Use of let-7a-5p in human umbilical cord mesenchymal stem cell exosomes

Abstract

The application discloses use of let-7a-5p in human umbilical cord mesenchymal stem cell exosomes, and relates to the technical field of biological medicines, and has the technical points that the let-7a-5p in the human umbilical cord mesenchymal stem cell exosomes is applied to preparation of a medicine for treating viral myocarditis; the let-7a-5p in the exosomes inhibits CVB3-induced myocardial cell ferroptosis through mediation of SMAD2 to promote a ZFP36 expression path, so that viral myocarditis is treated. The application proves how the let-7a-5p in the human umbilical cord mesenchymal stem cell exosomes relieves the process of CVB3-induced viral myocarditis, and provides a new and effective treatment method according to the mechanism of CVB3-induced viral myocarditis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to the use of let-7a-5p in human umbilical cord mesenchymal stem cell exosomes. BACKGROUND

[0002] Viral myocarditis (VMC) is one of the most common acquired heart diseases in children and adolescents, and it is a major cause of dilated cardiomyopathy and sudden cardiac death. The main cause of VMC usually comes from multiple viral infections, leading to subsequent myocardial inflammatory destruction, including enterovirus, adenovirus, parvovirus B19, human herpesvirus 6 and other microbial infections. VMC can cause severe heart disease, such as acute heart failure, ventricular arrhythmia and cardiogenic shock. At present, VMC has been identified as a threat to the heart health of young people, leading to up to 12% of sudden cardiac death, and it is urgent to find effective intervention programs to alleviate VMC.

[0003] Iron death is a genetically encoded cell death program caused by iron-dependent lipid peroxidation. Glutathione (GSH) is the substrate of glutathione peroxidase (GPX), which is responsible for reducing the level of soluble peroxide in cells and specifically targeting acyl peroxide. Impaired GSH synthesis or inhibited GPX4 activity can induce the occurrence of cellular iron death, leading to the accumulation of lipid peroxide and causing cell death. In addition, it is reported that during the iron death process of brain injury, the level of iron is elevated and the SMAD pathway is activated. ZFP36 is a well-characterized cytoplasmic mRNA decay activator that is highly expressed during epithelial mesenchymal transition. It has been identified that ZFP36 can bind to a variety of mRNAs, including tumor necrosis factor, interleukin (IL)-6, ILI7A, IL33 and prostaglandin G / H synthase 2 (PTGS2), and can regulate the post-transcriptional activity of target genes. ZFP36 is responsible for regulating lipid peroxidation, oxidative stress, apoptosis and immune stimulation response. Our previous studies have found that coxsackievirus B3 (CVB3) can induce iron death in cardiomyocytes (CMCs), and at the same time, myocardial cell iron death is considered to be actively involved in the regulation of the death process of CMCs. However, the precise mechanism has not yet been explored.

[0004] Exosomes are single-layer membrane vesicles actively secreted by cells, containing a variety of bioactive substances such as mRNA, lncRNA and miRNA. Exosomes secreted by human umbilical cord mesenchymal stem cells (hucMSCs) have therapeutic effects on a variety of diseases, including acute liver injury, intervertebral disc degeneration (IVDD) and inflammatory bowel disease (IBD). It has been reported that exosomes from hucMSCs can alleviate viral myocarditis by activating the AMPK / mTOR-mediated autophagy flux pathway. Exosome let-7a-5p can serve as an intermediary for cell communication and a biomarker for cardiovascular disease. Studies have shown that exosome let-7 can effectively improve pulmonary fibrosis and regulate cell proliferation, apoptosis and pyroptosis through different signaling pathways. Let-7a-5p is involved in SMAD2 inhibition of tumors and regulation of cell processes.

[0005] However, the use of let-7a-5p in exosomes for viral myocarditis has not been reported. Therefore, the present application aims to provide the use of let-7a-5p in human umbilical cord mesenchymal stem cell exosomes. SUMMARY

[0006] The purpose of the present application is to solve the above problems and provide the use of let-7a-5p in human umbilical cord mesenchymal stem cell exosomes. The present application demonstrates through research how let-7a-5p in human umbilical cord mesenchymal stem cell exosomes alleviates CVB3-induced viral myocarditis, providing a new and effective treatment method from the perspective of CVB3-induced myocardial cell ferroptosis.

[0007] To achieve the above purpose, the technical scheme of the present application is as follows:

[0008] The present application provides the use of let-7a-5p in human umbilical cord mesenchymal stem cell exosomes in the preparation of a drug for treating viral myocarditis.

[0009] Further, let-7a-5p in the exosomes inhibits CVB3-induced myocardial cell ferroptosis.

[0010] Further, let-7a-5p in the exosomes inhibits CVB3-induced myocardial cell ferroptosis by mediating the SMAD2 pathway to promote ZFP36 expression.

[0011] Compared with the prior art, the present application has the following beneficial effects:

[0012] The present application overcomes the deficiencies in the prior art, applies human umbilical cord mesenchymal stem cell exosomes to the treatment of viral myocarditis, and proves through research how let-7a-5p in human umbilical cord mesenchymal stem cell exosomes alleviates the process of CVB3-induced viral myocarditis, provides a new effective treatment method from the perspective of CVB3-induced myocardial cell ferroptosis, and simultaneously, the present application can also be applied to the preparation of drugs for treating viral myocarditis. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 is myocardial tissue injury and ferroptosis development of VMC mice in the embodiment of the present application ((A) Electron microscopy studies the structure of isolated myocardium of normal mice and VMC mice (B) Echocardiogram examination of normal mice and VMC mice (C) Comparison of LVEF% and LVFS% of normal mice and VMC mice (D and E) Expression of SMAD2 and ZFP36 in VMC model (F) Immunohistochemistry of SLC7A11 in VMC model *P<0.05);

[0014] Figure 2 is that exosome let-7a-5p can effectively inhibit ferroptosis in CMCs ((A) Cell viability of CMCs in different treatment groups (B, C) TUNEL detection shows the apoptosis rate of CMCs in different groups (D) Expression of GXP4 under different treatments (E-H) Changes of GSH, MDA, ROS, Fe 2+ levels under different treatments *P<0.05);

[0015] Figure 3 is that the expression of SMAD2 is positively correlated with the development of ferroptosis in CMCs ((A) IP detection of SMAD2 and ZFP36 in CMCs (B) Cell viability of CMCs when SMAD2 is knocked out and ZFP36 is overexpressed (C) mRNA expression and protein level of SMAD2 and ZFP36 in CMCs treated by erastin (D) Changes of mitochondrial morphology and structure of CMCs under different treatments under electron microscopy (E) Expression of GXP4 under different treatment conditions (F-I) Changes of GSH, MDA, ROS, Fe 2+ levels under different treatments *P<0.05);

[0016] Figure 4Is the embodiment of the present application let-7a-5p and SMAD2 interaction, down-regulation of its expression in CMCs ((A) let-7a-5p and SMAD2 mRNA interaction prediction. (B) By dual luciferase reporter gene detection, it is proved that let-7a-5p and SMAD2 mRNA interaction. (C-D) let-7a-5p and SMAD2 level. (E) let-7a-5p and SMAD2 expression is negatively correlated. * P < 0.05);

[0017] Figure 5 Is the embodiment of the present application let-7a-5p down-regulation of SMAD2 to inhibit CMCs ferroptosis. ((A) the expression of SMAD2 and let-7a-5p under different treatments. (B, C) EdU method detects the proliferation of CMCs. (D) flow cytometry detects the apoptosis rate of CMCs under different treatments. (E) the expression of SLC7A11 and GXP4 under different treatments. (F-I) GSH, MDA, ROS, Fe 2+ level under different treatment methods. * P < 0.05);

[0018] Figure 6 Is the embodiment of the present application exosome let-7a-5p inhibits SMAD2 promoted cell ferroptosis. ((A) the change of LVEF% and LVSF% level under different treatments. (B) the change of VMC mouse SMAD2, ZFP36 and GXP4 expression under different treatments. (C, D) the expression change of SLC7A11, PTGS2, p53 under different treatment conditions. (E and F) the apoptosis rate of CMCs isolated from in vivo model is affected by SMAD2 and let-7a-5p. * P < 0.05);

[0019] Figure 7 Is the embodiment of the present application mesenchymal stem cells and exosomes ((A) the morphology of human umbilical cord mesenchymal stem cells. (B) flow cytometry identifies human umbilical cord mesenchymal stem cells by detecting biomarkers. (C) osteogenic induction differentiation of human umbilical cord mesenchymal stem cells. (D) adipogenic induction differentiation of human umbilical cord mesenchymal stem cells. (E) nanoparticle tracking analysis. (F) identification of exosomes under electron microscope. (G) WB identification of exosome surface biomarkers. (H) let-7a-5p in hucMSCs and exosomes is quantified by qRT-PCR;

[0020] Figure 8are exosomes from hucMSCs in the embodiments of the present application inhibit ferroptosis of CMCs. ((A) The proliferation of CMCs was detected at 12h, 24h, 48h, respectively. (B, C) The apoptosis rate of CMCs. (D) The expression of GPX4 protein. (E-H) The levels of GSH, MDA, ROS, Fe 2+ DETAILED DESCRIPTION

[0021] In order to enable persons skilled in the art to better understand the present application, the technical solutions of the present application will be further described in detail below in combination with the embodiments of the present application and the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by persons skilled in the art without creative labor should belong to the protection scope of the present application.

[0022] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below in combination with the embodiments.

[0023] Embodiments

[0024] The scheme provided in the embodiments of the present application is that let-7a-5p in human umbilical cord mesenchymal stem cell exosomes is applied to the preparation of a drug for treating viral myocarditis.

[0025] The exosome let-7a-5p in the present application inhibits CVB3-induced myocardial cell ferroptosis, and inhibits CVB3-induced myocardial cell ferroptosis by mediating SMAD2 to promote ZFP36 expression.

[0026] The following are specific experiments of the embodiments of the present application:

[0027] Methods and materials

[0028] Isolation and culture of human umbilical cord mesenchymal stem cells

[0029] As described previously, human umbilical cord mesenchymal stem cells are isolated from the fresh umbilical cord at the placental end with informed consent. This study has obtained ethical approval. Cells are cultured in DMEM at 37℃. After 3-5 generations, human umbilical cord mesenchymal stem cells (hucMSCs) are used for further experiments.

[0030] Flow cytometry

[0031] ​For the characterization of hucMSCs, CD44, CD45, CD90 and CD105 were identified as previous reports, cells were collected and fixed with 4% paraformaldehyde. Anti-CD44 (#12-0441-82, eBioscience, USA), anti-CD45 (#12-0459-42, eBioscience, USA), anti-CD90 (#12-0909-42, eBioscience, USA) and anti-CD105 (#12-1057-42, eBioscience, USA) were added and incubated for 30 min. Cells were detected by flow cytometry (Beckman Coulter, USA). The apoptosis rate was determined by Annexin V-APC / PI apoptosis detection kit (#KGA1030-100, KeyGEN BioTECH, China) according to the manufacturer's instructions. Annexin V-FITC and propidium iodide (PI) working solution were added for 10 min, and the apoptosis rate was analyzed by flow cytometry.

[0032] Osteogenic and adipogenic differentiation

[0033] hucMSCs were induced for osteogenic and adipogenic differentiation. Alizarin red and oil red O staining were used to evaluate the osteogenic and adipogenic differentiation potential of hucMSCs.

[0034] Exosome extraction

[0035] hucMSCs culture supernatant was collected. Exosomes were isolated using ExoQuick exosome precipitation solution (#EX0Q5A-1, SBI, USA) according to the manufacturer's instructions. Exosomes were resuspended with 100 μL of PBS. Nanoparticle tracking analysis (NTA) was used to detect the size of exosomes, and transmission electron microscopy (TEM) was used to observe the morphology of exosomes.

[0036] Animals and treatment

[0037] A mouse model of VMC in vivo was generated using coxsackievirus B3 (CVB3) (ATCC, Manassas, VA, USA). BALB / c mice were randomly divided into 7 groups, namely normal group, VMC group, husMSCs-exo group, hucMSCs-exo-inhibitor NC group, hucMSC-exo-let-7a-5p inhibitor NC group, hucMSC-exo-let-7a-5p mimic NC group, and hucMSC-exo-let-7a-5p mimic group. BALB / c mice were injected intraperitoneally with 0.1 mL of 1X103TCID50CVB3 in PBS to induce the VMC model, and PBS was injected intraperitoneally as a control (normal). The mice in the husMSCs-exo, hucMSCs-exo-inhibitor NC, hucMSC-exo-let-7a-5p inhibitor NC, hucMSC-exo mimic NC, and hucMSC-exo-let-7a-5p mimic groups were injected intraperitoneally with 0.1 mL of CVB3 in PBS, 50 μg of husMSCs-exo-exo-inhibitor NC, hucMSC-exo-let-7a-5p mimic NC, hucMSC-exo-let-7a-5p mimic NC, hucMSC-exo-mimic NC, and hucMSC-exo-let-7a-5p mimi. After 7 days, all mice were subjected to echocardiography, and the cardiac function parameters were measured. The entire operation and animal-related experiments were approved by the Animal Ethics Committee.

[0038] Transmission electron microscopy scanning

[0039] The heart and cell samples were fixed with glutaraldehyde. After being treated with different concentrations of alcohol, the samples were permeated with propylene oxide and epoxy resin. Then, the epoxy resin-embedded samples were ultrathin sectioned. After being stained with uranyl acetate and lead citrate, the samples were observed using a transmission electron microscope.

[0040] Cell experiments

[0041] Primary CMCs were isolated from neonatal BALB / c mice, and CVB3 was maintained by HeLa cells. HeLa cells and CMCs were cultured in Dulbecco's modified Eagle's medium (DMEM). The virus titer in HeLa cells was determined using the 50% tissue culture infective dose (TCID50) method. The control group used HeLa cells infected with CVB3 to culture CMCs, and the normal group did not use any treatment.

[0042] Transfection and viral infection

[0043] Inhibitor NC, let-7a-5p inhibitor, mimic NC and let-7a-5p mimic were purchased from GenePharma (China). HucMSCs were transfected with inhibitor NC, let-7a-5p inhibitor, NC mimic NC and let-7a-5p mimic with Lipofectamine 2000 for 24 h. Exosome extraction was performed after reaching 80% confluent cells. si-SMAD2, oe-SMAD2, oe-ZFP36, let-7a-5p mimic and their negative controls (NC) were purchased from GenePharma (China) and transfected into CMCs. After 24 h, cells were treated with erastin to induce ferroptosis and CVB3 infection.

[0044] Immunoblotting

[0045] Proteins were extracted from cells and exosomes using radioimmunoprecipitation (RIPA) buffer. Protein concentration was detected using BCA protein assay kit (#P0012, Beyotime, China). Proteins were separated in 10% polyacrylamide gel with sodium dodecyl sulfate (SDS-PAGE). 5% skim milk was used to block nitrocellulose filter membrane. The filter membrane was incubated with primary and secondary antibodies for 90 min at room temperature. Protein expression was evaluated with chemiluminescence imaging system (Chemiscope 6100, CLINX, China). Primary antibodies cd63 (#25682-1-AP), anti-tsg101 (#28283-1-AP), anti-SMAD2 (#12570-1-AP), anti-zfp36 (#12737-1-AP), anti-gpx4 (#14432-1-AP), anti-slc7all (#26832-1-ap), anti-p53 (#10442-1-AP), anti-ptgs2 (#12375-1-AP) and anti-gapdh (#60004-1-Ig) were purchased from proteintech (USA). Anti-calnexin (#ab22595) was purchased from abcam (UK). The secondary antibodies used were HRP goat anti-mouse IgG (#SA00001-1, proteintech, USA) and HRP goat anti-rabbit IgG (#SA00001-2, proteintech, USA). GAPDH was used as a negative control for protein content.

[0046] Real-time quantitative PCR (qRT-PCR)

[0047] RNA samples were extracted with TRIzol (#15596062, Thermo, MA, USA). RNA was reverse transcribed into cDNA using HiFi Script cDNA Synthesis Kit (#CW2569, cwbiotech, China) and miRNA cDNA Synthesis Kit (#CW2141, cwbiotech, China). PCR amplification was performed with the addition of UltraSYBR Mix (#CW2601, cwbiotech, China). The relative RNA quantity was calculated with 2 -△△Ct Methods were normalized after primers. See Supplementary Table 1.

[0048] Immunohistochemistry

[0049] Myocardial tissue slides were incubated with anti-SLC7A11 primary antibody (#26864-1-AP, Proteintech, USA) at 4°C overnight, and then with HRP-conjugated secondary antibody at 37°C. The next 30 min. The sections were developed with DAB working solution and stained with hematoxylin for microscopic observation.

[0050] CCK8 assay

[0051] CCK8 assay was performed using CCK8 kit (#NU679, DOJINDO, Japan). According to the manufacturer's instructions, 30 μL of CCK8 solution was added to each well. Cells were incubated at 37°C and 5% CO2 for 4 hours, and cell viability was evaluated by absorbance at 450 nm.

[0052] TUNEL staining

[0053] Paraffin sections were deparaffinated and 50 μL of TUNEL solution was added. The conversion agent-peroxidase was added and developed with DAB. The sections were treated with hematoxylin, 95% ethanol, anhydrous ethanol and xylene. The results were analyzed under a light microscope.

[0054] Measurement of GSH, MDA and Fe 2+ levels

[0055] GSH, MDA and Fe 2+ levels were detected in trophoblast cells. GSH (#A006-2-1) and MDA (#A003-1) kits were purchased from Nanjing Jiancheng Bioengineering Institute (China). Fe 2+ kit (#ab83366) was purchased from abcam (UK).

[0056] ROS detection

[0057] ROS level was detected by ROS assay kit (#: S0033S, Beyotime Biotech, China). Cells were collected after removing growth medium, and diluted DCFH-DA solution was added to immerse cells. Cells were incubated at 37°C for 20 min, and then washed with serum-free medium for 3 times. Flow cytometry was used to detect after trypsin digestion.

[0058] IP assay

[0059] Cells were used for anti-SMAD2 (12570-1-AP, proteintech, USA), and cells were lysed with cell lysis buffer for Western and IP (#AWB0144a, abiowell, China). The supernatant was mixed with anti-SMAD2 at 4°C overnight, and then co-cultured with magnetic beads (Santa Cruz) at 4°C for 2 h. Western blot was used to detect the expression of SMAD2 and ZFP36 (#12737-1-AP, proteintech, USA).

[0060] Dual-luciferase reporter assay

[0061] pGL3-M vectors with SMAD23-untranslation region (UTR), miR-133a mimics and mimics NC of wild type (wt) or mutant (mut) sequences were purchased from Honorgene (China). These vectors were co-transfected into CMCs with miR-133a mimics or mimics NC by Lipofectamine 2000. Luciferase activity was detected after 48 h.

[0062] EdU staining

[0063] EdU staining was used to analyze cell proliferation. 50 μM EdU working solution was added to cultured cells. Cells were fixed with 4% paraformaldehyde. Fluorescence microscope was used for observation.

[0064] Statistical analysis

[0065] Statistical data analysis was performed using GraphPad Prism 8.0.2 software (GraphPad Software, San Diego, USA). Data were presented as mean ± standard deviation (SD) of three independent experiments. Two-tailed Student's t-test was used to evaluate the differences between groups. Pearson correlation coefficient was used to analyze the correlation between genes. P value < 0.05 was considered significant.

[0066] Results

[0067] Iron death of CMCs was found and confirmed in CVB3-induced VMC mice.

[0068] We established a CVB3-induced in vivo VMC mouse model. The pathological damage of myocardial tissue of VMC model was detected (e.g. Figure 1 in FIG. 2A). Compared with normal myocardial tissue, the myocardial tissue of VMC model showed damaged structures under electron microscopy. Further echocardiography showed that the cardiac structure of VMC mice was damaged (e.g. Figure 1 in FIG. 2B). In addition, the modeling group showed left ventricular ejection fraction (LVEF) attenuation and left partial ventricular shortening (LVFS) (e.g. Figure 1 in FIG. 2C).

[0069] Both parameters were significantly reduced compared with the normal group. The LVEF of the VMC group was about half of the Normal group, while the LVFS was even lower, only 40% of the Normal group. The mRNA expression of SMAD2 and ZFP36 was quantified by qRT-PCR (e.g. Figure 1 in FIG. 2D). Due to ferroptosis, the expression of SMAD2 in the VMC group was more active than the control group, while the expression of ZFP36 was greatly inhibited. In addition, the relative expression of the two proteins in VMC mice showed a similar trend to the mRNA expression (e.g. Figure 1 in FIG. 2E). SMAD2 maintained a high level, while ZFP36 was inhibited at a low level. In VMC mice, according to the immunohistochemical results, the expression of SLC7A11 in myocardial tissue was weakened, indicating the presence of persistent ferroptosis (e.g. Figure 1 in FIG. 2F). Therefore, based on these data, the ferroptosis of CMCs in VMC mice can be induced by CVB3 with high expression of SMAD2 but low expression of ZFP36.

[0070] Exosome let-7a-5p can inhibit CVB3-induced ferroptosis of CMCs

[0071] The marker proteins (CD44, CD45, CD90 and CD105) of hucMSCs were separated and identified by flow cytometry (e.g. Figure 7 in FIG. 3A and Figure 7 in FIG. 3B). The osteogenic and adipogenic differentiation potential of hucMSCs was evaluated by alizarin red and oil red O staining (e.g. Figure 7 in FIG. 3C and Figure 7 in FIG. 3D). Next, exosomes were isolated from the culture supernatant of hucMSCs. Then, the size and concentration of exosomes were detected by nanoparticle tracking analysis (NTA), and the morphology of exosomes was observed by transmission electron microscopy (TEM) (e.g. Figure 7 in FIG. 3E and Figure 7 in FIG. 3F), while the markers of exosomes and hucMSCs (Calnexin, CD63 and TSG101) were detected by western blotFigure 7 G). Then, an in vitro model was established by infecting CMCs with CVB3, and the model group was treated with hucMSCs-exo. Compared with the control group, the proliferation activity of CMCs in the hucMSCs-exo group increased ( Figure 8 A), the apoptosis rate decreased ( Figure 8 B and Figure 8 C), the levels of GPX4, GSH and SLC7A11 in the hucMSCs-exo group were higher than those in the control group, while the levels of MDA, ROS, free ions (Fe 2+ ), p53 and PTGS2 decreased ( Figure 8 D- Figure 8 DI) in the hucMSCs-exo group. Since exosomes have low immunogenicity and no self-replication, they are qualified transporters that can be used to ensure intercellular communication. It was found that the expression of let-7a-5p was enriched in hucMSC-exosomes (hucMSC-exo) ( Figure 7 H).

[0072] In addition, in order to detect whether the transferred let-7a-5p in hucMSCs-exo plays an important role in the progression, the model cells were treated with hucMSCs-exo or hucMSCs-exo+let-7a-5p mimic, respectively. The inventors quantified the cell proliferation of CMCs under different treatments ( Figure 2 A). When infected with the virus, the proliferation of CMCs decreased, and this trend was reversed by hucMSCs-exo, and the cell proliferation of the hucMSCs-exo+let-7a-5p mimic group was higher than that of the hucMSCs-exo+mock NC group. At the same time, the TUNEL experiment was operated, and the apoptosis rate of CMCs in the VMC model increased ( Figure 2 B and Figure 2 C), and it could be seen that let-7a-5p mimic enabled CMCs to inhibit cell death caused by CVB3. In addition, let-7a-5p could balance the redox changes induced by CVB3 ( Figure 2 D- Figure 2 F). The levels of GPX4 and GSH in the control group were lower than those in the normal group, but the levels of GPX4 and GSH in the let-7a-5p mimic group were restored ( Figure 2 D and Figure 2 E). The restoration amounts of GPX4 and GSH levels were about 40% and 30%, respectively. MDA increased after infection, and let-7a-5p mimic could reduce the level of MDA ( Figure 2 F). At the same time, the level of ROS in the VMC model increased ( Figure 2G). In contrast, when let-7a-5p mimic was applied, ROS levels decreased. Fe 2+ accumulation in VMCs, while let-7a-5p mimic significantly alleviated the increase of ion accumulation in VMCs Figure 2 H). Therefore, in the in vitro model, virus induced a series of ferroptosis phenomena in VMCs, while let-7a-5p mimic could inhibit CVB3-induced ferroptosis.

[0073] Inhibition of SMAD2 expression up-regulated ZFP36 levels and inhibited ferroptosis in CMCs

[0074] To better understand the role of SMAD2 and ZFP36 in ferroptosis of CMCs, the inventors first characterized the interaction between SMAD2 and ZFP36 by immune-precipitation (IP) Figure 3 A). In the VMC model, we induced ferroptosis in CMCs using Erastin. si-SMAD2 or oe-ZFP36 was transfected into CMCs and the transfection efficiency was determined Figure 3 C). After silencing SMAD2, cell proliferation was significantly promoted Figure 3 B). Overexpression of ZFP36 further increased the proliferative activity of CMCs. In addition, changes in mitochondria, an indicator of ferroptosis, were observed by TEM Figure 3 D). In CMCs that developed ferroptosis, mitochondria volume was reduced and the number of mitochondrial cristae was reduced. In contrast, siSMAD2 showed inhibitory effect on ferroptosis, thus promoting the remodeling of normal morphology of mitochondria. In addition, the enrichment of GPX4 and the expression of GSH in CMCs were also regulated by SMAD2 and ZFP36 Figure 3 E and Figure 3 F). Down-regulation of SMAD2 and overexpression of ZFP36 increased their expression levels. MDA, ROS, Fe 2+ levels in SMAD2-inhibited CMCs, Erastin+si-SMAD2+oe-NC group was lower than Erastin+siNC+oe-NC group Figure 3 G- Figure 3 I). Overexpression of ZFP36 also helped to reduce the levels of these parameters. In summary, si-SMAD2 or oe-sfp36 can restore erastin-induced ferroptosis, indicating that these two proteins are key regulators of ferroptosis.

[0075] The target of let-7a-5p is the E3 ubiquitin ligase SMAD2

[0076] SMAD2 is a key regulator of the E3 ubiquitination pathway, and according to the data of the present inventors, SMAD2 expression in normal CMCs is relatively low. Therefore, the relationship between let-7a-5p and SMAD2 was characterized. First, the interaction between let-7a-5p and the 3’utr sequence of SMAD2 mRNA was predicted (A in FIG. 10). Then, the interaction between let-7a-5p and SMAD2 was determined by dual luciferase reporter gene assay (B in FIG. 10). When mRNA expression was detected in the VMC model, the mRNA expression of SMAD2 was still significantly down-regulated by let-7a-5p overexpression (C in FIG. 10). Similarly, the protein level of SMAD2 was also lower in the let-7a-5p mimic group than in the mock NC (D in FIG. 10). From the above data, it can be seen that let-7a-5p is negatively correlated with SMAD2 expression by establishing the relationship between let-7a-5p and SMAD2 expression (E in FIG. 10). Therefore, let-7a-5p interacts with SMAD2 to down-regulate its expression in CMCs. Figure 4 Figure 4 Figure 4 Figure 4 Figure 4

[0077] let-7a-5p can down-regulate SMAD2 in vitro to inhibit ferroptosis of CMCs

[0078] To further elucidate the functional association of let-7a-5p with SMAD2 and its regulatory effect on ferroptosis of CMCs, the expression of let-7a-5p and SMAD2 in an in vitro model was quantified preferentially, and let-7a-5p mimic or oe-SMAD2 treatment was used (A in FIG. 11). Consistent with the in vivo situation, let-7a-Sp expression was down-regulated and SMAD2 expression was up-regulated in the Erastin group in the in vitro experiment. Through the EdU experiment, we captured the changes in the proliferation of CMCs cells in different groups (B in FIG. 11). Compared with the control group, let-7a-5p mimic can promote the proliferation of CMCs. Interestingly, even though SMAD2 was overexpressed, let-7a-5p was proven to have a role in promoting the proliferation of CMCs (C in FIG. 11). The results of flow cytometry more clearly reflect the inhibitory effect of let-7a-5p on ferroptosis in CMCs overexpressing SMAD2 (D in FIG. 11). Figure 5 Figure 5 Figure 5 Figure 5 ​​​​​​​​D) in FIG. 1. Once let-7a-5p was used, cell death was significantly inhibited even with overexpression of SMAD2. In line with this, GPX4 and SLC7A11 were expressed higher in the Erastin + oe-NC + let-7a-5p mimic group than in the Erastin + oe-NC + mimic NC, and lower in the Erastin + oe-SMAD2 + let-7a-5p mimic group than in the Erastin + oe-NC + let-7a-5p mimic group Figure 5 E) in FIG. 1. The expression of GSH was increased in the treatment of le-7a-5p mimic compared to mimic NC Figure 5 F) in FIG. 1. The present inventors also compared the changes in MDA, ROS, and Fe 2+ content in SMAD2 overexpressing CMCs in the presence or absence of let-7a-5p Figure 5 G- in FIG. 1 Figure 5 D) in FIG. 1. It is clear that the use of let-7a-5p reduced the number of these indicators, proving that this miRNA is effective in inhibiting SMAD2-promoted ferroptosis. Based on these findings, let-7a-5p can inhibit ferroptosis, at least partially, by directly downregulating SMAD2.

[0079] Let-7a-5p in hucMSCs exosomes exhibits therapeutic effects on cardiomyocyte ferroptosis in vivo

[0080] The inhibition of ferroptosis by let-7a-5p modulated our motivation to continue the study in vivo. hucMSCs-exo, hucMSCs-exo-let-7a-5p inhibitor, and hucMSCs-exo-let-7a-5p mimic were injected into VMC mice, respectively. To examine the recovery of function in the VMC model, we measured the LVEF and LVFS of the different treated mice Figure 6 A) in FIG. 2. Both of these parameters were extremely low in the VMC group, suggesting a defect in cardiac function. When treated with hucMSCs-exo, both LVEF and LVFS were significantly increased, which is a sign of recovery of cardiac function. Next, we analyzed the expression of SMAD2 and ZFP36 in the model mice with or without let-7a-5p mimic Figure 6VMC mice exhibited high expression of SMAD2 and low expression of ZFP36, suggesting active ferroptosis. However, the internalization of let-7a-5p favored the expression of ZFP36 and inhibited the expression of SMAD2 in VMC mice. In the let-7a-5p mimic treatment group, the ZFP36 protein level was also up-regulated, while the SMAD2 protein level was down-regulated. Consistent with the results of in vitro experiments, GPX4 was mainly down-regulated in VMC mice, but hucMSCs-exo-let-7a-5p mimic could induce the recovery of GPX4 Figure 6 VMC mice exhibited high expression of SMAD2 and low expression of ZFP36, suggesting active ferroptosis. However, the internalization of let-7a-5p favored the expression of ZFP36 and inhibited the expression of SMAD2 in VMC mice. In the let-7a-5p mimic treatment group, the ZFP36 protein level was also up-regulated, while the SMAD2 protein level was down-regulated. Consistent with the results of in vitro experiments, GPX4 was mainly down-regulated in VMC mice, but hucMSCs-exo-let-7a-5p mimic could induce the recovery of GPX4 Figure 6 VMC mice exhibited high expression of SMAD2 and low expression of ZFP36, suggesting active ferroptosis. However, the internalization of let-7a-5p favored the expression of ZFP36 and inhibited the expression of SMAD2 in VMC mice. In the let-7a-5p mimic treatment group, the ZFP36 protein level was also up-regulated, while the SMAD2 protein level was down-regulated. Consistent with the results of in vitro experiments, GPX4 was mainly down-regulated in VMC mice, but hucMSCs-exo-let-7a-5p mimic could induce the recovery of GPX4 Figure 6 VMC mice exhibited high expression of SMAD2 and low expression of ZFP36, suggesting active ferroptosis. However, the internalization of let-7a-5p favored the expression of ZFP36 and inhibited the expression of SMAD2 in VMC mice. In the let-7a-5p mimic treatment group, the ZFP36 protein level was also up-regulated, while the SMAD2 protein level was down-regulated. Consistent with the results of in vitro experiments, GPX4 was mainly down-regulated in VMC mice, but hucMSCs-exo-let-7a-5p mimic could induce the recovery of GPX4 Figure 6 VMC mice exhibited high expression of SMAD2 and low expression of ZFP36, suggesting active ferroptosis. However, the internalization of let-7a-5p favored the expression of ZFP36 and inhibited the expression of SMAD2 in VMC mice. In the let-7a-5p mimic treatment group, the ZFP36 protein level was also up-regulated, while the SMAD2 protein level was down-regulated. Consistent with the results of in vitro experiments, GPX4 was mainly down-regulated in VMC mice, but hucMSCs-exo-let-7a-5p mimic could induce the recovery of GPX4 Figure 6 VMC mice exhibited high expression of SMAD2 and low expression of ZFP36, suggesting active ferroptosis. However, the internalization of let-7a-5p favored the expression of ZFP36 and inhibited the expression of SMAD2 in VMC mice. In the let-7a-5p mimic treatment group, the ZFP36 protein level was also up-regulated, while the SMAD2 protein level was down-regulated. Consistent with the results of in vitro experiments, GPX4 was mainly down-regulated in VMC mice, but hucMSCs-exo-let-7a-5p mimic could induce the recovery of GPX4

[0081] Through the above experiments, the interaction between SMAD2 and ZFP36 was determined by IP. Inhibition of SMAD2 can up-regulate the expression of ZFP36. Similarly, overexpression of ZFP36 can reduce the expression level of SMAD2. At the same time, silencing SMAD2 and overexpressing ZFP36 further inhibited the ferroptosis of VMC cells. Through the prediction and characterization of the interaction between SMAD2 and let-7a-5p, we further confirmed the targeting relationship between them. let-7a-5p inhibits the expression of SMAD2 by targeting SMAD2 mRNA. This upstream inhibition ultimately leads to the inhibition of the ferroptosis process, which is manifested as the change in the level of biomarkers. In the established VMC mouse model, the effect of SMAD2 overexpression on the development of ferroptosis is largely reversed by the internalization of let-7a-5p. Therefore, let-7a-5p enriched in exosomes can alleviate CVB3-induced cellular ferroptosis by regulating the SMAD2 signaling pathway.

[0082] In conclusion, let-7a-5p in exosomes from hucMSCs can alleviate CVB3-induced ferroptosis of cardiomyocytes by regulating the SMAD2 signaling pathway. The determination of the effectiveness of let-7a-5p in exosomes opens a door for the proper design and development of treatment methods for VMC and expands the research on the treatment of VMC.

[0083] Therefore, through the above embodiments of the present application, the present application overcomes the deficiencies in the prior art, applies the exosomes let-7a-5p of human umbilical cord mesenchymal stem cells to the treatment of viral myocarditis, and proves through research how the exosomes let-7a-5p of human umbilical cord mesenchymal stem cells alleviate the process of CVB3-induced viral myocarditis, provides a new and effective treatment method from the perspective of CVB3-induced ferroptosis of cardiomyocytes, and at the same time, the present application can also be applied to the preparation of drugs for treating viral myocarditis.

[0084] The above specific embodiments are only an explanation of the present application, and are not a limitation of the present application, and those skilled in the art can make modifications to the present embodiments without creative contribution after reading the present specification, but as long as the present application is within the scope of the claims, it is protected by the patent law.

Claims

1. Use of let-7a-5p in human umbilical cord mesenchymal stem cell exosomes in the preparation of a drug for treating viral myocarditis induced by CVB3.

2. Use according to claim 1, characterized in that: The let-7a-5p in the exosomes inhibits CVB3-induced myocardial cell ferroptosis.

3. Use according to claim 2, characterized in that: The let-7a-5p in the exosomes inhibits CVB3-induced myocardial cell ferroptosis by mediating the SMAD2 to promote the ZFP36 expression pathway.

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