Objective To investigate the role and mechanism of RNA-Specific adenosine deaminase 3 (Adar3) in regulating macrophage polarization during Coxsackievirus B3(CVB3)-induced viral myocarditis (VM). Methods Bone marrow-derived macrophages (BMDM) from mice were cultured in vitro and induced into M1/M2 macrophages using interferon-gamma (IFN-γ)/lipopolysaccharide (LPS) or interleukin 4 (IL-4), respectively. The mRNA expression levels of Adar1, Adar2, and Adar3 in each group of cells were assessed by real-time quantitative PCR (qRT-PCR). Specific siRNAs targeting the Adar3 gene were designed, synthesized, and transiently transfected into M2 macrophages. The mRNA levels of M2 polarization-related marker genes-including arginase 1 (Arg1), chitinase 3-like molecule 3 (YM1/Chi3l3), and resistin-like molecule alpha (RELMα/FIZZ1)-were detected by qRT-PCR. RNA sequencing was performed to analyze the signaling pathways affected by Adar3. The expression levels of Wnt/β-catenin signaling pathway were further validated using qRT-PCR and Western blot. The adeno-associated virus overexpressing Adar3 was designed, synthesized, and injected into mice via tail vein. Three weeks later, a myocarditis mouse model was established. After an additional week, the phenotype and function of cardiac macrophages, as well as multiple indicators of VM (including echocardiography, body weight, histopathology and serology) were examined. Additionally, the protein levels of the Wnt/β-catenin signaling pathway were assessed. Results Compared to M0-type macrophages, the expression level of Adar3 was significantly increased in M2-type macrophages. After transfection of Adar3 siRNA, the mRNA levels of Arg1, YM1 and FIZZ1 in M2 macrophages were downregulated. RNA sequencing revealed 149 upregulated genes and 349 downregulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and subsequent validation experiments indicated that Adar3 modulated the Wnt/β-catenin signaling pathway. In vivo experiments demonstrated that Adar3 overexpression alleviated the cardiac dysfunction of VM mice. The proportion of M1 macrophages in the heart decreased, while the proportion of M2 macrophages increased. At the same time, the Adar3 overexpression activated the Wnt/β-catenin signaling pathway. Conclusion Adar3 promotes macrophage polarization toward the M2 phenotype by activating the Wnt/β-catenin signaling pathway, thereby alleviating VM.
Animals ; Adenosine Deaminase/metabolism* ; Macrophages/immunology* ; Wnt Signaling Pathway/genetics* ; Myocarditis/immunology* ; Mice ; Coxsackievirus Infections/metabolism* ; Male ; Mice, Inbred BALB C ; Enterovirus B, Human/physiology* ; beta Catenin/genetics*

OBJECTIVE: To report on a child with B-cell-negative severe combined immunodeficiency (B-SCID) manifesting as fulminant myocarditis and carry out genetic testing for her. METHODS: A child with B-SCID who presented at Fujian Maternity and Child Health Care Hospital on January 31, 2021 was selected as the subject. Whole exome sequencing was carried out for her. Candidate variant was verified by Sanger sequencing. RESULTS: The female infant had developed recurrent skin and lung infections soon after birth, and was admitted due to fulminant myocarditis. Serological examination has disclosed a remarkable reduction in immunoglobulins. Flow cytometric analysis revealed that her peripheral blood T and B lymphocytes and NK cells were significantly reduced. Whole exome sequencing revealed that she has harbored a homozygous c.C3007T (p.Q1003X) nonsense variant of the RAG1 gene, for which both of her parents were heterozygous carriers. The variant has not been recorded in normal population databases. Based on the guidelines from the American College of Medical Genetics and Genomics, the variant was predicted to be pathogenic. CONCLUSION A case of RAG1 gene associated B-SCID has been diagnosed. Above finding has enriched the spectrum of RAG1 gene variants and enabled early diagnosis and intervention of the disease.
Female ; Humans ; Pregnancy ; Genetic Testing ; Homeodomain Proteins/genetics* ; Mutation ; Myocarditis/genetics* ; Phenotype ; Severe Combined Immunodeficiency/diagnosis* ; Infant

Objective To investigate the effect of viral myocarditis serum exosomal miR-320 on apoptosis of cardiomyocytes and its mechanism. Methods The model of viral myocarditis mice was established by intraperitoneal injection of Coxsackie virus B3. Serum exosomes were extracted by serum exosome extraction kit and co-cultured with cardiomyocytes. The uptake of exosomes by cardiomyocytes was detected by laser confocal microscopy. Cardiomyocytes were transfected with miR-320 inhibitor or mimic, and the expression level of miR-320 was detected by real-time quantitative PCR. Flow cytometry was used to detect cardiomyocyte apoptosis rate, and the expression levels of B cell lymphoma 2 (Bcl2) and Bcl2-related X protein (BAX) were tested by Western blot analysis. The prediction of miR-320 target genes and GO and KEGG enrichment analysis were tested by online database. The relationship between miR-320 and its target gene phosphoinositide-3-kinase regulatory subunit 1(Pik3r1) was examined by luciferase reporter gene. The effect of miR-320 on AKT/mTOR pathway protein was detected by Western blot analysis. Results Viral myocarditis serum exosomes promoted cardiomyocyte apoptosis, and increased the level of BAX while the level of Bcl2 was decreased. miR-320 was significantly up-regulated in myocardial tissue of viral myocarditis mice, and both pri-miR-320 and mature of miR-320 were up-regulated greatly in cardiomyocytes. The level of miR-320 in cardiomyocytes treated with viral myocarditis serum exosomes was significantly up-regulated, while transfection of miR-320 inhibitor counteracted miR-320 overexpression and reduced apoptosis rate caused by exosomes. Pik3r1 is the target gene of miR-320, and its overexpression reversed cardiomyocyte apoptosis induced by miR-320 up-regulation. The overexpression of miR-320 inhibited AKT/mTOR pathway activation. Conclusion Viral myocarditis serum exosome-derived miR-320 promotes apoptosis of mouse cardiomyocytes by inhibiting AKT/mTOR pathway by targeting Pik3r1.
Mice ; Animals ; Myocytes, Cardiac ; Phosphatidylinositol 3-Kinase/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Myocarditis/pathology* ; Exosomes/metabolism* ; bcl-2-Associated X Protein/metabolism* ; MicroRNAs/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Apoptosis/genetics*

Viral myocarditis (VMC) is a disease characterized by inflammation of myocardial cells caused by viral infection. Since the pathogenesis mechanism of VMC has not been fully elucidated, the diagnosis and treatment of this disease remains extremely challenging. Non-coding RNAs (ncRNAs) are a class of RNAs that do not encode proteins. An increasing number of studies have shown that ncRNAs are involved in regulating the occurrence and development of VMC, thus providing potential new targets for the treatment and diagnosis of VMC. This review summarizes the possible roles of ncRNAs in the pathogenesis and diagnosis of VMC revealed recently.
Coxsackievirus Infections ; Enterovirus B, Human ; Humans ; Inflammation ; Myocarditis/genetics* ; Virus Diseases/genetics*

OBJECTIVETo investigate the effect of tranilast on myocardial fibrosis in mice with viral myocarditis (VMC).

METHODSMale balb/c mice (n=72) were randomly divided into control, VMC and tranilast groups (n=24 each). In the VMC and tranilast groups, the mice were infected with Coxsackie virus B3 (CVB3) to prepare VMC model, while the control group was treated with Eagle's medium. After modeling, the tranilast group was administrated with tranilast [200 mg/(kg.d)] until the day before sampling. On days 7, 14 and 28 after CVB3 or Eagle's medium infection, heart specimens (n=8) were taken and examined after Toluidine blue staining and Nissl staining for counts of mast cells (MC), hematoxylin-eosin staining for myocardial pathological changes, and Masson staining for myocardial fibrosis. The expression of CTGF and type I collagen (Col I) in the myocardial tissue was measured by RT-PCR and Western blot. The correlations of CTGF mRNA expression with MC counts and Col I expression were analyzed.

RESULTSThe myocardial pathological changes and collagen volume fraction in the VMC group were significantly higher than in the control group at all three time points (P<0.05). Tranilast treatment significantly decreased the myocardial pathological changes and collagen volume fraction compared with the VMC group (P<0.05). The mRNA and protein expression of CTGF and Col I increased in the VMC group compared with the control group, and the increases were reduced with tranilast treatment (P<0.05). The number of MC was positively correlated to CTGF mRNA expression on the 7th day and 14th day (r=0.439, P=0.049) in the VMC group. There were positive correlations between the mRNA expression of Col I and CTGF on the 7th day and 14th day (r=0.646, P=0.007) and the 28th day (r=0.326, P=0.031).

CONCLUSIONSTranilast may inhibit the aggregation of MC and down-regulate the expression of CTGF, relieving myocardial fibrosis of mice with VMC.

Animals ; Collagen Type I ; genetics ; Connective Tissue Growth Factor ; genetics ; Coxsackievirus Infections ; drug therapy ; Enterovirus B, Human ; Fibrosis ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; drug therapy ; Myocardium ; pathology ; RNA, Messenger ; analysis ; ortho-Aminobenzoates ; pharmacology

OBJECTIVETo study the role of tranilast in the pathogenesis of myocardiac fibrosis in viral myocarditis.

METHODSSeventy-two BALB/C mice were randomly divided into control, model and intervention groups (n=24 each). Mice in the model and intervention groups were infected with Coxsackievirus B3 to induce viral myocarditis. The intervention group was given with tranilast (200 mg/kg) by gavage until sacrifice for sampling, while the other two groups were administered with the same volume of normal saline. Cardiac tissues were obtained from 8 mice on 7, 14 and 28 days after modeling. The mast cell number was observed by toluidine blue staining and thionine staining. The cardiac tissues were stained with hematoxylin and eosin as well as masson trichrome to observe the pathological changes in cardiac tissues. The mRNA and protein expression of osteopontin and transforming growth factor-β1 was measured by RT-PCR and immunohistochemistry respectively.

RESULTSIn the model group, the mRNA and protein expression of osteopontin reached the highest level on the 7th day, decreasing from the 14th day, and became to the least on the 28th day; while the expression of TGF-β1 increased from the 7th day, reaching a peak on the 14th day, and decreased slightly on the 28th day. The mRNA and protein expression of TGF-β1 and OPN was lower in the intervention group than the model group (P<0.05), but higher than the control group (P<0.05). The expression of OPN mRNA was positively correlated to the number of mast cells.

CONCLUSIONSTranilast can reduce myocardial fibrosis by decreasing the number of mast cells, inhibiting the expression of TGF-β1 and OPN.

Animals ; Fibrosis ; Male ; Mast Cells ; drug effects ; Mice ; Mice, Inbred BALB C ; Myocarditis ; complications ; Myocardium ; pathology ; Osteopontin ; analysis ; genetics ; Transforming Growth Factor beta1 ; analysis ; genetics ; ortho-Aminobenzoates ; pharmacology ; therapeutic use

OBJECTIVETo investigate the role and significance of cardiac mast cells and Toll-like receptor 4 (TLR4) in the development and progression of viral myocarditis (VMC).

METHODSForty-eight Balb/c mice were randomly divided into a control group (n=24) and a model group (n=24). Coxsackievirus B3 was intraperitoneally injected into the model group mice to establish a VMC model. In each group, cardiac tissues were collected from 8 mice at 7, 14 and 28 days after the model was established. The cardiac tissues were stained with hematoxylin and eosin as well as Masson trichrome to observe pathological changes in cardiac tissues. The number and degranulation of cardiac mast cells at each time point were measured and evaluated by toluidine blue staining and transmission electron microscopy. The mRNA and protein expression of TLR4 in cardiac tissues was measured by RT-PCR and immunohistochemistry. In the model group, the correlation between number of cardiac mast cells and mRNA expression of TLR4 at all time points was analyzed.

RESULTSThe model group had significantly higher pathological scores of cardiac tissues than the control group at all time points (P<0.05). The myocardial collagen volume fraction in the model group at 28 days was significantly higher than in the control group at all time points and higher than in the model group at 7 and 14 days (P<0.05). At each time point, the model group had a significantly increased number of mast cells (P<0.05), and significantly increased mRNA and protein expression of TLR4 (P<0.05) compared with the control group. In the model group, the number of cardiac mast cells was positively correlated with the mRNA expression of TLR4 at all time points (R²=0.877, P<0.05).

CONCLUSIONSMice with VMC have significantly increased numbers of cardiac mast cells and expression of TLR4 compared with control mice at all time points, suggesting that mast cells and TLR4 may play important roles in the inflammatory response and fibrosis of VMC.

Animals ; Coxsackievirus Infections ; immunology ; Enterovirus B, Human ; Female ; Mast Cells ; physiology ; Mice ; Mice, Inbred BALB C ; Myocarditis ; immunology ; Myocytes, Cardiac ; pathology ; Toll-Like Receptor 4 ; analysis ; genetics ; physiology

To evaluate the effects of lentivirus-delivered short hairpin RNA (shRNA) on CVB3 infection in an animal model by RNA interference technique, we constructed a recombinant lentivirus expressing shRNA-3753 against the viral genome region 3753-3771, then transduced Lenti-sh3753 into mice infected with CVB3. We evaluated the antiviral ability of lenti-sh3753 by cytopathic effect (CPE), viral plaque assay and histological analysis of mice hearts. The results showed that Lenti-sh3753 exhibited a significant protective effect on cell viability and reduction of viral titers in supernatant of cell culture by specific inhibition on viral replication. Lenti-sh3753 also prolonged the mice survival and limited the viral production in mice hearts. These data proposed that Lenti-sh3753 can effectively inhibit CVB3 infection in a coxsackievirus-induced myocarditis model, suggesting its potential role in prevention and therapy of viral diseases.
Animals ; Coxsackievirus Infections ; drug therapy ; virology ; Down-Regulation ; Enterovirus B, Human ; genetics ; physiology ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; drug therapy ; virology ; RNA Interference ; RNA, Small Interfering ; genetics ; therapeutic use ; RNA, Viral ; genetics ; Virus Replication

OBJECTIVE: To study the mechanism of osteopontin (OPN) in viral myocarditis by observing the expression of OPN and collagen I (Col I) in mice myocardium. METHODS: The viral myocarditis models were achieved by infection with myocarditic coxsackievirus B3 (CVB3). The myocardium of mice was stained by HE and Masson staining, and the pathological scores and the collagen volume fraction (CVF )of myocardium were tabulated. The expression of Col I mRNA was measured by RT-PCR. The expression of OPN was detected by RT-PCR and ELISA. RESULTS: The histopathological examination revealed a prevalence of myocardial cell necrosis and obvious inflammation changes at the 7th day post-infection. Subsequently the inflammatory lesions were gradually absorbed. At the 28th day, the inflammatory cells had almost disappeared and obvious fibrosis occurred. The pathological scores and the expression of OPN mRNA were higher than those of the control group (P<0.05), and reached the highest level at the 7th day (P<0.05). From the 14th day, these parameters decreased,reflected also in the ELISA results. At the 7th day and the 14th day, the Col I expression was similar to that of control. Col I expression at the 21th and 28th days was higher than those of the control (P<0.05), and correlated positively to the CVF results. CONCLUSION The OPN mRNA expression increased in acute stage of VMC, and higher than that of the control group when in recovery stage, suggesting that OPN might be related to the inflammatory response in acute stage of, and promote the collagen synthesis of recovery stage.
Animals ; Collagen Type I ; genetics ; metabolism ; Coxsackievirus Infections ; metabolism ; Enterovirus B, Human ; isolation & purification ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; metabolism ; virology ; Osteopontin ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo study the effects of astragaloside on the expression of insulin-like growth factor-1 (IGF-1) and associated proteins in mice with viral myocarditis.

METHODSSixty-five 4-week-old BALB/C mice were randomly divided into 5 groups: normal control, astragaloside control, untreated myocarditis, low-dose and high-dose astragaloside-treated myocarditis. The BALB/C mice in the later three groups were intraperitoneally injected with CVB3. The low-dose and high-dose astragaloside-treated myocarditis groups were given astragaloside of 0.07 and 0.6 mg/kg•d, respectively by intragastric administration. Fifteen days later, the samples of blood and muscular tissues were obtained. The expression of IGF-1 in plasma was measured using ELISA. The levels of IGF-1 and associated proteins in muscular tissues were measured by immunohistochemistry. The expression of IGF-1 mRNA in muscular tissues was examined by RT-polymerase chain reaction (RT-PCR).

RESULTSThe expression of IGF-1 and associated proteins increased significantly in mice infected with CVB3. High-dose astragaloside treatment reduced the expression of IGF-1 and associated proteins, but low-dose astragaloside did not.

CONCLUSIONSHigh-dose astragaloside may reduce the expression of IGF-1 and associated proteins in mice with acute viral myocarditis, possibly thus providing protective effects on muscular tissues.

Acute Disease ; Animals ; Coxsackievirus Infections ; drug therapy ; metabolism ; Drugs, Chinese Herbal ; therapeutic use ; Enterovirus B, Human ; Insulin-Like Growth Factor Binding Protein 3 ; analysis ; Insulin-Like Growth Factor I ; analysis ; genetics ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; drug therapy ; metabolism ; Myocardium ; chemistry ; RNA, Messenger ; analysis ; Receptor, IGF Type 1 ; analysis ; Saponins ; therapeutic use ; Triterpenes ; therapeutic use