Application of tRNA-Ser-GCT in the preparation of products for treating alcoholic cardiomyopathy

Abstract

This invention discloses the application of tRNA-Ser-GCT in the preparation of products for treating alcoholic cardiomyopathy. In this invention, tRNA-Ser-GCT is highly expressed in alcoholic cardiomyopathy and mediates myocardial immune inflammatory response and cell damage by regulating the PI3k-Akt signaling pathway. In vivo experiments show that inhibiting tRNA-Ser-GCT can effectively reduce alcohol-induced myocardial inflammation and cardiac function impairment, providing a novel molecular target for the treatment of ACM.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of tRNA-Ser-GCT in the preparation of products for treating alcoholic cardiomyopathy. Background Technology

[0002] Alcoholic cardiomyopathy (ACM) is an acquired dilated cardiomyopathy caused by long-term excessive alcohol consumption and is a major cause of heart failure and cardiac death worldwide. Epidemiological studies have shown a strong correlation between long-term daily alcohol intake of ≥80 g and a significantly increased risk of ACM. The main pathological features of ACM include left ventricular dilation, ventricular wall thinning, decreased myocardial contractility, and progressive heart failure, often complicated by severe arrhythmias and even sudden death in its later stages. Although the causal relationship between excessive alcohol consumption and myocardial damage has been widely established, the molecular pathological mechanisms of ACM remain incompletely understood. Current clinical diagnosis relies primarily on medical history, imaging findings, and exclusionary diagnoses, lacking specific molecular markers, leading to difficulties in early identification and missed intervention windows. Therefore, in-depth analysis of the molecular regulatory network of ACM and the identification of reliable early diagnostic and intervention targets have significant basic research and clinical translational implications.

[0003] Recent studies have shown that chronic inflammation, oxidative stress, and mitochondrial dysfunction are core pathological links in the development of acute myocardial infarction (ACM). Abnormal activation of inflammatory signaling pathways not only exacerbates cardiomyocyte damage but also participates in myocardial remodeling and fibrosis. However, the upstream regulatory mechanisms of the inflammatory response remain unclear, especially the epigenetic and non-coding RNA regulatory networks, which require further investigation. With the development of high-throughput sequencing technology, a class of non-coding RNAs derived from tRNA-specific cleavage—tRNA-derived small RNAs (tsRNAs)—has gradually attracted attention. tsRNAs can be classified into subtypes such as tRF-5, tRF-3, tRF-1, and stress-induced tiRNAs based on their cleavage sites. These small RNAs not only possess high stability but can also bind to Ago proteins through a miRNA-like mechanism, targeting specific mRNAs and regulating their translation or stability, thereby participating in post-transcriptional gene expression regulation. Previous studies have confirmed that tsRNAs play important regulatory roles in cardiovascular diseases such as myocardial infarction, heart failure, and myocardial fibrosis. However, their function and molecular mechanisms in ACM remain poorly understood. Summary of the Invention

[0004] The purpose of this invention is to provide an application of tRNA-Ser-GCT in the preparation of products for treating alcoholic cardiomyopathy. In this invention, tRNA-Ser-GCT is highly expressed in alcoholic cardiomyopathy and mediates myocardial immune inflammatory response and cell damage by regulating the PI3k-Akt signaling pathway. In vivo experiments show that inhibiting tRNA-Ser-GCT can effectively reduce alcohol-induced myocardial inflammation and cardiac function impairment, providing a novel molecular target for the treatment of ACM.

[0005] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: The first aspect of this invention provides the application of tRNA-Ser-GCT in the preparation of products for treating alcoholic cardiomyopathy.

[0006] Preferably, the use of tRNA-Ser-GCT inhibitors in the preparation of products for treating alcoholic cardiomyopathy.

[0007] Preferably, the tRNA-Ser-GCT inhibitor comprises tRNA-Ser-GCT inhibitor, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0008] A second aspect of the present invention provides a medicament for treating alcoholic cardiomyopathy, the medicament comprising a therapeutically effective amount of a tRNA-Ser-GCT inhibitor and pharmaceutically acceptable excipients.

[0009] A third aspect of the present invention provides the application of tRNA-Ser-GCT in the preparation of products for treating myocarditis.

[0010] Preferably, the use of tRNA-Ser-GCT inhibitors in the preparation of products for treating myocarditis.

[0011] Preferably, the tRNA-Ser-GCT inhibitor comprises tRNA-Ser-GCT inhibitor, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0012] A fourth aspect of the present invention provides a medicament for treating myocarditis, the medicament comprising a therapeutically effective amount of a tRNA-Ser-GCT inhibitor and pharmaceutically acceptable excipients.

[0013] The fifth aspect of this invention provides the application of tRNA-Ser-GCT in the preparation of products for diagnosing alcoholic cardiomyopathy.

[0014] Preferably, the reagent for detecting tRNA-Ser-GCT is used in the preparation of products for diagnosing alcoholic cardiomyopathy.

[0015] Preferably, the reagent for detecting tRNA-Ser-GCT includes primers for amplifying tRNA-Ser-GCT.

[0016] Compared with the prior art, the beneficial effects of the present invention include at least the following: Studies have shown that alcohol exposure significantly impairs cardiac function in mice, manifested as decreased LVEF (Left Ventricular Ejection Fraction) and FS (Fractional Shortening), and increased LVIDd (Left Ventricular Internal Dimension in Diastole) (P < 0.05), accompanied by myocardial fiber disorder, inflammatory cell infiltration, and increased fibrosis. Whole transcriptome sequencing and RT-qPCR analysis revealed that tRNA-Ser-GCT was significantly upregulated in the alcohol-exposed group (fold change > 2, P < 0.01). KEGG analysis indicated that it is mainly involved in the phosphatidylinositol 3 kinase-protein kinase B (PI3K-AKT) pathway related to inflammation and oxidative stress. Cellular experiments further demonstrated that overexpression of tRNA-Ser-GCT can upregulate tumor necrosis factor-α (TNF-α). The expression of TNF-α, interleukin-1β (IL-1β), and interleukin-6 (IL-6) is inhibited, and Igf1 nuclear translocation is promoted. Inhibition of tRNA-Ser-GCT has the opposite effect. RNA pull-down assays have confirmed that tRNA-Ser-GCT can directly bind to Igf1. Therefore, in this invention, tRNA-Ser-GCT is highly expressed in alcoholic cardiomyopathy and mediates myocardial immune inflammatory response and cell damage by regulating the PI3k-Akt signaling pathway. Inhibition of tRNA-Ser-GCT can effectively reduce alcohol-induced myocardial inflammation and cardiac function impairment, providing a novel molecular target for the treatment of ACM and a new approach for the auxiliary diagnosis of ACM. Attached Figure Description

[0017] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0018] Figure 1 This describes the establishment of the ETOH-induced alcoholic cardiomyopathy model and the changes in cardiac function in Example 1 of the present invention. Figure 2 The results of screening and functional analysis of differentially expressed tsRNAs in the myocardial tissue of mice with alcoholic cardiomyopathy (ACM) in Example 2 of this invention; Figure 3 The expression levels of differentially expressed tRNAs in the CON group and ACM group in Example 3 of this invention; Figure 4 This describes the expression location and changes of tRNA-Ser-GCT in myocardial tissue in Example 4 of the present invention. Figure 5 In Example 5 of this invention, tRNA-Ser-GCT regulates the expression and secretion levels of inflammatory factors. Figure 6 The results of screening and functional verification of tRNA-Ser-GCT targeting Igf1 in Example 6 of this invention; Figure 7 This is the result of the study on the effect of inhibiting tRNA-Ser-GCT on improving cardiac function and ventricular remodeling in mice with alcoholic cardiomyopathy in Example 7 of the present invention. Detailed Implementation

[0019] The embodiments of the technical solution of the present invention will be described in detail below with reference to the examples. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore only examples, and should not be used to limit the scope of protection of the present invention.

[0020] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0021] This invention provides an application of tRNA-Ser-GCT in the preparation of products for treating alcoholic cardiomyopathy.

[0022] In one embodiment, the tRNA-Ser-GCT inhibitor is used in the preparation of a product for treating alcoholic cardiomyopathy.

[0023] In one embodiment, the tRNA-Ser-GCT inhibitor includes a tRNA-Ser-GCT inhibitor, the nucleotide sequence of which is shown in SEQ ID NO: 1, specifically AAACAUGGAAGCAUGAAUUAGCAGUUCUUGCAAUCUUUCUU.

[0024] Another embodiment of the present invention provides a medicament for treating alcoholic cardiomyopathy, the medicament comprising a therapeutically effective amount of a tRNA-Ser-GCT inhibitor and pharmaceutically acceptable excipients.

[0025] Another embodiment of the present invention provides the application of tRNA-Ser-GCT in the preparation of products for treating myocarditis.

[0026] In one embodiment, the tRNA-Ser-GCT inhibitor is used in the preparation of a product for treating myocarditis.

[0027] In one embodiment, the tRNA-Ser-GCT inhibitor comprises tRNA-Ser-GCT inhibitor, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0028] In another embodiment of the present invention, a medicament for treating myocarditis is provided, the medicament comprising a therapeutically effective amount of a tRNA-Ser-GCT inhibitor and pharmaceutically acceptable excipients.

[0029] Another embodiment of the present invention provides the application of tRNA-Ser-GCT in the preparation of products for diagnosing alcoholic cardiomyopathy.

[0030] In one embodiment, the reagent for detecting tRNA-Ser-GCT is used in the preparation of a product for diagnosing alcoholic cardiomyopathy.

[0031] In one embodiment, the reagent for detecting tRNA-Ser-GCT includes primers for amplifying tRNA-Ser-GCT.

[0032] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0033] (1) Male mice (C57BL / 6J background, 8 weeks old) were first paired and fed either a modified Lieber-DeCarli alcoholic liquid diet or an isocaloric maltodextrin control liquid diet, following a stepwise feeding protocol. Eight weeks after model induction, echocardiography was performed on mice in the alcoholic cardiomyopathy (ACM) group and the control group to assess cardiac function. Myocardial tissue from both groups of mice was collected for whole transcriptome sequencing and bioinformatics analysis. (2) The differentially expressed tRNAs in myocardial tissue after alcohol exposure were identified by whole transcriptome sequencing results, and their expression changes in heart tissue were verified by quantitative real-time PCR (RT-qPCR). It was found that the differential expression of tRNA-Ser-GCT was the most significant. Combined with KEGG enrichment analysis, it was found that the main functional pathway related to tRNA-Ser-GCT under alcohol exposure conditions is immune inflammatory response. (3) By constructing tRNA-Ser-GCT overexpression and inhibition vectors, H9c2 cardiomyocytes were transfected, and their regulatory effects on cellular inflammation level and cytokine secretion under alcohol-induced pathological conditions were evaluated using Western blot (WB), RT-qPCR, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining. (4) The interaction proteins of tRNA-Ser-GCT were identified by RNA pull-down assay and RT-qPCR and were negatively regulated by tRNA-Ser-GCT.

[0034] Example 1 This example demonstrates the construction and phenotypic confirmation of an ETOH-induced ACM model. To determine whether long-term ethanol (ETOH) treatment successfully induced an alcoholic cardiomyopathy (ACM) model, this invention systematically evaluated cardiac structure and function using echocardiography and serological indicators.

[0035] Male mice (C57BL / 6J background, 8 weeks old) were paired and fed either a modified Lieber-DeCarli alcoholic liquid diet or an isocaloric maltodextrin control liquid diet, following a stepwise feeding protocol. Eight weeks after model induction, echocardiography was performed on mice in both the alcoholic cardiomyopathy (ACM) group and the control group to assess cardiac function. Myocardial tissue from both groups was collected for whole-transcriptome sequencing and bioinformatics analysis. The results are as follows Figure 1 As shown, Figure 1In the study, (A) representative M-mode echocardiographic images of mice in the CON and ETOH groups; (B) statistical analysis of serum BNP levels, left ventricular ejection fraction (EF), and fractional shortening (FS); (C) comparison of left ventricular end-systolic diameter (LVIDs), end-diastolic diameter (LVIDd), and heart rate (HR); and (D) comparison of left ventricular posterior wall diastolic thickness (LVPWd) and interventricular septal thickness (IVSd). Data are presented in [data missing]. ±s indicates the mean. ns indicates no statistically significant difference; *P<0.05, ***P<0.001, ****P<0.0001; M-mode echocardiography was used to detect left ventricular systolic function in mice. Figure 1 Compared with the control group (CON), the ETOH group mice showed significantly decreased left ventricular ejection fraction (EF) and fractional shortening (FS) (P<0.0001), indicating significant impairment of myocardial contractile function. Figure 1 (B) Meanwhile, serum brain natriuretic peptide (BNP) levels were significantly elevated in the ETOH group (P<0.001), further indicating decreased cardiac function and the formation of a heart failure phenotype. Regarding ventricular structural parameters, both left ventricular end-systolic diameter (LVIDs) and end-diastolic diameter (LVIDd) were significantly increased in the ETOH group (P<0.001), suggesting ventricular enlargement and ventricular remodeling. Figure 1 (C). Heart rate (HR) showed no significant difference between the two groups (P>0.05), indicating that changes in cardiac function were not caused by changes in heart rate. Furthermore, ventricular wall thickness analysis showed that the ETOH group exhibited significantly decreased diastolic left ventricular posterior wall thickness (LVPWd) and interventricular septal thickness (IVSd) (P<0.05 or P<0.001). Figure 1 The presence of BNP (indicating myocardial thinning and structural remodeling) suggests that long-term ETOH treatment leads to typical ventricular dilation, decreased systolic function, and myocardial structural remodeling in mice, accompanied by elevated BNP levels, consistent with the pathological characteristics of alcoholic cardiomyopathy. These results demonstrate that this invention successfully established a stable and reliable animal model of ACM, laying the foundation for subsequent molecular mechanism research.

[0036] Example 2 This example demonstrates the screening of differentially expressed tsRNAs in the myocardial tissue of mice with alcoholic cardiomyopathy: To systematically screen for tsRNAs associated with alcoholic cardiomyopathy, this invention performed high-throughput sequencing analysis on myocardial tissues from control (CON) and ACM groups of mice; the analysis results are as follows: Figure 2 As shown, Figure 2In the diagram, (A) Principal component analysis (PCA) of tsRNA expression profiles; (B) Venn plot showing the number of shared and specifically expressed tsRNAs in the two groups, indicating significant differences in some tsRNAs between groups; (C) Hierarchical clustering heatmap of differentially expressed tsRNAs, clearly separating tsRNA expression patterns among different samples, with upregulated tsRNAs indicated in red and downregulated tsRNAs in blue; (D) scatter plot of tsRNA expression, with the horizontal and vertical axes representing the normalized expression levels of the CON and ACM groups, respectively, upregulated tsRNAs marked in red, downregulated tsRNAs marked in blue, and tsRNAs with no significant difference indicated in gray; (E) Volcano plot showing the significance (-log10 P-value) and fold change (log2FC) of differentially expressed tsRNAs; and (F) Bar chart of KEGG pathway enrichment analysis of differentially expressed tsRNA target genes.

[0037] Depend on Figure 2 It can be seen that principal component analysis (PCA) Figure 2 (A) shows clear separation of the two groups of samples in tsRNA expression profiles, with good intra-group reproducibility, indicating reliable sequencing data and systematic differences between groups. Venn diagram ( Figure 2 Figure B further illustrates the common and specific tsRNA molecules expressed in both groups, indicating that most tsRNAs show some expression differences between the two groups. Differential expression analysis shows that multiple tsRNAs are significantly upregulated or downregulated in the ACM group. Heatmap ( Figure 2 The scatter plot (C) visually illustrates the clustering of differentially expressed tsRNAs across samples, showing a clear separation of expression patterns between groups for upregulated and downregulated tsRNAs. Figure 2 The middle (D) plot further reflects the expression distribution trend of tsRNA between the two groups. Differentially expressed tsRNAs are distributed in the upregulated (red), downregulated (blue), and stable (gray) regions, showing that the tsRNA expression profile in the ACM group was significantly reshaped. Volcano plot ( Figure 2 The significance and fold change of differentially expressed tsRNAs were quantified by (E), facilitating the screening of candidate molecules for subsequent studies. Functional enrichment analysis ( Figure 2 The GO / KEGG pathway analysis of potential target genes of differentially expressed tsRNAs (F) showed that these tsRNAs may be involved in inflammation, cell signal transduction and cardiomyopathy-related pathways, providing a theoretical basis for subsequent research.

[0038] Example 3 This example demonstrates qRT-PCR validation analysis of differentially expressed tRNAs: To further verify the reliability of the sequencing results, this invention selected six differentially expressed tRNAs for qRT-PCR detection, and the detection results are as follows: Figure 3 As shown, Figure 3 In the study (A–F), the relative expression levels of six candidate tRNAs in the control group (CON) and the ACM group were detected by RT-qPCR. These included tRNA-Pro-CGG (A), tRNA-Met-CAT (B), tRNA-Glu-CTC (C), tRNA-Arg-CCT (D), tRNA-Ser-GCT (E), and tRNA-Tyr-GTA (F). Data are expressed as mean ± standard deviation (Mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns indicates no statistically significant difference.

[0039] The results showed that, compared with the CON group, the expression of tRNA-Ser-GCT, tRNA-Pro-CGG, tRNA-Met-CAT, tRNA-Tyr-GTA, and tRNA-Arg-CCT was increased in the ACM group, with tRNA-Ser-GCT showing the most significant differential expression (P<0.0001). Figure 3 (E). Furthermore, there was no statistically significant difference in tRNA-Glu-CTC expression between the two groups (P > 0.05). Figure 3 (C). Overall, most candidate tRNAs showed a significant upregulated expression trend in the ACM group, which was largely consistent with previous sequencing results, suggesting that these tRNAs may be involved in ACM-related molecular regulatory processes and may play a potential role in the occurrence and development of the disease.

[0040] Example 4 This example illustrates the expression localization and dynamic changes of tRNA-Ser-GCT in an ACM model: To investigate the expression changes and potential role of tRNA-Ser-GCT in alcoholic myocardial injury, this invention first observed the expression of tRNA-Ser-GCT in cardiac tissue using immunofluorescence co-staining. Figure 4 (A). In the control group (CON), cTNT-labeled myocardial tissue showed intact structure and weak tRNA-Ser-GCT (red) signal; while in the alcoholic cardiomyopathy model group (ACM), myocardial structure was significantly disordered, and tRNA-Ser-GCT signal was significantly enhanced and diffusely distributed. Further magnification showed that the interfibrillary spaces of the myocardium in the ACM group were enlarged, accompanied by a significant increase in red fluorescence signal. Quantitative analysis results ( Figure 4 (Figure B) showed that the relative fluorescence intensity in the ACM group was significantly higher than that in the control group (****, P < 0.0001), suggesting that tRNA-Ser-GCT was significantly upregulated in alcoholic myocardial injury. Further comparison of tRNA-Ser-GCT expression in different cell types (Figure B) Figure 4 The results showed that the expression level in cardiomyocytes was significantly higher than that in cardiomyocytes (*, P < 0.05), suggesting that it may mainly function in cardiomyocytes. Subsequently, the expression changes of related mRNAs were detected by treating cells with different concentrations of ethanol. Figure 4 The results showed that the expression of the target mRNA first increased and then decreased with increasing ethanol concentration (0–400 mmol / L), reaching a peak at 200 mmol / L, which was significantly different from the control group (****, P < 0.0001). Furthermore, time-gradient experiments were conducted under 200 mmol / L ethanol treatment conditions (…). Figure 4 The results showed that mRNA expression reached its highest level at 24 h (*, P < 0.05), and the overall trend suggests that ethanol-induced gene expression is time-dependent. In conclusion, tRNA-Ser-GCT was significantly upregulated in alcoholic myocardial injury, and its expression was regulated by ethanol concentration and treatment time, suggesting that it may play an important role in the development and progression of alcoholic cardiomyopathy.

[0041] Example 5 This embodiment validates the function of tRNA-Ser-GCT in regulating the inflammatory response of cardiomyocytes: By constructing tRNA-Ser-GCT overexpression and inhibition-related nucleic acid molecules, the expression level of tRNA-Ser-GCT in cardiomyocytes was regulated, thereby evaluating its role in alcohol-induced inflammatory responses. Specifically, this invention provides a tRNA-Ser-GCT overexpression mimic, which is a chemically synthesized double-stranded oligonucleotide used to mimic the high expression state of endogenous tRNA-Ser-GCT. Its single-stranded nucleotide sequence is shown in SEQ ID NO: 2, specifically AAGAAAGAUUGCAAGAACUGCUAAUUCAUGCUUCCAUGUUU. Simultaneously, an tRNA-Ser-GCT inhibitor is provided, which is a single-stranded oligonucleotide complementary to the tRNA-Ser-GCT sequence, used to specifically inhibit its function. Its nucleotide sequence is shown in SEQ ID NO: 1, specifically AAACAUGGAAGCAUGAAUUAGCAGUUCUUGCAAUCUUUCUU. Preferably, the present invention also includes corresponding negative control nucleic acid molecules, wherein the inhibitor negative control (inhibitor NC) is a random sequence, the nucleotide sequence of which is shown in SEQ ID NO: 3, specifically CAGUACUUUUGUGUAGUACAA, and a mimic negative control (mimic NC) is set up to exclude non-specific interference. The above nucleic acid molecules are transfected into H9c2 cardiomyocytes, preferably using liposome transfection reagent. Based on this, an in vitro alcoholic cardiomyocyte injury model is established by ethanol (ETOH) stimulation. After transfection, the expression level of tRNA-Ser-GCT is first detected by RT-qPCR to verify the transfection efficiency. Further, the regulatory effects on cellular inflammation levels and cytokine secretion under alcohol-induced pathological conditions are evaluated by Western blot, real-time quantitative PCR (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining. To verify transfection efficiency, the expression level of tRNA-Ser-GCT was detected. The results are as follows: Figure 5 As shown, Figure 5The results are as follows: (A) Validation of tRNA-Ser-GCT expression after mimic transfection. (B) qRT-PCR detection of mRNA expression levels of TNF-α, IL-6, and IL-10 after mimic and ETOH treatment. (C) Validation of tRNA-Ser-GCT expression after inhibitor transfection. (D) qRT-PCR detection of mRNA expression levels of TNF-α, IL-6, and IL-10 after inhibitor and ETOH treatment. (E) ELISA detection of secretion levels of IL-6, TNF-α, and IL-10 after mimic and ETOH treatment. (F) ELISA detection of secretion levels of IL-6, TNF-α, and IL-10 after inhibitor and ETOH treatment. Data are expressed as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0042] The results showed that, compared with the Control group and the mimic-NC group, the expression of tRNA-Ser-GCT was significantly increased in the mimic group (P < 0.001). Figure 5 In the inhibitor group, expression was significantly decreased (P < 0.001). Figure 5 The presence of C indicates good transfection efficacy. Regarding the expression levels of inflammatory cytokines mRNA, ETOH stimulation significantly upregulated TNF-α and IL-6 expression while decreasing the expression of the anti-inflammatory cytokine IL-10. Further analysis revealed that under ETOH conditions, tRNA-Ser-GCT overexpression (mimic+ETOH group) further enhanced the expression levels of TNF-α and IL-6 (P < 0.01 or P < 0.001) and significantly inhibited IL-10 expression (P < 0.001). Figure 5 (Middle B). Conversely, inhibition of tRNA-Ser-GCT (inhibitor+ETOH group) significantly reduced TNF-α and IL-6 expression levels and partially restored IL-10 expression ( Figure 5 (D). To further verify its regulatory role in the inflammatory response, ELISA was used to detect the secretion levels of inflammatory factors. The results showed that compared with the mimic-NC+ETOH group, the secretion of IL-6 and TNF-α was significantly increased in the mimic+ETOH group, while the secretion of IL-10 was significantly decreased. Figure 5 Conversely, in the inhibition experiment, the levels of IL-6 and TNF-α secretion were significantly decreased and the level of IL-10 secretion was increased in the inhibitor+ETOH group. Figure 5(F). In summary, overexpression of tRNA-Ser-GCT exacerbates alcohol-induced inflammatory responses, while inhibiting its expression alleviates inflammatory damage, suggesting that tRNA-Ser-GCT plays a pro-inflammatory role in the regulation of ACM inflammation. This result is consistent with the aforementioned finding of its significant upregulation in ACM models, further supporting its potential role as a key regulatory molecule in ACM.

[0043] Example 6 This example demonstrates the screening and validation of tRNA-Ser-GCT targeting Igf1: To further elucidate the molecular mechanism of action of tRNA-Ser-GCT in alcoholic cardiomyopathy (ACM), based on the aforementioned results confirming that tRNA-Ser-GCT is significantly upregulated in myocardial tissue in the ACM model and can regulate the expression and secretion of inflammatory factors, we focused on screening its potential target genes and performing functional verification.

[0044] First, four bioinformatics databases—miRanda, RNAhybrid, TargetScan, and RNA22—were used to predict and analyze the potential target genes of tRNA-Ser-GCT. The results are as follows: Figure 6 As shown, Figure 6 In the diagram, (A) Venn diagram analysis of potential target genes for tRNA-Ser-GCT. (B) Enrichment analysis of the KEGG pathway for co-predicted target genes and screening results of genes crossing the AMPK, MAPK, PI3K-AKT, and Wnt signaling pathways. (C) RNA pull-down assay to detect the enrichment of candidate gene mRNA in the Bio-tRNA-Ser-GCT group. (D) Expression level of candidate gene mRNA after tRNA-Ser-GCT mimicry staining. (E) Expression level of candidate gene mRNA after tRNA-Ser-GCT inhibitor transfection. (F) Dual-luciferase reporter gene assay to verify the direct binding relationship between tRNA-Ser-GCT and Igf1 3′UTR. (G) Sequence pairing and secondary structure prediction diagram of the binding site between Igf1 3′UTR and tRNA-Ser-GCT. (H) Note: ns indicates no statistically significant difference; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Data are expressed as follows: ±s represents.

[0045] The Venn plot results showed that the four algorithms collectively identified 1084 commonly predicted target genes. Figure 6(A) This provides a candidate gene set for subsequent functional enrichment analysis. KEGG pathway enrichment analysis was then performed on the aforementioned common target genes. The results showed that the candidate target genes were significantly enriched in the AMPK signaling pathway, MAPK signaling pathway, PI3K-Akt signaling pathway, and Wnt signaling pathway. Figure 6 (B) Further analysis of the intersection genes of the four signaling pathways identified six core genes: Creb1, Csf1, Pik3cb, Prkaa1, Mapk1, and Igf1. Among them, Igf1, as an important factor regulating cardiomyocyte growth, metabolism, and inflammatory response, has a clear protective effect in cardiovascular diseases and was therefore selected as a key research target. To verify the binding relationship between tRNA-Ser-GCT and the above candidate genes, an RNA pull-down experiment was first performed. The results showed that, compared with the NC group, the Bio-tRNA-Ser-GCT group showed significantly enriched expression of Igf1, Prkaa1, and Mapk1 mRNA ( Figure 6 The presence of C in the middle suggests that it may directly bind to target genes. Further gain-of-function experiments were conducted to verify its regulatory relationship. After transfection with tRNA-Ser-GCTmimic, the mRNA expression levels of Igf1, Pik3cb, Prkaa1, and Mapk1 significantly decreased ( Figure 6 (D); while after transfection with tRNA-Ser-GCT inhibitor, the expression of Igf1, Prkaa1 and Mapk1 was significantly upregulated ( Figure 6 (E). The above results indicate that tRNA-Ser-GCT has a negative regulatory effect on genes such as Igf1. To further clarify whether Igf1 is a direct target gene of tRNA-Ser-GCT, a dual-luciferase reporter vector containing Igf1 3′UTR wild-type (WT) and mutant (Mut) binding sites was constructed. The results showed that tRNA-Ser-GCT mimic significantly reduced the activity of the WT-Igf1 3′UTR reporter gene, while having no significant effect on the Mut vector (E). Figure 6 The results suggest that tRNA-Ser-GCT can inhibit Igf1 expression by specifically binding to the 3′UTR. Secondary structure prediction at the binding site shows a stable base pairing relationship between the two, with a minimum free energy of −30.6 kcal / mol. Figure 6 The presence of tRNA-Ser-GCT further supports its direct targeting relationship. Overexpression of tRNA-Ser-GCT in H9c2 cells led to a decrease in Igf1 expression (…). Figure 6(H). In summary, tRNA-Ser-GCT can inhibit Igf1 expression by directly binding to the 3′UTR and may participate in cardiomyocyte inflammatory responses and the development of alcoholic cardiomyopathy by affecting signaling pathways such as AMPK, MAPK, and PI3K-Akt. These results provide important experimental evidence for elucidating the molecular mechanism of tRNA-Ser-GCT in ACM.

[0046] Example 7 This embodiment studies the therapeutic effect of tRNA-Ser-GCT on an ACM model: Building upon the aforementioned results confirming that tRNA-Ser-GCT is significantly upregulated in the ACM model and participates in inflammatory regulation, this invention further evaluates the effect of inhibiting tRNA-Ser-GCT on alcoholic myocardial injury through in vivo intervention experiments. Eight-week-old male C57BL / 6J mice were randomly divided into a Control group, an ETOH group, an AAV-sh-NC+ETOH group, and an AAV-sh-tRNA-Ser-GCT+ETOH group. The ETOH group and the intervention group were fed a modified Lieber-DeCarli alcoholic liquid diet to establish an alcoholic cardiomyopathy model, while the control group was given an isocaloric maltodextrin control liquid diet. Modeling was performed according to a stepwise alcohol acclimatization feeding protocol. Previous results have shown that long-term ETOH treatment can significantly reduce EF and FS, increase LVIDd and LVIDs, and lead to ventricular wall thinning, successfully establishing an ACM model. Based on this, in vivo intervention was performed via tail vein injection of adeno-associated virus (AAV) vector. The AAV-sh-tRNA-Ser-GCT+ETOH group received an AAV vector carrying a tRNA-Ser-GCT interference sequence, the AAV-sh-NC+ETOH group received a negative control vector, and the ETOH group received no targeted intervention. ETOH treatment continued after intervention. At the experimental endpoint, M-mode echocardiography was used to assess cardiac function in each group, obtaining short-axis images of the left ventricle, and measuring parameters such as EF, FS, LVIDd, LVIDs, LVPWd, and LVPWs to evaluate the effect of tRNA-Ser-GCT inhibition on improving central dysfunction and ventricular remodeling in the ACM model. Results are as follows: Figure 7 As shown, Figure 7(A) Representative M-mode echocardiographic images of mice in each group, including the control group, ethanol-treated group (ETOH), AAV-sh-NC+ETOH group, and AAV-sh-tRNA-Ser-GCT+ETOH group. (B) Statistical analysis of left ventricular ejection fraction (EF) and fractional shortening (FS). (C) Analysis of left ventricular structural parameters, including end-diastolic diameter (LVIDd), end-systolic diameter (LVIDs), diastolic posterior wall thickness (LVPWd), and systolic posterior wall thickness (LVPWs). Data are expressed as mean ± standard deviation (mean ± SD). ns indicates no statistically significant difference; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0047] Depend on Figure 7 It can be seen that M-mode echocardiography can be used to observe the cardiac contractile function of mice in each group. Figure 7 (A). Compared with the control group, the ETOH group showed significantly weakened left ventricular contractile activity, indicating impaired cardiac function; no significant improvement was observed in the AAV-sh-NC+ETOH group, while the AAV-sh-tRNA-Ser-GCT+ETOH group showed significantly enhanced ventricular wall motion amplitude, suggesting some recovery of cardiac function. Further quantitative analysis showed ( Figure 7 In the middle B group, ETOH treatment significantly reduced left ventricular ejection fraction (EF) and fractional shortening (FS), indicating significant impairment of myocardial contractile function. Compared with the ETOH group, AAV-sh-tRNA-Ser-GCT intervention significantly improved EF and FS levels, while no significant improvement was observed in the AAV-sh-NC group, suggesting that inhibiting tRNA-Ser-GCT can effectively improve alcohol-induced cardiac dysfunction. Regarding ventricular structure ( Figure 7 In the ETOH group, both the left ventricular end-diastolic diameter (LVIDd) and end-systolic diameter (LVIDs) were significantly increased (P<0.05 or higher), indicating ventricular dilation and remodeling. Simultaneously, the left ventricular posterior wall thickness (LVPWd, LVPWs) was significantly decreased, indicating myocardial thinning. AAV-sh-tRNA-Ser-GCT intervention significantly reduced LVIDd and LVIDs, and increased LVPWd and LVPWs, demonstrating its significant ameliorative effect on alcohol-induced ventricular dilation and myocardial structural remodeling. However, there were no significant differences in any indicators between the AAV-sh-NC group and the ETOH group.

[0048] In summary, in vivo inhibition of tRNA-Ser-GCT significantly improved cardiac systolic function in mice with alcoholic cardiomyopathy and partially reversed ventricular structural remodeling, further supporting the key role of tRNA-Ser-GCT in the development of ACM. This result is consistent with its previously mentioned roles in inflammation regulation and Igf1 signaling, suggesting that it may participate in myocardial injury through the "tRNA-Ser-GCT / Igf1 / inflammation" axis.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. Application of tRNA-Ser-GCT in the preparation of products for treating alcoholic cardiomyopathy.

2. The application according to claim 1, characterized in that, Application of tRNA-Ser-GCT inhibitors in the preparation of products for the treatment of alcoholic cardiomyopathy.

3. The application according to claim 2, characterized in that, The tRNA-Ser-GCT inhibitor includes tRNA-Ser-GCT inhibitor, the nucleotide sequence of which is shown in SEQ ID NO:

1.

4. A drug for treating alcoholic cardiomyopathy, characterized in that, The drug comprises a therapeutically effective amount of a tRNA-Ser-GCT inhibitor and pharmaceutically acceptable excipients.

5. Application of tRNA-Ser-GCT in the preparation of products for treating myocarditis.

6. The application according to claim 5, characterized in that, Application of tRNA-Ser-GCT inhibitors in the preparation of products for the treatment of myocarditis.

7. The application according to claim 6, characterized in that, The tRNA-Ser-GCT inhibitor includes tRNA-Ser-GCT inhibitor, the nucleotide sequence of which is shown in SEQ ID NO:

1.

8. A drug for treating myocarditis, characterized in that, The drug comprises a therapeutically effective amount of a tRNA-Ser-GCT inhibitor and pharmaceutically acceptable excipients.

9. Application of tRNA-Ser-GCT in the preparation of products for diagnosing alcoholic cardiomyopathy.

10. The application according to claim 9, characterized in that, Application of reagents for detecting tRNA-Ser-GCT in the preparation of products for diagnosing alcoholic cardiomyopathy.

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