Pharmaceutical composition for treating myocardial dysfunction caused by sepsis and application thereof

Abstract

The invention discloses a pharmaceutical composition for treating myocardial dysfunction (SICM) caused by sepsis. The pharmaceutical composition comprises a component capable of inhibiting expression or activity of ADAM8 in macrophages. The component capable of inhibiting the expression or the activity of the ADAM8 in the macrophage is a lentivirus transfection plasmid aiming at the ADAM8. A series of experiments prove that expression up-regulation of ADAM8 in macrophages is associated with SICM, CKO (Conditional Knockout) of macrophage specificity ADAM8 can improve cardiac dysfunction after SICM, ADAM8 hinders intercellular effect of cardiac macrophages after SICM, and sMer in-situ cardiac injection can aggravate cardiac dysfunction of CKO mice, so that the CKO mice can be prevented from being infected with the CKO mice. Important theoretical basis and potential targets are provided for diagnosis and treatment of related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to a pharmaceutical composition for treating myocardial dysfunction caused by sepsis and its application. Background Technology

[0002] Sepsis-associated cardiomyopathy (SICM) is a serious complication of sepsis, characterized by acute cardiac dysfunction and high mortality. Currently, treatment options for SICM are limited and primarily focused on supportive care, lacking effective interventions targeting its pathogenesis. The ADAM family (integrin-metalloproteinases) plays a crucial role in cardiovascular and inflammatory diseases. In inflammatory diseases, macrophages are one of the main cell types functioning within the ADAM family. By regulating the shedding of macrophage surface molecules, ADAM proteases affect macrophage activation, migration capacity, and interactions with other cells, thereby modulating the degree and duration of the inflammatory response. Studies have shown that ADAM8, as a metalloproteinase, plays a significant role in inflammation and cardiovascular disease. It can cleave and detach various cell surface molecules associated with vascular function, thrombosis, and inflammatory responses, thus affecting multiple aspects such as vascular permeability, leukocyte migration, and thrombosis, thereby regulating disease progression. However, its specific role in the pathogenesis of SICM remains unclear, and related molecularly targeted drugs have not yet been effectively developed. Summary of the Invention

[0003] The purpose of this invention is to provide a pharmaceutical composition for treating myocardial dysfunction caused by sepsis and its application therein, which aims to reduce cardiac inflammatory response, reduce myocardial cell apoptosis, and improve cardiac function.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] In a first aspect, the present invention provides a pharmaceutical composition for treating myocardial dysfunction caused by sepsis.

[0006] A pharmaceutical composition for treating myocardial dysfunction caused by sepsis, comprising an ingredient capable of inhibiting the expression or activity of ADAM8 in macrophages.

[0007] Preferably, the component capable of inhibiting ADAM8 expression or activity in macrophages is a lentiviral transfection plasmid targeting ADAM8.

[0008] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.

[0009] Preferably, the dosage form of the pharmaceutical composition is an injection, an oral preparation, a topical preparation, or a locally administered preparation.

[0010] Secondly, the present invention provides the application of the pharmaceutical composition.

[0011] The use of the pharmaceutical composition in the preparation of a medicament for treating myocardial dysfunction caused by sepsis.

[0012] Thirdly, the present invention provides the application of a substance that inhibits the expression or activity of ADAM8 in macrophages.

[0013] Application of substances that inhibit ADAM8 expression or activity in macrophages in the preparation of drugs to improve cardiac function in septic cardiomyopathy.

[0014] Preferably, the substance is a lentiviral transfection plasmid targeting ADAM8.

[0015] Preferably, the drug can achieve at least one of the following:

[0016] Increases left ventricular ejection fraction (LVEF) and shortens left ventricular ejection fraction (FS), while decreasing left ventricular end-diastolic diameter (LVID; d) and left ventricular end-systolic diameter (LVID; s);

[0017] It reduces the levels of serum aspartate aminotransferase (AST) and lactate dehydrogenase (LDH);

[0018] It reduces the expression of the pro-apoptotic protein Bax in cardiac tissue and decreases the Bax / Bcl2 ratio;

[0019] It reduces the expression of Ccl4, Ccr7, Bmp4, Ccl22 and Ebi3 in cardiac tissue.

[0020] Fourthly, the present invention provides a kit for detecting the expression level of ADAM8 in macrophages associated with sepsis-induced myocardial dysfunction.

[0021] A kit for detecting the expression level of ADAM8 in macrophages associated with sepsis-induced myocardial dysfunction, comprising reagent components for performing one or more of the following methods: Western blotting, immunofluorescence staining, or qRT-PCR;

[0022] The kit is designed for use with isolated cardiac tissue samples or isolated macrophage samples from patients with spontaneous myocardial infarction (SICM) or animal models.

[0023] Furthermore, the kit assesses the severity of myocardial dysfunction (SICM) or the effectiveness of treatment by comparing the detected ADAM8 expression level with the ADAM8 expression level of a control reference range, which is determined by testing samples from healthy individuals.

[0024] Fifthly, the present invention provides a kit for studying the role of macrophage ADAM8 in myocardial dysfunction caused by sepsis.

[0025] A kit for studying the role of macrophage ADAM8 in sepsis-induced myocardial dysfunction, comprising:

[0026] Reagent components used to construct macrophage-specific ADAM8 conditional knockout (CKO) animal models or to perform macrophage gene knockdown;

[0027] A reagent component used to detect one or more of the following indicators: cardiac function, myocardial injury, cardiomyocyte apoptosis, expression of inflammatory factors, or survival rate.

[0028] Furthermore, correlation analysis was conducted to investigate the potential mechanism of action of ADAM8 in SICM by comparing changes in ADAM8 expression or activity levels with the detection results of the above indicators.

[0029] Unless otherwise specified, the term "ADAM8" refers to ADisintegrin and Metalloprotease 8, a metalloproteinase belonging to the ADAM family.

[0030] Beneficial effects: This invention has confirmed the association between upregulation of ADAM8 expression in macrophages and SICM through a series of experiments. Macrophage-specific conditional knockout of ADAM8 can improve cardiac dysfunction after SICM. ADAM8 inhibits the burial of cardiac macrophages after SICM. Orthotopic cardiac injection of sMer exacerbates cardiac dysfunction in CKO mice. This provides important theoretical basis and potential targets for the diagnosis and treatment of related diseases. Attached Figure Description

[0031] Figure 1 To screen macrophage ADAM8 as a key molecule that significantly increases SICM expression and to verify the effect of ADAM8 CKO on cardiac function in LPS-induced SICM mice using bioinformatics techniques;

[0032] Figure 2 RNA transcriptome sequencing analysis of heart tissues from Flox and ADAM8 CKO mice suggests that cytokine-related pathways are key phenotype changes in SICM heart after ADAM8 knockout.

[0033] Figure 3 To validate in vivo RNA sequencing results with qRT-PCR to determine changes in the expression of key inflammatory factors;

[0034] Figure 4To investigate the effects of ADAM8 CKO on cardiac function and related parameters in a cecal ligation and punch (CLP) induced SICM mouse model;

[0035] Figure 5 To conduct RNA sequencing analysis on RAW264.7 cells at the cellular level after transfection with ADAM8 knockdown (KD) plasmid using lentiviral vector, under both LPS and non-LPS conditions, and to verify through experiments that endocytosis is a key pathway for ADAM8 to regulate macrophage function, this study aimed to investigate the role of endocytosis in regulating macrophage function. Detailed Implementation

[0036] The present invention will be further explained below with reference to the embodiments.

[0037] Example

[0038] (I) Laboratory Animals

[0039] Healthy, age- and weight-matched mice of specific strains were selected to establish a sepsis-induced myocardial dysfunction (SICM) model. Two types of mouse SICM models were established using either LPS or CLP. A macrophage-specific ADAM8 conditional knockout (CKO) mouse model was also established, with Flox mice serving as a control.

[0040] (II) Cell Line

[0041] The mouse macrophage cell line RAW264.7 was used for experimental studies at the cellular level.

[0042] II. Experimental Methods and Procedures

[0043] (I) Construction of SICM mouse model and grouping treatment

[0044] 1. CLP model establishment: Cecal ligation and puncture (CLP) surgery was performed on mice to induce a SICM model. Control mice underwent sham surgery.

[0045] 2. LPS-induced model: Some mice were induced to develop SICM by intraperitoneal injection of LPS, while the control group was injected with an equal volume of physiological saline.

[0046] 3. Grouping: Mice were divided into different groups, including a control group, an ADAM8 CKO group, and a Flox group. Heart tissue and serum samples were collected at appropriate time points (e.g., day 3 after CLP surgery) for subsequent testing.

[0047] (II) Bioinformatics Analysis

[0048] 1. This invention discloses RNA transcriptome sequencing of SICM mouse heart tissue and RNA sequencing at the RAW264.7 cell level, led by the applicant, to explore the specific mechanisms and phenotypes downstream of ADAM8.

[0049] 2. Obtain relevant datasets from public databases (such as GSE190856), process and analyze the data using bioinformatics software and tools, and determine the expression changes of ADAM8 in the macrophage subsets of SICM mice.

[0050] (III) Western blotting detection

[0051] 1. Sample preparation: Collect mouse heart tissue or cell samples, add an appropriate amount of lysis buffer to lyse, and extract total protein.

[0052] 2. Protein quantification: The extracted protein was quantified using the BCA protein quantification kit.

[0053] 3. Electrophoresis and transfer: The protein samples were separated by SDS-PAGE electrophoresis, and then the proteins were transferred onto a PVDF membrane.

[0054] 4. Blocking and antibody incubation: Block the PVDF membrane with 5% skim milk powder, and then add primary antibody (such as ADAM8, Bax, Bcl2, MerTK, etc.) and secondary antibody for incubation.

[0055] 5. Development and Analysis: Chemiluminescent reagents were used for development, and the bands were analyzed for grayscale using software such as ImageJ to calculate the relative expression levels of the protein.

[0056] (iv) Enzyme-linked immunosorbent assay (ELISA)

[0057] 1. Sample collection: Collect mouse serum samples and dilute them according to the kit instructions.

[0058] 2. Sample addition and incubation: Add the diluted serum sample to the wells of the ELISA plate, along with the standard, and incubate for a certain period of time.

[0059] 3. Washing and adding enzyme-labeled antibody: Wash away unbound substances, add enzyme-labeled antibody, and incubate again.

[0060] 4. Color development and termination: Add a colorimetric reagent to develop color, and then add a stop solution to terminate the reaction.

[0061] 5. Reading and Analysis: Use an ELISA reader to measure the absorbance of each well, and calculate the concentration of each indicator in the serum (such as AST, LDH, ALT, Cr, CRP, CKMB, etc.) according to the standard curve.

[0062] (V) Cardiac Function Testing

[0063] The cardiac function of mice was assessed using an echocardiogram system, and parameters such as left ventricular ejection fraction (LVEF), fractional shortening (FS), left ventricular end-diastolic diameter (LVID; d), and left ventricular end-systolic diameter (LVID; s) were recorded.

[0064] (vi) qRT-PCR validation

[0065] 1. RNA extraction: Collect mouse heart tissue or cell samples and extract total RNA using RNA extraction reagents.

[0066] 2. Reverse transcription: RNA is reverse transcribed into cDNA using reverse transcription reagents.

[0067] 3. qRT-PCR reaction: Using cDNA as a template, specific primers and SYBR Green dye are added to perform qRT-PCR reaction.

[0068] 4. Data Analysis: The relative expression levels of genes were calculated using the 2^-ΔΔCt method, and the effects of ADAM8 CKO on the expression of related genes (such as Ccl4, Ccr7, Bmp4, Ccl22, Ebi3, etc.) were analyzed.

[0069] (vii) Kaplan-Meier Survival Analysis

[0070] The survival time of mice in each group was recorded, survival curves were plotted using the Kaplan-Meier method, and the differences in survival rates between groups were compared using the Logrank test.

[0071] (VIII) Cell Experiments

[0072] 1. Lentiviral transfection: An ADAM8 knockdown (KD) lentiviral vector was constructed and transfected into RAW264.7 cells. A negative control (NC) group was also set up. The shRNA-Adam8 knockdown lentivirus was designed with the species mouse, Accession: NM_007403, and the target sequence was ccTAGAGTCATTCGTGACAAA.

[0073] 2. Immunofluorescence staining: After a certain period of time after transfection, the cells are fixed, permeabilized, and blocked. Then, specific antibodies are added for immunofluorescence staining to observe the ADAM8 knockdown effect.

[0074] 3. Transcriptome sequencing: RAW264.7 cell samples were collected after transfection and transcriptome sequencing analysis was performed. The gene expression differences between the ADAM8 KD group and the NC group were compared to identify the pathways and genes that were significantly upregulated or downregulated.

[0075] 4. Western blotting detection of cell-related protein expression: Cell samples were collected, proteins were extracted and analyzed by Western blotting to determine the effect of ADAM8 knockdown on the expression of proteins such as MerTK. Simultaneously, cell culture supernatant was collected to detect the level of soluble Mer (sMer).

[0076] (ix) Results Analysis

[0077] 1. Upregulation of ADAM8 expression in cardiac macrophages of SICM mice

[0078] Analysis of the SICM mouse cardiac single-cell transcriptome dataset GSE190856 revealed that ADAM8 expression was significantly increased in macrophage subsets in the cecal ligation and puncture (CLP) group on postoperative day 3 (e.g., Figure 1 (As shown in Figure A). Furthermore, in a wild-type (WT) mouse CLP model, cardiac macrophages were isolated and analyzed by Western blotting. The results showed that ADAM8 expression in cardiac macrophages of the CLP group was significantly increased compared to the control group (e.g., ...). Figure 1 (As shown in B).

[0079] 2. The effect of macrophage-specific ADAM8 conditional knockout (CKO) on improving cardiac dysfunction after SICM

[0080] To investigate whether ADAM8 in macrophages affects sepsis-induced cardiac dysfunction (SICM), this invention constructed a macrophage-specific ADAM8 CKO mouse model and induced sepsis using lipopolysaccharide (LPS) and cecal ligation (CLP). Experimental results showed that, compared with the control group, the CKO group mice exhibited significantly improved left ventricular ejection fraction (LVEF) and fractional shortening (FS). Figure 1 (As shown in C). Meanwhile, the serum levels of aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in the CKO group mice were significantly lower than those in the control group, while the levels of alanine aminotransferase (ALT), creatinine (Cr), C-reactive protein (CRP), and creatine kinase isoenzyme MB (CKMB) showed no significant differences (e.g., ...). Figure 1 (As shown in D). Western blotting analysis showed that, compared with the Flox group, Bax expression in the hearts of CKO group mice was significantly reduced, leading to a significant decrease in the Bax / Bcl2 ratio (as shown in D). Figure 1 (As shown in E). Furthermore, there was no significant difference in the expression of necrosis-related molecules, and the survival rate of mice in the CKO group was improved (e.g., ...). Figure 1 (As shown in F).

[0081] 3. Macrophage-specific cardiac transcriptomic profile analysis in ADAM8 CKO mice

[0082] To gain a deeper understanding of the transcriptional profile of the heart in macrophage-knockout mice, this invention performed transcriptome sequencing on the heart tissue of LPS-induced septic mice (e.g., Figure 2 (As shown in Figure A). KEGG enrichment analysis indicated that the cytokine-cytokine receptor interaction pathway was the most significant (e.g., ...). Figure 2 (As shown in B). Gene set enrichment analysis (GSEA) showed that the KO04060 pathway was significantly downregulated in the CKO group mice (e.g., as shown in B). Figure 2 (As shown in C). Dot plots and bar heatmaps show that the expression of key inflammatory molecules associated with the KO04060 pathway, including Bmpr1b, Ccl22, Bmp5, Bmp4, IL15ra, Ccl19, IL21r, Ebi3, IL1rn, Cxcr4, and Ccl4, was significantly reduced (e.g., as shown in C). Figure 2 (As shown in D). RNA was further extracted from the hearts of both groups of mice, and these inflammatory factors were verified using qRT-PCR. The results showed that compared with the Flox group, the expression of Ccl4, CcR7, Bmp4, Ccl22, and Ebi3 in the hearts of CKO group mice was significantly reduced, while other molecules showed no significant differences (e.g., ...). Figure 3 (As shown in Figure A). Overall, the levels of inflammation and apoptosis in the hearts of mice in the CKO group were reduced.

[0083] 4. Further verification of the effects of macrophage-specific ADAM8 CKO on cardiac function in SICM mice.

[0084] To further verify the effect of ADAM8 on cardiac function in SICM mice within macrophages, this invention again established a sepsis mouse model using cecal ligation and puncture (CLP). The experimental results were consistent with previous findings; cardiac function in the KO group mice was significantly improved compared to the control group (e.g., Figure 4 (As shown in Figure A). Meanwhile, the serum AST and LDH levels in the KO group mice were significantly lower than those in the control group (as shown in Figure A). Figure 4 (As shown in Figure B). Western blotting analysis further confirmed that, compared with the Flox group, Bax expression in the hearts of CKO group mice was significantly reduced, leading to a significant decrease in the Bax / Bcl2 ratio and improved survival rate (as shown in Figure B). Figure 4 (As shown in C). Compared with the Flox group, the expression of Ccl4 and Ebi3 in the hearts of CKO group mice was significantly reduced (as shown in C). Figure 4 (As shown in D). Furthermore, the survival rate of mice in the CKO group was significantly improved (e.g., ...). Figure 4 (As shown in E).

[0085] 5. The inhibitory effect of ADAM8 on cardiac macrophage burial after SICM

[0086] Given that ADAM8 knockout in macrophages improves the survival rate of SICM mice at the in vivo level, this invention further elucidates the functional regulatory role of ADAM8 on macrophages at the cellular level. A lentivirus with ADAM8 knockdown (KD) was constructed and transfected into RAW264.7 cells (e.g.,...). Figure 5 (As shown in A). After LPS intervention, transcriptome sequencing was performed on the ADAM8 negative control (NC) group and the KD group. The results showed that the KO04145 pathway was significantly upregulated after KD (e.g., Figure 5 (As shown in Figure B). Dot plots and bar heatmaps show the expression of common phagocytic bridging molecules, indicating that during the "eat me" signaling phase of phagocytosis, the expression of phagocytic receptor molecules such as MerTK and Axl increases, while the expression of "don't eat me" signals (such as Gas6) also increases (as shown in Figure B). Figure 5 As shown in Figure C, the overall level of cell burial effect is improved.

[0087] To validate these findings, primary bone marrow-derived macrophages (BMDMs) were extracted and subjected to Western blotting analysis. The results showed that compared to the Flox group, the KO BMDM group exhibited significantly increased MerTK expression, while the level of soluble Mer (sMer) in the BMDM culture supernatant was decreased (e.g., ...). Figure 5 (As shown in D). Based on these observations, the present invention hypothesizes that ADAM8 cleaves the TAM receptor (MerTK) to generate sMer, thereby inhibiting endocytosis and promoting inflammation.

[0088] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for treating myocardial dysfunction caused by sepsis, characterized in that: It contains components that can inhibit the expression or activity of ADAM8 in macrophages.

2. The pharmaceutical composition according to claim 1, characterized in that: The component that can inhibit the expression or activity of ADAM8 in macrophages is a lentiviral transfection plasmid targeting ADAM8.

3. The pharmaceutical composition according to claim 1, characterized in that: The pharmaceutical composition also includes a pharmaceutically acceptable carrier, excipient, or diluent.

4. The pharmaceutical composition according to claim 1, characterized in that: The dosage form of the pharmaceutical composition is an injection, oral preparation, external preparation, or topical administration preparation.

5. Use of the pharmaceutical composition of claims 1-4 in the preparation of a medicament for treating myocardial dysfunction caused by sepsis.

6. Application of substances that inhibit the expression or activity of ADAM8 in macrophages in the preparation of drugs to improve cardiac function in septic cardiomyopathy.

7. The application according to claim 6, characterized in that: The substance is a lentiviral transfection plasmid targeting ADAM8.

8. The application according to claim 6 or 7, characterized in that: The drug can achieve at least one of the following: Increases and shortens the left ventricular ejection fraction, and decreases the left ventricular end-diastolic diameter and left ventricular end-systolic diameter; Reduce serum levels of aspartate aminotransferase and lactate dehydrogenase; It reduces the expression of the pro-apoptotic protein Bax in cardiac tissue and decreases the Bax / Bcl2 ratio; It reduces the expression of Ccl4, Ccr7, Bmp4, Ccl22 and Ebi3 in cardiac tissue.

9. A kit for detecting the expression level of ADAM8 in macrophages associated with sepsis-induced myocardial dysfunction, characterized in that: It contains reagent components for performing one or more of the following methods: Western blotting, immunofluorescence staining, or qRT-PCR; The kit is designed for use with: isolated cardiac tissue samples or isolated macrophage samples from patients with myocardial dysfunction or animal models. The kit assesses the severity of myocardial dysfunction or treatment efficacy by comparing the detected ADAM8 expression level with the ADAM8 expression level of a control reference range, which is determined by testing samples from healthy individuals.

10. A kit for studying the role of macrophage ADAM8 in sepsis-induced myocardial dysfunction, characterized in that: Include: Reagent components used to construct macrophage-specific ADAM8 conditional knockout animal models or to perform macrophage gene knockdown; A reagent component used to detect one or more of the following indicators: cardiac function, myocardial injury, cardiomyocyte apoptosis, expression of inflammatory factors, or survival rate.

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