Application of raloxifene or pharmaceutical salt thereof in medicine for treating CryAB mutant cardiomyopathy

Abstract

The invention provides an application of raloxifene or a pharmaceutical salt thereof in a medicine for treating CryAB mutant cardiomyopathy. According to the invention, based on a CryAB R120G mutation model, core pathological characteristics of CryAB mutation cardiomyopathy, including formation of protein aggregates in myocardial cells and heart failure performance, are simulated. The raloxifene is used for relieving cardiac hypertrophy, inhibiting myocardial fibrosis and removing CryAB protein aggregate, and has the effect of inducing myocardial remodeling by CryAB mutation. The pharmacological action of the raloxifene is verified through multi-level experiments from in-vitro cells to in-vivo mice, and research results show that the raloxifene and the pharmaceutical salt thereof have potential application value in treatment of the conjunctin-related cardiomyopathy, and a new strategy and an experimental verification path are provided for research of the hereditary cardiomyopathy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical technology, in particular, the application of raloxifene or a pharmaceutically acceptable salt thereof in a drug for treating CryAB mutation cardiomyopathy. BACKGROUND

[0002] Desmin-related cardiomyopathy (DRC) is a genetic cardiomyopathy characterized by interfilament disease, which is an important subtype of idiopathic cardiomyopathy. With the progression of the disease, patients will gradually develop ventricular remodeling, impaired diastolic function, and even biventricular pump failure and other serious complications. Due to its irreversible pathological process, the clinical course often shows a progressive worsening trend. At present, DRC has become a major challenge to global public health. According to the statistics of American Heart Association (AHA) in 2023, the incidence of genetic cardiomyopathy worldwide is about 1 / 2500, and DRC accounts for a certain proportion in familial cases, and CryAB gene mutation is one of the common pathogenic mechanisms of DRC.

[0003] CryAB (αB-crystallin, αB-crystallin) mainly functions as a chaperone to prevent protein misfolding and aggregation, and prevent the toxic effects of proteins in cells. This protein can also inhibit various inflammatory responses and has anti-apoptotic effects. Under physiological conditions, wild-type CryAB can spontaneously form a stable dimeric structure through the interaction between D109 and R120. The human CryAB coding gene is located at 11q23.1, and current studies have confirmed that there are multiple different types of mutations in the CryAB gene, such as D109G, R120G, R123W, G154S, R157H, etc., showing autosomal dominant inheritance pattern. When the CryAB gene is mutated, especially mutations in key amino acid residues such as D109G and R120G, the normal function of CryAB can be disrupted. The gene mutation not only causes cataracts, but also causes severe damage to the myocardium and skeletal muscle. For example, in the myocardium, CryAB R120G mutation disrupts the D109-R120 salt bridge structure, leading to loss of chaperone function and triggering abnormal phase separation, resulting in accumulation of insoluble aggregates. These aggregates not only contain mutant CryAB, but also capture key structural proteins such as desmin, disrupting the integrity of the sarcomere.

[0004] Currently, clinical treatments for DRC (such as beta-blockers and ACEIs) can only alleviate symptoms or slow disease progression to some extent, but they cannot target and correct the protein toxicity stress caused by CryAB mutations. Especially in the late stages of the disease, patients still rely on heart transplantation. However, heart transplantation is difficult to widely implement due to donor shortages and the high incidence of postoperative rejection. Furthermore, although some progress has been made in the treatment of hereditary cardiomyopathy in recent years, specific therapeutic drugs for DRC remain scarce, especially in clearing protein aggregates, inhibiting myocardial fibrosis, and reversing myocardial remodeling—there are no mature treatment options yet. Based on these circumstances, developing novel therapeutic strategies that can clear misfolded proteins at the molecular level and improve myocardial pathological changes has become a key need in the field of DRC treatment. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide the application of raloxifene in the treatment of CryAB-mutant cardiomyopathy.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect is the use of raloxifene or its pharmaceutical salts in the treatment of CryAB-mutant cardiomyopathy.

[0007] Preferably, the pharmaceutical salt is an acid addition salt formed by raloxifene and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

[0008] Preferably, the application is based on the CryAB R120G mutation model to simulate the core pathological features of CryAB mutant cardiomyopathy, including the formation of intracellular protein aggregates, myocardial hypertrophy, and heart failure manifestations.

[0009] Preferably, the CryAB R120G mutation model uses a genomic sequence containing pα-MHC-CryAB-intron-BGHpa to drive heart-specific expression of the CryAB R120G mutation via the α-myosin heavy chain promoter.

[0010] Preferably, the primer sequences for specific expression include a forward primer and a reverse primer; the forward primer is 5'-GGCTTGTCCTGTTCCATCCTG-3', and the reverse primer is 5'-ATGTTCGTCCTGGCGTTCTTC-3'.

[0011] Preferably, the drug is used to reduce myocardial hypertrophy, inhibit myocardial fibrosis, clear CryAB protein aggregates, and counteract CryAB mutation-induced myocardial remodeling.

[0012] Preferably, the maximum safe concentration of raloxifene and its salt in primary neonatal rat cardiomyocytes is 5 μM.

[0013] Secondly, the application of raloxifene or its pharmaceutical salts in the preparation of drugs for treating cardiomyocyte hypertrophy.

[0014] Thirdly, the use of raloxifene or its pharmaceutical salts in the preparation of drugs for treating myocardial fibrosis.

[0015] Fourthly, the use of raloxifene or its pharmaceutical salts in the preparation of drugs for treating myocardial remodeling.

[0016] The present invention has the following beneficial effects: This invention systematically verified the efficacy of raloxifene in anti-protein aggregation, inhibition of fibrosis, and improvement of cardiac function through in vitro and in vivo experiments using mouse models and primary cardiomyocytes. Raloxifene significantly reduced the aggregation of CryAB (R120G) mutant protein in in vivo and in vitro pathological models and alleviated cell hypertrophy caused by CryAB mutation. In vivo experimental results showed that raloxifene reduced myocardial hypertrophy and myocardial fibrosis in CryAB (R120G) transgenic mice, demonstrating a significant cardioprotective effect against hereditary cardiomyopathy caused by CryAB (R120G) mutation. Meanwhile, raloxifene slowed down cardiomyocyte hypertrophy caused by CryAB (R120G) mutation and reduced the expression levels of ANP and BNP. Echocardiography, HE staining, and Masson staining revealed that raloxifene significantly reduced myocardial hypertrophy and fibrosis in CryAB (R120G) mutant mice, while also reducing the aggregation of insoluble CryAB protein. This indicates that raloxifene alleviates pathological myocardial remodeling induced by CryAB (R120G) mutation by reducing myocardial hypertrophy, inhibiting myocardial fibrosis, and clearing CryAB protein aggregates.

[0017] Furthermore, this invention points out the potential application value of raloxifene or its pharmaceutical salts in the treatment of desmin-related cardiomyopathy, providing a new strategy and experimental verification pathway for the research of hereditary cardiomyopathy. The pharmacological effects of raloxifene were verified through multi-level experiments ranging from in vitro cells to in vivo mice, forming a closed-loop research system combining computational models and experimental verification.

[0018] In addition to the objectives, features, and advantages described above, the present invention has other objectives, features, and advantages. The invention will now be described in further detail with reference to the figures. Attached Figure Description

[0019] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a validation of myocardial pathological remodeling in a CryAB (R120G) mutant mouse model; Figure 2 To validate the expression and aggregation of CryAB protein in CryAB (R120G) mutant mice; Figure 3 Raloxifene inhibits the aggregation of CryAB (R120G) protein in cardiomyocytes; Figure 4 Determine the maximum safe concentration for raloxifene in the CCK8 concentration gradient; Figure 5 Raloxifene inhibits CryAB (R120G) mutation-induced cardiomyocyte hypertrophy; Figure 6 Flowchart for animal experiments involving raloxifene hydrochloride injection; Figure 7 Injection of raloxifene hydrochloride significantly reduced abnormal myocardial hypertrophy induced by CryAB (R120G); Figure 8 Raloxifene hydrochloride significantly alleviated myocardial pathological remodeling in CryAB (R120G) transgenic mice; Figure 9 Raloxifene and its hydrochloride reduced the deposition of insoluble CryAB protein in vitro and in vivo, respectively. Detailed Implementation

[0020] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and specific experiments.

[0021] The experimental methods used are as follows: 1. Preparation of paraffin sections of tissue Mouse heart tissue was harvested and rapidly rinsed with pre-cooled physiological saline to remove blood. A complete mid-segment of the heart was then cut and immediately placed in 4% paraformaldehyde fixative at 4°C for 24-48 hours. The fixed heart was then dehydrated using a gradient of ethanol (70%, 80%, 90%, 95%, and anhydrous ethanol, 30-60 min each), followed by clearing with xylene I and xylene II for 20-30 min each. The cleared tissue was then immersed in molten paraffin for 45-60 min each, repeated three times. Finally, the tissue was embedded in a paraffin block and cooled to room temperature for solidification. After paraffin embedding, continuous sections of 4-6 µm thickness were cut along the short axis of the heart using a microtome and laid flat on polylysine-coated slides. The sections were then baked at 60°C for 30 min for later use.

[0022] 2. Establishment of CryAB (R120G) transgenic mouse model This invention uses CryAB (R120G) transgenic mice as an experimental model, which can significantly simulate the core pathological features of CryAB mutant cardiomyopathy, including the formation of intracellular protein aggregates and heart failure manifestations. Wild-type C57BL / 6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were male, free of specific pathogens, and housed in the animal facility of Peking University People's Hospital (SPF environment, room temperature 22 ℃±2 ℃, relative humidity 40%-45%, good ventilation, 12-hour light-dark cycle). Mice underwent a one-week acclimatization period after arrival before the formal experiments began. All animal experiments were approved by the Animal Use Ethics Committee of Peking University People's Hospital (approval number PHC051).

[0023] During the experiment, mice were euthanized by cervical dislocation after being anesthetized with isoflurane, and their hearts were removed for subsequent experiments. The CryAB(R120G) transgenic mouse model was constructed by Beijing Weishang Lide Biotechnology Co., Ltd. (Beijing, China) using the C57BL / 6J mouse background. This model uses a genomic sequence containing pα-MHC-CryAB-intron-BGHpa, which drives heart-specific expression of the CryAB(R120G) mutation through the α-myosin heavy chain (α-MHC) promoter. The transgenic DNA was introduced into C57BL / 6J mouse zygotes via microinjection. After successfully obtaining the first generation of transgenic positive mice, they were mated with wild-type C57BL / 6J mice to produce the F1 generation, and continued to breed to the F3 generation and beyond to ensure the stability of gene expression. In this study, genotyping of the tail DNA of offspring mice was performed using PCR technology. The specific primer sequences were: forward primer 5'-GGCTTGTCCTGTTCCATCCTG-3', and reverse primer 5'-ATGTTCGTCCTGGCGTTCTTC-3'. CryAB (R120G) transgenic mice (TG) were finally obtained for further experiments, while non-transgenic littermates (NTG) served as a baseline control.

[0024] 3. Extraction of soluble and insoluble proteins Extraction of soluble and insoluble proteins from tissue: Frozen mouse heart tissue was transferred to EP tubes. 500 μL of PBS buffer (2% Triton X-100, 5 mM EDTA) containing protease and phosphatase inhibitors was added to the tubes. The tissue was then minced with scissors and mechanically homogenized on ice for 5 min using a pre-chilled tissue homogenizer, followed by another 15 min on ice. After lysis, the tissue was centrifuged at 12000 rpm for 10 min at 4 °C using a pre-chilled centrifuge. The supernatant was carefully aspirated from the tube and transferred to a new labeled EP tube, designated as the soluble fraction. The precipitate was resuspended in 1× loading buffer (40 mmol / L Tris HCl [pH 8.8], 1% SDS, 8% glycerol), boiled for 5 min, and centrifuged at 3000×g for 10 min. The supernatant was then designated as the insoluble fraction.

[0025] Extraction of soluble and insoluble proteins from cells: Remove intracellular culture medium using a pipette or pipette tip. Gently wash cells 2-3 times with pre-chilled PBS, then discard the PBS. Add 400 μL of Cell-lysis buffer containing protease inhibitors and phosphatase inhibitors to the cells and scrape them off using a cell scraper. Transfer the cell lysis buffer to a pre-labeled, pre-chilled EP tube using a pipette tip and place on ice for 15-20 min of lysis. After lysis, centrifuge at 12000 rpm for 10 min using a pre-chilled 4 ℃ centrifuge. Carefully aspirate the supernatant from the tube and transfer it to a new, labeled EP tube, labeling it as the soluble fraction. Wash the precipitate with lysis buffer and centrifuge three times. Then resuspend in DNase I (1 mg / mL, 5 mM MgCl₂) containing protease inhibitors and phosphatase inhibitors. 2+ In a solution, sonicated on ice, the insoluble component is recorded.

[0026] 4. Extraction of primary neonatal rat cardiomyocytes and infection of H9c2 cells with CryAB (R120G) virus Primary neonatal rat cardiomyocytes (NRCMs) were isolated using the differential adhesion method: Hearts of 2-day-old SD rats were collected and carefully separated in pre-cooled Hank's balanced salt solution (HBSS, calcium and magnesium ion-free). The tissue was minced and digested with 0.5 mg / mL type II collagenase (Gibco, 17101015) at 37°C with shaking for 30 min. Digestion was terminated by adding DMEM containing 20% ​​fetal bovine serum (FBS). The cell pellet was collected by centrifugation at 1000 rpm for 5 min and resuspended in DMEM high-glucose medium containing 10% FBS and 10 μM BrdU. The cells were then seeded in 10 cm culture dishes and allowed to adhere at 37°C for 90 min to remove fibroblasts. Unadhered cardiomyocytes were then cultured at 1×10⁻⁶ cells per cell. 5 Cells were seeded at a density of / well in 6-well plates. After 24 hours, the medium was replaced with BrdU-free medium containing 2% FBS. The following day, cells were infected with wild-type / mutant CryAB adenovirus carrying the GFP tag (AAV GFP-CryABWT / R120G, MOI=50). The virus was removed 24 hours after infection. H9c2 cells were infected using the same virus and titer.

[0027] 5. BCA method for protein quantification and preparation of protein samples A modified detection procedure was established according to the BCA protein concentration assay kit instructions. The specific steps were as follows: 2 mg / mL bovine serum albumin (BSA) standard was serially diluted to six concentration points (2, 1, 0.5, 0.25, 0.125, and 0.0625 mg / mL) using phosphate-buffered saline (PBS, pH 7.4). The test sample was pre-diluted at a 1:10 ratio and simultaneously added to a 96-well plate with 25 μL precisely added to each well. The BCA working solution was freshly prepared at a reagent A:B ratio of 100:1. After vortexing and incubation in the dark for 5 min to fully chelate copper ions, 200 μL of the working solution was quickly added to each well. The plate was incubated at 37°C for 30 min, then transferred to room temperature for 15 min to stabilize the color development. The absorbance (OD value) was read at 562 nm using a microplate reader, with the detection time window strictly controlled within 20 min to minimize absorbance drift. A standard curve (R²) was constructed using linear fitting. 2 >0.99), the protein concentration of the sample was calculated based on the curve equation. For samples exceeding the linear range (>2 mg / mL), a second dilution was performed using the lysis buffer, followed by retesting. After quantification, the final sample concentration was adjusted to 1 μg / μL using lysis buffer, and 5× loading buffer (containing DTT) was added at a volume ratio of 4:1. The protein was thoroughly denatured by heating in a 95℃ metal bath for 10 min and stored at -20℃ until use.

[0028] 6. Western blotting and dot blot assays for proteins. (1) Gel preparation and electrophoresis: Select an appropriate concentration of separating gel according to the molecular weight of the target protein (e.g., 12% separating gel for 10-150 kDa proteins), and use a uniform concentration of 5% for stacking gel; fix the cleaned gel preparation glass plate in the transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) and pre-cool to 4°C. After activating the PVDF membrane with methanol for 30 s, assemble it with filter paper and gel into a "sandwich" structure (sequence: cathode sponge-filter paper-gel-membrane-filter paper-anodic sponge), avoiding air bubbles throughout the process. Transfer the membrane at a constant current of 300 mA for 100 min (cooled in an ice bath). After transfer, stain with Ponceau S for 1 min to observe the transfer efficiency. Wash the membrane 3 times with TBST (5 min / time). Prepare 1× electrophoresis working solution in advance by adding 50 ml of 10× electrophoresis buffer to 450 ml of distilled water. Assemble and fix the prepared gel plate in the electrophoresis tank, add the prepared electrophoresis solution, and slowly pull out the comb vertically upwards in the electrophoresis solution. Then load the samples, adding markers and samples to the wells, and fill in the gaps with 1× loading buffer to ensure the same sample volume in each well to guarantee electrophoresis quality. If any wells are empty, fill them with an equal volume of 1× protein loading buffer. Connect the power supply and adjust to a suitable voltage for constant voltage electrophoresis. The electrophoresis conditions are generally set as follows: 80 V constant voltage electrophoresis for 30 min to fill the sample gaps, then 120 V constant voltage electrophoresis for 50 min. Finally, stop electrophoresis when the bromophenol blue indicator reaches the appropriate position on the gel plate.

[0029] (2) Transfer: Prepare 1× electroporation working solution in advance. Take 100 ml of 10× electroporation buffer, add 200 ml of methanol and 700 mL of distilled water to prepare the solution, and place it in the chromatography cabinet for pre-cooling. If precipitation occurs, be sure to mix it well. Cut the PVDF membrane to the appropriate size according to the gel size in advance. The PVDF membrane needs to be activated in methanol before use. Prepare filter paper of appropriate size and thickness. After electrophoresis, pry open the thin plate, scrape off the stacking gel, and keep only the protein gel in the separating gel. Prepare a sandwich in the pre-prepared electroporation solution. From bottom to top, the sandwich consists of the black side of the transfer plate (cathode), sponge, thick filter paper, thin filter paper, protein gel, PVDF membrane, thin filter paper, thick filter paper, sponge, and the white transparent side of the transfer plate (anode). Note that there should be no air bubbles throughout the process. After preparation, place it in the electroporation tank, connect the power supply, and adjust the current to a suitable constant current for electroporation. Set the electroporation conditions to: 300 mA constant current for 100 min, and then stop the electroporation. A lot of heat is generated during the electroporation process. To avoid affecting the transfer effect, ice packs are placed in the electroporation tank and the electroporation tank is placed in an ice box.

[0030] (3) Blocking: Block the membrane with rapid blocking solution at a shaking speed of 40-60 r / min at room temperature for 10 min. After blocking, wash the membrane 2-3 times with TBST to avoid affecting the subsequent antibody recovery.

[0031] (4) Incubation of primary antibody: Dilute the antibody according to the antibody instructions. The dilution ratio is generally 1:1000. Centrifuge the antibody briefly before dilution. Add the diluted primary antibody to the box containing the membrane and incubate overnight in a chromatography cabinet at 4 ℃. Then recover the primary antibody and wash the membrane 3 times with TBST for 10 min each time.

[0032] (5) Incubation of secondary antibody: Select a secondary antibody of the same species as the primary antibody, dilute it at 1:5000, and incubate it on a shaker at room temperature (shaking speed 40-50 r / min) for 1 h. After that, recover the secondary antibody and wash the membrane 3 times with TBST for 10 min each time.

[0033] (6) Chemiluminescence: Prepare the working solution of the ultrasensitive luminescence solution, ensuring it is prepared fresh for each use and protected from light; place the membrane in the luminescence analyzer for chemiluminescence, and then photograph the luminescence results. Some membranes require removal of antibodies, re-blocking, and re-incubation with primary and secondary antibodies before luminescence.

[0034] (7) Gray value analysis: The gray values ​​of the luminescent protein bands were scanned using Image Lab software. The relative expression was analyzed by the ratio of the gray values ​​of the protein bands to their corresponding internal reference proteins.

[0035] For the Dot Blot experiment, 1× lysis buffer containing 0.1% SDS was used as the diluent. After denaturing the insoluble proteins, the buffer was successively diluted to four gradients: 1× (stock solution), 0.5×, 0.25×, and 0.125×. After vortexing and mixing, the buffer was kept on ice. The NC membrane (0.45 μm, Millipore) was placed on absorbent paper, and 5 μg of diluted sample was added to each spot. After spotting, the membrane was dried at 37 °C for 2 h, and then directly immersed in the rapid blocking buffer. The procedure was the same as for the Western Blot.

[0036] Example 1: CryAB (R120G) induces myocardial pathological remodeling and protein toxicity accumulation. In this embodiment, a heart-specific overexpression mouse model of α-MHC-CryAB (R120G) was successfully constructed using transgenic technology (TG group). After obtaining F0 generation chimeras via microinjection, a stable genetic line (C57BL / 6J background) was established through three consecutive backcrosses. Non-transgenic mice from the same littermate (NTG group) served as the control group. To assess whether the transgenic mice exhibited pathological changes in the myocardium, qPCR was used to detect the expression levels of markers of myocardial remodeling and heart failure, including ANP, BNP, MYH7, and ACTA1, in the myocardial tissue of 28-week-old TG mice.

[0037] Figure 1 To establish a CryAB(R120G) mutant mouse model and validate myocardial function, heart-specific CryAB(R120G) transgenic mice (TG group) were constructed, with littermate non-transgenic mice (NTG group) serving as controls. Figure 1 In Figure A, the relative expression levels of myocardial remodeling markers (ACTA1, MYH7, ANP, BNP) mRNA in myocardial tissue were detected by real-time quantitative PCR, with GAPDH as an internal control (n=5). In Figure B, the area of ​​myocardial collagen fibers (blue) was quantitatively detected by Masson's trichrome staining. In Figure C, the collagen volume fraction was quantitatively analyzed using ImageJ software (n=3 / group). The results showed that compared with the NTG control group, the expression levels of BNP, MYH7, and ACTA1 in the myocardial tissue of TG mice were significantly increased (P<0.05). The upregulation of ACTA1 reflects actin isoform switching and is associated with the progression of myocardial fibrosis, while the expression level of ANP showed no significant difference (P>0.05). This suggests that TG mice exhibit significant myocardial remodeling and heart failure at 28 weeks of age. Figure 1 (A). To further verify the pathological changes of myocardial fibrosis, Masson's trichrome staining was used to assess the degree of myocardial fibrosis in mice in the TG and NTG groups. The results showed that the area of ​​collagen fibers (blue) in the myocardial tissue of mice in the TG group was significantly increased ( Figure 1 China B and Figure 1 The difference between the C group and the NTG group was statistically significant (P<0.05). These results indicate that CryAB (R120G) overexpression leads to the pathological changes of myocardial fibrosis in mice.

[0038] Since CryAB (R120G) can lead to an increase in intracellular misfolded proteins, resulting in abnormal protein aggregation in myocardial tissue and the formation of insoluble protein aggregates, soluble and insoluble protein components in the myocardial tissue of TG and NTG mice were further separated by fractional extraction and detected by Western blotting and dot blot.

[0039] Figure 2To validate CryAB protein expression in CryAB(R120G) mutant mice, heart-specific CryAB(R120G) transgenic mice (TG group) were constructed, with littermate non-transgenic mice (NTG group) serving as controls. Western blotting was used to detect the levels of soluble (A, B) and insoluble (C) CryAB proteins in myocardial tissue, with GAPDH as an internal control. Insoluble protein fractions were analyzed by spot staining after serial dilution (1×, 0.5×, 0.25×, 0.125×). Data are expressed as mean ± SD, ns: no statistical difference (P>0.05), *: P<0.05, **: P<0.01. The results showed that the expression level of soluble CryAB protein in the myocardial tissue of mice in the TG group was significantly increased compared with the NTG control group. Figure 2 (A). Gray value analysis results show ( Figure 2 In the TG group (B group), the expression level of soluble CryAB protein was significantly higher than that in the NTG group (Negative group). After resuspending the insoluble fraction, the same amount (5 μg) of protein samples were analyzed by dot blot using a serial dilution method (1×, 0.5×, 0.25×, 0.125×). The results showed a significant increase in CryAB protein aggregates in the TG group (B group). Figure 2 (C)

[0040] Example 2: Raloxifene inhibits the aggregation of CryAB (R120G) protein in cardiomyocytes. Raloxifene, quinacrine, dasatinib, treprostine, and dexamethasone were selected for functional validation. Drug concentrations were set according to literature reports: raloxifene (5 μM), quinacrine (10 μM), dasatinib (20 μM), treprostine (1 μM), and dexamethasone (100 nM). Preliminary experiments confirmed cell viability >85% at each concentration. To further screen candidate drugs, this invention used an H9c2 cell model infected with AdV-GFP-CryAB (R120G). To evaluate the effects of candidate drugs on CryAB (R120G) protein aggregates, H9c2 cells were treated with raloxifene, treprostine, quinacrine, dasatinib, and dexamethasone for 48 h after 72 h of viral infection. Subsequently, GFP-labeled CryAB (R120G) protein aggregates were observed and recorded using fluorescence microscopy.

[0041] Figure 3 Raloxifene inhibits the aggregation of CryAB (R120G) protein in cardiomyocytes. Figure 3In Figures A and B, H9c2 cells were infected with AdV-GFP-CryAB (R120G) virus (MOI=50) to establish a pathological model (MUT group). Forty-eight hours after infection, raloxifene (5 μM), treprostrinone (1 μM), quinacrine (10 μM), dasatinib (20 μM), and dexamethasone (100 nM) were added, respectively, and the formation of fluorescent aggregates was observed (A). Quantitative analysis of aggregates was performed using ImageJ to calculate aggregate area / cell area (n=5 fields of view) (B). ns: P>0.05, *: P<0.05, **: P<0.01, ****: P<0.0001. The results showed that compared with the mutant group (MUT group), the number of aggregates (green punctate fluorescence) in the raloxifene treatment group was significantly reduced (P<0.0001), while the dexamethasone and dasatinib treatment groups showed a slight decreasing trend, but there was no statistically significant difference (P>0.05); no significant reduction was observed in the treprostyl group; secondly, quinacrine significantly increased the number of intracellular aggregates (P>0.05). Figure 3 China A and Figure 3 (B). The above results indicate that raloxifene, as a selective estrogen receptor modulator (SERM), has the potential to promote the degradation of CryAB (R120G) protein aggregates.

[0042] To confirm the pharmacological effects of raloxifene in promoting the clearance of CryAB (R120G) protein aggregates and alleviating cell hypertrophy, based on previous findings in H9c2 cells, this invention further validated its efficacy in a primary neonatal rat cardiomyocyte model. Through the CCK8 assay, the maximum safe concentration of raloxifene for use in primary cardiomyocytes was determined to be 5 μM. Figure 4 The maximum safe concentration of raloxifene was determined using the CCK8 assay for a concentration gradient. Primary cardiomyocytes were treated with different concentrations of raloxifene (100 μM, 50 μM, 25 μM, 10 μM, 5 μM) for 48 h, and then incubated with CCK8 for 2 h before measuring absorbance at 450 nm. The absorbance at 0 μM concentration was used as a control for statistical analysis, employing a one-way ANOVA. ns: P>0.05, **: P<0.01.

[0043] Example 3: Raloxifene inhibits CryAB (R120G)-induced cardiomyocyte hypertrophy in vitro. To investigate whether raloxifene can reverse CryAB (R120G)-induced cardiomyocyte hypertrophy, a pathological cardiomyocyte hypertrophy model was established by overexpressing CryAB (R120G) in primary neonatal rat cardiomyocytes based on the optimal raloxifene concentration confirmed by CCK8. The cardiomyocyte area was quantitatively analyzed using high-content cell imaging technology.

[0044] Figure 5Raloxifene inhibits CryAB (R120G)-induced cardiomyocyte hypertrophy. Figure 5 Image A and Image B show high-content imaging of NRCM cells infected with AdV-CryAB (R120G) virus treated with raloxifene (5 μM) (A), and ImageJ quantitative cardiomyocyte area statistical analysis. The average value of 50 cells from different fields of view in each group was used, n=4 / group. Image C shows the relative expression of cardiac remodeling markers ANP, BNP, and MYH7, based on 2... −ΔΔCT Using empty virus-carrying cells (NC group) as the baseline, relative number quantification was performed, with GAPDH as the internal reference gene. Multiple group analyses were conducted using one-way ANOVA and within-group comparisons; ns: P>0.05, *: P<0.05; data are expressed as mean ± SEM. Results showed that compared with the mutant group (MUT group), raloxifene treatment significantly reduced the area of ​​primary cardiomyocytes (P<0.0001). Figure 5 (A and B in the original text). This result suggests that raloxifene has a significant ameliorative effect on myocardial hypertrophy induced by CryAB (R120G) mutation. Mechanistically, by examining the changes in cardiomyocyte markers induced by CryAB (R120G) treatment after raloxifene treatment, RT-qPCR results showed that raloxifene treatment significantly reduced the expression levels of ANP and BNP in NRCMs overexpressing CryAB (R120G) (P<0.05), but the expression level of Myh7 did not show a statistically significant difference (P>0.05). Figure 5 (C)

[0045] Example 4: Raloxifene alleviates CryAB (R120G)-induced cardiac hypertrophy and pathological remodeling in vivo. To verify the in vivo therapeutic effect of raloxifene, this invention further conducted a 2-week raloxifene intervention experiment in a 28-week-old CryAB (R120G) transgenic mouse model. Considering in vivo absorption, raloxifene hydrochloride was selected as the main agent in the animal experiment. The experimental procedure is as follows: Figure 6 As shown. Figure 6 A flowchart for the animal experiment design of raloxifene hydrochloride injection is provided. The flowchart shows the design of the animal experiment for raloxifene, in which α-MHC-CryAB (R120G) heart-specific transgenic mice (28 weeks old) are intraperitoneally injected with raloxifene hydrochloride (10 mg / kg / d, for 2 weeks). The flowchart also shows the viral vector structure and the dosing time window.

[0046] To evaluate the effects of raloxifene hydrochloride on cardiac function, we performed echocardiography on mice in each group and quantified key cardiac structural and functional parameters. Representative M-mode ultrasound images are shown below. Figure 7 As shown.

[0047] Figure 7 Raloxifene hydrochloride injection significantly reduced CryAB (R120G)-induced abnormal myocardial hypertrophy. Figure 7 Image A shows representative M-mode echocardiograms of mice in each group. Image B shows the statistical analysis of left ventricular end-diastolic anterior wall thickness (LVAW;d), left ventricular end-systolic anterior wall thickness (LVAW;s), left ventricular end-diastolic posterior wall thickness (LVPW;d), left ventricular end-systolic posterior wall thickness (LVPW;s), fractional shortening (FS%), left ventricular end-diastolic diameter (LVID;d), left ventricular end-systolic diameter (LVID;s), left ventricular end-diastolic volume (LVEV;d), left ventricular end-systolic volume (LVEV;s), and ejection fraction (EF%) for each group. The sample sizes for NTG, NTG+Raloxifene, TG, and TG+Raloxifene were 7, 13, 8, and 7, respectively. Multiple group analyses were performed using one-way ANOVA and within-group comparisons; ns: P>0.05, *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001; data are expressed as mean ± SEM. Results showed that compared with the TG control group, the TG+Raloxifene group exhibited significantly reduced thickness of the left ventricular anterior and posterior walls at end-diastole and end-systole (LVAW;d, LVAW;s, LVPW;d, LVPW;s). However, there were no significant differences in left ventricular diameter (LVID;d, LVID;s) and left ventricular end-systolic diameter (LVEV;s), ejection fraction (EF%), and fractional shortening (FS%) between the NTG and TG groups, and between the TG and TG+Raloxifene groups, suggesting that the overall pump function of CryAB(R120G) transgenic mice was still compensatorily maintained at 28 weeks of age. However, the overall results showed that raloxifene hydrochloride treatment significantly improved left ventricular wall thickness. Meanwhile, there were no significant differences in parameters between the NTG+Raloxifene group and the NTG group, indicating that raloxifene has no significant negative impact on normal hearts.

[0048] In addition, to further evaluate whether raloxifene can alleviate abnormal myocardial remodeling caused by CryAB (R120G), tissue staining and qPCR were used for verification. Masson staining was used to assess the deposition of collagen fibers in myocardial tissue.

[0049] Figure 8 Raloxifene hydrochloride significantly alleviated myocardial pathological remodeling in CryAB (R120G) transgenic mice. Figure 8A represents representative images of the heart morphology of mice in each group, with DMSO solvent as a control; representative images of HE staining (Bar, 500μm), Masson staining (Bar, 200μm), and WGA staining (Bar, 20μm) for each group; B represents the ratio of heart weight to body weight in NTG and TG mice (7, 13, 8, and 7 samples for NTG, NTG+Raloxifene, TG, and TG+Raloxifene, respectively); C represents the difference between the body weight of mice in each group 2 weeks after intraperitoneal injection and the body weight on the day of injection, expressed as the body weight on the first day minus the body weight on the 14th day; D and E represent the area of ​​myocardial collagen fibers after Masson staining (D) and the area of ​​myocardial cells after WGA staining (E) in each group of mice, respectively, analyzed using ImageJ; F represents the relative expression levels of cardiac remodeling markers (ANP, BNP, MYH7, ACTA1) in each group of mice, normalized with the NTG group as a control, and β-actin as a control. Multiple group analyses were performed using one-way ANOVA and within-group comparisons; ns: P>0.05, *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001; data are expressed as mean ± SEM. Results showed that raloxifene treatment significantly reduced collagen fiber deposition in the TG group, and WGA staining showed that raloxifene reduced the cardiomyocyte area in the TG group. Furthermore, qPCR was used to detect the relative expression levels of ANP, BNP, MYH7, and ACTA1 in cardiac tissue. The results showed that raloxifene treatment significantly downregulated the expression of the aforementioned remodeling-related genes (BNP, MYH7, and ACTA1), consistent with previous cell experiments. Figure 8 (C). The above results indicate that raloxifene can reduce myocardial hypertrophy, inhibit myocardial fibrosis, and reverse pathological myocardial remodeling induced by CryAB (R120G) mutation.

[0050] Example 5: Raloxifene and its hydrochloride significantly reduced the deposition of insoluble CryAB protein induced by CryAB (R120G) mutation in vitro and in vivo. To further evaluate whether raloxifene hydrochloride helps alleviate the abnormal aggregation of insoluble CryAB protein in the myocardium of CryAB (R120G) transgenic mice. Figure 9 Raloxifene and its hydrochloride reduced the expression of insoluble CryAB protein in vitro and in vivo. Figure 9Image A shows immunofluorescence staining of myocardial CryAB two weeks after intraperitoneal injection of raloxifene hydrochloride (10 mg / kg / d) into 28-week-old CryAB (R120G) transgenic positive (TG) and negative (NTG) mice. Image B shows quantitative analysis of CryAB immunofluorescence staining, using ImageJ to analyze representative images of each sample, and taking the average area of ​​CryAB (red) in 10 different fields of view as the statistical value. Image C shows the expression of insoluble CryAB protein in Dot blot analysis. After dissolving and quantifying the insoluble protein components of the cardiac tissue of each sample, the samples were homogenized, and 1 μg was loaded into each well. Image D shows the quantitative statistical analysis of insoluble CryAB protein in Dot blot analysis, with 5 samples per group. Image J shows the aggregation formation observed by laser confocal microscopy two days after NRCM infection with AdV-GFP-CryAB (R120G) and the addition of raloxifene (5 μM). Image J was used to calculate the aggregation area / cell area (n=10) for NRCM quantitative analysis. Image G shows the expression of CryAB protein in Western blot analysis. Blot analysis showed the content of insoluble CryAB in NRCM cells, with α-Actinin as an internal control. Multiple group analyses were performed using one-way ANOVA and within-group comparisons; ns: P>0.05, *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001; data are expressed as mean ± SEM.

[0051] Analysis by immunofluorescence staining ( Figure 9 (A) The deposition of insoluble CryAB in each group was examined. The results showed that raloxifene hydrochloride treatment significantly reduced the abnormal expression of insoluble CryAB protein in the myocardium of the TG group. At the same time, Dot blot quantitative analysis showed that ( Figure 9 In the raloxifene hydrochloride treatment group, the expression of CryAB protein in the insoluble fraction was significantly reduced (C). Furthermore, this invention examined the pharmacological effect of raloxifene in clearing insoluble CryAB protein in primary cardiomyocytes in vitro. Primary cardiomyocytes were infected with AdV-GFP-CryAB (R120G) for 3 days, and then treated with raloxifene for 48 hours. Laser confocal imaging was then used (C). Figure 9 In vitro and Western blot (WB) results showed that raloxifene also promoted the clearance of insoluble CryAB protein in vitro. These results indicate that raloxifene significantly reduced the deposition of insoluble CryAB protein induced by the CryAB (R120G) mutation both in vivo and in vitro.

[0052] This invention systematically verified the efficacy of raloxifene in anti-protein aggregation, inhibition of fibrosis, and improvement of cardiac function through in vitro and in vivo experiments using mouse models and primary cardiomyocytes. The results indicate that raloxifene has potential application value in the treatment of desmin-related cardiomyopathy, providing a new strategy and experimental verification pathway for the research of hereditary cardiomyopathy.

[0053] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of raloxifene or its pharmaceutical salt in the treatment of CryAB-mutant cardiomyopathy.

2. The application according to claim 1, characterized in that, The pharmaceutical salt is an acid addition salt formed by raloxifene with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

3. The application according to claim 1, characterized in that, The application is based on the CryAB R120G mutation model and simulates the core pathological features of CryAB mutant cardiomyopathy, including the formation of protein aggregates in cardiomyocytes, myocardial hypertrophy, and heart failure manifestations.

4. The application according to claim 1, characterized in that, The CryAB R120G mutation model described uses a genomic sequence containing pα-MHC-CryAB-intron-BGHpa to drive heart-specific expression of the CryAB R120G mutation via the α-myosin heavy chain promoter.

5. The application according to claim 1, characterized in that, The primer sequences for specific expression include a forward primer and a reverse primer; the forward primer is 5'-GGCTTGTCCTGTTCCATCCTG-3', and the reverse primer is 5'-ATGTTCGTCCTGGCGTTCTTC-3'.

6. The application according to claim 1, characterized in that, The drug is used to reduce myocardial hypertrophy, inhibit myocardial fibrosis, clear CryAB protein aggregates, and counteract CryAB mutation-induced myocardial remodeling.

7. The application according to claim 1, characterized in that, The maximum safe concentration of raloxifene and pharmaceutical-grade salts in primary neonatal rat cardiomyocytes is 5 μM.

8. The use of raloxifene or its pharmaceutical salt in the preparation of drugs for treating cardiomyocyte hypertrophy.

9. The use of raloxifene or its pharmaceutical salt in the preparation of drugs for the treatment of myocardial fibrosis.

10. Use of raloxifene or its pharmaceutical salt in the preparation of drugs for the treatment of myocardial remodeling.

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