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. 2025 Nov 10;15:39207. doi: 10.1038/s41598-025-16941-2

Baicalin inhibits infectious bronchitis virus replication via MAVS-dependent interferon-β upregulation and mitochondrial function preservation

Jiongjie He 1, Huilin Guo 2, Ling Wang 1, Shengyi Wang 1,✉
PMCID: PMC12603211  PMID: 41214049

Abstract

The inhibitory effect of baicalin on infectious bronchitis virus (IBV) replication is closely related to expression of the mitochondrial antiviral signaling protein (MAVS) in chicken embryonic kidney (CEK) cells. Baicalin significantly upregulated MAVS expression dose-dependently and maintained mitochondrial function through MAVS. Baicalin increased expression of mitofusin-1 (Mfn1), anti-apoptotic mitochondrial import receptor subunit TOM70 (TOM70), voltage-dependent anion channel 1 (VDAC1), autophagy-related 5 (Atg5), and autophagy-related 12 (Atg12). Overexpression of Mfn1, VDAC1, TOM70, Atg5, and Atg12 enhanced baicalin-mediated interferon-β (IFN-β) expression, while knockdown of Mfn1, TOM70, VDAC1, TRADD, Atg5, or Atg12 significantly decreased IFN-β protein levels. In MAVS-knockdown cells, overexpression of Mfn1, mitofusin-2 (Mfn2), VDAC1, TNFR1-associated death domain protein (TRADD), Fas-associated death domain protein (FADD), Atg5, and Atg12 did not affect baicalin-mediated IFN-β expression. Thus, baicalin promotes mitochondrial fusion, permeability, metabolism, and autophagy. Baicalin upregulates IFN-β via Mfn1, VDAC1, TOM70, Atg5, and Atg12 dependent on MAVS. Moreover, baicalin inhibits key cytokines in the NF-κB pro-inflammatory pathway.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-16941-2.

Keywords: Baicalin, Infectious bronchitis virus, Mitochondrial antiviral signaling gene (MAVS), Mitochondrial function, Interferon-β, NF-κB pro-inflammatory pathway

Subject terms: Target validation, Antivirals

Introduction

Baicalin, with the molecular formula C21H18O11 (Fig. 1) and a molecular weight of 446.37, exhibits a wide range of pharmacological activities, including clearing heat and removing toxins; anti-tumor, anti-inflammation, anti-atherosclerosis, and neuroprotective effects; improvement of depressive behaviors; and regulation of the gut microbiota1. As a compound derived from traditional Chinese medicinal materials, baicalin has shown broad-spectrum antiviral activity2. Studies have shown that baicalin can inhibit the replication of hepatitis B and hepatitis C viruses to protect the liver3. With Zika virus, in vitro studies have demonstrated that baicalin can downregulate viral replication within 10 h post-infection4. Coxsackievirus is highly sensitive to baicalin, which can mitigate cell edema and necrosis while maintaining cellular function through its protective effect on Na+-K+-ATPase located in the cell membrane and calcium antagonistic activity5. The mechanism by which baicalin inhibits HIV may be linked to apoptosis6. In vivo studies have demonstrated that baicalin at a concentration of 100 pM exhibits the strongest inhibitory effect on rotavirus adsorption and enhances gluconeogenesis by upregulating the activity of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, mitigating the adverse effects of rotavirus on this metabolic pathway7. In respiratory viruses, baicalin has multiple mechanisms for inhibiting influenza virus activity, which include modulating the function of NS1 protein to enhance interferon biosynthesis and interacting with viral neuraminidase to improve symptoms in mice infected with influenza A H1N1 and H3N28. One study found that baicalin inhibits influenza viruses and is an effective inducer of IFN-γ9. Baicalin can effectively inhibit respiratory syncytial virus and reduce T-lymphocyte infiltration and proinflammatory factor gene expression, although the precise mechanisms underlying these effects require further systematic investigation10. Previous studies have shown that baicalin effectively inhibits the replication of infectious bronchitis virus (IBV), and this inhibitory effect is closely linked to the induction of IFN production11. Our preliminary results suggest that mitochondrial antiviral signaling protein (MAVS) has a crucial role in the inhibition of IBV replication by baicalin. Furthermore, baicalin can upregulate the expression of MAVS, which in turn activates production of interferon-β (IFN-β).

Fig. 1.

Fig. 1

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Powder and molecular formula of baicalin. (A) Powder form of baicalin. (B) Molecular formula of baicalin.

MAVS is a 540-amino acid protein encoded by a nuclear gene and is primarily located on the outer mitochondrial membrane, although it has also been found on peroxisomes and mitochondria-associated endoplasmic reticulum membranes9. MAVS comprises a proline-rich domain, an N-terminal caspase activation and recruitment domain (CARD), and a C-terminal transmembrane domain. The N-terminal CARD interacts with similar domains in retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) to thereby induce MAVS activation. The C-terminal domain is essential for MAVS localization to the outer mitochondrial membrane12. Innate immune signaling pathways make up a fundamental defense system that protects the body against pathogenic microorganisms. MAVS has a crucial role in innate immune signaling; the precise regulation of this protein is essential for defense against pathogenic microorganisms as well as maintaining homeostasis. MAVS expression can be modulated at transcriptional level through post-translational modifications, such as ubiquitination and phosphorylation, as well as via protein–protein interactions13. MAVS is also implicated in apoptosis and autophagy, with the latter potentially influencing MAVS levels14. In addition to the aforementioned regulation of MAVS, many viral proteins can prevent MAVS activation or downregulate its expression, which benefits viral replication in the host15. Upon infection, viruses can modulate innate immune signaling pathways by cleaving, degrading, or isolating MAVS, typically through viral non-structural proteins16. An outer mitochondrial membrane protein, MAVS has an important role in maintaining mitochondrial function17. Given that IBV is a respiratory pathogen that can seriously compromise mitochondrial structure and function, elevated expression of MAVS may help to maintain mitochondrial function in cells infected with IBV.

Building on the abovementioned studies and hypotheses, we investigated the role of MAVS in baicalin-mediated inhibition of IBV replication, focusing on activation of the IFN pathway and stabilization of mitochondrial function.

Results

Maximum nontoxic concentration of Baicalin

To determine the optimal dose of baicalin, cell viability was assessed using trypan blue staining with observation under an optical microscope, and further confirmed by MTT assay. The maximum non-toxic concentration (MNTC) of baicalin was determined to be 20 µg/mL, whereas concentrations > 20 µg/mL induced significant cell death (Fig. 2A). Cytotoxicity was corroborated by the MTT assay, which demonstrated nearly 100% cell viability following treatment with 20 µg/mL. Collectively, these results indicate that 20 µg/mL exerted no significant cytotoxic effects on cell viability (Fig. 2B).

Fig. 2.

Fig. 2

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The impact of baicalin on CEK cell viability. (A) Trypan blue staining was used to evaluate cell viability. Mock treated and baicalin treated cells (20 µg/mL) were stained with trypan blue. Cells excluding the trypan blue stain were considered to be viable. (B) Cell viability was determined by MTT assay after treatment with different concentrations of baicalin. A greater than 50% cell survival rate was considered to be the maximum non-toxic concentration of baicalin. Data are expressed as the mean ± SD of three independent experiments.

Antiviral effects of Baicalin in vitro

Given the rapid period of IBV replication occurs 24–36 h post infection, we chose to assess baicalin’s effects 24–36 h post infection. At indicated timepoints, the relative mRNA expression level of IBV was detected by qRT-PCR, viral titers of IBV were determined by TCID50, and the relative protein expression level of the IBV-N gene was detected by western blot to analyze the antiviral effects of baicalin (Fig. 3). Interestingly, treatment with 10 µg/mL or 20 µg/mL of baicalin could inhibit IBV replication, with the inhibitory effects associated with 20 µg/mL of treatment found to be the most significant (Fig. 3A). Further, increasing doses of baicalin led to significant decreases in IBV titers in a dose dependent manner, with 20 µg/mL baicalin having best anti-IBV action (Fig. 3B). Therefore, the concentration of 20 µg/mL baicalin was selected for subsequent experiments in vitro. The N protein is the main structural protein of IBV, is immunogenic, and has a large number of antigenic epitopes. The N gene is conserved in IBV, and as such is often used as a diagnostic reagent and a target protein for vaccine development19, as it positively correlates with IBV replication. In line with our results, the expression of the N protein could be inhibited by baicalin (Fig. 3C). These results indicate that baicalin can be used as a high quality natural drug to inhibit IBV infection.

Fig. 3.

Fig. 3

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The effect of baicalin on IBV mRNA expression levels, nucleocapsid N protein expression levels and viral titers of IBV in CEK cells. To investigate the impact of baicalin on IBV-infected cells, the relative mRNA expression levels of IBV (A), viral titers (B), and the relative expression levels of the IBV-N protein were detected (C). Untreated, infected cells was included as controls. Data are expressed as mean ± SD of three independent experiments (t-test, *p < 0.5, **p < 0.1, ***p < 0.05).

Confirming the inhibitory effects of Baicalin

In order to further confirm the inhibitory effects of baicalin on IBV-infected cells, the fluorescent signal of virus was detected by immunofluorescence assays (IFA) (Fig. 4). As expected, CEK cells infected with IBV produced a strong fluorescence signal 30 h after infection. On the contrary, the fluorescence signal of CEK cells infected with IBV and treated with baicalin was weakened, and the fluorescence signal decreased in a dose dependent manner with increases in the baicalin concentration. These findings further confirmed baicalin’s inhibitory effects on IBV infected cells.

Fig. 4.

Fig. 4

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The inhibitory effects of baicalin on infectious bronchitis virus (IBV) by immunofluorescence assays (IFA). (A) CEK cells were infected with 100 TCID50 IBV for 2 h at 37 °C followed by incubation with 20 µg/mL baicalin for 30 h. (B) CEK cells were infected with 100 TCID50 IBV for 2 h at 37 °C followed by incubation with 10 µg/mL baicalin for 30 h. (C) CEK cells were cultured in M199 media alone as negative control, (D) CEK cells were incubated with 100 TCID50 IBV as a positive control. The fluorescence intensity (×20) produced by IBV on CEK cells was photographed.

Effect of Baicalin on mRNA levels of IFN-α and IFN-β in vitro

The type I IFN response is an important immune defense mechanism against viral infection, which can induce infected cells to produce antiviral proteins, inhibit viral replication, or further induce T-cell-mediated acquired immune responses. Studies have found that once IBV infects the body, the host initiates the acquired immune response driven in large part by IFNs. Treatment of IBV-infected cells or animal models with type I IFNs can inhibit viral replication20. Therefore, we speculated that the inhibitory effect of Baicalin on viral replication may be related to its regulation of the type I IFN response. In order to study the effect of baicalin on Type I IFNs expression induced by IBV infection in CEK cells, the mRNA expression levels of IFN-α and IFN-β were determined. After 12–36 h post treatment, baicalin did not significantly affect the mRNA levels of INF-α after IBV infection, but slightly upregulated the expression of IFN-α at 48 h (Fig. 5A). In contrast, baicalin treatment significantly increased the expression of IFN-β 24–48 h post- IBV infection(Fig. 5B). These results suggest that baicalin could increase the expression of type I IFNs, specifically IFN-β, 36–48 h after IBV infection.

Fig. 5.

Fig. 5

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The effects of baicalin on mRNA expression levels of IFN-α and IFN-β in vitro. CEK cells were infected with 100 TCID50 IBV at 37 °C for 2 h, and then incubated with 20 µg/mL baicalin. CEK cells were treated with M199 media or 100 TCID50 IBV as controls. Total RNAs was extracted from CEK cell samples at 12, 24, 36, 48 and 60 h after treatment for analysis. Data are presented as mean ± standard deviation. *p < 0.5, **p < 0.1, ***p < 0.05.

Baicalin upregulates the expression of MAVS

To elucidate the regulatory effect of baicalin on key factors of the type I interferon (IFN) signaling pathway, we examined changes in the expression of these proteins at both transcriptional and translational levels in IBV-infected CEK cells treated with baicalin using qRT-PCR and western blot analysis. The results demonstrated that at the transcriptional level, baicalin significantly upregulated mitochondrial antiviral-signaling protein (MAVS) and significantly downregulated nuclear factor κB (NF-κB). At the translational level, baicalin still significantly upregulated MAVS and significantly reduced the IBV infection-induced phosphorylation of p65 and IκB-α. However, no significant alterations were observed in MDA5, tank-binding kinase 1 (TBK1), or toll-like receptor 3 (TLR3), or they exhibited no consistent regulatory pattern (Fig. 6). These findings suggest that baicalin likely inhibits IBV infection by modulating the MAVS-dependent interferon pathway and the NF-κB pro-inflammatory pathway.

Fig. 6.

Fig. 6

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Effects of baicalin on the expression of NF-κB pro-inflammatory pathway-related genes in vitro. Chicken embryonic kidney cells were incubated with infectious bronchitis virus (IBV) at a concentration of 100 TCID50 at 37 °C for 2 h. Cells were then incubated with 10 and 20 µg/mL baicalin for 36 h. Chicken embryonic kidney cells treated with M199 and 100 TCID50 IBV were used as controls. Expression of TNF-α, IL-1β, IL-6, TLR7, and MyD88 was detected at transcriptional (A) and translational (B) levels. Protein concentrations were measured using ImageJ software (C). Data are presented as mean ± standard deviation. *p < 0.5, **p < 0.1, ***p < 0.05. The symbols “+” and “−” in the figures indicate the addition and absence of components respectively.

Baicalin significantly suppressed the expression of key signaling factors within the pro-inflammatory pathway

We observed that both 10 and 20 µg/mL baicalin significantly inhibited NF-κB expression. This led us to hypothesize that baicalin might modulate the NF-κB pro-inflammatory pathway. Consequently, we assessed the mRNA and protein expression levels of crucial NF-κB pathway factors—TNF-α, IL-1β, IL-6, TLR7, and MyD88 in CEK cells (Fig. 6). The results demonstrated that baicalin significantly suppressed the expression of TNF-α, IL-1β, IL-6, TLR7, and MyD88 at both the transcriptional and translational levels. The inhibitory effect of baicalin on IL-6 and MyD88 expression was dose-dependent, with 20 µg/mL baicalin exerting a significantly stronger suppression than 10 µg/mL. In contrast, the inhibitory effects of 10 and 20 µg/mL baicalin on TNF-α and IL-1β expression showed no significant difference. Notably, 10 µg/mL baicalin suppressed TLR7 expression more potently than 20 µg/mL baicalin (Fig. 6A,B). Collectively, these findings indicate that baicalin, at therapeutic concentrations, inhibits key pro-inflammatory factors of the NF-κB pathway, thereby alleviating inflammatory responses induced by IBV infection in CEK cells.

Baicalin sustains mitochondrial homeostasis through MAVS-mediated regulation

Coronaviruses can potentially disrupt mitochondrial homeostasis and impair mitochondrial function. As a coronavirus, the impact of IBV on mitochondria is of special interest. Our findings indicate that following the infection of CEK cells with IBV, mitochondrial homeostasis and function were severely disrupted. Mitochondrial ATP levels (Fig. 7A ), mitochondrial oxygen consumption (Fig. 7B,C), and mitochondrial membrane potential (Fig. 7D) all showed significant declines. However, when baicalin was added to IBV-infected CEK cells, mitochondrial homeostasis, ATP levels, oxygen consumption, and membrane potential were significantly improved in comparison with untreated cells. The observed effects with 20 µg/mL baicalin were superior to those at 10 µg/mL (Fig. 7), indicating that baicalin has a dose-dependent effect on the maintenance of mitochondrial homeostasis.

Fig. 7.

Fig. 7

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Effects of baicalin on mitochondrial function in CEK cells. (A) Regulation of ATP production by baicalin in CEK cells infected with IBV. a: Under low glucose conditions, effects of baicalin at different concentrations on mitochondrial ATP production in CEK cells infected with IBV, with IBV-infected CEK cells without baicalin and wild-type CEK cells as positive and negative controls, respectively; b: under high glucose conditions, effects of baicalin at different concentrations on mitochondrial ATP production in CEK cells infected with IBV, with IBV-infected CEK cells without baicalin and wild-type CEK cells as positive and negative controls, respectively; c: under low glucose conditions, effects of baicalin at different concentrations on mitochondrial ATP production in MAVS-knockdown CEK cells infected with IBV, with IBV-infected MAVS-knockdown CEK cells without baicalin and MAVS-knockdown CEK cells as positive and negative controls, respectively; d: under high glucose conditions, effects of baicalin at different concentrations on mitochondrial ATP production in MAVS-knockdown CEK cells infected with IBV, with IBV-infected MAVS-knockdown CEK cells without baicalin and MAVS-knockdown CEK cells as positive and negative controls, respectively. (B) Effects of baicalin on mitochondrial oxygen consumption in CEK cells infected with IBV. a: Mitochondrial oxygen consumption in wild-type CEK cells infected with IBV and treated with different concentrations of baicalin; b: mitochondrial oxygen consumption in MAVS-knockdown CEK cells infected with IBV and treated with different concentrations of baicalin. (C) Four mitochondrial oxygen consumption parameters in CEK cells infected with IBV. a: Six mitochondrial oxygen consumption parameters in wild-type CEK cells infected with IBV and treated with different concentrations of baicalin; b: six mitochondrial oxygen consumption parameters in MAVS-knockdown CEK cells infected with IBV and treated with different concentrations of baicalin. (D) Mitochondrial membrane potential in CEK cells infected with IBV. a: Mitochondrial membrane potential measured by tetramethylrhodamine methylester staining in wild-type CEK cells infected with IBV and treated with different concentrations of baicalin; b: statistical analysis of mitochondrial membrane potential measured in a; c: mitochondrial membrane potential measured by tetramethylrhodamine methyl ester staining in MAVS-knockdown CEK cells infected with IBV and treated with different concentrations of baicalin; d: results of statistical analysis of mitochondrial membrane potential measured in c. Data are presented as mean ± standard deviation. *p < 0.5, **p < 0.1, ***p < 0.05.

MAVS is an outer mitochondrial membrane protein that is crucial for the innate immune response against RNA viruses and is regulated by mitochondrial dynamics and bioenergetics. Studies have shown that MAVS is closely related to the maintenance of mitochondrial homeostasis, and MAVS knockdown is detrimental to mitochondrial homeostasis17. To investigate whether the maintenance of mitochondrial homeostasis mediated by baicalin is associated with MAVS, the MAVS gene was knocked out. Mitochondrial ATP levels, oxygen consumption, and membrane potential were significantly decreased in MAVS-knockdown cells in comparison with wild-type cells, which confirmed that MAVS has a role in maintaining mitochondrial homeostasis. Furthermore, when MAVS-knockdown CEK cells were infected with IBV, mitochondrial ATP levels, oxygen consumption, and membrane potential were further decreased. The addition of 10 µg/mL baicalin did not enhance mitochondrial homeostasis in MAVS-knockdown cells, and 20 µg/mL baicalin led to a slight increase in mitochondrial ATP levels, oxygen consumption, and membrane potential (Fig. 7). These results indicated that baicalin-mediated maintenance of mitochondrial homeostasis is closely related to MAVS. Our findings demonstrate that 20 µg/mL baicalin treatment modestly enhanced ATP production and oxygen consumption rates even under MAVS knockdown conditions. This observation suggests that baicalin’s mitochondrial homeostasis maintenance may involve mechanisms beyond MAVS signaling. The residual mitochondrial protective effects could potentially be attributed to either through its established antiviral activity that reduces viral load, or alternatively, via modulation of other mitochondrial regulatory pathways independent of the MAVS axis. Further investigation is required to elucidate the precise compensatory mechanisms underlying this MAVS-independent mitochondrial stabilization.

Baicalin regulates genes involved in MAVS-mediated signaling

To further investigate the molecular mechanism by which baicalin maintains mitochondrial homeostasis, we systematically examined its regulatory effects on the expression of key genes involved in mitochondrial dynamics and quality control, including Mfn1, Mfn2, VDAC1, and TOM70. Additionally, we analyzed its influence on apoptotic regulators TNFR1-associated death domain protein (TRADD) and Fas-associated death domain protein (FADD), as well as autophagy-related proteins Atg5 and Atg12. These targets were selected based on their established roles in mitochondrial homeostasis maintenance and close functional association with mitochondrial antiviral-signaling protein (MAVS).

Studies have found that, as an outer mitochondrial membrane protein, MAVS is closely related to the mitochondrial fusion proteins Mfn1 and Mfn2, mitochondrial permeability and cellular energy metabolism regulatory proteins VDAC1 and TOM70, apoptosis proteins TNFR1-associated death domain protein (TRADD) and Fas-associated death domain protein (FADD), the autophagy protein Atg5 and Atg12. These proteins are involved in MAVS-mediated signaling, activation of downstream cytokines, promotion of IFN expression, and inhibition of pro-inflammatory cytokine expression. Mfn1 and Mfn2 regulate mitochondrial fusion, and TOM70 and VDAC1 enhance mitochondrial permeability. As a death domain protein, TRADD is localized in the cytoplasm and interacts with FADD to promote apoptosis. Atg5 and Atg12 located on the autophagosomal membrane promotes autophagy. MAVS has a crucial role in the inhibition of IBV replication by baicalin. Therefore, we investigated whether these MAVS-related proteins also contribute to the anti-IBV replication effect of baicalin. We examined the regulatory effects of baicalin on transcriptional and translational levels of genes encoding Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5 and Atg12. Our results showed that in IBV-infected CEK cells, Mfn1, VDAC1, TOM70, Atg5, Atg12, and Mfn2 were significantly upregulated. With the addition of baicalin, expression levels of Mfn1, VDAC1, TOM70, Atg5 and Atg12 were further upregulated; VDAC1 showed a dose-dependent response to baicalin, similar to MAVS expression in cells treated with baicalin. Baicalin had a minimal impact on Mfn2 expression. Under the influence of IBV, expression levels of TRADD and FADD were slightly downregulated, and baicalin did not significantly influence their expression (Fig. 8A–C). These results suggest that baicalin may facilitate mitochondrial fusion, improve mitochondrial permeability, increase cellular energy metabolism, and enhance autophagy and that these effects are closely related to the anti-IBV activity of baicalin.

Fig. 8.

Fig. 8

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Changes in expression of Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5 and Atg12 at transcriptional and translational levels in chicken embryonic kidney cells infected with infectious bronchitis virus and treated with baicalin. (A) Transcription of genes encoding Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5 and Atg12 in chicken embryonic kidney (CEK) cells incubated with infectious bronchitis virus for 2 h and then treated with 20 µg/mL baicalin. (B) Translation of mRNAs encoding Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5 and Atg12 in CEK cells incubated with infectious bronchitis virus for 2 h and then treated with 20 µg/mL baicalin. (C) Protein concentrations measured using ImageJ software. BA, baicalin. *p < 0.05, **p < 0.01, and ***p < 0.001. The symbols “+” and “−” in the figures indicate the addition and absence of components respectively.

Baicalin regulates IFN-β expression via Mfn1, TOM70, VDAC1, Atg5 and Atg12

Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5 and Atg12 are involved in regulating MAVS-mediated signaling and regulatory factors implicated in the downstream IFN signaling pathway, thereby promoting or inhibiting IFN expression. Our previous research showed that baicalin promoted the expression of IFN-β via MAVS and regulated the expression of genes encoding Mfn1, Mfn2, TOM70, VDAC1, Atg5 and Atg12. The remaining question was whether these genes are involved in the regulation of IFN-β by baicalin. We therefore overexpressed genes encoding MAVS, Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5 and Atg12 to determine whether their increased expression would influence the regulatory effect of baicalin on IFN-β. Our results showed that without the addition of baicalin, overexpression of MAVS, Mfn1, VDAC1, TOM70, Atg5 and Atg12 promoted the expression of IFN-β, with MAVS leading to the most significant increases. However, overexpression of Mfn2, TRADD, and FADD had little impact on IFN-β expression. In cells treated with 20 µg/mL baicalin, overexpression of MAVS, Mfn1, VDAC1, and TOM70 resulted in significantly greater upregulation of IFN-β in comparison with the control group; overexpression of Atg5 and Atg12 slightly upregulated IFN-β, and overexpression of Mfn2, TRADD, and FADD had little effect on IFN-β expression (Fig. 9).

Fig. 9.

Fig. 9

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Overexpression of genes encoding Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5 and Atg12 affects baicalin-induced IFN-β expression. (A) Transcriptional-level expression of IFN-β; (B) Expression of IFN-β at the translational level. (C) Protein concentrations measured using ImageJ software. Plasmids for overexpression of genes encoding Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5 and Atg12 were constructed and transferred into chicken embryonic kidney cells. Cells were incubated with infectious bronchitis virus for 2 h and then treated with 20 µg/mL baicalin. Wild-type chicken embryonic kidney cells incubated with infectious bronchitis virus for 2 h, followed by treatment or no treatment with 20 µg/mL baicalin, used as controls. BA, baicalin. *p < 0.05, **p < 0.01, and ***p < 0.001. The symbols “+” and “−” in the figures indicate the addition and absence of components respectively.

To confirm whether MAVS, Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5 and Atg12 mediate the regulation of IFN-β by baicalin, we knocked down the genes encoding these proteins. Specific knockdown of MAVS, Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5, and Atg12 was achieved using siRNA technology. Validation by Western blotting demonstrated effective knockdown, revealing significantly reduced protein expression levels of MAVS, Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5, and Atg12 compared to controls(Figure 10).Following IBV infection of CEK cells, knockdown of Mfn1, TOM70, VDAC1, Atg5 and Atg12 reduced IFN-β mRNA expression, while knockdown of Mfn2, TRADD had virtually no effect. conversely, knockdown of FADD promoted IFN-β mRNA expression (Fig. 11A). BA treatment elevated IFN-β mRNA levels in all knockdown groups above those of the IBV-infected control. However, this BA-induced enhancement was significantly attenuated in Mfn1, TOM70, VDAC1, FADD, Atg5, and Atg12 knockdown groups compared to BA-treated non-knockdown cells. Knockdown of Mfn2, FADD did not affect BA-mediated upregulation, whereas knockdown of TRADD potentiated the BA-induced increase in IFN-β mRNA (Fig. 11B). At the protein level, knockdown of Mfn2, TRADD, FADD minimally affected IFN-β expression, but knockdown of Mfn1, TOM70, VDAC1, Atg5, or Atg12 significantly decreased IFN-β protein levels (Fig. 11C,D). BA treatment increased IFN-β protein in all knockdown groups. Mirroring the mRNA results, the BA-induced protein increase was significantly lower in Mfn1, TOM70, VDAC1, Atg5, and Atg12 knockdown groups relative to BA-treated non-knockdown cells. Knockdown of Mfn2 and FADD again did not affect BA-mediated enhancement, while knockdown of TRADD moderately promoted IFN-β protein expression after BA treatment, demonstrating overall consistency between transcriptional and translational regulation (Fig. 11C,E). These findings demonstrate that reduced expression of Mfn1, TOM70, VDAC1, FADD, Atg5, and Atg12 negatively regulates IFN-β protein production, whereas reduced expression of TRADD positively regulates IFN-β.

Fig. 10.

Fig. 10

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Western blot analysis was performed to assess the protein expression levels of MAVS, Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5, and Atg12 following knockdown by siRNA. Cells transfected with a non-targeting siRNA served as the control group.

Fig. 11.

Fig. 11

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Knockdown of genes encoding Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5 and Atg12 affects baicalin-induced IFN-β expression. (A) After inducing overexpression of specific genes and adding BA, IFN-β transcript levels were measured; (B) After overexpression of each gene without BA but with an equivalent volume of M199, the transcriptional level of IFN-β was determined. (C) Following IBV infection of CEK cells, the expression of IFN-β at the translational level were assessed in the presence or absence of BA; (D,E) Protein concentrations measured using ImageJ software. BA, baicalin. *p < 0.05, **p < 0.01, and ***p < 0.001. The symbols “+” and “−” in the figures indicate the addition and absence of components respectively. SiRNAs targeting mRNAs encoding Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5 and Atg12 were constructed and transferred into chicken embryonic kidney cells. Cells were incubated with infectious bronchitis virus for 2 h and then treated with 20 µg/mL baicalin. Wild-type chicken embryonic kidney cells incubated with infectious bronchitis virus for 2 h, followed by treatment or no treatment with 20 µg/mL baicalin, used as controls. BA, baicalin. *p < 0.05, **p < 0.01, and ***p < 0.001.

MAVS mediates regulation of IFN-β by Baicalin via Mfn1, TOM70, VDAC1, Atg5 and Atg12

To investigate whether the impact of Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5 and Atg12 on the regulation of IFN-β by baicalin is related to MAVS, we knocked out the MAVS gene and overexpressed genes encoding these proteins. Our results showed that in MAVS-knockdown cells, overexpression of the genes encoding Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5 and Atg12 did not significantly affect the regulation of IFN-β by baicalin (Fig. 12). This result suggests that regulation of IFN-β by baicalin via Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5 and Atg12 requires MAVS as a mediator. Thus, MAVS is a critical component in modulation of the IFN signaling pathway by baicalin.

Fig. 12.

Fig. 12

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Overexpression of genes encoding Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5 and Atg12 affects baicalin-induced IFN-β expression in MAVS-knockdown cells. (A) Transcriptional-level expression of IFN-β; (B) Expression of IFN-β at the translational level; (C) Protein concentrations measured using ImageJ software. BA, baicalin. *p < 0.05, **p < 0.01, and ***p < 0.001. The symbols “+” and “−” in the figures indicate the addition and absence of components respectively. MAVS siRNA and eukaryotic expression plasmids for genes encoding Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5 and Atg12 were constructed and transferred to chicken embryonic kidney (CEK) cells. Cells were incubated with infectious bronchitis virus (IBV) for 2 h and then treated with 20 µg/mL baicalin. IBV-infected wild-type CEK cells, IBV-infected MAVS-knockdown CEK cells, and IBV-infected MAVS-knockdown CEK cells treated with 20 µg/mL baicalin after 2 h of incubation with the virus used as controls. BA, baicalin. *p < 0.05, **p < 0.01, and ***p < 0.001.

Discussion

MAVS is a key linker molecule downstream of RIG-I and MDA5 in the RIG-I-like receptor signaling pathway, and MAVS is crucial for the expression of type I IFNs18. MDA5 recognizes intracellular dsRNA viruses and initiates the production of type I IFNs. The binding of MDA5 to RNA causes a conformational change, allowing it to interact with the important adaptor protein MAVS in the downstream of the cytosolic pathway. Upon receiving signals from MAVS, TBK1 and inducible IκB kinase activate transcription factors IRF3 and NF-κB, which subsequently trigger production of type I IFNs and other antiviral factors19. Our previous research showed that baicalin can promote the expression of IFN-β, which is closely related to inhibition of IBV replication. MAVS is involved in upregulation of IFN-β by baicalin. Notably, baicalin can affect MAVS expression in a dose-dependent manner and enhance IFN-β expression via MAVS (Fig. 13). MAVS knockdown can significantly inhibit the expression of IFN-β. Our findings suggest that MAVS is an important mediator in the regulation of type I IFNs by baicalin. Concurrently, in our study examining the impact of betulinic acid (BA) on the expression of genes associated with the MAVS pathway, we observed that BA significantly suppressed the expression level of NF-κB. Given that NF-κB is a pivotal regulator of pro-inflammatory signaling, these findings suggest that BA may modulate the NF-κB-mediated pro-inflammatory pathway. Furthermore, investigation into the effect of BA on the expression levels of key downstream factors within this pathway—including TNF-α, IL-1β, IL-6, TLR7, and MyD88—revealed that BA inhibited inflammatory responses and alleviated inflammation in CEK cells. This provides a foundation for further research into the anti-inflammatory mechanisms of BA (Fig. 6).

Fig. 13.

Fig. 13

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Effects of baicalin on the expression of type I interferon-related genes in vitro. Chicken embryonic kidney cells were incubated with infectious bronchitis virus (IBV) at a concentration of 100 TCID50 at 37 °C for 2 h. Cells were then incubated with 10 and 20 µg/mL baicalin for 36 h. Chicken embryonic kidney cells treated with M199 and 100 TCID50 IBV were used as controls. Expression of MAVS, MDA5, TBK1, TLR3 and NF-κB was detected at transcriptional (A). MAVS, MDA5, TBK1, TLR3, p65, p-p65, IκB-α,p-IκB-αwas detected at translational (B) levels. Protein concentrations were measured using ImageJ software (C). Data are presented as mean ± standard deviation. *p < 0.5, **p < 0.1, ***p < 0.05. The symbols “+” and “−” in the figures indicate the addition and absence of components respectively.

MAVS is located in the outer mitochondrial membrane20, and its overexpression contributes to the maintenance of mitochondrial homeostasis17. As a typical coronavirus, IBV invades cells and targets the mitochondria, disrupting mitochondrial homeostasis and function21. We found that baicalin can reduce damage to mitochondrial function caused by IBV in CEK cells, potentially through upregulation of MAVS expression. Further research showed that in MAVS-knockdown cells, the effect of baicalin on maintaining mitochondrial homeostasis was significantly diminished, confirming the important role of MAVS in this process (Fig. 7). This result also indicated that disrupting mitochondrial function is a key feature of coronaviruses, and maintaining mitochondrial homeostasis via MAVS is an important mechanism underlying the anti-IBV activity of baicalin. Our findings demonstrate that baicalin treatment at 20 µg/mL moderately enhanced ATP production and oxygen consumption rate (OCR) even under MAVS knockdown conditions. This observation suggests that baicalin-mediated maintenance of mitochondrial homeostasis may involve mechanisms independent of the MAVS signaling axis. The residual mitochondrial protective effects could stem either from its well-documented antiviral activity (potentially reducing viral load) or through modulation of alternative mitochondrial regulatory pathways that bypass MAVS-dependent mechanisms. While our study could not definitively establish the mechanism underlying baicalin’s ability to mildly upregulate ATP synthesis and OCR post-MAVS knockdown, we subsequently investigated baicalin’s interaction with mitochondrial outer membrane-localized genes that functionally associate with MAVS and participate in mitochondrial homeostasis regulation.

MAVS mainly participates in the IFN signaling pathway22. Mfn1, Mfn223, TOM7024, VDAC125, TRADD, FADD26, Atg5 and Atg1227 regulate innate immunity through the modulation of MAVS-mediated signaling. Specifically, Mfn1 and Mfn2 regulate mitochondrial fusion28. Mfn1 promotes and Mfn2 inhibits the MAVS-mediated signaling29. In CEK cells with IBV, baicalin could enhance the expression of Mfn1 without significantly affecting Mfn2, indicating that baicalin has a role in promoting mitochondrial fusion (Fig. 8). TOM70 and VDAC1 regulate mitochondrial permeability and cellular energy metabolism30,31. In this study, baicalin upregulated TOM70 and VDAC1, indicating that baicalin has a role in promoting mitochondrial permeability and cellular energy metabolism (Fig. 3). TRADD is located in the cytoplasm, and its interaction with FADD is involved in apoptosis32. Baicalin could not significantly affect TRADD and FADD, indicating that it does not regulate apoptosis via these proteins (Fig. 8). Atg5 and Atg12 is a critical complex in the autophagy process and has an essential role in the expansion of the autophagosomal membrane33. Atg5 and Atg12 was significantly upregulated at a baicalin concentration of 20 µg/mL, indicating that baicalin at this concentration can promote autophagy (Fig. 8). Through functional integration analysis of Mfn1/2, VDAC1, TOM70, TRADD/FADD, and Atg5/Atg12, we revealed that mitochondrial morphological plasticity in response to energy demands is coordinately regulated by Mfn1/2-mediated fusion and Drp1 (Dynamin-related protein 1)-dependent fission. VDAC1 serves as a critical nexus bridging energy metabolism and apoptotic signaling, while TRADD/FADD facilitates the transduction of death receptor signals to mitochondria. TOM70 maintains mitochondrial proteostasis through its essential role in protein import machinery. The Atg5/Atg12 conjugation system orchestrates selective mitophagy to eliminate damaged organelles. This coordinated network establishes a “synthesis-repair-clearance” tripartite cycle that sustains mitochondrial homeostasis. Notably, our experimental data demonstrated that 20 µg/mL baicalin restored mitochondrial function in MAVS-knockdown models. This therapeutic effect is likely attributable to baicalin’s regulatory capacity in modulating the expression levels of key mitochondrial regulators including Mfn1, VDAC1, TOM70, Atg5, Atg12, and Mfn2, thereby rebalancing mitochondrial quality control mechanisms. Further validation experiments are currently underway to confirm these molecular interactions and pathway activations.

Past research has shown that Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5 and Atg12 are involved in the modulation of regulatory factors downstream of MAVS and MDA5, thereby affecting the expression of type I IFNs. Mfn1 and Mfn2, two mitochondrial fusion proteins, interact with the C-terminal domain of MAVS via their coiled-coil domains, forming a complex that stabilizes MAVS aggregates34. However, conflicting observations exist in certain cellular models. For instance, it is reported that Mfn1 overexpression disrupts MAVS signaling, likely by sequestering free MAVS monomers and preventing their aggregation. This discrepancy may arise from context-dependent differences in Mfn1 stoichiometry, subcellular localization, or post-translational modifications. Notably, our findings showing Mfn1 overexpression enhances IFN-β expression suggest a dual regulatory role of Mfn1 in MAVS signaling, potentially balancing aggregation dynamics and monomer availability under varying conditions35. TOM70, an outer mitochondrial membrane (OMM) protein, is indispensable for initiating MAVS-dependent antiviral responses. Upon viral infection, MAVS recruits TOM70 to form a signaling platform that facilitates the assembly of TBK1 and IRF3. This interaction enables TBK1-mediated phosphorylation and nuclear translocation of IRF3, driving transcription of type I interferons (IFN-β). Beyond immune activation, the MAVS-TOM70 complex also regulates BAX-dependent apoptosis during severe viral stress. Mechanistically, MAVS-TOM70 binding alters OMM permeability, promoting BAX oligomerization and cytochrome c release, thereby eliminating virus-infected cells through programmed cell death36. While not directly engaged in MAVS activation, VDAC1 indirectly regulates its signaling efficiency by maintaining mitochondrial metabolic homeostasis. As a gatekeeper of mitochondrial metabolites, VDAC1 ensures optimal energy states for MAVS signal transduction. Dysfunctional VDAC1 disrupts mitochondrial membrane potential, impairing MAVS aggregation and downstream IFN production. Furthermore, VDAC1-MAVS cross-regulation emerges during apoptosis: MAVS activation modulates VDAC1 permeability, while VDAC1-mediated apoptosisdestabilizes MAVS localization on Outer Mitochondrial Membrane (OMM), creating a feedback loop that integrates immune and cell death pathways37. The Atg5-Atg12 conjugate, classically associated with autophagosome formation, exhibits context-dependent roles in MAVS signaling. Under basal conditions, it stabilizes MAVS through direct interaction38. MAVS signaling intersects with apoptosis regulators such as TRADD and FADD, traditionally linked to TNF receptor signaling. Both pathways converge on NF-κB activation, highlighting a shared node for immune and inflammatory responses. Additionally, MAVS-mediated IFN production and TRADD/FADD-dependent apoptosis may synergistically eliminate infected cells while preventing excessive inflammation39. Our study findings showed that baicalin could significantly upregulate IFN-β in cells with overexpression of Mfn1, TOM70, VDAC1, Atg5 and Atg12 (Fig. 9). However, upregulation of IFN-β by baicalin was reduced in cells with downregulation of Mfn1, TOM70, VDAC1, FADD, Atg5 and Atg12 (Fig. 11). Moreover, MAVS knockdown could effectively diminish the upregulation of IFN-β by baicalin via Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5 and Atg12, indicating that Mfn1, TOM70, VDAC1, Atg5 and Atg12 rely on MAVS to mediate regulation of IFN-β by baicalin (Fig. 12). This result confirmed the important role of MAVS in the enhancement of IFN expression by baicalin. The present experiments identified MAVS as a target for enhancing anti-IBV activity in baicalin, providing a theoretical basis for the development of baicalin as an antiviral drug.

Conclusion

We found that the anti-IBV effect of baicalin is closely related to MAVS. In the present study, baicalin could upregulate the expression of MAVS and maintain mitochondrial homeostasis and function through MAVS. Baicalin could regulate Mfn1, TOM70, VDAC1, Atg5 and Atg12, which interact with MAVS to regulate the expression of genes associated with innate immunity. Our findings confirm that baicalin is involved in regulating mitochondrial fusion, enhancing mitochondrial permeability, increasing cellular energy metabolism, and promoting autophagy. Baicalin could upregulate the expression of Mfn1, VDAC1, TOM70, Atg5 and Atg12, thereby enhancing IFN-β expression, with this effect being dependent on MAVS. Ultimately, our findings demonstrate that baicalin inhibits key mediators of the NF-κB pro-inflammatory pathway, including NF-κB itself, TNF-α, IL-1β, IL-6, TLR7, and MyD88. This suggests that baicalin may alleviate inflammation associated with infectious bronchitis virus (IBV) infection.

Materials and methods

Virus strain, cell line, and reagents

We obtained the IBV M41 strain (GenBank: FJ904723.1) from the Gansu Institute of Animal Husbandry and Veterinary Medicine and identified by the China Institute of Veterinary Drug Control. Chicken embryos were inoculated with IBV and observed every 12 h; those that did not survive within 24 h were discarded. At 72 h post-inoculation, we collected allantoic fluid from the surviving embryos, which was stored at − 80 °C. Chicken embryonic kidney (CEK) cells were prepared using 10-day-old specific-pathogen-free chicken embryos. Fully confluent monolayer CEK cells were cultured at 37 °C with 5% CO2 in Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. Baicalin was purchased from Sigma (Shanghai, China) and diluted in dimethyl sulfoxide.

Treatment of virus-infected cells

To determine the effects of baicalin on IBV-infected cells, CEK cells cultured in 96-well plates were infected via incubation with IBV at a concentration of 100 TCID50 (50% tissue culture infectious dose) at 37 °C for 2 h. The cells were then treated with baicalin at a concentration of 10–20 µg/mL for 24 h at 37 °C, followed by three washes using D-Hank’s solution. At designated time points, the total RNA was extracted and reverse-transcribed into cDNA. Relative expression levels of IBV mRNA were detected using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR).

Cytotoxicity assay

CEK cell monolayers in 96-well culture plates were washed thrice with D-Hanks solution and then incubated with baicalin at concentrations of 10, 20, 30, 40, and 50 µg/mL (triplicate wells per concentration). Control cells were incubated without experimental compounds but with the same concentration of DMSO. Following incubation at 37 °C for 48 h, 20 µL of MTT (5 mg/mL in M199 medium) was added to each well and the cells were further incubated at 37 °C for 4 h. After washing with D-Hanks, 200 µL DMSO was added to each well and the cells were incubated at 37 °C for 10 min. The plates were then gently agitated at room temperature for 10 min to dissolve formazan precipitates, and the OD570 was measured. Using the mean OD570 values, the cell survival rate was calculated as: Survival rate = (OD570 drug / OD570 control) × 100%.

Immunofluorescence assay

An indirect immunofluorescence assay (IFA) was performed to evaluate the antiviral activity of baicalin at its optimal concentration. CEK cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (room temperature, 15 min), and permeabilized with 0.1% Triton X-100 for 10 min. After three PBS washes, nonspecific binding was blocked with 0.1% glycine. Cells were then incubated with a rabbit anti-infectious bronchitis virus (IBV) polyclonal antibody (1:200 dilution; prepared in-house, Northeast Agricultural University) for 1 h, followed by incubation with an Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (1:500 dilution; Zhongshan Golden Bridge Biotechnology, China) for 30 min under light-protected conditions. Fluorescence images were captured using an inverted fluorescence microscope (Nikon Ti-S, Japan). IBV-infected CEK cells and mock-infected controls were included in all assays.

Real-time fluorescence quantitative PCR

We analyzed the expression levels of core genes involved in the IFN signaling pathway, including those encoding MDA5, MAVS, TBK1, TLR3, NF-κB, and IFN-β, as well as the important regulatory genes involved in MAVS-mediated innate immune signaling, including those encoding Mfn1, Mfn2, VDAC1, TRADD, FADD, Atg5 and Atg12. PCR primers were designed using Primer 5.0 software and synthesized by Sangon Biotech (Shanghai, China). The primer sequences are listed in Supplemental Table e1. Based on previous research, we developed a PCR-based method to quantify relative mRNA expression levels of the aforementioned genes in cells, with the gene encoding β-actin as the internal reference gene. Relative expression levels of the target genes were calculated using the 2−ΔΔCt method. The PCR system (20 µL) comprised 10 µL 2× TS-INGKE Master qPCR Mix, 1 µL each of forward and reverse primers, 1.0 µg cDNA template, and ddH2O. The PCR program was as follows: 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s and 61 °C for 30 s.

Western blot analysis of protein expression levels

We extracted total protein from the cells following lysis using a buffer consisting of radioimmunoprecipitation assay buffer and phenylmethylsulfonyl fluoride at a volume ratio of 100:1. The protein was separated using Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, transferred to a polyvinylidene difluoride membrane at low temperature, and blocked with 5% skim milk at room temperature for 2 h. The membrane was washed with tris-buffered saline containing Tween 20 (TBST) and incubated with primary antibody at 4 °C overnight, followed by three washes with TBST. Next, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody at 37 °C for 2 h on a shaker, washed three times with TBST, and visualized using an enhanced chemiluminescence substrate.

Mitochondria isolation

A total of 1 × 107 wild-type or MAVS-knockdown CEK cells were seeded in two 10-cm diameter Petri dishes. After a 24-h incubation with 100 TCID50 IBV and 20 µg/mL baicalin, CEK cells were digested with trypsin and resuspended in 1 mL isolation buffer containing 210 mM mannitol, 70 mM sucrose, 5 mM 2-[4-(2-hydroxyethyl) piperazin-1-yl]ethanesulfonic acid, 1 mM ethylene glycol tetraacetic acid, and 0.5 g/L bovine serum albumin. The cells were homogenized on ice using a tissue homogenizer. We centrifuged the homogenate at 700×g for 10 min at 4 °C and the supernatant at 7000×g for 10 min at 4 °C. We then resuspended the pellet in isolation buffer for measurement of mitochondrial respiration.

Mitochondrial respiration measurement

We measured mitochondrial respiration at 37 °C using a high-resolution Oxygraph-2k respirometry system (Oroboros Instruments, Innsbruck, Austria). For whole cells, 2 × 105 cells were suspended in 2 mL DMEM per well. We measured respiration in response to oligomycin (2 µg/mL), mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (0.5 µM), rotenone (0.5 µM), and antimycin A (2.5 µM). Isolated mitochondria were suspended in MiR05 buffer containing 0.5 mM ethylene glycol tetraacetic acid, 3 mM MgCl2, 60 mM lactobionic acid, 20 mM taurine, 10 mM KH2PO4, 20 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, 110 mM D-sucrose, and 1 g/L fatty acid-free bovine serum albumin (pH 7.1, adjusted with KOH). Subsequently, we added 5 µM pyruvate, 2 µM malate, and 10 µM glutamate. To measure fatty acid oxidation, we suspended liver cells in Krebs-Henseleit buffer containing 111 mM NaCl, 4.7 mM KCl, 2 mM MgSO4, 1.2 mM Na2HPO4, and 0.5 mM L-carnitine. To this, we added 100 µM palmitic acid and 3 µM etomoxir.

Mitochondrial membrane potential measurement

CEK cells were incubated with tetramethylrhodamine methyl ester or 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide together with Hoechst in fetal bovine serum-free DMEM for 20 min. The cells were then washed with phosphate-buffered saline and examined using a TCS SP8 laser scanning confocal microscope (Leica, Germany). Cellular ATP levels were measured using a luminescent cell viability assay kit (Beyotime, China).

siRNA design and transfection

We designed siRNA sequences targeting mRNAs encoding Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5, Atg12, and MAVS (Supplemental Table e1). The siMfn1, siMfn2, siTOM70, siVDAC1, siTRADD, siFADD, siAtg5, siAtg12, and siMAVS were synthesized by Sangon Biotech (Shanghai, China). CEK cells were inoculated into 10-cm diameter Petri dishes at a cell density yielding a 70–90% confluence after 24 h for transfection. siRNA (2 µg) and lipofectamine (4 µL) were separately mixed with 200 µL of saline and allowed to stand for adequate mixing. Then the two mixtures were gently combined, incubated at room temperature for 15 min, and added to the Petri dish. The cells were incubated at 37 °C and 5% CO2.

Target gene plasmid construction and expression

We retrieved the open reading frame sequences of genes encoding Mfn1, Mfn2, TOM70, VDAC1, TRADD, FADD, Atg5, Atg12, and MAVS from the GenBank database; PCR primers and restriction sites were designed accordingly (Supplemental Table e1). The target sequences were amplified, digested with the appropriate restriction enzymes, and cloned into the p3×FLAG-CMV-7.1 digested with the same enzymes. Plasmids containing the target gene fragments were then transfected into CEK cells.

Statistical analysis

Each experiment was performed three independent times, and all data was analyzed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Results are expressed as the mean ± standard deviation (SD). Differences between groups were evaluated using the one-way analysis of variance (ANOVA) two tailed test. p < 0.05 was considered as statistically significant, p < 0.01 and P < 0.001 were considered as highly significant.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (182.5KB, pdf)
Supplementary Material 2 (244.5KB, pdf)
Supplementary Material 3 (12KB, xlsx)

Acknowledgements

We want to thank Lanzhou Institute of Husbandry and Pharmaceutical Sciences of Chinese Academy of Agriculture Sciences for kindly providing us the experimental equipments used in this work.

Author contributions

Conceptualization, J.H. and S.W.; data curation, H.G.; formal analysis, J.H. and L.W.; investigation, J.H. and S.W.; methodology, J.H. and H.G.; project administration, J.H.; resources, S.W. and H.G.; supervision, S.W.; validation, S.W. ,J.H. and L.W.; visualization, H.G. and L.W.; writing—original draft preparation, J.H and H.G.; writing review and editing, J.H. and S.W. All of the authors have made substantial contributions to the presented work. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by the Natural Science Foundation of Gansu Province. Project number:24JRRA024.

Data availability

Data is provided within the manuscript or supplementary information files.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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