
==== Front
Virol Sin
Virol Sin
Virologica Sinica
1674-0769
1995-820X
Wuhan Institute of Virology, Chinese Academy of Sciences

S1995-820X(24)00113-5
10.1016/j.virs.2024.07.003
Research Article
Baicalein suppresses Coxsackievirus B3 replication by inhibiting caspase-1 and viral protease 2A
Dong Yanyan a
Shao Enze a
Li Siwei a
Wang Ruiqi a
Wang Dan a
Wang Lixin a
Yang Hong a
He Yingxia a
Luan Tian a
Chen Yang b
Wang Yao a
Lin Lexun a
Wang Yan b
Zhong Zhaohua zhongzh@hrbmu.edu.cn
b⁎
Zhao Wenran zhaowr@hrbmu.edu.cn
a⁎
a Department of Cell Biology, Harbin Medical University, Harbin 150081, China
b Department of Microbiology, Harbin Medical University, Harbin 150081, China
⁎ Corresponding authors. zhongzh@hrbmu.edu.cnzhaowr@hrbmu.edu.cn
16 7 2024
8 2024
16 7 2024
39 4 685693
9 1 2024
12 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Myocarditis is an inflammatory disease of the cardiac muscle and one of the primary causes of dilated cardiomyopathy. Group B coxsackievirus (CVB) is one of the leading causative pathogens of viral myocarditis, which primarily affects children and young adults. Due to the lack of vaccines, the development of antiviral medicines is crucial to controlling CVB infection and the progression of myocarditis. In this study, we investigated the antiviral effect of baicalein, a flavonoid extracted from Scutellaria baicaleinsis. Our results demonstrated that baicalein treatment significantly reduced cytopathic effect and increased cell viability in CVB3-infected cells. In addition, significant reductions in viral protein 3D, viral RNA, and viral particles were observed in CVB3-infected cells treated with baicalein. We found that baicalein exerted its inhibitory effect in the early stages of CVB3 infection. Baicalein also suppressed viral replication in the myocardium and effectively alleviated myocarditis induced by CVB3 infection. Our study revealed that baicalein exerts its antiviral effect by inhibiting the activity of caspase-1 and viral protease 2A. Taken together, our findings demonstrate that baicalein has antiviral activity against CVB3 infection and may serve as a potential therapeutic option for the myocarditis caused by enterovirus infection.

Highlights

• Baicalein shows inhibition on CVB3 replication both in vitro and in vivo.

• Baicalein can dramatically alleviate myocarditis induced by CVB3.

• Baicalein targets the early stage of CVB3 infection.

• Baicalein inhibits viral 2A protease of CVB3 and suppresses caspase-1.

Keywords

Baicalein
Coxsackievirus B (CVB)
Myocarditis
2A protease
Caspase-1
==== Body
pmc1 Introduction

Coxsackieviruses are single-stranded RNA viruses belonging to the Picornaviridae family and the Enterovirus genus. Infections caused by Group B Coxsackievirus (CVB) are common worldwide (Garmaroudi et al., 2015). There are six known serotypes of CVB (CVB1–6), which are the causative pathogens for a variety of diseases including gastrointestinal illness, myocarditis, encephalitis, meningitis, and type I diabetes (Bopegamage et al., 2021; Chung et al., 2022; Gaaloul et al., 2014; Nekoua et al., 2022). CVB3 infection is the primary cause for viral myocarditis, which is the inflammation of the cardiac muscle. If not recovered, myocarditis may progress to dilated cardiomyopathy or even heart failure (Gaaloul et al., 2014; Yip et al., 2023).

The genome of CVB is a single-stranded, positive-sense RNA of approximately 7.4 ​kb nucleotides encoding 11 proteins in a single open reading frame (ORF), flanked by 5′untranslated region (5′UTR) and 3′UTR (He et al., 2001; Tu et al., 1995). CVB encoded 11 proteins, including four capsid proteins (VP1–VP4) and seven non-structural proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D) (Organtini et al., 2014; Wang et al., 2019). Structural proteins (VP1–VP4) form viral icosahedral capsid structure (Muckelbauer et al., 1995), while the non-structural proteins are involved in viral RNA replication, viral polyprotein processing, and the manipulation of cellular activities (Chau et al., 2007; Wu et al., 2016). 3D, the RNA-dependent RNA polymerase (3Dpol) of CVB, is responsible for the replication of viral genome (Gazina et al., 2011). Protease 2A and 3C (2Apro and 3Cpro) facilitate the processing of viral polyprotein, which is directly translated from the positive-sense viral genomic RNA (Palmenberg, 1987). Therefore, 2Apro and 3Cpro are essential for sustaining viral protein maturation and the assembly of viral particles (Kim et al., 2015; Laitinen et al., 2016). Moreover, viral proteases also cleave various cellular proteins, such as eukaryotic initiation factor 4G (eIF4G) (Chau et al., 2007), adenosine-uridine (AU)-rich element RNA binding factor 1 (AUF1) (Wong et al., 2013), mitochondrial antiviral signaling protein (MAVS), and Toll/IL-1 receptor domain containing adaptor inducing interferon-beta (TRIF) (Mukherjee et al., 2011). By cleaving these cellular proteins, CVB can manipulate the cellular biosynthesis environment and suppress antiviral innate immunity (Mukherjee et al., 2011; Stone et al., 2021; Zhou et al., 2022). Due to the lack of vaccine, effective antiviral medication is crucial for controlling CVB infection. However, despite the ongoing efforts to understand the pathogenesis and develop antiviral drugs, effective anti-CVB therapy remains available. Therefore, identifying or repurposing antiviral drugs is important for addressing the health issues associated with CVB infection.

The roots of Scutellaria baicaleinsis (S. baicaleinsis), a medicinal plant, have been utilized for centuries in traditional medicine to combat inflammation, bacterial infections, and various disorders (Ming et al., 2018; Zhao et al., 2019). Compounds isolated from S. baicaleinsis demonstrate diverse pharmacological activities, including antitumor, antioxidant, liver protection, and antiviral effects (Wang et al., 2018b). Baicalein (5,6,7-trihydroxyflavone) and baicalin (5,6-dihydroxy-7-O-glucuronide flavone) are the active ingredients of S. baicaleinsis. Baicalin is the glucuronide form of baicalein (Moghaddam et al., 2014) (Fig. 1A). An in vitro study showed that while both baicalein and baicalin exhibited antiviral activity against ZIKA virus replication, the two compounds seemed to function through distinct mechanisms (Oo et al., 2019). Oral administration of a novel crystal form of baicalein significantly suppressed SARS-CoV-2 replication and alleviated lung injury caused by virus infection (Song et al., 2021). A previous study demonstrated the inhibitory effect of baicalin on CVB3 replication (Wang et al., 2020a). However, it remains to be elucidated whether baicalein exhibits antiviral effects against CVB3 infection.Fig. 1 Baicalein inhibits CVB3 replication in vitro. A The chemical structure of baicalein and baicalin. B CC50 of baicalein was determined by measuring cell viability. HeLa and HEK293T cells were grown in the medium supplemented with baicalein at various concentrations for 24 ​h. Cell viability was measured using Cell Counting Kit-8 (n ​= ​6). The experiment has been repeated three times and one of the typical results was presented. C, D The dose-dependent inhibition of baicalein on virus replication was determined. HeLa cells were infected with CVB3 at MOI of 1 and treated with baicalein at various concentration for 24 ​h. Cells were collected and subjected to RT-qPCR (C) and Western blotting (D). E The EC50 of baicalein was determined by TCID50 assay. HeLa and HEK293T cells were infected with CVB3 at MOI of 1, and treated with baicalein at various concentrations for 24 ​h. Cells were collected and subjected to freeze–thaw three times to release viral particles, which were determined by TCID50 assay. F, G The antiviral effect of baicalein was determined in the cells infected with CVB3 at different MOI. HeLa (F) and HEK293T (G) cells were infected with CVB3 and treated with baicalein (50 ​μmol/L) for 24 ​h. Cells were harvested and subjected to RT-qPCR and Western blotting. H Overall production of viral particles at different time points after CVB3 infection. I, J The inhibition of baicalein on CVA16 replication was determined. HeLa cells were infected with CVA16 (MOI ​= ​1) for 24 ​h. Cells were harvested and subjected to the analysis of RT-qPCR (I) and Western blotting (J). The experiment has been repeated three times and one of the typical results was presented. Quantifications of Western blots were performed with ImageJ. ∗, P ​< ​0.05; ∗∗, P ​< ​0.01; ∗∗∗, P ​< ​0.001; hpi: hour of post-infection; BAI: baicalein.

Fig. 1

2 Materials and methods

2.1 Mice

Newborn Balb/c mice aged 2–7 days after birth were provided by the Laboratory Animal Center of Harbin Medical University (Harbin, China). Mice were housed in specific pathogen-free environments at 25 ​± ​1 ​°C with 40%–50% humidity and allowed to access food and water ad libitum. To generate a mouse model of viral myocarditis, newborn mice aged 7 days after birth were intraperitoneally inoculated with CVB3 (1 ​× ​106.5 TCID50). Baicalein (10 ​mg/kg body weight) was administered intraperitoneally once per day, starting at 24 ​h post-infection (p.i.). Control mice were treated with Dulbecco's Modified Eagle Medium (DMEM). Mice were euthanized at day 5 p.i. Mouse ventricles were subjected to histological examination and the extraction of RNA and protein. Changes in body weight of the mice per day were monitored.

2.2 Cell culture

HeLa and HEK293T cells were grown in a humidified incubator with 5% CO2 at 37 ​°C. Cells were maintained and passaged in DMEM (Thermo Fisher, Shanghai, China) supplemented with 10% (v/v) fetal bovine serum (FBS) (Bioindustry, Israel), penicillin (100 ​μg/mL) and streptomycin (100 ​μg/mL). After virus inoculation, cells were maintained in the medium containing 2% FBS.

2.3 Virus

CVB3 woodruff (GenBank: U57056.1) was kindly provided by the Scripps Research Institute (San Diego, USA). Viruses were amplified in HeLa cells and stored at −80 ​°C. The virus stock solution was subjected to three freeze–thaw cycles and centrifuged at 100×g for 5 ​min, and the supernatant was collected for virus titration. Viral titers were determined by a 50% tissue culture infectious dose (TCID50) assay as previously described (Wang et al., 2020b). To determine the half-maximal effective concentration (EC50), cells were infected with CVB3 (MOI ​= ​1) and treated with baicalein at various concentrations for 24 ​h. Cells were collected and subjected to TCID50 assay to determine the EC50.

2.4 Chemicals and antibodies

Baicalein (MCE, Shanghai, China) was dissolved and diluted with DMEM. Antibodies against GAPDH, EGFP, eIF4G, β-tubulin, β-actin, caspase-1, HRP-conjugated goat anti-mouse IgG (H ​+ ​L), and HRP-conjugated goat anti-rabbit IgG (H ​+ ​L) were purchased from Proteintech (Wuhan, China). Polyclonal antibodies against 3Dpol and VP1 of CVB3 was prepared in our laboratory.

2.5 Transfection

Sequence encoding CVB3 2A protein was constructed into pEGFP-C1 to construct plasmid pEGFP-2A as described previously (Wu et al., 2014). Plasmids were amplified in E. coli DH5α strain (Takara, Dalian, China). Transfection was performed with Lipofectamine 3000 (Thermo Fisher) and P3000 reagent (Thermo Fisher) according to the protocol recommended by the providers. After transfection, cells were cultured for 24 ​h in fresh medium, harvested, and analyzed by Western blotting.

2.6 Western blotting

Cells were cultured in 6 or 12-well plates and allowed to proliferate to 90% confluency and treated with 100 ​μL of RIPA Lysis Buffer (Beyotime, Beijing, China) containing the protease inhibitor PMSF (100:1 in v/v) (Beyotime) for 15 ​min on ice. Cells were scraped off the culture plates, and the cell lysates were centrifuged at 13,800 ×g for 20 ​min at 4 ​°C to collect the supernatant. To extract proteins from tissues, mouse ventricles were collected and washed in cold PBS three times, followed by the homogenization for 5 ​min with RIPA Lysis Buffer containing the protease inhibitor PMSF. The homogenates were centrifuged at 13,800 ×g for 20 ​min at 4 ​°C. The supernatant was collected and stored at −80 ​°C. Proteins were separated on 10% or 12.5% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidenedifluoride (PVDF) membranes (Millipore, USA). Blots were visualized with FluorChem R system (ProteinSimple, Santa Clara, CA) and analyzed with ImageJ.

2.7 RNA extraction and real-time quantitative PCR

Cells were cultured to 90% confluence in 6 or 12-well plates, and total RNA was extracted with TRIzol (Life Technologies, Carlsbad CA). Real-time quantitative PCR (RT-qPCR) was performed according to the manufacturer's instructions. Briefly, 1 ​μg of total RNA was reverse transcribed in a 20 ​μL reaction. Quantitative PCR was performed by adding 2 ​μL of cDNA, 0.4 ​μmol/L of forward and reverse primers (Supplementary Table S1), 10 ​μL of 2 ​× ​TransStart Top Green qPCRSuperMix (Transgen, Beijing, China), and RNase-free water to make a total reaction system of 20 ​μL. PCR reactions were performed in a LightCycler 96 (Roche) for 45 cycles with denaturation at 94 ​°C for 5 ​s, annealing at 60 ​°C for 15 ​s, and extension at 72 ​°C for 10 ​s. Relative RNA quantification was calculated using the 2−ΔΔCT method and normalized to GAPDH. All reactions were performed in triplicate.

2.8 Cytotoxicity assay

Cells were treated with baicalein at various concentrations of for 24 ​h using Cell Counting Kit-8 (Beyotime, Wuhan, China). The 50% cytotoxic concentration (CC50) was defined as the concentration of the compound that reduces cell viability by 50%.

2.9 Histopathology

Mouse hearts were collected, washed with PBS, and fixed in 4% formalin at 4 ​°C for 24 ​h. The fixed tissue was embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE).

2.10 Statistical analysis

All experiments were repeated three times. Quantitative data were expressed as mean ​± ​SD. Student's t-test was used to analyze the results. P value less than 0.05 is considered as statistically significant. Data were analyzed using Graphpad Prism 8.

3 Results

3.1 Baicalein inhibits CVB3 replication in vitro

Both baicalin and baicalein are active flavonoids derived from S. baicaleinsis (Akao et al., 2000). Previous study has indicated that baicalin has the ability to inhibit CVB3 replication (Wang et al., 2020a). However, it remains to be elucidated whether baicalein also exhibits anti-CVB3 effect. It is important to note that the structures of baicalin and baicalein are not identical (Fig. 1A). Prior to assessing the antiviral efficacy of baicalein, the cytotoxicity of baicalein was evaluated in HeLa and HEK293T cells, yielding CC50 values of 139.39 ​μmol/L and 112.55 ​μmol/L, respectively (Fig. 1B). The dose-dependent antiviral effect of baicalein was determined in HeLa cells (Fig. 1C and D). The results demonstrated that baicalein treatment significantly reduced the levels of viral RNA (Fig. 1C) and viral 3Dpol (Fig. 1D) in a dose-dependent manner.

The EC50 of baicalein against CVB3 was determined using the TCID50 assay (Fig. 1E). These results show that the EC50 of baicalein determined with HeLa and HEK293T cells is 45.01 ​μmol/L and 38.12 ​μmol/L, respectively. The selectivity index (SI, calculated by CC50/EC50) is 3.1 (139.39/45.01) and 2.95 (112.55/38.12). Therefore, a concentration of 50 ​μmol/L of baicalein, which did not significantly reduce cell viability (Fig. 1B), but markedly inhibited viral replication (Fig. 1C–E), was selected for further investigation of the antiviral efficacy of this compound.

The antiviral effect of baicalein was further assessed in HeLa and HEK293T cells infected with various titers of CVB3 and treated with 50 ​μmol/L baicalein. The results showed that even when cells were infected with high titer of CVB3, baicalein treatment still led to a significant reduction in both viral RNA and viral protein 3Dpol (Fig. 1G and F). Additionally, the overall production of viral particles (Fig. 1H) was reduced at different time points after infection. These results indicate that baicalein exhibits anti-CVB3 activity.

To investigate the inhibitory effect of baicalein on other enteroviruses, we assessed the replication of Coxsackievirus A16 (CVA16) in the presence or absence of baicalein (Fig. 1I and J). The results showed a significant reduction in both RNA and 3Dpol of CVA16 upon baicalein treatment, suggesting a potential inhibition of baicalein against other clinically relevant enteroviruses.

3.2 Baicalein targets the early stage of CVB replication

CVB replication cycle is a set of complex processes initiated by binding viral capsid protein to the host cell surface receptor, followed by uncoating and the release of viral RNA into the cytoplasm (Bergelson et al., 1997; Smyth and Martin, 2002). Once viral RNA is released into the cytoplasm, it immediately functions as mRNA, which is translated to produce viral proteins (Bonderoff and Lloyd, 2008). The period from virus adhesion to the release of viral RNA into the cytoplasm is approximately 2–3 ​h (Lopacinski et al., 2021; Baggen et al., 2018; Diep et al., 2019). To investigate the antiviral mechanism of baicalein, HeLa cells were infected with CVB3 at MOI of 1 and baicalein was added at different time points. Then the viral protein quantity was measured at 9 ​h after infection (Fig. 2A). Our results demonstrate that the production of viral 3Dpol was completely inhibited when baicalein was added at 0 or 1 ​h p.i. (Fig. 2B and C). When baicalein was added at 2 or 3 ​h p.i., the 3Dpol level remained lower compared to the untreated cells. Administration of baicalein at 4 or 5 ​h p.i., did not yield any significant difference in 3Dpol levels between treated and untreated cells (Fig. 2B and C). These results indicate that baicalein exerts antiviral effect at the early stage of CVB3 infection.Fig. 2 Baicalein targets the early stage of CVB3 replication. A The time-of-addition procedure in which CVB3-infected HeLa cells were treated with baicalein at various time points post infection (p.i.). B HeLa cells were infected with CVB3 (MOI ​= ​1) and treated with baicalein as described in (A). Cells were collected and analyzed by immunoblotting. C Quantitative analysis of the immunoblotting results (B) was carried out by ImageJ. D–E The duration of baicalein's antiviral activity. HeLa cells were cultured to 70% confluency. The culture medium was removed and cells were covered with CVB3 diluted with DMEM (MOI ​= ​1) and incubated at 37 ​°C for 1 ​h. The virus dilution was removed and cells were cultured in DMEM supplemented with 50 ​μmol/L of baicalein. Cells were collected at various time points p.i. (6, 9, 12, 24 ​h), and viral 3Dpol (D) and viral RNA (E) were determined by immunoblotting and RT-qPCR, respectively. The quantification of immunoblotting was performed with ImageJ. Three independent experiments were performed and representative results were presented. ∗∗, P ​< ​0.01; ∗∗∗, P ​< ​0.001; ns: not significant. BAI: baicalein; hpi: hour of post-infection.

Fig. 2

Furthermore, we determined the duration of baicalein's antiviral effect (Fig. 2D and E). HeLa cells were cultured to 70% confluency. The culture medium was removed and cells were covered with CVB3 diluted with DMEM (MOI ​= ​1) and incubated at 37 ​°C for 1 ​h. The virus dilution was removed and cells were cultured in DMEM supplemented with 50 ​μmol/L of baicalein. Cells were collected at different time points (6, 9, 12, 24 ​h p.i.), and viral 3Dpol (Fig. 2D) and viral RNA (Fig. 2E) were determined. We found that baicalein, which was added to the cell culture simultaneously with virus infection, almost completely blocked the production of viral protein 3Dpol at all-time points (Fig. 2D). Viral RNA levels were also markedly reduced in the cells treated with baicalein (Fig. 2E). These observations suggest that the antiviral effect of baicalein could last for at least 24 ​h.

3.3 Baicalein alleviates the myocarditis induced by CVB3

Viral myocarditis is one of the major diseases caused by CVB3 infection. Therefore, the antiviral activity of baicalein was further validated in mice infected with CVB3. Sucking Balb/c mice at day 7 after birth were infected with CVB3 (1 ​× ​106.5 TCID50) and treated with baicalein at 10 ​mg/kg (body weight) intraperitoneally. The first dose of baicalein was given at 24 ​h p.i. Mice were treated once a day for five consecutive days. The daily dose of baicalein was determined by pre-experiments which measured the no-effect dosage with the consideration of tolerability. Viral replication and myocardial damage were assessed at the end of day 5 p.i. The general condition and body weight of the mice were monitored and recorded per day. The surviving mice at day 5 p.i. are shown in Fig. 3A.Fig. 3 Baicalein alleviates myocarditis induced by CVB3 infection. A–C Newborn Balb/c mice were infected with CVB3 at 1 ​× ​106.5 TCID50 at day 7 after birth. The first dose of baicalein was given at 24 ​h post infection (p.i.). Mice were treated with baicalein once a day for five consecutive days. The overall health status of the mice was monitored. The mice that were alive on day 5 p.i. were presented (A). The body weight changes and mouse survival were recorded (B, C). D Histopathological changes of the mouse myocardium were analyzed by hematoxylin and eosin staining at day 5 p.i. E The production of inflammatory cytokines in mouse myocardium was determined by ELISA. (E–I) Total RNA and proteins of the mouse myocardium were extracted and analyzed by ELISA (E; n ​= ​3), RT-qPCR (F; n ​= ​3), and Western blotting (G; n ​= ​3). Three independent experiments were performed and representative results were presented (G). Western blots were quantified with ImageJ (H, I). ∗, P ​< ​0.05; ∗∗, P ​< ​0.01; ∗∗∗, P ​< ​0.001; BAI: baicalein.

Fig. 3

We found that CVB3-infected mice exhibited decreased daily activity, disrupted suckling behavior, and delayed weight gain (Fig. 3A and B). By day 5 p.i., over 90% (11/12) of CVB3-infected mice died, while 67% (8/12) of baicalein-treated virus-infected mice survived (Fig. 3C), with a body weight increase similar to sham-infected mice (Fig. 3B). Histopathological examination revealed severe myocarditis in the CVB3-infected mice characterized by damaged cardiomyocytes and diffuse infiltration of inflammatory cells, while baicalein treatment dramatically alleviated the myocardial damage (Fig. 3D). The production of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6, triggered by CVB3 infection was also significantly reduced in the myocardium treated with baicalein (Fig. 3E). Moreover, we found that baicalein treatment significantly reduced the levels of viral RNA (Fig. 3F) and viral protein 3Dpol in CVB3-infected myocardium (Fig. 3G and H). Furthermore, baicalein treatment also inhibited caspase-1 activation in CVB3-infected myocardium (Fig. 3I). These results demonstrate that baicalein effectively suppresses viral replication and alleviates myocarditis caused by CVB3 infection.

3.4 Baicalein inhibits viral protease 2A of CVB3

Upon virus entry into the host cells, the genomic RNA of CVB immediately functions as mRNA to translate viral polyprotein, which is autocatalytically processed into individual viral proteins. After the initial viral translation, viral proteases 2A and 3C are responsible for the polyprotein processing of CVB (Tapparel et al., 2013). According to our available data, baicalein exerts antiviral effects by inhibiting the early stage of CVB3 infection. However, we found that baicalein did not affect the entry of CVB3 into the cell (Supplementary Fig. S1). Therefore, we hypothesized that baicalein may target viral proteases, thereby inhibiting viral protein maturation and viral replication.

To validate the inhibitory effect of baicalein on viral 2Apro, we assessed the cleavage of eIF4G in the CVB3 2Apro expressing cells. Our results demonstrated that the cleavage of eIF4G induced by 2Apro of CVB3 was blocked by the treatment of baicalein at 10 ​μmol/L, whereas treatment with baicalein at higher concentrations (50 ​μmol/L or 100 ​μmol/L) completely blocked the cleavage of eIF4G (Fig. 4A and B). These observations indicate that baicalein effectively suppressed the activity of 2Apro.Fig. 4 Baicalein inhibits the activity of 2Apro of CVB3. A, B Dose-dependent inhibition of baicalein on 2Apro activity. HeLa cells were transfected with pEGFP-2A and treated with baicalein at various concentrations for 24 ​h. Cells were collected and cell lysates were analyzed by Western blotting. Control cells were transfected with empty vector pEGFP-C1. C–E The inhibitory effect of baicalein on CVB3 replication in 2Apro overexpression cells. HeLa cells were transfected with pEGFP-2A for 9 ​h, followed by the infection of CVB3 at MOI of 1 and baicalein treatment for 9 ​h. Control cells were transfected with pEGFP-C1. Cells were harvested and analyzed by Western blotting. Three independent experiments were performed and representative results were presented. Western blots were quantified with ImageJ. ∗, P ​< ​0.05; ∗∗∗, P ​< ​0.001; BAI: baicalein.

Fig. 4

To demonstrate the contribution of inhibited 2Apro to the antiviral mechanism of baicalein, HeLa cells were transfected with pEGFP-2A for 9 ​h, followed by the infection of CVB3 (MOI ​= ​1) for 9 ​h with baicalein treatment. Results showed that baicalein treatment blocked the cleavage of eIF4G and significantly reduced the VP1 level in CVB3-infected cells, while 2Apro overexpression resulted in the re-emergence of the cleaved eIF4G and a moderate increase in VP1 level. These observations indicate that inhibition of 2Apro contributes to the anti-CVB effect of baicalein. However, the activity of 3Cpro of CVB3 was unaffected by baicalein treatment (Supplementary Fig. S2).

3.5 Baicalein inhibits caspase-1 activation

Viral myocarditis is characterized by the infiltration of the inflammatory cells in the myocardium and the production of inflammatory cytokines, which play a critical role in the pathophysiology of myocarditis and dilated cardiomyopathy. Our previous study showed that caspase-1 inhibitor exhibited antiviral effect on CVB3 and Enterovirus A71 (EV-A71) (Wang et al., 2018a). Since baicalein significantly inhibited the production of proinflammatory cytokines, including IL-1b, in the myocardium infected with CVB3 (Fig. 3E), we hypothesized that this inhibition might be due to suppressed caspase-1 activity. To assess whether baicalein suppresses caspase-1 activation, we evaluated activated caspase-1 in the cells infected with CVB3. As shown in Fig. 5, baicalein treatment dramatically reduced the level of cleaved caspase-1 in CVB3-infected cells compared untreated cells (Fig. 5A and B). Furthermore, we observed that baicalein also inhibited caspase-1 activation induced by lipopolysaccharide (LPS) (Fig. 5C and D), suggesting that the inhibition of caspase-1 activation is the intrinsic property of baicalein.Fig. 5 Baicalein inhibits caspase-1 activation in the cells infected with CVB3. A, B The inhibitory effect of baicalein on caspase-1 activation in CVB3-infected cells. HeLa cells were infected with CVB3 at MOI of 1 and simultaneously treated with baicalein (50 ​μmol/L) for 12 ​h. Cell lysates were harvested and analyzed by Western blotting. C, D The inhibitory effect of baicalein on caspase-1 activation in the cells stimulated with LPS. HeLa cells were treated with LPS (100 ​ng/mL) in the presence or absence of baicalein (50 ​μmol/L) for 12 ​h. Cells were harvested and analyzed by Western blotting. E–G The inhibitory effect of baicalein on CVB3 replication in the cells with upregulated caspase-1 activation. HeLa cells were infected with CVB3 (MOI ​= ​1) and simultaneously treated with baicalein (50 ​μmol/L) or baicalein plus LPS (100 ​ng/mL). Cells were harvested and analyzed by Western blotting. Three independent experiments were performed and representative results were presented. Western blots were quantified with ImageJ. ∗, P ​< ​0.05; ∗∗, P ​< ​0.01; ∗∗∗, P ​< ​0.001. BAI: baicalein; procasp-1: procaspase-1; casp-1: caspase-1; LPS: lipopolysaccharide.

Fig. 5

To investigate whether inhibited caspase-1 activation contributes to the antiviral mechanism of baicalein, we examined viral VP1 and activated caspase-1 in CVB3-infected cells with enhanced caspase-1 activation induced by LPS treatment (Fig. 5E–G). HeLa cells were infected with CVB3 (MOI ​= ​1), and simultaneously treated with baicalein and LPS for 12 ​h. Our results demonstrate that LPS markedly elevated the levels of both cleaved caspase-1 and VP1, compared to CVB3-infected cells without LPS treatment (Fig. 5E, lane 5 vs lane 2; Fig. 5F and G). Baicalein treatment completely blocked the synthesis of VP1 in CVB3 infected cells (Fig. 5E: lane 3; Fig. 5G), whereas cells treated with both baicalein and LPS led to a slight but notable increase in VP1 level (Fig. 5E: lane 5; Fig. 5G). These findings indicate that caspase-1 activation promotes CVB3 replication, while suppressed caspase-1 contributes to the anti-CVB3 effect of baicalein.

4 Discussion

CVB3 is a significant pathogen associated with severe diseases, including viral myocarditis, meningitis, and encephalitis (Tao et al., 2012). CVB3 infection is also one of the causes for hand, foot, and mouth disease which primarily affect young children (Han et al., 2019; Liu et al., 2023). Due to the lack of vaccines against CVB3, antiviral therapy is critical for the control of viral infection.

The root of S. baicalensis has been used as herbal medicine for more than 2000 years. Over the past two decades, both S. baicalensis and its extracted chemical compounds have been reported to exhibit beneficial effects on various disorders such as tumor, hypertension, inflammation, and atherosclerosis (Wang et al., 2018b, 2023). A previous study demonstrated that baicalin, one of the flavonoids found in S. baicalensis, showed anti-CVB3 activity by inhibiting the formation of autophagosomes (Wang et al., 2020a), which are exploited by CVB3 replication (Wong et al., 2008). Given that both baicalin and baicalein are active flavonoids of S. baicalensis (Liang et al., 2017), we hypothesized that baicalein also has anti-CVB3 activity. Here we found that baicalein inhibited CVB3 replication and significantly alleviated the myocarditis caused by CVB3 infection. We demonstrated that the antiviral mechanism of baicalein is attributed to its inhibition on caspase-1 and viral 2Apro.

Both baicalin and baicalein are active flavonoids derived from S. baicalensis. A study showed that orally administrated baicalin was readily hydrolyzed to baicalein by rat intestinal bacteria and then restored to its original form after intestinal absorption (Akao et al., 2000). However, a pharmacokinetic study revealed that both baicalein and baicalin were detectable in plasma of rats followed by the intravenous administration of baicalein (Song et al., 2021), suggesting that the in vivo anti-CVB3 activity of baicalein, which was administered intraperitoneally in our study, is the combined effect of both baicalein and baicalin. Therefore, the antiviral property of baicalein against CVB3 should be attributed to both baicalein and baicalin. We demonstrated that baicalein showed antiviral effects during the early stages of CVB3 infection, and this antiviral effect of baicalein is associated with its inhibition of viral 2Apro and caspase-1. Whether or not this property is shared by baicalin remains to be investigated.

Despite their structural similarity, baicalein and baicalin, both active compounds of S. baicaleinsis, do not exhibit identical biological activities. Baicalein has been reported to inhibit SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) activity more potently than baicalin (Zandi et al., 2021). A cell-based study showed that baicalein potently inhibits intracellular Zika virus replication, whereas baicalin is most effective against virus entry (Oo et al., 2019). Molecular docking analysis suggests that baicalein differs from baicalin in the regulation of inflammation (Xiang et al., 2021). Moreover, the baicalein-rich fraction of S. baicalensis extract strongly induces the loss of mitochondrial membrane potential, in contrast to the baicalin-rich fraction. Baicalein (IC50 ​= ​2–3 ​μmol/L) also exerted stronger effect than baicalin (IC50 ​= ​5–26 ​μmol/L) in suppressing the accumulation of intracellular reactive oxygen species and reducing cytosolic Ca2+ ions (Shen et al., 2003). Therefore, we propose that in the case of CVB3 infection, the antiviral mechanism of baicalein may differ from that of baicalin. Nonetheless, further studies are needed to reveal the molecular basis of the antiviral properties of these flavonoids.

Our study further revealed that the suppression of caspase-1 activation contributes to the anti-CVB3 activity of baicalein. It has long been recognized that baicalein exhibits potent anti-inflammation effects (Chagas et al., 2022; Liao et al., 2021; Rui et al., 2020; Wang et al., 2021). Our previous study demonstrated that the activation of caspase-1, the critical regulator for the maturation and secretion of the pro-inflammatory cytokine interleukin 1-b (IL-1b) and IL-18 (Miao et al., 2011), is essential for the replication of EV-A71 and CVB3, with unknown mechanism. Furthermore, caspase-1 inhibitors such as VX765 and N-acetylcysteine exhibited antiviral effects against enterovirus infection (Wang et al., 2018a, 2020a). Based on these data, we hypothesize that the anti-CVB3 effect of baicalein is also associated with the suppression of caspase-1 activation. Our results indicate that, in the context of increased activation of caspase-1, the viral VP1 level is markedly upregulated. This finding supports our previous finding that caspase-1 activation favors CVB replication. When comparing CVB3-infected cells treated concurrently with LPS or LPS plus baicalein (Fig. 5E: lane 5 vs lane 4), we observed a dramatic decrease in both cleaved caspase-1 and VP1 levels following baicalein treatment. Since baicalein exhibits intrinsic inhibition on caspase-1 activation, suggesting that this inhibition contributes to its anti-CVB activity. The dual role of baicalein in relieving inflammation and inhibiting viral replication might explain the dramatically alleviated myocardial damage induced by CVB3 infection.

Targeting viral proteases is a typical strategy for antiviral therapy (Anirudhan et al., 2021). Both 2Apro and 3Cpro of CVB3 are viral proteases essential for virus replication (Chau et al., 2007). Moreover, 2Apro and 3Cpro also cleave multiple cellular proteins such as MAVS, RIG-I, and TRAF3 to suppress the innate immunity in response to CVB3 infection (Kemball et al., 2010). Therefore, blocking viral protease activity could not only inhibit viral replication, but may also relieve the suppression of the innate immunity. In this study, we showed that baicalein inhibited the protease activity of 2Apro but not 3Cpro of CVB3. However, further research is needed to determine whether this inhibition leads to improved innate immunity both in vitro and in vivo.

We also found that baicalein inhibits the replication of CVA16 (Fig. 1I and J), one of the important causative pathogens for hand, foot, and mouth disease (HFMD), likely by inhibiting caspase-1 and viral 2Apro. This is supported by the high amino acid homology of 2Apro between CVB3 and CVA16 (exceeding 80%), despite the average amino acid similarity of 2Apro among enterovirus is 50%–70% (Laitinen et al., 2016) (Supplementary Fig. S3). These data implicate that baicalein has the potential to be an antiviral agent for HFMD. Furthermore, it is worth investigating whether other flavonoids extracted from S. baicaleinsis also exhibit antiviral properties against CVB3 and other enteroviruses.

It is important to note that the very narrow application window of baicalein (from 0 ​h to 2 ​h after CVB3 infection) may limit its usage against enteroviruses infection, which is the limitation of the current study. Chemical modifications to baicalein may be necessary to enhance its pharmacological effect while minimizing adverse effects on the host.

5 Conclusions

In conclusion, our study demonstrates that baicalein inhibits viral replication and alleviates myocarditis caused by CVB. The antiviral mechanism of baicalein is related to its suppression on caspase-1 activation and the 2Apro of CVB.

Data availability

Raw data of this study can be found online at https://data.mendeley.com/datasets/2sr5bwb8hv/1 and ScienceDB (Doi:10.57760/sciencedb.11576).

Ethics statement

The Ethics Committee of Harbin Medical University approved the use of laboratory mice in this study. All experimental procedures were followed the guideline about the Use and Care of Laboratory Animals for Research of Harbin Medical University.

Author contributions

Yanyan Dong: Investigation, methodology, data curation, writing original draft. Enze Shao: investigation, methodology. Siwei Li and Yao Wang: methodology. Ruiqi Wang, Dan Wang, and Lixin Wang: methodology. Hong Yang, Yingxia He, and Yang Chen: methodology. Tian Luan and Lexun Lin: methodology. Yan Wang: Resources. Wenran Zhao and Zhaohua Zhong: conceptualization, funding acquisition, supervision, writing-review & editing.

Conflict of interest

The authors declare that there is no conflict of interests in this work.

Appendix A Supplementary data

The following are the Supplementary data to this article:Supplementary Material

Supplementary Material

Fig. S1 Baicalein does not block the entry of CVB3 into the cells. HeLa cells were cultured to 70% confluency. The culture medium was removed and cells were covered with virus diluted with DMEM (MOI = 100) supplemented with baicalein at 50 mol/L. Cells were incubated at 37 °C for 2 h to allow virus attachment and virus entry into the cells. Cells were then washed with PBS to remove attached viruses. Cells were collected and viral RNA was determined with RT-qPCR. Experiments were repeated three times. ns: non-significant. BAI: baicalein.

figs1

Fig. S2 Baicalein does not inhibit the activity of 3Cpro of CVB3. The cleavage of TDP-43 was determined to represent the activity of 3Cpro of CVB3. HeLa cells were transfected with pEGFP-3C for 24 h. Control cells were transfected with pEGFP-C1. Cell lysate was collected and subjected to Western blotting.

figs2

Fig. S3 The sequence homology of 2Apro between CVA16 and CVB3. The amino acid sequences of 2Apro between CVB16 and CVB3 are aligned. Amino acids with similar features are highlighted in blue. Amino acids with distinct features are highlighted in red. 2Apro sequence homology between CVA16 and CVB3 is 86%.

figs3

Acknowledgements

This study was supported by the 10.13039/501100001809 National Natural Science Foundation of China (82172247 and 81971920 to WZ, 82072278 to ZZ, and 82202493 to LL, 82302502 to Yao Wang), 10.13039/501100005046 Natural Science Foundation of Heilongjiang Province of China (YQ2023H003 ), 10.13039/501100002858 China Postdoctoral Science Foundation (2021M693818 ), and Heilongjiang Postdoctoral Fund, Heilongjiang Province of China (LBH-Z21023 ).

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.virs.2024.07.003.
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