate that farnesoid directly inhibits CD4+ T cell activation, proliferation and Th17 cell differentiation.

9) RNA-seq revealed that farnesoid influences CD4+ T cell transcriptional characteristics: RNA-seq was used to examine the effect and underlying mechanism of farnesin on CD4+ T cells with 5 or 10. Mu.M farnesin analysis of transcriptome profiles. Principal Component Analysis (PCA) showed that cells treated with 5 μ M and 10 μ M acacetin were spatially close but far away from cells without acacetin treatment, indicating that acacetin can cause significant changes in the CD4+ T cell transcriptome (a in fig. 10).

The differential gene expression was analyzed in cells with 5 or 10. Mu.M farnesin present. In farnesin 5 μ M treated cells, 157 genes were up-regulated and 158 genes down-regulated (fig. 10, B). Whereas in farnesin 10 μ M cells, 428 genes were up-regulated and 336 genes were down-regulated (C in fig. 9), the volcano plot shows 20 genes with the smallest regulated P-value.

Subsequently, changes in potential pathways of action were analyzed using GSEA algorithm. GSEA analysis showed that cells treated with either farnesoid 5 or 10 μ M induced a decrease in cell cycle-associated pathways, such as APC/C mediated cyclin degradation, sister chromatid isolation and DNA synthesis (D in fig. 10 and E in fig. 10), consistent with inhibition of cell proliferation (C in fig. 10). Furthermore, farnesin 5 μ M resulted in a significant reduction of mitochondrial respiration-related pathways, such as electron transport in the respiratory system and oxidative phosphorylation of OXPHOS system in mitochondria (D in fig. 10).

Acacetin 10 μ M causes a significant reduction in lipid metabolism-related pathways such as cholesterol biosynthesis and fatty acid β oxidation, as well as a reduction in the oxidative phosphorylation system of the electron transport chain in the mitochondrial, proteasome and PPAR signaling pathways (fig. 10E). GSEA and gene expression heatmaps also show that farnesoid decreases gene expression associated with cell cycle, electron transport and fatty acid beta oxidation (F in fig. 10 to H in fig. 10). These results indicate that farnesin mainly affects the cell cycle, mitochondrial respiration and lipid metabolism of CD4+ T cells.

10 Acacetin decreases T cell mitochondrial respiration: previous studies have shown that mitochondrial respiration plays a key role in T cell activation, proliferation and differentiation, and that decreased mitochondrial respiration impedes lipid metabolism and affects DNA and nucleic acid synthesis. GSEA analysis and gene expression heatmaps showed that farnesin 5 μ M caused extensive inhibition of the mitochondrial respiratory pathway (F in fig. 10). This suggests that farnesin first interferes with mitochondrial function, affecting T cell lipid metabolism and the cell cycle. Gene expression heat maps show that most of the down-regulated genes are subunits of the electron transport chain complexes I, II, III, IV and V, which mediate mitochondrial respiration and oxidative phosphorylation of OXPHOSPHOS (D in FIG. 10 to H in FIG. 10). These results indicate that the main target of acacetin action is mitochondrial energy metabolism by CD4+ T cells.

The role of farnesin regulating mitochondrial respiration was further investigated by measuring the oxygen consumption rate of T cells (OCR, measured by extracellular flux analysis) using the Seahorse System cell mitochondrial stress detection kit. Farnesin (5 or 10 μ M) significantly inhibited T cell oxygen consumption rate, basal and maximal respiration, reserve respiratory capacity and ATP production after mitochondrial separation from the proton carrier FCCP (a in fig. 11). These results indicate that farnesin mainly inhibits mitochondrial respiratory reserve, thereby limiting T cell activation energy.

11 Acacetin reduces CD4+ T cell mitochondrial ROS production and mitochondrial membrane potential: it is believed that mitochondrial oxidation of respiratory chain-generated ROS (mROS) promotes intracellular signaling and enhances T cell activation and proliferation. Given the inhibitory effect of farnesin on mitochondrial respiration, we investigated the effect of farnesin on mROS production. MitoSOX staining showed that farnesin significantly inhibited CD4+ T cell mROS levels (B in fig. 11).

Mitochondrial membrane potential MMP maintained the activated state of CD4+ T cells, so we measured the effect of farnesin on mitochondrial membrane potential in activated CD4+ T lymphocytes loaded with JC-1 fluorescence, and found that farnesin induced a significant decrease in mitochondrial membrane potential (indicated by a decrease in the PE/FITC ratio) (C in fig. 11). However, mitochondrial respiration impairment was not due to mitochondrial mass reduction, as mitoTracker staining showed that farnesin 10 μ M only slightly reduced mitochondrial mass (D in fig. 11). These results indicate that farnesin inhibits CD4+ T cell mROS generation and mitochondrial membrane potential.

12 Acacetin inhibits mitochondrial complex II activity: slight changes in mitochondrial mass of CD4+ T cells indicate that farnesoid may alter the activity of the electron transport chain. PharmMapper (a well-known pharmacophore matching and potential target recognition platform) was used to predict and analyze the farnesin target on the electron transport chain. Autodock Vina is a computational procedure that effectively predicts the non-covalent binding of a macromolecular receptor to a small molecule to significantly improve the accuracy of the prediction, the binding conformation and the binding affinity. Pharmmoper analysis showed that farnesin binds to succinate dehydrogenase (z-score = 2.562), which functions as mitochondrial complex II. Docking of farnesin with complexes I-V using Autodock Vina showed that complex II had the lowest binding energy (Kcal/mol = -9.2) compared to the other complexes and that farnesin had a stronger stable root mean square error RMSE with the first three binding conformations of complex II, indicating that the docking results were reliable (a in fig. 12).

Further analysis of the docking conformation at the lowest binding energy showed that farnesin could successfully bind to complex II through hydrogen bonding to ASP106 and ARG79 amino acid residues (B in fig. 12). Mitochondrial complex II, also known as succinate dehydrogenase, is a central vector that reprograms metabolic and respiratory adaptation in response to various intrinsic and extrinsic stimuli and abnormalities, complex II controls the production of mROS and ATP and regulates T cell activation. The effect of farnesoid on the activity of complex II was examined using a complex II activity assay kit to confirm the above molecular docking results. Farnesin was found to inhibit T cell complex II activity in a concentration-dependent manner (C in fig. 12).

Then, we measured the effect of complex II activity on activation and proliferation by treating CD4+ T cells with the complex II inhibitor, atpen A5, and the results showed that atpen A5 can reduce activation and proliferation of CD4+ T (D in fig. 12).

Finally, we tested the function of CD4+ T cells with the membrane permeable material DES of complex II, observing whether mitochondrial function could be restored by increasing complex II substrate. As expected, the use of DES reversed the decrease in CD44 expression and CD4+ T cell proliferation by farnesin (fig. 12, E). These results indicate that farnesin inhibits CD4+ T cell mitochondrial function by affecting complex II activity.

13 Therapeutic administration of acacetin may prevent the progression of dilated heart failure: the beneficial effect of farnesin on myocarditis mice was observed in previous experiments by oral administration (fig. 7). However, most patients who enter the rehabilitation process from acute myocarditis develop dilated heart failure with myocardial fibrosis, ventricular dilatation and cardiac insufficiency, and no drugs are clinically available to delay the development of the dilated heart failure at present. To investigate whether farnesoid can delay and/or prevent the progression of dilated heart failure, in another set of experiments, myocarditis mice were given oral gavage of farnesoid (100 mg/kg/day) for two weeks on day 7 after subcutaneous injection of α -MHC. Animals were sacrificed three weeks off until day 42 of the experiment after echocardiographic assessment of cardiac function (a in figure 13).

Echocardiographic analysis showed that oral gavage myocarditis mice had further decreased left ventricular EF% and FS% and significantly increased dilated heart failure parameters, i.e., left ventricular end-systolic diameter (LVEDD) and left ventricular end-diastolic diameter (LV EDD), compared to the results of the 21-day experiment (C in fig. 7), indicating that dilated heart failure occurred over a longer experimental period (42 days) and exhibited heart failure. Notably, cardiac insufficiency and increases in LVEDD and LVEDD were reversed in myocarditis mice receiving acacetin treatment (B in fig. 13).

After echocardiography, hearts were isolated and weighed, and mice in the myocarditis vehicle group had increased heart size and HW/BW ratio, while animals in the myocarditis acacetin group did not (C in fig. 13, D in fig. 13). In addition, masson stained ventricular sections showed that extensive fibrosis was observed in the myocardium of mice with oral vehicle myocarditis, while areas of fibrosis were significantly reduced in the myocardium of mice with oral farnesoid myocarditis (E in fig. 13, F in fig. 13). These results indicate that administration of acacetin in the acute phase of myocarditis can prevent the progression of dilated heart failure.

3. Conclusion of the experiment

In the present study, we demonstrated that farnesin inhibits myocarditis immune response, cardioinflammatory response, fibrosis and cardiac dysfunction, and improves cardiac remodeling and ventricular dilatation in dilated heart failure in rat immune myocarditis and mouse immune myocarditis induced by subcutaneous injection of α -MHC.

The beneficial effects of acacetin on myocarditis are mainly associated with the inhibition of CD4+ T cell activation, proliferation and Th17 cell differentiation. Mechanistically, farnesin inhibits CD4+ T cell activation, proliferation and Th17 cell differentiation by binding to mitochondrial complex II, limiting mitochondrial activity, thereby inhibiting mitochondrial respiration, mROS production and mitochondrial membrane potential MMP, thereby reducing levels of activation of proteins associated with inflammatory responses and fibrosis.

In summary, the present inventors have found for the first time a new pharmacological action of acacetin, that is, acacetin reduces the development of dilated heart failure caused by myocarditis by reducing the activity of CD4+ T cell mitochondrial complex II, inhibiting mitochondrial respiration, CD4+ T cell mitochondrial ROS and immune response, which indicates that acacetin may be a valuable new drug for treating CD4+ T cell mediated immune myocarditis.

The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims (10)

1. Application of acacetin or acacetin water-soluble prodrug in preparing medicine for preventing and treating immune myocarditis is provided.

2. Use according to claim 1, characterized in that: the immune myocarditis is myocarditis caused by immune response caused by different infectious or non-infectious diseases.

3. Use according to claim 1, characterized in that: symptoms of the immune myocarditis include reduced left ventricular pressure, left ventricular hypertrophy, left ventricular dilation, reduced left ventricular systolic function, reduced left ventricular diastolic function, or heart failure.

4. Use according to any one of claims 1 to 3, characterized in that: targets for farnesoid inhibition of the myocarditis immune response include mitochondrial energy metabolism of CD4+ T cells.

5. Use according to any one of claims 1 to 3, characterized in that: farnesoid can be used for preventing and treating immune myocarditis by reducing the activity of a mitochondrial complex II, inhibiting mitochondrial respiratory function, energy metabolism and immune response in CD4+ T cells, inhibiting activation and proliferation of CD4+ T lymphocytes and Th17 cell differentiation, further inhibiting activity and expression of YAP, TAZ or MST1, inhibiting cardiac inflammatory reaction, fibrosis and cardiac function damage, and improving cardiac remodeling and ventricular dilatation of dilated heart failure.

6. Use according to any one of claims 1 to 3, characterized in that: the dosage of the mouse orally taking the Acacia sericata or the water-soluble prodrug of the Acacia sericata is 5-100 mg/kg.

7. Use according to any one of claims 1 to 3, characterized in that: farnesin prevents and treats immune myocarditis by inhibiting the activation and release of late inflammatory mediators.

8. Use according to any one of claims 1 to 3, characterized in that: the administration mode of the medicine is subcutaneous injection, intramuscular injection, intravenous injection, oral administration or spray inhalation.

9. Application of acacetin or acacetin water-soluble prodrug in preparing medicine for preventing and treating immune myocarditis complication is provided.

10. A medicine for preventing and treating immune myocarditis is characterized in that farnesol or a water-soluble prodrug of the farnesol is used as a main component.

Images