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: Autophagy is a highly conserved intracellular degradation pathway. Many picornaviruses induce autophagy to benefit viral replication, but an understanding of how autophagy occurs remains incomplete. In this study, we explored whether coxsackievirus B3 (CVB3) infection induced autophagy through endoplasmic reticulum (ER) stress. METHODS: In CVB3-infected HeLa cells, the specific molecules of ER stress and autophagy were detected using Western blotting, reverse transcription polymerase chain reaction (RT-PCR), and confocal microscopy. Then PKR-like ER protein kinase (PERK) inhibitor, inositol-requiring protein-1 (IRE1) inhibitor, or activating transcription factor-6 (ATF6) inhibitor worked on CVB3-infected cells, their effect on autophagy was assessed by Western blotting for detecting microtubule-associated protein light chain 3 (LC3). RESULTS: CVB3 infection induced ER stress, and ER stress sensors PERK/eIF2α, IRE1/XBP1, and ATF6 were activated. CVB3 infection increased the accumulation of green fluorescent protein (GFP)-LC3 punctuation and induced the conversion from LC3-I to phosphatidylethanolamine-conjugated LC3-1 (LC3-II). CVB3 infection still decreased the expression of mammalian target of rapamycin (mTOR) and p-mTOR. Inhibition of PERK, IRE1, or ATF6 significantly decreased the ratio of LC3-II to LC3-I in CVB3-infected HeLa cells. CONCLUSION CVB3 infection induced autophagy through ER stress in HeLa cells, and PERK, IRE1, and ATF6a pathways participated in the regulation of autophagy. Our data suggested that ER stress may inhibit mTOR signaling pathway to induce autophagy during CVB3 infection.
Activating Transcription Factor 6 ; metabolism ; Autophagy ; Coxsackievirus Infections ; metabolism ; Endoplasmic Reticulum Stress ; Endoribonucleases ; metabolism ; Enterovirus B, Human ; HeLa Cells ; Humans ; Protein-Serine-Threonine Kinases ; metabolism ; Signal Transduction ; eIF-2 Kinase ; metabolism

OBJECTIVE: To investigate the effects of total flavonids of astragalus(TFA) on arrhythmia, endoplasmic reticulum stress and connexcin in mice with viral myocarditis and to clarify the mechanisms of TFA against viral myocarditis complicated with arrhythmia. METHODS: Thirty-six male Balb/c mice were randomly divided into control group, viral myocarditis group and total flavonoids group (=12). The mice of viral myocarditis were intraperitonealy injected with 0.1 ml/day 10-950 TCID CVB3 for 3 days. The mice of TFA group were intraperitoneal injected with 0.1 ml/day 10-950 TCID CVB3 for 3 days and treated with 0.1ml, 20 mg/L TFA by tail vein injection. At the end of the experiment, arrhythmia was detected by electrocardiogram, the heart of mice were stained by HE, the expressions of glucose-regulated protein 78(GRP78), endoplasmic reticulum stress signaling pathway factor activating transcription factor 4(ATF4) and connexcin 43(Cx43) were detected by Western blot. RESULTS: The expressions of GRP78 and ATF4 were increased and the expression of Cx43 was decreased in viral myocarditis, while TFA inhibited these effect of viral myocarditis in heart of mice. CONCLUSIONS The antiarrhythmic effect of TFA may be related to the alleviation of endoplasmic reticulum stress and the increase of Cx43 expression.
Activating Transcription Factor 4 ; metabolism ; Animals ; Arrhythmias, Cardiac ; drug therapy ; Astragalus Plant ; chemistry ; Connexin 43 ; metabolism ; Coxsackievirus Infections ; drug therapy ; Drugs, Chinese Herbal ; pharmacology ; Endoplasmic Reticulum Stress ; drug effects ; Flavonoids ; pharmacology ; Heat-Shock Proteins ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; drug therapy ; virology ; Myocardium

OBJECTIVETo investigate the myocardial protective effect of L-carnitine in children with hand, foot and mouth disease (HFMD) caused by Coxsackie A16 virus and possible mechanisms.

METHODSA total of 60 HFMD children with abnormal myocardial enzyme after Coxsackie A16 virus infection were enrolled and randomly divided into L-carnitine group and fructose-1,6-diphosphate group (fructose group), with 30 children in each group. The two groups were given L-carnitine or fructose diphosphate in addition to antiviral and heat clearance treatment. Another 30 healthy children who underwent physical examination were enrolled as control group. The changes in myocardial zymogram, malondialdehyde (MDA), superoxide dismutase (SOD), and apoptosis factors sFas and sFasL after treatment were compared between groups.

RESULTSThere was no significant difference in treatment response between the L-carnitine group and the fructose group (P>0.05). One child in the fructose group progressed to critical HFMD, which was not observed in the L-carnitine group. Before treatment, the L-carnitine group and the fructose group had significantly higher indices of myocardial zymogram and levels of MDA, sFas, and sFasL and a significantly lower level of SOD than the control group (P<0.05), while there were no significant differences in these indices between the L-carnitine group and the fructose group (P>0.05). After treatment, the L-carnitine group and the fructose group had significant reductions in the indices of myocardial zymogram and levels of MDA, sFas, and sFasL and a significant increase in the level of SOD (P<0.05); the fructose group had a significantly higher level of creatine kinase (CK) than the control group and the L-carnitine group, and there were no significant differences in other myocardial enzyme indices, MDA, sFas, and sFasL between the L-carnitine group and the fructose group, as well as between the L-carnitine and fructose groups and the control group (P>0.05). SOD level was negatively correlated with aspartate aminotransferase, lactate dehydrogenase (LDH), CK, and creatine kinase-MB (CK-MB) (r=-0.437, -0.364, -0.397, and -0.519 respectively; P<0.05), and MDA level was positively correlated with LDH and CK-MB (r=0.382 and 0.411 respectively; P<0.05).

CONCLUSIONSL-carnitine exerts a good myocardial protective effect in children with HFMD caused by Coxsackie A16 virus, possibly by clearing oxygen radicals and inhibiting cardiomyocyte apoptosis.

Carnitine ; therapeutic use ; Child, Preschool ; Coxsackievirus Infections ; complications ; Female ; Hand, Foot and Mouth Disease ; drug therapy ; etiology ; metabolism ; Heart ; drug effects ; Humans ; Infant ; Male ; Malondialdehyde ; analysis ; Myocardium ; metabolism ; pathology ; Superoxide Dismutase ; metabolism

OBJECTIVETo investigate the effect of total flavonoids of astragalus on the expression of endoplasmic reticulum chaperone, calumenin and connecxin 43 (CX43) in suckling mouse myocardium with myocarditis caused by coxsackievirus B3 (CVB3).

METHODSThe primary culture of suckling mouse myocardium cells were randomly divided into control group, CVB3 infected group and total flavonoids of astragalus group. Firstly, to confirm the identity of the suckling mouse myocardium, α-SMA was monitored by immunohistochemistry method. Then the protein expression changes of endoplasmic reticulum chaperone-glucose regulatory protein 78 ( GRP78), calumenin and CX43 were detected by Western blot.

RESULTS(1) Compared with that of the control group, the GRP78 expression level in CVB3 infected group was improved, the expression levels of calumenin and CX43 were all reduced. (2) Compared with that of CVB3 infected group, GRP78 expression level was decreased, and the expression levels of calumenin and CX43 were increased in total flavonoids of astragalus group.

CONCLUSIONCVB3 infection may cause endoplasmic reticulum stress of rat myocardium cells by increasing the expression of GRP78 and decreasing the expression of calumenin and CX43. On the other hand, total flavonoids of astragalus can reduce the expression of GRP78 and increase the expression of calumenin and CX43.The results of this experiment may be closely related to the effects of anti-arrhythmia with viral myocarditis caused by CVB3.

Animals ; Astragalus Plant ; chemistry ; Blotting, Western ; Calcium-Binding Proteins ; metabolism ; Cells, Cultured ; Connexin 43 ; metabolism ; Coxsackievirus Infections ; drug therapy ; Endoplasmic Reticulum ; metabolism ; Endoplasmic Reticulum Stress ; drug effects ; Flavonoids ; pharmacology ; Heat-Shock Proteins ; metabolism ; Mice ; Myocarditis ; drug therapy ; virology ; Myocardium ; cytology ; Myocytes, Cardiac ; drug effects ; virology ; Rats

OBJECTIVETo investigate the dynamic expression and role of vitamin D receptor (VDR) in the myocardium of mice with viral myocarditis (VMC).

METHODSOne hundred and twenty 4-week-old male BALB/c mice were selected and assigned into control (n=40) and experimental groups (n=80). The mice in the experimental group were injected intraperitoneally with Coxsackievirus B3 to establish the model of VMC, while the mice in the control group were injected intraperitoneally with an equal volume of DMEM solution. Fifteen mice in the experimental group and ten mice in the control group were sacrificed at 3, 7, 14, or 28 days after injection, and the myocardial specimens were obtained. The dynamic expression of VDR in the myocardium was determined by the immunohistochemical technique. The pathological changes in the myocardium were examined using hematoxylin and eosin staining.

RESULTSIn the experimental group, the mice had significantly increased expression of VDR after virus injection (P<0.01); the expression of VDR reached the peak at 7 days after injection, and then declined gradually; the expression of VDR remained high at 28 days after injection. At 3, 7, 14, and 28 days after injection, the expression of VDR in the experimental group was significantly higher than that in the control group (P<0.01). Moreover, in the experimental group, the changes in the pathological score of the myocardium were in accordance with the changes in the expression of VDR; the expression level of VDR in the myocardium was positively correlated with the pathological changes in the myocardium in the experimental group (P<0.01).

CONCLUSIONSVDR may be involved in the inflammatory-immune process in the pathogenesis of VMC.

Animals ; Coxsackievirus Infections ; metabolism ; Enterovirus B, Human ; Immunohistochemistry ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; metabolism ; pathology ; Myocardium ; chemistry ; pathology ; Receptors, Calcitriol ; analysis ; physiology

OBJECTIVE: Astragaloside is a simple substance of saponin and the active constituent of astragali. It was reported that the astragaloside exerted therapeutical eff ect on viral myocarditis and dilated cardiomyopathy. The purpose of this study was to investigate the effect of astragaloside on TL1A expression in viral myocarditis. METHODS: A total of 100 BALB/c mice were randomly divided into 6 groups: the normal control group (group A, n=10), the high-dose control group (group B, n=10), the myocarditis control group (group C, n=20), the low-dose group (group D, n=20), the middle-dose group (group E, n=20) and the high-dose group (group F, n=20). Mice in group A and group B were injected intraperitoneally with 0.1 mL EMEM solution, while mice in group C, D, E and F were treated with 0.1 mL of 1×102 TCID50 CVB3 (diluted in EMEM). Then, mice in group A and group B were treated with carboxymethycellulose solution and 9% astragaloside for 1 week, respectively. At the same time, mice in group C, D, E and F were treated with sodium carboxymethycellulose solution, 1% [0.07 g/(kg.d)], 3% [0.2 g/(kg.d)] and 9%[0.6 g/(kg.d)] astragaloside for 1 week, respectively. After 14 days, the mice were sacrificed and their hearts were collected. The expression levels of TL1A mRNA and protein in the myocardium were examined by RT-PCR and immunohistochemistry, respectively. RESULTS: There was no death in the group A and B. The mortality in the group C, D, E and F was 45% (9/20), 30% (6/20), 25% (5/20) and 10% (2/20), respectively. Compared with the group C, the mortality in the group F was significantly decreased (P<0.05), but there no significant difference in mortality between the group C and the group D or E (P>0.05). There was no any pathological lesion in the group A and B. The TL1A mRNA and protein expression in the myocardium of mice in the group A and B was at low level, with no difference between them (P>0.05). Compared with the group A, the expression levels of TL1A mRNA and protein in the group C were markedly up-regulated (P<0.01), which was dramatically attenuated by the intervention of astragaloside at high dosage (the group F, P<0.01) but not at low (the group D) or middle-dosage (the group E) (P>0.05). CONCLUSION Astragaloside may play a pivotal role in protection of the heart injury in viral myocarditis by suppressing the expression of TL1A.
Acute Disease ; Animals ; Cardiomyopathy, Dilated ; drug therapy ; Coxsackievirus Infections ; drug therapy ; Drugs, Chinese Herbal ; pharmacology ; Mice ; Mice, Inbred BALB C ; Myocarditis ; drug therapy ; virology ; Myocardium ; metabolism ; pathology ; RNA, Messenger ; Saponins ; pharmacology ; Tumor Necrosis Factor Ligand Superfamily Member 15 ; metabolism ; Up-Regulation

OBJECTIVETo explore the effects of emodin in myocardial protection in mice with viral myocarditis (VMC) and explore molecular mechanisms.

METHODSFifty-five male 4-week-old BALB/c mice were randomly divided into control group (n=15), model group (n=20), and emodin group (n=20). The mice in model and emodin groups were inoculated with 0.1 ml Eagle's solution containing coxsackievirus B3 intraperitoneally, and those in the control group were given only 0.1 ml Eagle's solution. From the day of inoculation, the mice in emodin group received intragastric administration with 0.1 ml of 3 mg/ml emodin solution once daily for 21 consecutive days, and those in the control and model groups received 0.1 ml distilled water only. On day 7 after inoculation, 5 mice from each group were sacrificed to determine the viral titers in the cardiac tissues. All the mice were sacrificed on day 22 for measurement of the heart weight and histopathological inspection of the heart with HE staining. The mRNA and protein expression levels of myocardial interleukin-23 (IL-23) and interleukin-17 (IL-17) were detected by real-time quantitative PCR and Western blotting, respectively, and serum IL-23 and IL-17 levels were examined using enzyme linked immunosorbent assay (ELISA). Th17 cell frequencies were analyzed by flow cytometry. The expression levels of myocardial nuclear factor-κB (NF-κB) p65 in the cardiomyocyte nuclei were examined using Western blotting, and myocardial interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) contents were detected by ELISA.

RESULTSThe mortality, myocardial histopathologic scores and virus titers in emodin group were all significantly lower than those in the model group (P<0.05). The heart-to-body weight ratio, myocardial IL-23 and IL-17 expressions, serum IL-23 and IL-17 levels, Th17 cell frequencies, cardiomyocyte nuclear NF-κB p65 expression, and myocardial contents of IL-1β, IL-6 and TNF-α were all significantly increased in the model group as compared to the control group (P<0.01) but reduced significantly in emodin group as compared to model group (P<0.05).

CONCLUSIONEmodin can protect against VMC by inhibiting IL-23/IL-17 inflammatory axis, Th17 cell proliferation and viral replication in mice.

Animals ; Coxsackievirus Infections ; immunology ; Cytokines ; immunology ; Emodin ; pharmacology ; Enterovirus ; physiology ; Interleukin-17 ; immunology ; Interleukin-23 ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; immunology ; virology ; Th17 Cells ; cytology ; drug effects ; Transcription Factor RelA ; metabolism ; Virus Replication

OBJECTIVETo explore the apoptotic pathway mediated by endoplasmic reticulum stress in the mouse myocardium with heart failure induced by acute viral myocarditis caused by B-3 Coxsackie virus.

METHODSForty BALB/c male mice were randomly divided into 2 groups (n = 20): the control group and the virus infection group. The BALB/c mouse myocarditis was induced by B-3 Coxsackie virus and the mouse behavior was observed conventionally. All the mice were sacrificed on day 7 and the changes of left ventricular pressure (LVP) and the rate of change of left ventricular pressure (LV dp/dt) were measured. The cardiomyocytic apoptosis was analyzed by TUNEL method and the mRNA expression level of endoplasmic reticulum haperones glucose-regulated protein (GRP)78 and GRP94 was detected by RT-PCR.

RESULTS(1) Compared with those of control group, the parameters of cardiac hemodynamics in the virus infection group were significantly decreased (P < 0.01); (2) Compared with that of control group, myocardial apoptosis was significantly increased in the myocardial cells from mice with heart failure induced by acute viral myocarditis (P < 0.01); (3) The mRNA expression level of GRP78 and GRP94 were increased significantly in the virus infection group compared with the control group.

CONCLUSIONThese findings suggest the endoplasmic reticulum stress may mediate the apoptosis of myocardial cells in the mice myocardium of heart failure induced by acute viral myocarditis caused by B-3 Coxsackie virus.

Animals ; Apoptosis ; Coxsackievirus Infections ; physiopathology ; Endoplasmic Reticulum Stress ; Heart ; physiopathology ; Heart Failure ; physiopathology ; virology ; Heat-Shock Proteins ; metabolism ; Male ; Membrane Glycoproteins ; metabolism ; Mice ; Mice, Inbred BALB C ; Myocarditis ; physiopathology ; virology ; Myocardium ; pathology ; Myocytes, Cardiac ; cytology

OBJECTIVE: To discuss the myocardial expression of Spry1 and MAPK proteins of viral myocarditis (VMC), to reveal its mechanism of sudden death, and to provide guides for forensic identification of sudden cardiac death. METHODS: Thirty Balb/c male mice were randomly divided into VMC group and control group, inoculated intraperitoneally with Coxsackievirus B3 and Eagel's solution, respectively. After the mice were sacrificed, the cardiac tissues of the mice were taken to proceed regular pathological examination. The changes of Spry1 protein, Spry1 mRNA and MAPK protein were detected by immunohistochemistry, Western blotting and real-time PCR. RESULTS: Under light microscope, the pathologic changes included myocardial interstitial edema, inflammatory cells infiltration, myocardial necrosis, and focal and patchy necrosis of myocardial fiber in VMC group. The expression of Spry1 protein in VMC group was lower than that in control group (P < 0.05). There was slightly decreased expression of Spry1 of the mRNA level in VMC group (P > 0.05). But the MAPK protein expression in VMC group was higher than that in control group (P < 0.05). CONCLUSION The pathway of MAPK/ERK involving Spry1 protein accelerates the expression of collagen, which may contribute to arrhythmia, heart failure and even sudden cardiac death.
Adaptor Proteins, Signal Transducing/metabolism* ; Animals ; Coxsackievirus Infections/pathology* ; Death, Sudden, Cardiac/pathology* ; Disease Models, Animal ; Immunohistochemistry ; Male ; Membrane Proteins/metabolism* ; Mice ; Mice, Inbred BALB C ; Mitogen-Activated Protein Kinases/metabolism* ; Myocarditis/virology* ; Myocardium/pathology* ; Phosphoproteins/metabolism* ; RNA, Messenger/metabolism* ; Random Allocation ; Real-Time Polymerase Chain Reaction