Icariin ameliorates viral myocarditis by inhibiting TLR4-mediated ferroptosis

Objective:To explore the mechanism by which icariin alleviates viral myocarditis.Methods:CVB3-induced cardiomyocytes were used as an in vitro model of viral myocarditis to assess the effects of icariin treatment on cell viability,inflammation,and apoptosis.Moreover,the effects of icariin on ferroptosis and TLR4 signaling were assessed.After AC16 cells were transfected with TLR4 overexpression plasmids,the role of TLR4 in mediating the regulatory effect of icariin in viral myocarditis was investigated.Results:Icariin significantly elevated cell viability and reduced inflammatory factors TNF-α,IL-1β,IL-6,and IL-18.Flow cytometry revealed that icariin decreased apoptosis rate,and the protein expression of Bax and cleaved caspase 3 and 9 in CVB3-induced cardiomyocytes.Additionally,it suppressed ferroptosis including lipid peroxidation and ferrous ion,as well as the TLR4 signaling.However,TLR4 overexpression abrogated the modulatory effects of icariin.Conclusions:Icariin mitigates CVB3-induced myocardial injury by inhibiting TLR4-mediated ferroptosis.Further animal study is needed to verify its efficacy.

Impairment of Myocardial and Skeletal Mitochondria in Mice with Viral Myocarditis and Their Correlation

In order to investigate the impairment of mitochondrial membrane phospholipid local- ization and DNA3867 (mtDNA3867) deletion and the correlation between cardiac and skeletal muscle cells in mice with viral myocarditis, 50 BALB/c mice were divided into two groups randomly. In ex- perimental group (n=40), the mice were intraperitoneally injected with 0.1 mL Eagle liquid with CVB3 (TCID50=108), while in the control group (n=10), the mice were subjected to equal volume of Eagle liquid. The impairment of mitochondrial membrane phospholipid localization and mtDNA3867 deletion rate of cardiac and skeletal muscle were detected separately at day 3, 11 and 24 after injec- tion. The correlation of mitochondrial membrane phospholipid localization and mtDNA3867 deletion rate between cardiac and skeletal muscle cells cells was analyzed using Spearman method. At the day 3 after injection, in both cardiac and skeletal muscle cells, mtDNA3867 deletion rate was significantly higher in experimental group than in control group (P<0.05), but the localization of mitochondrial membrane phospholipid showed no difference between two groups (P>0.05). At day 11 after injec- tion, the mtDNA3867 deletion rate of both cells in experimental group was increased to the peak level (P<0.05), and the impairment of mitochondrial membrane phospholipid localization of both cells also increased markedly in experimental group as compared with control group (P>0.05). At the day 24 after injection, the impairment of mitochondrial membrane phospholipid localization and mtDNA3867 deletion of both cells showed a recovery tendency, but still severer than those at the day 3 after injec- tion (P<0.05). The impairment of mitochondrial membrane phospholipid localization and mtDNA3867 deletion were consistent and synchronistic between cardiac and skeletal muscle cells, and showed good correlations (P<0.05). The impairment of mitochondria plays an important role in the patho- genesis of viral myocarditis, and the skeletal muscle cells might act as a peripheral 'window' to re- flect the mitochondrial damage of cardiac myocytes.

Effect of Astragalus Injection on Levels of Blood Selenium and Immunity Function in Children with Viral Myocarditis

Objective: To observe the effect of Astragalus Injection (AD on levels of blood selenium (Se) and cytokines, and T cellular immune function with viral myocarditis (VM) in children. Methods: Eighty children with VM were randomly divided into 2 groups. The control group consisted of 38 patients, to whom conventional therapy, including energy mixture, vitamin C and coenzyme Q10, etc. were given. The treated group (n = 42), to whom combination therapy of conventional therapy and Al were given. The levels of blood Se and cytokine, including interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and also evaluation of T lymphocyte subsets and cardiac function were observed. Results: The results showed that after treatment, the levels of blood Se were significantly higher (P<0.01), while IL-1,IL-6 and TNF-a were significantly lower (P<0.01) than those before treatment in the control group. The left ventricular end diameter (LVED) were significantly decreased (P<0.01), left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were significantly increased than those before treatment in the treated group(P<0.01, P<0.05). T lymphocyte subsets got normalized (P<0.01), and compared with the control group, the difference was significant (P<0.01). Conclusion: Astragalus membranaceus possesses anti-viral effect, adjusts the balance of cytokine and T cellular immunity, and improves the clinical manifestation and cardiac function. It is an effective approach in treating viral myocarditis.

Effect of Shengmal Powder(生脉散) on Symptoms and Markers of Inflammatory Damage of Patients with Acute Viral Myocarditis

Objective: To evaluate the efficacy of Sheng-mai Power (SMP) in treating acute viral myocarditis. Methods: 102 patients with acute viral myocarditis were randomized to SMS group (n = 52) and placebo control group (n = 50 ). Semiquantitative integral methods were taken to observe changes of clinical symptoms such as dyspnea, palpitation and chest pain after 4 weeks of treatment, simultaneously EKG, 24h Holter, concentration of serum cardiac troponin-I, cardiac troponin T and neutralizing antibody test to the Coxsackie B virus were determined. Results: Dyspnea improved much more obvious in SMP group than in the placebo

Observation on Therapeutic Effects of Shengmai Powder (生脉散)in Treating Acute Viral Myocarditis

Objective: To evaluate the clinical efficacy of Shengmai Powder (SMP, 生脉散) in treating a-cute viral myocarditis objectively. Methods: One hundred and twenty-four patients with acute viral myocarditis were randomized into the treated group (SMG, n = 64) and the control group(CG, n = 60 ). Such myo-cardial nutrient medicine as ATP, CoA , Vit-C, were given to both groups. And to the treated group, 40 ml of Shengmai Injection per day was given intravenously for 2 weeks, which was followed by oral intake of Shengmai granule, one package three times daily for another 2 weeks in total. The same anti-arrhythmia agents were applied to both groups, and no fructose-1, 6-diphosphate (FDP) for either. Semi-quantitative scoring method was adopted to observe such symptoms as chest stuffiness, palpitation and chest pain before treatment and four weeks after treatment. Meanwhile, EGG, dynamic ECG by Holter monitor, left ventricular end-diastolic dimension (LVEDD), left ventricular ejection fraction (LVEF), serum neutralizing antibody of virus Coxsackie B, cardiac troponin I (cTnl) and cardiac troponin T (cTnT) were examined. Results: (1) Compared with the control group, more significant improvement was got in SMG in respects of chest stuffiness, palpitation, chest pain and arrhythmia (P<0.05 or P<0.01). (2) Negative converting rates of cTnl ,cTnT in the two groups were 59.46% vs35.48%, 68.75% vs42.31% respectively ( P<0. 05). (3) LVEDD before and after treatment in SMG was 52. 44 ± 3. 40 mm and 48. 81 ± 2. 23mm respectively, while that in the control group was 52. 31 ± 3. 74 mm and 49. 92 ± 2. 67mm respectively; LVEF before and after treatment in SMG was 60.67 ± 4. 62 % and 65. 02 ± 4. 16 % respectively, while that in the control group was 60.91 ± 4. 26 % and 63. 67 ± 3.17 % . There was obvious improvement in the two parameters in both groups, but the improvement in SMG was superior to that in the control group ( P<0. 05). Conclusion: SMP shows a good effect in improving clinical symptoms and signs, heart function, abnormal ECG and inflammatory injury indexes in patients with acute viral myocarditis.

STUDY OF PERSISTENT VIRAL INFECTION IN AN ANIMAL MODEL OF VIRAL MYOCARDITIS BY PCR

Objective To study the role of persistent viral infection in the mechanism of viral myocarditis. Methods A mice model of CVB3m viral myocarditis wes made and the viral RNA in mice myocardium and whole bind sample wes tested by using polymerase chain reaction (PCR ) technique. The pathological changes in mice myocardium were determined. Results De day 3, the viral gene in whole bind and myocardium wes found, which portly became negative on day 8, but the change of myocardial anthology borne obvious. Although the bind specimens wers tested negatively on day 12, the viral gene in mice myocardium remained positive within 120d. Conclusion This study indicates that persistent viral infection plays a role in the pathogenesis of viral myocarditis.

Experience in Treating Viral Myocarditis with Master Lei's Huoxue Decoction

Modem medical treatment of viral myocarditis by suppressing viral replication and improving cardiomyocyte metabolism has a good effect on the acute infectious period of viral myocarditis,but the curative rate is poor,especially the sequelae of viral myocarditis,which often lingers for years to decades.The use of traditional Chinese medicine can achieve a certain curative effect by promoting the positive and dispelling evil spirits as well as balancing yin and yang.After long-term observation,it is found to have clear eff ect on improving myocardial injury caused by viral myocarditis.

Effects of Shenmai Injection(参麦注射液)Combined with Meglumine Adenosine Cyclophosphate Injection on Cardiac Function and Peripheral Serum Levels of TNF-α,TGF-β1 and IFN-γ in Patients with Viral Myocarditis

Objective: To explore effects of Shenmai Injection(参麦注射液) combined with Meglumine Adenosine Cyclophosphate Injection on cardiac function and peripheral serum levels of tumor necrosis factor-α(TNF-α), transforming growth factor-β1(TGF-β1) and interferon-γ(IFN-γ) in patients with viral myocarditis.Methods: A total of 70 patients with viral myocarditis admitted in Cardiovascular Medicine Department of our hospital from June 2016 to June 2018 were selected and divided into a control group(treated with Meglumine Adenosine Cyclophosphate Injection) and an observation group(additionally treated with Shenmai Injection(参麦注射液) on the treatment basis of the control group) according to random number method, with 35 cases in each group.Before and after the treatment, cardiac functional indexes like cardiac index(CI), left ventricular ejection fraction(LVEF) and left ventricular shortening fraction(FS), serum levels of TNF-α, TGF-β1 and IFN-γ, clinical efficacy and adverse reactions occurred in the 2 groups were recorded and compared.Results: Therapeutic effective rate in the observation group was 88.57%, higher than 68.57% in the control group(P < 0.05).After the treatment, cardiac functional indexes like CI, LVEF and FS were higher than those in the control group(P < 0.05).After the treatment, the serum expression levels of TNF-α and TGF-β1 were lower than those in the control group(P < 0.05), while the serum expression level of IFN-γ was higher than that in the control group(P < 0.05).There was no statistically significant difference in the adverse reaction rates between the 2 groups(P > 0.05).Conclusion: The treatment of patients with viral myocarditis by Shenmai Injection(参麦注射液) combined with Meglumine Adenosine Cyclophosphate Injection is more effective, reducing inflammation and restoring cardiac function.

Astragalus Membranaceus Injection Combined with Conventional Treatment for Viral Myocarditis:A Systematic Review of Randomized Controlled Trials

Objective：To assess the efficacy and safety of Astragalus membranaceus Injection combined with conventional therapy in the treatment of viral myocarditis.Methods：Randomized controlled trials（RCTs） of A.membranaceus Injection combined with conventional treatment compared with conventional treatment alone were included.Study population characteristics and outcome results were extracted independently by two assessors.Meta-analysis was performed for data available.Results：Six RCTs,involving 639 participants,were included in this study.The methodological quality of the included trials was generally low,and there was high risk of publication bias in the included trials.The total effective rate of A.membranaceus Injection combined with conventional treatment was significantly higher than that of conventional treatment alone.Compared with conventional treatment,the cointervention treatment group showed significant recovery in myocardium enzyme levels and electrocardiography.Two RCTs reported there were no adverse effects from A.membranaceus Injection combined with conventional treatment.Conclusion：A.membranaceus Injection combined with conventional treatment appeared to be more efficacious compared with conventional treatment alone for treating viral myocarditis.However,this conclusion should be cautiously interpreted due to low methodological quality,small sample size,limited number of trials,and high risk of publication bias and other unidentified risks of bias.The safety of A.membranaceus Injection combined with conventional treatment remains uncertain.

Dynamic changes in myocardial matrix metalloproteinase activity in mice with viral myocarditis

Background Matrix metalloproteinases (MMPs) are the major regulators of collagen degradation involved in the pathogenesis of several diseases of the heart The purpose of this study was to investigate the dynamic changes in myocardial MMP activity in mice with viral myocarditis (VM), the relationship between MMP activity and both cardiac function and the quantity of myocardial collagen, and the role MMPs playing in the pathological lesions of VM KH*2/5DMethods Sixty-five six-week-old male DBA/2 mice were divided into two groups Mice in the infected group (n=50) were inoculated intraperitoneally with 0 14 ml of Coxsackievirus B 3 (CVB 3, Nancy strain) Control mice (n=15) were inoculated intraperitoneally with 0 14 ml of Eagle’s medium Eight infected mice and three control mice were sacrificed on each of days 3, 7, 10, 21 and 30 after inoculation MMP activity was measured on an SDS-PAGE substrate gel embedded with type Ⅰ gelatin (zymography) Echocardiographic studies were performed under anesthesia with 3% chloralhydrate administered intraperitoneally (0 01 ml/g-0 015 ml/g) Cardiac systolic function indices, such as peak velocity of the aorta (V p), flow velocity integral of the aorta (V i), ejection fraction (EF), and fractional shortening (FS) were determined by echocardiography Histological cross sections of the hearts were stained with hematoxylin-eosin and myocardial histopathological scores were determined under an optical microscope The amount of myocardial collagen was measured by means of hydroxyproline quantification Results In virus-infected mice, both MMP-2 and MMP-9 activities were significantly higher than in control mice, reaching a peak on day 10 ( P <0 01) On day 10, cardiac systolic function indices (EF, FS, V p, and V i) were all significantly lower compared both to other stages following viral inoculation and to the control group ( P <0 05) In the acute stage, the amount of myocardial collagen in mice with VM was not significantly different from normal control mice ( P >0 05) However, the amount of myocardial collagen in infected mice at the recovery stage (on days 21 and 30) was significantly greater than those of the control mice MMP-2 and MMP-9 activities positively correlated with myocardial histopathological scores ( r =0 801,0 821, P <0 01) and negatively correlated with V p ( r =-0 649, -0 683, P <0 01) and V i ( r =-0 711, -0 755, P <0 01) However, V p negatively correlated with myocardial histopathological scores ( r =-0 756, P <0 01) Conclusions In mice with VM, the activities of myocardial MMP-2 and MMP-9 increase significantly during the acute stage, and the total quantity of myocardial collagen increases by the time of recovery These changes are associated with myocardial interstition remodeling and cardiac dysfunction MMP activity is an important reference marker for myocardial pathological lesions and can be used to evaluate the severity of myocardial interstitial damage and cardiac dysfunction

MicroRNA-324-3p Plays A Protective Role Against Coxsackievirus B3-Induced Viral Myocarditis

Viral myocarditis(VM) is an inflammatory disease of the myocardium associated with heart failure, which is caused by common viral infections. A majority of the infections are initiated by coxsackievirus B3(CVB3). Micro RNAs(mi RNAs)have a major role in various biological processes, including gene expression, cell growth, proliferation, and apoptosis, as well as viral infection and antiviral immune responses. Although, mi RNAs have been found to regulate viral infections,their role in CVB3 infection remains poorly understood. In the previous study, mi RNA microarray results showed that mi R-324-3 p expression levels were significantly increased when cells and mice were infected with CVB3. It was also found that miR-324-3p downregulated TRIM27 and decreased CVB3 replication in vitro and in vivo. In vitro, analysis of downstream signaling of TRIM27 revealed that, miR-324-3p inhibited CVB3 infection, and reduced cytopathic effect and viral plaque formation by reducing the expression of TRIM27. In vivo, miR-324-3p decreased the expression of TRIM27,reduced cardiac viral replication and load, thereby strongly attenuating cardiac injury and inflammation. Taken together,this study suggests that miR-324-3p targets TRIM27 to inhibit CVB3 replication and viral load, thereby reducing the cardiac injury associated with VM.

A Randomized Clinical Study on Optimum Proposal of Integration of Disease and Syndrome to Treat Viral Myocarditis

Objective：To determine the optimum treatment for viral myocarditis（VMC）.Methods：A total of 126 VMC patients were randomly divided into the control group（42 cases） that was treated with conventional Westem medicine,and the intervention group（84 cases） that was treated with a combination of Chinese medicine（CM）and Western medicine intervention termed optimum proposal of integration of disease and syndrome（OPIDS）.Before and after 4 weeks of treatment,the integral of CM syndrome,self-rating depression and anxiety scales（SDS and SAS,respectively）,echocardiograms（ECGs）,heart rate variability and left ventricular systolic function were observed.Results：Compared with the control group,the intervention group showed significant reductions on the SDS and SAS（P〈0.05）;improvement of premature ventricular beats,atrioventricular blocks,ST-segment abnormalities,and significant T wave changes（P〈0.05）;greater reductions in standard deviation of NN intervals（SDNN）,standard deviation for per 5 min averages NN intervals（SDANN）,and root-mean-square of successive difference of NN intervals（rMSSD）（P〈0.05）;and increases in cardiac output,stroke volume,and ejection fraction,the last of which was statistically significant（P〈0.05）.Overall,the treatment efficacy rate was significantly better（P〈0.05） in the intervention group（75.61%） compared with the control group（69.70%）.Conclusion：OPIDS is quite effective in treating VMC and improves symptoms such as anxiety and depression,left ventricular systolic dysfunction,premature ventricular contraction,and cardiac autonomic nervous system dysfunction.[Registration：Chinese clinical trial center（No.ChiCTR-TRC-00000298）]

DETECTION OF CYTOMEGALOVIRUS GENOME BY IN SITU HYBRIDIZATION IN PARAFFIN EMBEDDED ENDOMYOCARDIAL BIOPSY SPECIMENS OF VIRAL MYOCARDITIS

Cytomegalovirus (CMV) genes were detected by in situ hybridization in 25 Chinese patients with viral myocarditis (VMC). The positive hybridization signals werre found in cardiomyocytes (6 cases, 24%), capillary endothelial cells (4 cases, 16%) and interstitial cells (7 cases, 28%). The difference between VMC and control group (16 cases died of brain trauma and 10 cases of congenital heart diseases was statistically significant. There was no definite pathomorphological relationship between the detection of CMV genes and myocardial lesions. The results suggest that CMV infection may be one of the causes of myocarditis and chronic stimulation of the immune system induced by CMV may be a possible pathogenesis of this disease.

EXPRESSION OF MHC-Ⅰ AND MHC-Ⅱ ANTIGENS IN ENDOMYOCARDIAL BIOPSIES FROM PATIENTS WITH VIRAL MYOCARDITIS AND CARDIOMYOPATHY

In situ expression of major histocompatibility complex (MHC) Ⅰ and Ⅱ antigens were examined in endomyocardial biopsy samples from 35 patients with viral myocarditis (VMC) and dilated cardiomyopathy (DCM). Increased expressions of these 2 antigens were observed on endocardia, capillary endothelia, dentritic and mononuclear cell membranes. The expression of sarcolemma was also found in specimens of VMC and DCM. Thus the abnormal expression of MHC is a new marker of autoimmune reactivity directed against cardiac structures.

Effect of Shenmai Injection on Cardiac Function and Cellular Immunity of Patients with Infantile Viral Myocarditis

Objective: To evaluate the clinical effect of Shenmai injection (SMI) on infantile viral myocarditis. Methods: Fifty patients were treated with SMI combined with routine Western medicines, the effects were observed and compared with those treated with Western medicines alone or compound Salviae liquid. Results: The cardiac function and cellular immunity of patients treated with SMI were significantly improved. The effect of SMI was superior to that of control groups. Conclusion: Using SMI as supplementary treatment in treating infantile viral myocarditis was helpful in recovery of cardiac function and cellular immunity.

Clinical and Experimental Study on Huodan Tablet (藿丹片) in Treating Infantile Viral Myocarditis

Objective: To study the effect of Huodan tablet (藿丹片, HDT) in treating infantile viral myocarditis. Methods: Clinical manifestations and physical signs as well as laboratory examinations have been observed. Results: The markedly effective rate was 68%, and total effective rate 91.67% in the treated group, while in the control group, the markedly effective rate was 30.83%, and the total effective rate 70.84%. According to Ridit analysis, significant difference was shown between the two groups. r-treated group=0.5000, r-control group=0.295+2×0.025 respectively. Conclusion: HDT has no toxic side-effect and can be taken safely and conveniently, it conforms to the demands of WHO on new drug for prevention and cure of myocarditis.

Clinical Observation on the Treatment of Infantile Viral Myocarditis with Puerarin

Objective: To observe the clinical effect of puerarin in treatment of infantile viral myocarditis. Methods: Puerarin was administered intravenously together with conventional treatment in the treated group, while conventional treatment alone was given to the control group. Creatine kinase isoenzyme, lactate dehydrogenase isoenzyme, cardiac function and clinical manifestations before and after treatment were observed. Results: Puerarin could significantly relieve the symptoms in patients of infantile viral myocarditis, enhance the metabolism of myocardium and improve the cardiac function. The total effective rate in the treated group was 87.04%, significantly higher than that of the control group, the difference between them was significant (P<0.05). Conclusion: Puerarin can be used to treat patients with infantile viral myocarditis with satisfactory results.

Clinical Observation on Shuanghuanglian Powder (双黄连粉剂) in Treating Viral Myocarditis of Children

Objective: To study immune function of children viral myocarditis and to evaluate the clinical effect of Shuanghuanglian Powder (SHLP) by injection. Methods: The 62 patients of viral myocarditis were divided into two groups randomly, the SHLP group (n=32) treated with conventional therapy plus SHLP and the conventional treatment group (n=30) with conventional therapy alone. Their serum antibody of Coxsackie virus group B (COXB-IgM), T-lymphocyte subsets including CD+3, CD+4, CD+8 and CD+4/CD+8 were determined with ELISA and indirect immunofluorescent assay. Results: COXB-IgM was positive in 39 of the 62 patients, which was significantly different with those of normal controls (P<0.001). Patients' serum level of CD+4 cells and CD+4/CD+8 ratio decreased while CD+8 increased. After treatment, the recovery of symptoms, signs and immune function in patients of the SHLP group were significantly better than those in patients treated with conventional treatment alone (P<0.01). Conclusion: Immunoregulatory disturbance is involved in children with viral myocarditis and SHLP is an effective drug in the treatment of children viral myocarditis

Effects of Astragaloside in Treating Myocardial Injury and Myocardial Sarco/Endoplasmic Ca^( 2+)-ATPase of Viral Myocarditis Mice

Objective: To investigate the effects on myocardial injury and sarcoplasmic reticulum (SR) Ca^2+-ATPase of viral myocarditis mice treated with Astragaloside (AS) and Astragalus Injection (AI). Methods: Viral myocarditis model was created by intraperitoneal inoculation with coxsackievirus B 3m (CVB 3m ) solution and were divided into model, AS, AI and normal control groups. The mortality, myocardial pathological changes, serum cardiac troponin I (cTnI) and the activity of myocardial Sarco/Endoplasmic Ca 2+ ATPase (SERCA) were observed. Results: The mortality of model was higher than that of the normal control (P=0.0042), AS and AI (P<005). The serum level of cTnI of model was significantly higher than that of the normal control (P<0001), AS (P<0025) and AI (P<005). The myocardial necrosis and inflammatory changes of AS and AI groups were alleviated than that of model (P<001). The activity of myocardial SERCA of model were significantly lower than that of normal control (P<0001), AS (P<001) and AI (P<005). Conclusions: AS and AI have some protecting effects on myocardial injury of viral myocarditis mice. AS is the effective component of Astragalus membranaceus in treating viral myocarditis. One of the mechanisms of Astragalus membranaceus and AS for viral myocarditis mice depriving of the myocardial injury may be due to improve the activity of myocardial SERCA in the mice.