Application of CFB inhibitor in preparation of medicine for preventing and / or treating arrhythmic cardiomyopathy

Abstract

The invention provides application of a CFB inhibitor in preparation of a medicine for preventing and / or treating arrhythmic cardiomyopathy, and belongs to the technical field of biological medicine. According to the invention, by constructing an ACM model mouse, activation of a complement system is found on the model, a complement bypass pathway is activated in the early stage of a disease, and the expression content of a key factor CFB of the complement bypass pathway is obviously improved. Besides, after a CFB inhibitor is applied to the model, pathological phenotypes such as heart failure, cardiac inflammation and fibrosis of model mice are obviously improved, it is indicated that CFB is an important target spot for treating ACM, and the disease progress of ACM patients can be effectively improved through targeted inhibition of CFB in the body.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of a CFB inhibitor in preparing a drug for preventing and / or treating arrhythmogenic cardiomyopathy. Background Art

[0002] Arrhythmogenic cardiomyopathy (ACM) is a hereditary myocardial disease characterized by malignant arrhythmias and progressive ventricular systolic dysfunction, and is one of the main causes of sudden cardiac death. Currently, the treatment of ACM patients mainly focuses on alleviating life-threatening arrhythmias and heart failure symptoms, but there is a lack of drug intervention measures based on the pathological progression of the ACM heart.

[0003] ACM is mainly caused by LP / P variants or their binding partners in genes encoding desmosomal proteins, which alter a series of signaling pathways, including WNT 72 signaling, YAP pathway, TGF β signaling, and NF-κB signaling, thereby affecting the pathological remodeling of the heart. Previous studies have shown that inflammation is an important hallmark of ACM. SB216763 is an inhibitor of GSK3β, which can improve cardiac remodeling and improve cardiac function in vivo. However, long-term use of these inflammatory inhibitors can cause side effects and may pose a risk of carcinogenesis, so these inhibitors have not yet been used in clinical practice. The complement system is an important component of innate immunity. Abnormal activation of the complement system in many diseases can aggravate the inflammatory response and lead to tissue damage, but its activation pathways and mechanisms have not been studied in depth in ACM, and there are no reports on the use of complement factor B in the prevention and / or treatment of ACM. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide the use of CFB inhibitors in the preparation of drugs for preventing and / or treating arrhythmogenic cardiomyopathy. Inhibiting CFB can effectively alleviate pathological damage such as cardiac inflammation and fibrosis, and alleviate abnormal cardiac function in ACM model mice.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions: The present invention provides the use of a CFB inhibitor in preparing a drug for preventing and / or treating arrhythmogenic cardiomyopathy.

[0006] Preferably, the CFB inhibitor includes one or more of an enzyme that degrades CFB protein, an agent that silences CFB gene, and an agent that inhibits NF-κB pathway.

[0007] Preferably, the agent for silencing the CFB gene comprises siRNA or shRNA.

[0008] Preferably, the agent that inhibits the NF-κB pathway comprises BAY 11-7082.

[0009] Preferably, the CFB inhibitor comprises LNP023.

[0010] Preferably, the CFB inhibitor inhibits the expression of C5b-9 protein in arrhythmogenic cardiomyopathy tissue.

[0011] Preferably, the CFB inhibitor reduces cardiac inflammation, fibrosis and alleviates cardiac contractile dysfunction.

[0012] The present invention provides a drug for preventing and / or treating arrhythmogenic cardiomyopathy, comprising the CFB inhibitor and a pharmaceutically acceptable carrier.

[0013] The present invention provides a method for screening drugs for preventing and / or treating arrhythmogenic cardiomyopathy, comprising the following steps: administering a test drug to an arrhythmogenic cardiomyopathy recipient animal, detecting the expression level of CFB protein and its cascade protein in the recipient animal, and judging whether the test drug is a drug for treating arrhythmogenic cardiomyopathy according to the expression level of CFB protein and its cascade protein.

[0014] Preferably, the judgment criteria include: if the expression level of CFB protein and its cascade proteins in the arrhythmogenic cardiomyopathy recipient animal is reduced compared with the negative control group, the test drug is a drug for preventing and / or treating arrhythmogenic cardiomyopathy.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides for the first time the use of CFB inhibitors in the preparation of drugs for preventing and / or treating arrhythmogenic cardiomyopathy, by constructing myocardial cell desmosome genes Dsg2 exon11-specific knockout mice (abbreviated as " Dsg2 The ACM-cKO mouse ("ACM-cKO mouse") model simulates the disease phenotype of ACM patients. Compared with the control mice, the experiment found that the complement alternative pathway of the model mice was significantly activated, and the transcription and translation levels of CFB were significantly upregulated in both heart and cardiomyocytes.

[0016] Furthermore, the present invention uses CFB inhibitor to treat Dsg2 -cKO mice, the results showed that CFB inhibitor treatment significantly reduced Dsg2 -cKO mice C5b-9 protein expression, indicating that LNP023 effectively inhibited the complement system; CFB inhibitor treatment Dsg2-cKO mice showed significantly reduced cardiac inflammation and fibrosis, and the mRNA expression levels of fibrosis-related genes were downregulated, indicating that CFB inhibitors alleviated cardiac inflammation and fibrosis; in terms of cardiac function, CFB inhibitor treatment Dsg2 -cKO mice significantly alleviated cardiac systolic dysfunction, as shown by significant improvement in LVEF and LVFS, while Myh6 was significantly upregulated, and Myh7 and Anp were significantly downregulated, which proved that inhibition of CFB can effectively improve Dsg2 -cKO mice. These data suggest that the alternative complement pathway Dsg2 CFB plays an essential role in the pathological progression of ACM caused by defects. CFB is a promising therapeutic target. Inhibition of CFB can effectively prevent and / or treat arrhythmogenic cardiomyopathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Flow chart of the experimental implementation of the present invention.

[0018] Figure 2 for Dsg2 -cKO mouse phenotypic detection results. A. RT-PCR detection of control mice and Dsg2 Knockout of exon 11 in myocardial tissue of cKO mice; B. qRT-PCR detection of relative expression level of exon 11 in control mice and Dsg2 cKO mice; C. Western blotting detection Dsg2 The relative expression levels in myocardial tissues of control mice and Dsg2 cKO mice; E. Immunofluorescence staining to detect the relative expression levels in myocardial tissues of control mice and Dsg2 cKO mice Dsg2 Protein expression level results; FH. control mice and Dsg2 qRT-PCR results of genes related to myocardial inflammation (F), fibrosis (G), and heart failure (H) in cKO mice; I. Echocardiography and statistical results of LVEF and LVFS in Dsg2-cKO mice at 4 and 16 weeks; J. Dsg2 Representative electrocardiograms of cKO mice; n = 3, mean ± SEM, * indicates p value less than 0.05, ** indicates p value less than 0.01, *** indicates p value less than 0.001.

[0019] Figure 3 for Dsg2 -cKO mouse cardiomyocyte complement alternative pathway activation detection results. A. qRT-PCR detection of the expression of exon 11 of the Dsg2 gene in the cardiomyocytes of control and Dsg2 cKO neonatal mice; B. Western blotting detection Dsg2Relative expression levels of complement genes in cardiomyocytes of control and Dsg2 cKO neonatal mice; D. qRT-PCR detection of relative expression levels of complement genes in cardiomyocytes of control and Dsg2 cKO neonatal mice; EF. Western blotting detection of relative expression levels of complement genes in cardiomyocytes of control and Dsg2 cKO neonatal mice; n=3, mean±SEM, * indicates p value less than 0.05, ** indicates p value less than 0.01, *** indicates p value less than 0.001.

[0020] Figure 4 The results verified that NF-κB activation mediated the upregulation of CFB expression caused by Dse2 deficiency. Among them, AB. Western blotting was used to detect the expression level of p-NF-κB protein in myocardial tissues of control mice and Dsg2 cKO mice; CD. Western blotting was used to detect the expression level of p-NF-κB protein in cardiomyocytes of control and Dsg2 cKO mice; E. qRT-PCR was used to detect the relative expression levels of Il-1β and Tnfα in cardiomyocytes of control and Dsg2 cKO mice; FqRT-PCR was used to detect the relative expression levels of Cfb and Il-1β in cardiomyocytes of wild-type neonatal mice treated with PBS and TNFα; GH. Western blotting was used to detect the relative expression levels of Cfb and I1-1β in cardiomyocytes of wild-type neonatal mice treated with PBS and TNFα; I. qRT-PCR was used to detect the relative expression levels of Cfb in cardiomyocytes of control and cKO neonatal mice treated with DMSO and BAY11-7082; J. Western blotting was used to detect the relative expression levels of Cfb in cardiomyocytes of DMSO and BAY Expression levels of CFB, pNF-κB, and NF-κB in cardiomyocytes of control and cKO neonatal mice treated with 11-7082; n = 3, mean ± SEM, * indicates p value less than 0.05, ** indicates p value less than 0.01, *** indicates p value less than 0.001.

[0021] Figure 5 To suppress CFB Dsg2-cKO mice. A. Schematic diagram of the administration of CFB inhibitor LNP023 to mice; B. Western blotting to detect the relative expression level of C5b-9 in the hearts of control mice, Dsg2 cKO mice and LNP023-Dsg2 cKO mice; DH&E staining to detect the infiltration of inflammatory cells in the myocardial tissues of control mice, Dsg2 cKO mice and LNP023-Dsg2 cKO mice; E. Masson staining to detect the deposition of collagen fibers in the myocardial tissues of control mice, Dsg2 cKO mice and LNP023-Dsg2 cKO mice; F. H. Echocardiography (F) and LVEF (G) and LVFS (H) statistical results of control mice, Dsg2 cKO mice and LNP023-Dsg2 cKO mice; I. qRT-PCR to detect the infiltration of inflammatory cells in the myocardial tissues of control mice, Dsg2 cKO mice and LNP023-Dsg2 cKO mice. Relative expression levels of molecular marker genes for cardiac fibrosis and heart failure in cKO mice; n = 3, mean ± SEM, * indicates p value less than 0.05, ** indicates p value less than 0.01, *** indicates p value less than 0.001. DETAILED DESCRIPTION

[0022] The present invention provides the use of CFB inhibitors in the preparation of drugs for preventing and / or treating arrhythmogenic cardiomyopathy. The present invention constructs ACM model mice, and after administering CFB inhibitors, it is found that the pathological phenotypes such as heart failure, cardiac inflammation and fibrosis in the model mice are significantly improved, and the abnormal cardiac function of the ACM model mice is alleviated, indicating that inhibiting the expression of CFB gene or protein can effectively prevent and / or treat arrhythmogenic cardiomyopathy.

[0023] In the present invention, the CFB inhibitor includes an enzyme that degrades CFB protein, an agent that silences CFB genes, and an agent that inhibits the NF-κB pathway. The agent that silences CFB genes in the present invention includes siRNA or shRNA. The agent that inhibits the NF-κB pathway in the present invention includes BAY 11-7082, and the BAY 11-7082 is an NF-κB inhibitor. The CFB inhibitor in the present invention is preferably LNP023 (Iptacopan, iptacopan hydrochloride).

[0024] In the present invention, by constructing myocardial cell desmosome gene Dsg2 The exon11 specific knockout mouse model simulates the disease phenotype of ACM patients. Compared with the control mice, the activation of the complement system was found in this model, and the complement alternative pathway was activated in the early stage of the disease. At the same time, the expression level of CFB, a key factor in the complement alternative pathway, was significantly increased. The present invention applies CFB inhibitors in the above mouse model, which significantly reduces Dsg2 -cKO mice C5b-9 protein expression, while the model mice heart failure, cardiac inflammation and fibrosis and other pathological phenotypes were significantly improved, relieving the abnormal cardiac function of ACM model mice, indicating that CFB is an important target for the treatment of ACM, targeted inhibition of CFB in ACM patients can effectively improve the disease progression of ACM patients, and targeting CFB can be used as an important basis for the development of ACM drugs. The above experimental implementation flow chart of the present invention is as follows Figure 1 shown.

[0025] The present invention also provides a drug for preventing and / or treating arrhythmogenic cardiomyopathy, the drug comprising the CFB inhibitor and a pharmaceutically acceptable carrier. The dosage form of the drug of the present invention includes but is not limited to spray, injection, lyophilized powder, granules, tablets, capsules, and emulsions.

[0026] The present invention also provides a method for screening drugs for preventing and / or treating arrhythmogenic cardiomyopathy, comprising the following steps: administering a test drug to an arrhythmogenic cardiomyopathy recipient animal, detecting the expression level of CFB protein and its cascade protein in the recipient animal, and if the expression level of CFB protein and its cascade protein in the arrhythmogenic cardiomyopathy recipient animal is reduced compared with the negative control group, the test drug is a drug for preventing and / or treating arrhythmogenic cardiomyopathy.

[0027] In the present invention, unless otherwise specified, all components, reagents or culture media are commercially available products well known to those skilled in the art.

[0028] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1 1. Establishment of ACM mouse model Cardiomyocyte specificity Dsg2 exon11 knockout mice ( Dsg2 cKO ) was provided by Dongguan First Hospital Affiliated to Guangdong Medical University. Dsg2 cKO model mice Dsg2 cKO mice and cre-negative mice born in their littermates were used for subsequent experiments. Dsg2 The cKO model mice were the cKO group, and the cre-negative mice were the negative control group (Ctrl group).

[0030] Among them, Dsg2 cKO mice were designed based on the homozygous variant of the East Asian founder (p.Phe531Cys). The design steps included: constructing ES cell targeting vectors by infusion method, Dsg2 Two forward lox P sites were introduced on both sides of exon 11, and pGK-Neo was introduced between exon 11 and 3' lox P. The linearized targeting vector was then electroporated into mouse embryonic stem cells (ES cells). Positive ES cell clones with correct homologous recombination were obtained through screening. After amplification, they were injected into C57BL / 6J mouse blastocysts to obtain chimeric mice. The chimeric mice were hybridized with Flp mice 39 (C57BL / 6J background), and the Neo gene cassette was removed to obtain Neo-free positive F1 mice. Dsg2 The exon 11F / F mouse model was crossed with the cardiomyocyte-specific Cre transgenic mouse (αMHC-Cre) to obtain Dsg2 cKO mouse model.

[0031] 2. Detection Method 2.1 RNA extraction, reverse transcription and quantitative PCR detection (1) Tissue / cell RNA extraction For each mouse tissue, collect the tissue in an RNase-free centrifuge tube, add 1 mL Trizol and several steel beads, shake at 1500 rpm for 3 min, and then immediately place on ice for 2 min. Repeat three times and place on ice for 10 min to allow the tissue to be fully lysed. Centrifuge at 4°C, 12000 rpm for 5 min, and collect the supernatant.

[0032] For cells, the cell pellet was obtained after digestion and collection, 1 mL of Trizol was added, lysed on ice for 5 min, 200 µL of chloroform was added, vortexed for 10-15 s, centrifuged briefly, left at room temperature for 10 min, centrifuged at 4°C, 12,000 rpm for 15 min, and the supernatant was collected.

[0033] Transfer the collected tissue / cell supernatant to a new RNase-free centrifuge tube; add 300µL chloroform, mix by inverting, centrifuge at 4°C, 12000rpm for 10min, transfer about 400µL supernatant to a new RNase-free 2mL centrifuge tube; add an equal amount of chloroform, mix by inverting, centrifuge at 4°C, 12000rpm for 10min; transfer the supernatant to a new RNase-free 2mL centrifuge tube, add an equal volume of pre-cooled isopropanol, place at -20°C for 1h, centrifuge at 4°C, 14000rpm for 20min, and discard the supernatant; add 1mL pre-cooled 75% ethanol (prepared with DEPC water), wash the precipitate by inverting, centrifuge at 4°C, 14000rpm for 5min, discard the supernatant, and repeat the wash twice; leave the centrifuge tube open for 10min, then add 30µL DEPC water was placed on ice for 10 min to fully dissolve the RNA, and the RNA concentration and purity were determined. Finally, the RNA was stored at -80°C for later use.

[0034] (2) Reverse transcription of tissue / cell RNA The RNA sample concentration was measured by Nanodrop spectrophotometer and Prime Script TM RT Master Mix was reverse transcribed into cDNA. The reverse transcription system and reaction conditions are shown in Table 1. The reversed cDNA was diluted to 5 ng / µL with DEPC water and stored at -80°C.

[0035] Table 1 Reverse transcription system and reverse transcription conditions

[0036] (3) Real-time quantitative PCR (qRT-PCR) Using PowerUp TM SYBR TM Green PCR premix was used for PCR amplification to prepare the qRT-PCR reaction system, see Table 2, and the primers involved in the reaction system are shown in Table 3. After centrifugation, the qRT-PCR program was run, see Table 4. After the amplification, the temperature was lowered to 60°C, and then heated to 95°C to denature the DNA product. The relative expression of the target gene was calculated using the relative quantitative algorithm based on the CT value, with the expression of GAPDH as the standard.

[0037] Table 2 qRT-PCR amplification system

[0038] Table 3 Primers involved in qRT-PCR amplification system

[0039] Table 4 qRT-PCR amplification procedure 2.2 Protein extraction and Western blot detection (1) Protein extraction For the extraction of myocardial tissue protein, the mouse heart was taken, the atria and auricles were removed, and the ventricles were retained. About 20 mg was taken and placed in 200 µL of RIPA lysis buffer containing 1% PMSF and temporarily stored on ice; the tissue was fully homogenized using a homogenizer until no tissue blocks were visible to the naked eye; centrifuged at 4°C and 12000 rpm for 5 min, and the supernatant was aliquoted to obtain the total protein sample in the heart tissue, which was stored in a -80°C refrigerator for future use to avoid repeated freezing and thawing.

[0040] For the extraction of cell proteins: collect the cell pellet and wash the cells with PBS; then add 100µL RIPA and 1µL PMSF lysis buffer, lyse on ice for 30 minutes, and centrifuge at 4°C, 12000rpm for 5 minutes; collect the supernatant to obtain the total protein sample in the cardiomyocytes, and store it in a -80°C refrigerator for later use to avoid repeated freezing and thawing.

[0041] (2) Protein concentration determination and denaturation The protein concentration was determined using an Enhanced BCA Protein Assay Kit. The standard with a concentration of 0.5 mg / mL was added to the standard wells of a 96-well plate at 0, 1, 2, 4, 8, 12, 16, and 20 µL in sequence, and each standard well was filled with PBS to 20 µL, which was equivalent to the standard concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml, respectively; the protein sample to be tested was appropriately diluted with PBS, and 20 µL of the diluted protein sample to be tested was added to a 96-well plate, and 3 replicates were made for each sample; BCA reagent A and BCA reagent B were prepared with BCA working solution at a ratio of 50:1, mixed thoroughly, and 200 µL of BCA working solution was added to each well of the standard and sample, and placed at 37°C for 20-30 min; the absorbance value of each well at a wavelength of 570 nm was measured in an ELISA reader, and a standard curve was drawn based on the absorbance and concentration of the standard, and the protein concentration of the protein sample to be tested was calculated based on the standard curve and the protein dilution multiple. 40 µg of protein sample was taken and mixed thoroughly with 5× protein loading buffer, centrifuged instantaneously, denatured at 99°C for 10 min, and immediately placed on ice for later use.

[0042] (3) Western blotting The protein sample obtained in step (2) and the prestained protein marker were added to the 10% SDS-PAGE gel wells in turn, and electrophoresis was performed at 60V constant voltage until the sample entered the separation gel, and then at 90V constant voltage for 1.5-2h; then the membrane was transferred at 4℃ constant current 200mA for 2h. After the transfer, the PVDF membrane was placed in 5% skim milk powder and incubated at room temperature for 2h. After the blocking was completed, the PVDF membrane was incubated with the primary antibody at 4℃ overnight [the primary antibodies are as follows: GAPDH (1:1000, Biyuntian, AG019), DSG2 (1:500, Abcam, ab150372), BAX (1:1000, Cell Signaling Technology, E4U1V), CFB (1:1000, A1706), C5b-9 (1:1000, Santa Cruz Biotechnology, sc-66190), phospho-NF-κB (Ser536) (1:1000, Cell Signaling Technology 93H1), NF-κB (1:1000, Cell Signaling Technology D14E12), IL-1β (1:1000, Cell Signaling Technology 3A6)]. After overnight incubation, the membrane was washed with PBST (3 times, 10 min each time), incubated with the corresponding secondary antibody at room temperature for 1 h, and washed with PBST (3 times, 10 min each time). TM The reaction was carried out in West PicoPLUS chemiluminescent substrate (Thermo, USA), and the membrane was placed in a developer for development. The images were subsequently quantitatively analyzed using ImageJ.

[0043] 2.3 Histopathology and Immunofluorescence The tissues were fixed with 4% paraformaldehyde solution for more than 36 hours, and then dehydrated in ethanol with a concentration gradient of 50%-100%. The dehydrated specimens were transparentized with xylene, embedded in paraffin, cut into 5-6μm sections, and subjected to H&E, Masson trichrome staining, immunofluorescence, and immunohistochemistry.

[0044] (1) H&E staining After the tissue sections were baked at 65°C for 1 hour, they were dewaxed and hydrated (xylene 7min×2 times, anhydrous ethanol 5min×2 times, 95% ethanol 5min, 75% ethanol 5min, distilled water 5min), then stained with hematoxylin solution for 5min, differentiated for about 40s, rinsed with tap water for 10min, immersed in eosin stain for 30s-2min, washed, and then immersed in tap water for 2-5min. Finally, they were dehydrated and transparent (95% ethanol 2-3s×2 times, anhydrous ethanol I 2-3s, anhydrous ethanol II 1min, xylene 1min×2 times), sealed with neutral gum, and dried naturally.

[0045] (2) Masson trichrome staining For details on dewaxing and hydration of paraffin sections, please refer to the H&E staining section; remove the sections from distilled water, add Bouin solution to the tissue and incubate for 1 hour, rinse with running water for about 10 minutes; use celestine blue staining solution for 2-3 minutes, wash with distilled water twice, each time for 10-15 seconds; drip stain with Mayer hematoxylin staining solution for 2-3 minutes, wash with distilled water twice, each time for 10-15 seconds; differentiate with acid ethanol differentiation solution for a few seconds, rinse with running water for 10 minutes; drip stain with Ponceau fuchsin staining solution for 5 minutes, wash with distilled water twice, each time for 10-15 seconds; treat with phosphomolybdic acid solution for about 10 minutes, shake off the liquid on the section, and directly add aniline blue staining solution for 2 minutes; after washing off the aniline blue staining solution with acetic acid solution, continue to add acetic acid for 2 minutes; the dehydration and transparency method is the same as H&E, use neutral gum to seal the slides, and dry naturally.

[0046] (3) Immunofluorescence staining For detailed procedures of paraffin section dewaxing and hydration, see the H&E staining section; heat the pH 6.0 sodium citrate antigen retrieval solution in a microwave oven until boiling, and place the slices in the microwave oven and heat at high heat for 20 minutes. During this period, distilled water was added every 5 minutes to prevent water evaporation during heating and cause dry slices; cool naturally to room temperature, wash off the retrieval solution with PBS, add blocking agent and incubate at room temperature in the dark for 10 minutes; rinse the slices with PBS 3 times, 5 minutes each time; add blocking solution and incubate at 37°C for 1 hour; shake off the blocking solution, directly add primary antibody and incubate at 4°C overnight; rewarm at room temperature for 30 minutes, rinse the slices with PBS 3 times, 5 minutes each time, add diluted fluorescent secondary antibody and incubate at 37°C for 1 hour; rinse the slices with PBS 3 times, 5 minutes each time; add DAPI to stain the cell nucleus and react at room temperature for 5 minutes; rinse the slices with PBS 3 times, 5 minutes each time; seal the slices with anti-fluorescence quenching sealing solution and observe under a fluorescence microscope or confocal fluorescence microscope.

[0047] 2.4 Ultrasonic testing Mice were anesthetized with 1.5% isoflurane inhalation and fixed in a supine position. The hearts of mice were examined by ultrasound using a Visualsonic Vevo 3100 ultra-high resolution ultrasound instrument and an MS400 ultrasound probe (probe frequency 30-MHz). Parasternal long-axis and short-axis sections were recorded to obtain M-mode and B-mode echocardiograms, and an average of three cardiac cycles was measured to obtain left ventricular ejection fraction (LVejection fraction, LVEF) and left ventricular fractional shortening (LV fractional shortening, LVFS).

[0048] 2.5 ECG Recording Electrocardiograms were recorded from sterile needle electrodes inserted subcutaneously in the lateral forelimbs and hindlimbs. Signals were then collected and analyzed using a digital acquisition and analysis system (Power Lab 8 / 35; AD Instrumentss; Lab Chart Reader v8.1.22). ECG parameters were quantified after recording for 10 minutes after the signal stabilized 1-2 minutes after anesthesia induction.

[0049] 2.6 Isolation of neonatal rat cardiomyocytes Prepare pancreatic enzyme digestion solution: add 25 mg pancreatic enzyme to 50 mL HBSS, mix thoroughly, and filter with a 0.22 µm filter for sterilization; collect ventricular tissues from P1-P3 day old rats and wash them in pre-cooled PBS; take 7-8 hearts as an example, place the ventricles in pre-cooled pancreatic enzyme digestion solution (7-8 hearts can be placed in a 15 mL centrifuge tube), and place the centrifuge tube flat in a 4°C refrigerator for digestion overnight.

[0050] Remove half the volume of trypsin digestion solution from the heart digestion solution with a pipette, then add half the volume of preheated Light medium, and incubate in a 37°C water bath with shaking for 3 minutes; remove the supernatant, add 10mL 160U / mL collagenase 2 digestion solution, and incubate in a 37°C water bath with shaking for 2 minutes; remove the supernatant, add 8mL 160U / mL collagenase 2 digestion solution, and incubate in a 37°C water bath with shaking for 7 minutes; gently blow the digestion solution to blow the tissue blocks into a cell suspension; transfer the cell suspension to a centrifuge tube containing precooled Dark medium and place on ice; continue to add 8mL 160U / mL collagenase 2 digestion solution to the undigested tissue, and incubate in a 37°C water bath with shaking for 7 minutes. Repeat this operation 2-3 times until all the tissues are digested into a cell suspension. Filter all cell suspensions with a 100µm cell mesh and centrifuge at 800rpm for 5min; discard the supernatant, suspend the cells in 10mL Dark medium, transfer to a 10cm culture dish, and culture in a 37℃ constant temperature incubator for 1.4h; gently shake the culture dish, transfer the cell suspension to a new 10cm culture dish and continue culturing, replace with fresh Dark medium after 24h, and treat cardiomyocytes with drugs (treat the cells with 5ng / mL TNFα and 2.5nmol / μL BAY11-7082 for 24h, then extract protein and RNA).

[0051] 3. Experimental verification and result analysis 3.1 Determination of ACM model mice To determine Dsg2 Myocardial tissue of cKO mice Dsg2 The knockout effect of exon 11 (about 228 bp) at the transcriptional level is Dsg2 qRT-PCR detection was performed using cDNA from myocardial tissue of cKO mice as a template, and the results showed that compared with negative control mice, Dsg2 The cDNA of myocardial tissue of cKO mice lacks 228 bp bases ( Figure 2 A) In addition, Dsg2 cKO mice Dsg2 The mRNA expression level of exon 11 was significantly decreased ( Figure 2 B). Western blotting and immunofluorescence staining were used to detect mouse myocardial tissue samples, further confirming that Dsg2 Dsg2 cKO mice lacked Figure 2 CE).

[0052] right Dsg2 qRT-PCR was performed on the heart tissues of cKO mice and negative control mice to detect the expression of Cd68 and IL-1β in the Dsg2 The mRNA expression levels in the hearts of cKO mice were increased ( Figure 2F) Similarly, the same method was used to detect fibrosis-related genes Col3a1 , Col1a1 , Col1a2 , Tgfβ1 , Tgfβ2 and Tgfβ3 The mRNA expression in Dsg2 cKO mice showed a significant increase in the heart Figure 2 G). In addition, markers associated with heart failure were detected by qRT-PCR ( Anp , BnP , Myh6 , Myh7 ) showed that the cardiac mRNA levels Myh6 Significantly downregulated, Myh7 , Anp and BnP Significantly upregulated ( Figure 2 H), indicating Dsg2 Cardiac stress in cKO mice.

[0053] To further determine the effect of Dsg2 protein deficiency on the cardiac structure and function of mice, we performed 4-week-old (1 month) and 16-week-old (4 month) Dsg2-deficient mice. Dsg2 Ultrasound examination of cKO mice and negative control mice showed that Dsg2 The left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) of cKO mice were significantly decreased, indicating that their cardiac contractile function was impaired and gradually deteriorated with age ( Figure 2 I) To characterize Dsg2 To investigate the occurrence and severity of ventricular arrhythmias in cKO mice, electrocardiograms were recorded. Dsg2 Prolonged and frequent ventricular premature beats were observed in cKO mice, manifested by wide and malformed QRS complexes and absent P waves ( Figure 2 J).

[0054] 3.2 Verification of activation of the alternative complement pathway in ACM The above method was used to isolate P1-P3 neonatal rat cardiomyocytes from the negative control group and cKO group in order to detect the complement system status at the early stage. Dsg2 The mRNA expression level of exon 11 and the expression of Dsg2 protein were significantly decreased ( Figure 3 AC). In addition, Dsg2 In the cardiomyocytes of cKO neonatal mice, qRT-PCR detected that the mRNA expression of CFB and Cfd, the key components of the bypass pathway, was significantly upregulated ( Figure 3D). Western blotting analysis further verified that the protein level of CFB increased significantly, and the content of C5b-9, the terminal effector product of the complement pathway, also increased significantly ( Figure 3 EF). The increase in C5b-9 protein level is caused by the activation of the complement alternative pathway, and CFB is a key factor in the complement alternative pathway and is more concentrated upstream. Therefore, these results show that in the early stage of ACM, the complement alternative pathway has been activated in myocardial cells, and the mRNA of CFB, a key factor in the complement alternative pathway, is significantly upregulated and the protein of CFB has a significant increase trend, indicating that the differential expression of CFB may play an important role in arrhythmogenic cardiomyopathy.

[0055] 3.3 Verification of NF-κB pathway-mediated Dsg2 overexpression in ACM mice NF-κB pathway and complement activation are early events. Dsg2 The cardiac tissues and cardiomyocytes of cKO mice (cKO group) and negative control mice (Ctrl) were tested. Dsg2 In the hearts and cardiomyocytes of cKO mice, the phosphorylation level of NF-κB was significantly increased ( Figure 4 AD), IL-1β and TNFα levels were upregulated ( Figure 4 E), indicating Dsg2 Activation of NF-κB signaling pathway in cKO cardiomyocytes.

[0056] In order to further determine the regulatory relationship between p-NF-κB and CFB expression in cardiomyocytes. TNFα was used to activate the NF-κB pathway in wild-type neonatal rat cardiomyocytes, that is, wild-type neonatal rat cells were treated with 5 ng / mL TNFα, and PBS was used as the control group. Protein and RNA were extracted after 24 hours. qRT-PCR was used to detect cardiomyocytes activated by TNFα and cardiomyocytes added with PBS. The results showed that the mRNA and protein expression levels of Cfb and IL-1β were significantly increased under TNFα induction ( Figure 4 FH). 2.5nmol / μL BAY 11-7082 (MCE, Catalog No.: HY-13453) was further used to inhibit Dsg2 NF-κB in cKO mouse cardiomyocytes, protein and RNA were extracted 24 hours later. qRT-PCR and Western blotting showed that BAY 11-7082 inhibitor inhibited NF-κB, resulting in Dsg2 CFB is downregulated in cardiomyocytes of cKO mice ( Figure 4 IJ). These results indicate that Dsg2In the early stages of ACM caused by defects, activation of the alternative complement pathway in cardiomyocytes depends on the NF-κB signaling pathway.

[0057] 3.4 Validation of CFB inhibition in ACM mice Dsg2-cKO mice aged 6-8 weeks were gavaged with 20 mg / kg of LNP023 (Iptacopan, Iptacopan hydrochloride, manufacturer MCE, product number HY-127105) and an equal volume of PBS twice a day for 35 consecutive days. All mice were then evaluated for cardiac function and their hearts were collected for subsequent experiments. At least 4 mouse hearts were taken from each group for research. Schematic diagram of gavage of CFB inhibitor LNP023 in mice is shown in Figure 5 A. Among them Dsg2 The cre-negative mice born in the same littermates of cKO were the negative control group (Ctrl group), the Dsg2-cKO mice gavaged with PBS were the cKO group, and the Dsg2-cKO mice gavaged with LNP023 were the LNP023-cKO group.

[0058] Western blotting was used to detect negative control mice. Dsg2 cKO mice and LNP023- Dsg2 The relative expression level of C5b-9 in the hearts of cKO mice showed that LNP023 inhibitor treatment significantly reduced Dsg2 The protein expression of C5b-9 in cKO mice, C5b-9 is also called membrane attack complex, which is the result of the combined action of CFB and other factors and is the end product of the complement system cascade reaction, which can reflect the activation state of the complement system. This shows that LNP023 inhibitor effectively inhibits the complement system ( Figure 5 BC).

[0059] H&E staining was used to detect negative control mice. Dsg2 cKO mice and LNP023-treated Dsg2 Inflammatory cell infiltration in myocardial tissue of cKO mice, and Masson staining detection of negative control mice, Dsg2 cKO mice and LNP023-treated Dsg2 Collagen fiber deposition in myocardial tissue of cKO mice. Histological analysis based on H&E and Masson staining showed that after LNP023 treatment, Dsg2 Cardiac inflammation and fibrosis were significantly reduced in cKO mice ( Figure 5 DE).

[0060] In terms of cardiac function, ultrasound detected negative control mice, Dsg2 cKO mice and LNP023-treated Dsg2Echocardiography, LVEF and LVFS of cKO mice. The results showed that LNP023 treatment Dsg2 cKO mice showed significantly reduced systolic dysfunction, as evidenced by significant improvements in LVEF and LVFS ( Figure 5 FH).

[0061] At the same time, qRT-PCR detected negative control mice, Dsg2 cKO mice and LNP023-treated Dsg2 The relative expression levels of fibrosis and heart failure molecular marker genes in cKO mice. Dsg2 In cKO mice, Myh6 was significantly upregulated, while Myh7 and Anp were significantly downregulated ( Figure 5 I), this experiment shows that inhibiting CFB can effectively improve Dsg2 Cardiac function of cKO mice.

[0062] These data suggest that the complement alternative pathway plays an essential role in the pathological progression of ACM caused by Dsg2 deficiency, and CFB is a promising therapeutic target. Inhibition of CFB can effectively improve Dsg2 Cardiac function of cKO mice.

[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of CFB inhibitors in the preparation of drugs for preventing and / or treating arrhythmogenic cardiomyopathy.

2. The use according to claim 1, characterized in that The CFB inhibitor includes one or more of an enzyme that degrades CFB protein, an agent that silences the CFB gene, and an agent that inhibits the NF-KB pathway.

3. The use according to claim 2, characterized in that The reagent for silencing the CFB gene includes siRNA or shRNA.

4. The use according to claim 2, characterized in that The agent that inhibits the NF-KB pathway includes BAY 11-7082.

5. The use according to claim 1, characterized in that The CFB inhibitors include LNP023.

6. The use according to claim 1, 2 or 5, characterized in that: The CFB inhibitor inhibits the expression of C5b-9 protein in arrhythmogenic cardiomyopathy tissue.

7. The use according to claim 1, 2 or 5, characterized in that: The CFB inhibitors reduce cardiac inflammation, fibrosis and alleviate cardiac contractile dysfunction.

8. A drug for preventing and / or treating arrhythmogenic cardiomyopathy, characterized in that: The drug comprises the CFB inhibitor according to claim 1 or 2 or any one of claims 5 to 7 and a pharmaceutically acceptable carrier.

9. A method for screening drugs for preventing and / or treating arrhythmogenic cardiomyopathy, characterized in that: The method comprises the following steps: administering a test drug to an arrhythmogenic cardiomyopathy recipient animal, detecting the expression level of CFB protein and its cascade protein in the recipient animal, and judging whether the test drug is a drug for treating arrhythmogenic cardiomyopathy according to the expression level of CFB protein and its cascade protein.

10. The method according to claim 9, characterized in that The judgment criteria include: if the expression level of CFB protein and its cascade protein in the arrhythmogenic cardiomyopathy recipient animal is reduced compared with the negative control group, the test drug is a drug for preventing and / or treating arrhythmogenic cardiomyopathy.

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