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ABSTRACT
Porcine reproductive and respiratory syndrome virus (PRRSV) causes abortion and respiratory disease in swine, hindering the development of the pig farming industry worldwide. However, at present, there are no effective vaccines or drugs for PRRSV control. In this study, we evaluated the inhibitory effect of hyperoside on PRRSV replication in vitro and in vivo and explored the underlying mechanisms. Our results revealed that hyperoside significantly inhibited PRRSV infection in MARC-145 and porcine alveolar macrophages (PAMs). This inhibition was linked to the hyperoside-induced attenuation of pro-inflammatory cytokine (IL-1β, IL-6, IL-8, and TNF-α) upregulation induced by PRRSV infection, which was mediated by the suppression of the Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-kB) signaling pathway. Moreover, hyperoside alleviated the autophagy induced by PRRSV via p62/Nrf2/Keap1 signaling pathway activation. In vivo, hyperoside treatment led to an obvious decrease in PRRSV replication in piglets. Therefore, hyperoside may be a useful antiviral agent against PRRSV.
IMPORTANCE
Porcine reproductive and respiratory syndrome virus (PRRSV) causes abortion and respiratory disease in swine, which induces huge economic losses every year. However, there have been no effective vaccines or drugs for PRRSV control until now. Our present study found that the inhibitory effect of hyperoside on PRRSV replication in vitro and in vivo. Furthermore, we demonstrate that hyperoside inhibits PRRSV proliferation via inhibiting inflammation and autophagy through the Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) and p62-Nrf2-Keap1 signaling pathways. Hence, we believe that hyperoside may be a useful antiviral agent to control PRRSV.
INTRODUCTION
Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped, positive-sense single-stranded RNA virus with a genome size of 14.9–15.5 kb, and it belongs to the Arteriviridae family (1, 2). This virus causes reproductive disorders in sows and severe respiratory disease in swine, generating massive economic losses in the global pig farming industry (3, 4). Since its emergence in the 1980s, PRRSV has undergone significant mutation and recombination (5, 6). However, effective vaccines or drugs that prevent or control PRRSV have yet to be developed, and there is an urgent need for new effective anti-PRRSV drugs.
Hyperoside is a natural flavonoid that can be extracted from various plants, including Cuscuta chinensis Lam., Forsythia suspensa, and Crataegus pinnatifida Bge (7). Previous studies have shown that hyperoside possesses a broad spectrum of biological activities, including anti-inflammatory, antiviral, antioxidant, antihyperglycemic, and anticancer activities (8–11). In recent years, hyperoside has also been found to inhibit several human and animal viruses, such as herpes simplex virus type 1 (12), hepatitis B virus (13), infectious bronchitis virus (14), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (15), African swine fever virus (16), porcine epidemic diarrhea virus (PEDV) (10), and equine herpesvirus type 8 (EHV-8) (17). However, information regarding the anti-PRRSV activity of hyperoside and the potential molecular mechanisms underlying its effects on this virus is limited.
Both inflammatory cytokines and autophagy play crucial roles in viral infection and pathogenesis (18). PRRSV infection significantly increases the levels of inflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α in porcine alveolar macrophages (PAMs) and swine (19, 20), with uncontrolled inflammation aggravating lung damage (21). Recent studies have shown that compounds that downregulate pro-inflammatory factors (22), such as the itaconate derivative 4-OI22 and allicin (23), can effectively inhibit PRRSV infection. Similarly, autophagy—an evolutionarily conserved cellular process that maintains homeostasis (24–26)—can either promote or inhibit viral replication, depending on the virus type (27, 28). Previous research has demonstrated that PRRSV-induced autophagy facilitates viral self-replication (29, 30), while disrupting this autophagy response can suppress PRRSV infection (31). Notably, hyperoside has been shown to inhibit inflammatory responses (32) and suppress autophagy (33, 34) in various disease models, although its effects on PRRSV-induced inflammation and autophagy remain unexplored.
In the present study, the anti-PRRSV activity of hyperoside was investigated, both in susceptible cells and in piglets. Moreover, the potential anti-PRRSV mechanisms of hyperoside were explored. Our results showed that hyperoside could effectively confer resistance against PRRSV in vitro at multiple replication stages. Further analysis revealed that hyperoside could also attenuate the overexpression of pro-inflammatory factors induced by PRRSV by suppressing the Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling pathway. Additionally, our findings showed that hyperoside alleviated PRRSV-induced autophagy by activating the p62/Nrf2/Keap1 signaling pathway. Finally, we demonstrated that hyperoside was effective in reducing the PRRSV load and lung injury in piglets in vivo. Our data indicate that hyperoside may serve as a promising therapeutic agent against PRRSV.
MATERIALS AND METHODS
Cells, viruses, and reagents
MARC-145 cells were obtained from the China Center for Type Culture Collection (Wuhan, China) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin at 37°C under 5% CO2. PAMs were obtained from 8-week-old PRRSV-negative pigs as previously described and cultured in RPMI-1640 medium supplemented with 10% FBS.
The PRRSV-1 strains GZ11-G1 (GenBank: KF001144.1) and P073-3 (GenBank: MK214314.1) and the PRRSV-2 strains SD16 (GenBank: JX087437.1), CH-1a (GenBank: AY032626), JXA1 (GenBank: EF112445.1), and NADC30-like (GenBank: KX766379) were used in this study. These PRRSV isolates were allowed to proliferate in MARC-145 cells and titrated via a plaque formation assay, as described previously (35, 36).
Hyperoside was obtained from Sigma?Aldrich (Saint Louis, USA) and dissolved in dimethylsulfoxide (DMSO). 3-Methyladenine (3-MA, an autophagy inhibitor) was obtained from Shandong Sparkjade Biotechnology Co., Ltd. (Jinan, China). Finally, porcine PRRSV convalescent serum, which contained monoclonal antibodies against the PRRSV-1 and PRRSV-2 N proteins, was prepared in our laboratory.
Cytotoxicity assay
The cytotoxicity of hyperoside to MARC-145 and PAM cells was examined as described in previous studies (17). Briefly, MARC-145 and PAMs were preseeded into 96-well plates overnight and then treated with various concentrations of hyperoside (0, 15, 30, 60, 120, and 200 μM) for 24 h. Then, the medium was removed, and the cells were resuspended in 50 μL of DMEM or RPMI-1640 containing 10% Cell Counting Kit-8 (CCK-8; Shandong Sparkjade Biotechnology Co., Ltd., Jinan, China) and incubated at 37°C for 2 h. To calculate cell viability, the optical density (OD) of each well was measured at 450 nm via an Epoch microplate spectrophotometer (Biotek, USA) and analyzed via GraphPad Prism 8.0. DMSO was used as the negative control for these experiments.
RNA extraction and quantitative real-time polymerase chain reaction
All the cell samples were harvested at the indicated time points, and total RNA was extracted via SparkZol Reagent (Jinan, China); subsequently, 1 μg of RNA extracted from each group of cells was reverse transcribed into cDNA via the PrimeScript RT Master Mix (Takara, Japan). Then, quantitative PCR (qPCR) was performed to quantify target gene expression via the 2???Ct method, as described previously (37). GAPDH transcripts were amplified to normalize the total RNA input. The primer information is listed in Table 1.
TABLE 1
| Genes | Forward primer (5′?3′) | Reverse primer (5′?3′) |
|---|---|---|
| IL-6 | ?CAGCCCTGAGAAAGGAGACA | CCAGGCAAGTCTCCTCATTG |
| IL-8 | ?ACTCCAAACCTATCCACCCC | CCACAACCCTAGACACCCAT |
| TNF-α | ?TCCCCAGGAAGACAGCG | GCAGCAGACAGAAGAGCGT |
| IL-1β(Marc-145) | ?GGAAGACAAATTGCATGC | CCCAACTGGTACATCAGCAC |
| IL-1β(PAMs) | ?ACCTGGACCTTGGTTCTCTG | CATCTGCCTGATGCTCTTGT |
| GAPDH | ?GTCTCCTCTGACTTCAACAGCG | ACCACCCTGTTGCTGTAGCCAA |
| ORF7(PRRSV-1) | ?ATGGCCGGTAAAAATCAGAGCC | TTAATTCGCACCCTGACTGG |
| ORF7(PRRSV-2) | ?ATGCCAAATAACAACGGCAAGC | TCATGCTGAGGGTGATGCTGTG |
| siTLR4a | ?GCAAATGCCTCTGTGATTT | AAATCACAGAGGCATTTGC |
| siTLR4b | ?GTGCAGGCATAATCTTCAT | ATGAAGATTATGCCTGCAC |
a
Swine.
b
Monkey.
Absolute quantification was also performed to evaluate the RNA genome copy numbers of PRRSV-1 and PRRSV-2 in the cell culture supernatant, as described previously (35). Two recombinant plasmids, pMD18-T-N-1 and pMD18-T-N-2, containing the PRRSV-1 ORF7 gene and PRRSV-2 ORF7 gene, respectively, were used. These plasmids served as templates for qPCR at the indicated times.
Western blot analysis
The cells were collected at the indicated time points and lysed with 2× Laemmli sample buffer. The lysates were then boiled, and equal amounts of samples were separated via 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes as described previously (38, 39). The PVDF membranes were further blocked with 5% serum albumin and incubated with specific antibodies to detect different target proteins. α-Tubulin served as the loading control. Finally, the membranes were imaged via a ChemiDoc XRS imaging system (Bio-Rad, USA). The primary antibodies used in the present study included antibodies against TLR4, NF-kB p65, p-NF-kB p65, IkB, p-IkB, and LC3 (Cell Signaling Technology, USA); anti-p62 and anti-Nrf2 antibodies (Servicebio, Wuhan, China); and anti-α-Tubulin and anti-Keap1 antibodies (Abcam, Cambridge, UK). Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG were purchased from Thermo Fisher Scientific (Massachusetts, USA).
Viral titration
MARC-145 cells were grown to approximately 80%–90% confluency in 96-well plates. The viral suspensions were serially diluted 10-fold, and 100 μL of each dilution was added to the wells (n = 8) and incubated for 1 h. Subsequently, the cells were incubated with DMEM containing 3% FBS for 5 days. The 50% tissue culture infectious dose (TCID50) was calculated via the Reed-Muench method according to previous reports (36) and analyzed via GraphPad Prism 8.0.
Antiviral activity assay
MARC-145 cells and PAMs were seeded into 12-well plates and cultured until they reached 90% confluence. These cells were subsequently treated with various concentrations of hyperoside (15, 30, 60, or 120 μM) or the indicated medium containing 0.1% DMSO for 2 h. Following incubation, the cells were infected with PRRSV SD16 for 1 h at a 0.1 multiplicity of infection (MOI). The medium was subsequently replaced with fresh DMEM or RPMI-1640 containing 3% FBS and the indicated concentration of hyperoside. These cellular supernatants and cells were collected at 24 hours post-infection (hpi) for further qPCR and western blot analysis.
Time-of-addition analysis
To further determine which stage of the PRRSV life cycle was disrupted by hyperoside, MARC-145 cells were seeded into 12-well plates and divided into pretreated, cotreated, and post-treated groups according to the timing of the 120 μM hyperoside treatment relative to PRRSV SD16 (MOI = 0.1) inoculation. Specifically, the pretreated group was incubated with 120 μM hyperoside for 2 h and then infected with PRRSV SD16 (MOI = 0.1). Moreover, the cotreated group was treated with a mixture of hyperoside (120 μM) and PRRSV SD16 (MOI = 0.1) for 1 h. Finally, the post-treated group was infected with PRRSV SD16 (MOI = 0.1) for 1 h, and the medium was then replaced with 3% FBS DMEM containing 120 μM hyperoside. After 24 h, qPCR and western blot assays were performed to examine PRRSV replication in these groups.
To further test whether hyperoside interacts with PRRSV directly, a virucidal activity assay was performed as follows. A mixture of hyperoside (120 μM) and PRRSV SD16 (0.1 and 1 MOI) was incubated together at 37°C for 2 h. Then, MARC-145 cells were treated with this mixture at 37°C for another 1 h. At 24 hpi, the cells and cellular supernatants were collected to assess PRRSV replication via western blot and qPCR assays.
Viral binding, internalization, replication, and release assays
For the binding assay, MARC-145 cells were cultured in 12-well plates and preincubated at 4°C for 1 h. Then, the cells were inoculated with PRRSV SD16 (MOI = 1) and hyperoside (120 μM) or 0.1% DMSO and incubated at 4°C for 2 h to facilitate viral attachment. These cells were then washed three times with prechilled phosphate-buffered saline (PBS) and harvested to extract viral RNA for qPCR quantification.
For the internalization assay, MARC-145 cells were seeded into 12-well plates overnight. These cells were then pretreated with hyperoside (120 μM) for 2 h before infection with PRRSV SD16 (MOI = 1) at 4°C for 1 h to allow viral attachment. The cells were then washed with ice-cold PBS to remove the unbound virus and incubated with 3% FBS DMEM and 120 μM hyperoside or DMSO at 37°C for 1 h. The cells were treated with citrate buffer (pH 3.0) to remove any noninternalized virus particles. Finally, viral RNA was extracted and quantified via qPCR.
For the viral replication assay, MARC-145 cells were cultured in 6-well plates and infected with PRRSV SD16 (MOI = 1) for 6 h. These cells were washed with PBS and then incubated with 3% FBS DMEM containing hyperoside (120 μM) or DMSO at 37°C. The cells were collected at 7, 8, 10, and 16 hpi to quantify PRRSV genome copies via qPCR.
For the viral release assay, MARC-145 cells were cultured in 12-well plates and infected with PRRSV SD16 (MOI = 1) for 24 h. The cells were washed with PBS and incubated with 3% FBS DMEM containing hyperoside (120 μM) or DMSO for 30 or 60 min at 37°C. Finally, the cell supernatants were collected to detect progeny virus particles via qPCR.
Small-interfering RNA assays
The siRNA knockdown assay was performed as described previously. MARC-145 cells were transfected with siRNAs targeting Nrf2 or the siRNA negative control (siNC) for 12 h and then treated with hyperoside (120 μM) for 2 h. Subsequently, the cells were infected with PRRSV SD16 (MOI = 0.5) and harvested at 24 hpi to analyze the protein expression of p62, Nrf2, Keap1, LC3I, LC3II, and PRRSV N via western blotting. MARC-145 cells or PAMs were transfected with siTLR4 or the siRNA negative control (siNC) for 12 h and then treated with hyperoside (120 μM) for 2 h. Subsequently, the cells were infected with PRRSV SD16 (MOI = 0.1) for 24 h. The levels of IL-6, IL-8, IL-1β, and TNF-α mRNA in the cells were analyzed via qPCR, and the production of IL-6, IL-8, IL-1β, and TNF-α in the supernatants from the PAM cultures was analyzed via enzyme-linked immunosorbent assay (ELISA).
ELISA
The supernatants from the PAM cultures were harvested. IL-6, IL-8, IL-1β, and TNF-α production in the supernatants was analyzed via porcine IL-6, IL-8, IL-1β, and TNF-α ELISA kits (Jianglai, Shanghai, China) following the manufacturer’s instructions.
mCherry-GFP-LC3B puncta assay
A mCherry-GFP-LC3B puncta assay was performed to detect the degree of autophagy induced by PRRSV infection, as described in previous studies (40). Briefly, MARC-145 cells were seeded on coverslips and placed in 12-well plates. Then, these cells were transfected with the mCherry-GFP-LC3 plasmid (Miaoling plasmid, Wuhan, China) for 12 h, treated with different concentrations of hyperoside (60 μM and 120 μM) or 3-MA (30 μM), and infected with PRRSV SD16 (MOI = 0.5) for 24 h. The cells were fixed with 4% paraformaldehyde and stained with DAPI (Invitrogen, USA) for 15 min. Finally, the cells were observed and imaged via an inverted fluorescence microscope (DMi8, Leica, Germany).
Animal experiments
Sixteen 6-week-old PRRSV-negative piglets purchased from Xilingjiao Science and Technology Ltd. (Jinan, China) were randomly divided into four groups (four piglets/group). Group 1 was infected with PRRSV SD16 only (3 × 105 TCID50/piglet), while Group 2 was treated with hyperoside (75 mg/kg) and then challenged with PRRSV SD16 (3 × 105 TCID50/piglet). Group 3 was treated with hyperoside (150 mg/kg) and challenged with PRRSV SD16 (3 × 105 TCID50/piglet). Finally, Group 4 served as the negative control group (mock group). At ?1, 1, 3, 5, and 8 days post-challenge (DPC), piglets from Group 2 and Group 3 received the same oral dose of hyperoside. Moreover, piglets from Group 1 and Group 4 received an equal volume of MEM orally (including 0.1% DMSO). Piglets from each group were housed in separate rooms to prevent cross-infection. Clinical symptoms and rectal temperature were monitored daily.
All the piglets were euthanized, and their lungs were collected for pathological analysis and immunohistochemistry examinations at 28 DPC. Necropsy and pathological analysis were performed as described in our previous study (36), and pathological scores were calculated. The PRRSV loads in the serum were detected at 4, 7, 10, 14, 21, and 28 DPC, as described previously (41).
Statistical analysis
The data are presented as the means ± SDs and were analyzed with GraphPad Prism 8.0 software. The intensities of the western blot bands were analyzed via ImageJ 1.8.0 software (National Institutes of Health). Differences among the groups were analyzed via the unpaired Student’s t-test. Statistically significant and very significant differences were defined as those with P < 0.05 and P < 0.01, respectively.
RESULTS
Hyperoside suppresses PRRSV infection in various susceptible cells
The chemical structure of hyperoside is depicted in Fig. 1A. In this study, the potential cytotoxicity of hyperoside to MARC-145 and PAM cells was detected via the CCK-8 assay. As shown in Fig. 1B, at concentrations up to 120 μM, hyperoside did not reduce cell viability.
Fig 1
MARC-145 and PAM cells were subsequently preincubated with different concentrations of hyperoside and infected with PRRSV SD16. These cellular supernatants and cells were collected to evaluate PRRSV replication at 24 hpi. The results revealed that hyperoside clearly decreased progeny virus production and PRRSV N protein expression in MARC-145 (Fig. 1C and D) and PAMs (Fig. 1E and F) (***, P < 0.001). These data indicate that hyperoside effectively inhibits PRRSV infection in vitro.
Hyperoside exhibits broad-spectrum antiviral activity against different PRRSV strains
To further determine whether hyperoside has antiviral effects on different PRRSV-1 and PRRSV-2 strains, MARC-145 and PAM cells were preincubated with hyperoside (120 μM) and infected with various PRRSV-1 strains (GZ11-G1 or P073-3). The cells and cellular supernatants were collected at 24 hpi to assess PRRSV replication via western blot and TCID50?analyses. The results revealed that N protein expression was significantly lower in the hyperoside treatment group than in the DMSO treatment group (Fig. 2A). Moreover, progeny virus production was significantly lower in the hyperoside treatment group than in the DMSO treatment group (Fig. 2B) (***, P < 0.001). Notably, hyperoside had similar antiviral effects against the GZ11-G1 and P073-3 strains of PRRSV-1 in PAMs (Fig. 2C and D) (***, P < 0.001). MARC-145 and PAMs were subsequently pretreated with hyperoside (120 μM) and infected with different PRRSV-2 strains (CH-1a, JXA1, and NADC30-like). Western blot and TCID50 assays were employed to assess PRRSV replication at 24 hpi. Hyperoside significantly decreased PRRSV N protein expression and progeny virus production in both MARC-145 cells (Fig. 2E and F) and PAMs (Fig. 2G and H) (***, P < 0.001). These results showed that hyperoside exerts broad-spectrum antiviral effects on different PRRSV-1 and PRRSV-2 strains.
Fig 2
Hyperoside inhibits PRRSV infection at multiple stages
To investigate which stage of the PRRSV life cycle is affected by hyperoside, a time-of-addition assay was performed, as illustrated in Fig. 3A. MARC-145 cells were pretreated, cotreated, or post-treated with hyperoside prior to PRRSV inoculation. These cells and cellular supernatants were collected at 24 hpi to analyze PRRSV N protein expression and progeny virus production. As shown in Fig. 3B, N protein expression was significantly lower in the hyperoside pretreated, cotreated, and post-treated groups than in the PRRSV infection-only group. Notably, a lower viral load was observed in all stages (Fig. 3C) (***, P < 0.001).
Fig 3
To further determine whether hyperoside can directly inactivate PRRSV, hyperoside was mixed with different loads of PRRSV SD16 (MOI = 0.1 or 1) and preincubated at 37°C for 2 h. Then, MARC-145 cells were infected with PRRSV SD16, and qPCR and western blotting were used to evaluate PRRSV replication. As shown in Fig. 3D and E, the protein and mRNA expression levels of PRRSV N did not significantly differ between the hyperoside-treated group and the DMSO-treated group.
Previous studies have reported that the infectious cycle of PRRSV involves various stages, such as adsorption, entry, replication, and release (42, 43). Therefore, we subsequently performed a viral adsorption assay, a viral entry assay, a viral replication assay, and a viral release assay to determine the specific phase during which hyperoside exerts anti-PRRSV effects in MARC-145 cells. As depicted in Fig. 3F, compared with DMSO treatment, hyperoside treatment obviously decreased the copy number of PRRSV progeny viruses in MARC-145 cells during the adsorption stage. Similar anti-PRRSV effects were also observed in MARC-145 cells during entry (Fig. 3G), replication (Fig. 3H), and release (Fig. 3I) (**, P < 0.01; ***, P < 0.001). These data suggest that hyperoside can suppress PRRSV infection at multiple stages, although it does not exert any direct virucidal effect on PRRSV.
Hyperoside significantly attenuates PRRSV-induced pro-inflammatory cytokine production in susceptible cells
Numerous studies have shown that PRRSV infection induces the release of pro-inflammatory factors, promoting pathogenicity and lung damage (22, 44). Moreover, evidence from recent research suggests that hyperoside can exert potent anti-inflammatory effects under some pathogenic conditions by regulating inflammatory cytokine production (34, 45). To test whether hyperoside modulates the expression of pro-inflammatory factors induced by PRRSV infection, MARC-145 cells and PAMs were treated with hyperoside (60 or 120 μM) and then infected with PRRSV SD16. The cells were collected at 24 hpi to analyze IL-6, IL-8, IL-1β, and TNF-α expression. As depicted in Fig. 4A through D, hyperoside significantly reduced the IL-6, IL-8, IL-1β, and TNF-α levels in MARC-145 cells (**, P < 0.01; ***, P < 0.001). Similar results were also observed in PAMs (Fig. 4E through H). Consistent with the mRNA levels, hyperoside significantly reduced the production of IL-6, IL-8, IL-1β, and TNF-α in the supernatants of PRRSV-infected PAMs (Fig. S1) (**, P < 0.01; ***, P < 0.001). These findings indicate that hyperoside can attenuate the pro-inflammatory cytokine responses induced by PRRSV infection.
Fig 4
Hyperoside inhibits the TLR4/NF-κB signaling pathway to suppress PRRSV infection
The TLR4/NF-κB signaling pathway plays important roles in pro-inflammatory responses, cell differentiation, proliferation, and apoptosis (46). More specifically, TLR4, which is expressed on the cell surface, serves as an innate immune receptor and plays a pivotal role in initiating innate immune responses (47). NF-κB, which is downstream of TLR4, is a key transcription factor involved in inflammatory responses in mammalian cells (48). To explore the mechanisms underlying the anti-inflammatory effects of hyperoside during PRRSV infection, TLR4/NF-κB signaling pathway-related protein expression was detected via western blotting. As depicted, the levels of phosphorylated NF-κB p65 (p-NF-κB p65) and phosphorylated IκB (p-IκB) in MARC-145 cells were significantly greater in the infection group than in the mock group (Fig. 5). Hyperoside treatment at the indicated concentrations decreased the expression of TLR4 and the levels of p-NF-κB p65 and p-IκB both in the presence (Fig. 5) and absence (Fig. S2) of PRRSV infection (**, P < 0.01; ***, P < 0.001). However, the levels of NF-κB p65 and IκB were significantly increased by hyperoside treatment in a dose-dependent manner (Fig. S2). Thus, TLR4 is the primary receptor that mediates the anti-inflammatory effects of hyperoside. siRNA-mediated knockdown of TLR4 in MARC-145 or PAM cells reversed the inhibitory effects of hyperoside on pro-inflammatory factors (Fig. S3) (***, P < 0.001). These data indicate that hyperoside treatment attenuates the expression of pro-inflammatory factors induced by PRRSV via the inhibition of the TLR4/NF-κB signaling pathway.
Fig 5
Hyperoside reduces PRRSV-induced autophagy
The autophagy induced by viral infection can promote viral replication. Previous studies have shown that PRRSV can hijack the autophagy pathway to increase viral self-replication (29, 49). Interestingly, hyperoside has been found to attenuate rotenone-induced neuronal injury and sepsis-induced acute lung injury via the regulation of autophagy (33, 34). To test whether hyperoside inhibits PRRSV infection via the suppression of autophagy, MARC-145 cells were pretreated with different concentrations of hyperoside (60 or 120 μM) or 3-AM (30 μM) and infected with PRRSV SD16 (MOI = 0.5). As depicted in Fig. 6, compared with mock-infected MARC-145 cells, PRRSV-infected MARC-145 cells presented an obvious band pattern transformation from LC3I to LC3II, and the ratio of LC3II/LC3I was increased, leading to degradation of p62. Moreover, hyperoside significantly inhibited PRRSV-induced autophagy in a dose-dependent manner. Notably, both hyperoside and 3-MA significantly decreased PRRSV N protein expression (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
Fig 6
Fluorescence microscopy was performed to confirm the effect of hyperoside on PRRSV-induced autophagy. The results revealed that PRRSV infection promoted autophagic plaque formation, but these changes were reversed by hyperoside in PRRSV-infected MARC-145 cells (Fig. 7) (*, P < 0.05; **, P < 0.01; ***, P < 0.001). These findings demonstrated that hyperoside can inhibit PRRSV replication by attenuating virus-induced autophagy.
Fig 7
Hyperoside attenuates PRRSV-induced autophagy through p62/Nrf2/Keap1 signaling axis activation
The p62/Nrf2/Keap1 signaling pathway has been implicated in numerous cellular functions, such as autophagy, apoptosis, and ferroptosis (50–53). In this study, we first tested whether hyperoside could promote p62/Nrf2/Keap1 activation. MARC-145 cells were treated with different concentrations of hyperoside for 24 h, and the total cell lysates were harvested and analyzed via western blotting. As shown in Fig. 8A, hyperoside increased the accumulation of p62 and total Nrf2 in MARC-145 cells in a dose-dependent manner. Moreover, it induced a corresponding decrease in Keap1 expression. Moreover, p62, Nrf2, and Keap1 expression in MARC-145 cells incubated with hyperoside were also detected at different time points via western blotting. As shown in Fig. 8B, hyperoside increased the accumulation of p62 and total Nrf2 in MARC-145 cells in a time-dependent manner, and Keap1 expression also decreased after 2 h of incubation with hyperoside. A previous study reported that hyperoside decreases rotenone-induced neuronal injury via the inhibition of autophagy (33). To further test whether the anti-PRRSV activity of hyperoside is related to autophagy regulation by the p62/Nrf2/Keap1 axis activation, MARC-145 cells were treated with different concentrations of hyperoside and infected with PRRSV SD16. These cells were collected at 24 hpi to analyze p62, Nrf2, Keap1, LC3II/I, and PRRSV N protein expression via western blot analysis. As shown in Fig. 8C, hyperoside decreased the ratio of LC3II/I and downregulated N protein expression in MARC-145 cells while increasing p62 and Nrf2 expression. In addition, we found that p62 and Nr2 were colocalized in the nucleus in PRRSV-infected cells treated with hyperoside (Fig. S4).
Fig 8
These data suggest that hyperoside may promote the nuclear translocation of Nrf2 to increase the transcription and expression of p62.
In another set of experiments, MARC-145 cells were transfected with siNrf2 or siNC, treated with hyperoside, and subsequently infected with PRRSV. Nrf2, Keap1, LC3I, LC3II, p62, and N protein expression in these cells were subsequently analyzed. As expected, siNrf2 attenuated the effects of hyperoside on the LC3II/I ratio and N protein expression (Fig. 8D). Collectively, these findings demonstrated that hyperoside exerts anti-PRRSV effects by suppressing PRRSV-induced autophagy via the p62/Nrf2/Keap1 signaling pathway.
Hyperosides exhibit anti-PRRSV activity in piglets
To further analyze the anti-PRRSV activity of hyperoside in vivo, piglets were challenged with PRRSV SD16 and treated with hyperoside (Fig. 9A). The rectal temperatures of all piglets were monitored daily after the PRRSV challenge. As shown in Fig. 9B, the average temperatures of the piglets in the PRRSV SD16 challenge-only group began to rise at 3 DPC, peaking at approximately 41°C by 8 DPC and remaining higher than 40°C until 17 DPC. In contrast, the rectal temperatures of the 75 mg/kg hyperoside-treated group rose slowly from 5 DPC, peaking at approximately 40.3°C by 14 DPC. Moreover, the rectal temperature of the 150 mg/kg hyperoside-treated group rose slowly from 7 DPC to a peak temperature of approximately 40.1°C at 10 DPC. In this group, high temperatures above 40°C were detected at 8–10 DPC (*, P < 0.05; **, P < 0.01; ***, P < 0.001). All the piglets exhibited various clinical signs of viral infection, including lethargy, a rough coat, poor appetite, dyspnea, and periocular edema. The clinical scores of piglets treated with 75 and 150 mg/kg hyperoside were significantly lower than those of untreated piglets challenged with PRRSV SD16 at 1–7, 8–14, 15–21, and 22–28 DPC (Fig. 9C) (***, P < 0.001). Consistent with the clinical symptom scores, the serum copy numbers of PRRSV in piglets treated with 75 or 150 mg/kg hyperoside were also obviously lower than those in untreated piglets challenged with PRRSV SD16 (Fig. 9D) (***, P < 0.001).
Fig 9
The therapeutic action of hyperoside against PRRSV-induced lung damage was also examined. We observed gross lung lesions in the piglets, and untreated piglets infected with SD16 presented more severe pulmonary consolidation than those treated with hyperoside. In contrast, no pathological lesions were identified in the negative control group (Fig. 9E) (***, P < 0.001). Microscopic lung lesion analysis revealed severely thickened alveolar septa and high infiltration of inflammatory cells in the SD16-only group, but these pathologies were milder in the piglets treated with 75 or 150 mg/kg hyperoside (Fig. 9F) (***, P < 0.001). Moreover, more PRRSV proteins were distributed in the lung tissue of the SD16-only group than in those of the hyperoside 75 mg/kg and hyperoside 150 mg/kg groups (Fig. 9G). The mean gross lesion and microscopic lesion scores of the lungs were obviously greater in the SD16-only group than in the negative control group and significantly greater than those in the hyperoside 75 mg/kg and hyperoside 150 mg/kg groups (Fig. 9E and F). However, 150 mg/kg hyperoside was more effective than 75 mg/kg hyperoside in reducing the clinical score.
DISCUSSION
PRRSV is an important pathogen that causes serious disease in pigs and hinders the development of the global swine industry. Current vaccines and drugs do not provide adequate protection against this virus, and new antiviral therapies are urgently needed. In the present study, hyperoside was found to exert potent anti-PRRSV effects in susceptible cells (Fig. 1 and 2) and piglets (Fig. 9). Furthermore, our mechanistic evaluation revealed that hyperoside alleviates pro-inflammatory responses by inhibiting the TLR4/NF-kB signaling pathway. Additionally, it suppresses PRRSV-induced autophagy via p62/Nrf2/Keap1 axis activation (Fig. 10).
Fig 10
Viral infection involves multiple phases, namely, binding, internalization, replication, and release (54). Recent studies have reported that hyperoside exerts antiviral effects at different stages of viral infection. For example, Wang et al. reported that hyperoside inhibits EHV-8 infection by disrupting the adsorption and internalization phases (17). In contrast, in another study, hyperoside was found to suppress SARS-CoV-2 infection by disrupting viral entry and replication (55). Furthermore, Wang et al. reported that hyperoside inhibits PEDV infection by preventing the interaction between the N protein and p53 in the replication phase (10). Notably, the results of our study demonstrated that hyperoside prevents PRRSV infection at the binding, entry, replication, and release stages without causing direct virus inactivation (Fig. 3).
The inflammatory response represents the host’s defence against viral infections. However, uncontrolled inflammatory responses usually cause severe damage to the host (56). PRRSV infection triggers the uncontrolled production of pro-inflammatory cytokines (IL-6, IL-8, IL-1β, and TNF-α) to promote self-replication (57). Interestingly, our study revealed that hyperoside can attenuate the upregulation of IFN-β, IL-6, IL-8, and TNF-α induced by PRRSV infection in susceptible cells (Fig. 4). NF-κB is a crucial mediator of inflammatory responses (58), and previous studies have shown that HP-PRRSV can induce pro-inflammatory cytokine production by regulating the TAK-1/NF-κB signaling pathway (59). Kim et al. reported that hyperoside exerts anti-inflammatory effects by blocking NF-κB activation in mouse peritoneal macrophages (60). In the present study, we explored the anti-inflammatory mechanisms of hyperoside against PRRSV infection. Our results revealed that hyperoside treatment reduces TLR4, p-NF-κB p65, and p-IκB expression in virus-infected cells (Fig. 5). Our data suggest that hyperoside can attenuate PRRSV-induced inflammatory storms by inhibiting the TLR4/NF-κB signaling pathway.
The relationship between viral replication and autophagy has been explored extensively. Autophagy can serve as a pro- or antiviral process during virus infection (61). Diao et al. demonstrated that PRRSV infection induces autophagy via endoplasmic reticulum stress-induced calcium signaling, which promotes viral replication (40). In the present study, we demonstrated that hyperoside obviously decreased the ratio of LC3II/I expression in PRRSV-infected cells in a dose-dependent manner (Fig. 6). Additionally, we found that hyperoside can inhibit PRRSV-induced autophagic plaque formation in susceptible cells (Fig. 7). In addition, our findings revealed that hyperoside has powerful anti-PRRSV activity both in vitro and in vivo. However, the present study has several limitations, and further investigations are needed to explore the direct targets of hyperoside.
Conclusions
In conclusion, our results revealed that hyperoside inhibited viral binding, entry, replication, and release during PRRSV infection in a dose-dependent manner. In addition, hyperoside alleviated the inflammatory responses and autophagy caused by PRRSV by suppressing the TLR4/NF-κB pathway and activating the p62/Nrf2/Keap1 axis. Our findings suggest that hyperoside has potential as a promising therapeutic candidate for PRRSV control, warranting further investigations and preclinical studies to evaluate its efficacy and safety.
ACKNOWLEDGMENTS
We thank Shujing Wang from Shandong Agricultural University for providing support in performing the laser confocal experiments in the revised manuscript.
This work was funded by the Shandong Province Pig Industrial Technology System (SDAIT-08-01); Shandong Provincial Natural Science Foundation (ZR2024MC162); the Innovation and Entrepreneurship Program for College Students (CXCY307); the Key Research and Development Project in Shandong Province (2022CXPT010, 2022TZXD0041); and the Taishan Scholars Program.
T.W., L.C., and A.W.: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing—original draft, Writing—review and editing.
Y.X., Y.Z., Q.Z., Q.R., and S.L.: Data curation, Formal analysis, Investigation, Methodology, and Writing—review and editing. Y.L. and J.W.: Writing—review and editing, supervision, and validation.
L.L., Y.L., and J.W.: Conceptualization, Funding acquisition, Validation, Methodology, Project administration, Resources, Software, Supervision, Writing—original draft, Writing—review and editing.
All the authors have read and approved the published version of the manuscript.
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Information & Contributors
Information
Published In
Microbiology Spectrum
Volume 13 ? Number 8 ? 5 August 2025
eLocator: e03107-24
Editor: Holly Ramag, Thomas Jefferson University, Philadelphia, Pennsylvania, USA
PubMed: 40503831
Copyright
? 2025 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
History
Received: 2 December 2024
Accepted: 13 May 2025
Published online: 12 June 2025
Keywords
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All the data generated or analyzed during this study are included in this published article and its additional files.
Contributors
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Holly Ramag
Editor
Thomas Jefferson University, Philadelphia, Pennsylvania, USA
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This study adhered to ARRIVE guidelines, and all experimental protocols were approved by the Liaocheng University Animal Care and Use Committee (permit number LC2024-03) and complied with the Animal Ethics Procedures and Guidelines of the People’s Republic of China. No other specific permissions were required for these activities. This study did not involve endangered or protected species.
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2025.
Hyperoside inhibits PRRSV proliferation via the TLR4/NF-κB and p62-Nrf2-Keap1 signaling pathways, mediating inflammation and autophagy. Microbiol Spectr
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