Kaempferol inhibits pseudorabies virus replication in vitro through regulation of MAPKs and NF-κB signaling pathways

Pseudorabies virus (PRV), belonging to the family Herpesviridae, is a pathogen of Aujeszky’s disease leading great economic losses to pig industry. Re-outburst of pseudorabies implies that new control measures are urgent needed. The present study provides a candidate drug for PRV infection that kaempferol possesses the ability to inhibit PRV replication in a dose-dependent manner in vitro. Kaempferol at a concentration of 52.40 μM could decrease PRV-induced cell death by 90%. Kaempferol with a IC50 of 25.57μM is more effective than acyclovir (Positive control) with a IC 50 of 54.97 μM. Mode of action study indicated that kaempferol inhibited viral penetration and replication stages and virus load was decreased by 4-fold and 30-fold, respectively. Addition of kaempferol within 16 hours post infection (hpi) could signicantly inhibit virus replication, and the DNA copies were decreased by almost 15-fold when kaempferol was added at 2 hpi. Kaempferol could regulate NF-κB and MAPKs signal pathways involved in PRV infection and change the levels of the target genes of MAPKs (ATF-2 and c-Jun) and NF-κB (IL-1α, IL-1β and IL-2) signaling pathways. All the results indicated that kaempferol has the ability to be an alternative control measure for PRV infection.

measure for Aujeszky's disease. Besides, acyclovir, exhibiting dramatically anti-herpesvirus activity, was selected as a positive control.

Cytotoxicity and antiviral activity of kaempferol and acyclovir
The cytotoxicity of different concentrations of ethyl alcohol and DMSO on PK-15 cells were tested since kaempferol was dissolved in ethyl alcohol and acyclovir was dissolved in DMSO during the anti-PRV activity evaluation. The results indicated that when the nal concentrations of ethyl alcohol and DMSO were below 1%, there were no cytotoxicity and no anti-PRV activity on PK-15 cells (data not shown). Therefore, throughout the tests, the concentrations of ethyl alcohol or DMSO were no more than 1%.
There was no toxicity for PK-15 cells when the concentration of kaempferol was below 104.81 μM and the CC 50 was 212.97 μM. The CC 50 of acyclovir was 348.85 μM. kaempferol at the concentration of 52.40 μM could inhibit PRV-induced cell death by 90% (Fig 1). In contrast, the inhibition rate of acyclovir could reach to 90%, when the concentration is 113.23 μM (Fig 1). The IC 50 of kaempferol was 25.57μM, which had a selectivity index (SI) of 8. 33. The IC 50 of acyclovir was 54.97 μM, with a SI of 6.34 (Table 1). a Cytotoxic concentration 50% (CC50), concentration required to reduce PK-15 viability by 50%, was measured by CCk-8 method.
b Inhibition concentration 50% (IC50), concentration required to reduce inhibit 50% cell death caused by PRV-infection. c SI: Selectivity index is de ned as the ratio of CC50 to IC50 (SI = CC50/IC50).
In order to test whether kaempferol could also show anti-PRV activity when the initial amount of infected virus was increased, PK-15 cells were infected with MOI = 1 PRV/well in the presence or absence of kaempferol or acyclovir. Kaempferol could signi cantly inhibit PRV replication in the concentrations ranged from 52.40 to 13.10 μM (Fig 2). When the concentration of kaempferol was 52.40 μM, the DNA copies were inhibited by about 50-fold in comparison with untreated group. In contrast, acyclovir could reduce the viral DNA copies by about 6-fold at a concentration of 113.23 μM (Fig 2). Kaempferol exhibited higher anti-PRV activity than acyclovir when the input virus was increased to MOI = 1.

Mode of action
For elucidating the mode of action of kaempferol, according to different phases of viral life cycle, ve independent experiments were performed. In antiviral tests, kaempferol and acyclovir sometimes directly incubated with the virus during infection. Inactivation was conducted to evaluate whether the observed antiviral effects were due to their inactivation, but no activity was detected (data not shown). In pretreatment assay, the drug was incubated with cells prior to virus infection, which was designed to detect whether the kaempferol could bind to virus receptors on the cell surface involved in the initial virus entry. However, neither kaempferol nor acyclovir exhibited inhibitory activity (data not shown). During virus adsorption, PRV and kaempferol / acyclovir were simultaneously incubated with cells. The results also showed that kaempferol and acyclovir could not inhibit virus adsorption (data not shown). After attachment to cell surface, penetration was triggered. Kaempferol could inhibit PRV penetration and the viral DNA copies were reduced by almost 4-fold (Fig. 3A), while acyclovir exhibited no activity (data not shown

Discussion
PRV infection is a constant threat for many countries [33,34], especially occur of the emerging PRV variant. However, there is no speci c medicine for PRV infection but vaccine which cannot provide full protection against variants [11]. Herein an alternative natural compound kaempferol is demonstrated to possess potent anti-PRV activity though regulating the NF-κB and MAPKs signaling pathways, which exhibits the potential for control of PRV infection.
As the common indexes to evaluate the antiviral activity of compounds [35], CC 50 and IC 50 were always tested in antiviral study. The CC 50 of kaempferol is lower than that of acyclovir, indicating that acyclovir had higher safe level to PK-15 cells. The IC 50 and SI of kaempferol were higher than those of acyclovir, which suggested that kaempferol exhibited a higher anti-PRV activity on PK-15 cells than acyclovir. For further con rmation of anti-PRV e cacy, the initially amount of infected virus were increased, kaempferol decreased virus reproduction by an order of magnitude.

Just as herpesvirus infection is initiated by attachment of virions to target cells and fusions of the virion
envelope and the cellular cytoplasmic membrane [36], the similar processes were taken in the PRV infection of cells. The PRV attachment is made by interaction of virion gC with heparin sulfate proteoglycans at the cell surface, which formes a primary, relatively labile interaction, followed by interaction of gD with its cellular receptor which mediates the conversion of the labile interaction into the stable binding [37]. A tight contact is needed between the cellular cytoplasmic membranes and the viral envelop to complete their fusion which at least requires four viral glycoproteins: gB, gH, gL and gD [36]. Nucleocapsid is translocated into the cytoplasm of the cell, then transported to the nuclear membrane and locates next to nuclear pores. Then a cascade-like fashion started with the expression of only one immediate-early gene to regulate PRV transcription [38]. In the present study, the capability of kaempferol to reduce virus titer, a basic index to evaluate the antiviral activity of samples [39], at different infection stages was tested. The study con rmed that kaempferol had ability to decrease the virus titer, especially at penetration and replication stages, which implied that kaempferol could inhibit the function of gB, gH, gL or gD [36] and the cascade-like fashion of PRV transcription [40]. Time of addition study implied that the inhibition of kaempferol on PRV occurs at early stage of PRV replication cycle. Therefore, kaempferol exhibited the possibilities to inhibit the transcription of immediate-early and early genes of PRV. Acyclovir only inhibited replication stage of PRV. The previous studies implied that acyclovir is targeted at thymidine kinase, whose activity could be activated by a HSV-or VZV-speci ed TK [41]. Besides, acyclovir is similar with deoxynucleoside triphosphate and has ability to compete with deoxyguanosine triphosphate for the viral DNA polymerase [41]. That's why the anti-PRV activity of acyclovir mainly occurred at replication stage. However, antiviral activity of kaempferol was higher dramatically than that of acyclovir, which is consistent with the values of IC 50 and SI.
NF-κB and MAPKs signaling pathways take part in innate immunity [42,43], which plays an important role in virus infection [44]. The acute response against virus infection should be a general and natural biological effect and NF-κB was activated by PRV infection at 4hpi by a luciferase reporter assay [45]. In the present study, at 4hpi, as the early stage of the PRV infectious cycle [45], the levels of P65 and P-P65 were increased signi cantly. However, at 8 hpi and 12 hpi the levels of P65 and P-P65 were decreased signi cantly. Besides, after kaempferol treatment, the levels of P65 were increased signi cantly, which indicated that NF-κB mediates PRV-induced immunity, but kaempferol was not able to inhibit the activation of NF-κB. Virus infection changes the expression of ERK1/2. The previous study implied that KSHV induces ERK1/2 very early in infection and the authors speculated that the activation probably allowed it to overcome the restriction on viral gene transcription imposed by the host cell and facilitated the viral gene expression and thus the establishment of infection [46].The study by zhao et.al indicated that PRV infection activated ERK1/2 [35]. In the present study, ERK1/2 was activated by PRV infection at early stage (4hpi) and inhibited at 8hpi and 12hpi, and kaempferol treatment was able to recover the level of ERK1/2 changed by PRV infection. The activation of P38 signaling takes part in PRV-induced apoptosis [47]. Compared with the levels of P38 and P-P38 on blank control cells, those were enhanced after PRV infection at 4hpi and 12hpi, while those were decreased at 8hpi. The levels of P38 and P-P38 changed by PRV infection were recovered with kaempferol treatment. Perhaps the anti-PRV activity of kaempferol on PK-15 cells generated by the effect on MAPKs rather than NF-κB. Based on the previous studies [48], the PRV-induced apoptosis in cultural cells was clari ed. NF-κB and MAPK signal pathways also mediate cell apoptosis [45,49,50], which perhaps is one of the reasons that PRV induced the changes of NF-κB and MAPKs signal pathways.
Pro-in ammatory cytokines were always increased by virus infection [51]. In the study, the expressions of IL-1α [52], IL-1β [53] and IL-2 [54,55], as the target genes of NF-κB, were detected to further con rm the NF-κB signal pathway that was regulated by kaempferol. The results implied that the levels of IL-1α, IL-1βand IL-2 were up-regulated and kaempferol treatment down-regulated signi cantly the levels of IL-1βand IL-2, which is consistent with the previous study [35].Virus infection always changes the target genes of MAPKs signal pathway [46]. In the study, the expressions of c-Jun, c-Fos, ATF-2, MEF2A, c-Myc and STAT1, as the target genes of MAPKs signal pathway [46], were changed by PRV infection, which is consistent with the previous study [56]. Kaempferol treatment decreased signi cantly the expressions of ATF-2 and c-Jun which were increased by PRV infection, but did not recover the expressions of c-Fos, MEF2A, c-Myc and STAT1. The c-Jun, as the most extensively studied protein of activator protein-1(AP-1) complex, takes part in variety cell activities, including proliferation, apoptosis, survival, tumorigenesis and tissue morphogenesis [57]. In the study, the expression of c-Jun was increased by PRV infection, which possibly due to c-Jun mediated the apoptosis of cells induced by PRV infection, and kaempferol possesses the ability to inhibit the effects induced by c-Jun.
The study on the anti-cancer activity of kaempferol disclosed that kaempferol exerts protective effects for non-mutated cells and triggers apoptosis for mutated cells [20]. In the present study, the regulation effects of kaempferol were also different on normal cells and PRV-infected cells. Based on the innate immunity and anti-apoptosis effects of NF-κB and MAPKs signal pathways, we speculated that kaempferol exhibited anti-PRV activity by regulating innate immunity and protecting cells from PRVinduced apoptosis, which probably attributed to the antioxidant effects of kaempferol on inhibition of ROS generation and lipid peroxidation, then protecting the cells from damaged in a broad-spectrum activity [58].

Conclusion
In conclusion, kaempferol exhibited anti-PRV activity in PK-15 cells, which is better than acyclovir. Kaempferol mainly inhibited PRV replication through regulation of NF-κB and MAPKs signal pathways and their target genes (Fig 7). Further study should be conducted to evaluate antiviral activity of kaempferol against PRV in vivo.

Compounds
Kaempferol and acyclovir were bought from Sigma with a purity of 98% and kept at 4°C from light. For antiviral assays, kaempferol was dissolved in 95% ethanol at the concentration of 349.36μM and acyclovir was dissolved in dimethyl sulfoxide (DMSO) at concentration of 444.05μM as the stored solutions. The stored solutions were diluted by at least 100-fold to different concentrations in Dulbecco's modi ed eagle medium (DMEM, HyClone) for antiviral assays.
Cell and virus PK-15 cell was preserved in Natural Medicine Research Center Sichuan Agricultural University (Chengdu, China) and grown in DMEM (high glucose) supplemented with 10% (v/v) fetal calf serum (Hyclone), 100U/mL penicillin and 100μg/mL streptomycin. For maintenance medium (MM), the serum concentration was reduced to 2%.

PRV (RA strain) was bought from China Veterinary Culture Collection Center (Beijing, China) and
propagated in PK-15 cells. The 50% tissue culture infective dose (TCID 50 ) was determined as 10 -6.1 /mL.

50% cytotoxic concentration assay
The cytotoxicity of kaempferol and acyclovir was evaluated by determination of cell viability with cell counting kit-8 (CCK-8; Dojindo, Kumamoto, Japan) assay according to the manufacturer's instructions. PK-15 monolayers grown in 96-well plates were incubated with MM containing two-fold dilutions of kaempferol or acyclovir. After incubation at 37℃ for 48 h, 10 μL of the CCK-8 solution was added to each well of the plate. The plates were re-incubated for 30 min at 37 ℃, followed by measurement of absorbance values in a microplate reader (Bio-Rad) at 450 nm. The 50% cytotoxic concentration (CC 50 ), the concentration of kaempferol or acyclovir required to reduce cell viability by 50%, was calculated through the Reed-Muench method [59].

50% inhibition concentration assay
The PK-15 monolayer cells in 96-well plates were infected with 200TCID 50 PRV/well with or without a series of two-fold dilutions of kaempferol or acyclovir. After incubation at 37℃ for 1 h, the medium was removed and MM containing a corresponding dose of kaempferol or acyclovir was added. The virusinfected cells with ethanol treatment (1%, 0.5% and 0.25%) or DMSO treatment (1%, 0.5% and 0.25%) were used as solvent controls. When cells in control groups showed approximately 80% cytopathic effect (CPE), CCK-8 assays was performed as described above. The 50% inhibitory concentration (IC 50 ), the concentration of kaempferol or acyclovir required to inhibit 50% cell death caused by viral infection [60], was calculated through the Reed-Muench method [59].

MOI assay
The PK-15 cells in 6-well plates were infected with MOI = 1 PRV at present or absent of different concentrations of kaempferol or acyclovir for 1h at 37 ℃, then the medium were removed and the plates were washed thrice with PBS. The different concentrations of kaempferol or acyclovir were added again and MM were added to cells of control groups. When cells in control groups showed approximately 80% CPE, the sample were subjected to twice freezing and thawing, total DNA was extracted from each well by using DNAiso Reagent (D305; Takara, China) according to the manufacturer's instructions. For determination of viral gene copies, uorescent quantitative polymerase chain reaction (FQ-PCR) was performed by using a Bio-Rad CFX96 Connect TM real-time PCR detection system (CA, USA) according to the method described by Zhao et al., 2017 [35].

Inhibitory action assay
Pre-treatment assay. Kaempferol and acyclovir were added to cell monolayers and the plates were incubated at 37℃ for 1h. Then the medium were removed and the monolayers were incubated with 200 TCID 50 PRV for 1h at 37℃. After incubation for 48h, the total DNA was extracted from PRV-infected cells and the virus titers in each well were measured by FQ-PCR method as described above.
Adsorption assay.A PK-15 monolayer grown in 6-well plates was infected with 200TCID 50 PRV/well in the presence of dilutions of kaempferol or acyclovir. After incubation at 4 ℃ for 1h, the cells was washed thrice with cold PBS to remove unadsorbed viruses and then incubated with MM at 37℃. After incubation for 48h, the virus titers in each well were measured by FQ-PCR method as described above.
Penetration assay. A PK-15 monolayer cells grown in 6-well plates was infected with 200TCID 50 PRV/well and incubated at 4 ℃ for 1h. The medium was aspirated and monolayers were washed twice with cold PBS to remove unadsorbed viruses. Dilutions of kaempferol were then added to each well and acyclovir were added as positive group, then plates were incubated at 37 ℃ for 1h to allow penetration. After removal of medium, the monolayer was washed with a citrate buffer (pH = 3.0) to inactivate virion that had not penetrated the cells. MM was added and the plates were incubated at 37 ℃ for 48h. The virus titers in each well were measured by FQ-PCR method as described above.
Replication assay. 6-well plates containing PK-15 monolayers were infected with 200TCID 50 PRV/well and incubated at 37 ℃ for 1h to allow adsorption and penetration. After washing, MM containing dilutions of kaempferol or acyclovir were then added to each well and plates were incubated at 37 ℃.
After incubation for 48h, the virus titers in each well were measured by FQ-PCR method as described above.
Inactivation assay. MM containing dilutions of kaempferol or acyclovir were incubated with an equal volume of concentrated virus suspension (2 × 10 6 TCID 50 PRV) at 37 ℃ for 1 h. The solution was then diluted 10 4 times and then added into PK-15 monolayer to allow infection of the residual infectious virus. After incubation for 48h, the virus titers in each well were measured by FQ-PCR method as described above.
Time of addition assay. The PK-15 monolayers in 6-well plates were infected with 1 mL/well 200TCID 50 PRV for 1h at 37℃, then the plates were washed with PBS for 3 times. The MM were added and the plates were re-incubated. Then kaempferol was added at 2  Table 2. The PCR cycling was 3min at 95℃, then 40 cycles of 10 s at 95℃, 30 s at 59.8℃, and 55℃ for 5 s. At the end of the cycling a melt curve analysis was done. Expression of β-actin was used to normalize the differences in total mRNA expression in each sample. Data analysis was performed using Bio-Rad CFX Manger software. Availability of data and materials All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.   The DNA copies of PRV-infected cells treated with different concentrations of kaemepferol and acyclovir.

Funding
The PK-15 cells were infected with MOI=1 PRV, followed by treatment with kaemepferol and acyclovir for 24h. The viral DNA copies were determined by FQ-PCR. Control, the PRV-infected group without any treatment. ***, P<0.001; **, P<0.01; *P<0.05 vs Control.  Anti-PRV activity of Kaempferol at different addition times. PK-15 cells were infected with 200TCID50 PRV/mL. Then kaempferol (52.40 μM) was added at 2hpi, 4hpi, 8hpi and 16hpi, respectively. The viral DNA copies of each sample was tested by FQ-PCR. Control, the PRV-infected group without any treatment. ***, P<0.001 vs Control.