The Molecular Mechanism For Astragalus In The Treatment Of Viral Myocarditis

"Viral myocarditis (VMC) characterized by myocardial inflammation represents one of the most challenging clinical problems in cardiology, associated with a broad spectrum of pathological triggers and a wide range of clinical presentations that vary from mild dyspnea to acute heart failure. It has been estimated to account for up to12%of sudden death in patients under40years of age and become a leading cause of dilated cardiomyopathy which may result in death for50%of patients one to two years after diagnosis. The dominant etiology of viral myocarditis is considered to be the enteroviruses of picornavirus family, with coxsackievirus B3(CVB3) being the most common one. The exact mechanisms of pathological damage of heart induced by VMC are not well understood. The likely mechanisms involve immune-mediated and direct viral cytotoxicity, especially the reactive inflammatory responses which significantly contribute to cardiac damage.To date, no specific treatment options are available to control the disease.The conventional treatment of symptoms in VMC is by using beta-blockers, calcium channel blockers, diuretics or angiotensin converting enzyme (ACE) inhibitors. Several proposed clinical treatment strategies that target specific points have been studied, including the application of immunosuppressive agents (azathioprine, prednisone, and cyclosporine), intravenous immunoglobulin (IVIG) which may replace antibodies, neutralize pathogens, and enhance clearance of inflammatory cytokines that contribute to myocytes destruction, and antiviral agents, such as interferons and pleconaril, which may target the causative organism, possibly halting the cascade of myocyte destruction.In addition to that, Traditional Chinese Medicine (TCM) which has been used for many centuries in China plays an increasingly important role in clinical treatment. Astragalus, the extracts of dried root from Astragalus membranaceus, is one of the most important medical herbs in TCM. It is widely used for the treatment of heart failure. Extensive clinical and experimental studies have demonstrated that Astragalus shows effective in alleviating syndrome, protecting myocardium and improving cardiac function. Currently, multiple forms of Astragalus remedies are commercially produced in China and become the commonly used Chinese patent medicine for the treatment of viral myocarditis in clinical practice. Although regarding the mechanism of Astragalus in treating viral myocarditis has been carried out from cellular and molecular levels with some progress that immune modulation was reported to be involved in, the underlying mechanism remains largely unknown. To better utilize Astragalus clinically and optimize the therapeutic strategies for VMC, in this study we sought to explore the molecular mechanism for Astragalus in the treatment of VMC. Therefore, we confirmed the therapeutic efficacy of Astragalus on human acute viral myocarditis and examined the proinflammatory cytokines expression in VMC patients before and after therapy to investigate whether Astragalus treatment may modulate inflammatory response in viral myocarditis. The molecular mechanism was further explored in mouse viral myocarditis model.Part I Astragalus was effective for the clinical treatment of viral myocarditisTo evaluate the therapeutic effect of Astragalus on human viral myocarditis,60eligible acute VMC patients were enrolled in the study and divided into two groups: Astragalus treatment Group and Routine treatment Group. The Astragalus Group contained30cases including Male18and Female12. The average age of the group was31.7±12.9. The Routine Group contained30cases including Male17and Female13. The average age of the group was32.3±13.6. There was no significant difference of age and gender between the two groups. The patients had similar baseline characteristics. At admission, the most common presenting features were fever [n=56(93.3%)], followed by mental status change, chest pain and tachypnea. The majority of subjects had both left ventricular dilatation and systolic dysfunction at diagnosis by echocardiography. More than half of patients presented with sinus tachycardia. The Astragalus Group patients were intravenously injected with commercial Injectio Astragali (20g/10ml)40g/d for two weeks, then orally administrated with Astragalus infusion (15g/bag)30g/d for three months. Meanwhile, taurine tablets, CoQ10, antiarrhythmic drugs beta-blockers (sotalol or propafenone), calcium channel blockers (mexiletine or verapamil) were orally administrated for3months. The Routine Group patients were intravenously injected with GIK (500ml glucose+10ml potassium chloride+6U insulin) daily for two weeks, then orally administrated with Vitamin C (0.2g/piece)0.6g/d for three months. Taurine tablets, CoQ10and antiarrhythmic drugs were orally administrated for3months just as Astragalus Group. The mean follow-up period for both groups was6months (6.56±1.89VS6.78±2.12).The results showed that in the Astragalus treatment group,21of the30patients had a clinically significant improvement in symptoms. Only9subjects were unchanged in symptoms. While in the Routine treatment group,15subjects were improved and13were unchanged in symptoms.2patients’conditions were even worsened and progressed to dilated cardiomyopathy. These data suggested that Astragalus treatment significantly improved the clinical manifestation of VMC patients. Ventricular rhythm was recorded and quantified with24-hour continuous electrocardiographic Holter recordings. The results showed that in the Astragalus treatment group,18of20patients in total reduced ventricular rhythm≥50%and10of them reduced≥85%.90%subjects had effective improvement in premature ventricular contraction. While in the Routine treatment group, only9of21patients (46.86%) in total reduced ventricular rhythm≥50%,12of them (57.15%) reduced<50%and had no effective improvement in premature ventricular contraction. These data indicated that Astragalus treatment was more effective to improve the premature ventricular contraction compared with Routine treatment. Ejection fraction (EF) and Left ventricular end systolic dimension (LVEDs) was detected by Echocardiography. EF (%)<50%was presented as abnormal cases. Increased EF (%) after therapy was presented as effective treatment. The results showed that the EF values were significantly increased both in Astragalus treatment group (P<0.01) and Routine treatment group (P<0.05) when compared between pre-treatment and post-treatment. However, there were no significant difference between Astragalus treatment group and Routine treatment group. The similar result was obtained for the LVEDs, which was significantly decreased after treatment, but no significant difference was observed between the two groups. Serological indices of cardiac damage cardiac troponin I (cTnI)>0.1ng/ml, creatine kinase-MB (CK-MB) activity CK-MB>30U/L were as positive. After Astragalus treatment,100%cases were recovered from cTnI within2 weeks.75%cases were recovered from CK-MB within2weeks. While in the Routine treatment group,60%and45.46%were recovered from cTnI and CK-MB respectively within2weeks.40%and54.54%were recovered in more than two weeks. Astragalus treatment could reduce the recovery time from cardiac damage caused by VMC, which indicated that Astragalus treatment had more significant protective effect on the cardiac tissues. The viral genome (CVB-RNA) and cardiac autoantibodies (CVB-IgM) in serum were also examined. We analyzed the percentage of CVB-RNA and CVB-IgM negative conversion cases within half a year. The data showed that in the Astragalus treatment group,80%patients effectively clear virus. While only50%patients did that in Routine treatment group, which suggested that Astragalus treatment could have more effective function to eradicate virus.All the above data indicated that Astragalus treatment efficiently alleviated symptoms and improved cardiac functions of VMC patients, and showed better therapeutic effect than Routine treatment.Part II Astragalus modulating proinflammatory response conferred to its therapeutic effect on VMC1. The proinflammatory cytokines expression were remarkably increased in VMC patients and mouse model, and positively correlated with the severity of VMC1.1The proinflammatory cytokines expression in serum of VMC patientsViral myocarditis is a serious clinical condition characterized by cardiac inflammation that impaired cardiac function. Inflammatory cytokines are being increasingly recognised as essential mediators of pathological immune responses. Therefore, we analyzed the proinflammatory cytokines expression, including tumor necrosis factor-a (TNF-α), interleukin (IL)-1β, IL-6and monocyte chemotactic protein-1(MCP-1) in serum of60VMC patients before treatment. Serum samples from healthy controls (HC) who had normal results from physical examinations were as controls. The results showed that TNF-a (10pg/ml) was detected in38of60VMC patients (63.3%). The highest expression level was up to305.4pg/ml and the average expression level was94.18pg/ml. It could not be detected in healthy controls (<10pg/ml). IL-6(>2pg/ml) was detected in57of60(95%) VMC patients. The highest expression level was up to1240pg/ml and the average expression level was 302.2pg/ml. It was negative in healthy controls (<2pg/ml). IL-1β (>0.1pg/ml) was detected in51of60(85%) VMC patients. The average expression level was59.37pg/ml. It was also negative in healthy controls (<0.1pg/ml). MCP-1(>7pg/ml) could be detected in all the serum samples including healthy controls. But the highest expression level in VMC patients was up to2064pg/ml and the average expression level was392.0pg/ml. While the the average expression level in healthy controls was52.52pg/ml. The concentration of MCP-1in VMC patients was notablely higher than that in healthy controls. These data indicated that the expression of proinflammatory cytokines was much more significantly increased in the majority of VMC patients when compared with healthy controls.1.2The proinflammatory cytokines expression in VMC mouse modelTo further analyze the proinflammatory cytokines expression in the progression of VMC. We used CVB3induced mouse VMC model which shared similar characteristics with human disease. Male BALB/c mice were administered intraperitoneal injection with10TCID50CVB3at day0to generate acute viral myocarditis model. The body weight changes were monitored daily post-infection. And the histological analysis of heart sections was performed at day0,4,7,10respectively. The hearts of mice were removed daily and homogenized daily post-infection for ELISA assay of detecting proinflammatory cytokines expression kinetically. The results showed that the body weight of CVB3infected mice were significantly and continuously decreased since day3when compared with normal mice. Consistently, histopathology of cardiac tissues showed that the myocardial injury was apparently observed at day4and increasingly severe in the following days. The expression of pro-inflammatory cytokines were robustly up-regulated at day4and persistently increasing from day4to day10in CVB3infected mice, which had similar variation tendency with cardiac injury. These data suggested that the pro-inflammatory cytokines might contribute to the pathogenesis of viral myocarditis.1.3The correlation between proinflammatory cytokines expression and the severity of VMC.To further investigate the involvement of pro-inflammatory cytokines in the pathological process of viral myocarditis, we analyzed the correlations between the proinflammatory cytokines expression levels in serum of VMC patients and cardiac function class (I as "1", II as "2", III as "3’", IV as "4"). The results showed that TNF-α IL-6, IL-1β, MCP-1levels were significantly positively correlated with cardiac function class (TNF-a, r=0.6834, P<0.0001; IL-6, r=0.5944, P<0.0001; IL-1β, r=0.4922, P=0.0002; MCP-1, r=0.4771, P=0.0001). In mouse VMC model, we also observed that the myocardial TNF-a, IL-6, IL-1(3, MCP-1levels were positively correlated with the body weight loss (TNF-a, r=0.6414, P=0.0005; IL-6, r=0.6185, P=0.001; IL-1β, r=0.5148, P=0.0085; MCP-1, r=0.6169, P=0.0010) and heart pathological score (TNF-a, r=0.8771; IL-6, r=0.9009; IL-1β, r=0.7935; MCP-1, r=0.8174; all P<.001), which confirming the essential participation of pro-inflammatory cytokines in the pathology of viral myocarditis.2. Astragalus treatment could modulate inflammatory response in VMC2.1Proinflammatory cytokines expression in VMC patients after Astragalus treatmentThe above results indicated that proinflammatory cytokines were essential mediators of pathological immune responses. To investigate the effect of Astragalus treatment on the inflammatory response in VMC patients, we analyzed the proinflammatory cytokines expression, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6and monocyte chemotactic protein-1(MCP-1) in serum of VMC patients after therapy. The results showed that there were no significant difference between Astragalus treatment group (n=30) and Routine treatment group (n=30) in the serum expression levels of TNF-a (96.13+6.14VS92.01±17.56), IL-6(364.6+67.77VS241.9+52.22), IL-1β (15.08±3.485VS15.1±3.094) and MCP-1(407.0±76.31VS376.9±75.29) before treatment. Two weeks after Astragalus intravenously injection, TNF-α (>10pg/ml) could be detected in10of30(33.3%) patients. The concentration was significantly decreased compared with that of pre-treatment. However, there was no significant change in the mean of TNF-a expression level of10detectable patients in Routine group between pre-treatment and2W post-treatment. IL-6was still detected in15of30(50%) patients, but the average expression levels were decreased from364.56pg/ml to39.36pg/ml after Astragalus treatment. The concentration of IL-6in the patients of Routine group was decreased to123.288pg/ml2W post-treatment. IL-1β was only (>0.1pg/ml) detected in5of30patients, and the mean of concentration was decreased to5.37pg/ml after Astragalus treatment, while there was no significant difference in the mean of IL-1β expression level of15detectable patients in Routine group between pre-treatment and2W post-treatment. MCP-1expression level was also remarkably lower2W post-treatment in Astragalus group (407.0±76.31pg/ml VS150.8±40.85pg/ml). No obvious change was observed between pre-treatment and2W post-treatment in Routine group. After a treatment course (more than3months post-treatment), nearly all the proinflammatory cytokines expression were significantly decreased both in Astragalus group and Routine group. The expression of TNF-α and IL-1β were undetectable in all the treating patients in Astragalus group. All the above data suggested that Astragalus treatment could efficiently reduce these cytokines expressions in a shorter time, in corresponding to a shorter recovery time from cardiac injury with Astragalus treatment, indicating that Astragalus treatment may modulate inflammatory response in VMC patients, consequently preventing the cardiac damage caused by inflammation.2.2The effect of Astragalus treatment on cardiac inflammation in VMC mouse modelTo explore the underlying mechanism of Astragalus for anti-inflammation in viral myocarditis precisely, we administrated a purified small molecular saponin Astragalosidc IV (AST-IV, C41H68O14,MW784),3-0-bcta-Dxylopyranosyl-6-0-beta-D-glucopyranosylcycloastragenol, the main active ingredient of Astragalus, in mouse viral myocarditis model. Male BALB/c mice were intraperitoneally injected with10J TCID50CVB3at day0to generate acute viral myocarditis model. Mice without inoculation were used as normal controls. The inoculated mice were received intragastric injections of two doses AST-Ⅳ(20mg/kg/d and40mg/kg/d) immediately after CVB3inoculation or the dissolvent of AST-Ⅳ as vehicle group. To examine the effect of AST-Ⅳ on proinflammatory response in CVB3mice, the proinflammatory cytokines expression including TNF-α, IL-6, IL-1(3and MCP-1in cardiac tissues and serum were assayed by ELISA on day7after AST-Ⅳdministration. The results showed that AST-Ⅳ treatment significantly decreased TNF-α, IL-1β, IL-6, and MCP-1expression levels both in cardiac tissues and serum when compared with vehicle group in dose dependent manner. Further, the inflammatory cell infiltrates were characterized by immunohistochemical analyses. Cardiac sections at day7were immunostained with immune cells marker anti-CD3and anti-CD11b. The results showed that upon infection the number of infiltrative CD3positive cells and CD11b positive cells were strongly increased within the myocardium at day7upon infection. However, reduced recruitment of the inflammatory cells at day7was observed in cardiac tissue of CVB3mice with AST-Ⅳ treatment. Obviously, higher dose AST-Ⅳ treatment (40mg/kg) showed more significantly decreasing recruitment of inflammatory cells. These results suggested that AST-Ⅳ treatment attenuated the activated inflammatory response in VMC by reducing proinflammatory cytokines expression and cardiac inflammatory cells infiltration, in corresponding with the clinical observation.We also evaluated the therapeutic efficiency of AST-IV on mouse viral myocarditis. Parameters of the severity of myocarditis, including body weight loss, survival rate, CK-MB activity, cTnI level, pathological features of cardiac sections and cardiac function were carefully studied. The results showed that AST-IV treatment could efficiently prevent CVB3mice from body weight loss, increase mice survival rate, relieve myocardial injury and improve cardiac function in dose-dependent manner.All the above data indicated that Astragalus modulating proinflammatory response conferred to its therapeutic effect on VMC.Part Ⅲ Astragalus modulating proinflammatory response in VMC was associated with suppression of NF-κB signalingIt is well accepted that NF-κB was a prototypical proinflammatory signaling pathway, based on the activation of large proinflammatory genes including cytokines, chemokines and adhesion molecules. We hypothesized that AST-Ⅳ would inhibit CVB3activated NF-κB signaling to restrict the inflammatory response. To determine the effect of AST-Ⅳ on NF-κB signaling, mice were intragastrically injected with40mg/kg/d AST-Ⅳ or vehicle immediately after CVB3inoculation. The cytoplasmic and nuclear protein was extracted from heart homogenates at day4. There was a significant increase in the levels of phosphorylation of IκBα and p65-NF-κB subunit which are indicators of NF-κB signaling activation, in heart tissues of CVB3-infected mice treated with vehicle, as well as an increase in the binding activity of heart nuclear extracts to a NF-κB consensus sequence compared with control mice. However, heart tissues from CVB3-infected mice treated with AST-IV showed lower levels of the phosphorylation of IκBα and p65when compared with vehicle group mice. The NF-κB DNA binding activity was also significantly decreased after AST-Ⅳtreatment. These data showed that AST IV effectively inhibited NF-κB signaling activation induced by CVB3in vivo.To further investigate whether AST-Ⅳ could inhibit CVB3-induced NF-κB signaling activity in vitro, the primary cardiac myocytes were purified from neonatal BALB/c mice and pre-treated with100μg/ml AST-Ⅳ or its solvent DMSO as control. Then the cardiac myocytes were treated with CVB3for0,1,2,4,6,8h. Cell lyses and nuclear extracts were prepared at the indicated time points and subjected to western blot analysis and ELISA-based transcription factor assay. It was found that AST-Ⅳ treatment inhibited phosphorylation of cytosolic IκBα, and p65triggered by CVB3infection. The result of NF-κB DNA binding activity also showed that AST-Ⅳ treatment significantly decreased CVB3-induced NF-κB p65DNA binding activity. Proinflammatory cytokines expression mediated by NF-κB in the cells with AST-Ⅳ treatment was also significantly decreased. The same experiments were preformed in CVB3-infected immune cells isolated from spleen and the results also showed the inhibitory effect of AST-Ⅳ on CVB3activated NF-κB signaling.All the above data indicated that AST-Ⅳ significantly inhibited NF-κB signaling activation in viral myocarditis, which might be one underlying mechanism contributing to its anti-inflammation effect.Part Ⅳ A20was a potential target for Astragalus in inhibiting NF-κB signaling activation in VMC1. Astragalus upregulated A20expressionUbiquitination is a post-translational modification of proteins that is recognized as a fundamental regulatory mechanism of signal transduction. Polyubiquitination has been demonstrated to be involved in NF-κB signaling. K63polyubiquitin chain linkages or linear polyubiquitination mediate a low affinity binding of other proteins that contain specific ubiquitinbinding domains. This can stabilize transient protein-protein interactions and is widely used in NF-κB signaling. Ubiquitination is reversed by Deubiquitinases (DUB) which are proteases that cleave ubiquitin chains from their substrate, making ubiquitination a very dynamic process. Several DUBs such as A20, CYLD, USP21and Cezanne, have been demonstrated to remove K63-linked ubiquitin chains from specific target substrates in the NF-κB signaling pathway, thus negatively regulating NF-κB activation. To investigate whether DUBs were involved in the inhibitory effect of AST-Ⅳ on CVB3-induced NF-κB signaling. We analyzed the mRNA expression of A20, CYLD, USP21and Cezanne in AST-Ⅳ (100μg/ml) treated cardiomyocytes with CVB3infection. The results showed that the expression of A20, CYLD, USP21and Cezanne were9.45,4.44,3.13,1.85folds higher than that of uninfected cells with AST-IV treatment6h after CVB3infection. A20expression could sustain high steady level, suggesting that AST-IV could significantly upregulate A20expression. The protein expression of A20assayed by western blot was also significantly increased after AST-IV treatment, which was in dose-dependent manner.Further, we analyzed the mRNA and protein expression of A20daily in the cardiac tissue of CVB3mice with AST-IV treatment (40mg/kg). The results showed that A20mRNA was8.33folds higher than control group2days following CVB3infection and sustained elevated expression until8days, which suggested that AST-IV treatment resulted in high and sustained A20expression, as compared with normal mice and vehicle treated mice. The same results were obtained in the protein expression of A20.All these data indicated that AST-IV could upregulate and sustain the elevation of cardiac A20expression.2. A20was physiologically required for inhibiting CVB3-induced NF-κBsignaling by restricting TRAF6K63ubiquitination2.1A20inhibited CVB3-induced NF-κB signaling in vitroTo investigate whether A20could restrict CVB3-induced NF-κB signaling activity in vitro, the primary cardiac myocytes were purified from neonatal BALB/c mice and pre-infected with adenovirus (Ad-A20/Ad-LacZ) to overexpress A20or lentivirus (LV-shA20/LV-ctrl) to knock down A20for48h. Then the cardiac myocytes were treated with CVB3for0,1,2,4,6,8h. Cell lyses and nuclear extracts were prepared at the indicated time points and subjected to western blot analysis and ELISA-based transcription factor assay. It was found that CVB3triggered the phosphorylation of IκBα and p65-NF-κB subunit. A20over-expression mediated by Ad-A20inhibited phosphorylation of cytosolic IκBα and p65. But the phosphorylation levels were elevated after A20knock down. The result of NF-κB DNA binding activity also showed that CVB3significantly increased NF-κB p65DNA binding activity, A20over-expression significantly attenuated this effect. But the binding activity was exaggerated after A20knock down. We also analyzed the expression of proinflammatory cytokines, which was decreased in A20overexpressed cells and increased in A20knock down cells following CVB3infection. The same results were observed in CVB3-infected immune cells isolated from spleen and showed the inhibitory effect of AST-IV on CVB3activated NF-κB signaling.Because phosphorylation of IκBα is mediated through IκBα kinase (IKK) activation, then the activity of IKK signalosome was analyzed by performing kinase assays on lysates from CVB3stimulated cardiac myocytes, which were pre-infected with Ad-A20/Ad-LacZ or LV-shA20/LV-ctrl. The results showed that Ad-A20infected cells displayed impaired IKK activity when compared to Ad-LacZ infected cells and LV-shA20infected cells augmented IKK activity when compared to LV-ctrl cells.These findings suggested that A20was physiologically required for inhibting CVB3-induced NF-κB signaling in vitro.2.2A20restricted endogenous TRAF6K63ubiquitinationTo better understand the molecular mechanism by which A20inhibited CVB3-induced NF-κB signaling, we considered that A20is an ubiquitin-editing enzyme that has been reported to directly remove K63-linked polyubiquitin chains of receptor interacting protein1(RIP1), TNF receptor associated factor6(TRAF6) and RIP2, which are critical in signaling to the IKK complex activation and downstream phosphorylation of IκBα The above data showed that A20was able to restrict IKK activity and the phosphorylation of IκBα induced by CVB3. To determine which targeting protein for A20may involve in its inhibitory effect on CVB3activated NF-κB signaling, cardiac myocytes pre-infected with Ad-LacZ or Ad-A20were treated with CVB3, lysed at the indicated time point, immunoprecipitated with specific antibodies for RIP1, TRAF6and RIP2respectively, and the K63-linked ubiquitination status of these proteins were tested by immunoblotting for ubiquitin. The whole cell lysates (WCLs) were directly subjected to immunoblot analysis of RIP1, TRAF6, RIP2, A20and (3-actin as loading control. These experiments revealed that endogenous TRAF6, rather than RIP1or RIP2was obviously ubiquitylated in CVB3infected cardiac myocytes. Strikingly, the ubiquitylated levels of TRAF6were reduced in A20over-expressed cardiac myocytes, suggesting that A20may deubiquitylate TRAF6to inhibit CVB3activated NF-κB signaling. To further examine whether A20may physiologically regulate TRAF6ubiquitylation in CVB3-induced NF-κB signaling, the same endogenous TRAF6ubiquitylation assay was performed in A20knock down cardiac myocytes with lentivirus (LV-shA20) infection. The results showed that A20knock down led to an increase of CVB3-induced ubiquitylation of TRAF6. All these data indicated that A20restricted endogenous TRAF6ubiquitylation, by which it suppressed CVB3-induced NF-κB signaling.2.3A20inhibited CVB3-induced NF-κB signaling activation in vivo To investigate the inhibitory effect of A20on CVB3induced NF-κB signaling activation in vivo, mice were intravenously injected with saline or3×109pfu of either Ad-A20or Ad-LacZ virus3days before CVB3inoculation. The cytoplasmic and nuclear protein was extracted from heart homogenates at day4. There was a significant increase in the levels of the phosphorylation of IκBα and p65-NF-KB subunit in heart tissues of CVB3-infected mice treated with saline, which are indicators of NF-κB signaling activation, as well as an increase in the binding activity of heart nuclear extracts to a NF-κB consensus sequence compared with control mice (P<0.001). However, heart tissues from CVB3-infected mice treated with Ad-A20showed lower levels of the phosphorylation of IκBα and p65when compared with saline or Ad-LacZ treated group mice. The NF-κB DNA binding activity was also significantly decreased after Ad-A20treatment (P<0.01). These results indicated that A20significantly inhibited NF-κB signaling activation in VMC mouse model.3. A20was important for the inhibitory effect of Astragalus on NF-κB signaling in VMCTo investigate the effect of A20on NF-κB signaling in viral myocarditis mice with AST-Ⅳ treatment, we overexpressed or knock down A20expression in vivo. Mice were intravenously injected with3×109pfu of either Ad-A20or Ad-LacZ to overexpress A20, or2×10’pfu of either LV-shA20or LV-ctrl to knock down A203days before103TCID50dose of CVB3inoculation. Mice without inoculation were used as normal controls. The CVB3mice were intragastrically administrated with20mg/kg/d AST-IV in adenovirus injected mice and administrated with40mg/kg/d AST-Ⅳ in lentivirus injected mice immediately after CVB3inoculation. The cytoplasmic and nuclear protein was extracted from heart homogenates at day4. The results showed that the phosphorylation of IκBα and p65-NF-κB subunit was inhibited in CVB3mice with AST-IV treatment. And their phosphorylation levels were more significantly lowered in A20overexpression mice with low dose20mg/kg AST-Ⅳ treatment. By contrast, the phosphorylation levels were enhanced in A20knock down mice with high dose40mg/kg AST-Ⅳ treatment, indicating that A20knock down could abrogate the inhibitory function of AST-IV on CVB3-induced NF-κB signaling. The NF-κB DNA binding activity was also much more lower in A20overexpressed mice and higher in A20knock down mice with AST-IV treatment, in line with the above results.These data indicated that Astragalus inhibited NF-κB signaling in viral myocarditis, at least in part, through upregulation of A20, suggesting that A20was a potential target for Astragalus in inhibiting NF-κB signaling activation in VMC.Part V A20alleviated viral myocarditis and was critical for the therapeutic effect of Astragalus on VMC1. A20overexpression impaired cardiac inflammation and mediated protectionagainst CVB3-induced myocarditis1.1The effect of A20on cardiac inflammation in VMCTo investigate the effect of A20on cardiac inflammation in CVB3-infected mice, mice were injected with3×109pfu of either Ad-A20or control (Ad-LacZ) virus intravenously immediately after CVB3infection. Heart tissues were collected at two day intervals and A20expression in heart homogenates was analyzed by western blot. The results showed that Ad-A20administration resulted in high and sustained cardiac A20protein expression from day0to10, as compared with normal mice and CVB3mice treated with saline or Ad-LacZ. The pro-inflammatory cytokines expression was measured by ELISA on day0,4,7,10respectively following CVB3infection. Compared with CVB3mice treated with saline or Ad-LacZ, significant decreases of TNF-a (P<0.01), IL-6(P<0.01) and IL-1β(P<0.05) were observed on both day4,7,10in the cardiac tissues of Ad-A20injected mice and the expression level of MCP-1was significantly lower on day4(P<0.01) and7(P<0.05) in the Ad-A20treated CVB3mice. These results indicated that Ad-A20treatment in vivo efficiently inhibited the production of pro-inflammatory cytokines in CVB3infected mice. Immunohistology was performed with cardiac sections at day7immunstained with anti-CD3and anti-CD11b antibody. The results showed that in Ad-A20treated mice there was a significantly reduced recruitment of both CD3+and CD11b+at day7. We also did flow cytometry analysis with cardiac cells. The results showed that A20could effectively reduced CD45+, CD3+, CD11b+inflammatory cells such as T lymphocytes and monocytes infiltrating into heart in CVB3infected mice. These data indicated that A20overexpression significantly impaired cardiac inflammation in VMC.1.2The protective effect of A20against VMCWe then investigated the protective effect of A20against CVB3-induced acute myocarditis. The results showed that Ad-A20adminstration in vivo prevented mice from body weight loss, improved survival rate, decreased serological CK, CK-MB, cTnl levels and attenuated myocardical inflammation showing few restricted mononuclear inflammation foci and tiny necrosis (P<0.05). All these data indicated that Ad-A20treatment could effectively protect mice from lethal myocarditis caused by CVB3infection.2. A20was critical for the therapeutic efficacy of Astragalus on VMC2.1The role of A20in the inhibitory effect of Astragalus on cardiac inflammationTo examine the role of A20on the proinflammatory response in viral myocarditis mice with AST-Ⅳ treatment, mice were intravenously injected with3×109pfu of Ad-A20or Ad-LacZ to overexpress A20, or2×107fu of either LV-shA20or LV-ctrl to knock down A203days before103TCID50dose of CVB3inoculation. Mice without inoculation were used as normal controls. The CVB3mice were intragastrically administrated with20mg/kg/d AST-IV in a...