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Rosmarinic acid-mediated downregulation of RIG-I and p62 in microglia confers resistance to Japanese encephalitis virus-induced inflammation

BMC Veterinary Research volume 20, Article number: 555 (2024) Cite this article

Abstract

Background

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic pathogen that causes encephalitis in humans and reproductive failure in pigs. The transmission of JEV between humans and animals poses a significant public health threat and results in substantial economic losses. Excessive inflammation in the central nervous system of JEV-infected patients is a major cause of mortality and disability. Rosmarinic acid (RA), a polyhydroxyphenolic compound isolated from medicinal herbs, has been preliminarily shown to possess anti-inflammatory properties and significantly inhibit JEV-induced neuroinflammation in mice.

Results

This study investigated the antiviral capacity and potential mechanisms of RA in JEV-infected cells. The results demonstrated that RA could inhibit JEV replication in vitro. Furthermore, the expression levels of inflammatory cytokines (including IL-6, IL-1β, CCL-2, and TNF-α), membrane receptors (including RIG-I, TLR3, TLR4, TLR7, and TLR8), NF-κB complex and p62/SQSTM1 were assessed using qPCR, ELISA, and Western blot, respectively. The findings indicated that RA significantly suppressed the expression of IL-6, IL-1α, TNF-α, and CCL-2 in JEV-infected BV-2 cells in a dose-dependent manner. Additionally, RA treatment downregulated the expression levels of RIG-I and p62, while p62 silencing inhibited the upregulation of inflammatory cytokines in JEV-infected BV-2 cells.

Conclusion

Our present study highlights the important role of RA-mediated reduction of RIG-I and p62 in microglia, conferring resistance to Japanese encephalitis virus-induced inflammation.

Peer Review reports

Background

Japanese encephalitis virus (JEV), first identified in Japan in 1870, belongs to the genus Flavivirus within the family Flaviviridae and is a zoonotic pathogen primarily transmitted by Culex tritaeniorhynchus mosquitoes [1,2,3,4]. Japanese encephalitis (JE), caused by JEV, is characterized by perivascular cuffing and parenchymal infiltration of inflammatory cells, accompanied by microglial nodules, edema, neuralgia, and necrosis in the central nervous system (CNS) [5,6,7]. It is reported that over 2 billion people live in JE-endemic areas, with approximately 70,000 cases of JE infection annually, resulting in around 10,000 deaths and 30–50% of survivors suffering from post-infection neurological deficits [8, 9]. Recently, the risk of JE outbreaks has been increasing due to the migration of infected birds and the expansion of Culex mosquito populations [10, 11]. However, there are currently no effective vaccines or therapeutic drugs available for the treatment of JE.

When Japanese encephalitis virus (JEV) invades the host’s central nervous system (CNS), it induces neuroinflammation and neuronal cell damage. JEV has been shown to stimulate the release of inflammatory mediators from CNS endothelial cells and astrocytes, leading to brain inflammation [12, 13]. Concurrently, peripheral immune cells are recruited to the CNS by inflammatory and chemotactic factors, resulting in a cytokine storm that exacerbates severe neuroinflammation and neuronal death [14, 15]. Microglia, the most abundant mononuclear phagocytes in the CNS, are capable of immune surveillance, antigen presentation, and phagocytosis. Upon activation, microglia release a plethora of inflammatory cytokines and chemokines, further contributing to neuroinflammation.

Rosmarinic acid (RA), a phenolic compound (3,4-dihydroxyphenyllactic acid, C8H8O4), is predominantly found in the subfamily Nepetoideae of the Boraginaceae and Lamiaceae families [16]. RA has been reported to exhibit antiviral, antibacterial, anti-inflammatory, and antioxidant activities [17, 18]. Studies have shown that RA can significantly ameliorate damage to the mammary structure in mice by inhibiting the LPS-activated TLR4/MyD88/NF-κB pathway and reducing myeloperoxidase activity, thereby exerting its anti-inflammatory effects [19]. Additionally, RA has been found to suppress JEV-induced inflammatory responses in mouse models, although the precise mechanisms remain unclear [20].

In this study, we validated the antiviral effects of RA against JEV infection using the BV-2 cell line as an in vitro cell model. We investigated the specific mechanisms by which RA counteracts JEV-induced inflammatory responses, aiming to elucidate the pathways through which RA exerts its anti-inflammatory effects.

Results

BV-2 cells and Neuro-2a were susceptible to JEV

Microglial cells are prevalent in the nervous system and play a critical role in responding to inflammatory signals, thereby contributing to the regulation of various physiological processes [21]. In this study, BV-2 and Neuro-2a cells were infected with Japanese encephalitis virus (JEV) at a multiplicity of infection (MOI) of 5. RNA samples were collected at 6, 12, and 18 h post-infection (hpi) to assess the mRNA levels of the JEV envelope protein (E) using quantitative PCR (qPCR). The results indicated that viral load increased over the course of JEV infection (Fig. 1A and C). Additionally, morphological changes were observed in the cells following JEV infection. Notably, at 12 hpi, significant cytopathic effects (CPE) were evident in both BV-2 (Fig. 1B) and Neuro-2a (Fig. 1D) cells. Collectively, these findings demonstrate that both BV-2 and Neuro-2a cells are susceptible to JEV infection.

Fig. 1
figure 1

Establishment of JEV-infection cell models. (A) BV-2 cells were infected with JEV at a MOI of 5. RNA samples were collected at 6, 12, and 18 hpi to assess the mRNA levels of the JEV E protein using quantitative PCR (qPCR). (B) CPEs were noted in BV-2 cells at 12 hpi following JEV infection at a MOI of 5. (C) Neuro-2a cells were similarly infected with JEV at a MOI of 5, and RNA samples were collected at 6, 12, and 18 hpi to measure the mRNA levels of JEV E by qPCR. (D) CPEs were observed in Neuro-2a cells at 12 hpi following infection with JEV at a MOI of 5

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The levels of inflammatory cytokines were elevated in BV-2 cells infected with JEV

BV-2 and Neuro-2a cells were infected with JEV, and the levels of IL-6, IL-1β, TNF-α, and CCL-2 were measured to assess the JEV-induced inflammatory response. As the result, compared to Neuro-2a cells, JEV infection at a dose of 5 multiplicity of infection (MOI) significantly induced the mRNA expression levels of IL-6, IL-1β, TNF-α, and CCL-2 in BV-2 cells after 12 hpi (Fig. 2A). Subsequently, we further evaluated the JEV-induced cellular inflammatory response by infecting BV-2 cells with different MOI of JEV for different times. BV-2 cells were infected with JEV at MOI of 1, 2, 5, and 10, with positive control (LPS) and blank control group. The results showed that the expression of inflammatory cytokines increased with the infection dose at 12 hpi (Fig. 2B). Additionally, using a MOI of 5 for JEV infection, the inflammatory response in BV-2 cells was more pronounced at 12 hpi compared to 24 hpi (Fig. 2C).

Fig. 2
figure 2

The effect of JEV infection on BV-2 cells inflammation. (A) Neuro-2a and BV-2 cells were infected with JEV at a multiplicity of infection (MOI) of 5. The MOCK group served as a blank control, while LPS was used as a positive control. The mRNA expression levels of IL-6, IL-1β, TNF-α, and CCL-2 were quantified by qPCR. (B) BV-2 cells were treated with JEV at varying doses. The mRNA expression levels of IL-6, IL-1β, TNF-α, and CCL-2 were quantified by qPCR. (C) BV-2 cells were infected with JEV at a MOI of 5, and the mRNA expression levels of IL-6, IL-1β, TNF-α, and CCL-2 were quantified by qPCR at different time points post-infection (12 hpi and 24 hpi)

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RA inhibited the replication of JEV in microglial cells

The structure formula of Rosmarinic acid (RA) was shown in Fig. 3A and the cytotoxicity of RA on BV-2 cells was assessed using the MTT assay. The results indicated that RA at various concentrations (25, 50, 100, and 200 µg/mL) did not exhibit significant cytotoxicity to BV-2 cells (Fig. 3B). The inhibitory effect of RA on JEV in BV-2 cells was investigated by detecting the expression of JEV NS3 protein using Western blot (WB) and the expression of JEV E mRNA using qPCR. The results demonstrated that RA significantly inhibited the proliferation of JEV at 12 hpi (Fig. 3C and D). Together, these results indicated that RA significantly reduced viral RNA replication and NS3 protein production in JEV-infected BV-2 cells in a dose-dependent manner.

Fig. 3
figure 3

RA inhibited the proliferation of JEV in BV-2 Cells. (A) The molecular structure of RA. (B) The BV-2 cells were treated with different concentrations of RA for 12/24 hours, and cell viability was detected using the MTT assay. (C-D) BV-2 cells were infected with JEV and then treated with different concentrations of RA (50, 100, and 200 µg/mL). At 12 hpi, RNA and protein samples were collected. The changes in the protein level of JEV NS3 and the mRNA level of JEV E were detected using WB and qPCR, respectively

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RA inhibited JEV-induced cellular inflammation

To investigate the anti-inflammatory effects of RA, BV-2 cells infected with JEV were treated with different concentrations of RA. Samples were collected at 12 hpi, and the changes in the expression of inflammatory cytokines, including IL-6, IL-1β, TNF-α, and CCL-2, were measured using qPCR and ELISA. The results indicated that the levels of IL-6, IL-1β, TNF-α, and CCL-2 in JEV-infected BV-2 cells significantly decreased following RA treatment in a concentration-dependent manner (Fig. 4A and B).

Fig. 4
figure 4

RA reduced JEV-induced cellular inflammation. The BV-2 cells were treated with different concentrations of RA (50, 100, and 200 µg/mL) for 12 h after JEV infection. (A) The mRNA expression levels of IL-6, IL-1β, TNF-α, and CCL-2 were detected using qPCR. (B) The expression levels of IL-6, IL-1β, TNF-α, and CCL-2 were detected by ELISA

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Effects of RA on Upstream Signaling molecules of NF-κB

NF-κB has been reported as a key mediator of inflammatory responses [22, 23]. Therefore, to investigate the anti-inflammatory mechanism of RA in JEV-infected cells, the upstream receptors of the NF-κB pathway were examined. BV-2 cells were first infected with JEV at a MOI of 5, followed by treatment with 200 µg/mL of RA. At 12 hpi, RNA and protein samples were collected. The expression levels of TLR3, TLR4, TLR7, and TLR8, RIG-I and p62 were then detected using qPCR and WB. The results showed no significant differences in the expression levels of TLR3, TLR4, TLR7, and TLR8 among the RA-treated groups in BV-2 cells. However, the expression level of RIG-I and p62 were elevated in the JEV-infected group, while it was downregulated after RA treatment (Fig. 5A and B). The activation of the NF-κB complex leads to the transcription of many NF-κB target genes, including cytokines such as IL-1β and IL-6, which are involved in promoting pro-inflammatory responses [24]. We further investigated the effect of RA on the activation of the NF-κB complex following JEV infection. As the results, compared to the JEV-infected group, the RA treatment exhibited a 2-fold and 3-fold downregulation of NF-κB1 and IkBα expression, respectively (Fig. 5C). These findings suggest RA-induced the change of NF-κB signaling may alleviate JEV-induced inflammatory responses.

Effects of p62 on the expression of inflammatory cytokines

To further investigate the effects of p62 on inflammatory cytokines, p62 was silenced (Fig. 6A), and the changes in the expression levels of inflammatory cytokines IL-6, IL-1β, TNF-α, and CCL-2 were detected using qPCR and ELISA. The results showed that the expression levels of IL-6, IL-1β, TNF-α, and CCL-2 were significantly decreased in the si-p62 group compared to the control group (Fig. 6B and C).

Fig. 5
figure 5

Effects of RA on upstream signaling molecules of NF-κB. BV-2 cells were infected with JEV at an MOI of 5, followed by treatment with 200 µg/mL RA. At 12 hpi, RNA and protein samples were collected. (A) The mRNA expression levels of TLR3, TLR4, TLR7, TLR8, RIG-I and p62 were analyzed using qPCR. (B) The protein expression levels of TLR3, TLR4, TLR7, TLR8, RIG-I and p62 were detected using WB. (C) The mRNA expression levels of NF-κB1 and IkBα were analyzed using qPCR

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Fig. 6
figure 6

Effects of p62 on the Expression of Inflammatory Cytokines. (A) The effect of si-p62 on p62 expression was detected using WB. (B) BV-2 cells were transfected with si-p62 for 6 h, followed by infection with JEV at a MOI of 5 and treatment with RA. The expression levels of IL-6, IL-1β, TNF-α, and CCL-2 were detected using qPCR. (C) BV-2 cells were transfected with si-p62, followed by infection with JEV at a MOI of 5 and treatment with RA. The expression levels of IL-6, IL-1β, TNF-α, and CCL-2 were detected using ELISA

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Discussion

Japanese encephalitis virus (JEV) and other neurotropic flaviviruses can affect the nervous system by crossing the host’s blood-brain barrier, leading to viral encephalitis [20, 25]. During viral encephalitis, dysregulated microglia produce cytokines that lead to inflammatory neuronal damage, resulting in neurocognitive impairments [26]. Therefore, timely control of microglial activation may be an effective strategy for treating viral encephalitis. In the present study, we established an inflammatory model of JEV infection and observed that, compared to Neuro-2a cells, JEV infection in BV-2 cells significantly upregulated the expression of inflammatory cytokines IL-6, IL-1β, TNF-α, and CCL-2. Furthermore, the herbal extract rosmarinic acid (RA) was found to dose-dependently limit the JEV-induced upregulation of these inflammatory cytokines.

RA, an active component of many herbal plants, exhibits anti-inflammatory, antioxidant, and antiviral activities [27, 28]. When BV-2 cells infected with JEV were treated with RA, a significant reduction in viral load was observed at 12 hpi, indicating the potential antiviral effect of RA. Upon invading the central nervous system, JEV stimulates glial cells, leading to the release of numerous inflammatory cytokines such as IL-1β, TNF-α, IL-6, and CCL-2, resulting in a cytokine storm [29, 30]. Studies have shown that a derivative of RA, RAG, could inhibit the inflammatory response induced by the influenza virus [31]. In this study, we used BV-2 cells as an in vitro infection model to confirm that JEV induced a strong inflammatory response. Additionally, treatment with RA significantly downregulated JEV-induced expression levels of inflammatory cytokines IL-6, IL-1β, TNF-α, and CCL-2. This suggests that RA may exert its antiviral effect by inhibiting the inflammatory response induced by JEV.

Increasing evidence indicates that the release of pro-inflammatory cytokines primarily occurs at the initial stage of transcription, mainly through the activation of various transcription factors, including NF-κB [32, 33]. Toll-like receptors (TLRs), as upstream signaling molecules of NF-κB, can participate in activating the body’s inflammatory response [34,35,36]. Studies have shown that TLR4-deficient mice exhibited stronger resistance to JEV, while TLR7-deficient mice were more susceptible to JEV infection [37, 38]. Additionally, NF-κB could regulate various inflammatory factors, such as NO, IL-6, IL-1β, COX-2, and TNF-α [39, 40]. As an important pattern recognition receptor, RIG-I can mediate inflammation and activate the transcription factor NF-κB [41,42,43]. Researchers have reported that JEV could regulate the production of pro-inflammatory cytokines in neurons through RIG-I, and RIG-I-deficient mice exhibited significantly increased viral loads in the brain after JEV infection [13]. In this study, we found that RA had no significant effect on the expression of TLR3, TLR4, TLR7, and TLR8 receptors, but it downregulated the expression of the RIG-I and p62. The lack of significant changes in the expression of TLR3, TLR4, and TLR7 upon JEV infection was inconsistent with previous studies, which might be due to differences between in vivo and in vitro studies [28]. However, the specific molecular mechanism by which RA regulates cellular inflammation through downregulation of the RIG-I and p62 receptor requires further investigation.

The multi-domain scaffold (adapter) protein p62 is involved in the activation of NF-κB in several cellular systems, participates in ubiquitination regulation, and provides scaffolding for various signaling proteins [44,45,46,47]. Studies have also found that p62 exerted its biological functions by regulating the inflammatory response [48,49,50]. In this study, we observed a significant increase in p62 expression levels in JEV-infected BV-2 cells, which was markedly inhibited by RA treatment. Furthermore, in p62-silenced BV-2 cells, RA failed to inhibit the upregulation of inflammatory cytokines induced by JEV infection, compared to the NC group. The p62 was reported to antagonize the inflammasome pathway through autophagy-dependent degradation of ubiquitinated inflammasome proteins [51]. Additionally, p62 regulated other pro-inflammatory and anti-inflammatory pathways, including the cell-protective transcription factor Nrf2, the pro-inflammatory transcription factor NF-κB, and the kinase mTOR [51]. Given the significant upregulation of p62 following JEV infection, it is worthwhile to further investigate the interaction between JEV proteins and p62 to elucidate the virus-host interplay.

Conclusion

In conclusion, this study underscores the critical role of RA-mediated downregulation of RIG-I and p62 in microglia, conferring resistance to Japanese encephalitis virus-induced inflammation. These findings lay a foundation for the future development of anti-inflammatory drugs.

Methods

Reagents

RA was purchased from Shanghai Aladdin biochemical technology Co., Ltd. (purity ≥ 98%; Shanghai, China). LPS (Escherichia coli 055: B5) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Neuro-2a and BV-2 cell lines were purchased from the Wuhan Yipu Biotechnology Co. Ltd. (Wuhan, China). The MTT kits were obtained from the Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China). The mouse IL-1β, IL-6, CCL-2, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were obtained from Proteintech (Wuhan, China). All the antibodies used in this study were provided by Abcam. Other chemicals were of reagent grade.

Virus and cells

Neuro-2a and BV-2 cells were grown in a minimum essential medium supplemented with 10% fetal bovine serum (FBS) and 0.1% antibiotic cocktail (Gibco). The HW1 strain of JEV was grown in Neuro-2a cells and its infectivity was measured by titration and calculated as the median tissue culture infective dose (TCID50).

The cell viability

The effect of RA on BV-2 cell viability was measured by the MTT kits. In brief, 1 × 105 BV-2 cells were seeded into 96-well plates for 24 h, RA (25, 50, 100, 200, and 400 µg/mL), and PBS were added or were not added, and incubation was continued for an additional time of 12/24 h. RA was dissolved in PBS, and PBS was served as the control. MTT was added (to 5% w/v) to each well, and the culture was continued for 3 h. The absorbance was acquired at 450 nm using a microplate reader.

Experimental design

The BV-2 cell model of JEV infection was established, and RA (50, 100, and 200 µg/mL) treatment group, JEV infection group, LPS control group, and blank control group were set up. The transcription and expression levels of related inflammatory factors (IL-6, IL-1β, CCL-2, and TNF -α) and signal molecules (RIG-I, TLR3, TLR4, TLR7, TLR8, p62) were detected by using qPCR, ELISA, and WB.

RNA interference

Short interference RNA (siRNA) against murine p62 (si-mp62) mRNA were purchased from Gima. Briefly, after subculture, BV-2 cells were resuspended in Opti-MEM (Thermo Fisher) and plated in 6-well plates at a density of 3 × 105 cells/ml. This was followed by adding 500 µL Optimem with 10 µL siRNA and 4 µL lipofectamine dropwise in the above well. The cells were incubated with the siRNA mix for 8 h and then the medium was replaced with MEM with 2% FBS without antibiotics and incubated for another 16 h for cell extraction to check the knockdown efficiency by western blot.

Quantitative PCR (qPCR) assay

Total RNA was extracted using the Trizol isolation kit with a spin column following the procedure described previously [52]. The qPCR assay was conducted using TransStart® Green qPCR SuperMix with the Roche LightCycler96 Real-Time PCR System. The amplification parameters were 95 ℃ for 5 min, followed by 40 cycles of 95 ℃ for15 s and 60 ℃ for 30 s. Each sample was analyzed in triplicate, and the relative expression of mRNA was calculated after normalization to GAPDH. All primer sequences used are listed in Table 1.

Table 1 Primers used for qPCR
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Enzyme-linked immunosorbent assay (ELISA)

The contents of TNF-α, CCL-2, IL-6, and IL-1β in serum and kidney tissues were determined by an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions. The optical densities (OD) were detected using a microplate reader at 450 nm. ELISA was determined in duplicate for each sample. The concentrations of TNF-α, CCL-2, IL-6, and IL-1β in the samples were calculated from a standard curve generated from the standards of TNF-α, CCL-2, IL-6, and IL-1β.

Western blot

Cells were seeded in a 6-wells cell culture plate, and treated as mentioned above. Cultured cells were lysed with RIPA buffer supplemented with protease and phosphatase inhibitors, scraped off the plate, and collected for protein extraction. The lysates were incubated on ice for 10 min and centrifuged at 12,000 rpm for 5 min at 4℃, and the supernatants were collected. The protein concentration was determined using a BCA kit. Proteins (30 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes for 60 min at 15 V. After blocking with 5% non-fat milk or BSA for 2 h in TBS-T, the membrane was incubated overnight with primary antibodies (RIG-I, 1:1000; TLR3, 1:1000; TLR4, 1:1000; TLR7, 1:1000, TLR8, 1:1000; p62, 1:1000; β-actin, 1:1000) in TBS-T at 4 ℃. On the next day, the membrane was washed three times for 10 min each with TBS-T buffer and incubated for 60 min with anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated immunoglobulin G (IgG) secondary antibodies diluted in TBS-T (1:3000). Finally, the membrane was washed three times for 10 min each with TBS-T buffer. The protein bands were detected with enhanced chemiluminescence (ECL).

Statistical analysis

The mean and standard errors of each group of data were calculated by using the software GraphPad Prism 9. Each group of data was expressed by means ± SD, and the significance of the mean difference of each group of data was analyzed by Student t-test. The statistical value of *P < 0.05 is considered as significant difference; **P < 0.01 is considered as extremely significant difference; ***P < 0.001 is considered as very significant difference.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

We thank the Shengbo Cao research group from Huazhong Agricultural University for providing suggestions on the data analysis.

Funding

This work was supported by National Key Research and Development Project (2016YFD0500402), National Natural Science Foundation of China (31602082) and Hubei Province Innovation Centre of Agricultural Sciences and Technology (No. 2024-620-000-001-013).

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Author notes

  1. Yuxin Yang and XianWang Hu contributed equally to this work.

Authors and Affiliations

  1. College of Animal Science, Yangtze University, Jingzhou, 434025, China

    Yuxin Yang & Yuying Yang

  2. Key Laboratory of Prevention and Control Agents for Animal Bacteriosis (Ministry of Agriculture), Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan, 430064, China

    XianWang Hu, Shuangshuang Wang, Yongxiang Tian, Keli Yang, Chang Li, Qiong Wu, Wei Liu, Ting Gao, Fangyan Yuan, Rui Guo, Zewen Liu & Danna Zhou

  3. Hubei Provincial Key Laboratory of Animal Pathogenic Microbiology, Wuhan, 430064, China

    XianWang Hu, Shuangshuang Wang, Yongxiang Tian, Keli Yang, Chang Li, Qiong Wu, Wei Liu, Ting Gao, Fangyan Yuan, Rui Guo, Zewen Liu & Danna Zhou

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  1. Yuxin Yang
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Contributions

Conceptualization, Y.Y., X.H., S.W., and D.Z.; Methodology, Y.Y., X.H., S.W., and D.Z.; Writing-Original Draft Preparation, X.H., Y.Y., and D.Z.; Writing-Review & Editing, C.L., Q.W., and D.Z.; Investigation, X.H., Y.Y., S.W., Y.T., K.Y., C.L., F.Y., W.L., T.G., R.G., Z.L and., Y.Y.; Visualization, C.L. and S.W.; Project Administration, D.Z.; Supervision, Y.Y., and D.Z.; Funding Acquisition, Y.Y., and D.Z. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Yuying Yang or Danna Zhou.

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Yang, Y., Hu, X., Wang, S. et al. Rosmarinic acid-mediated downregulation of RIG-I and p62 in microglia confers resistance to Japanese encephalitis virus-induced inflammation. BMC Vet Res 20, 555 (2024). https://doi.org/10.1186/s12917-024-04397-x

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BMC Veterinary Research

ISSN: 1746-6148

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