NF-κB p65 signal pathway and protects mice from CVB3-induced virus myocarditis

Accepted Manuscript Astragalus polysaccharide from Astragalus Melittin ameliorates inflammation via suppressing the activation of TLR-4/NF-κB p65 signal pathway and protects mice from CVB3-induced virus myocarditis

Tianlong Liu, Mingjie Zhang, Haiyan Niu, Jing Liu, M.A. Ruilian, Yi Wang, Yunfeng Xiao, Zhibin Xiao, Jianjun Sun, Yu Dong, Xiaolei Liu PII: DOI: Reference:

S0141-8130(18)35541-7 https://doi.org/10.1016/j.ijbiomac.2018.12.207 BIOMAC 11366

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

16 October 2018 18 December 2018 21 December 2018

Please cite this article as: Tianlong Liu, Mingjie Zhang, Haiyan Niu, Jing Liu, M.A. Ruilian, Yi Wang, Yunfeng Xiao, Zhibin Xiao, Jianjun Sun, Yu Dong, Xiaolei Liu , Astragalus polysaccharide from Astragalus Melittin ameliorates inflammation via suppressing the activation of TLR-4/NF-κB p65 signal pathway and protects mice from CVB3-induced virus myocarditis. Biomac (2018), https://doi.org/10.1016/ j.ijbiomac.2018.12.207

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ACCEPTED MANUSCRIPT Astragalus polysaccharide from Astragalus Melittin ameliorates inflammation via suppressing the activation of TLR-4/NF-κB p65 signal pathway and protects mice from CVB3-induced virus myocarditis

Tianlong Liu1a, Mingjie Zhang1a, Haiyan Niu2, Jing Liu1, Ruilian MA1, Yi Wang1, Yunfeng Xiao2, Zhibin Xiao2, Jianjun Sun1, Yu Dong3*, Xiaolei Liu2*

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Author affiliations: a contributed equally 1 Department of Pharmacy, Affiliated Hospital of Inner Mongolia Medical University, 010059 Hohhot, PR China. 2 Department of Pharmacology, College of Pharmacy, Inner Mongolia Medical University, 010059 Hohhot, PR China. 3 Department of Natural Medicinal Chemistry, College of Pharmacy, Inner Mongolia Medical University, Hohhot 010110, PR China.

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*Correspondence to:

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Xiaolei Liu

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E-mail: [email protected] Phone: Tel: +86-0471-4306348;

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Department of Pharmacology, Inner Mongolia Medical University, Xinhua street, Huimin District, 010059 Hohhot, PR China.

Yu Dong

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Department of Natural Medicinal Chemistry, College of Pharmacy, Inner Mongolia Medical University, Hohhot 010110, PR China.

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E-mail: [email protected] Phone: Tel: +86-13644887629;

Running Title: AP protected heart from CVB3-induced myocarditis

ACCEPTED MANUSCRIPT Astragalus polysaccharide from Astragalus Melittin ameliorates inflammation via suppressing the activation of TLR-4/NF-κB p65 signal pathway and protects mice from CVB3-induced virus myocarditis

Abstract Inflammation plays a crucial role in regulating cardiomyopathy and injuries of coxsackievirus B3 (CVB3)-induced viral myocarditis (VM). It has been reported that Astragalus polysaccharide (AP) from Astragalus Melittin could inhabit inflammatory gene expression under a variety of pathological

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conditions. However, the functional roles of AP in CVB3-induced VM still remain unknown. Here, we found that AP significantly enhanced survival for CVB3-induced mice. AP protected the mice

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against CVB3-induced myocardial injuries characterized by the increased body weight and depressed serum level of creatine kinase-MB (CK-MB), aspartate transaminases (AST) and lactate dehydrogenase

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(LDH), enhanced left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS). At the pathological level, AP ameliorated the mice against CVB3-induced myocardial

damage, dilated cardiomyopathy and chronic myocardial fibrosis. We subsequently found that AP

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significantly suppressed CVB3-induced expression of inflammation marker (IL-1β, IL-6, TNF-α, INF-γ and MCP-1) in heart. Furthermore, we confirmed that AP suppressed the CVB3-induced expression of TLR-4 and phosphorylated NF-κB p65 in heart. Taken together, the data suggest that AP

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protects against CVB3-induced myocardial damage and inflammation, which may partly attribute to the regulation of TLR-4/NF-κB p65 signal pathway, moreover, suppressive effect of AP on

CVB3-induced activation of TLR-4/NF-κB p65 signal was TNF-α-independent.

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Keywords: Astragalus polysaccharide from Astragalus Melittin; Virus myocarditis; Inflammation;

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TLR-4/NF-κB p65 signal pathway

ACCEPTED MANUSCRIPT 1.Introduction VM (VM) is an important cause of heart failure and sudden death in young, previously healthy individuals(Corsten et al., 2012). Myocarditis has been estimated to account for up to 12% of sudden cardiac deaths in patients ,40 years of age, and 10% of biopsies from patients with unexplained heart failure had Signs of VM(Corsten et al., 2015). Previous study showed that VM was based on an adverse immune response evoked by infection of the cardiac muscle by cardiotropic viruses, such as the enteroviral CVB3, Human Herpesvirus 6, or Parvovirus B19 (PVB19)(Valaperti et al., 2013). Adverse immune response led to viral elimination as well as cardiac myocyte destruction, reparative

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fibrosis, and heart failure. Despite it remains elusive whether broad and a specific inhibition of the immune response will result in patient benefit, the overexpression of pro-inflammatory cytokines (such

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as TNF-α, IL-1 and IL-6) in heart significantly aggravated CVB3-induced myocarditis (Mason et al., 1995). Meanwhile, inhibiting the expression of the inflammatory cytokines by gene knockout,

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significantly improved myocardial injury(Li-Sha et al., 2017). The pathogenesis of VM is still unclear, so evidence-based therapies for VM are currently lacking in clinical(Cooper, 2009). Natural products from traditional Chinese medicine provided a lot of resources to exploit new

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therapeutic drugs of multiple diseases, such as cardiovascular diseases(Hao et al., 2017), immune system disease(Zhou et al., 2016) and cancer(Martel et al., 2017). Meanwhile, kinds of new biotechnology had been exploited to screen active compounds, including silico techniques(Liu et al.,

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2014), luminescent chemosensors techniques(Ma et al., 2017) and related database for Structure-activity relationship studies(Ito et al., 2018). Radix Astragali was primarily prescribed for the treatment of ‘Xin Ji’ which referred to ‘palpitation’ in Traditional Chinese Medicine, and a traditional Chinese medicinal herb derived from the root of Astragalus membranaceus with polysaccharides,

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flavonoids and saponins being the active constituents(2003; Song et al., 2008). Among those active constituents, polysaccharides was reported to have biological activities in myocardial preservation,

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such as two-way regulations of immunologic function, anti-virus, limiting myocardial infarction size, regulating metabolism of cardiac energy metabolism and inhibiting cardiac fibrosis(Yuan and Jing, 2011). Previous study showed that balancing the antiviral and inflammatory response in different phase will be important therapeutic approach of VM(Kuhl and Schultheiss, 2009; Tschope and Kuhl, 2016).

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Thus, we hypothesized that AP could regulate the CVB3-induced virus myocarditis. In the present study, we aimed to investigate the effects of Astragalus polysaccharide (AP) from Astragalus Melittin on CVB3-induced myocardial inflammation and injuries. Our study provides

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evidence to support a novel role of AP in protecting against CVB3-induced VM by suppressing the activation of TLR-4/NF-κB p65 signal pathway. 2. Materials and methods 2.1 Experimental animals and Coxsackievirus B3 plaque assay All animal protocols were approved by the Committee of affiliated Hospital of Inner Mongolia medical university on Ethics of Animal Experiments. Sixty male C57BL/6 mice (8–9 weeks) were purchased from Model Animal Research Center Of Nanjing University (Nanjing, China). All mice were housed in a specific pathogen-free environment under a 12 h/12 h light-dark cycle and fed rodent diet ad libitum. CVB3 (Nancy strain) was maintained by passage through HeLa cells. HeLa cells was used to perform

ACCEPTED MANUSCRIPT virus titer assay according to previous report(Van Linthout et al., 2011). Virus titer was determined prior to infection by a 50% tissue culture infectious dose (TCID50) assay on HeLa cell monolayer. Mice were infected by an intraperitoneal injection with 0.1 ml of PBS containing 103 TCID50 CVB3. 2.2 Induction of VM and experimental protocols C57BL/6 mice were randomly divided into three groups, namely control, VM and VM with AP treatment. AP were purchased from Pharmagenesis Inc., USA(Wang et al., 2015). Before VM was

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induced, AP treated mice received 200 mg/kg of AP (300 μL) per day by gavage to performed a pretreatment(Lv et al., 2017). Control and VM mice received 300 μL of saline orally per day. After 2 weeks pretreatment, VM and VM with AP treated mice were inoculated intraperitoneally with 0.1mL

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103 TCID50 of Coxsackievirus B3 to induced VM and control mice were inoculated intraperitoneally saline(Van Linthout et al., 2011). After virus was injection, VM with AP treatment mice continuously

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received 200 mg/kg of AP (300 μL) per day by gavage until the end of experiment.

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2.3 Echocardiography assessment

Echocardiography was used to evaluate cardiac hypertrophy, systolic and diastolic function after virus

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injection for 3 weeks. A Visual sonics high-resolution Vevo 2100 system (VisualSonics Inc., Toronto, Canada) was used. In brief, mice were anesthetized with3.0% isoflurane (Airflow velocity: 1L/min). After the pain reflex disappears and the heart rate stabilized at 400 to 500 beats per minute. Parasternal long-axis images were acquired in B-mode with appropriate positioning of the scan head and the

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maximum LV length identified. The M-mode cursor was positioned perpendicular to the maximum LV dimension in end-diastole and systole, and M-mode images were obtained for measuring wall thickness

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and chamber dimensions. Apical four-chamber view was acquired and the peak flow velocities during early diastole (E wave) were measured across the mitral valve. Early-diastolic peak velocity (E’ wave) of mitral valve ring was also measured in this view, then E/E’which reflected the left ventricular

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diastolic function were calculated. 2.4 Histological Analysis

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For histological analysis, hearts were arrested with a 10% potassium chloride solution at end-diastole and then fixed in 4% paraformaldehyde. Fixed hearts were embedded in paraffin and cut transversely into 5 μm sections. Serial heart sections were stained with hematoxylin and eosin (H&E) to measure degree of cardiac dilation and damage. The degree of collagen deposition was detected by picrosirius red (PSR) staining, and images were analyzed using a quantitative digital image analysis system (Image-Pro Plus 6.0). 2.5 Western Blotting and Quantitative Real-Time PCR Protein were extracted with radioimmunprecipitation assay (RIPA) buffer (50 mM Tris-HCl PH 7.4, 150 mM NaCl, 1mM EDTA, 0.25% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail). Homogenates were sonicated and centrifuged at 4°C for 15 minutes, and the supernatants were used for

ACCEPTED MANUSCRIPT western blotting. 20-50 μg of protein were separated by SDS-PAGE and transferred to a NC membrane (Millipore). After being blocked with 5% non-fat milk, the membranes were incubated with the following primary antibodies overnight at 4°C: anti-mouse p65-NF-κB, anti-phospho-p65-NF-κB (Cell Signaling Technology) and anti-GAPDH (Cell Signaling Technology). Subsequently, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology), and exposed to ECL reagent for detection of protein expression. Total RNA was extracted using TRIzol reagent (Invitrogen), and first-stand cDNA was synthesized using reverse transcriptase (Takara, Japan). Real-time PCR with SYBR Green (Takara, Japan) was performed to examine the relative mRNA levels of indicated genes. Sequences for real-time PCR

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primers are shown in supplemental table 1.

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2.6 ELISA assay

Cardiac phospho-NF-κB p65 expression level was measured by ELISA kit (7834, Cell Signaling

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Technology, Inc.). Blood was collected from the eye sockets at the end of experiment and separated into serum to measure the activities of lactic dehydrogenase (LDH), aspartate transaminases (AST),

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creatine kinase (CK) by using commercially available kits to identify myocardial injury. 2.7 TNF-α blockade with etanercept to study the effect of AP on CVB3-induced activation of

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TLR-4/NF-κB p65 signal

To further verified whether the suppressed effect of AP on CVB3-induced activation of TLR-4/NF-κB p65 signal associated with inhibiting TNF-α expression, TNF-α inhibitor etanercept (Et) was used to block TNF-α-induced activation of TLR-4/NF-κB p65 signal in heart. C57BL/6 mice were randomly

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divided into four groups, namely blank, VM, AP treatment (CVB3+AP) and AP treatment with TNF-α blockade (CVB3+AP+Et). AP treated mice (CVB3+AP group and CVB3+AP+Et group) were

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pretreated with 200 mg/kg of AP (300 μL) per day by gavage for 2 weeks before CVB3 injection. CVB3+AP+Et group mice were firstly injected with etanercept (1 mg/mL) subcutaneously 2 day prior to CVB3 injection, others were given PBS alone. Subsequently, etanercept or PBS were injected twice weekly until experimental end(Dufton et al., 2017; Matsumori et al., 2004). After 2 weeks pretreatment, 3

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VM mice, VM+AP mice and CVB3+AP+Et mice were inoculated intraperitoneally with 0.1mL 10

TCID50 of Coxsackievirus B3 to induced VM and control mice were inoculated intraperitoneally

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saline.

2.8 Statistical analysis Data were presented as mean±S.E.M. Differences among all groups were assessed using a one-way analysis of variance (ANOVA) followed by the Newman-Keuls post hoc test. A p-value less than 0.05 was considered statistically significant. 3. Results 3.1 AP ameliorates CVB3-induced virus myocarditis We firstly examined the effect of AP on CVB3-induced model of VM in mice. Within 3 weeks after

ACCEPTED MANUSCRIPT the CVB3 injection, Kaplan–Meier analysis revealed that there was a marginally significant difference in survival curves between the control and VM groups (p = 0.05), while AP treatment mice had a significant decreased mortality rate compared with VM mice (Figure 1A). Compared to control mice, VM mice a dramatic and continuous loss of bodyweight as maximal to 18.08%, while VM with AP treatment mice had a little fluctuation in bodyweight (Figure 1D). CK-MB, AST and LDH as critical myocardial enzyme were released to plasma when cardiomyocytes was destroyed under multiple Pathological conditions. Therefore, CK-MB, AST and LDH level in plasma were defined as biomarker of cardiac damage. In our study, CK-MB, AST and LDH level were significantly increased in plasma of VM mice, those were significantly ameliorated in VM with AP treatment mice (Figure 1C).

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Ultrasonic results show the overall echo of the heart is attenuated in heart of CVB3-induced mice, while those was improved in heart of AP treatment mice (Figure 2D). Mouse heart function exhibited a

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decrease in the cardiac systolic function (EF%) and diastolic function (E/E‘ IVS) in heart of CVB3-induced mice, while heart of AP treatment mice exhibited a better heart function than

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CVB3-induced heart. Meanwhile, a compensatory increase in left ventricular posterior wall diameter (LVPWd) was found in heart of CVB3-induced mice compared to control mice, while LVPWd has a significant increase in heart of AP treated mice compared to CVB3-induced mice. Left ventricular

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fractional shortening (FS) value were much lower in heart of VM mice than control mice, a significant increased FS was found in heart of AP treated mice compared with CVB3-induced mice (Figure 1E). These results demonstrate that AP treatment attenuated CVB3-induced mouse weight loss, cardiac

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damage and function dysfunction.

3.2 AP protect mice heart from CVB3-induced dilated cardiomyopathy and fibrosis

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To advanced illustrate the role of AP on CVB3-induced virus myocarditis, Histological analysis of heart sections was performed to examine degree of cardiac dilation and fibrosis.

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Histological analysis with haematoxylin and eosin (H&E) staining of heart sections revealed that the heart of VM mice appeared a significant dilation and cardiac damage after virus injection for 3 weeks, while those were significantly attenuated in heart of VM with AP mice (Figure 2A), the quantified data of cardiac damage area were showed in Figure 2B. The degree of collagen deposition in heart was

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detected by picrosirius red (PSR) staining. Compared with control mice, cardiac fibrosis level was found a significant increase in heart of VM mice, while those were ameliorated in heart of AP treated mice (Figure 2C), the quantified data of cardiac fibrosis was showed in Figure 2D. These results fibrosis.

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demonstrate that AP treatment had significant effect on CVB3-induced dilated cardiomyopathy and

3.3 AP treatment attenuated pro-inflammatory response in heart of CVB3-induced mice VM is characterized by cardiac inflammation that contributes to the impaired cardiac function, particular in subacute and chronic phase. Therefore, we examined the effect of AP treatment on pro-inflammatory factors expression in heart. The results illustrated that the expression level of TNF-α, IL-1β, IL-6 and MCP-1 had a significant increase in heart of CVB3-induced mice, while those were inhabited in heart of AP treated mice (Figure 3A). Consistent with these data, the expression level of TNF-α, IL-1β in heart were verified by immunohistochemical analyses (Figure 3B and C). These results demonstrated that AP treatment attenuated CVB3-induced pro-inflammatory factor expression

ACCEPTED MANUSCRIPT in heart. 3.4 AP treatment suppressed CVB3-induced activation of TLR-4/NF-κB p65 signal pathway Previous study showed that TLR-4/NF-κB p65 signal pathway could regulation inflammation reaction in heart(Gui et al., 2015). Therefore, we examined the expression level of TLR-4 and NF-κB p65 in heart. Quantitative real-time PCR analysis showed that the expression level of TLR-4 was significantly increased in heart of CVB3-induced mice, while those were inhabited in heart of AP treated mice (Figure 4A), which were verified by western blot (Figure 4B and C). We detected increased

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phosphor-NF-κB p65 in heart of CVB3-induced mice compared with control mice and a sharply increased phosphor-NF-κB p65 levels was found in heart of CVB3-induced mice compared with blank

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mice, which were inhibited in heart of mice with AP treated mice (Figure 4D). The expression level of inhibitor of kappa Bα (IκBα) was not found a significant difference among different treated mice

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(Supplemental figure 1). Besides, the expression level of phosphor-NF-κB p65 in heart was detected by ELISA kit, the result was consistent with western blot (Figure 4E). Together, these data show that AP

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suppressed CVB3-induced activation of TLR-4/NF-κB p65 signal pathway in heart. 3.6 Suppressive effect of AP on CVB3-induced activation of TLR-4/NF-κB p65 signal in heart was

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TNF-α-independent.

TNF-α also could activated NF-κB p65 signal by a positive feedback pathway(Cheng et al., 2015; Daniluk et al., 2012), we further verified whether the suppressed effect of AP on CVB3-induced activation of TLR-4/NF-κB p65 signal associated with inhibiting TNF-α expression. Whether

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etanercept-treatment or not, AP could protect heart from CVB3-induced mouse death, while TNF-α blockade did not significantly attenuate CVB3-induced mouse death comparing with AP treated mice

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(Supplemental figure 2). Both AP-treated mice and AP-treated mice with TNF-α blockade displayed reduced level of LDH and CK-MB in serum comparing with CVB3-induced mice, but no significant difference was found between them (Supplemental figure 3). TNF-α blockade had not a significantly synergistic effect of AP on protecting heart from CVB3-induced cardiac damage (Figure 6A) and

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fibrotic level (Supplemental figure 4). qPCR and western blot results showed that a significantly increased expression level of TLR-4 in heart was found in CVB3-induced mice comparing with blank mice, while AP could significantly inhibit CVB3-induced TLR-4 expression in heart, TNF-α blockade

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had not influence on AP bioactivity (Figure 6B and C). The expression level of Phospho-p65-NF-κB was significantly increased in heart of CVB3-indueced mice comparing with blank mice, which illustrated that Activation level of TLR-4/NF-κB p65 signal in heart of CVB3-induced mice was higher than blank mice, while this was attenuated in heart of AP treated mice, TNF-α blockade had not influence on AP inhibiting CVB3-induced expression of Phospho-p65-NF-κB (Figure 6D and E). Together, Suppressive effect of AP on activation of TLR-4/NF-κB p65 signal in heart was TNF-α-independent. 4. Discussion Inflammation reaction appeared after virus infected heart and followed through Pathological process of VM, including from acute phase (virus infection for 3–4 days), subacute phase (virus infection for 5–14

ACCEPTED MANUSCRIPT days) to chronic phase (after virus infection for 14 days)(Badorff and Knowlton, 2004; Mann, 2011;

Papageorgiou and Heymans, 2012). In acute and subacute phase of VM, myocytes, fibroblasts, endothelial cells, and dendritic cells (DCs) expressed a lot of pro-inflammatory cytokines, including interleukin-1b (IL-1β), IL-6, IL-18, tumor necrosis factor-α (TNF-α), and type I and type II interferons (IFNs) due to cardiac damage from virus infection and pathogen-induced immune reaction (Fuse et al., 2005). Those cytokines contributed to irreversible cardiac damage by downregulating protein synthesis and stimulating p53-mediated apoptosis(Shi et al., 2009). In chronic phase, regulatory T cells and alternatively activated (M2) macrophages secreted anti-inflammatory cytokines such as transforming growth factor-β (TGF-β) and IL-10 to promote resolution of the immune response and replacement of dead tissue by a

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fibrotic scar(Esfandiarei and McManus, 2008). Therefore, inhibiting virus-induced abnormal inflammation reaction was considered as an effectively therapeutic approach for virus myocarditis, especially for chronic

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phase (Jensen and Marchant, 2016). Indeed, many biological molecules, including protein, non-code RNA and compounds from natural product were reported to protect heart from CVB3-induced virus myocarditis

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via inhibiting inflammation reaction(Corsten et al., 2015; Jiang et al., 2017; Weithauser et al., 2013). In our study, a significantly decreased expression of pro-inflammation cytokines was found in heart of AP treated

mice compared with VM mice, which was an important reason for protective effect of AP on

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CVB3-induced cardiac dysfunction and fibrosis.

Before myocardial tissue was infiltrated by innate immune cells, pathogen-associated molecular

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patterns (PAMPs) as conserved motifs of pathogens and damage associated molecular patterns (DAMPs) were recognized by Toll-like receptors (TLRs) as pattern recognition receptors (PRRs) (Mann, 2011). Among TLRs, TLR4 as the highest expressional level for TLR mRNAs in the human heart was involved in multiple pathogens-induced inflammation reaction(Hori and Nishida, 2008). After the double-stranded RNAs of CVB3 virus as PAMPs was recognized by TLR4, TLR4 could activate NF-kB through MyD88 dependent pathway, activated NF-κB as a transcription factor transferred into the nucleus and facilitated the expression of pro-inflammation or inflammation cytokines (Cheng et al., 2015). Therefore, blockade of TLR4 was an effective approach to keep tissue structure and function, especially for disease associated with inflammation in heart (Dange et al., 2014). Meanwhile, some study had showed that NF-κB signaling could mediated

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cardiac inflammation reaction and metabolic remodeling in acute virus myocarditis, while inhibiting NF-κB signaling activation attenuated multiple pathogens-induced myocarditis via suppressed inflammation reaction(Matsumori et al., 2004; Remels et al., 2018). Our results showed that the

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expression level of TLR-4 and phospho- NF-κB p65 were found a significant increase in heart of CVB3-induced mice compared with control mice, while those were inhibited in heart of AP treated mice. Suppressed CVB3-induced activation of TLR-4/NF-κB p65 signal pathway may be an

important mechanism on inhibiting expression of inflammation cytokines in heart of AP treated mice.

Besides, expressed cytokines could also activate the NF-κB by a positive feedback pathway, including TNF-α(Cheng et al., 2015; Daniluk et al., 2012). TNF-α interacted with cell-surface receptor (TNFR1 and TNFR2) to facilitate expression of NF-κB, p38 mitogen activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) via TNFR-associated factor 2 (TRAF2)-dependent mode(Yao et al., 2014). However, the effect of TNF-α -induced NF-κB activation on cardiomyopathy was controversial. Some study showed that blockade of NF-κB

ACCEPTED MANUSCRIPT activation to attenuate inflammation reaction improved the TNF-α-induced cardiomyopathy(Kawamura et al., 2005). Tariq Hamid’s study showed that TNF-α interacting with TNFR1 and TNFR2 had opposing effects on remodeling, hypertrophy, NF-κB related inflammation response and apoptosis, TNFR1 exacerbates these events, whereas TNFR2 was improved(Hamid et al., 2009). In our study, the expression level of TNF-α was up-regulated in heart of CVB3-induced mice compared with normal mice, however, we didn’t know which receptors interacted with increased TNF-α in heart and what role it played.

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Astragalus species, the dry roots of Astragalus membranaceus, was widely used in traditional Chinese medicine as an antiperspirant, antihypertensive, diuretic, and tonic treatments(Wang et al., 2018). Previous study showed that Astragalus species and its extracts could protect heart from Ischemia/reperfusion injury(Jin et al., 2014), oxidative stress(Huang et al., 2016), and modulate immune reaction and cardiac energy metabolism(Han et al., 2017). For virus myocarditis, Gui J’s study showed that Astragaloside IV protected heart from CVB3-induced VM via modulating inflammatory response(Gui et al., 2015). Other monomeric compounds from Astragalus species were not reported the protective effect on virus myocarditis. Our results showed that AP could inhibit CVB3-induced activation of TLR-4/NF-κB p65 signal to protect heart from VM, moreover, suppressive effect of AP on CVB3-induced activation of TLR-4/NF-κB p65 signal was TNF-α-independent.

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In summary, our results demonstrated AP inhibits the progression of CVB3-induced cardiac dysfunction, dilated cardiomyopathy and fibrosis via suppressed activation of TLR-4/NF-κB p65 signal to ameliorate inflammation reaction.

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Acknowledgements

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Conflict of interest: none declared. Funding

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The study was supported by National Natural Science Foundation of China (81460066 to X.L.L).

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Figure 1 AP ameliorates CVB3-induced virus myocarditis. (A) Survival of mice was monitored within

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3 weeks post infection (n=20 per group). (B) The body weight change of mice was monitored within 3 weeks post infection (n=20 per group). (C)The levels of CK-MB, AST and LDH in serum

were measured by kit (n=10 per group). (D) Representative echocardiographic recordings from

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three groups. (E) E/E’ IVS , LVPWd and FS were assessed by echocardiographic examination after *

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CVB3 injection for 3 weeks (n=10 per group). P<0.05, P<0.01 and

with Blank group,

△

P<0.05,

△△

P<0.01 and

△△△

***

P<0.001 as compared P<0.001 as compared with CVB3 group.

Figure 2 AP protect mice heart from CVB3-induced dilated cardiomyopathy and fibrosis. (A) H&E staining of heart sections (scale bars:100μM). (B) Cardiac damage was quantified on haematoxylin-eosin staining (n=10 per group). (C) Picrosirius red-stained transverse sections of the left ventricles from the indicated groups (Scale bars:50 μM, n=6 per group). (D) Quantitative analysis of *

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cardiac fibrosis (n=10 per group). P<0.05, P<0.01 and

group,

△

P<0.05,

△△

P<0.01 and

△△△

***

P<0.001 as compared with Blank P<0.001 as compared with CVB3 group.

Figure 3 AP treatment attenuated pro-inflammatory response in heart of CVB3-induced mice. (A)

ACCEPTED MANUSCRIPT Expression level of different pro-inflammation cytokines in different groups after CVB3 injection for 3 weeks (n=10 per group). (B)Immunohistochemical analysis with cardiac sections of cardiac TNF-α and IL-6 expression after CVB3 injection for 3 weeks (Scale bars:50 μM). (C) Quantitative analysis of *

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positive area in cardiac sections (n = 10 per group). P<0.05, P<0.01 and

with Blank group,

△

P<0.05,

△△

P<0.01 and

△△△

***

P<0.001 as compared P<0.001 as compared with CVB3 group.

Figure 4 AP treatment suppressed CVB3-induced activation of TLR-4/NF-κB p65 signal pathway. (A) Expression level of TLR-4 in heart was measured by qPCR after CVB3 injection for 3 weeks (n=10 per group). (B) Western blot analysis of TLR-4 expression level in heart after CVB3 injection for 3 weeks.

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(C) Quantitative analysis of Western blot results (n=6 per group). (D) Western blot analysis of Phospho-p65-NF-κB expression level in heart after CVB3 injection for 3 weeks and quantitative analysis of Western blot results (n=6 per group). (E) Phospho-p65-NF-κB expression level in heart was

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*

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detected by ELISA after CVB3 injection for 3 weeks (n=5 per group). P<0.05, P<0.01 and

P<0.001 as compared with Blank group, compared with CVB3 group.

△

P<0.05,

△△

P<0.01 and

△△△

P<0.001 as

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***

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Figure 5 Suppressive effect of AP on CVB3-induced activation of TLR-4/NF-κB p65 signal was TNF-α-independent. (A) H&E staining of heart sections and cardiac damage was quantified on haematoxylin-eosin staining (scale bars:100μM, n=7-10 per group). (B) Expression level of TLR-4 in

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heart was measured by qPCR after CVB3 injection for 3 weeks (n=10 per group). (C) Western blot analysis of TLR-4 expression level in heart after CVB3 injection for 3 weeks and quantitative analysis of Western blot results (n=6 per group). (D) Western blot analysis of Phospho-p65-NF-κB expression level in heart after CVB3 injection for 3 weeks and quantitative analysis of Western blot results (n=6

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per group). (E) Phospho-p65-NF-κB expression level in heart was detected by ELISA after CVB3

P<0.05, **P<0.01 and ***P<0.001 as compared with △ △△ △△△ Blank group, P<0.05, P<0.01 and P<0.001 as compared with CVB3 group, ns: no significance.

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injection for 3 weeks (n=5 per group).

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Supplemental table 1 The primers of Real-time PCR Supplemental figure 1 Western blot analysis of IkBα expression level in heart after CVB3 injection for 3 weeks and quantitative analysis of Western blot results (n=6 per group).

Supplemental figure 2 Survival of mice was monitored within 3 weeks post infection (n=20 per group)

Supplemental figure 3 The levels of CK-MB (D,n=10 per group) and LDH (E, n=10 per *

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group) in serum were measured by commercially available kits. P<0.05, P<0.01 and

P<0.001 as compared with Blank group, △P<0.05, compared with CVB3 group, ns: no significance. ***

△△

P<0.01 and

△△△

P<0.001 as

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Supplemental figure 4 Picrosirius red-stained transverse sections of the left ventricles from the indicated groups (Scale bars:2cm, n=6 per group) and quantitative analysis of cardiac fibrosis (n=10 per

P<0.001 as compared with Blank group, △P<0.05, P<0.01 and △△△P<0.001 as compared with CVB3 group, ns: no significance. *

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***

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group). P<0.05, P<0.01 and

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